# RECENT ADVANCES IN BIOREMEDIATION/ BIODEGRADATION BY EXTREME MICROORGANISMS, 2nd Edition

EDITED BY : Edgardo Donati, Rajesh K. Sani, Kian Mau Goh and Kok Gan Chan PUBLISHED IN : Frontiers in Microbiology

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ISSN 1664-8714 ISBN 978-2-88971-167-3 DOI 10.3389/978-2-88971-167-3

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# RECENT ADVANCES IN BIOREMEDIATION/ BIODEGRADATION BY EXTREME MICROORGANISMS,

2nd Edition

Topic Editors:

Edgardo Donati, National University of La Plata, Argentina Rajesh K. Sani, South Dakota School of Mines and Technology, United States Kian Mau Goh, University of Technology Malaysia, Malaysia Kok Gan Chan, University of Malaya, Malaysia

Publisher's note: This is a 2nd edition due to an article retraction.

Citation: Donati, E., Sani, R. K., Goh, K. M., Chan, K. G., eds. (2021). Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms, 2nd Edition. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88971-167-3

# Table of Contents

*04 Editorial: Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms*

Edgardo Rubén Donati, Rajesh Kumar Sani, Kian Mau Goh and Kok-Gan Chan

*06 Low-Abundance Members of the* Firmicutes *Facilitate Bioremediation of Soil Impacted by Highly Acidic Mine Drainage From the Malanjkhand Copper Project, India*

Abhishek Gupta, Avishek Dutta, Jayeeta Sarkar, Mruganka Kumar Panigrahi and Pinaki Sar

*24 Living at the Frontiers of Life: Extremophiles in Chile and Their Potential for Bioremediation*

Roberto Orellana, Constanza Macaya, Guillermo Bravo, Flavia Dorochesi, Andrés Cumsille, Ricardo Valencia, Claudia Rojas and Michael Seeger

*49 Enzymatic Bioweathering and Metal Mobilization From Black Slate by the Basidiomycete* Schizophyllum commune

Julia Kirtzel, Soumya Madhavan, Natalie Wielsch, Alexander Blinne, Yvonne Hupfer, Jörg Linde, Katrin Krause, Aleš Svatoš and Erika Kothe

*59 Extremophilic Microfactories: Applications in Metal and Radionuclide Bioremediation*

Catarina R. Marques


Maximiliano Figueroa, Valentina Fernandez, Mauricio Arenas-Salinas, Diego Ahumada, Claudia Muñoz-Villagrán, Fabián Cornejo, Esteban Vargas, Mauricio Latorre, Eduardo Morales, Claudio Vásquez and Felipe Arenas


# Editorial: Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms

#### Edgardo Rubén Donati <sup>1</sup> \*, Rajesh Kumar Sani <sup>2</sup> , Kian Mau Goh<sup>3</sup> and Kok-Gan Chan4,5

<sup>1</sup> Facultad de Ciencias Exactas, CINDEFI (CCT, La Plata-CONICET, UNLP), Universidad Nacional de la Plata, La Plata, Argentina, <sup>2</sup> Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City, SD, United States, <sup>3</sup> Faculty of Sciences, Universiti Teknologi Malaysia, Skudai, Malaysia, <sup>4</sup> Division of Genetics and Molecular Biology, Faculty of Science, Institute of Biological Sciences, University of Malaya, Kuala Lumpur, Malaysia, <sup>5</sup> International Genome Centre, Jiangsu University, Zhenjiang, China

Keywords: pollutants, bioremediation, biodegradation, biomineralization, extremophiles

#### **Editorial on the Research Topic**

#### **Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms**

#### Edited by:

Jean Armengaud, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), France

#### Reviewed by:

Rafael Bosch, University of the Balearic Islands, Spain

\*Correspondence: Edgardo Rubén Donati donati@quimica.unlp.edu.ar

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

> Received: 26 June 2019 Accepted: 26 July 2019 Published: 14 August 2019

#### Citation:

Donati ER, Sani RK, Goh KM and Chan K-G (2019) Editorial: Recent Advances in Bioremediation/Biodegradation by Extreme Microorganisms. Front. Microbiol. 10:1851. doi: 10.3389/fmicb.2019.01851 To remediate polluted sites, biological processes have many advantages from economic, environmental, and practical aspects. Adsorption and biodegradation of organic contaminants and the immobilization, mobilization, and/or transformation of metal(loid)s are the main remediation processes that can be mediated by the action of several microorganisms especially those extremophiles surviving in hostile environments with high concentrations of pollutants. The aim of this Research Topic of Frontiers in Microbiology is to provide an appropriate platform to publish the latest results on the bioremediation of various pollutants by extremophilic pure cultures or microbial consortia. This Research Topic consists of 4 reviews and 7 original articles.

Marques reviewed the theme of extremophiles as microfactories which are able to provide genetic or metabolic mechanisms as controlled services to the clean-up of environmental pollution. The review focuses on metal and radionuclides pollution, and includes a discussion about the use of synthetic biology to improve the bioremediation processes.

Two articles in this Research Topic are focused on heavy metal(loid)s contaminants. Figueroa et al. described that several microorganisms exhibited high resistance to 19 metal(loid)s. Most of those strains displayed metal or metalloid reducing activity, and have been successfully used for the biological synthesis of nanostructures containing metal(loid)s. Tellurium and gold nanostructures showed antibacterial properties, which inhibited E. coli and L. monocytogenes growth. Acid mine drainage (AMD) is considered a severe environmental problem provoked by the microbial oxidation of sulfidic minerals. Gupta et al. explored the abundance and role of indigenous microorganisms displaying sulfate- and metal(loid)- reducing activity in the natural attenuation of an AMD impacted soil (AIS). The addition of nutrients (e.g., cysteine and lactate) to AIS increased the activity of such microorganisms achieving an increase in pH from 3.5 to 6.6, and reduction of sulfate (95%), iron (50%), and other heavy metals. In this way, Gupta et al. demonstrated that addition of nutrients could biostimulated the growth of some members of phylum Firmicutes (e.g., sulfate- and iron- reducing microorganisms) and bioremediate AMD impacted sites.

Orellana et al. reviewed extensively the most recent research on polyextremophilic microorganisms isolated from a wide range of extreme environments including salars, geothermal springs, deserts, ice fields, and diverse zones in Chile such as Altiplano, Atacama Desert, Central Chile, Patagonia, and Antarctica. This review also discussed the molecular and physiological capabilities of many of these isolates which were beneficial for bioremediation processes.

**4**

Diverse anthropogenic activities, particularly the emission due to the burning of fossil fuels, have triggered an alarming rise of CO<sup>2</sup> in the environment. A description of the measures of greenhouse gases emission is reviewed by Bose and Satyanarayana. In this review, authors discussed the merits and demerits of various approaches with extensive bibliographical material. Finally, a deep description of the use of carbonic anhydrases (CA) for biomineralization of CO<sup>2</sup> was included. This methodology was proposed as one of the most economical methods to mitigate global warming.

The other six articles in this Research Topic are related to the bioremediation of organic pollutants. Park and Park described the strategies for alkane degradation under extreme conditions (e.g., low and high temperatures, high salt, and acidic and anaerobic conditions). Alkane degraders seem to possess exclusive metabolic pathways and survival strategies. The thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus uses a novel alkylsuccinate synthase for longchain alkane degradation, and the thermophilic Candidatus Syntrophoarchaeum butanivorans anaerobically oxidizes butane via alkyl-coenzyme M formation. In addition to alkane degradation, extremophiles produce energy via the glyoxylate shunt and the Pta-AckA pathway when grown on a diverse range of alkanes under stress conditions.

Blázquez et al. focused on the bioremediation of aromatic compounds such as toluene and xylenes. The degradation of such pollutants is relevant due to their carcinogenic and neurotoxic effects to humans. This article provided evidences that the bss and bbs genes are not only essential for anaerobic degradation of toluene but also for m-xylene oxidation in the beta-proteobacterium Azoarcus sp. The peripheral pathway for the anaerobic oxidation of toluene would consist of an initial activation catalyzed by a benzylsuccinate synthase and a subsequent modified oxidation of benzylsuccinate to benzoyl-CoA and succinyl-CoA (both pathways encoded by bbs genes). Su et al. determined the crystal structure of the dibenzothiophene (DBT) sulfone monooxygenase (BdsA) from Bacillus subtilis at the resolution of 2.2 Å. This is one of the key enzymes in the 4S desulfurization pathway catalyzing the oxidation of DBT sulfone to 2′ -hydroxybiphenyl 2-sulfonic acid. The structure of the BdsA-FMN complex at 2.4 Å was also determined. Finally, Su et al. showed that mutations in the residues involved in catalysis or flavin substrate-binding, resulted in a significant loss of enzymatic activity.

Meier et al. studies the catabolism of hydroxylated aromatic acids in A. adeninivorans and showed that the genes encoding enzymes involved catabolic pathway of gallic acid are induced using aromatic acid substrates as inducers. Through the construction of gallic acid decarboxylase disruption mutants, the authors showed that gallic acid decarboxylase Agdc1p was the only enzyme responsible for the transformation of gallic acid. They suggest that this enzyme might have useful industrial applications not only in bioremediation processes but also in synthesis of chemicals.

Chandra et al. detected that effluents discharged from the pulp and paper industry contain various refractory and androgenic compounds, even after secondary treatment by activated processes. Most of these compounds are classified as endocrinedisrupting chemicals and are environmental toxicants. This study also assessed the degradability of such compounds by biostimulation. The results suggested that pulp and paper mill wastewater, after this secondary detoxification process, could be safe for disposal.

Lastly, Kirtzel et al. investigated the ability of Schizophyllum commune to degrade black slates (e.g., metamorphic rocks rich in sulfides, heavy metals and organic matters). S. commune is a filamentous basidiomycete possesses a broad range of enzymes including multicopper oxidases such as laccases and laccaselike oxidases. Both life forms (haploid monokaryotic and mated dikaryotic strains) were able to degrade the slate releasing metals at the same time.

This Research Topic of Frontiers in Microbiology shows how bioremediation by means of extremophiles is an active research theme.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

## FUNDING

ED is Superior Researcher at CINDEFI (CONICET, Argentina) and acknowledges the financial support from ANPCYT (PICT 2015 0463 and PICT 2016 2535). This project was co-financially supported by Universiti Teknologi Malaysia RU grant (Grant number: 16H89) and (UK-SEA-NUOF) with project number 4B297. K-GC thanked University of Malaya for financial support (PPP grants: PG136-2016A, PG133-2016A, HIR grant: H50001-A-000027). RS acknowledges the support from National Science Foundation in the form of BuG ReMeDEE initiative (Award # 1736255).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Donati, Sani, Goh and Chan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Low-Abundance Members of the Firmicutes Facilitate Bioremediation of Soil Impacted by Highly Acidic Mine Drainage From the Malanjkhand Copper Project, India

Abhishek Gupta<sup>1</sup> , Avishek Dutta1,2, Jayeeta Sarkar<sup>1</sup> , Mruganka Kumar Panigrahi<sup>3</sup> and Pinaki Sar<sup>1</sup> \*

#### Edited by:

Rajesh K. Sani, South Dakota School of Mines and Technology, United States

#### Reviewed by:

Virginia Helena Albarracín, Center for Electron Microscopy (CIME), Argentina Christopher Anthony Abin, University of Oklahoma, United States

> \*Correspondence: Pinaki Sar sarpinaki@yahoo.com; psar@bt.iitkgp.ac.in

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

Received: 31 December 2017 Accepted: 12 November 2018 Published: 11 December 2018

#### Citation:

Gupta A, Dutta A, Sarkar J, Panigrahi MK and Sar P (2018) Low-Abundance Members of the Firmicutes Facilitate Bioremediation of Soil Impacted by Highly Acidic Mine Drainage From the Malanjkhand Copper Project, India. Front. Microbiol. 9:2882. doi: 10.3389/fmicb.2018.02882 <sup>1</sup> Environmental Microbiology and Genomics Laboratory, Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India, <sup>2</sup> School of Bioscience, Indian Institute of Technology Kharagpur, Kharagpur, India, <sup>3</sup> Department of Geology and Geophysics, Indian Institute of Technology Kharagpur, Kharagpur, India

Sulfate- and iron-reducing heterotrophic bacteria represented minor proportion of the indigenous microbial community of highly acidic, oligotrophic acid mine drainage (AMD), but they can be successfully stimulated for in situ bioremediation of an AMD impacted soil (AIS). These anaerobic microorganisms although played central role in sulfateand metal-removal, they remained inactive in the AIS due to the paucity of organic carbon and extreme acidity of the local environment. The present study investigated the scope for increasing the abundance and activity of inhabitant sulfate- and ironreducing bacterial populations of an AIS from Malanjkhand Copper Project. An AIS of pH 3.5, high soluble SO<sup>4</sup> <sup>2</sup><sup>−</sup> (7838 mg/l) and Fe (179 mg/l) content was amended with nutrients (cysteine and lactate). Thorough geochemical analysis, 16S rRNA gene amplicon sequencing and qPCR highlighted the intrinsic metabolic abilities of native bacteria in AMD bioremediation. Following 180 days incubation, the nutrient amended AIS showed marked increase in pH (to 6.6) and reduction in soluble -SO<sup>4</sup> <sup>2</sup><sup>−</sup> (95%), -Fe (50%) and other heavy metals. Concomitant to physicochemical changes a vivid shift in microbial community composition was observed. Members of the Firmicutes present as a minor group (1.5% of total community) in AIS emerged as the single most abundant taxon (∼56%) following nutrient amendments. Organisms affiliated to Clostridiaceae, Peptococcaceae, Veillonellaceae, Christensenellaceae, Lachnospiraceae, Bacillaceae, etc. known for their fermentative, iron and sulfate reducing abilities were prevailed in the amended samples. qPCR data corroborated with this change and further revealed an increase in abundance of dissimilatory sulfite reductase gene (dsrB) and specific bacterial taxa. Involvement of these enhanced populations in reductive processes was validated by further enrichments and growth in sulfate- and iron-reducing media. Amplicon sequencing of these enrichments confirmed growth of Firmicutes members

**6**

and proved their sulfate- and iron-reduction abilities. This study provided a better insight on ecological perspective of Firmicutes members within the AMD impacted sites, particularly their involvement in sulfate- and iron-reduction processes, in situ pH management and bioremediation.

Keywords: acid mine drainage, bioremediation, Firmicutes, biostimulation, quantitative PCR, metagenomics, dissimilatory sulfate reduction

#### INTRODUCTION

fmicb-09-02882 December 7, 2018 Time: 16:20 # 2

Acid mine drainage (AMD) is considered to be a global environmental problem faced by mining industries due to the biological oxidation of sulfidic minerals (Johnson and Hallberg, 2005; Neculita and Zagury, 2008; Qian et al., 2017). Owing to its highly toxic nature manifested through acidic pH, elevated levels of heavy metals and sulfate, AMD is not only a threat to aquatic and terrestrial ecosystems but considered to be a major contributor in long term degradation of environmental quality (Johnson and Hallberg, 2005; Chandra and Gerson, 2010; Hallberg, 2010). Despite its extreme nature, a diverse range of microorganisms inhabit AMD systems (Méndez-García et al., 2015; Chen et al., 2016; Huang et al., 2016). The most dominant bacterial populations residing in AMD are highly acidophilic, chemolithoautotrophic iron and sulfur oxidizers such as Acidithiobacillus, Leptospirillum, Ferrithrix, and Ferritrophicum etc. (Baker and Banfield, 2003; Chen et al., 2015, 2016; Méndez-García et al., 2015; Huang et al., 2016; Mesa et al., 2017; Teng et al., 2017). These acidophilic, autotrophic and Fe/S oxidizing microorganisms mainly contribute toward AMD generation and were studied extensively for their physiology, molecular mechanisms and ecological relevance (Denef et al., 2010; Kuang et al., 2013; Méndez-García et al., 2014; Chen et al., 2015; Goltsman et al., 2015; Chen et al., 2016), whereas the small heterotrophic populations thriving in the same niches could be of great significance in reducing AMD generation process and attenuating the overall hazard of these systems remain less explored.

Microbial sulfur- and iron-metabolisms through redox transformations coupled with or without energy generation constitute the major biochemical reactions within AMD (Baker and Banfield, 2003; Druschel et al., 2004). These transformation reactions facilitate generation of acidity and contribute toward raising the soluble -sulfate or -iron concentrations, while on the other hand could lead to reversal of such processes and aid to restoration of such environments. Sulfate- and iron-reductions are the two key reactions carried out by heterotrophic sulfateor iron-reducing bacteria (SRBs or IRBs) that could reverse the AMD generation, metal precipitation and thus decrease the soluble metal concentrations and facilitate in raising the pH of AMD or AMD impacted ecosystems (Kaksonen et al., 2004; Church et al., 2007; Bijmans et al., 2009, 2010; Giloteaux et al., 2013). Bioremediation of AMD or AMD impacted ecosystems have been a subject of intense research in last decades (Kaksonen et al., 2004; Luptakova and Kusnierova, 2005; Church et al., 2007; Hiibel et al., 2008; Becerra et al., 2009; Bijmans et al., 2009; Hiibel et al., 2011; Burns et al., 2012; Moreau et al., 2013; Xingyu et al., 2013; Lefticariu et al., 2015; Sahinkaya et al., 2015; Deng et al., 2016; Zhang et al., 2016; Kefeni et al., 2017). In particular, enhancing the activities of indigenous microorganisms capable of sulfate- and/or iron-reduction and generation of alkalinity have gained interest for developing in situ bioremediation strategies (Neculita et al., 2007; Hiibel et al., 2008, 2011; Becerra et al., 2009; Bijmans et al., 2009; Burns et al., 2012; Xingyu et al., 2013; Lefticariu et al., 2015).

It is interesting to note that AMD or AMD impacted environment harbors SRBs and/or IRBs, but generally with low abundance and they remained metabolically less active at pH < 5.0 (Church et al., 2007; Sánchez-Andrea et al., 2011, 2012a; Giloteaux et al., 2013; Méndez-García et al., 2015). The limited presence and activities of these bacteria in AMD could be due to the presence of low organic carbon/other environmental variables and thermodynamic limitations as dissimilatory sulfateand/or iron-reduction are energetically expensive (Church et al., 2007; Muyzer and Stams, 2008; Bird et al., 2011; Johnson, 2012; Giloteaux et al., 2013). Nevertheless, metabolic versatility of SRB has been exploited in bioremediation of AMD with different approaches, among which amendment of suitable carbon and electron sources, nitrogen, phosphorus compounds etc. are important (Kaksonen et al., 2004; Church et al., 2007; Neculita et al., 2007; Hiibel et al., 2008, 2011; Becerra et al., 2009; Bijmans et al., 2009; Burns et al., 2012; Xingyu et al., 2013; Zhang and Wang, 2014; Lefticariu et al., 2015; Zhang et al., 2017).

During the past decades, microbiology of AMD has been studied extensively, particularly the cultivation-independent deep sequencing studies have resolved the community composition and biogeochemical functions of previously unknown microorganisms (Bertin et al., 2011; Kuang et al., 2013; Méndez-García et al., 2014; Chen et al., 2015; Goltsman et al., 2015; Hua et al., 2015). In contrast, exploration of AMD communities with special reference to heterotrophic SRBs and IRBs or other metal reducing populations remained less explored (Giloteaux et al., 2013). In situ bioremediation of these hazardous wastes is limited due to paucity of knowledge on the diversity of SRBs/IRBs and factors that promote their activities.

In the present study we aimed to explore the abundance and role of indigenous sulfate- and/or metal-reducing bacterial populations in natural attenuation of an AMD impacted soil designated as AIS. Soil impacted with highly acidic, sulfate- and multiple heavy metal-rich AMD from Asia's largest open-cast copper mine of Malanjkhand Copper Project (MCP) was used in this study. Microcosm based approach was adopted to promote presence and activities of indigenous sulfate- and/or metalreducing bacteria using cysteine and lactate as biostimulation agents. A thorough assessment of microbial populations involved

in sulfate/metal reduction and their characterization was done through 16S rRNA gene based amplicon sequencing coupled with qPCR and DGGE. The study was structured to answer the following questions: (i) How far it is possible to enhance the presence and activities of indigenous sulfate- and iron-reducing microbial populations present within an AMD impacted soil? (ii) What is the effect of such treatment(s) in the improvement of local physicochemical conditions, particularly the pH, concentrations of soluble -sulfate, -iron and -other heavy metals present therein? and (iii) Is it possible to enrich and cultivate the specific populations responsible for sulfate- and iron-reduction and management of the local physicochemical condition? The study demonstrates a comprehensive composition of microbial community residing in AIS and investigates the scope for in situ bioremediation.

#### MATERIALS AND METHODS

#### Sampling Site

The AMD impacted soil was collected in a sterile container from 5–10 cm below the top layer of a field flooded with AMD from a neighboring sump of Malanjkhand Copper Project (MCP), Balaghat district, Madhya Pradesh, India (N 21◦ 59.91<sup>0</sup> , E 080◦ 41.879<sup>0</sup> ) in the year 2014. The soil is exposed to AMD for over 10 years. The AMD water is released (as overflow) from the adjacent sump which receives AMD continuously from the mine areas. Selected physicochemical parameters such as oxidation reduction potential (ORP), pH and conductivity were measured on-site using multiparameter (Orion Star A329 portable Multiparameter, Thermo Fisher Scientific). All samples were collected following aseptic techniques, stored immediately at 4◦C, brought to the laboratory and stored at −80◦C till further processing.

## Microcosm Preparation

The microcosm setup was prepared with 5 g of AMD contaminated soil (AIS) using 20 ml filter sterilized distilled water in 30 ml glass vial. Three sets of microcosms were prepared. The first microcosm was amended with 0.1% (w/v) cysteine hydrochloride and designated as C. The second microcosm was amended with both 0.1% (w/v) cysteine hydrochloride and 0.1% (w/v) lactate (as sodium lactate), designated as C+L. The third microcosm was not amended with anything extra and designated as H (H stands for H2O, since only filter sterilized distilled water was present with AIS). Killed control was prepared for each setup by adding 2% (w/v) HgCl<sup>2</sup> as biocide. The glass vials were sealed with gas-tight rubber stoppers and aluminum crimp seals. To mimic the natural environment nitrogen was not purged into the microcosm vials. The microcosms were incubated in dark for 180 days at 30◦C. Each microcosm was set up in duplicate. Since the microcosms were of sacrificial type (i.e., the vial once opened was not reused in the same study) three experimental replicates were prepared: one for 4 months (120 days) incubation and marked as C\_4M, C+L\_4M, and H\_4M; second for 5 months (150 days) incubation and marked as C\_5M, C+L\_5M, and H\_5M and third for 6 months (180 days) incubation and marked as C\_6M, C+L\_6M, and H\_6M. Physicochemical parameters were measured from each microcosm setups (at 120 and 180 days of incubation). Samples were withdrawn from each of the setup in triplicates and used for measuring the physicochemical parameters. The major physicochemical parameters such as pH and ORP of the slurry were measured by Orion Multi parameter (Orion Star A329 portable Multiparameter, Thermo Fisher Scientific). The slurry samples were taken out from the microcosm setup and centrifuged at 4000 rpm to settle down the soil particles. SO<sup>4</sup> 2− estimation was performed with the supernatant through BaCl<sup>2</sup> turbidometric spectroscopy based method (Chesnin and Yien, 1951) while for Fe2<sup>+</sup> estimation, samples were acidified to avoid any oxidation and Fe2<sup>+</sup> concentration was measured by Ferrozine method (Viollier et al., 2000). The major elements such as Fe, Cu, As, Cr, Ni, and Zn were estimated from the slurry using atomic absorption spectroscopy (Perkin Elmer). In short, the slurry was centrifuged at 4000 rpm and supernatant was passed through 0.22 µm filter membrane and 2% HNO<sup>3</sup> was added to prevent any oxidation.

#### Metagenome Extraction, Library Preparation, and Sequencing

The microbial diversity analysis based on 16S rRNA gene amplicon targeted sequencing was performed with 6M setups (i.e., with 180 days incubation). Original AIS sample (0\_Day) was also used for comparison. From the three microcosms and the 0\_Day AIS, samples were withdrawn in triplicates and metagenome was extracted from each of the withdrawn samples using Power Soil DNA Isolation Kit (MoBio laboratories) according to the manufacturer's protocol. Metagenome from the replicate samples were pooled, mixed thoroughly and used for amplification of V4 region of 16S rRNA gene. V4 region of 16S rRNA gene was amplified with V4 specific primers (Bates et al., 2011). The following amplification conditions: 95◦C for 5 min, 35 cycles of 95◦C for 40 s, 50◦C for 45 s and 72◦C for 40 s with final extension at 72◦C for 7 min were used for amplification of V4 region. Thereafter amplicons were purified using 2% E-gel (E-Gel SizeSelect II Agarose Gel, Thermo Fisher Scientific) and sequencing was performed with Ion S5TM System (Thermo Fischer Scientific). In order to understand the microbial diversity at 5M setups (i.e., with 150 days incubation), Denaturing gradient gel electrophoresis (DGGE) was performed with H\_5M, C+L\_5M, and C\_5M samples. Metagenome was extracted in triplicates from these setups and were pooled together to amplify the V4 region using GC-clamp forward primer as described above. A DCode Universal Mutation Detection system (Bio-Rad, United States) was used to perform DGGE with similar protocol as described by Paul et al. (2015). The denaturing gradient from 35 to 70% was used for the present study. Twenty-three distinct bands in DGGE profile were excised and eluted by keeping it in 20 µl DNase free PCR water at 4◦C for overnight. These gel eluted products were re-amplified by using without GC clamp 515F and 806R primers (V4 region) and were cloned into the pTZ57RT vector for sequencing. EzTaxon<sup>1</sup> and SILVA 119

<sup>1</sup>www.ezbiocloud.net

reference database<sup>2</sup> were used for the taxonomic assignment of the obtained sequences.

## Quantification of Bacterial/Specific Taxa and dsrB Copy Number

Quantification of bacterial abundance and remarkably shifted taxa; Firmicutes, Acidobacteria, Actinobacteria as well as dsrB gene involved in sulfate reduction were performed for all the samples (0\_Day, H\_6M, C\_6M, and C+L\_6M). The bacterial abundance was quantified through bacterial specific 16S rRNA gene copy number. Similarly, abundance of Actinobacteria, Acidobacteria, and Firmicutes were quantified through specific 16S rRNA gene specific to these taxa. Copy numbers of functional gene dsrB were also quantified using qPCR based technique to estimate the sulfate-reducing populations. Real-time primers for bacterial 16S rRNA gene was taken from Muyzer et al. (1993), primers specific to Actinobacteria and Firmicutes was taken from Mühling et al. (2008), primer used for Acidobacteria as described by Lee and Cho (2011) and dsrB was taken from Purkamo et al. (2013). The qPCR was performed in Quant Studio 5 Real-Time PCR System (Thermo Fisher Scientific) with Power SYBR green PCR Mastermix (Invitrogen), with a total volume of 10 µl containing primer concentration of 5 picomoles and 2 µl of metagenomic DNA. All the reactions were set in triplicates. The following amplification conditions: 95◦C for 10 min, 40 cycles of 95◦C for 15 s, 55◦C for 30 s and 72◦C for 30 s was followed for bacterial and dsrB gene while 63◦C, 59◦C, and 57◦C annealing temperature were used for Actinobacteria, Acidobacteria, and Firmicutes, respectively. Melting curve analysis was run after each assay to check PCR specificity. Bacterial 16S rRNA gene copy numbers were determined in each sample by comparing the amplification result to a standard dilution series ranging from 10<sup>2</sup> to 10<sup>8</sup> of plasmid DNA containing the 16S rRNA gene of Achromobacter sp. MTCC 12117. Firmicutes gene copy number was calculated from plasmid DNA containing 16S rRNA gene from Bacillus. Whereas 16S rRNA gene of Actinobacteria and Acidobacteria as well as dsrB gene were cloned from metagenome and different dilution series of plasmid DNA copy number were used to prepare the standard curve for comparing the amplification result. The efficiency of qPCR was calculated using formula E = 10 (−1/ − Slope) − 1. The standard curve was linear for all the taxa specific and dsrB gene. R 2 value was greater than 0.993 for all the standard curve while efficiency was ranges from 84 to 112% (**Supplementary Table S1**).

#### Enrichment of Firmicutes Specific Members and Their Potential Role in Fe3<sup>+</sup> and SO<sup>4</sup> <sup>2</sup><sup>−</sup> Reduction

Firmicutes specific populations were enriched in facultative anaerobic medium (Stieglmeier et al., 2009) and Clostridium specific medium containing following ingredients in g/L NaCl 2.0, K2HPO<sup>4</sup> 5.0, MgCl<sup>2</sup> 0.2, ferric citrate 0.2, yeast extract 1.0, lysine 0.5 and cellulose 7.0 at pH 7.0 in 50 ml glass serum vials. Both the media were purged with filtered N<sup>2</sup> gas for 15–20 min to remove the oxygen and cysteine HCl (0.025%) was added as a mild reducing agent. Serum bottles were sealed with rubber stoppers. Two ml slurry from both C\_6M and C+L\_6M was used as inoculum in both the media and incubated at 30◦C for 2 weeks. The enrichment was subcultured three times in the same media before transferring into sulfate reducing medium (SRM) (modified from Postgate, 1963) and iron reducing medium (IRM) (containing ferric citrate 5 mM, NH4Cl 1.50 g/L, NaH2PO<sup>4</sup> 0.60 g/L, KCl 0.10 g/L, sodium acetate 2.50 g/L and yeast extract 0.05 g/L). Nitrogen gas was flushed for 15–20 min and cysteine HCl (0.025%) was added as a mild reducing agent in both the media to make the environment anaerobic. The pH of these two media was set to 7.0 using 1N NaOH/1N HCl and incubated at 30◦C for 2 weeks. Enriched population was sub-cultured thrice in same media after seeing the visual changes in the media (iron containing medium turned colorless, sulfate reducing medium turned black due to precipitation of iron sulfide). Remaining sulfate and increased iron (Fe2+) concentrations were measured for assessing the reduction of sulfate and iron (Fe3+) using BaCl<sup>2</sup> turbidometric method and Ferrozine method, respectively. Briefly, 2 ml samples were taken out and bacterial cells were pelleted down to use supernatant for estimation of SO<sup>4</sup> <sup>2</sup><sup>−</sup> and Fe2<sup>+</sup> concentration.

#### DNA Extraction From Enrichment

Total DNA from enriched populations was extracted from 4 ml of each enrichment. Equal volume of 0.5 M ammonium oxalate was added in iron enrichment to dissolve iron precipitates. The culture was pelleted at high speed for 5 min at room temperature. The cell pellet was dissolved in 500 µL TNE buffer (Tris HCl-10 mM, NaCl-2.0 M, EDTA-1 mM), 1/10 volume silica bead was added and vortexed for 15–20 min. 100 µL lysozyme (100 mg/ml) was added in the cell suspension, vortexed briefly to mix and incubated at 37◦C for 2 h. 30 µL proteinase K (20 mg/ml) and 50 µL SDS (10%) were added and incubated at 37◦C for 45 min. DNA was then extracted using chloroform:isoamyl alcohol (24:1). DNA pellet was washed twice with ice-cold 70% ethanol and the pellet was air dried. DNA was resuspended in PCR grade water. 16S rRNA gene amplicon from the DNA was prepared as described above for microcosm treatments (see section "Metagenome Extraction, Library Preparation, and Sequencing"). To understand the microbial diversity of these enrichments, amplicon based analysis was performed with Clostridium and facultative enrichments from both C\_6M and C+L\_6M setups but to identify the main iron and sulfate reducing populations, enrichments from C\_6M was considered.

#### Diversity Analysis and Statistical Tool

Ion Torrent data analysis of V4 region of 16S rRNA gene was performed with QIIME 1.9.1 pipeline (Caporaso et al., 2010). Quality filtering of reads and bioinformatics were performed as described by Gupta et al., 2017. In brief, quality filtering was performed for raw reads to remove primers, sequences with homopolymers run of >6 bp and read length beyond the range of 230–300 bp. Only 3 primer mismatches

<sup>2</sup>www.arb-silva.de/documentation/release-119

were allowed due to degeneracy of primer set in this step. Denovo OTU picking was performed with uclust and SILVA 119 reference database<sup>3</sup> was used for taxonomy assignments of reads as mentioned in QIIME pipeline. The OTU level analysis was performed by sub-sampling the samples to the lowest number of reads obtained in any of the samples through QIIME 1.9.1 pipeline. Venn diagram was generated in InteractiVenn<sup>4</sup> (Heberle et al., 2015) for top 100 OTUs. Microbial metabolic pathways were estimated based on the 16S rRNA gene data from the closed OTU picking method using PICRUSt software package (Langille et al., 2013) on the web-based Galaxy server<sup>5</sup> . For PICRUSt analysis, Greengenes database<sup>6</sup> was used for taxonomy assignment. One-way ANOVA was performed to assess the changes in the microbial diversity between the treatments using PAST software version 3.20 (Hammer et al., 2001). Weighted pair group mean arithmetic (WPGA) based hierarchical clustering was performed with Bray–Curtis distance dissimilarity matrix. Ternary plot was generated using PAST software to assess difference in diversity pattern among the treatments. All the data represented for physicochemical parameters were mean of its triplicates with standard deviation.

#### Nucleotide Accession Number

Metagenomic sequences are available under the NCBI BioProject ID PRJNA416924. The SRR number for each samples are SRR6320797 (C+L\_6M), SRR6320796 (C\_6M), SRR6320800 (0\_Day), SRR6320884 (H\_6M), SRR6320885 (FA\_C\_6M), SRR6320921 (Clos\_C\_6M), SRR6320922 (Clos\_IRM), SRR6320919 (FA\_IRM), SRR6320923 (Clos\_SRM), SRR6320920 (FA\_SRM), SRR7865998 (FA\_C+L) and SRR7865999 (Clos\_C+L). Sequence of DGGE bands were submitted in Genbank under accession numbers MH938427-MH938447.

#### RESULTS

#### Change in Physicochemical Parameters After the End of Incubation

Nutrient amendments to AIS facilitated a considerable improvement of its physicochemical conditions (**Table 1**). At the onset of the study (0\_Day), major physicochemical parameters of the soil slurry were measured. This sample was found to be of highly acidic (pH 3.51) nature; rich in soluble SO<sup>4</sup> <sup>2</sup><sup>−</sup> (7838 mg/l) and Fe (179 mg/l). Following incubation with nutrients, significant increase in pH (up to pH 6.61) but decrease in ORP (up to 110 mV) were observed coupled with considerable changes in concentrations of SO<sup>4</sup> <sup>2</sup>−, Fe, Fe2<sup>+</sup> and heavy metals. Control set (H\_6M) with only water addition showed slight change with respect to the test physicochemical parameters while killed control did not show any shift at all. Incubation with only water (H\_6M) could initiate reactions responsible for the observed shift in pH and ORP, presence of nutrients favored such reactions significantly. Following cysteine and cysteine + lactate amendment, soluble sulfate concentration was greatly reduced along with Fe (total Fe as well as Fe2+), Cu, Zn and Ni. Compared to H\_6M that showed nearly 50% decrease in SO<sup>4</sup> <sup>2</sup><sup>−</sup> (to that of its initial level), cysteine + lactate addition could led to a 95% reduction. Microcosm amended with only cysteine showed only up to 76% lowering of SO<sup>4</sup> 2− (compared to 0\_Day). Soluble Fe level presented an interesting trend: concentrations of both total Fe and Fe2<sup>+</sup> were enhanced in H\_6M (2.3-fold for Fe and 2.5-fold Fe2+) and C\_6M (5.6-fold for Fe and 4.8-fold for Fe2+), while the values decreased significantly (0.5-fold for Fe and 0.6-fold for Fe2+) in C+L\_6M. Although an overall enrichment experiment showed a strong role of the test nutrients in improving the local physicochemical condition of the AMD impacted soil, lactate + cysteine was identified as a better stimulant than cysteine alone.

#### Shift in Microbial Community Composition

16S rRNA gene amplicon sequencing and estimated diversity indices revealed an assessable shift in microbial community composition of AIS following incubation with nutrient amendments (**Table 2**). Both estimated Chao1 and observed OTUs were increased coupled with distinct shifts in microbial community composition (**Table 2**). The most abundant bacterial phyla within the AIS at 0\_Day were Proteobacteria (42%), WD272 (20%), Actinobacteria (14%), Acidobacteria (11%), Chloroflexi (9%), and Firmicutes (1.5%) (**Figure 1**). Following incubation, a distinct shift in community composition with great enhancement of Firmicutes coupled with the striking decrease in abundance of Proteobacteria, Acidobacteria, and Actinobacteria were detected (**Figure 1**). Abundance of the members of Firmicutes affiliated to Clostridia, OPB54, Negativicutes, and Bacilli was increased in both C+L\_6M and C\_6M. The extent of enhancement of Firmicutes was up to 36.5-fold in C\_6M and 35.4-fold in C+L\_6M (**Figure 1**). Proteobacteria [Gammaproteobacteria (35%), Alphaproteobacteria (6%), Betaproteobacteria (1%), and Deltaproteobacteria (0.07%)] that constituted the major phylum at 0\_Day was found to be considerably less prevalent within the communities enriched with various amendments (**Figure 1**). The noteworthy decrease in abundance of Gammaproteobacteria and Alphaproteobacteria was observed in all the setup whereas abundance of Betaproteobacteria and Deltaproteobacteria was increased in C\_6M (**Figure 1**). Members of the phylum Chloroflexi (Ktedonobacteria and KD4-96) also showed a substantiate increase in their abundance in C\_6M (19.0%) while it got reduced in C+L\_6M (0.03%). The other major classes such as Acidobacteria, Acidimicrobiia, Actinobacteria, and Thermoleophilia showed decrease in their abundance in C\_6M and C+L\_6M (**Figure 1**).

Family level analysis within the 0\_Day, H\_6M, C\_6M and C+L\_6M microcosms showed increase in abundance of several families. The abundance of facultative and/or strict anaerobic members of Clostridiales (Clostridiaceae 1, Family XIII,

<sup>3</sup>www.arb-silva.de/documentation/release-119

<sup>4</sup>http://www.interactivenn.net

<sup>5</sup>https://huttenhower.sph.harvard.edu/galaxy

<sup>6</sup>http://greengenes.secondgenome.com



All the units are represented in ppm except ORP (mV) and pH (SI unit). H, C and C+L denote unamended, cysteine amended and cysteine and lactate amended microcosms while 4M (120 days incubation) and 6M (180 days incubation) represent the time of incubation. The values are represented mean of three independent experiments with standard deviations.

TABLE 2 | Details of 16S rRNA gene reads and non-parametric diversity indices of microbial communities from microcosms and enrichments.


<sup>∗</sup>Denotes the microcosm setup while rest denotes the enrichments. Enrichment was performed from both the setup (C\_6M and C+L\_6M) in facultative anaerobic and Clostridium specific media. FA, facultative anaerobic enrichment; FA\_SRM, facultative anaerobic enrichment in sulfate reducing medium; FA\_IRM, facultative anaerobic enrichment in iron reducing medium; Clos, Clostridium specific enrichment; Clos\_SRM, Clostridium specific enrichment in sulfate reducing medium; and Clos\_IRM, Clostridium specific enrichment in iron reducing medium. ∗∗Denotes the enrichment was performed for C\_6M.

Christensenellaceae, Lachnospiraceae, Gracilibacteraceae, Peptococcaceae, Peptostreptococcaceae, Ruminococcaceae, and VadinBB60), Bacillales (Alicyclobacillaceae and Bacillaceae), Veillonellaceae, uncultured OPB54 and Coriobacteriaceae was increased in both C\_6M and C+L\_6M. Heat map analysis (**Figure 2**) of the distribution of major genera (also considering taxa classified up to family level) under Clostridiales indicated considerable enhancement in abundance of several taxa commonly attributed to sulfate- and iron-reduction following nutrient amendments. In contrast to this, only water amendment (control; H\_6M) allowed enhancement of mostly known taxa involved in iron and sulfur oxidation [Acidobacteriaceae (Subgroup 1), Gallionellaceae, Xanthomonadaceae] and few other taxa such as OPB35 soil group, KD4-96, Ktedonobacteria\_JG30- KF-AS9, BSV26 and Cystobacteraceae. Overall the successful enrichment of diverse fermentative and anaerobic populations was achieved, suppressing the growth of acidophilic members following creation of anoxic environment and supply of metabolizable C- and N-sources. A ternary plot was generated to understand the distribution of top 50 genera across 0\_Day, C\_6M and C+L\_6M microcosm samples

(**Figure 3**). The result showed that acidophilic genera such as Ferrithrix, Metallibacterium, Acidobacterium, uncultured –Acidomicrobiales and -Acidobacteriaceae Subgroup 1 etc. were more prevalent at 0\_Day. In contrast, taxa affiliated to Firmicutes; Desulfitobacterium, Clostridium Sensu Stricto 1, Desulfosporosinus, Desulfurispora, uncultured -Christensenellaceae, -OPB54, -Clostridiales Family XVII, -Ruminococcaceae, -Lachnospiraceae, -Peptococcaceae etc. capable of sulfate- and iron-reduction dominated in C\_6M and C+L\_6M. One-way ANOVA analysis confirmed that microbial diversity among the treatments was significantly different (P < 0.05).

#### Microbial Shift at OTUs Level

In order to understand the dynamics of microbial community composition beyond the taxonomic level, most abundant OTUs (top 100 OTUs) from each of the microcosms were analyzed (**Figures 4A–D**). Top 100 OTUs from each microcosm contributed 79–85% of the total reads of the respective samples. The interesting finding was that when considering top 100 OTUs of one treatment, the same OTUs in another treatment

contributed less percentage of the total reads, clearly indicating the effect of treatments (**Figures 4E–H**). Venn diagram depicted the pattern of sharing of OTUs among the treatments (**Figure 4I**) and signified that how the abundance of OTUs was significantly changed during the treatments. Taxonomic identities of these OTUs were determined to find their affiliation to 32 different taxa (**Figure 5**). Out of 100 OTUs from each of the microcosms, OTUs affiliated to Firmicutes were dominant in C+L\_6M (80 OTUs) and C\_6M (64 OTUs) while OTUs assigned to Proteobacteria were high in 0\_Day (40 OTUs) and H\_6M (33 OTUs) (**Figure 5**). These results were perfectly in line with our taxonomy based observation of increasing abundance of Clostridia in C+L\_6M and C\_6M. Total 26 OTUs (out of top 100 OTUs) were found to be shared between 0\_Day and H\_6M (**Figure 6A**). These common OTUs were affiliated mostly to acidophilic taxa. Among the C\_6M and C+L\_6M communities 24 shared OTUs were detected and these were affiliated to iron/sulfate reducing, fermentative and anaerobic Firmicutes taxa (**Figure 6B**).

phylum using Bray–Curtis dissimilatory matrix is represented. U denotes uncultured member.

## qPCR Based Quantification of Bacterial/Specific Taxa and dsrB Gene

Quantitative estimation of the major taxa (Firmicutes, Acidobacteria, and Actinobacteria) as well as dsrB gene (involved in dissimilatory sulfate reduction) was performed for 0\_Day, C\_6M, C+L\_6M and H\_6M communities using qPCR based approach. Total bacterial 16S rRNA gene copies indicated a marginal reduction in bacterial abundance following microcosm amendments (**Figure 7**). The estimation of 16S rRNA gene copies for Actinobacteria, Acidobacteria, and Firmicutes corroborated with the amplicon based community data suggesting the decrease in abundance of Actinobacteria and Acidobacteria but increase in Firmicutes following nutrient amendment (**Figure 7**). The involvement of sulfate reducing bacteria in nutrient amended microcosms was highlighted by a remarkable increase in dsrB gene copy number from 7.8 × 10<sup>4</sup> to 3.9 × 105–1.0 × 10<sup>6</sup> (**Figure 7**).

## PICRUSt Based Functional Prediction of the Community

Metabolic functions of the microbial communities were established through PICRUSt analysis. Using the genome-wide analysis tools integrated in PICRUSt, we could look into the genomic inventories related to sulfate and cysteine metabolism and other major biogeochemical processes of the enriched communities (**Supplementary Figure S1**). The result showed abundance of genes involved in dissimilatory sulfate metabolism (aprAB and dsrAB), cysteine metabolism (cysteine desulfhydrase, cysteine synthase, cystathionine synthase and cystathionine lyase) along with hydrogenases, metal tolerance/transporter gene for As, Fe, Cu, Zn, Co, etc., nitrogen metabolism and other major categories of metabolic functions. Considerable change in the abundance of dsrAB (involved in dissimilatory sulfate reduction)


FIGURE 2 | Heat map based relative abundance of distribution of top 50 Clostridiales members in all the microcosm setup. U and O denote uncultured member and others, respectively.

and cysteine desulfhydrase (involved in cysteine utilization) was observed in nutrient amended microcosms. The analysis clearly indicated that enriched microbial populations were genetically equipped for dissimilatory sulfate reduction following cysteine amendment.

## Enrichment of Firmicutes Members Using Specific Medium From Microcosm Setup

The 16S rRNA gene based investigation indicated that following cysteine amendment abundance of members of Firmicutes were enriched considerably. Most of these taxa were known for their role in anaerobic sulfate- and iron-reduction. Although the qPCR and PICRUSt supported the role of these members in the observed physicochemical changes within our AIS microcosms, final validation of their biogeochemical role was done by enrichment of the Firmicutes members under specific culture conditions. Cultures from both C\_6M and C+L\_6M microcosms were sub-cultured in two specific media: Clostridium specific and facultative anaerobic. Following three repeated sub-culturing in the respective media, taxonomic identities of the enriched populations were established by 16S rRNA gene based amplicon sequencing (**Figure 8A**). The results indicated that our culture conditions were highly supportive for the enrichment of Firmicutes members in both the Clostridium specific and facultative anaerobic media. Members of this phylum contributed 95.33–99.85% while Proteobacteria, Actinobacteria, and Bacteroidetes constituted very small populations (**Figure 8A**). The most dominant genera detected in the Clostridium specific enrichment belonged to Clostridiales and Bacillales members such as Clostridium sensu stricto 1, Lysinibacillus, Ruminococcaceae\_incertae sedis, Clostridium sensu stricto 10, Clostridium sensu stricto 12, Paenibacillus, Clostridiales\_Family XIII, uncultured Planococcaceae and uncultured Veillonellaceae members (**Figure 8B**). In the facultative anaerobic medium sub cultured from C\_6M microcosm, Bacillus was the most dominant genera (75.70%) followed by Clostridium sensu stricto 1 (20.63%), Clostridium sensu stricto 12 (1.01%) and Sporolactobacillus (0.68%) (**Figure 8B**). In contrast, Clostridium sensu stricto 12 and

Clostridium sensu stricto 11 accounted for more than 95% of the community grown in the same medium but subcultured from C+L\_6M (**Figure 8B**).

#### Sulfate- and Iron-Reduction Potential of the Microorganisms Enriched in Clostridium Specific and Facultative Anaerobic Media

Bacterial cultures enriched in Clostridium specific and facultative anaerobic media were further inoculated in sulfate reducing and iron reducing media (designated as SRM and IRM) for assessing their potential toward sulfate- and iron-reduction. Following three repeated sub-culturing, 16S rRNA gene amplicon sequencing was done for all the four sets derived originally from C\_6M microcosm. The amplicon sequencing result of Clostridium specific enrichment grown in SRM showed that uncultured Veillonellaceae members, Ruminococcaceae incertae sedis and Clostridium sensu stricto 12 accounted for 92.25% (**Figure 8C**). The same enrichment culture grown in IRM showed the abundance of Lysinibacillus (80.43%) along with Bacillus, uncultured Planococcaceae member, Clostridium sensu stricto 12 and Clostridium sensu stricto 10 (**Figure 8C**). Similar study performed with facultative anaerobic enrichment indicated proliferation (cumulative abundance of 98%) of Bacillus, Brevibacillus, Clostridium sensu stricto 10, Fictibacillus, Anoxybacillus, and Clostridium sensu stricto 1 in SRM (**Figure 8C**) while Bacillus, Paenibacillus, Clostridium sensu stricto 10 and Fictibacillus accounted for 97.71% of the IRM culture (**Figure 8C**). Metabolic abilities of the enriched

populations derived from microcosms C\_6M and C+L\_6M microcosms toward sulfate- and iron-reduction were confirmed by quantitative estimation of SO<sup>4</sup> <sup>2</sup><sup>−</sup> and Fe2<sup>+</sup> ions. Nearly complete reduction of SO<sup>4</sup> <sup>2</sup><sup>−</sup> (15 mM) and Fe3<sup>+</sup> (5 mM) were noticed following 10–14 days of incubation, confirming their abilities for reduction of these terminal electron acceptors. The formation of black precipitates of iron sulfide in SRM and change in color of the IRM from yellow to light green or colorless with precipitation of Fe (Ferric citrate as redox indicator) (Pan et al., 2017) was also noted (**Supplementary Figure S2**). In order to confirm the presence of these SRB and IRB after 4 months of incubation where a decline in soluble iron concentration (Fe2+) was observed due to precipitation with sulfide produced by sulfate reducing activity in the treatments, DGGE based microbial community analysis was performed with 5 months incubated microcosms (H\_5M, C\_5M, and C+L\_5M). The banding pattern obtained for C\_5M and C+L\_5M communities showed enrichment of almost similar types of microbial populations (**Figure 9**). The enrichment of Clostridium sp., Themincola sp., Bacillus sp., Steroidobacter sp., as well as members of Acidobacteriaceae, Ruminococcaceae, and Coriobacteriaceae in these treatments clearly indicated their potential toward both iron- and sulfate-reduction (**Figure 9**). These groups were also detected in the same treatments through amplicon based sequencing after 6 months of incubation. These known iron and sulfate reducing populations were also detected in both iron and sulfate reducing media hence confirmed their involvement in reduction of iron and sulfate during 5 months of incubated setups.

#### DISCUSSION

Geomicrobiology of AMD including the nature of microorganisms and biogeochemical functions of various acidophilic microorganisms is well established. In contrast to that, the broader ecological roles of AMD organisms in terms of the attenuation of the hazardous nature of such acidic environment remain less explored. Our study demonstrated that it is possible to enhance the activities of indigenous sulfate- and iron-reducing bacteria of an AIS to achieve improvement of its major physicochemical parameters desirable for bioremediation. With respect to the major questions we posed during this study, our results proved that (a) it is possible to enhance the abundance and activities of autochthonous sulfate- and iron-reducing bacteria of an AIS and (b) this altered microbial community could lead toward changing the physicochemical conditions favorably, thus decreasing the hazardous nature of the studied sample considerably.

There are reports highlighting the presence of heterotrophic sulfate- and iron-reducing bacteria (Clostridiaceae, Peptococcaceae, and Bacillaceae members) within highly acidic AMD systems (Sánchez-Andrea et al., 2012a,b). Our biostimulation based approach was successful in enhancing the abundance of Firmicutes members capable of anaerobic sulfate-/iron-reduction. In the native AIS, these bacterial taxa constituted only 1.5% which (low abundance of heterotrophic reducing taxa) corroborated the earlier reports on different AMD environments (Chen L.X. et al., 2013; Kuang et al., 2013). Increase in abundance of these anaerobic/facultative anaerobic populations surpassing the acid producing-, sulfurand metal-oxidizing microorganisms with nutrient amendments was impressive. All these members of the phylum Firmicutes were well known for their facultative to strict anaerobic metabolism, but not so much for sulfate- and iron-reduction except few taxa such as Clostridium, Desulfosporosinus, Desulfotomaculum etc. (Chockalingam and Subramanian, 2006; Church et al., 2007; Sánchez-Andrea et al., 2012b; Pan et al., 2017). The increased abundance of gene encoding dissimilatory sulfite reductase (dsrB) and Firmicutes specific 16S rRNA gene detected in

enrichment in iron reducing medium. U and O denote uncultured member and others, respectively.

qPCR, reduction of -sulfate/-iron and rise in pH were all in strong agreement. Our results demonstrated that a number of sulfate- and iron-reducing bacterial taxa present in AMD impacted environment can be proliferated and implicated with the desirable reductive processes successfully. The PICRUSt analysis confirmed that the enriched bacterial populations were genetically equipped for dissimilatory sulfate reduction processes.

The effect of cysteine (alone or along with lactate) as successful proxy to provide required metabolic resources and thus biostimulate the target groups of microorganisms could be attributed to its dual characteristics. Cysteine could be used by the microbes as a carbon and nitrogen source, and also might act as a reducing agent (that helps in scavenging the dissolved oxygen) to facilitate reduction of iron and sulfate. Microbes catabolize cysteine for their fermentative mode of metabolism through two enzymes (i) cysteine desulfhydrase which produces NH3, pyruvate and H2S and (ii) cystathionine-γ-lyase which utilizes an oxidized form of cysteine (Morra and Dick, 1991). Microbe mediated H2S production was possible from selective enrichment of soil amended with cysteine through cysteine desulfhydrase enzyme. Wang et al. (2000) demonstrated that the genes encoding cysteine desulfhydrase and serine acetyltransferase may be used to develop a metabolically engineered Escherichia coli that can carry out aerobic sulfate reduction. Suitability of carbon sources rich in amino acids, but low in lignin in promoting sulfate

reduction was also reported (Coetser et al., 2006). Recently, Zhang et al. (2017) established the role of tryptone and yeast extract in the remediation of mine tailings by promoting the growth of SRB.

Microbial taxa enriched during this study were reported to be of facultative- or strict-anaerobic nature, involved in anaerobic hydrolytic fermentation, cysteine utilization, acetateand H2S-production and metal reduction (Petrie et al., 2003; Church et al., 2007; Finke and Jørgensen, 2008; Kosaka et al., 2008; Li et al., 2011; Bertel et al., 2012; AlAbbas et al., 2013; Hausmann et al., 2016; Peng et al., 2016; Pan et al., 2017). The major genera identified in this study such as Clostridium, Clostridium sensu stricto members, Lutispora, Sporobacter, Acetanaerobacterium, Caldicoprobacter, Gracilibacter, Oxobacter, Fonticella, Papillibacter, as well as unclassified members of Ruminococcaceae, Lachnospiraceae, and Christensenellaceae were all reported as anaerobic, fermentative, cellulose- and cysteinemetabolizing, acetogenic, and iron reducing members (Grech-Mora et al., 1996; Defnoun et al., 2000; Chen and Dong, 2004; Lee et al., 2006; Shiratori et al., 2008; Bouanane-Darenfed et al., 2011; Chen M. et al., 2013; Fraj et al., 2013; Peng et al., 2016). Presence of these organisms was reported from diverse sulfur-rich environments including hot spring (Zavarzina et al., 2007; Bouanane-Darenfed et al., 2011; Fraj et al., 2013), mine tailings/drainage/soil (Sánchez-Andrea et al., 2011; Gupta et al., 2017), constructed wetland (Lee et al., 2006) and AMD treatments sites (Clarke et al., 2004; Kaksonen et al., 2004; Pruden et al., 2007; Bijmans et al., 2009; Hiibel et al., 2011;

Lu et al., 2011; Martins et al., 2011; Sánchez-Andrea et al., 2014; Deng et al., 2016). Considering the known metabolic characteristics of these taxa and their ecological relevance, we could attribute their abundance to the observed sulfate- and ironreduction. In accordance with previous reports, the other known strict anaerobic sulfate reducing taxa such as Desulfurispora, Desulfotomaculum, Desulfosporosinus, and Desulfitobacterium was also enriched during our study (Kaksonen et al., 2004; Church et al., 2007; Hiibel et al., 2008, 2011; Bijmans et al., 2010). The enhanced abundance of facultative anaerobic fermentative and strictly anaerobic sulfate reducing populations following cysteine amendments highlights the synergistic role of these metabolically dependent organisms confirming the fermentation coupled with sulfate reduction phenomenon (Finke and Jørgensen, 2008). We hypothesize that in the presence of cysteine, fermentative organisms become activated, producing metabolites and deplete the dissolved oxygen rapidly and thereby creating more anoxic niches. Within these anoxic micro-niches strict anaerobic populations proliferate, making use of the sulfate as preferred terminal electron acceptor thus facilitates sulfate reduction and rise in pH (Church et al., 2007). Our attempt to confirm the physiological abilities of the enriched populations toward sulfate- and iron-reduction by using culture media specific for Clostridium and facultative anaerobic bacteria supported the above hypothesis. We were successful in identifying the facultative and strict anaerobic sulfate- and iron-reducing populations with conformity through specific enrichment and deep sequencing.

The potential involvement of individual members of the enriched populations toward reductive processes was validated by a third level of enrichment wherein sulfate- and ironreducing populations were grown more selectively in two specific media. These sulfate- and iron-reducing bacteria specific enrichments were meant to segregate and identify the organisms responsible for individual terminal electron acceptor utilization (iron as Fe3<sup>+</sup> and sulfate as SO<sup>4</sup> 2 ). 16S rRNA gene sequencing of metagenomes retrieved from these enrichments revealed that members of the families Clostridiaceae, as well as Bacillaceae (genera Lysinibacillus, Bacillus and Paenibacillus etc.), Veillonellaceae and Ruminococcaceae etc. specifically contributed toward sulfate- or iron-reduction. Presence of these members in both C\_5M and C+L\_5M microcosms through DGGE further confirmed their potential of sulfate and iron reduction. Clostridiaceae and Bacillaceae members were previously reported in different AMD bioremediation studies or in sulfate-/ironreducing enrichments/AMD environment (Clarke et al., 2004; Scala et al., 2006; Hiibel et al., 2008, 2011; Sánchez-Andrea et al., 2011; Yi et al., 2012; Giloteaux et al., 2013; Zhang and Wang, 2016; Zhang et al., 2016). The predominance of metal reducing Pelosinus (member of Veillonellaceae) on lactate amendment was reported by Mosher et al. (2012). Metal reduction and fermentative mode of metabolism of Veillonellaceae members were reported by earlier investigators including the whole genome sequence analysis of uncultured Veillonellaceae strain RU4 that confirmed presence of genes for sulfate reduction as well as polysulfide reduction (Brown et al., 2012; Shah, 2013; Kwon et al., 2016). Zhao et al. (2010) reported the role of Ruminococcus spp. (member of Ruminococcaceae) in sulfate reduction. Thus in our study, these enriched members confirmed their involvement in iron and sulfate reduction.

#### CONCLUSION

An acidic, sulfate-, iron- and other heavy metal-rich AMD impacted soil harbored low proportion of heterotrophic, sulfateand iron-reducing anaerobic bacterial populations. These redox active members can be successfully stimulated by cysteine and lactate amendment. These enriched microbial groups can facilitate dramatic change in physiochemical condition. The microorganisms which got enriched with nutrient amendment belonged to the fermentative and strict anaerobic sulfateand iron-reducing populations affiliated to Clostridiaceae, Veillonellaceae, Bacillaceae, Ruminococcaceae etc. Increased abundance of these organisms as evident from 16S rRNA amplicon sequencing and taxon-specific qPCR; enhancement of dsrB gene, change in genomic composition suitable for carrying out the required catabolic function corroborated with reduction in soluble sulfate- and iron-reduction and pH management. This study enabled us to gain a better insight on ecological perspective of the members of phylum Firmicutes indigenous to AMD impacted sites and more importantly, their involvement in sulfate- and, iron-reduction processes. The study also demonstrated the suitability of amino acid/protein rich natural substances as potent biostimulation agent for bioremediation of AMD/AMD impacted sites and provided us the specific microbial populations capable of anaerobic sulfate-, and/or iron-reduction which could be used as a potent bioaugmentation agent for future bioremediation applications.

## AUTHOR CONTRIBUTIONS

PS conceived and designed the experiments and arranged funds. AG performed the major experiments. PS and AG were responsible for manuscript preparation. MP, PS, and AG arranged sampling from MCP. AG and JS performed the qPCR. AG and AD performed the bioinformatics analysis for deciphering microbial diversity. AG and AD performed the 16S rRNA gene amplicon sequencing in Ion S5 sequencer.

## FUNDING

The authors are grateful to Department of Biotechnology, Government of India for funding the project (BT/PR 7533/BCE/8/959/2013, Dated 10/12/2013). The authors are thankful to IIT Kharagpur for providing the NGS facility (Ion S5 sequencer) through SGBSI challenge grant (IIT/SRIC/BT/ODM/2015-16/141). AG thanks the Department of Biotechnology, Government of India for providing fellowship under DBT-JRF category (DBT/2014/IITKH/113). Financial support to AD (IIT/ACAD (PGS&R)/F.II/2/14/BS/91R01) and JS (IIT/ACAD(PGS&R)/F.II/2/13/BT/91P01) from IIT Kharagpur and institutional fellowship was acknowledged.

#### ACKNOWLEDGMENTS

fmicb-09-02882 December 7, 2018 Time: 16:20 # 16

The generous help from Malanjkhand Copper Project, Hindustan Copper Limited authority for sample collections was acknowledged.

#### REFERENCES


#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02882/full#supplementary-material

ecosystems. Curr. Opin. Biotechnol. 38, 150–158. doi: 10.1016/j.copbio.2016. 01.013



gene sequences. J. Microbiol. Methods 86, 195–203. doi: 10.1016/j.mimet.2011. 05.003



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Gupta, Dutta, Sarkar, Panigrahi and Sar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Living at the Frontiers of Life: Extremophiles in Chile and Their Potential for Bioremediation

Roberto Orellana1,2, Constanza Macaya<sup>1</sup> , Guillermo Bravo<sup>1</sup> , Flavia Dorochesi<sup>1</sup> , Andrés Cumsille<sup>1</sup> , Ricardo Valencia<sup>1</sup> , Claudia Rojas<sup>1</sup> and Michael Seeger<sup>1</sup> \*

<sup>1</sup> Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química and Centro de Biotecnología Daniel Alkalay Lowitt, Universidad Técnica Federico Santa María, Valparaíso, Chile, <sup>2</sup> Departamento de Biología, Facultad de Ciencias Naturales y Exactas, Universidad de Playa Ancha, Valparaíso, Chile

#### Edited by:

Jean Armengaud, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), France

#### Reviewed by:

Camila Fernandez, UMR7621 Laboratoire d'Océanographie Microbienne (LOMIC), France Karima Hezbri, National Institute of Applied Science and Technology, Tunisia

> \*Correspondence: Michael Seeger michael.seeger@usm.cl

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

Received: 30 April 2018 Accepted: 10 September 2018 Published: 30 October 2018

#### Citation:

Orellana R, Macaya C, Bravo G, Dorochesi F, Cumsille A, Valencia R, Rojas C and Seeger M (2018) Living at the Frontiers of Life: Extremophiles in Chile and Their Potential for Bioremediation. Front. Microbiol. 9:2309. doi: 10.3389/fmicb.2018.02309 Extremophiles are organisms capable of adjust, survive or thrive in hostile habitats that were previously thought to be adverse or lethal for life. Chile gathers a wide range of extreme environments: salars, geothermal springs, and geysers located at Altiplano and Atacama Desert, salars and cold mountains in Central Chile, and ice fields, cold lakes and fjords, and geothermal sites in Patagonia and Antarctica. The aims of this review are to describe extremophiles that inhabit main extreme biotopes in Chile, and their molecular and physiological capabilities that may be advantageous for bioremediation processes. After briefly describing the main ecological niches of extremophiles along Chilean territory, this review is focused on the microbial diversity and composition of these biotopes microbiomes. Extremophiles have been isolated in diverse zones in Chile that possess extreme conditions such as Altiplano, Atacama Desert, Central Chile, Patagonia, and Antarctica. Interesting extremophiles from Chile with potential biotechnological applications include thermophiles (e.g., Methanofollis tationis from Tatio Geyser), acidophiles (e.g., Acidithiobacillus ferrooxidans, Leptospirillum ferriphilum from Atacama Desert and Central Chile copper ores), halophiles (e.g., Shewanella sp. Asc-3 from Altiplano, Streptomyces sp. HKF-8 from Patagonia), alkaliphiles (Exiguobacterium sp. SH31 from Altiplano), xerotolerant bacteria (S. atacamensis from Atacama Desert), UV- and Gamma-resistant bacteria (Deinococcus peraridilitoris from Atacama Desert) and psychrophiles (e.g., Pseudomonas putida ATH-43 from Antarctica). The molecular and physiological properties of diverse extremophiles from Chile and their application in bioremediation or waste treatments are further discussed. Interestingly, the remarkable adaptative capabilities of extremophiles convert them into an attractive source of catalysts for bioremediation and industrial processes.

Keywords: extremophile, Chile, Atacama Desert, Altiplano, Patagonia, Antarctica, bioremediation

## INTRODUCTION

Most the well-described forms of life are mainly adapted to face environments with "physiological conditions," a term described in literature as moderate temperature (10–37◦C), pH ∼ 7, salinity ranging from 0.15 to 0.5 M NaCl, pressure 1 atm and enough water availability (Aguilar et al., 1998; Antranikian et al., 2005). However, there is still a large under-examined group of organisms, known as extremophiles, that are capable of adjust,

**24**

survive or thrive in hostile habitats that were previously thought to be inhospitable or even lethal for life (Rampelotto, 2013). In general, extremophiles are divided in two categories: extremophiles, which require one or more extreme conditions to grow, and extremotolerant organisms, which can tolerate extreme and/or toxic conditions, although they grow optimally at "physiological" conditions (Canganella and Wiegel, 2011). The study of extremophiles is a rather difficult field, mainly constrained by the complexity of reaching their ecological niches and isolating these microbes. Most extremophiles are still part of the microbial dark matter that has not been discovered yet (Bernard et al., 2018). Hunting microbes in extreme environment is a huge challenge for microbiologists worldwide, which could provide microorganisms, enzymes and biomolecules for diverse applications in biotechnology, biomedicine and industrial processes. The knowledge of the adaptation mechanisms of microbes to extreme environments provides metabolic networks, regulation circuits and pieces for systems biology and synthetic biology (Chen and Jiang, 2018). Extreme conditions drive the evolution of their inhabitants, highlighting the role of extremophiles as models for the study of the evolution of biological entities.

Newly developed technologies have allowed research on extreme environments to gain knowledge on microorganisms and provide significant insights about the origin of life on Earth. Recent studies have uncovered that the first terrestrial life form, known as LUCA (Last Universal Common Ancestor), was a thermophilic anaerobe capable of gaining energy from geochemical sources (Weiss et al., 2016). Basic research has provided valuable insights on how extremophiles can survive such challenging environments. However, the presence of highly sophisticated mechanisms of adaptation together with the availability of a sweet of novel biochemical pathways sustaining peculiar physiological metabolic capabilities converts extremophiles into a current focus of applied research to exploit their biotechnological potential (Rampelotto, 2013). Further attention has also been devoted to identification, isolation and characterization of biomolecules, most of them enzymes named as extremozymes, which are well adapted to be active also at extreme conditions (Rampelotto, 2013).

Extremophiles, can be classified according to the conditions in which they grow: thermophiles and hyperthermophiles (organisms growing at temperatures of 45–80◦C and >80◦C, respectively) (Madigan et al., 2000; Berenguer, 2011), psychrophiles (organisms that grow at <10◦C) (Siddiqui et al., 2013), acidophiles and alkaliphiles (organisms optimally adapted to pH < 5 and pH > 9, respectively), halophiles (organisms that require NaCl for growth, in concentration of 200–5,900 mM) (Edbeib et al., 2016), microorganisms that survive in dry environments (water activity < 0.75) (Connon et al., 2007) and radiotolerant (UV resistant) extremophiles that are resistant to the permanent exposure to damaging solar radiation (Gabani et al., 2014) (**Table 1**). Additionally, it is worth mentioning that extremophiles are usually defined by one extreme condition, nevertheless, many natural environments possess two or more extreme conditions. The microbiota living on those ecosystems, also known as polyextremophiles, is adapted to an additional extreme condition to the one condition that characterizes them, such as temperature, pH, salinity (Urbieta et al., 2015b).

Chile occupies a long strip of territory in the south west of South America and with other countries shared part of the Antarctica. It has been referred to be a "biogeographic island" (Scherson et al., 2017), due to a series of geological events that have formed the current natural barriers Altiplano, Atacama Desert, Los Andes Mountains, Pacific Ocean, Patagonia, and Antarctica (Villagrán and Hinojosa, 1997; Villagrán and Armesto, 2005). Due to its extremely diverse geography and singular geochemical and climatic conditions, Chile gathers many extreme environments, which may result intolerably hostile or even lethal for most life forms, except for extremophiles (Rampelotto, 2013). In order to enable growth under these harsh conditions, extremophiles have been subjected to several adaptations, which have been only partially characterized to date. Altiplano, Atacama Desert, Central Chile, Patagonia, and Antarctica correspond to main geographical areas in Chile that harbor multiple extreme biotopes (**Figure 1**). Specific extreme biotopes of Chile are attractive scenarios to study the evolution of microorganisms under extreme conditions, which resemble the islands of the Galapagos Archipelago that provide Darwin inspiring ideas to build up the Theory of Evolution by Natural Selection of macroorganisms based on the divergence of species of birds, especially the Galapagos finches.

The preservation of the natural ecosystems and the restauration of polluted sites are crucial for a sustainable


TABLE 1 | General description of extremophiles present in diverse extreme environments of Chile.

development. Bioremediation is an important technology for the clean up of environments contaminated with persistent organic pollutants [e.g., pesticides, polychlorobiphenyls (PCBs), petroleum hydrocarbons] and heavy metals (e.g., Hg, Cd) (Morgante et al., 2010; Saavedra et al., 2010; Rojas et al., 2011; Fuentes et al., 2014; Bravo, 2017). Mining of valuable metals such as copper, gold and silver is historically one the most relevant economic activities in Chile, mainly in the Northern and Central regions. Copper mining is the main activity in Chile, contributing with one third of the world's production. Unfortunately, this mining activity has been associated with heavy metal pollution in diverse sites. Microorganisms are main biocatalysts for bioremediation of polluted environments and waste treatment processes (Hernández et al., 2008a,b; Saavedra et al., 2010; Rojas et al., 2011; Fuentes et al., 2016; Méndez et al., 2017) and extremophilic microbes are required as catalysts for the bioremediation of polluted extreme environments. The aims of this review are to describe extremophiles that inhabit the main extreme biotopes in Chile, and their molecular and physiological capabilities that may be advantageous for the design and development of bioremediation processes.

## BIOTOPES AND ECOLOGICAL NICHES

Chilean territory is spanned within a long altitudinal and latitude range that contributes to the origin of a suite of landscapes and geological features that are extreme habitats for microorganisms. Some of these ecosystems will be described briefly.

## Atacama Desert

The Atacama Desert is located along the western border of South America and is the driest and oldest desert on Earth's, exhibiting similar conditions described for Mars. This ecosystem provides a unique collection of habitats, ideal for studying microbial dynamics in several extreme conditions, such as alkaline or acidic pH, high temperature, water stress, and UV radiation (**Figure 1**). Its distinctive climate is the result of the confluence of a subtropical high-pressure zone, the cold Humboldt Current on the coast, offshore winds, as well as the Andean rain-shadow effect and latitudinal position of the region (Houston and Hartley, 2003).

## Altiplanic Ecosystems

The Andean Altiplano occupies part of Peruvian, Bolivian, and Chilean territory with a mean altitude of 3,700 m above sea level and covers an area that is 300 km wide and 1,500 km long (Muñoz and Charrier, 1996). The Altiplano is surrounded by volcanoes and mountains rising up to 6,700 m and represents one of the largest plateaus in the world. The Altiplano possess several extremes environments, such as thermal waters and geysers, some basins, salars, large lakes in the north and salt flats in the south. Some of the more characteristic Altiplano ecosystems are described below.

#### Parinacota Region

The paleolakes Chungará Lake, Parinacota Wetland, and Piacota Lagoon are located 4,300–4,500 m above sea level. The microbial community composition is highly variable between the different wetlands, but also between water and sediment samples (Dorador, 2007). Each of these environments supported a unique community of Bacteria and Archaea, revealing a differentiation between high altitude lakes, freshwater wetlands, and saline wetlands.

#### Geothermal Springs and Geysers

The extreme conditions (high temperature and pressure) are characteristic of hot springs and geysers located in the Northern region and is the result of the permanent interaction of groundwater with magma and hot igneous materials stemming from near the rather abundant volcanic areas (Jones and Renaut, 1997; Fernandez-Turiel et al., 2005; Rafferty, 2010).

The Geothermal site El Tatio (Kunza language, meaning "crying grandfather") is the largest geyser field in the Southern hemisphere and one of highest geysers around the world (∼4,200 m.a.s.l.) (Glennon and Pfaff, 2003). Located on the Andean Altiplano, it harbors over 100 erupting springs and is surrounded by high volcanoes. The water discharged by the Tatio Geyser is very rich in silica, with the highest concentrations reported for a natural surface water, of both arsenic (∼0.5 mM) and antimony (∼0.02 mM) at a nearly neutral pH (Landrum et al., 2009). These properties differ from other well-studies geothermal sites, such as basins in Yellowstone National Park, United States, and Dallol Volcano, Ethiopia, for which their waters can reach extreme pH values (Rowe et al., 1973; Barbieri and Cavalazzi, 2014). Therefore, El Tatio in an environment with unique physicochemical characteristics.

The North regions of Chile harbor many hydrothermal fields that remained less explored than El Tatio. For instance, the Surire hydrothermal system, at 4,000 m.a.s.l., is located in the south part of Surire Salar (Procesi, 2014). The water discharges are characterized by higher concentration of sulfate. Analogous situation occurs with Lirima Geothermal field that is located at an altitude of 3,900 m.a.s.l., 25 km southwest of the Sillajhuay volcanic chain. The Lirima area contains bubbling pools with temperatures between 38◦C and 80◦C (Tassi et al., 2010; Procesi, 2014).

#### Salars

Chile harbors a huge diversity of salars along its territory. Saline ecosystems are located in Altiplano, Atacama Desert, Central Chile, and Patagonia. Specific salars that has been subjected to microbial studies will be described.

#### Athalassohaline Ecosystem: Huasco Salar

Athalassohaline systems are saline ecosystems with a non-marine origin but originated from evaporation of fresh water in a system dominated by calcium, magnesium and sulfate, in contrast with sodium and chloride that are prevalent in the ocean (Grant, 2006). At 3,800 m above sea level, Huasco Salar is a good example of an athalassohaline system (Dorador et al., 2010). These systems exhibit extreme conditions such as low temperatures

and atmospheric pressure, high solar radiation, negative water balance, and a wide range of salt concentrations (Castro-Severyn et al., 2017).

#### Acidic Salars: Ignorado Salar and Gorbea Salar

Two acidic salars in Chile are located in the Andes Mountains (Risacher et al., 2003). Gorbea Salar is located in a basin with extreme acidic brines, with pH ranging from 2 to 4 and NaCl concentrations from 0.03 to 1.3 M (Quatrini et al., 2017). Ignorado Salar is an acid saline lake, with surface waters with pH ranging from 3.3 to 4.1 and a total dissolved solids concentration from 0.5 to 3% (Karmanocky and Benison, 2016).

#### Borderline Salar: Atacama Salar

Salar de Atacama is located between the Atacama Desert and the westernmost margin of the Altiplano at 2,300 m above sea level (Zúñiga et al., 1991). It is the largest and oldest evaporating basin in Chile, and the largest Quaternary halite deposit worldwide (Warren, 2010; Lara et al., 2012). Salar de Atacama has some shallow lakes with high salt concentration (Zúñiga et al., 1991) and with distinct geological features compared to the Altiplano basins (Demergasso et al., 2010).

#### Central and Southern Salars

Other saline ecosystems are also located in the Central Region and the Southern Patagonia (De Los Rios-Escalante and Gajardo, 2010; Plominsky et al., 2014). Cáhuil Lagoon is a saline ecosystem located at the coast close to Pichilemu in Central Chile (**Figure 1**).

#### Patagonia

Patagonia is comprised by an extensive structure of cold fjords (e.g., Comau Fjord) and channels, and by oligotrophic cold lakes in Southern Chile, characterized by low temperatures, low nutrient concentration and low dissolved organic carbon (Gutiérrez et al., 2015; Aguayo et al., 2017). Cold lakes in Patagonia (e.g., Grey Lake, **Figure 1**) are affected by seasonal temperature variations, ranging from 4◦C in winter to 20◦C in summer (Aguayo et al., 2017).

Volcanic activity has been also reported with a high eruption frequency (Carrillo et al., 2018), which has formed numerous hydrothermal fields including Porcelana Hot Spring and Geyser, and Cahuelmo Hot Spring. Cahuelmo Hot Spring is a geothermal site located at the sea level coast of Cahuelmo Fjord, that contains waters rich in metallic minerals and elements such as pyrite, polonium, magnetite, and chalcopyrite (Mackenzie et al., 2013). On the other hand, Porcelana hot spring is located ∼100 m above sea level in Northern Patagonia. Porcelana is a pristine spring characterized by a rather extensive thermal gradient (∼38–69◦C) and neutral pH (Alcamán et al., 2015).

#### Antarctica

Antarctica displays extreme climates and environmental conditions above and below the water surface. This environment is dominated by strong gradients in temperature (−10◦C to −2 ◦C), salinity (35–150%), and irradiation (<0.1% to 1–5% UV radiation), properties highly variable and ultimately governed by air temperature and snow cover (Cirés et al., 2017). The search of new pigments, antibiotics, and enzymes has become a main research focus in the Antarctic continent (Loperena et al., 2012; Órdenes-Aenishanslins et al., 2016; Lavin et al., 2017). In spite of been the coldest continent on Earth, surprisingly, Antarctica harbors many geothermal sites.

## THERMOPHILES

Early life in Earth was initially dominated by thermophilic anaerobes that had chemoheterotrophic or chemolithoautotrophic metabolism capable of been sustained by hydrothermal energy sources (Woese et al., 1990; Konhauser et al., 2003; Weiss et al., 2016). Thermophiles are organisms, mainly prokaryotes, whose optimum growth temperatures are >44◦C (Madigan et al., 2000). Hyperthermophiles are thermophiles that grow at temperatures >80◦C (Berenguer, 2011) (**Table 1**). Thermophiles and hyperthermophiles are present in several natural ecosystems such as geothermal waters (**Figure 2**), hot springs, mud pots, fumaroles, geysers, deep-sea hydrothermal vents, volcanoes, and also in engineered environments, such as compost facilities and anaerobic reactors (Ahring, 1995; Rastogi et al., 2010; Urbieta et al., 2015b).

A general mechanism of thermophiles to safeguard their cellular components at high temperature is the adaption of thermophilic proteins through amino acid changes in their primary structure, increasing their thermal stabilities (Xu et al., 2018). Thermophilic proteins possess a larger fraction of amino acid residues in α-helices and have shorter amino acid length (Urbieta et al., 2015a; Xu et al., 2018). A main mechanism in thermophiles is the role of heat shock proteins (HSPs) including the chaperones DnaK, GroEL, and GroES to assist protein folding (**Figure 3**). DNA-repair systems (e.g., SOS system) are also active to respond to DNA damage. For the stabilization of the membranes, thermophiles use branched chain fatty acids and polyamines (e.g., spermidine). Another mechanism of thermophiles is the use of compatible solutes to stabilize cell components (Urbieta et al., 2015a). In addition, upregulated glycolysis pathway (e.g., pyruvate dehydrogenase complex) proteins provide immediate energy to cope with heat stress (Wang et al., 2015).

An increase in the use of (hyper)thermophilic microorganisms has been observed, especially for the need of the industry to couple biological solutions at high-temperature industrial processes (Urbieta et al., 2015a). Industrial waste reactors containing living microorganisms required to be pre-cooled down due to the fact that several industrial processes are carried out at temperatures >100◦C (Gavrilescu, 2010). Therefore, (hyper)thermophiles offer a suitable solution due to their capabilities to grow at such high-temperature and also resist and metabolize several pollutants from contaminated industrial wastewaters (Vieille and Zeikus, 1996). Previous studies have demonstrated the high potential of (hyper)thermophilic pure cultures and consortia for bioremediation of heavy

metal-contaminated surface and groundwaters, including biosorption and immobilization of radionuclides and heavy metals (Chatterjee et al., 2010; Sar et al., 2013), removal of heavy metals (Ilyas et al., 2014) and for degradation of persistent organic compounds such as aliphatic and (poly)aromatic hydrocarbons (Mnif et al., 2014; Zhou et al., 2018), and synthetic

FIGURE 3 | Molecular mechanisms of extremophiles for their adaptation to extreme environmental conditions. Acidophiles. (i) Potassium antiporter releases protons towards the extracellular medium, (ii) ATP synthase, (iii) membrane highly impermeable to protons, (iv) Chaperones, and (v) DNA-repair proteins. Thermophiles. (i) Upregulated glycolysis proteins (e.g., pyruvate dehydrogenase complex (PDC)), (ii) Lipids with iso-branched chain fatty acids and long chain dicarboxylic fatty acids, (iii) polyamines (spermidine), and (iv) Chaperones. Halophiles. (I) High salt-in strategy: (i) chloride transporters (primary or secondary), (ii) potassium uptake into cells by concerted action of bacteriorhodopsin and ATP synthase. (II) Low-salt strategy: (i) de novo synthesis or uptake of osmoprotectants (proline-betaine, ectoine) that maintain osmotic balance and establish the proper turgor pressure under different salt concentration. Psychrophiles. (i) high degree of unsaturated, cyclopropane containing fatty acids and short chain fatty acids, (ii) Cold shock proteins (CSP) (iii) Chaperones, (iv) Anti-freeze proteins (AFP) restrict the ice growth on protein surfaces, (v) Mannitol and other compatible solutes accumulate in the cell cytoplasm as cryo-protectants to prevent protein aggregation, and (vi) Carotenoids (star symbols) support maintenance of membrane fluidity and prevent cell damage by UV radiation. UV resistance. (i) Manganese accumulation and reduced iron levels, (ii) Antioxidants (glutathione), (iii) Chaperones, and (iv) DNA-repair proteins. Xeric resistance. (I) Evasion mechanism: (i) bacteria sporulation. (II) Adaptation mechanism: (i) increased extracellular polymeric substances (EPS), (ii) DNA-repair proteins, and (iii) accumulation of osmoprotectants (glycine, trehalose). Alkaliphiles. (i) Electrochemical gradient of Na<sup>+</sup> and H<sup>+</sup> by electrogenic antiporters for proton accumulation, (ii) Na+-solute uptake system, and (iii) Cytochrome c-552 enhance terminal oxidation function by electron and H<sup>+</sup> accumulation.

dyes (Deive et al., 2010). Increasing research is conducted into evaluating the applications of thermostable enzymes in waste treatment and remediation (Kataoka et al., 2014; Wang et al., 2015; Rigoldi et al., 2018).

A high portion of the Chilean territory is superimposed on the subducting Nazca plate, the largest tectonic relief, that drives displacement and rearrangements of geological structures and generates routinely megathrust earthquakes (Armijo et al., 2015). In those areas, the compression and decompression of magma may modulate volcanic activity on land, in the seafloor, and in the deep subsurface, generating abundant natural formations that are suitable for survival and growth of (hyper)thermophilic microbial life forms. Geothermal fields are usually main contributors of arsenic to both surface and subsurface water (Ballantyne and Moore, 1988; Smedley and Kinniburgh, 2002). The water influx is particularly important at the water discharged at the Tatio Geyser, which achieved in surface water a arsenic concentration of 45 mg L −1 (Landrum et al., 2009). The effluent discharges caused high arsenic levels (0.1–1.5 mgL−<sup>1</sup> ) throughout the basin of Loa River. Besides the potential human health risks (Yañez et al., 2005), those arsenic levels impact also the microbiota. Pseudomonas fluorescens and Serratia odorifera strains isolated from an arsenicpolluted river in the Atacama Desert showed tolerance to arsenic 800–1,000 mM (Campos et al., 2009; Escalante et al., 2009). Due to their physicochemical properties, the water discharged by the Tatio Geyser is an unique environment to study (hyper)thermophiles. A fraction of the water is very rich in silica, contains high antimony concentration (∼0.02 mM), low sulfite concentration (∼0.5 mM) at a nearly neutral pH (Landrum et al., 2009). Most of the worldwide studies efforts directed to the biotransformation of arsenic by (hyper)thermophiles have been restricted to sulfidic waters at extreme pH conditions, including high arsenic geothermal sites in Yellowstone National Park in United States and Dallol Volcano in Ethiopia (Rowe et al., 1973;

Barbieri and Cavalazzi, 2014). Research has been focused toward isolation and characterization of (hyper)thermophiles tolerant to As, or capable of playing a role on its biogeochemical cycle. The geothermal water discharges arsenite As(III), which is the most mobile and toxic form of arsenic that progressively oxidizes to arsenate As(V) downstream (Engel et al., 2013). Several prospections have shown that nearly all of the geyser features and streams at El Tatio are covered by biofilms and colorful microbial mats dominated by photosynthetic bacteria (Chloroflexi and cyanobacterial communities) (Fernandez-Turiel et al., 2005; Engel et al., 2013; Plenge et al., 2017). Microbial community analysis based on 16S rRNA gene of the geyser-discharge water revealed that Chloroflexi, Deinococcus–Thermus, Aquificales, and Chlorobi were the most prevalent microorganisms in the zones where arsenite reduction takes place (Engel et al., 2013). Indeed, the survey of functional genes revealed that most of the arsenite oxidase aioA-like gene found within the community showed high identity to genes belonging to strains of the thermophilic anoxygenic phototroph Chloroflexus aurantiacus. Chloroflexi-like aioA gene sequences obtained from the microbial community of El Tatio clustered on two separated clades likely representing additional diversity that could be associated with either novel groups of arsenic-resistant bacteria, or novel mechanisms of As resistance. It is also noteworthy to mention that Chloroflexus aurantiacus can either grow phototrophically under anaerobiosis or chemotrophically under aerobic and dark conditions, providing enough metabolic flexibility to function as a potential strain for bioremediation of As-contaminated environments, but also during carbon sequestration efforts (Tang et al., 2011).

Besides arsenic, the microbial communities associated to El Tatio cope with a wide variety of environmental stressors, such as the presence of other toxic metals (e.g., antimony) and the impact of extreme UV radiation. Living at such selective pressure has fostered the development and retention of a suite of metabolic and physiological adaptations that has enormous potential to be used for biotechnological applications. On one hand, multi-resistant microbial isolates may be useful in bioremediation. Furthermore, a second area of interest is the identification and characterization of the mechanisms of resistance to multiple stressors. Indeed, enzymes synthesized by (hyper)thermophiles display high levels of thermostability, converting them in candidates to explore potential novel applications for industrial processes. For example, the archaeon isolated from a solfataric pool located in El Tatio, Methanofollis tationis (**Figure 2**, first described as Methanogenium tatii) (Zabel et al., 1984; Zellner et al., 1999) produced its own particular pterin, a type of biomolecule involved in immune modulation, cellular signaling, metabolism and pigmentation. Two years after its isolation, the identification and structure of the novel pterin produced by Methanofollis tationis, named tatiopterin, were elucidated. Afterward, it has been shown that tatiopterin specifically enhances photostability of materials, preventing the bleaching of photosynthetic pigments due to irradiation (Elshahawi et al., 2015). More efforts in order to understand the role of such molecules under those conditions should be further explored. Specifically, one area of interest in this field is the increase of exploration efforts of novel thermozymes that can be applied for production of biofuels from starch and lignocellulosic waste materials. Due to their complex structure, lignocellulosic materials are degraded by physicochemical treatments to obtain cellulose, hemicellulose, and lignin, which can be further treated with hydrolytic enzymes (Urbieta et al., 2015a).

Previous prospections in the Surire Salar have described the presence of arsenic-precipitating bacteria, suggesting that may be also present in the Surire hydrothermal vents (Demergasso et al., 2007). The culturable mesophiles in Lirima wetland were reported, whereas the hot spring remain unexplored (Scott et al., 2015). Special attention should be devoted in this site due to the fact that water contain high chloride and boron concentrations, becoming a source of microorganisms capable of bioremediation of boron-polluted wastewaters. Microbes associated to the hydrothermal systems such as Puchuldiza-Tuja (4,100 m.a.s.l.), Colpitas (4,000 m.a.s.l.), Apacheta (4,500 m.a.s.l.) have not been explored yet.

Porcelana hot spring in Northern Patagonia is characterized by abundant and colorful microbial mats widespread along a rather extensive thermal gradient (∼38–69◦C) (Alcamán et al., 2015). The microbial community associated to Porcelana hot spring is dominated by cyanobacteria, particularly of the diazotrophic Mastigocladus (Stigonematales) genus. Members of the same genus were also identified as part of biofilms in Copahue geothermal field at the North west corner of Neuquén province, Argentina (Urbieta et al., 2015b), indicating its cosmopolitan character (Miller, 2007). At Porcelana hot spring, Mastigocladus sp. strain CHP1 was the most dominant contributor of nitrogen through nitrogen fixation (∼87% of the nifH gene transcripts) (Alcamán et al., 2015). Physiological studies revealed that Mastigocladus isolates gather a broad range of metabolic plasticity regarding the nitrogen metabolism (Alcamán et al., 2017). Mastigocladus sp. strain CHP1 is capable to fix nitrogen at 60◦C, the highest temperature reported for the activity of N2-fixing filamentous cyanobacteria (Alcamán et al., 2015). Further studies should explore the metabolic versatility of Mastigocladus sp. strain CHP1 in the treatment of nitrate-contaminated industrial waters at high temperature.

Environments are so dynamic that surprise us. An iconographic example of such diversity is depicted by the fact that Antarctica, a continent well known as the coldest place on earth, also harbors many geothermal sites suited for the growth of (hyper)thermophile microorganisms (Flores et al., 2013). The uppermost temperatures of those sites range from 40 to 110◦C, and has extremely low concentrations of nutrients, such as N and P, and high concentrations of heavy metals (Cu, Zn, Cd, Pb, and Hg) (Muñoz et al., 2011). Although environmental conditions seem to be harsh for microbial growth, recent studies have shown that the environment is suitable for thermophilic organisms. Bacteria from the Geobacillus, Bacillus, Brevibacillus, Thermus genera and uncultured sulfate reducing bacteria were abundant in a fumarole at 90–110◦C from Deception Island, an active strato volcano located in the South Shetland Islands (Muñoz et al., 2011). Some of these microbes can carry metal redox interactions that have further implications for ore formation and might be capable to recover metals from ore-containing

materials. For instance, the thermophilic Geobacillus sp. strain ID17 is capable to synthesize gold nanoparticles when exposed to Au(III). The nanoparticles were found to be intracellularly accumulated raising the potential applications in bioremediation of gold-bearing wastes (Correa-Llantén et al., 2013). The synthesis of gold nanoparticles by bacteria can be useful for diverse biotechnological and medical applications (Montero-Silva et al., 2018). Geobacillus species are also capable to degrade organic pollutants. Indeed, members of the same clade including Geobacillus thermoleovorans T80 degrade hexadecane (70%) during bioremediation at ∼60◦C. Archaea have also been isolated from soils extracted at Deception Island. Dennett and Blamey (2016) isolated the hyperthermophilic archaea Pyrococcus sp. M24D13, and characterized a novel thermostable cyanide-degrading nitrilase. This thermostable enzyme may be useful to remediate cyanide contaminated waste streams (Dennett and Blamey, 2016). (Hyper)thermophiles and their novel thermostable enzymes are of considerable biotechnological interest to find novel alternatives for bioremediation and other bioprocesses to be carried out at high temperatures.

## ACIDOPHILES

Acidophiles are defined as organisms that grow at an optimum pH < 5 (Johnson, 2008). Extreme acidophiles are microorganisms that showed an optimum growth at pH 3 or less, whereas moderate acidophiles are those that grow with optimum pH between 3 and 5. Acid-tolerant microorganisms, have optimum pH > 5, but are still active in low pH environments (Johnson, 2007, 2008). Extreme acidophilic organisms are exclusively microbial and distributed in Archaea, Bacteria, and Eukarya domains (Sharma et al., 2016).

Acidophiles maintain the cytoplasmic pH close to neutrality to safeguard the acid-labile cellular constituents, which require the generation of a large pH gradient. Three main mechanisms are involved in the adaptation to an acidic environment (**Figure 3**). A first mechanism is an active pumping of protons to the maintenance of 1pH, by proton flux systems. The role of proton efflux via transport pumps in the electron transport chain alongside the influx of protons through the F0F1-type ATP synthase has been described in Bacillus acidocaldarius, Thermoplasma acidophilum, and Leptospirillum ferriphilum (Michels and Bakker, 1985). Additional proton flux systems include primary proton pumps (symporter) and secondary proton pumps (e.g., cation/H<sup>+</sup> antiporter), and proton-consuming reactions. A carbonic anhydrase and amino acid decarboxylases that aid in pH homeostasis by consuming protons have been reported in L. ferriphilum (Christel et al., 2018). A second mechanism is a decreased permeability of the cell membrane to suppress the entry of protons into the cytoplasm. The influx of protons is inhibited by the inside positive membrane potential formed by K<sup>+</sup> ions (Christel et al., 2018). A wide repertoire of genes related to cell membrane biosynthesis that may be associated with acid tolerance was identified in L. ferriphilum. The presence of tetrapetric lipids in the cell membrane that provide tolerance to acidic pH has been reported in Archaea Ferroplasma acidiphilum and Sulfolobus solfataricus. A third mechanism is an improved protein and DNA-repair systems in acidophiles compared to neutrophils. An external pH shift from 3.5 to 1.5 induced proteins that are involved in the heat shock response such as chaperones in the acidophile At. ferrooxidans (Amaro et al., 1991).

Acidophilic microorganisms play an important role in biomining of metals from low grade sulfur minerals (Bustos et al., 1993; Seeger and Jerez, 1993; Seeger et al., 1996; Okibe et al., 2003; Demergasso et al., 2005, 2010; Acosta et al., 2017). Previous studies have demonstrated the role of acidophiles in bioremediation of polluted soils and waters, through (i) metal reductive processes (Kolmert and Johnson, 2001; Suzuki et al., 2003; Leigh et al., 2015), (ii) metal adsorption onto jarosites (Natarajan, 2008; Asta et al., 2009), (iii) metal biosorption (Liu et al., 2004; Chakravarty and Banerjee, 2012), and (iv) degradation of petroleum hydrocarbons (Stapleton et al., 1998; Margesin and Schinner, 2001; Christen et al., 2012; Arulazhagan et al., 2017). Additionally, enzymes from acidophilic microorganisms are explored due to their tolerance to low pH, which favors their industrial applications in starch, fruit juices, feed and baking industries (Matzke et al., 1997; Nakayama et al., 2000; Serour and Antranikian, 2002; Sharma et al., 2012). More recently novel applications of acidophiles including electric generation have been explored (Sulonen et al., 2015; Ni et al., 2016).

Acidophilic microorganisms are present in several natural habitats, such as solfataric fields and geothermal sulfur rich sites (Sharma et al., 2016). These sites are niches for a variety of acidophilic microorganisms with unique adaptations for survival in the hostile low pH environments such as Sulfolobus solfataricus and Sulfolobus acidocaldarius (Yellowstone National Park, United States) (Brock et al., 1972). Acidophilic microorganisms have also been reported in anthropogenic environments such as acidic mine drainage (AMD), which are associated with heavy metals and coal mining. The acidophilic microorganisms mobilize metals and generate AMD. AMD generates most of the extremely acidic niches on Earth and disseminates heavy metals in the environment (Panda et al., 2016).

In Chile, an important area where acidophiles have been studied is located in the Atacama Desert. The microbial solubilization of metals in acidic environments has been successfully used in bioleaching for the extraction of metals. In Escondida mine located 170 km south-east from Antofagasta, the analyses of the microbial community of a low-grade copper sulfide leach pile indicated the presence of At. ferrooxidans (**Figure 2**), At. thiooxidans, L. ferriphilum, and F. acidiphilum (Galleguillos et al., 2008; Remonsellez et al., 2009; Demergasso et al., 2010; Acosta et al., 2017). Prokaryotic acidophile microarray (PAM) analysis showed members of Sulfobacillus genus in samples from heap leaching (Remonsellez et al., 2009). 16S rRNA genes phylogenetic analysis, real time PCR and metagenomics analysis revealed in the heap leaching the bacteria At. ferrooxidans, At. thiooxidans, At. caldus, At. ferrivorans, L. ferriphilum, S. acidophilus, S. thermosulfidooxidans, and Acidiphilium spp., and the archaea Ferroplasma acidiphilum, Ferroplasma acidarmanus, and Sulfolobus spp.

(Demergasso et al., 2005, 2010; Soto et al., 2013; Acosta et al., 2017). Acidithiobacillus, Leptospirillum, and Sulfobacillus strains have also been reported in the copper tailings of La Andina mine in Central Chile (**Figure 2**). Molecular methods revealed the presence of heterotrophic acidophiles associated to Acidobacterium capsulatum, Acidobacterium-like bacterium and Acidiphilum sp. (Diaby et al., 2007). In Chile, solfataras throughout Andes Mountains (e.g., Purico Complex and Nevados de Chillán) that harbor acidophiles have been described. The moderate acidophile archaea S. solfataricus was isolated from a hot spring in Nevados de Chillán (Valdebenito-Rolack et al., 2017). To date, acidophilic microbes have been scarcely studied in diverse acidic environments in Chile including solfataras.

In the last decades, the bioremediation potential of acidophiles in acidic environments have been studied. Acidophiles capable to degrade phenol at low pH have been reported. The acidophile S. solfataricus degrades phenol (Christen et al., 2011). Zhou et al. (2016) reported the degradation by S. acidophilus of phenol, and methylphenols. Acidophilic bacteria able to grow in presence of alkanes have been described (Hamamura et al., 2005). Acidophiles including Acidisphaera, Acidiphilium, and Acidithiobacillus strains were capable to degrade 50% hexadecane in hydrocarbon-amended soil/sand mixtures. Acidophiles have been used for the treatment of petroleum-polluted wastewater under acidic condition (Arulazhagan et al., 2017). Acidophilic microorganisms such as Acidithiobacillus and Sulfobacillus strains may be useful for the remediation of hydrocarbonpolluted acidic sites (Ivanova et al., 2013).

The removal of heavy metals by acidophiles from Chile has been reported. At. ferrooxidans has been used for removal of different heavy metals at laboratory scale. The addition of At. ferrooxidans to an AMD increased precipitation kinetics of heavy metals and decreased water iron content, accelerating heavy metal removal (Darkwah et al., 2005). At. ferrooxidans has been used for the ex situ bioremediation of uranium (VI), removing up to 50% of uranium (100 mg L−<sup>1</sup> ) from polluted mine water (Romero-González et al., 2016). Takeuchi and Sugio (2006) reported that mercury was almost completely removed by volatilization in mercury-polluted soil by At. ferrooxidans strains SUG 2- 2 and MON-1. Also MON-1 cells immobilized in PVA resins efficiently volatilize mercury from mercurypolluted wastewater. Bioremediation by bioaugmentation with heavy metal-resistant bacteria of mercury-polluted waters has been reported (Rojas et al., 2011). This bioremediation process was scaled up (Bravo, 2017) and may be applied in mercury-polluted sites closed to metal mining activities. At. ferrooxidans and Leptospirillum ferrooxidans have been used for arsenite removal. Bioremoval occurs through the adsorption of arsenic (III) onto the jarosites generated during microbial growth (Natarajan, 2008). Furthermore, At. ferrooxidans and At. thiooxidans strains are able to reduce chromium (VI). The almost complete removal (93%) of chromium (VI) from electroplating waste by A. thiooxidans has been reported (Cabrera et al., 2007). At. thiooxidans is able to reduce uranium by polythionates that are synthesized during oxidative sulfur metabolism (Gargarello et al., 2010). At. ferrooxidans has been applied for removing sulfur from solids and gases and heavy metals from electric wastes and sludge (Zhang et al., 2018). In addition, the removal of Ni and Hg from port sediments through bioaugmentation with a consortium of ironoxidizing acidophilic bacteria (A. thiooxidans, A. ferrooxidans, and L. ferrooxidans) in microcosm was observed (Beolchini et al., 2009).

Metal mining is a key player in the Chilean economy. Therefore, diverse acidophiles useful for bioleaching have been isolated, characterized and applied for bioleaching in Chile. Interestingly, some of these acidophilic microorganisms have been used at laboratory scale for the bioremediation of heavy metals and organic pollutants. However, the application of these acidophiles in bioremediation at industrial scale is still an important challenge. Acidophilic microbes from extreme environments are attractive biocatalysts for bioremediation processes under acidic conditions, especially for heavy metals.

#### HALOPHILIC MICROORGANISMS

Microorganisms belonging to the three domains of life are present over the whole range of salt concentrations in the environment (Oren, 1999). Halophiles are microorganisms that obligately require salt to grow (Margesin and Schinner, 2001). They are classified based on their optimal NaCl concentration for growth as slight halophiles (0.2 M), moderate halophiles (0.5–2.5 M), borderline extreme halophiles (>2.5–4.0 M) or extreme halophiles (>4.0–5.9 M) (Edbeib et al., 2016). Halophilic microorganisms are ubiquitous in salars, saline lakes, oceans, polar ice, and coastal areas (Edbeib et al., 2016). Halotolerants are microorganisms that grow in the presence and absence of NaCl, and those that grow in presence of >2.5 M NaCl are considered extremely halotolerant (Margesin and Schinner, 2001).

Halophilic and halotolerant microorganisms have adapted and evolved to survive in saline environments. They gather unique metabolic properties toward maintaining more water in the cytoplasm than in their surroundings, avoiding water losses. Halophilic or halotolerant microorganisms have evolved two main strategies (**Figure 3**). The first strategy is maintaining an intracellular salt concentration equivalent to the environment, and consequently, all intracellular systems have been adapted. This is achieved with chloride and potassium uptake into the cells by transporters (primary or secondary) and concerted action of bacteriorhodopsin and ATP synthase. The other strategy is maintaining low intracellular salt concentration, and therefore osmotic pressure is balanced by organic compatible solutes, such as betaine and ectoine. Due to its nature, the latter strategy does not require a global adaptation of the intracellular machinery (Oren, 1999; Margesin and Schinner, 2001).

Chile gathers several high saline environments. Most of them are concentrated in the Northern region, where 52 saline lakes and salt crusts are distributed alongside The Andes, spanning an area of over 200,000 km<sup>2</sup> of the Atacama Desert. The saline ecosystems found in the Northern region can be classified in (i) Borderline Salars, (ii) Athalassohaline ecosystems, which are located in the Altiplano, and (iii) Acidic Salars (Risacher et al., 2003; Risacher and Fritz, 2009). Particularly, in Lejía

Lake, an extreme saline lake nested at the base of Lascar Volcano in the Chilean Altiplano, two halotolerant bacteria closely related to Halomonas alkaliantarctica strain CRSS and capable to grow at 15% NaCl were isolated (Mandakovic et al., 2018). Other saline lakes are also located in Central region (Cáhuil Lagoon), and the Southern Patagonia (De Los Rios-Escalante and Gajardo, 2010; Plominsky et al., 2014). One third of bacterial isolates from different niches in the Atacama Salar were classified as moderate halophilic and halotolerant bacteria. These isolates belong to Marinomonas, Vibrio, Alteromonas, Marinococcus, Acinetobacter, Micrococcus, Bacillus, Pseudomonas, Deleya, Staphylococcus genera and also some Cyanobacteria, such as members of the Cyanothece, Gloeocapsa, and Gloeobacter genera (Zúñiga et al., 1991; Dorador, 2007). Saltern ecosystems are commonly dominated by Archaea, where members of the family Halobacteriaceae are the most common halophiles (Oren, 2002). Metagenomic studies performed on samples extracted from a salt crystallizer pond located in the Cáhuil Lagoon, revealed that 61% of the gene sequences belonged to Archaea, mainly from the Halobacteriaceae family, followed by 19% sequences from viruses and 16% from bacteria (Plominsky et al., 2014). Microbial communities in the Atacama Desert ecosystems, such as halites, saline soils and salt lakes, are also dominated by members of the Halobacteriaceae family (Robinson et al., 2015; Finstad et al., 2017).

Arsenic is a metalloid that is widely distributed at high concentrations along the Atacama Desert. The sources of arsenic are often associated with volcanic-hydrothermal springs, metal ores and anthropogenic activities. Microbial communities from Atacama Salar contributed to arsenic reduction (Lara et al., 2012), which is a process that has been widely studied in salinealkaline systems (Oremland et al., 2004; Kulp et al., 2007). Shewanella sp. strains Asc-3 and CC-1 isolated from Ascotán low-pH salt flats (Demergasso et al., 2007; Lara et al., 2012) were capable of precipitating arsenic (**Table 2**). Surprisingly, arsenic-precipitating bacteria accounted for 50% of the bacterial communities, suggesting that arsenic-based processes in this ecosystem are significant in the formation of arsenic minerals (Demergasso et al., 2007).

Exiguobacterium sp. SH31 isolated from a moderate saline environment in Huasco Salar was able of grow in presence of As(III) 10 mM and As(V) 100 mM, highlighting its natural resistance to arsenic. Exiguobacterium strains capable of reducing arsenate and chromium have been reported. Interestingly, Exiguobacterium strains have been explored for bioremediation applications. An E. aurantiacum strain isolated from a lake in Southern Spain degrades pesticides (López et al., 2005). Microbacterium sp. CGR1 (**Table 2**) from the Atacama Desert tolerates NaCl 1.2 M, and possesses arsenic resistance (Mandakovic et al., 2015). An additional survey of metal resistance in halotolerant and halophilic isolates from the Atacama Desert showed that most of them were capable to cope with the presence of heavy metals such as Cd, Zn, Ni, Cu, and Co. For example, Thalassobacillus devorans showed high tolerance to cadmium and nickel (Moreno et al., 2012).

Other Chilean ecosystems such as Cahuil marine salterns are sources for strains capable to be used in bioremediation platforms. The extreme halophile Haloferax sp. CL47 that was isolated in Cahuil degrades several polyaromatic hydrocarbons (PAHs) such as naphthalene, anthracene, phenanthrene, pyrene and benzanthracene (Bonfá et al., 2011). In addition, Haloferax sp. CL47 has been used for petroleum degradation in wastewater. The extreme halophile Halomonas nitroreducens 11S<sup>T</sup> (**Figure 2** and **Table 2**) showed the capability to reduce selenite to elemental selenium (González-Domenech et al., 2008).

Halotolerant strains belonging to the phylum Actinobacteria were isolated from marine sediment samples from the Comau Fjord in Northern Patagonia (Undabarrena et al., 2016). These strains showed tolerance to up to NaCl 1.7 M. Streptomyces sp. H-KF8 (**Figure 2**) tolerates different heavy metals, such as Ni (15 mM), Cu (0.75 mM), Co (6 mM), Zn (50 mM), Cd (1.5 mM), Hg (60 µM), Te (40 µM), Cr (20 mM), and As (100 mM). The genome sequencing of the Streptomyces sp. H-KF8 strain revealed the presence of 49 heavy metal resistance genes (Undabarrena et al., 2017).

Due to industrial activities or natural sources, saline environments frequently have a high concentration of organic compounds (Castillo-Carvajal et al., 2014; Edbeib et al., 2016). Industrial wastewaters frequently possess high salt concentration, and high levels of organic matter and pollutants, for which a wide variety of conventional biological treatments are not currently suitable (Bonfá et al., 2011). The presence of high levels of aromatic compounds in saline wastewater during crude


oil extraction has been described (Le Borgne et al., 2008). The biodegradation of hydrocarbon-derived pollutants in both soils and groundwater is impaired under elevated salt concentration (Lay et al., 2010). Thus, the application of halotolerant pollutantdegrading bacteria is essential for the bioremediation processes in saline environments.

Halophilic microorganisms capable of degrading hydrocarbons, lignocellulosic materials, chlorophenols, formaldehyde, nitroaromatic compounds have been reported (Oren et al., 1992; García et al., 2004). The halophile Halomonas organivorans degrades a wide range of aromatic compounds (García et al., 2004). Halomonas sp. KHS3 is capable to degrade diverse aromatic hydrocarbons and to produce extracellular rhamnolipids (Corti Monzón et al., 2018). Rhamnolipids are biosurfactants that are used for bioremediation and enhanced oil recovery (Rahman et al., 2003; Sachdev and Cameotra, 2013; Sekhon Randhawa and Rahman, 2014). Additionally, halophilic microorganisms capable of degrading lignocellulosic and nitroaromatic substrates, and chlorophenols have been reported (Oren et al., 1992). The applications of halotolerant bacteria for bioremediation of natural and industrial saline environments is an attractive challenge (Edbeib et al., 2016).

Climate change, chemical fertilizers and saline water used for irrigation are increasing salinity in agricultural soils (Valipour, 2014). Salt toxicity is a major restrictive factor in crop productivity and 20% of the cultivated land worldwide are seriously affected by salinity (Zhu, 2001; Farhangi-Abriz and Nikpour-Rashidabad, 2017). In response to salt stress, reactive oxygen species (ROS) are accumulated in plant tissues, damaging the photosynthetic apparatus and cellular membranes (Bose et al., 2014; Oukarroum et al., 2015). For preserving osmotic and ionic homeostasis under salt stress, plants accumulate osmolytes such a glycine betaine and proline (Farhangi-Abriz and Torabian, 2017). Halophilic and halotolerant microorganisms produce glycine betaine (Lamark et al., 1991) that may protect plants in saline soils. More recent studies have shown that other halotolerant bacterial mechanisms are also involved in plant protection: (i) increase production of extracellular hydrolytic enzymes (Rohban et al., 2009), (ii) increase activity of 1-aminocyclopropane-1 carboxylic acid (ACC) deaminase that reduced plant ethylene levels, which are typically increased by salt stress (Siddikee et al., 2010), (iii) increase indole-3-acetic acid (IAA) levels that enables plant to increase nutrient uptake under salt stress (Vacheron et al., 2013). These studies support the potential application of halotolerant plant growth promoting bacteria to protect crops in saline soils.

## ALKALIPHILES

Alkaliphiles are organisms that grow on alkaline habitats (pH > 9), usually showing an optimal growth within pH ∼10 (Horikoshi, 1999). These extremophiles are classified in two main physiological groups: obligate and facultative alkaliphiles. Facultative alkaliphiles are capable of growing in the pH range of 7.0–9.5, whereas obligate alkaliphiles (e.g., Bacillus krulwichiae) showed an optimal growth between pH 10.0 and 12.0 (Krulwich and Guffanti, 1989). Alkaliphiles may coexist with neutrophilic microorganisms under mild basic pH conditions, and also live in specific extreme environments.

To cope with high pH, alkaliphile bacteria possess molecular mechanisms, which comprise the activation of both symporter and antiporter systems (**Figure 3**). Electrochemical gradient of Na<sup>+</sup> and H<sup>+</sup> is produced by electrogenic antiporters, and the symporter system allows the uptake of Na<sup>+</sup> and other solutes into the cells (Krulwich, 1995; Krulwich et al., 1998). The function of cytochrome c-552 in electron and H<sup>+</sup> accumulation enhances the function of terminal oxidation in respiratory system (Matsuno and Yumoto, 2015). These systems enable the influx of protons and solutes inside the cell due to the alteration in the distribution of ions (e.g., Na+), maintaining the hydrosaline homeostasis and thermodynamic stability of the cell. The transporters are controlled, probably by signaling from a transmembrane pH sensor (Krulwich, 1995).

Diverse alkaliphilic microorganisms, including bacteria belonging to Bacillus, Micrococcus, Pseudomonas, and Streptomyces genera and eukaryotes, such as yeast and filamentous fungi, have been isolated from alkaline environments, including highly alkaline hyper-saline lakes (e.g., Lake Natron, Tanzania) and alkaline soda lakes (e.g., Lake Mono, CA, United States) (Duckworth et al., 1996; Groth et al., 1997; Horikoshi, 1999; Yakimov et al., 2001). Alkaliphiles are also present in highly alkaline enrichments generated by industrial activities (e.g., indigo dye plants) or soils with high alkalinity (e.g., estuaries with long periods of evaporation, clay particles with highly abundant alkaline crevices) (Grant, 2003; Sorokin et al., 2006).

Alkaliphilic microorganisms have been studied as novel sources for several biotechnological applications, including the treatment of highly toxic wastewater (e.g., dye-containing effluents). Textile effluents are characterized by the presence of high salt and alkaline pH along with the presence of toxic dyes (Maier et al., 2004; Khalid et al., 2012; Prasad and Rao, 2013). Several alkaliphiles have been isolated from these hostile environments and explored as biocatalysts for the treatment of dye-containing effluents (Hou et al., 2017). Nesterenkonia lacusekhoensis EMLA3 that was isolated from a highly alkaline textile effluent (pH ∼13) degrades the toxic azo dye methyl red in the presence of high salt concentration and heavy metals (Ni (II), Cr (VI), and Hg (II) (Bhattacharya et al., 2017). Alkaliphilic microorganisms have been applied to remove ammonia from N-rich saline wastewater, mainly produced by coke plants, fuel refining and fertilizer industries. Chemolithoautotrophic alkalophilic microorganisms have been applied to clean up ammonia pollution in effluents (Sheela et al., 2014; Cui et al., 2016). Complete ammonia oxidation into nitrate by bacteria could be useful for waste water treatment and to engineer ecosystems (Lawson and Lücker, 2018).

In Chile, alkaliphiles have been isolated mainly in the Northern Region, specifically along salt deposits from Atacama Desert and Altiplano. This region has aridic to semi-aridic climate regimes and comprises many different (hyper)saline deposits, including evaporitic basins, known as saltflats, and athalassohaline ponds (Chong, 1984; Demergasso

et al., 2004). Microorganisms in saltflats from Llamará Salar (Central Depression), Atacama Salar (Pre-Andean Depression), Ascotán Salar and Huasco Salar (Altiplano) have been reported (Demergasso et al., 2004; Dorador et al., 2008; Castro-Severyn et al., 2017), revealing insights of the special metabolic capabilities of these alkaliphiles and their responses to multiple stressors. Mandakovic et al. (2018) isolated from Lejía Lake in the Chilean Altiplano Microbacterium sp. CGR2, Planococcus sp. strains ALS7 and ALS8 that are tolerant to pH 12. However, the extremophiles from several alkaline ecosystems in Chile have not been explored. For example, Amarga Lake (Torres del Paine National Park, Patagonia) is a shallow cold lake with hypersaline water and an alkaline pH of 8.9 (Solari et al., 2004), which should harbor polyextremophile bacteria. However, to date in Amarga Lake only the presence of stromatolites has been reported.

Several heavy metal-resistant alkaliphiles have been isolated in Chile. The microbiota associated to the athalassohaline ecosystem Huasco Salar in the Atacama Desert showed resistance/tolerance to copper, tellurium, and arsenic (Castro-Severyn et al., 2017). Exiguobacterium isolates from this site carry a number of stress-related genetic determinants, including metal/metalloid resistance genes. For example, the arsenic resistant strain Exiguobacterium sp. SH31 has a set of genes encoding proteins required for arsenic resistance, including the arsenic efflux pump Acr3, growing in presence of arsenite (10 mM) and arsenate (100 mM) (Ordoñez et al., 2015; Castro-Severyn et al., 2017). Dorador et al. (2008) analyze ammonia-oxidizing bacteria (AOB) from four sites in Huasco Salar. A phylotype exhibited 98% sequence similarity to the extremely alkalitolerant ammoniaoxidizing Nitrosomonas europaea/Nitrosococcus mobilis (Squeo et al., 2006; Dorador et al., 2008). AOB play a key role in the nitrogen cycle. Nitrosomonas and Nitrosococcus genera belong to Nitrosobacteria that are AOB involved in the aerobic oxidation of ammonia into nitrite in agricultural soils (Hernández et al., 2011).

Alkaliphiles have been also found in unusual habitats. Quiroz et al. (2015) reported the presence of Halomonas alkaliphila in a brine shrimp Artemia that was collected from salty lagoons scattered in saltflats of the Atacama Desert (Browne, 1980; Abatzopoulos et al., 2002; Riddle et al., 2013; Quiroz et al., 2015). Halomonas alkaliphila is an alkaliphilic halotolerant bacterium that grows aerobically at pH 9, which has been previously isolated from a salt pool located in Montefredane, Italy (Romano et al., 2006). Recently, two Halomonas strains were isolated from Lejía Lake soil in Atacama Desert (Mandakovic et al., 2018). Due to its adaptation to a wide range of salt concentrations and alkaline pH, Halomonas may be useful for the clean up of polluted saline habitats (Berendes et al., 1996). Additional studies of novel alkaliphilic bacteria and archaea are required to understand their metabolism and physiology and to assess their bioremediation potential.

#### MICROBES IN DRY ENVIRONMENTS

An increase of dry environments in diverse regions due to low rainfall, high temperatures and drought has been associated to Climate change (Mukherjee et al., 2018). Water is essential for all living organisms (Robinson et al., 2015; Lebre et al., 2017). Arid environments like deserts are considered to be at the dry limit for life (Navarro-González et al., 2003; Bull and Asenjo, 2013). Extremophile xerotolerant organisms can survive in dry environments with water activity < 0.75 (Connon et al., 2007; Lebre et al., 2017). Water activity is calculated as the ratio of the vapor pressure in an environment relative to pure water, and it is considered to be the amount of water available to organisms in the environment (Lebre et al., 2017). Arid areas are biomes with a ratio of mean annual rainfall to mean annual evaporation of <0.05 mm year−<sup>1</sup> , and <0.002 mm year−<sup>1</sup> for extreme hyperarid areas (Mohammadipanah and Wink, 2016). In addition to the low rainfall, other factors such as high and low temperatures, low water activity, high salinity, low concentrations of organic carbon and intense radiation intensify the xeric conditions, constraining the survival of microorganisms (Dose et al., 2001; Crits-Christoph et al., 2013).

Xerotolerant bacteria have developed two strategies to survive in dry environments: evasion of environmental stress and adaptative mechanisms (**Figure 3**). Evasion of dry environmental implies the conversion of cells into a state of non-replicative viability through the formation of spores (Crits-Christoph et al., 2013). Adaptative mechanisms are associated to preventing water loss and increasing water retention through the accumulation of osmoprotectants (trehalose, L-glutamate, glycine betaine), production of extracellular polymeric substances (EPS), modifications on the cell membrane to retain intracellular water, and synthesis of DNA-repair proteins (Dose et al., 2001; Lebre et al., 2017).

The Atacama Desert is the oldest desert on Earth with hyper-arid soils with mean rainfall <5 mm year−<sup>1</sup> to 2.4 mm year−<sup>1</sup> in the Yungay sector (Warren-Rhodes et al., 2006; Azua-Bustos et al., 2015). Nevertheless, microorganisms are capable to inhabit this extreme environment. Previous studies using culture-dependent methods have reported low numbers of bacteria in Atacama Desert soils, ranging from not detectable to 10<sup>6</sup> CFU per g of soil, with a high degree of spatial heterogeneity (Crits-Christoph et al., 2013). Analysis through culture-independent methods on the subsurface layers of the hyper-arid core showed limited abundance of microbial communities including Proteobacteria, Actinobacteria, Cyanobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes, Planctomycetes, and Thermomicrobia phyla (Dong et al., 2007; Crits-Christoph et al., 2013; Azua-Bustos et al., 2015; Piubeli et al., 2015; Azua-Bustos et al., 2017).

Proteobacteria from Atacama Desert showed a high bioremediation potential. Pseudomonas arsenicoxydans strain VC-1 isolated from arsenic-polluted site is able to tolerate high concentration of As(III) (5 mM), and also capable of oxidizing As(III) to As(V) at high rates (Campos et al., 2010). Actinobacteria are the most abundant culturable phylum present in Atacama Desert soils, including strains from Streptomyces, Nocardia, Microlunatus, Prauserella, Microcella, Arthrobacter, Cryobacterium, Frigobacterium, Dietzia, Nocardioides, Propionibacterium, Luteococcus, Kocuria, and Patulibacterium genera (Bull and Asenjo, 2013). Actinobacteria plays a relevant ecological role including resistance to heavy

metals, recycling of substances, degradation of complex polymers and organic persistent pollutants, and synthesis of bioactive molecules (Claverías et al., 2015; Alvarez et al., 2017; Undabarrena et al., 2017), which may explain its capability to still predominate in such harsh environmental conditions. The extreme desiccating condition of deserts has been the main driving force in the evolution of several desert-derived Actinobacteria, which showed a wide range of biotechnological applications including the removal of organic and inorganic pollutants (Mohammadipanah and Wink, 2016; Alvarez et al., 2017). Chilean desert-Actinobacteria research has focused on obtaining microorganisms able to synthesize novel bioactive molecules (Bull and Asenjo, 2013; Azua-Bustos and González-Silva, 2014). Xerotolerant Streptomyces bulli and S. atacamensis synthesize ansamycin and 22-membered macrolactones with antibacterial and antitumor activities (Santhanam et al., 2013; Mohammadipanah and Wink, 2016). However, the potential of Atacama Desert-Actinobacteria toward bioremediation of pollutants and waste treatment remained underexplored.

As Atacama Desert conditions are too dry to support higher plants and most other photoautotrophic microorganisms, it gathers multiple features to ensure proliferation of primary producers. It has been shown that translucent rocks served as "refuge" for photosynthetic microbial communities, that are capable to colonize the underside of the rocks, where sufficient moisture depending of fog and dew is retained, and light intensities are not lethal. Indeed, hypolithic cyanobacteria are present in small spatially isolated islands on hyper arid Atacama Desert soil (Dong et al., 2007). These hypolithic Cyanobacteria communities are composed mainly of the Chroococcidiopsis genus, which is one of the most primitive representant of the phylum (Warren-Rhodes et al., 2006). Chroococcidiopsis strains are members of the hydrocarbon-degrading biofilms during bioremediation of hydrocarbon pollutants in aquatic environments (Al-Bader et al., 2013). Although Atacama Cyanobacteria has not been used in bioremediation processes, the use of xerotolerant bacteria of dry environments is an attractive strategy for bioremediation under hydric resource limitation.

Desertification and the demand for residential space in sites with water availability limitation have caused an increase of pollution in dry environments. Therefore, technologies for the bioremediation of polluted dry sites are required. Bioremediation of oils spills in desert soils has been reported (Godoy-Faúndez et al., 2008; Benyahia and Embaby, 2016). Strategies to alleviate the xeric stress are relevant to improve bioremediation processes in dry environments. Hereby, xerotolerant bacteria play a crucial role for bioremediation of soils in extreme dry conditions.

#### UV- AND GAMMA-RESISTANT MICROORGANISMS

Ultraviolet (UV) light is radiation with a wavelength between 40 and 400 nm. UV radiation is divided into five different ranges: (i) Vacuum UV (40–190 nm), (ii) Far UV (190–220 nm), (iii) Short-wavelength UVC (220–290 nm), (iv) Medium-wavelength UVB (290–320 nm), and (v) Relatively long-wavelength UVA (320–400 nm) (Marizcurrena et al., 2017). Life without an assured level of radiation is impossible (Pikuta et al., 2007). However, this type of radiation can be one of the most detrimental abiotic factors causing serious damages in organisms at community and cellular levels. On one hand, UV light constrains the microbial diversity and dynamics of ecosystems to only those UV-resistant (micro)organisms. On the other hand, UV radiation can affect cell survival, producing DNA damage and mutations, oxidative stress and protein denaturation (Marizcurrena et al., 2017; Pérez et al., 2017). UVB and UVC are the most harmful radiation to life (Paulino-Lima et al., 2016). UVB causes direct and indirect DNA damage. UVC may cause direct DNA damage by the formation of pyrimidine dimers and pyrimidine photoproducts (Marizcurrena et al., 2017). However, UVC is completely filtered by the atmosphere and does not reach the Earth's surface. UVB is mostly filtered by the atmosphere, but in places where the ozone layer is thinner, the protective filter activity of the atmosphere is progressively reduced, causing a higher penetration of UVB radiation (Yang et al., 2008).

In Chile, several places have exposure to extreme solar radiation levels and high doses of UVB due to the ozone layer hole (Cordero et al., 2016; Paulino-Lima et al., 2016; Pérez et al., 2017). Atacama Desert is characterized by its high altitude, prevalent cloudless conditions, extreme dryness, relatively low columns of ozone and water vapor and intense solar UV radiation (Cordero et al., 2016; Paulino-Lima et al., 2016), which converts this desert in a key region for international astrobiology studies. The microorganisms that are capable of living under these extreme conditions are known as UV resistant extremophiles (Gabani et al., 2014). The study of microorganisms living in these hostile environments is currently focused to gain understanding on the origin of life and early evolution on Earth, to develop models for predicting consequences of future Climate change and to exploit their potential for biotechnological applications (Pérez et al., 2017).

Ultraviolet resistant extremophiles have developed different strategies to resistant UV stress (**Figure 3**). These strategies are related to efficient machinery for DNA-repair, induction of chaperones and active defense against UV-induced oxidative stress (e.g., glutathione accumulation) (Pérez et al., 2017). The capability of these microorganisms to repair DNA damage has been associated to radiation resistance, since it has been suggested that radiotolerant bacteria accumulate high intracellular manganese and reduced iron levels (Pikuta et al., 2007), conferring them resistance to UV radiation (Paulino-Lima et al., 2016). These types of microorganisms are usually polyextremophiles, since it has been noticed that DNA damage that accumulates during desiccation is critical also for desiccation tolerance (Mattimore and Battista, 1996).

Novel species of the genus Deinococcus has been isolated, mainly from environments with high UV radiation, such as deserts and arctic zones (Hirsch et al., 2004; Rainey et al., 2005; Bruch et al., 2015; Guerra et al., 2015). Strains isolated from these environments have been proposed as biocatalysts for bioremediation of radioactive waste sites (Daly, 2000; Mrazek, 2002; Ghosal et al., 2005). UV resistant strains of the Deinococcus genus have been associated to

radiation resistance (Cox and Battista, 2005). D. radiodurans has been engineered for bioremediation of heavy metals and persistent organic pollutants (Daly, 2000; Cox and Battista, 2005). Deinococcus peraridilitoris strains KR-196, KR-198 and KR-200t (**Table 1**) were isolated from an arid desert soil collected in the north of Antofagasta, Chile. These isolates showed a high tolerance to ionizing radiation > 10 kGy, radiation even higher than reported for the model Deinococcus radiodurans strain R1 (10 kGy) (Cox and Battista, 2005; Rainey et al., 2007). Deinococcus sp. UDEC-P1 was isolated in Témpanos Lake, an oligotrophic lake in the Chilean Patagonia (Guerra et al., 2015). Due to their natural resistance to high levels of radiation, Deinococcus species isolated in Chile are potential candidates for bioremediation of radioactive wastes and environments.

High altitude lakes have the potential to be fertile niche for the isolation of novel UV-resistant microorganisms with potential for bioremediation processes. Escudero et al. (2007) have studied the diversity of microorganisms of high lakes Aguas Calientes (5,870 m above sea level) and Licancabur (5,916 m above sea level) located in the Northern Andes of Chile. These areas have high rates of UVB radiation (48.5 MW/cm<sup>2</sup> ). Analysis of sediments and brine of Aguas Calientes lake show that the microorganisms belong mainly to the phylum Proteobacteria, and 70% of the sequences could not be assigned to a phylogenetic group. Licancabur Lake showed a dominance of Cytophaga–Flavobacteria–Bacteroidetes and Proteobacteria. Plankton analysis of Licancabur Lake shows poor diversity and abundance in water samples, reporting Cyanobacteria, Chrysophyceae, Euglenophyta, and Chlorophyceae strains (Albarracín et al., 2015). In spite of these pioneering reports, the microbial communities and UV-resistant microorganisms of Chilean sites with extreme solar radiation levels such as high altitude deserts and lakes and Antarctica are still mainly unknown.

## PSYCHROPHILES

Psychrophiles refer to microbes that are capable to grow at temperatures ranging from −20 to 20◦C, with an optimal growth temperature of <15◦C (Morita, 1975; Clarke et al., 2013). Psychrophilic bacteria possess diverse molecular mechanisms to survive at low temperatures (**Figure 3**). On one side, an increase of unsaturated fatty acids, cyclopropane-containing fatty acids and short chain fatty acids in membranes prevents the loss of membrane fluidity (Feller and Gerday, 2003; D'Amico et al., 2006). A second mechanisms is the high synthesis of coldshock proteins (CSPs) and chaperones that protect the synthesis of RNA and proteins (Godin-Roulling et al., 2015). A third mechanism is the synthesis of anti-freeze proteins (AFPs) that binds to ice crystals and generates a state of thermal hysteresis (Kristiansen and Zachariassen, 2005; Muñoz et al., 2017). A fourth mechanism is the accumulation of mannitol and other compatible solutes as cryo-protectants to prevent cell damage by UV radiation. An additional mechanism is the transport of compatible solutes such as mannitol to stabilize the cytoplasmatic environment and prevent ice formation (Baraúna et al., 2017). Nevertheless, the most general mechanism is the adaptation of psychrophilic enzymes through inherent modifications of their primary structure. Some of these amino acid changes are minimal in comparison with mesophilic proteins, playing a role in catalytic regions or stabilization of the proteins (Feller and Gerday, 2003; D'Amico et al., 2006; Cavicchioli et al., 2011).

In Chile, psychrophiles have been isolated from several cold environments, such as the high mountains of Los Andes Mountain, vast ice regions, such as the Northern and Southern Ice Field, Southern extreme zones, such as the Patagonia, and the Antarctic continent. Antarctica became a subject of intense research in several fields, including the exploration of novel microbes and their pigments, antibiotics and enzymes. The development of special research programs that supports long-timed expedition logistics have facilitated the access of researchers and increase the research on the Antarctica (Loperena et al., 2012; Órdenes-Aenishanslins et al., 2016; Lavin et al., 2017). However, in-depth research conducted on psychrophiles in cold environments, such as a report in Alpine glacier (Ferrario et al., 2017), remain to be conducted.

Antarctica plays also a huge role in two mainstream research topics, global Climate change and global contamination assessment. The pollution in Antarctica is partly explained by the grasshopper effect: pollutants are moved across the globe in the atmosphere without suffering major changes and are later deposited in continuous-forming ice sheets across Antarctica surface. Other pollution source on Antarctica resides on human activity from military and scientific bases (ASOC, 2007). The detection of diverse organic and inorganic contaminants in Antarctic territory is of increasing concern. Since Antarctica is a region where the introduction of foreign microorganisms is forbidden, the only strategy for bioremediation processes is the application of native microbes (Poland et al., 2003; Gran-Scheuch et al., 2017).

Bacteria capable to degrade crude oil at low temperatures even below the freezing point have been reported (Gerdes et al., 2005; Bowman and Deming, 2014). Alkane hydroxylases are key enzymes for the degradation of alkanes, activating them through conversion into alcohols (van Beilen and Funhoff, 2007; Fuentes et al., 2014). A survey of alkane hydroxylases genes in psychrophilic bacteria reveals the adaptability of these proteins, giving valuable insights on the degradation of long chain hydrocarbons at low temperature. Mostly enzymes were related to the AlkB and cytochrome P450 alkane hydroxylases, but also LadA and AlmA enzymes involved in long-chain alkanes degradation were reported (Bowman and Deming, 2014). The primary structure of psychrophile enzymes differ from their mesophile homologs in the preferences for specific amino acids and increased flexibility on loops, bends, and α-helices. Notably, various novel genes were reported from Psychrobacter, Octadecabacter, Glaciecola, Terriglobus and Photobacter genera (Bowman and Deming, 2014). Obligated hydrocarbonoclastic microorganisms (i.e., microbes that use exclusively hydrocarbons as carbon sources) isolated near to Antarctic coast have been described. For example, Oleispira antarctica, whose genome

reveals a diversity of alkane degradation genes and a system of proteins that acts as cold barrier to circumvent low temperature effects that could stop hydroxylase reaction (Kube et al., 2013).

Studies performed in the Chilean Antarctic territory are mainly focused on Fildes Peninsula, where the human activity in scientific and military bases had caused several contamination events, including hydrocarbon spills. The estimation of the polluted area impacted is about 3.5 km<sup>2</sup> , corresponding to 12% of the total area of the Peninsula (ASOC, 2007). However, bioremediation plans for these sites have not yet been reported. Remarkably, the hydrocarbon degradation potential of native microorganisms from the near Argentinian Carlini Station has been described. Polaromonas naphthalenivorans, a strain capable of naphthalene degradation and N<sup>2</sup> fixation, and strains belonging to Nocardioides genus were dominant in soils with higher hydrocarbon pollution (Vázquez et al., 2017). The hydrocarbonoclastic strains Rhodococcus sp. D32AFA, Pseudomonassp. E43FB and Sphingobium sp. D43FB that degrade phenanthrene were isolated from diesel contaminated Antarctic soils (Gran-Scheuch et al., 2017). The Antarctic soil strain Streptomyces sp. So3.2 is able to produce a biosurfactant, which may be useful for hydrocarbon bioremediation (Lamilla et al., 2018).

Psychrophiles seem to be naturally adapted to cope with a wide-range of heavy metals in the environment (De Souza et al., 2006; Tomova et al., 2015; Gonzalez-Aravena et al., 2016). This adaptive trait may be explained by the presence of heavy metals in Antarctica's sediments. In addition a high occurrence of plasmids in Antarctic bacteria has been reported, which are probably related to the transfer of resistance/tolerance genes to heavy metals such as mercury, tellurium, cadmium, copper, chromium, and lead (Mangano et al., 2013; Gonzalez-Aravena et al., 2016; Rodriguez-Rojas et al., 2016a). Interestingly, a high proportion of these bacterial isolates are multiple heavy metalresistant bacteria. Mercury- and tellurite-resistant strains were isolated from Greenwich Island (Rodriguez-Rojas et al., 2016b). Pseudomonas putida strain ATH-43 (**Figure 2** and **Table 2**) revealed high tolerance to cadmium, copper, chromium, and selenium, whereas Psychrobacter sp. ATH-62 is resistant to mercury and tellurite (Rodriguez-Rojas et al., 2016a). Heavy metal-resistant bacteria were also isolated from invertebrates from the Antarctic shores. Mercury-resistant Pseudoalteromonas sp. gw196 and Colwellia sp. gw172, and zinc and cadmiumresistant Arthrobacter and Psychrobacter strains were isolated from a marine sponge (Mangano et al., 2013). Flavobacterium and Psychrobacter strains with resistance to mercury and zinc were isolated from a sea urchin (Gonzalez-Aravena et al., 2016).

Antarctic bacteria have been also studied for additional biotechnological applications. An Antarctic psychrophilic Pseudomonas strain was able to produce quantum dots from Cd salts (Gallardo et al., 2014). A psychrophilic Pseudomonas mandelli able to produce alginate has also been reported (Bayat et al., 2015; Vasquez-Ponce et al., 2017). Alginate is useful to encapsulate microbes to improve the bioremediation of persistent organic pollutants such as pesticides in polluted soils (Morgante et al., 2010). Encapsulation of microbes in alginate beads may prevent stress or predation of the inocula (Morgante et al., 2010).

The diversity of bacteria from the Antarctica that showed wide range heavy metal resistance and hydrocarbon degradation capabilities revealed a complex community that include adapted cosmopolitan bacteria such as Pseudomonas and other native strains, which may be useful for bioremediation in cold environments.

## CONCLUDING REMARKS AND FUTURE PERSPECTIVES

Chile has been referred to be a "biogeographic island" due to a succession of natural barriers, including Altiplano, Atacama Desert, Los Andes Mountains, South Pacific Ocean, Patagonia, and Antarctica. Geological processes and climatic conditions have driven the current geography of Chile into a set of diverse ecosystems that reassemble heaven for both extremophilic and polyextremophilic microorganisms. Those biotopes include a multiplicity of ecosystems with more than one extreme environmental condition, such a high salinity, low humidity, high UV radiation, high or low temperature, high or low pH, and high concentration of heavy metals. These extreme environmental conditions exert a strong pressure limiting the biodiversity of each niche, since every condition is associated with its own set of adaptations for stability on its environment. At specie-level, microbes that are capable to persist in such (multi) extreme conditions gather (multiple) metabolic capabilities that are not present in mesophilic microbes, and thus, represent a great potential for biotechnological and environmental applications (Liu et al., 2018). At an ecological level, it has been previously shown that microbial communities in extreme environments evolved at a faster rate than mesophiles living in benign environments, highlighting particularities of the evolutionary dynamic of such environments (Li et al., 2014). For these reasons, diverse research efforts have been initially focused on analyzing natural microbial communities of those sites and studying physiological and genomic characteristics of the microbial isolates.

Next generation sequencing (NGS) and next generation proteomics (NGPs) provide powerful techniques to gain insights about the molecular mechanisms involved in microbial adaptation to extreme conditions (Armengaud, 2016). These type of studies and methods will reveal the mechanisms and strategies used by microbes to adapt to extreme conditions and are useful to understand the evolution of microorganisms subjected to extreme conditions.

In recent years, there are significant advances in the field of extremophiles at the global level. Similar trend has also been reported by researchers from Chile, whose discoveries have made the field to flourish. This progress relied upon the advances of physico-chemical characterization of those biotopes together with the advent of high-throughput molecular tools to understand the complexity of microbial communities and improved tools and techniques for microbial isolation. Many of those studies have gained significant insights on the

molecular mechanisms involved in extremophiles' adaptations to extreme environments and their co-evolutionary processes, and also contributed to current biotechnological developments, such as Swissaustral, a company with headquarters in Chile, which performs functional enzymatic screening for industrial and biotechnological applications (Blamey et al., 2017). Additional research efforts must be invested into broadening the taxonomic breadth of extremophiles and expanding towards the study of extremolyes, metabolites utilized as biological defense mechanisms to combat extreme environmental stresses (such as ectoine).

The restoration of polluted environment is a crucial task for sustainable development. For the clean up of polluted sites, bioremediation is an attractive alternative to physicochemical treatment processes. Microbes are main catalysts for the bioremediation of polluted environments. Extremophiles has been isolated in diverse zones in Chile that possess extreme conditions such as Altiplano, Atacama Desert, Central Chile, Patagonia, and Antarctica. Interestingly, the remarkable adaptative capabilities of extremophiles convert these organisms into an attractive source of catalysts for bioremediation and industrial processes.

Future research will carry out more detailed studies on extremophiles already described in order to deepen their knowledge and optimize their use in industrial processes including bioremediation. On the other side, a large number of extreme environments in Chile are still unexplored or underexplored. These habitats harbor microbial communities with metabolic capabilities useful for bioremediation processes and other applications. Therefore, future research should focus on the isolation, identification and characterization of a higher number of extremophiles from ecosystems with extreme conditions. Thereafter, studies should explore the metabolic potential of microbial isolates for application in biotechnology including the bioremediation of heavy metals and persistent organic pollutants. The basic research on

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extremophiles combined with the scale up of the biotechnological process including bioremediation and waste treatments will offer a bright future to Chilean economy and an increasing welfare to its inhabitants.

## DEDICATION

This article is dedicated to the memory of Professor Dr. Burkhard Seeger Stein (1929–2016), who inspired the study of natural resources in Chile through his pioneering research and teaching.

## AUTHOR CONTRIBUTIONS

RO, CM, GB, FD, AC, RV, CR, and MS wrote and discussed the article.

#### ACKNOWLEDGMENTS

The authors gratefully acknowledge Pedro Serrano, Alejandra Pemjean, Enrique Bialoskorski, Michael Seeger, and Sebastian Fuentes for the landscape photographies shown in **Figure 1**. The authors acknowledge Conicyt Ph.D. fellowships (CM, FD, and GB) and financial support of CONICYT Programa de Investigación Asociativa (PIA) Anillo ACT172128 GAMBIO (MS), FONDECYT 1151174 (http://www.fondecyt.cl) (MS), USM (http://www.usm.cl) (MS), CONICYT/PAI Concurso Nacional de Apoyo al Retorno de Investigadores desde el Extranjero 82140028 (http://www.conicyt.cl) (RO and MS), and Apoyo a la Formación de Redes Internacionales para Investigadores en Etapa Inicial 170600 (http://www.conicyt.cl) (RO and MS). The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.




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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Orellana, Macaya, Bravo, Dorochesi, Cumsille, Valencia, Rojas and Seeger. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Enzymatic Bioweathering and Metal Mobilization From Black Slate by the Basidiomycete Schizophyllum commune

Julia Kirtzel<sup>1</sup> , Soumya Madhavan<sup>1</sup> , Natalie Wielsch<sup>2</sup> , Alexander Blinne<sup>3</sup> , Yvonne Hupfer<sup>2</sup> , Jörg Linde<sup>4</sup> , Katrin Krause<sup>1</sup> , Aleš Svatoš<sup>2</sup> and Erika Kothe<sup>1</sup> \*

<sup>1</sup> Microbial Communication, Institute of Microbiology, Friedrich Schiller University, Jena, Germany, <sup>2</sup> Research Group Mass Spectrometry/Proteomics, Max Planck Institute for Chemical Ecology, Jena, Germany, <sup>3</sup> Helmholtz Institute Jena, Jena, Germany, <sup>4</sup> Hans Knöll Institute, Jena, Germany

#### Edited by:

Edgardo Donati, National University of La Plata, Argentina

#### Reviewed by:

Eric D. van Hullebusch, UMR7154 Institut de Physique du Globe de Paris (IPGP), France John Stephen Horton, Union College, United States

> \*Correspondence: Erika Kothe erika.kothe@uni-jena.de

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

> Received: 26 January 2018 Accepted: 05 October 2018 Published: 24 October 2018

#### Citation:

Kirtzel J, Madhavan S, Wielsch N, Blinne A, Hupfer Y, Linde J, Krause K, Svatoš A and Kothe E (2018) Enzymatic Bioweathering and Metal Mobilization From Black Slate by the Basidiomycete Schizophyllum commune. Front. Microbiol. 9:2545. doi: 10.3389/fmicb.2018.02545 Schizophyllum commune is a filamentous basidiomycete causing white-rot in many wood species with the help of a broad range of enzymes including multicopper oxidases such as laccases and laccase-like oxidases. Since these enzymes exhibit a broad substrate range, their ability to oxidatively degrade black slate was investigated. Both haploid monokaryotic, and mated dikaryotic strains were able to grow on black slate rich in organic carbon as sole carbon source. On defined media, only the monokaryon showed growth promotion by addition of slate. At the same time, metals were released from the slate and, after reaching a threshold concentration, inhibited further growth of the fungus. The proteome during decomposition of the black slate showed induction of proteins potentially involved in rock degradation and stress resistance, and the gene for laccase-like oxidase mco2 was up-regulated. Specifically in the dikaryon, the laccase gene lcc1 was induced, while lcc2 as well as mco1, mco3, and mco4 expression levels remained similar. Spectrophotometric analysis revealed that both life forms were able to degrade the rock and produce smaller particles.

Keywords: Schizophyllum commune, bioweathering, proteome, laccases, multicopper oxidases, rock, black slate

#### INTRODUCTION

In lignin degradation performed by white-rot fungi like Schizophyllum commune, peroxidases and multicopper oxidases are involved. The lignin degrading enzymes (FOLymes, Levasseur et al., 2008) can be identified from fungal genomes, and 16 FOLymes including two laccases and four other multicopper oxidases were identified for S. commune (Ohm et al., 2010). Compared to other whiterot fungi, S. commune lacks genes encoding peroxidases and thus features a slim FOLyme repertoire (Ohm et al., 2010).

Multicopper oxidases, including true laccases, show a wide range of substrate utilization and thus are involved in various degradative and biosynthesis processes. With two laccases (lcc1 and lcc2) and four laccase-like multicopper oxidases (mco1, mco2, mco3, and mco4), each containing copper binding domains with the typical four laccase signature sequences (Madhavan et al., 2014), S. commune seems well equipped for acting on an array of organic carbon fractions occurring in nature including wood, but also bitumen or kerogen. Kerogen is the altered form of lignin present

**49**

in sedimentary rocks like shales and slates. Indeed, previous studies have confirmed the ability of S. commune to degrade black slate material resulting in a release of heavy metals and carbon as dissolved organic matter (Wengel et al., 2006; Kirtzel et al., 2017).

Black slates are low-grade metamorphic rocks rich in sulfides, heavy metals and high amounts of organic matter altered into kerogen. Black slates and shales are important to nature as they are a prominent reservoir of organic carbon and experience several changes when they are exposed to oxygen. Weathering of such organic matter is essential for the geochemical cycles of carbon and oxygen, and fungal involvement in this process may lead to fast weathering of the rock material (Fischer et al., 2007). Due to their metal contents, black slates have been mined for aluminum, copper or vitriol production, but also for production of uranium. In Thuringia, Germany, the former vitriol mines at Saalfeld and Morassina and the former uranium mine near Ronneburg are examples for black slate mining activities. The high sulfide contents, especially at the former Ronneburg mining site, has led to acid mine drainage representing a major environmental challenge. The weathering leading to metal release and acidification through sulfide (pyrite) oxidation is often linked to bacterial activity (Mustin et al., 1992; Uroz et al., 2009; Konhauser et al., 2011).

Nevertheless, the activity of fungi can largely contribute to such bacterial activities as fungi are able to secrete organic and inorganic acids, and widen cracks in the rock and thus expose fresh surfaces to bacterial activity (Burford et al., 2003; Hoffland et al., 2004). This will be specifically important in dense rock material like black slates. However, in order to exert a force on the rock, in addition to directional growth, the fungus requires enzymatic degradation of the rock material to induce surface roughness which in turn enables hyphal attachment (Willmann and Fakoussa, 1997; Burford et al., 2003).

The structure of kerogen in black slates has been suggested to be similar to lignin components, explaining the observed lytic activities (Catcheside and Mallett, 1991; Hofrichter et al., 1997a,b). In accordance with this observation, fungi have been identified to depolymerize or transform coal (Hofrichter et al., 1997a,b; Petsch et al., 2001; Wengel et al., 2006). Here, we focus on the differential expression and enzymes active in the degradation of black slate.

The life cycle of basidiomycetes involves germination and haploid, monokaryotic mycelial growth from sexually formed basidiospores. If two compatible mates fuse, a dikaryon with two parental nuclei per cell is formed (Freihorst et al., 2016). This dikaryon can then form fruiting bodies under optimal environmental conditions, in which haploid basidiospores are produced, which makes it a good model to study sexual development (Raper and Miles, 1958; Kothe, 1997). S. commune is able to complete this life cycle on water agar with black slate as the only carbon and nutrient source within 2–3 weeks (**Supplementary Figure S1**).

The significance of fungi in bioweathering of rocks and minerals gained considerable attention during the last decades. However, only very few investigations deal with basidiomycetes and their enzymatic biodegradation. In the present study, we investigated the ability of S. commune to degrade black slate and use it as a carbon source. The effect of black slate on mono- and dikaryotic strains was examined, as well as the influence of the rock material on fungal growth and metabolism. Since the two life stages show different enzyme expression patterns, we performed proteome analyses to identify secreted proteins participating in black slate degradation. To the best of our knowledge, these results provide a first insight into possible rock decay mechanisms for S. commune. Detected multicopper oxidases were further investigated via an enzymatic assay and differential expression analysis during growth of both S. commune life stages on black slate.

## MATERIALS AND METHODS

#### Strains and Culture Conditions

Pre-cultures of S. commune monokaryon 4–39 and dikaryon 12–43 × 4–39 (Jena Microbial Resource Collection, Jena, Germany) were grown in liquid complex yeast extract medium (Raper and Hoffman, 1974). After 5 days, the mycelium was washed twice with water, homogenized and resuspended in 100 ml half diluted minimal medium (Raper and Hoffman, 1974). Of this, 7 ml were used to inoculate 200 ml half diluted minimal medium containing 3 g powdered black slate (0.5 g < 63 µm, 2.5 g 63 µm-1 mm fractions obtained by sieve analysis, collected from Schmiedefeld, a former alum mining area in Thuringia, Germany) and incubated at 28◦C and 130 rpm. The specific surface area of black slate has been shown to vary between 10 and 20 m<sup>2</sup> /g (Fischer and Gaupp, 2004, 2005).

## Growth Rate

The effect of black slate on biomass production was determined for monokaryon and dikaryon in liquid medium using three biological replicates according to the above-mentioned culture conditions in presence and absence of black slate. Dry weight was determined over a period of 4 weeks and every black slate containing sample was normalized to the corresponding control containing no black slate. Fungal growth and the interaction with black slate were observed microscopically (Stemi 2000-C and LSM 780, Zeiss, Germany) 14 days after inoculation. At different points in time, mycelium and black slate were harvested by centrifugation at 11,000 rpm for 30 min and dried at 60◦C. After subtracting the weight of black slate that had been added to the culture, the fungal biomass was calculated. To determine the effect of black slate for every replicate, the control biomass was subtracted from the appropriate biomass treated with black slate and the final change in growth was expressed in mg.

#### Metal Release

Black slate containing liquid culture medium was analyzed to examine fungal-induced metal release from the rock material. Black slate containing medium inoculated with S. commune monokaryon was sampled after different points in time. Samples were filtered (0.22 µm, Rotilabo, Roth, Germany), acidified using HNO<sup>3</sup> and analyzed by ICP-MS (X Series II, Thermo Fisher Scientific). Measurements were performed with three biological and three technical replicates using non-inoculated samples for control.

## Spectrophotometric Assay for Quantification of Black Slate Solubilization

fmicb-09-02545 October 22, 2018 Time: 18:39 # 3

Fungal solubilization of black slate was spectrophotometrically quantified by measuring organic substances such as (dissolved) humic substances released from black slate. Liquid cultures of monokaryon and dikaryon as well as controls without fungal biomass were incubated with and without powdered black slate and centrifuged for 5 min at 8000 rpm to allow settling of mycelium and large rock particles. The supernatant was transferred into cuvettes (Greiner Bio-One, Germany) and the absorbance was measured at 450 nm (Ultrospec 2100pro, Amersham Biosciences, Germany), the most suitable wavelength to determine dark-colored organic substances from black slate. Measurements were performed with three biological replicates and statistical analysis was achieved using paired, two-tailed t-test. Degraded black slate particles were microscopically examined (Axioplan 2 microscope, Zeiss, Germany) and their sizes were measured using SPOT advanced (Diagnostic Instruments, Germany).

## Protein Extraction

For preparation of protein extracts, liquid culture medium of four biological replicates was separated from fungal cultures and, when added, supplemented black slate 7 days after inoculation by centrifugation and 50 ml of the supernatant were concentrated to 1.5 ml (Amicon Ultra-15 Centrifugal Filter Units, 3 kDa, Merck Millipore, Germany). The supernatant was centrifuged at 12,000 × g for 30 min to remove cell debris or insoluble compounds. Secreted proteins were precipitated by the addition of 10 % (w/v) trichloroacetic acid.

After centrifugation at 13,000 × g for 10 min at 4◦C, the supernatant was discarded, the pellet washed twice in 90% acetone (−20◦C) and subsequently dried at room temperature.

For shotgun analysis, the pellet was resuspended in 50 mM NH4HCO<sup>3</sup> and prior trypsin (Roche) digestion the samples were reduced with 10 mM DTT at 56◦C and alkylated with 55 mM iodoacetamide in the dark. A trypsin/protein ratio of 1:30 at 37◦C was applied for approximately 12 h.

## NanoUPLC-MS/MS Analysis

The samples were acquired on UPLC M-class system (Waters Corporation) online coupled to a Synapt G2-si mass spectrometer equipped with a T-WAVE-IMS device (Waters Corporation). The peptides were first on-line preconcentrated and desalted using a UPLC M-Class Symmetry C18 trap column (100 Å, 180 µm × 20 mm, 5 µm particle size) at a flow rate of 15 µl/min (0.1% aqueous formic acid) and then eluted onto a ACQUITY UPLC HSS T3 analytical column (100 Å, 75 µm X 200, 1.8 µm particle size) at a flow rate of 350 nl/min with the following gradient: 1–9% B over 10 min, 9–19% B over 10 min, 19–32% B over 10 min, 32–48% B over 10 min, 48–58% over 5 min, 70–95% over 5 min, isocratic at 95% B for 4 min, and a return to 1% B over 1 min (phases A and B composed of 0.1% FA and 100% acetonitrile in 0.1% FA, respectively). The analytical column was re-equilibrated for 9 min prior to the next injection. The eluted peptides were transferred into the mass spectrometer operated in V-mode with a FWHM resolving power of at least 20000. All analyses were performed in a positive ESI mode. A 100 fmol/µl human Glu-Fibrinopeptide B in 0.1% formic acid/acetonitrile (1:1 v/v) was infused at a flow rate of 1 µl/min through the reference sprayer every 45 s to compensate for mass shifts in MS and MS/MS fragmentation mode.

During HDMS<sup>E</sup> analysis, a wave height of 40 V was applied in IMS past of TriWave, and the traveling wave velocity was ramped from 1000 m/s to 600 m/s. Wave velocities in the trap and transfer cell were set to 311 m/s and 175 m/s and wave heights to 4 V and 4 V, respectively. For fragmentation, the collision energy was linearly ramped in the transfer region of TriWave from 20 to 45 V. The acquisition time in each mode was 0.5 s with a 0.05 s interscan delay. HDMS<sup>E</sup> data were collected using MassLynx v4.1 software (Waters Corporation).

## Processing of LC-MS Data

Data analysis was performed using ProteinLynx Global Server (PLGS) version 2.5.2 (Waters Corporation). The thresholds for low/high energy scan ions and peptide intensity were set at 300, 30 and 750 counts, respectively. The processed data were searched against the S. commune protein database obtained from the Joint Genome Institute, and combined with a subdatabase containing common contaminants (human keratins and trypsin). The database searching was performed at a False Discovery Rate (FDR) of 2%, following searching parameters were applied for the minimum numbers of: fragments per peptide (1), peptides per protein (1), fragments per protein (3), and maximum number of missed tryptic cleavage sites (1). Searches were restricted to tryptic peptides with a fixed carbamidomethyl modification for Cys residues. For functional description of proteins, the Functional Catalog (FunCat) was used as classification system (Ruepp et al., 2004).

#### Laccase Activity Assays

Extracellular laccase activity was determined from the culture fluid of liquid medium in presence and absence of black slate 7 days after inoculation (dpi, Sánchez et al., 2004), the point in time at which S. commune has been shown to release organic carbon from black slate (Wengel et al., 2006). Black slate powder was removed by centrifugation and extracellular enzymes were concentrated using membrane filters (Amicon Ultra-15 Centrifugal Filter Units, 10 kDa cutoff, Merck Millipore, Germany). Laccase activity was observed as increase in absorbance at 420 nm and expressed as units per liter (1 U = 1 µmol 2,2<sup>0</sup> -azino-bis (3-ethylbenzothiazoline-6 sulphonic acid), ABTS, oxidized per minute) with 1 mM ABTS in 100 mM sodium acetate buffer at pH 4.5 (Linke et al., 2005). Protein concentration was determined (Bradford, 1976). All tests were performed with three biological and three technical replicates and statistically analyzed using paired, two-tailed t-test.

#### Expression Analyses

fmicb-09-02545 October 22, 2018 Time: 18:39 # 4

Approximately 100 mg of 7-day-old mycelium from monokaryotic or dikaryotic strains were harvested from the liquid black slate culture and washed properly. After grinding in liquid nitrogen, total RNA was isolated (Qiagen RNeasy Plant Mini Kit, Qiagen, Germany). For quantitative realtime PCR (qRT-PCR), the laccases lcc1 (DOE JGI protein ID 2509814), lcc2 (protein ID 1194451), laccase-like mco1 (protein ID 2621035), mco2 (protein ID 2634619), mco3 (protein ID 2516955), mco4 (protein ID 2483752) and reference genes act1 (protein ID 1194206), tef1 (protein ID 1037126), and ubi (protein ID 71656) were analyzed. Primers were used after Madhavan et al. (2014), and efficiencies were calculated from calibration curves (cDNA concentrations from 0.001 to 200 ng cDNA were used per 25 µl of PCR mixture). The qRT-PCR was performed using the Miniopticon Real Time PCR System (BioRad, Germany) and Maxima SYBR Green qPCR (Fisher Scientific, Germany) with initial denaturation at 94◦C for 10 min, followed by 35 cycles of 94◦C for 20s, 65◦C for 20s (lcc2 and mco3) or 60◦C (for all other genes), and 72◦C for 20 s. A melting curve analysis was performed for every run and each reaction was conducted with at least three biological and three technical replicates including negative controls, one without template and one without reverse transcription. PCR products were sequenced to verify the gene identity. The C<sup>t</sup> values of target genes were normalized with respect to the reference genes, and calculated for relative and normalized fold change compared to treatments without black slate by the term 2−11CT (Pfaffl, 2001; Vandesompele et al., 2002).

#### RESULTS

#### Fungal Growth With Black Slate

To test the impact of black slate on fungal growth, S. commune was grown with and without powdered rock supplemented to the minimal medium. Both mono- and dikaryons of S. commune were able to grow in liquid media with powdered black slate. The growth of the monokaryon was promoted by the addition of rock material during the first 21 days (with a peak in biomass production after 14 days, **Figure 1**). After that point, growth promotion of black slate decreased and a reverse effect, where black slate reduced the biomass production, was observed at 28 days. The dikaryon consistently exhibited a better growth in absence of the rock material, and no black slate induced change in growth was observed between 7 to 28 dpi.

During growth in liquid medium, the mycelium of S. commune grew in mycelial balls which enclosed black slate particles (**Figure 2**). Most rock grains and mainly larger particles were located in the center of the grown mycelium, while smaller particles were found at lower incidence at the rim. A closer examination of exterior hyphae indicated the

FIGURE 1 | Growth rate of Schizophyllum commune strains 4–39 and 12–43 × 4–39 in presence of black slate normalized to the corresponding control without black slate supplement. Positive values indicate a black slate induced burst of growth and negative values reveal a black slate induced inhibition of growth; dpi days after inoculation. Error bars represent standard deviation of three biological replicates.

FIGURE 2 | S. commune growing in half diluted minimal medium without and with fractionized black slate (A), hyphal ball stained with Lactophenol Cotton Blue with inclusions of black slate (B), and hypha of monokaryon 4–39 with attached small black slate particles (C) 14 dpi.

attachment and slight adherence of very small particles with an average size of 0.7 µm (**Figure 2C**).

#### Metal Release

The ability of S. commune to release metals from black slate was confirmed with a significantly higher release of Al, Fe, and U and a slight release of Pb in presence of the fungus (**Figure 3**). A reverse effect, at which the monokaryon caused lower metal concentrations in the medium, was seen for Cu. Especially in presence of the fungus, increasing element concentrations were detected for all metals over time.

#### Black Slate Degradation

The effect on rock degradation was verified by investigating the supernatants of liquid culture media with black slate. Bioweathering of black slate by S. commune cracked the rock into smaller pieces and caused the elevated release of darkcolored organic substances that may dissolve in the medium and

can be spectrophotometrically measured. Both monokaryon and dikaryon caused a significantly higher absorbance at 450 nm due to organic substances and suspended black slate particles (**Figure 4**). The dikaryon S. commune 12–43 × 4–39 caused the highest turbidity of medium and the highest increase in absorbance 5–7 dpi; a less rapid but still escalating effect was seen up to 21 dpi, followed by a constant absorbance to 28 dpi. The monokaryon S. commune 4–39 showed a weaker, but similarly increasing absorbance pattern with a sharp rise in absorbance 5 dpi, and a slight increase in absorbance until 28 dpi.

Microscopic analyses demonstrated that both fungal strains caused a decomposition of black slate grains into smaller particles with an average size of 0.7 to 0.8 µm after 14 dpi. In presence of S. commune, these particles were present in the supernatant to a larger extent as compared to samples not inoculated with the fungus.

#### Secretome Analysis

The secreted proteins of S. commune monokaryon 4–39 and dikaryon 12–43 × 4–39 were analyzed during cultivation with and without black slate. Both strains revealed the occurrence of 269 proteins, about 60% showed secretion motifs. For functional annotation, 157 proteins could be annotated. With 887 functions within 17 FunCat main categories, multiple function of most proteins was assigned.

For the monokaryon S. commune 4–39, 38 of the 241 proteins were exclusively detected in presence of black slate (**Supplementary Table S1**). Their assigned functions mainly belonged to metabolism, to proteins with binding function or cofactor requirement, or to cellular transport, transport facilitation and transport routes (**Figure 5A**). Seventy proteins were detected in absence of black slate and exhibited functions in transport facilitation and transport routes, metabolism, protein fate, and protein with binding function or cofactor requirement (**Figure 5B** and **Supplementary Table S2**).

The analysis of the dikaryon S. commune 12–43 × 4– 39 revealed 142 proteins, 37 of them were solely present with black slate (**Supplementary Table S3**). They mainly belonged to metabolism and proteins with binding

function or cofactor requirement. The 25 proteins exclusively identified without black slate were associated to metabolism, protein fate and protein with binding function or cofactor requirement (**Figure 6** and **Supplementary Table S4**).

Between both life stages, 79 identical proteins were detected. Of these, 12 were exclusively produced in presence of black slate. When considering only predicted secreted proteins, they showed assigned functions in metabolism and cell rescue, defense and virulence. In absence of black slate, ten proteins matched for both strains, and the predicted secreted proteins possessed functions in metabolism or proteins with binding function or cofactor requirement.

From the assigned functions, insights into biological processes during fungal growth with black slate could be gained. To achieve the identification of proteins involved in the process, only proteins exclusively produced during black slate degradation in monokaryon, dikaryon or both were considered. From these, proteins involved in biomass degradation, C-compound metabolism, and stress response were characterized. Many peptidases and glycoside hydrolases (GH) including proteins of GH family 92 and 93, which are generally participating in plant biomass degradation, were secreted. Furthermore, enzymes specifically involved in the degradation of polysaccharides (GH families 5 and 61) or lignin (multicopper oxidases) were secreted. Aside from an intracellular aconitase A/isopropylmalate dehydratase involved in the tricarboxylic acid cycle, proteins associated with carbohydrate metabolism (GH families 5 and 6), polysaccharide metabolism, as well as sugar, glucoside, polyol and carboxylate catabolism (GH family 43, carbohydrate esterase family 4) were detected. Besides these extracellular enzymes, proteome analysis also revealed proteins which are usually intracellularly located and involved in sugar, glucoside, polyol and carboxylate anabolism (phosphoglucose isomerase, aldo/keto reductase, aconitase A/isopropylmalate dehydratase).

Furthermore, proteins involved in stress response (heat shock proteins, peptidyl-prolyl cis-trans isomerase) including detoxification processes by export (ABC transporter) and degradation (multicopper oxidase), or thioredoxin, glutaredoxin and glutathione metabolism (gamma-glutamyltranspeptidase) were proven as well as the

occurrence of a defense-related protein (osmotin; thaumatin-like protein).

#### Differential Regulation of Multicopper Oxidases During Black Slate Utilization

At the level of enzyme activity, laccases from culture supernatant was measured 7 dpi with an extracellular activity of 0.48 U/l when the dikaryotic strain was grown with black slate (**Figure 7**). In contrast, no laccase activity was detected in dikaryotic cultures without black slate, or for the monokaryon, neither in absence nor in presence of black slate.

The expression of all laccase and laccase-like multicopper oxidase genes of S. commune was tested for regulation on mRNA level. After growth in half diluted minimal medium with black slate powder, up-regulation of multicopper oxidase mco2 was found in both life-stages (**Figure 8**). The other laccase and multicopper oxidase genes did not show changes in expression during treatment with black slate.

#### DISCUSSION

#### S. commune Causes Degradation of Black Slate

The influence of S. commune on black slate degradation was tested over a period of 4 weeks. A previous study had suggested the involvement of S. commune in release of heavy metals and organic carbon from black slate, when the rock is enzymatically attacked as a substrate (Wengel et al., 2006). Etch pits formed by S. commune on the surface of black slate supported the hypothesis of bioweathering (Kirtzel et al., 2017). Here, we could

show that both life-stages of the fungus, the haploid monokaryon and the mated dikaryon, caused a decay of rock particles, resulting in a significantly higher turbidity and discoloration of the medium measurable at an absorbance at 450 nm. The dikaryon showed a stronger effect as compared to the monokaryon.

When fungi or bacteria are inoculated with rock samples, an elevated turbidity due to rock compounds like humic substances has been described (Fakoussa, 1988; Hofrichter et al., 1999). Black slates contain high amounts of organic matter which can be, according to their solubility, divided into bitumen or kerogen. They might be rich in humic substances and cause the dark color of the rock. During fungal attack, such organic fractions within the black slate undergo degradation, might dissolve in the medium, and cause its dark colorization. Furthermore, fungal bioweathering might crack solid bitumen or kerogen grains to an average size of 0.7–0.8 µm. Those grains either stick to the hyphae and might be more susceptible to biochemical weathering or remain in the supernatant, are unaffected by centrifugation and thus lead to a further increase in absorbance. Thus, absorbance measurements could be correlated with intensities of fungal-induced black slate degradation.

#### Black Slate Influences Fungal Metabolism

During growth of S. commune in liquid medium supplemented with black slate, rock grains were attached to the hyphae. Like many organisms, fungi are known to produce extracellular polymeric substances which facilitate the attachment to solid surfaces (Warscheid and Braams, 2000; Gadd, 2007). S. commune is a well-known producer of several exopolysaccharides

including the glucan schizophyllan (Hao et al., 2010). Therefore, small black slate particles can attach to the mycelial surfaces via interaction with the oligo- or polyglucans. Moreover, extracellular polymeric substances frequently contain complexforming agents and acidic metabolites which in turn can cause an additional accelerated dissolution of black slate (Hoffland et al., 2004; Gorbushina, 2006; Gadd, 2007). Proteome analyses revealed that black slate influenced the tricarboxylic acid cycle where several organic acids like citric, succinic, fumaric and malic acid are produced. They are hypothesized to be deeply involved in rock weathering and could therefore promote black slate degradation (Palmer et al., 1991; Landeweert et al., 2001; Scheerer et al., 2009).

The growth of S. commune monokaryon 4–39 was accelerated by black slate. By access to organic materials and released essential nutrients, energy metabolism and biosynthesis are increased allowing for faster growth (Hochella, 2002; Gadd, 2007). However, after 21–28 days, a growth inhibition was scored, likely due to exceeding (heavy) metal concentrations caused by bioweathering of the stone (Gadd, 1993; Baldrian, 2003). Indeed, we could demonstrate that S. commune released Pb, U, and exceedingly high concentrations of Al and Fe from black slate. Wengel et al. (2006) have shown an increase in Fe concentrations from 220 µg/l to 330 µg/l and Mn concentrations from 1300 µg/l to 2430 µg/l by different strains of S. commune over a period of 84 days. Due to high Al and Fe contents in the black slate used in our study, the release of Fe by S. commune is much higher and increases from 140 µg/l to 1180 µg/l within 14 days. Based on these results, S. commune might be of interest for industrial bioleaching when addressing the extraction of metals from carbonaceous rocks.

#### Wood-Degrading Enzymes Are Responsible for Black Slate Degradation

As a wood-rotting fungus, S. commune secretes a broad range of enzymes crucial for degrading complex biomolecules (Ohm et al., 2010). The black slate-specific proteome included proteins involved in C-compound and carbohydrate metabolism, as well as extracellular polysaccharide and lignin degradation. Our results thus link wood-degrading proteins to bioweathering of black slate. In particular, several glycoside hydrolase family proteins and a multicopper oxidase seemed to be involved in degradation of the organic carbon fraction of the rock material. Specifically, mco2 was up-regulated, which may also be explained by a large number of xenobiotic response elements in the mco2 promoter (Madhavan et al., 2014). Extracellular enzymes have previously been suggested to participate in fungal rock degradation (Willmann and Fakoussa, 1997; Hofrichter et al., 1999; Silva-Stenico et al., 2007). During the formation of sedimentary organic matter, lignin and lipids were presumed to withstood degradation processes (Durand, 1980) and lignin could be converted into aromatic hydrocarbons (Shen et al., 2016). The organic matter fraction of black slate mainly consists of aromatic hydrocarbons and might thus be susceptible to degradation by ligninolytic enzymes such as laccases (Seifert et al., 2011). Since lignin does not provide a primary source of carbon and energy for fungal growth, it had to be derived from other sources such as cellulose and hemicellulose which provide glucose and other sugars, respectively (Fritsche and Hofrichter, 2005). Black slate organic matter could contain, besides lignin, cellulose and other plant residues (Swanson, 1960), thus explaining the detection of proteins able to degrade plant constituents other than lignin.

#### Rock Degradation Causes Stress in S. commune

Bioweathering of black slate by S. commune was accompanied by a release of metals which might cause stress in fungi. Stress response was visible in significant changes of the proteome, including the multicopper oxidase gene regulation. This enzyme has been shown to be involved in heavy metal stress and could consequently be involved in stress response (Baldrian and Gabriel, 2002; Lorenzo et al., 2006). Since laccases and multicopper oxidases contain four Cu atoms in the active center, the specific sequestration of Cu may be associated with the increased production of laccases/multicopper oxidases. This assumption could also be supported by the metal analysis of the culture medium. Here, the Cu concentrations released from black slate were lower in presence of the fungus. Released Cu was probably accumulated and used for synthesis of the laccase protein.

As a defense mechanism to metal and oxidative stress, fungi are known to produce antioxidants like glutathione or superoxide dismutase (Bellion et al., 2006; Jozefczak et al., 2012). During growth on black slate, the proteome as well as microarray data of S. commune showed an up-regulation of antioxidants like thioredoxin, glutaredoxin and glutathione which could protect the fungus from oxidative stress induced by the released (heavy) metals (Carmel-Harel and Storz, 2000; Belozerskaya and Gessler, 2007; Jozefczak et al., 2012; Kirtzel et al., 2017). Furthermore, these extracellular antioxidants could quench the ABTS reaction and prevent a color development during the enzyme assay (Re et al., 1999). This would also explain the low laccase activity in the dikaryon in presence of black slate as well as the inhibition of the ABTS reaction in the monokaryon although proteomics revealed the presence of multicopper oxidase in this strain under black slate treatment.

The complexity and variation in the organic carbon content as well as the presence of high levels of metals in black slate certainly impact bioweathering through S. commune. The investigation could show a diverse pattern of secreted proteins and an altered expression for a multicopper oxidase, encoded by the gene mco2, which could explain bioweathering and substrate utilization. This proof-of-principle allows a better understanding of the degradation of recalcitrant organic matter through fungal metabolism.

#### AUTHOR CONTRIBUTIONS

fmicb-09-02545 October 22, 2018 Time: 18:39 # 9

JK, KK, and EK conceived and designed the study. SM performed laccase assay and gene expression analyses. JK and YH conducted the protein extraction. NW and AS performed MS analyses and data processing. AB cross-referenced protein data with JGI online databases and JL functionally described the proteins according to the Functional Catalog. JK conducted the rest of the experiments, analyzed and interpreted the data, and wrote the manuscript. EK participated in the critical review of the manuscript.

#### FUNDING

JL was supported by the Deutsche Forschungsgemeinschaft CRC/Transregio "Pathogenic fungi and their human host: Networks of interaction" sub-project INF and the Free State of

#### REFERENCES


Thuringia and the European Regional Development Fund. We acknowledge the German Research Foundation support through GRK 1257 and JSMC.

#### ACKNOWLEDGMENTS

We would like to thank Dirk Merten and Georg Büchel for ICP-MS analysis.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02545/full#supplementary-material



Lett. Appl. Microbiol. 38, 483–487. doi: 10.1111/j.1472-765X.2004.01 517.x


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kirtzel, Madhavan, Wielsch, Blinne, Hupfer, Linde, Krause, Svatoš and Kothe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Extremophilic Microfactories: Applications in Metal and Radionuclide Bioremediation

#### Catarina R. Marques\*

Departamento de Biologia and Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, Aveiro, Portugal

Metals and radionuclides (M&Rs) are a worldwide concern claiming for resilient, efficient, and sustainable clean-up measures aligned with environmental protection goals and global change constraints. The unique defense mechanisms of extremophilic bacteria and archaea have been proving usefulness towards M&Rs bioremediation. Hence, extremophiles can be viewed as microfactories capable of providing specific and controlled services (i.e., genetic/metabolic mechanisms) and/or products (e.g., biomolecules) for that purpose. However, the natural physiological plasticity of such extremophilic microfactories can be further explored to nourish different hallmarks of M&R bioremediation, which are scantly approached in the literature and were never integrated. Therefore, this review not only briefly describes major valuable extremophilic pathways for M&R bioremediation, as it highlights the advances, challenges and gaps from the interplay of 'omics' and biological engineering to improve extremophilic microfactories performance for M&R clean-up. Microfactories' potentialities are also envisaged to close the M&R bioremediation processes and shift the classical idea of never 'getting rid' of M&Rs into making them 'the belle of the ball' through bio-recycling and bio-recovering techniques.

#### Edited by:

Kian Mau Goh, University of Technology, Malaysia

#### Reviewed by:

M. Oves, King Abdulaziz University, Saudi Arabia Navanietha Krishnaraj, National Institute of Technology, Durgapur, India

#### \*Correspondence:

Catarina R. Marques crmarques@ua.pt

#### Specialty section:

This article was submitted to Extreme Microbiology, a section of the journal Frontiers in Microbiology

Received: 28 January 2018 Accepted: 16 May 2018 Published: 01 June 2018

#### Citation:

Marques CR (2018) Extremophilic Microfactories: Applications in Metal and Radionuclide Bioremediation. Front. Microbiol. 9:1191. doi: 10.3389/fmicb.2018.01191 Keywords: extremophilic bacteria and archaea, meta-'omics', genetic engineering, synthetic biology, mine wastes, metal-radionuclide recycling/recovering

## INTRODUCTION

Metals and radionuclides (M&Rs) are problematic pollutants sourced from different industrial sectors, nuclear power plants, electronic waste and, especially, from mining activities. Undoubtedly, mining industry nurtures a plethora of human needs (Carvalho, 2017), but past and ongoing mining activities represent a huge footprint of environmental (Marques et al., 2014) and health injuries (Pereira et al., 2014; Venkateswarlu et al., 2016). Still, future societal demands will force industrial growth, thus making M&R pollution a never-ending threat, mainly due to their persistence and non-biodegradability (Gupta and Diwan, 2017).

Bioremediation, though being often viewed as an 'old fashion' and slow-acting technology, it has been thriving for M&R reclamation. Its sustainability, low-cost and efficiency totally rival conventional remediation techniques (Li and Zhang, 2012). In particular, microbially-based bioremediation relies on microbes abilities, biogenic products and/or components to scavange, transform or immobilize M&Rs (Lloyd and Lovley, 2001; Voica et al., 2016). Despite the interesting nature-based self-healing character of this approach, the tolerance of microbes to high M&R

levels, extreme physical (e.g., radiation), chemical (e.g., acidic pH) and climate changing conditions may constrain (in situ) bioremediation efficiencies (Ramos et al., 2011). Extremophilic microbes, though, have attractive skills as bioremediation tiny factories (microfactories), which performance can even be improved and customized for M&R clean-up. This review will hence focus: (1) the peculiar mechanisms of extremophilic bacteria and archaea exploitable as 'microfactories' for M&R bioremediation, (2) the groundbreaking opportunities leveraged from nature and science ('omics' plus biological engineering) interplay to enhance extremophilic-microfactories-based bioremediation, (3) M&R recovery and recycling conducted by extremophilic microfactories as a sustainable and valueadded management of M&R-bioremediation-resulting wastes. Overall, this is an innovative overview on the potentialities of extremophilic bacteria/archaea in three M&R bioremediation hallmarks: application, optimization, end-waste management (**Figure 1**).

### EXTREMOPHILES AS MICROFACTORIES FOR M&R BIOREMEDIATION

Extremophilic bacteria and archaea evolved specialized mechanisms to endure physical and/or geochemical extreme environments (Rothschild and Mancinelli, 2001). Acidophilic and/or metalophilic microorganisms are especially appealing agents for bioremediation, given their defense mechanisms against M&Rs and acidity. They synthesize extremophilic enzymes (extremozymes) (Elleuche et al., 2014) and biomolecules (Raddadi et al., 2015) that keep active and/or stable under harsh conditions. The stability of thermophilic enzymes, like the esterase EstATII (Mohamed et al., 2013), helps facing acidity and metal stress, thanks to stiff folds sustained by ion-pair networks, to compact hydrophobic cores and aminoacid arrangements/packing (Elleuche et al., 2014). Additionally, extremophiles evolved sharpened metal detoxification pathways (Voica et al., 2016) due to fast-adapting transcriptional and translational mechanisms that activate and/or inhibit many anti-oxidative stress, metal-binding, metal-transport, and membrane-permeability responses (Mukherjee et al., 2012; Dekker et al., 2016). Their membranes exhibit a specific structure (Singh and Singh, 2017), composition and a positively-charged inner layer that promote metaltransporters functioning (Dekker et al., 2016) and minimize metals and protons entrance, thereby controlling acidity and M&R toxicity (Slonczewski et al., 2009; Krulwich et al., 2011; Navarro et al., 2013; Zhang et al., 2016). Notwithstanding, since extremophiles can express defense mechanisms active against multiple extremes simultaneously (Rothschild and Mancinelli, 2001), thermophilic, halophilic, radiophilic, and polyextremophilic bacteria/archaea are additional M&R cleanup agents (Amoozegar et al., 2012; Wheaton et al., 2015). Either as whole-cells or providers of economically-valuable bio-services and/or biomolecules (**Figure 2**), extremophiles can be explored as tiny factories (microfactories) for M&R remediation.

### M&R Sensing

Sensing M&Rs presence is the alarm button to trigger the activation/repression of specific regulatory proteins and downstream cascades that control the expression of resistance genes/operons encoding enzymes and biomolecules for M&R detoxification (Das et al., 2016). Along this, the uptake/efflux of M&Rs across extremophiles cell wall and membranes may occur through energy-dependent P-type ATPases (copA1Af , copA2Af , and copBAf ), resistance nodulation cell division carriers (cusAAf , cusBAf , and cusCAf ) and/or chaperones (cusFAf and copCAf ) as in the copper-resistant Acidithiobacillus ferrooxidans (Navarro et al., 2009). A comprehensive description of the genetic characteristics and functioning of regulatory and M&R uptake/efflux systems was performed namely for thermoacidophilic, acidophilic, and metalophilic bacteria and/or archaea (Nies, 2000; Orell et al., 2010, 2013; Navarro et al., 2013; Wheaton et al., 2015).

## Biomolecules for M&R Biosorption

Extremophiles-derived cell wall/capsule, S-layer proteins, extracellular polymer substances (EPS) and siderophores present unusual structural and functional properties for M&Rs biosorption under severe stress. Llorens et al. (2012) suggested uranium sorption by S-layer proteins on the metalophilic Cupriavidus metallidurans at pH1; while acidophilic (Masaki et al., 2015) and moderate halophilic bacteria/archaea adsorbed M&Rs onto cell surface (Amoozegar et al., 2007, 2008, 2012). EPS are the most relevant structures for M&R biosorption with proved efficiency namely on psychrotolerant (Pseudoalteromonas sp.; Qin et al., 2007) and acidophilic (A. ferroxidans; Sand and Gehrke, 2006) bacteria. Either attached on the cell surface or extracellularly released, extremophilic EPS promote cell adhesion, biofilm formation, and M&Rs biosorption (Poli et al., 2011) due to a greater abundance of negatively-charged metalbinding sites of its components (e.g., polysaccharides, proteins, nucleic acids, and lipids) (Nies, 2000; Navarro et al., 2013; Shukla et al., 2017). An improved structure/composition favors enzyme stabilization and protein anchoring, hence boosting bacteria/archaea proliferation (Qin et al., 2007). Some halophilic archae can synthesize EPS with specialized jellifying properties due to abundant glucuronic acids and sulfates that stabilize EPS matrix and enhances M&R sorption (Singh and Singh, 2017). Siderophores are small biomolecules released to the environment for metal scavaging. De Serrano et al. (2016) reviewed regulatory mechanisms and chemical features of siderophores synthesized by extremophiles, evidencing their potential as bioremediation agents for metal chelation.

#### Biocatalysts for M&R Biotransformation and Bioprecipitation/Bioaccumulation

Extremophiles mediate the chemical transformation of M&R through the biosynthesis of enzymes that intracellularly or extracellularly catalyze M&Rs oxidation (solubilization) or reduction (precipitation) (Das et al., 2016). Overall, the redox reactions may occur through direct or indirect activity of extremophiles (e.g., sulfur-/iron-oxidizing or sulfate-reducing), under aerobiosis and/or anaerobiosis (Zhang et al., 2016;

Shukla et al., 2017). Normally, M&R reduction leads to the bioprecipitation/bioaccumulation of organic- and mineral-M&R complexes, and M&R-based nanocrystals. Halophilic (Amoozegar et al., 2012; Srivastava et al., 2013), thermotolerant (Özdemir et al., 2017a,b), thermophilic (Kashefi and Lovley, 2000), metalophilic (Lovley et al., 1991), radiophilic (Appukuttan et al., 2011) and acidophilic bacteria and archaea (Orell et al., 2012; Masaki et al., 2015), proved to precipitate tellurite, uranium, iron, silver, chromium, or copper as elemental nanocrystals, carbonate complexes, magnetite and metal-sulfides. Inorganic polyphosphate granules enzymatically-transformed by acidophilic and metalophilic bacteria (e.g., Caulobacter crescentus, Acidithiobacillus sp.) and archaea (e.g., Sulfolobus metallicus) can intracellularly and extracellularly accumulate M&Rs (Hu et al., 2005; Navarro et al., 2009; Orell et al., 2012). M&R bioaccumulation/bioprecipitation was indeed favored by highly reductive pili (Cologgi et al., 2011) and c-cytochromes (Marshall et al., 2006; Shelobolina et al., 2007) that increase electron transport, which attached to Geobacter sp. or Shewanella oneidensis outer membranes, promote the extracellular reduction of U(VI) into the precipitable U(IV) along with cell viability and growth. These strategies are excellent towards in situ

bioremediation, as already applied for uranium clean-up in groundwater (Coates and Anderson, 2000).

Despite the great biotechnological potential of extremophilic microfactories, remediation efficiency, biomass productivity, and economic profitability may be challenging, especially for in situ applications due to extreme optima, interspecies competition and interaction/communication inhibiting bioremediation, and limited proliferation (Ramos et al., 2011; Johnson et al., 2015). Consequently, extremophiles-based M&R bioremediation may become less attractive considering mining industry demands, hence requiring function-directed improvements.

## UPGRADING EXTREMOPHILIC MICROFACTORIES FOR ENHANCED M&R BIOREMEDIATION

'Omics' and biological engineering are leading the achievement of more proficient extremophilic microfactories for M&R bioremediation to keep pace with current economical, technological, social, and climate change defiance.

#### 'Omics' Package: Unraveling New Genes and Functions of Extremophiles

A fundamental step is to untie the genomic and metabolic complexity governing extremophiles-M&R interactions. Therefore, genomic approaches involving amplicon and wholegenome sequencing, comparative genomics and in silico genome mining have been employed to target essential genes encoding regulatory proteins, promoters, metal-binding proteins, enzymes, and biomolecules involved in radionuclides (e.g., Makarova et al., 2001; Methé et al., 2003; Zivanovic et al., 2009) and metals (e.g., Nies, 2000; Osorio et al., 2008; Cárdenas et al., 2010; Guo et al., 2014; Kernan et al., 2017) detoxification in extremophilic bacteria (e.g., Geobacter sulfurreducens, Acidithiobacillus spp., Deinococcus radiodurans, Sulfobacillus thermosulfidooxidans) and archaea (e.g., Thermococcus gammatolerans, Metallosphaera sedula).

Despite the relevance of this knowledge, the study of M&R-constrained gene expression and regulation through transcriptomics assumes a pivotal role for bioremediation purposes. DNA microarrays applied to the As-resistant Thiomonas spp. evidenced genomic rearrangements and horizontal gene transfer (Arsène-Ploetze et al., 2010), which can be exploited to transfer M&R resistance. Other studies observed overexpression of metallochaperones and enzymes mediating metal-phosphate granules formation in acidophilic bacteria (A. ferooxidans) (Navarro et al., 2009) and (thermo)acidophilic archaea (Sulfolobus metallicus, Metallosphaera sedula) challenged with M&Rs (Orell et al., 2013; Wheaton et al., 2016).

Nevertheless, many transcripts keep with unassigned functions or activities. Proteomics, through 2-DE gel coupled with mass spectroscopy (e.g., LC–MS, MALDI-ToF, and ICPL) tools and protein libraries, is thereby elucidating the type and abundance of proteins synthesized under M&R stress (Desai et al., 2010). Among them, those associated with EPS and biofilm production, oxidative stress responses, regulation of RND-type Cus system and metal uptake/efflux pumps (Zivanovic et al., 2009; Almárcegui et al., 2014; Martínez-Bussenius et al., 2016) are hugely relevant.

Additionally, metabolomics enlightens the metabolic fingerprinting of M&R-induced responses on extremophiles (Mosier et al., 2013). A diverse toolbox of chromatographic (GC and HPLC) and spectroscopic (FTIR and NMR) techniques have hence been employed to harness the genetic, transcriptional and translational processes sustaining the cellular biomolecules/metabolite fluxes (i.e., fluxomics) (Hold et al., 2009; Desai et al., 2010) leading to M&R sequestration. In silico reconstruction of unknown metabolic networks relevant for M&R bioremediation based on sequenced genomes (Sun et al., 2009), robust bioinformatics and libraries, is becoming a powerful fine-tuning approach, though requiring a cautious analysis of metal-microbe interactions to prevent misinterpretations (Johnson et al., 2015). In this context, community-level meta-omics (metagenomics, metatranscriptomics, and metaproteomics) offer great avenues by avoiding extremophiles culturing hurdles, while uncovering pools of novel biomolecules/enzymes for M&R bioremediation without previous sequence data (Mohamed et al., 2013), and providing ecologically-relevant analysis of community shifts and functions (Cowan et al., 2015; Garris et al., 2016; Mirete et al., 2016) along the bioremediation processes. Multi-'omics' integration, either at individual or community levels, constitutes another robust approach to unravel new M&R-bioremediation agents or monitoring bio-devices. Indeed, Wilkins et al. (2011) used a proteogenomic analysis to develop a biomarker of G. sulfurreducens activity during uranium bioremediation.

#### Engineering Extremophic Microfactories for M&R Bioremediation

The advent of DNA recombinant techniques for tailoring existent abilities or to create synthetic ones, in natural or newly designed extremophiles, is a doorway to boost M&R bioremediation.

#### Genetic/Metabolic Engineering of Whole-Microbes and Communities

The peculiar mechanisms of extremophiles can be customized through the insertion of genes or gene clusters, as to enhance their skills and robustness for M&R attenuation (Cárdenas et al., 2010). Metal-resistant (Cupriavidus metallidurans; Rojas et al., 2011), radiophilic (D. radiodurans; Brim et al., 2000) and thermoacidophilic (D. geothermalis; Brim et al., 2003) bacteria were transformed with genes/clusters encoding enzymes involved in individual- or multi-M&Rs reduction/oxidation. This is biotechnologically worthy given the mixture of M&Rs in mine/nuclear wastes. In order to enhance M&R bioprecipitation, D. radiodurans was indeed manipulated to express a Cd(II) binding synthetic phytochelatin (EC20) and metallothionein (smtA) (Chaturvedi and Archana, 2014), and a periplasmic acid phosphatase (PhoN), which hydrolysed organic into inorganic phosphates to precipitate uranium (Appukuttan et al., 2006, 2011). However, some extremophiles are more resistant to transformation due to their robust protective mechanisms (Chen

et al., 2011; Liu et al., 2011), hence forcing the construction of adapted vectors (Meng et al., 2013).

Recently, the innovative project NANOBINDERS (PTDC/AAG-REC/3004/2014) is enrolled in the creation of biogenic nanopolymers functionalized to bind M&Rs (the NANOBINDERS) from uranium mine effluents. These M&R-binding nanopolymers are self-assembled inside a host transformed with new constructs harboring nanopolymer- and metal-binding-peptides-encoding genes, originally obtained from microbes isolated in a uranium mine. Such functional nanobeads are a revolutionary option to engineered microbes, which in situ release still raises regulatory concerns, besides genetic instability and limited efficiency under real scenarios (de Lorenzo, 2009). Alternatively, manipulated extremophiles may house suicidal genes (Paul et al., 2005) activated upon undetectable M&R levels, for future in situ application.

Native microbial communities have been used to rehabilitate M&R-contaminated areas by natural attenuation (Shukla et al., 2017), which can be engineered through biostimulation (i.e., supplementation of compounds/substrates enhancing microbial proliferation and activities) and/or bioaugmentation measures (i.e., addition of microbes endowed with particular functional mechanisms). In mesocosms, Ñancucheo and Johnson (2011) reduced acid mine drainage production when consortia of metabolically-cooperating microalgae, acidophilic and/or acid-tolerant bacteria were added to promote iron- and/or sulfate-reduction of tailings. The biostimulation of Geobacter sp. activity by acetate enrichment had enhanced uranium reduction, thereby proving successful in situ bioremediation (Anderson et al., 2003). However, metabolic fluxes modeling (Hold et al., 2009) should be applied in the future to design novel extremophilic consortia with tailored co-activities for M&R clean-up.

#### Synthetic Biology

Synthetic biology (SynBio) moves beyond the simple transformation of genes or operons to the insertion of newly designed and constructed synthetic biological parts and circuits to create robust, profitable, programmable and customized microfactories (Martínez-Garcia and de Lorenzo, 2016). The genetic instability and limited performance of engineered microbes for M&R bioremediation, can be overcome by DNA de novo synthesis and genome editing tools [e.g., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)- and protein Cas; Mougiakos et al., 2016, 2017]. Hence, extremophiles can be engineered as novel platforms (or chassis) housing stable and multiple designed functions together with the transcriptional and translational machinery to be active from laboratory- to field-scale extreme scenarios (Adams, 2016). Likewise, Gerber et al. (2015) took advantage of Deinococcus genetic plasticity and robustness to construct a chassis towards different applications. Deinococcus spp. was also successfully engineered with metal-resistance genes, demonstrating their genetic flexibility for heterologous and co-expression of different metal-resistance determinants under extreme temperature and radiation (Brim et al., 2000, 2003). Thus, constructing extremophilic-microfactories chassis is an appealing new wave for enhanced M&R bioremediation, especially if universal genetic toolkits could be created (Adams, 2016).

The joint endeavor of SynBio and metabolic engineering of communities is indeed enabling the design and building of synthetic consortia (Shong et al., 2012) for M&R remediation. Developing synthetic consortia demands the engineering of cell–cell communication, i.e., quorum-sensing (QS) systems (Brenner et al., 2008). QS modulates behavioral, metabolic and structural dynamics in microbial communities through signaling molecules [e.g., acyl homoserine lactones family (AHL)] (Shong et al., 2012). QS is pivotal namely in EPS synthesis and biofilm formation (Farah et al., 2005; McDougald et al., 2012), as well as AHLs can mediate copper resistance in A. ferrooxidans (Wenbin et al., 2011). Thus, putting effort on the modulation or creation of new QS systems and signaling molecules biosynthesis can fine-tune the pool of relevant functions in extremophilic consortia for M&R removal (e.g., biomass production and EPS synthesis) (Brune and Bayer, 2012; Shong et al., 2012).

## GENERATING RICHNESS FROM BIOREMEDIATED M&Rs

A discouraging issue in M&R bioremediation is the disposal of final wastes. Nevertheless, extremophilic microfactories can be successfully explored for M&Rs recycling and/or recovery after being remediated, thereby sustainably closing M&R bioremediation processes.

#### Biomining

Biomining is usually conducted by extremophilic bacteria and archaea for the recovery of M&R from ores and mine/metalrich/nuclear wastes, through biooxidation (minerals oxidation for metal release) and/or bioleaching (metal solubilization) (Brune and Bayer, 2012; Johnson, 2014). These bioprocesses take advantage of the anaerobic metabolism, redox pathways, intercellular communication, biofilm formation (Vera et al., 2013), and resistance to heat, acid and metals of (thermo)acidophiles consortia (e.g., Leptospirillum spp., Acidithiobacillus spp., Sulfobacillus spp., Ferroplasma spp., Acidiplasma spp.) (Orell et al., 2010, 2013). Insoluble metal sulfides are converted into soluble metal sulfates by iron- and sulfur-oxidizing extremophiles, thereby facilitating the recovery of economically-relevant metals (e.g., Cu, Fe, Au, Ni, and Zn) (Watling et al., 2015; Wheaton et al., 2015).

## Microbial Fuel Cells (MFCs)

Microbial fuel cells (MFCs) can be a valuable recovery method since it uses the catalytic activity of microbes to generate electrical energy from the oxidation of organic substrates (Mathuriya and Yakhmi, 2014). They are composed by an anode that captures electrons from substrate oxidation, a cathode and an intermediate cation-specific membrane. Since M&Rs have a high redox potential, they can be reduced and precipitated by receiving electrons from the

cathode. Geobacter sulfurreducens biofilms possessing electronconductive nanowires (pili) have been exploited for increased electricity generation into MFCs (Reguera et al., 2006). The generated electrons can be further used to reduce U(VI) into removable U(IV), hence synergizing Geobacter sp. skills for bioremediation-derived-waste recycling. An anoxic sludge containing Geobacter spp. recovered the noble metal silver, while generating electrical energy in a MFC (Ho et al., 2017). Other extremophiles have indeed been used in MFCs (Shrestha et al., 2018), especially towards metal recovery (Dopson et al., 2015).

#### Recycling Through Nanoparticles (NPs) Production

Extremophiles biosynthesize economically-lucrative inorganic NPs through intracellular or extracellular transformation/precipitation of M&Rs (Ulloa et al., 2016), hence providing another profitable M&R recycling strategy. The metaltolerant Cupriavidus metallidurans CH34 mineralized Au and precipitated it as nanoparticulate Au<sup>0</sup> in the periplasm (Reith et al., 2009), whilst the alkalotolerant Rhodococcus sp. accumulated Au-NPs in the cytoplasm and cell wall (Ahmad et al., 2003). Spherical Ag-NPs were also extracellularly synthesized at 80◦C by the thermophilic Ureibacillus thermosphaericus (Juibari et al., 2011). CdS-NPs with enhanced stability under acidic conditions were produced by Acidithiobacillus spp., being a gainful option for Cd turnover (Ulloa et al., 2016). Uraninite, which is a worthy biogenic NP for uranium in situ remediation (Bargar et al., 2008), was synthesized by Geobacter metallireducens upon extracellular U(VI) reduction (Gorby and Lovley, 1992). The precious metal Pd(II) used in many industrial sectors was precipitated in G. sulfurreducens biofilms as Pd(0)-NPs and easily extracted by centrifugation (Yates et al., 2013).

#### CONCLUDING REMARKS

Extremophiles enclose a pool of genetic and metabolic opportunities that can be harnessed as microfactories for multiple M&R-bioremediation hallmarks. Nevertheless, the complexity of extremophiles' cellular mechanisms, interspecies relationships

#### REFERENCES


and M&R interplay under real scenarios of co-occurring extreme conditions may rise some challenges. Multi-'omics' applied side-by-side with genetic engineering techniques are covering knowledge gaps, allowing gene expression and metabolic pathways customization for improved bioremediation. Synthetic biology, however, through an iterative design-build-test-analyze framework is further revolutionizing M&R bioremediation and opening new perspectives on the creation of robust extremophilic recombinants or chassis with de novo-designed biologically-, energetically-, and economically-viable traits for specific M&R removal processes. Future trends will hence target adaptation or construction of multi-skilled extremophilic microfactories to sustainably empower M&R bioremediation and end-waste recycling, while keeping up with global changes, natural resources availability, and cost-efficiency requirements.

## AUTHOR CONTRIBUTIONS

CM was responsible for doing all the tasks concerning the development of the work: conceptualized the idea and goals of the mini-review, performed literature search and revision, and wrote the manuscript.

## FUNDING

This work was funded by the Portuguese Foundation for Science and Technology (FCT) and by the European Regional Development Fund (ERDF) through the Portugal 2020 Partnership Agreement between Portugal and the European Union, the Competitiveness and Internationalization Operational Program (COMPETE 2020) and the Regional Operational Program Lisboa (POR-Lisboa), under the project NANOBINDERS (PTDC/AAG-REC/3004/2014). CM was granted as an Invited Scientist within the project NANOBINDERS (ref. BCC/UI88/5626/2016). Thanks are due for the financial support to CESAM (UID/AMB/50017 – POCI-01- 0145-FEDER-007638), to FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.



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**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Marques. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Survival and Energy Producing Strategies of Alkane Degraders Under Extreme Conditions and Their Biotechnological Potential

Chulwoo Park and Woojun Park\*

Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, Korea University, Seoul, South Korea

Many petroleum-polluted areas are considered as extreme environments because of co-occurrence of low and high temperatures, high salt, and acidic and anaerobic conditions. Alkanes, which are major constituents of crude oils, can be degraded under extreme conditions, both aerobically and anaerobically by bacteria and archaea of different phyla. Alkane degraders possess exclusive metabolic pathways and survival strategies, which involve the use of protein and RNA chaperones, compatible solutes, biosurfactants, and exopolysaccharide production for self-protection during harsh environmental conditions such as oxidative and osmotic stress, and ionic nutrient-shortage. Recent findings suggest that the thermophilic sulfate-reducing archaeon Archaeoglobus fulgidus uses a novel alkylsuccinate synthase for long-chain alkane degradation, and the thermophilic Candidatus Syntrophoarchaeum butanivorans anaerobically oxidizes butane via alkyl-coenzyme M formation. In addition, gene expression data suggest that extremophiles produce energy via the glyoxylate shunt and the Pta-AckA pathway when grown on a diverse range of alkanes under stress conditions. Alkane degraders possess biotechnological potential for bioremediation because of their unusual characteristics. This review will provide genomic and molecular insights on alkane degraders under extreme conditions.

#### Edited by:

Kian Mau Goh, Universiti Teknologi Malaysia, Malaysia

#### Reviewed by:

Rachel Narehood Austin, Columbia University, United States Natsuko Hamamura, Kyushu University, Japan

> \*Correspondence: Woojun Park wpark@korea.ac.kr

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

> Received: 30 December 2017 Accepted: 07 May 2018 Published: 25 May 2018

#### Citation:

Park C and Park W (2018) Survival and Energy Producing Strategies of Alkane Degraders Under Extreme Conditions and Their Biotechnological Potential. Front. Microbiol. 9:1081. doi: 10.3389/fmicb.2018.01081 Keywords: extremophiles, alkane oxidizer, survival strategies, energy production, bioremediation

## BACKGROUND

Extremophiles are microorganisms that can not only survive but also prefer to grow under extremely harsh conditions (Rampelotto, 2013). Based on their habitat, extremophiles are classified as acidophiles (pH 0.5–5), alkaliphiles (pH 9–12), barophiles (approximately 0.1 MPa), psychrophiles (0◦C–20◦C), thermophiles (50◦C–70◦C), halophiles (5–20% salt). Extremophiles have evolved to possess special features for adapting to extreme conditions. For example, psychrophilic bacteria synthesize cold-shock proteins, which function as transcriptional enhancers and RNA-binding proteins, to protect RNA from numerous stressors (Wouters et al., 1999). In addition, many oligotrophs increase their surface area by synthesizing appendages for easy nutrient absorption and utilization of a wide range of substrates (Huyop and Cooper, 2012). Halophilic bacteria assimilate glycine betaine from the environment and produce osmoprotectants, such as trehalose or N-acetylglutaminylglutamine amide (Sagot et al., 2010).

n-alkanes, the major components of petroleum, can be metabolized aerobically or anaerobically by numerous microorganisms. Several alkane oxidation mechanisms have been documented

till date, namely, terminal oxidation, subterminal oxidation, biterminal oxidation, and the Finnerty pathway. Alkane oxidation by aerobic bacteria produces fatty acids, which are subsequently metabolized via β-oxidation to generate acetyl-CoA (**Figure 1**). On the contrary, anaerobic alkane degradation is initiated by the addition of fumarate onto the terminal or subterminal carbon of alkane molecules, yielding alkylsuccinate derivatives as intermediates (**Figure 1**). Anaerobic alkane degradation was first proposed by So and Young, who demonstrated anaerobic alkane metabolism by fumarateaddition in the sulfate-reducing Desulfatibacillum alkenivorans AK-01 (So and Young, 1999; Rojo, 2009). Furthermore, recent studies also suggested that initial hydrocarbon activation by fumarate-addition occurs in syntrophic bacteria which produces acetate, CO2, and/or H<sup>2</sup> that are utilized by methanogens (Berdugo-Clavijo and Gieg, 2014). Anaerobic hydrocarbon-degrading syntrophic bacteria possess genes encoding an alkylsuccinate (assA) or a benzylsuccinate synthase (bssA) as well as genes for syntrophic processes, such as H<sup>2</sup> and acetate production (Washer and Edwards, 2007; Kato S. et al., 2009; Walker et al., 2009; Fowler et al., 2012; Aitken et al., 2013; Cheng et al., 2013). However, several questions regarding the mechanism underlying anaerobic alkane degradation persist, such as the nature of all key enzymes and metabolic pathways for methanogenic alkane degradation by syntrophic bacteria and methanogen (Tan et al., 2015a).

Alkane monooxygenases have unique substrate ranges, which are classified into three categories based on the alkane chain length (van Beilen and Funhoff, 2007). Methane (C1) to butane (C4) conversion is catalyzed by methane monooxygenase (MMO)-like enzymes such as soluble MMO, particulate MMO, propane monooxygenase (PMO), and butane monooxygenase (BMO). In addition, integral membrane non-heme iron (AlkB) or cytochrome P450 enzymes (CYP153) oxidize pentane (C5) to hexadecane (C16), and alkanes longer than heptadecane are metabolized by a novel alkane dioxygenase, putative flavin-binding monooxygenase (AlmA), or long-chain alkane monooxygenase (LadA) (Maeng et al., 1996; Feng et al., 2007; Throne-Holst et al., 2007). However, a recent study showed that a novel Rieske-type alkane monooxygenase degrades pentane (C5) to tetracosane (C24) using NADH as a cofactor (Li et al., 2013). In addition, many bacteria possess multiple alkane hydroxylases (AH), and therefore, it is possible that a wide range of alkanes can be degraded by one microorganism (Whyte et al., 1998; Rozhkova-Novosad et al., 2007; Amouric et al., 2010; Liu et al., 2011; Park et al., 2017).

Biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated, and nitrated compounds, occurs in various extreme habitats. In addition, biodegradation of petroleum hydrocarbons by extremophiles has also been demonstrated under several conditions. For example, Rhodococcus sp. strain Q15, a psychrophile, showed short- and medium-alkane (C<sup>10</sup> to C21) degradation at 5 ◦C. In addition, solid-phase microextraction–gas chromatography-mass spectrometry showed the co-appearance of 1-hexadecanol and 2-hexadecanol when strain Q15 utilized hexadecane, suggesting that Q15 assimilate alkanes via both the terminal and subterminal oxidation pathways (Whyte et al., 1998). A novel halophilic species, Amycolicicoccus subflavus, retains defensive genes against high salinity, osmotic stress, and poor nutrient availability, resulting in growth in the presence of 1–12% NaCl. Furthermore, genome analysis revealed that Amycolicicoccus subflavus possesses four AH (AlkB, CYP153, LadA, and PMO), which allows growth on C<sup>10</sup> to C<sup>36</sup> alkanes and propane (Nie et al., 2013). Thermophilic Geobacillus and Aeribacillus species, isolated from petroleum reservoirs and a hot spring, respectively, grew from 38◦C to 68–70◦Ñ, with maximum growth at 60◦Ñ, and utilized C10–C<sup>30</sup> alkanes as carbon source. Both strains are proven to have an alkBtype alkane monooxygenase. Two other Geobacillus strains (G. toebii B-1024 and 1017) also possess genes homologous to ladA and show activity similar to that of G. toebii B-1027 during long-chain alkane metabolism (Tourova et al., 2016).

Alkane-degrading extremophiles can be used in bioremediation of diverse oil-contaminated environments because of their special capabilities under extreme conditions. Cold-adapted hydrocarbon degraders have been applied to oil-polluted cold soil (Margesin et al., 2003; Aislabie et al., 2004; Wang J. et al., 2015) and wastewater (Margesin and Schinner, 1998; Gratia et al., 2009). In addition, psychrophiles from alpine habitats showed the highest degradation rate at 10◦C within 8 days (40–60%) and at 4◦C after 8 days (20–40%), showing high biodegradation efficiency (Margesin et al., 2000; Das and Chandran, 2011). Another well-studied example of bioremediation is the utilization of halophiles. Hydrocarbon biodegradation in the presence of high salinity is substantially valuable, not only from the economical aspect, but also in ecological and scientific studies. Recently, four Pseudomonas aeruginosa strains isolated from oil-contaminated saline soil were used to treat 10 g kg−<sup>1</sup> of crude oil with more than 40% biodegradation efficiency in the presence of 300 mM NaCl after 120 days (Ebadi et al., 2017). Marinobacter hydrocarbonoclasticus showed high biodegradation activities of n-alkanes such as hexadecane (100%), eicosane (91%), and heneicosane (84%) in the presence of 4.6–20% NaCl (Gauthier et al., 1992). In another study, artificially weathered crude oil was proven to be degraded by microorganisms in a sandy salt marsh with a nitrogen–phosphorus–potassium (NPK) fertilizer (Lin et al., 1999). Several studies have demonstrated the feasibility of applying extremophiles to bioremediation and provided useful information for optimization of biodegradation efficiency.

However, despite the versatile potential of extremophiles in biodegradation, the molecular mechanisms and biological characteristics of these microorganisms have not been wellstudied. This review discusses recent studies on alkane monooxygenase and 16S rRNA-based phylogenetic analysis of extremophiles, genotypic and phenotypic characteristics of pollutant-degrading extremophiles, and the ecological/advanced aspects of bioremediation. Overall, this review presents an updated overview of petroleum hydrocarbon degradation by microorganisms in different ecosystems.

#### PHYLOGENETIC AFFILIATION AND DISTRIBUTION OF ALKANE-UTILIZING EXTREMOPHILES

#### Extremophiles Possessing AlkB and CYP153 Family Alkane Hydroxylases

The genetic diversity of alkane hydroxylases has been extensively investigated under several conditions, including various oilpolluted and non-polluted environments. In particular, AlkB and members of the CYP153 family are widely distributed and have been extensively investigated in soil, marine environments, and groundwater (Sotsky et al., 1994; Knaebel and Crawford, 1995; Stapleton and Sayler, 1998; Luz et al., 2004; Kuhn et al., 2009).

#### Psychrophiles

Biodegradation of several petroleum components by indigenous cold-adapted microbial populations has been observed at low temperatures in hydrocarbon-contaminated Arctic and Antarctic soils. A large population of Pseudomonas species was observed in Arctic soil after diesel contamination. Furthermore, gene expression analysis confirmed that Pseudomonas and Rhodococcus species induce hydrocarbon degradation genes in Arctic biopile soils during bioremediation, indicating the importance of both species in oil-contaminated Arctic soil (Yergeau et al., 2012). DNA-based study showed that rhodococcal alkB genotypes (Rh alkB) are generally distributed in both contaminated and pristine Antarctic soils, whereas alkB from Pseudomonas putida (Pp alkB) was detected more in contaminated soils than in non-contaminated soils, implying that Rhodococcus spp. may be the predominant alkane-degradative bacteria in both polar soils, but Pseudomonas spp. may be enriched after contamination (Whyte et al., 2002). Populations of γ-proteobacteria such as Colwellia, Marinomonas, and Glaciecola appeared to be predominated in oil-contaminated Arctic fjord ice cores, (Brakstad et al., 2008). The 16S rRNA gene libraries of both non-contaminated and crude oil-contaminated seawater from sub-Antarctic areas showed the predominance of Roseobacter, Sulfitobacter, Staleya, Glaciecola, Colwellia, Marinomonas, Cytophaga, and Cellulophaga. However, non α- and γ- proteobacteria (ε-proteobacteria and Bacteroidetes) such as Arcobacter, Polaribacter, Ulvibacter, and Tenacibaculum started to appear when seawater was contaminated with hydrocarbons (Prabagaran et al., 2007). Therefore, α-, and γ-proteobacteria contribute maximally to alkane degradation in cold marine environments, whereas non α- and γ- proteobacteria have minor contribution.

#### Halophiles

The metagenomic database of a seawater sample from the Sargasso Sea (Venter et al., 2004) revealed the presence of alkB and cyp153 (van Beilen and Funhoff, 2007), implying wide

distribution of halophilic and halotolerant alkane degraders in the ocean environment. Other PCR-based study examining the Atlantic Ocean surface seawater revealed that both alkB and cyp153 genes coexist in Alcanivorax and Salinisphaera species, whereas all Parvibaculum species possess only cyp153 and discovered new culturable alkane-degraders belonging to Brachybacterium, Idiomarina, Leifsonia, Martelella, Kordiimonas, Parvibaculum, and Tistrella (Wang W. et al., 2010; Supplementary Table S1). Characterization of culturable Alcanivorax strains, Marinobacter, Nocardioides, Parvibaculum strains, originating from deep-sea hydrothermal vents, also revealed that only Parvibaculum strain possesses an alkaneoxidizing cytochrome P450 (CYP)-like protein to degrade alkanes (Bertrand et al., 2013). In subtropical seawater, alkB was detected in Gallaecimonas, Castellaniella, Paracoccus, and Leucobacter species, which shows a completely different bacterial community compared to the pelagic area (Wang L. et al., 2010), indicating that the distribution of alkane degraders varies with ocean conditions and geographical location. The number of α- and γ-proteobacteria (dominated by Sphingomonadaceae, Rhodobacteraceae, and Chromatiales) increased when crude oil was spilled in the supratidal and intertidal zones, on the other hand, γ- and δ-proteobacteria were more abundant in subtidal zones. The phylum Actinobacteria, particularly the genus Rhodococcus, was a key player in microbial response to the spillage especially in the supratidal sediment (Acosta-González et al., 2015). Likewise, no alkB sequence from Actinobacteria was detected in the marine metagenome despite a high proportion of alkB sequences from Actinobacteria in terrestrial and freshwater metagenomes (Nie et al., 2014).

#### Acidophiles

Although information on acidophilic alkane degraders is limited, some studies on alkane degradation at low pH have indicated the existence of hydrocarbon-degrading acidophiles. 16S rRNA library analysis of acidic soil from natural hydrocarbon seeps (pH 2.8–3.8) showed that majority of sequences were from heterotrophic acidophilic bacteria, including Acidisphaera and Acidiphilium species of α-proteobacteria, as well as the iron-and sulfur-oxidizing chemolithotroph Acidithiobacillus strain. A novel Acidisphaera-related strain C197 possesses alkB homologs (92.5% similarity with an alkB fragment from Xanthobacter flavus) and performs hexadecane metabolism (Hamamura et al., 2005). Mycobacterium is also a predominant bacterial genus found in extreme acidic sulfur block environments and alkane degradation of strain AGS10, an acidophilic Mycobacterium, was demonstrated. Interestingly strain AGS10 has both alkB and cyp153 genes, implying the possibility of two alkane hydroxylase families in many acidophilic Mycobacteria strains (Ivanova et al., 2014).

#### Extremophiles Possessing AlmA and LadA Family Alkane Hydroxylases

Flavin-binding family alkane hydroxylase (AlmA) and long-chain alkane monooxygenase (LadA) have recently been investigated; however, little is known about the distribution of either gene in the environment. Studies on functional diversity of alkane hydroxylase genes, including almA and ladA homologs, provided evidence that some psychrophiles possess AH for long-chain alkane degradation. Alignments with combined Pfam data sets showed the presence of almA homologs in Paraglaciecola psychrophila 170 (five copies) and Psychrobacter cryohalolentis K5 (one copy). On the contrary, a ladA candidate appears in the genomes of Octadecabacter antarcticus 307, Octadecabacter arcticus 238, and Terriglobus saanensis SP1PR4 (Bowman and Deming, 2014). These results hint at the occurrence of longchain alkane metabolism at low temperatures, although the functionality of almA and ladA genes has not been shown experimentally. The diversity of almA in marine bacteria and oil-enrichment bacterial communities was investigated in three surface seawater samples from the South China Sea, Indian Ocean, and Atlantic Ocean (Wang and Shao, 2012). The acquired DNA sequences suggested two clades of almA gene. Class I almA contained a few sequences represented by the Salinisphaera and Parvibaculum genera, whereas class II was larger and more diverse and had many proteobacteriarelated sequences, mainly from Alcanivorax and Marinobacter. However, metagenomic studies for detecting ladA in marine or saline conditions have not been conducted so far, although several marine hydrocarbonoclastic bacteria and halophilic hydrocarbon-utilizing bacteria harboring either AlmA- or LadAtype homologs, or both, have been reported (Liu et al., 2011; Nie et al., 2013; Meier et al., 2016). Thus, further investigation of long-chain alkane metabolism, especially the involvement of almA and ladA, under conditions of high salinity, are warranted.

Plasmid-born ladA gene was first investigated in Geobacillus thermodenitrificans NG80-2, indicating high possibility of horizontal gene transfer (HGT) of the ladA gene (Feng et al., 2007; Li et al., 2008). However, other member of Geobacillus, G. thermoleovorans B23 harbors three genes encoding LadAtype alkane hydroxylases (ladAαB23, ladAβB23, and ladBB23, collectively named the ladAB region) on its chromosome. Comparative genome analysis of relevant Geobacillus strains confirmed the absence of the ladA gene on the chromosome of G. thermodenitrificans NG80-2, but the presence of the ladAB gene island in many other Geobacillus strains (Boonmak et al., 2014).

We retrieved whole genome data of extremophiles from the National Center for Bio-technology Information (NCBI)<sup>1</sup> and Integrated Microbial Genomes (IMG)<sup>2</sup> databases. Total 66 identified genomes were aligned and analyzed (Supplementary Table S1). alkB genes are widely distributed in aerobic alkanedegrading extremophiles (psychrophiles, halophiles, acidophiles, and thermophiles), although their association with HGT was not clear; nonetheless, the different evolution patterns of cyp and 16S rRNA, shown in **Figure 2**, indicate the occurrence of HGT. Interestingly, alkB, cyp, and almA were mostly detected in psychrophiles, whereas halophiles and thermophiles possess ladA (**Figure 3**). In addition, acidophilic alkane degraders possess only

<sup>1</sup>https://www.ncbi.nlm.nih.gov/

<sup>2</sup>https://img.jgi.doe.gov/

alkB, which is not present in the genome of thermophilic alkane degraders (**Figure 2** and Supplementary Table S1).

## Methanotrophic and Anaerobic Extremophiles

Anaerobic methane oxidation (AOM) via reverse methanogenesis commonly occurs in anaerobic methaneoxidizing archaea (ANME) under anoxic environments, and was first identified in marine sediments where AOM was coupled to sulfate reduction. Therefore, ANME usually formed a community with sulfate-reducing bacteria (SRB) of δ-proteobacteria. ANME are generally classified into three categories: ANME-1 (sub-clusters a, and b) related to Methanomicrobiales and Methanosarcinales, ANME-2 (sub-clusters a, b, and c) related to cultivated members of Methanosarcinales, and ANME-3 related to Methanococcoides spp. (Timmers et al., 2017). However, all mcr genes (encoding methyl-coenzyme M reductase) in the class Methanomicrobia, such as Methanoculleus, Methanoregula, and Methanosaeta showed over 60% identity each other, which is indicative of its high conservation (**Figure 4**). Compared to samples containing only methane, samples from natural freshwater gas sources containing methane and sulfate, harbored ANME-2a/b such as Methanolobus, and AOM-associated archaea such as Candidatus Methanoperedens nitroreducens, as determined by archaeal 16S rDNA analysis (Timmers et al., 2016).

Methanogenic hydrocarbon-degrading δ-proteobacteria (Syntrophus/Smithella, Desulfosarcina/Desulfococcus), Firmicutes (Desulfotomaculum, Desulfosporosinus, and Pelotomaculum), and relatively few bacteria belonging to Spirochaetes, Bacteroidetes, Chloroflexi, and β-proteobacteria have been identified in hydrocarbon-contaminated aquifers (Tan et al., 2015a). In addition, previous reports showed that the γ-proteobacterial genus Marinobacter, and δ-proteobacterial genera Syntrophus and Smithella, are the predominant hydrocarbon degraders in samples from estuarine sand and oil sand tailings under methanogenic conditions (Gray et al., 2011; Siddique et al., 2012). Hydrocarbon degradation in marine environments is predominantly conducted by SRB, and on-site stable isotope probing revealed that Desulfosarcina/Desulfococcus (DSS) of δ-proteobacteria actively oxidize short- (butane) and long-chain (dodecane) alkanes. Although few SRB alkane degraders have been identified, several culture-dependent and independent studies at oil-contaminated seeps have indicated the presence of more diverse species than the currently known SRB (Kleindienst et al., 2014). In experimental condition, cultures enriched from oil sands tailings ponds included predominant δ-proteobacteria, such as Desulfoglaeba, Desulfobacteraceae, and Geobacter when short-chain alkane-degrading culture with a mixture of C6, C7,

blue, Firmicute; dark blue, Thermus–Deinococcus.

FIGURE 3 | AlmA and LadA phylogenetic tree of extremophiles. Collected sequences were aligned and phylogenetic tree was drawn using the MEGA 6 program. Colored words indicate the following environmental types of microorganisms: blue, psychrophile; red, thermophile; black, halophile; yellow, acidophile. Colored backgrounds indicate the following phylogenetic groups: yellow, Actinobacteria; purple, Alphaproteobacteria; green, Gammaproteobacteria.

C8, and C<sup>10</sup> n-alkanes as predominant organic carbon sources was transferred into sulfate-supplemented medium (Tan et al., 2015b).

## SURVIVAL STRATEGIES OF EXTREMOPHILES UNDER HARSH CONDITION

#### Psychrophiles

Many DNA-binding proteins are present in extremophiles such as psychrophiles. For example, cold-shock proteins (Csp) play essential roles in DNA or RNA stabilization and membrane rigidity by regulating unsaturated fatty-acid synthesis (D'Amico et al., 2006). Interestingly, Methanogenium frigidum, a stenopsychrophilic archaea, possesses csp family of genes, which are generally not detected in archaeal (such as thermophilic and hyperthermophilic) genomes (Giaquinto et al., 2007). The Csp sequences of M. frigidum and Escherichia coli, are highly similar, and complementation of csp-knockout E. coli strain with csp gene from M. frigidum, results in growth recovery at low temperature, showing compatibility between archaeal and bacterial Csps. We observed that Pseudomonas fragi strains A22 and B25 (Supplementary Table S1) have the most abundant csp genes (6 genes) among putative alkane-degrading psychrophiles. However, six csp genes were also present in the genome of the

mesophilic alkane degrader Acinetobacter oleivorans DR1. Thus, it is assumed that other strategies facilitate cold adaptation in psychrophiles regardless of the number of csp genes.

Most cold-adapted proteins in psychrophiles also show other characteristics, including high structure flexibility, low proportion of acyl chains in the cell membrane, and increased protein volume (Siddiqui and Cavicchioli, 2006). Chemical and biological reactions are slower at low temperatures, and therefore, the synthesis of cold-active enzymes with higher activity than mesophilic enzymes are required for maintaining an appropriate rate for essential metabolic reactions. To achieve high enzyme activity in cold conditions, the catalytic center must be flexible and unstable. Therefore, cold-adapted enzymes are heat labile and possess lower Gibbs free energy (1G) than mesophilic or thermophilic enzymes (Feller and Gerday, 2003; Siddiqui and Cavicchioli, 2006). Comparative sequence alignments of putative alkane hydroxylases from mesophiles and psychrophiles revealed that substitution by specific amino acids, such as alanine or glycine, contributes to increased flexibility of loops, bends, and α-helical structures of psychrophilic alkane hydroxylases. Furthermore, reports show that the flexible loops, bends, and helical regions of P450 from Glaciecola psychrophila are located in the center of three methionine residues, which is known to act as an alternative heme-binding site at low-temperatures (Bowman and Deming, 2014).

Trehalose production plays significant roles in resistance to freezing in cold environments. Observations from neutron diffraction, Raman spectroscopy, and inelastic neutron scattering revealed that trehalose disrupts the tetrahedral intermolecular network of water formed at low temperatures and thereby protects from ice damage. Image analysis based on field-emission transmission electron microscopy showed that trehalose was better than sucrose in preventing ice formation (Magazù et al., 2012). Currently, only psychrophilic Rhodococcus is known to produce trehalose lipid as a biosurfactant during alkane degradation (Rapp and Gabriel-Jürgens, 2003). However, there is a high possibility of trehalose lipid production by psychrophilic alkane degraders as trehalose synthesis can be induced by low temperature (Phadtare, 2004). Taken together, trehalose can be utilized as a cryoprotectant or biosurfactant by psychrophiles under cold conditions.

#### Halophiles

Many halophilic microorganisms develop strategies for surviving in highly saline environments, such as transportation of inorganic ions (mainly potassium) for stabilizing external osmotic pressure or synthesis of metabolism-compatible osmolytes. Genome and expression analyses of the moderately halophilic alkane degrader, Amycolicicoccus subflavus DQS3-9A1<sup>T</sup> showed upregulation of the KdpD/KdpE two component system (high-affinity K<sup>+</sup> uptake system), Trk-type K<sup>+</sup> transporter and Na+/K<sup>+</sup> antiporter, MtrA/MtrB two component system (BetP regulator), and glycine/betaine transporter (BetP) in response to high salt stress (Nie et al., 2013). Trk-type K<sup>+</sup> transporter, one of main K<sup>+</sup> transporters, requires three genes for cellular K<sup>+</sup> uptake, namely, trkA (1,374 bp, encoding cytoplasmic NAD+/NADH binding protein), trkH (1,449 bp, encoding Trk transporter), and trkI (1,479 bp, encoding Trk transporter). Genome data show that several halophilic alkane degraders, such as Paracoccus saliphilus, Marinobacterium lutimaris, Salinisphaera, and Marinobacter hydrocarbonoclasticus, possess multiple Trk potassium uptake proteins, implying that Trk transporter provides osmolarity balance to halophiles during alkane degradation under high salinity conditions.

Several classes of organic compatible solutes, such as amino acids, alcohols, sugars, and derivatives act as organic osmoprotectants, which can be classified into three chemical categories: zwitterionic, non-polar, and anionic solutes (Roberts, 2005). These organic osmoprotectants are regulated by extracellular salt concentration. The synthesis rate of trehalose in Haladaptatus paucihalophilus decreases with increase in salinity; on the contrary, the intracellular concentration of glycine betaine increases due to import at high salinities, suggesting dynamic regulation of two compatible solutes for osmoadaptation (Youssef et al., 2014). The obligate hydrocarbon bacterium, Alcanivorax borkumensis SK2, synthesizes and accumulates ectoine to maintain osmolality under hyperosmosis, which promotes active metabolism and maintains cell integrity albeit low hydrocarbon degrading performance, thereby protecting cells from damage due to hyperosmosis (Scoma and Boon, 2016).

Undesirable interactions that destroy internal microbial proteins by dehydration can be avoided by controlling net charge. Compared to non-halophilic proteins, halophilic proteins contain several glutamate and aspartate residues on their surfaces, which provides high water solubility in the presence of salinity (Edbeib et al., 2016). Although comparative sequence alignment and structural studies of halophilic alkane hydroxylases have not been performed so far, we speculate that large numbers of glutamate and aspartate on their surfaces distributed to the stabilization of alkane hydroxylases.

#### Acidophiles

Specific adaptation to and metabolism of alkane degradation have not been extensively studied in acidophiles. Acidophiles possess several systems for surviving in highly acidic conditions (Baker-Austin and Dopson, 2007), such as a cytoplasmic buffering, DNA protection and repair, blocking of extracellular proton uptake, and organic acid utilization (Babu et al., 2015). Buffer capacity in the cytoplasm is determined mainly by amino acids, such as glutamate and arginine, which sequester protons. An acid-resistant E. coli strain showed that decarboxylated amino acids which require intracellular proton consumption enhanced internal pH, resulting in maintenance of pH homeostasis. However, the buffering capacities of acidophilic bacteria Bacillus acidocaldarius and other neutrophilic bacilli were not significantly different, suggesting that the survival mechanism encompasses several ways via which acidophilic bacteria adapt to low pH (Richard and Foster, 2004).

A large number of DNA and protein repair genes in acidophiles contribute to preservation of pH homeostasis (Dopson et al., 2005). A DNA-binding protein in starved cells (Dps), which protects DNA from reactive oxygen species (ROS) generated by the Fenton reaction, plays a significant role when cells are exposed to oxidative and nutritional stress. Studies

showed that the E. coli O157:H7 wild-type strain survives better than dps::nptI mutants in the initial 1 h of exposure to acidic conditions. Furthermore, complementation of wild type dps gene to dps::nptI mutants restored survival (Choi et al., 2000). However, available genome data showed that Dps is not present in Acidiphilium, suggesting that Dps synthesis does not occur by all alkane-degrading acidophiles for surviving under acidic conditions.

Upregulation of an outer membrane porin (Omp40) in Acidithiobacillus ferrooxidans was observed when pH was shifted from 3.5 to 1.5 (Amaro et al., 1991). Further studies illustrated that the positively charged L3 loop of the porin protein controls pore size and ion selectivity at pH 2.5 (Guiliani and Jerez, 2000). In addition, the decrease in membrane fluidity of E. coli O157:H7 may confer resistance to protons under acidic conditions (Yuk and Marshall, 2004). When lipids of acidophilic strains were adapted to acidic condition, palmitic acid (C16:0) concentrations increased, whereas that ofcis-vaccenic acid (C18:1ω7c) decreased, resulting in reduced membrane fluidity, which regulated porin pore size. Low membrane fluidity confers resistance to toxicity from hydrocarbons and acidic condition, but compromises alkane uptake.

#### Thermophiles

Most proteins with catalytic and regulatory functions in thermophiles are thermostable and are structurally more compact than their mesophilic counterparts. Hyperthermostable proteins in Thermococcus onnurineus NA1, hydperthermophilic archaea, incubated at 100◦C include intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors (Yun et al., 2011). Furthermore, transcriptomic and proteomic analyses of thermophiles revealed that many thermostable proteins of certain categories [heat stable proteins, chaperonin, metalloenzyme, putative ribosomal associated proteins, and superoxide dismutase (SOD)] are translated at high temperatures, implying that they are essential for thermophile survival at those temperatures (Li et al., 2010; Shih and Pan, 2011; Wang W. et al., 2014). These proteins have large numbers of disulfide bonds and positively charged amino acids, such as lysine and arginine, which are critical for their stability (Brocchieri, 2004; Beeby et al., 2005; Ma et al., 2010). Additionally, in silico studies illustrated that disulfide bonds are ubiquitous among thermophiles. BLAST revealed the presence of nine genes encoding FAD-dependent pyridine nucleotide-disulfide oxidoreductase, a potential key enzyme in thermophilic intracellular disulfide-bond formation, in the thermophilic alkane degrader Geobacillus sp. Y4.1MC1 (Beeby et al., 2005). Increase in the number of small residues such as Gly, Ala, Ser, and Val, and decrease in the number of Cys and polar residues (Asp, Asn, Glu, Gln, and Arg) were observed in membrane proteins of many thermophiles, which increases stringent membrane hydrophobicity at high temperatures (Meruelo et al., 2012). The amino acid preference might occur in membrane proteins of thermophilic alkane degraders.

Proteome analyses in most thermophiles has demonstrated the presence of genes encoding highly thermostable proteins such as antioxidants, antitoxins, heat-shock proteins (Hsps), and enzymes involved in carbon metabolism pathways, including glycolysis (Wang Q. et al., 2015). Several antioxidants and antitoxins, such as the VapBC complex, are translated in response to high-temperature stress (Shih and Pan, 2011). Global transcriptomic analysis demonstrated that VapBC proteins, the virulence-associated proteins in the hyperthermophilic crenarchaeon Sulfolobus solfataricus, are induced at elevated temperatures ranging from 80–90◦C. Furthermore, a 1vapBC S. solfataricus knockout strain showed a highly altered transcriptomic profile and susceptibility to heat shock (Cooper et al., 2009). However, S. solfataricus VapBC homologues do not exist in any thermophilic alkane degrading bacteria (Geobacillus species, Thermus brockianus, and Aeribacillus pallidus) and archaea (Archaeoglobus fulgidus DSM 4304), indicating that the VapBC system is not a ubiquitous heat-shock defense mechanism. Small Hsps (sHSPs) bind to proteins and confer protection from denaturation under high temperatures (Li et al., 2012). Unlike Csp proteins, Hsp proteins are abundant in many thermophilic alkane degraders. Five Hsp-encoding genes are present in the Geobacillus thermodenitrificans NG 80-2 genome, and most Geobacillus species possess more than four genes. Thus, Hsp proteins in alkane-degrading thermophiles may play an important role in survival at high temperatures.

### ENERGY PRODUCTION OF EXTREMOPHILES DURING ALKANE METABOLISM

Most alkane-degrading extremophiles have ubiquitous aerobic or anaerobic alkane oxidation pathways represented by ω-oxidation or fumarate addition, respectively (**Figure 1**). It is assumed that most aerobic psychrophiles, halophiles, acidophiles, and thermophiles degrade alkanes through general alkane oxidation pathways, as demonstrated by the possession of AlkB, CYP, AlmA, and LadA-type alkane hydroxylases (Bertrand et al., 2013; Nie et al., 2013; Ivanova et al., 2014). However, some extremophiles oxidize alkanes via a unique pathway and produce energy via alternative pathways to avoid stress under harsh conditions.

#### Novel Aerobic Alkane Degradation Systems in Extremophiles

The aerobic alkane degrader, Dietzia DQ12-45-1b, grown at 4–45◦C, pH 6.0–12.0, and 0–20% (w/v) NaCl (Wang et al., 2011), possesses an alkW1-alkX system, which is co-expressed and induced by fatty acids (Liang et al., 2016). The proposed mechanism is that alkX, a TetR family regulator, competes with DNA polymerase at the alkW1 promoter region and represses the expression of alkW1, an alkB-type alkane hydroxylaserubredoxin fusion gene, in the absence of alkanes. However, fatty acids produced by alkane oxidation directly bind to AlkX, removing it from the alkW1 promoter when alkanes are present, indicating that this system is energy-cost effective. Furthermore,

phylogenetic and gene alignment analyses showed that alkW1 alkX may be ubiquitous in Actinobacteria such as Rhodococcus and Mycobacterium. Thus, alkW-alkX system confer benefits to Actinobacteria living in harsh conditions characterized by high pH, osmotic stress, and lack of nutrients (Liang et al., 2016).

Recent genome sequencing analysis revealed that the moderate halophile, Amycolicicoccus subflavus DQS3-9A1<sup>T</sup> possesses four different alkane hydroxylation systems (propane monooxygenase, AlkB, CYP, and LadA) (Nie et al., 2013). A previously described halophile, Alcanivorax dieselolei B-5, harbors multiple copies of alkane hydroxylases (two AlkB-, one CYP153-, and AlmA-type alkane hydroxylase) and showed broad range of alkane-degrading capability (C5–C36) (Liu et al., 2011). Furthermore, the available complete genome sequence of Marinobacter aquaeolei VT8 shows the existence of three AlkB-, two CYP153-, and one AlmA-like alkane hydroxylase enzymes in the VT8 genome. Although the evolutionary reasons for possession of multiple alkane hydroxylase systems are not yet clear, it probably facilitates utilization of diverse hydrocarbon sources by halophilic bacteria under high saline environments. Illumination and addition of casamino acid as an organic fertilizer were tested to measure biodegradation efficiency of the extremely halophilic archaeal strains, Haloferax, Halobacterium, and Halococcus in highly saline soil (>22% salinity) and pond water (>16% salinity), which resulted in significantly enhanced biodegradation by halophilic archaea in the presence of casamino acid and illumination (Al-Mailem et al., 2012). It was hypothesized that archaea in hypersaline environment can synthesize ATP using a red pigment, perhaps a bacteriorhodopsin-like system, under low oxygen tension. Casamino acid also confers better growth and degradation of halophilic archaea, indicating promotion of biodegradation efficiency by addition of organic nitrogen source. Further studies are required to understand enhanced hydrocarbon degradation by haloarchaea in the presence of light and N source.

## Alternative Pathways Producing Energy in Extremophiles

The glyoxylate shunt in the mesophilic alkane-degrading bacteria, Acinetobacter oleivorans DR1, is activated to generate energy during triacontane metabolism (Park et al., 2017). The glyoxylate shunt requires isocitrate lyase (encoded by aceA) and malate synthase (encoded by aceB or glcB), which are activated under oxidative stress or in the presence of C<sup>2</sup> carbon sources (Ahn et al., 2016). Among psychrophiles, upregulation of glyoxylate cycle-related genes in Nesterenkonia sp. AN1, isolated from Antarctic soil, was observed at 5◦C. Furthermore, a large number of glyoxylate shunt-related genes belonging to the psychrophilic bacteria Colwellia and Neptuniibacter were observed in samples from the area surrounding the Deepwater Horizon oil spill (Rivers et al., 2013). In Colwellia maris, the expression level of icl was higher at 0◦C than at 15◦C (Watanabe et al., 2002), implying that Colwellia possess the advantage of surviving in cold environment by activating the glyoxylate shunt during alkane degradation.

Little is known about complete energy producing metabolism of alkane-degrading psychrophiles. The presence of methylglyoxal synthase in psychrophilic bacteria, Exiguobacterium sibiricum, was proposed as an important alternative catabolic pathway for glyceraldehyde phosphate (Rodrigues et al., 2008). In addition, the glyoxalase family proteins in Planococcus halocryophilus Or1 have been emphasized as key enzymes for utilization of carbon sources under cold environments. In addition, repression of energy metabolism at low temperature (−15◦C) in Planococcus halocryophilus Or1, but increased expression of succinic semialdehyde dehydrogenase, alcohol dehydrogenase, and several oxidoreductases indicate maintenance of energy metabolism and ATP levels (Mykytczuk et al., 2013). Similarly, transcriptomic analysis of Pseudomonas extremaustralis showed activation of the ethanol oxidation pathway, involving a pyrroloquinoline quinone (PQQ) dependent ethanol dehydrogenase, cytochrome c550, and an aldehyde dehydrogenase (encoded by exaA, exaB, and exaC, respectively) although genes associated with tricarboxylic acid (TCA) and cytochrome synthesis are repressed under cold conditions (Tribelli et al., 2018). Proteomics study suggested that enzymes participating in the fatty acid degradation pathway (β-oxidation) were more abundant at low temperatures (4–10◦C) in cold adapting marine bacterium, Sphingopyxis alaskensis under artificial seawater medium (Ting et al., 2010). Cold adaptation strategy which uses fatty acid metabolism was also observed in Pseudomonas putida KT2440 at 10◦C in Luria-Bertani (LB) medium. Analyses of proteomics and RNA sequencing data in KT2440 strain showed upregulation of the 2-methylcitrate and branched amino acid degradation pathways under cold temperature, implying conversion of propionate or propionyl-CoA to succinate and pyruvate via 2-methyl citrate under cold condition (Fonseca et al., 2011).

Proteomic analysis of the halophilic bacteria, Alcanivorax borkumensis SK2, also showed upregulation of AceA (ABO\_2741) and GlcB (ABO\_1267) and downregulation of TCA cycle enzymes, including isocitrate dehydrogenase (icd, ABO\_1281) and 2-oxoglutarate dehydrogenase (lpdG, ABO\_1494), when bacteria were grown in hexadecanesupplemented media, suggesting that the glyoxylate shunt is the main pathway for hexadecane metabolism (Sabirova et al., 2006). Similarly, another strategy for adapting to high temperatures involves a shift in the carbohydrate metabolism pathways. Transcriptomic and proteomic analysis of Thermus filiformis at 63◦C, 70◦C, or 77◦C revealed that oxidative stress generated by high temperature induced genes related to the pentose phosphate (PP) pathway, whereas genes participating in glycolysis and the TCA cycle were downregulated (Mandelli et al., 2017), indicating evasion of ROS generation. In addition, metabolomic analysis revealed accumulation of ROS scavengers such as oxaloacetate and α-ketoglutarate, indicating preservation of ROS homeostasis. It has been reported that the PP pathway, glyoxylate cycle, and ROS defense systems are essential for detoxifying petroleum hydrocarbons (Feng et al., 2007; Kato T. et al., 2009; Shih and Pan, 2011). Thus, exposure to heat shock probably assists the growth of thermophilic alkane degraders at high temperatures in the presence of alkanes. The same pattern was observed in

the genomic and proteomic analysis of thermophilic bacteria Geobacillus thermodenitrificans NG80-2, which showed activated glyoxylate shunt instead of the TCA cycle in the presence of hexadecane (Feng et al., 2007).

The interface between alkane degraders and hydrocarbon compounds may form microoxic or anoxic niches because of hydrophobicity (Suter et al., 2017). In addition, alkane oxidation generates excessive acetyl-CoA (Sabirova et al., 2006; Schönfeld and Wojtczak, 2016; Park et al., 2017), creating appropriate conditions for aerobic fermentation, which is also called the Crabtree effect (Zhou et al., 2017). Acetate assimilation via the Pta-AckA pathway occurs in the presence of long-chain alkanes (triacontane), producing ATP (Park et al., 2017). Although studies on the Pta-Ack pathway during alkane metabolism are limited, evidence shows that extremophiles can synthesize ATP via the Pta-AckA pathway. Phenotypic analysis of Psychrobacter arcticus 273-4 shows that acetic acid can be diffused into cells without energy consumption, whereas energy-consuming glucose could not be utilized at low temperatures (Bergholz et al., 2009). In addition, transcriptomic and proteomic analysis of Psychrobacter sp. PAMC 21119, which was grown under cold conditions, revealed upregulation of acetyl-CoA metabolism, although the central energy production and conversion pathways were downregulated (Koh et al., 2017). Geobacillus thermodenitrificans NG80-2 harbors pta and ackA, which are indicative of possible methods of ATP synthesis during alkane assimilation (Feng et al., 2007). Methanogenic archaea and bacteria also possess pta and ackA genes. Furthermore, methanogenic and halophilic archaea have the same ancestral Pta lineage (Barnhart et al., 2015), implying the possibility of acetate production via the Pta-AckA pathway during alkane metabolism at high salinity.

#### Novel Alkane Degradation Pathways Under Anaerobic Conditions

Although many alkane hydroxylase genes involved in aerobic alkane degradation were detected in extremophiles, anaerobic alkane metabolism may have advantages, particularly because extreme environments confer oxidative stress on bacteria. The thermophilic sulfate-reducing archaeon, Archaeoglobus fulgidus strain VC-16, which degrades C10–C<sup>21</sup> alkanes with thiosulfate or sulfate as a terminal electron acceptor, possess pyruvate formate lyase (encoded by pflD), which is homologous to alkylsuccinate synthase (encoded by assA). Phylogenetic analysis showed that homologs of PflD and PflC mainly belong to bacteria such as Firmicutes, δ-proteobacteria, γ-proteobacteria, and Actinobacteria, indicating that these genes are acquired from bacteria via HGT. Furthermore, three-dimensional simulation suggested that PflD might be a potential alkylsuccinate synthase, which is supported by the upregulation of pflD gene expression in the presence of hexadecane but not fatty acid (Khelifi et al., 2014). An anaerobic hyperthermophile, Geoglobus acetivorans, isolated from deep-sea hydrothermal vents, requires ferric iron (Fe (III)) as an electron acceptor during alkane degradation. Genome analysis revealed that this archaeon also harbors close homologs of Archaeoglobus fulgidus PflC and PflD (69 and 57% identity, respectively). However, Gace\_0240 (homolog to PflC in Archaeoglobus fulgidus) shares features with alkyl and benzylsuccinate synthases, rather than with pyruvate formate lyase-activating enzymes (Mardanov et al., 2014).

A methanogenic culture, enriched from freshwater hydrocarbon-contaminated aquifer sediments, consumed octacosane (C28) and generated methane. Smithella was predominant and assA was abundant in the presence of octacosane, furthermore, phylogenetic analysis showed that assA copies in the culture were closely related to assA of Smithella, indicating that fumarate addition in octacosane is mainly performed by Smithella. Interestingly, the detection of α,ω-dicarboxylic acids in the octacosane-only media implies simultaneous activation by fumarate addition, although the mechanism remains unclear (Oberding and Gieg, 2017). Another alkane degradation pathway has been suggested in Verrucomicrobia methylotrophs, which grow optimally at pH 2.0–2.5 and consume methane. Interestingly, analysis of the draft Verrucomicrobia genome detected genes encoding pMMO, which is homologous to the pMMO in methanotrophic proteobacteria. However, genes for subsequent methanol and formaldehyde oxidation were incomplete or missing, suggesting that the bacterium uses novel methylotrophic pathways (Dunfield et al., 2007). Further analysis showed that three pmoA genes in Verrucomicrobia were located in a different clade of proteobacterial pmo homologs, indicating divergent evolution with proteobacterial methanotrophs. Recent metatranscriptome, metaproteome, and metagenome analyses of Candidatus Syntrophoarchaeum butanivorans showed that genes encoding β-oxidation enzymes, carbon monoxide dehydrogenase, and reversible C<sup>1</sup> methanogenesis enzymes [reverse methyl coenzyme M (Mcr)] are also expressed, indicating that enzymes for complete butane metabolism exist in Ca. Syntrophoarchaeum butanivorans. Furthermore, ultra-high resolution mass spectrometry data showed that Ca. Syntrophoarchaeum butanivorans generates alkyl-coenzyme M. Phylogenetic analysis showed that MCR was present in the draft genomes of Bathyarchaeota and pan-genome of uncultivated Hadesarchaea, indicating that uncultivated alkane degraders may perform non-methane alkane degradation (Laso-Pérez et al., 2016).

## BIOTECHNOLOGICAL APPLICATION OF EXTREMOPHILES IN BIOREMEDIATION

In situ bioremediation of petroleum, mainly applied to soil and groundwater, includes biostimulation and bioventing. Biostimulation was recently performed in desert soil near the Arabian Gulf region (Al-Mailem et al., 2015). Community analysis revealed that 19 thermophilic hydrocarbonoclastic bacterial taxa were present in the soil sample. Furthermore, predominant species belonged to Amycolatopsis, Chelativorans, Isoptericola, Nocardia, Aeribacillus, Aneurinibacillus, Brevibacillus, Geobacillus, Kocuria, Marinobacter, and Paenibacillus, which showed better growth and hydrocarbon consumption rate at 50◦C than at 30◦C. However, enhanced

growth and hydrocarbon consumption were observed after supplementation with Ca2<sup>+</sup> (∼2.5 M CaSO4) or addition of 8% w/v dipicolinic acid (DPA), implying that Ca2<sup>+</sup> and DPA-mediated increased heat resistance of alkane degraders may stimulate biodegradation at high temperatures (Radwan et al., 2017). A bioelectrochemical system was recently applied to marine sediments for hydrocarbon biodegradation, wherein a graphite rod (snorkel) is placed between the anoxic contaminated sediment and the O2-containing water. The snorkel acts as an electron acceptor and shuttles electrons generated by anaerobic hydrocarbon oxidation using reducing elements, such as S<sup>2</sup> and Fe2+. Electrons move to the water phase along the graphite and fuse with oxygen and protons to form water (Viggi et al., 2015). This device serves as a sustainable respiratory electron acceptor in anaerobic oxidation of hydrocarbons, and can stimulate hydrocarbon degradation by SRB by scavenging toxic sulfide (Mapelli et al., 2017). Psychrophilic bacteria can potentially be used to degrade organic pollutants from soil and water at low temperatures, mainly, psychrophilic petroleum degraders such as Acinetobacter, Arthrobacter, Colwellia, Cytophaga, Halomonas, Marinobacter, Marinomonas, Pseudoalteromonas, Oleispira, Rhodococcus, and Shewanella (Brakstad et al., 2008). Acinetobacter spp. isolated from oil-polluted soils of King George Island were inoculated into a microcosm of contaminated soil, resulting in reduction of contaminant concentration by 75% after 50 days, compared to NP addition, which had no significant influence (Ruberto et al., 2003). Rhoder, composed of hydrocarbon-degrading bacteria Rhodococcus ruber and R. erythrococcus, oil, and gas, was used for removing oil pollution (Murygina et al., 2000). Over 99% bioremediation rate was obtained in open aquatic surfaces with light contamination (<20 g L−<sup>1</sup> ); however, primary mechanical collection of oil and triple Rhoder treatment resulted in a 94% bioremediation rate in heavily polluted aquatic systems (thickness of oil film > 3 mm) and wetland (2,000 m<sup>2</sup> ) in Urai, indicating that complex bioremediation strategies for effective oil-degradation are required.

Biopile bioremediation involves excavation of polluted soil, followed by supply of nutrients and air for increasing microbial hydrocarbon degradation and promoting bioremediation. Biopile remediation has recently been applied to polluted extreme environments such as cold regions (Gomez and Sartaj, 2014; Dias et al., 2015; Whelan et al., 2015; Martínez Álvarez et al., 2017). In a recent study, a previously suggested nutrient ratio (C:N:P ratio of 100:17.6:1.73) for biological activity (as FDA levels) was used to treat 0.4-ton geomembrane-covered biopiles from diesel fuel storage tanks of Antarctic expedition (Martínez Álvarez et al., 2017). Results showed a higher hydrocarbon removal rate (75.9%) in biostimulated soil within 40 days than in the control biopile (49.5%). In another study, an optimized biopile condition was obtained using surface response methodology (RSM) − a combination of 3 mL m−<sup>3</sup> microbial consortia and 5% mature compost. After 94 days, 90.7% petroleum hydrocarbon reduction was observed, whereas hydrocarbon was reduced by only 56% in control soil (excavated from Val-des-Bois, in the Outaouais region of Quebec, Canada) between 1 and 8◦C, indicating synergistic interaction between bioaugmentation and biostimulation for bioremediation enhancement. Additionally, biostimulation of Antarctic soils with fishmeal reduced 71% of total hydrocarbon and facilitated a bacterial community shift, increasing Actinobacteria over the 50-day study period (Dias et al., 2015). Landfarming has also been demonstrated as an effective bioremediation method in polar regions, which involves aggressive soil tilling and selective fertilization with a mixture of fertilizers (ammonium phosphate and urea) over a span of 70 days (McCarthy et al., 2004). However, soil concentrations of diesel range organics in northeast (380 mg kg−<sup>1</sup> ) and southwest soil (430 mg kg−<sup>1</sup> ) were already reduced to the target concentrations (500 mg kg−<sup>1</sup> ) at 31 and 55 days, respectively.

#### CONCLUDING REMARKS

Although many alkane degraders living in extreme environment has been isolated and characterized, there is a great number of uncharacterized extremophiles that appear to be alkanedegraders, such as Brachybacterium, Idiomarina, and Leifsonia. Thus, further investigation of such novel alkane-degrading extremophiles will provide new metabolic pathways and survival strategies under harsh environments. Recent studies on discovery of new alkane metabolic systems in aerobic-, anaerobic extremophiles, such as AlkW1-AlkX system, Pfl homologues, and alkyl-coenzyme M indicate that there are several questions to be solved regarding alkane degradation metabolism. The evidences demonstrated the activation of several alternative pathways, such as glyoxylate shunt, Pta-AckA, or alcohol/fatty acid metabolism, instead of TCA cycle when extremophiles survive under extreme condition, implying that they are beneficial during alkane degradation. Because of high adaptability, strong stress resistance, and unique biodegradation capability, extremophiles are promising bioremediation-reagents to clean up polluted environments with low cost and high efficiency.

#### AUTHOR CONTRIBUTIONS

CP and WP designed and coordinated the study. CP collected the data. CP wrote the first complete draft of the manuscript. WP provided substantial modifications. All authors contributed to and approved the final version of the manuscript.

## FUNDING

This work was supported by a National Research Foundation of Korea (NRF) grant to WP, funded by the Korea Government (MSIP) (No. NRF-2017R1A2B4005838).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 01081/full#supplementary-material

#### REFERENCES

fmicb-09-01081 May 23, 2018 Time: 11:52 # 12


National Park. Appl. Environ. Microbiol. 71, 5943–5950. doi: 10.1128/AEM.71. 10.5943-5950.2005




**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Park and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Synthesis and Antibacterial Activity of Metal(loid) Nanostructures by Environmental Multi-Metal(loid) Resistant Bacteria and Metal(loid)-Reducing Flavoproteins

Maximiliano Figueroa1†, Valentina Fernandez 1†, Mauricio Arenas-Salinas <sup>2</sup> , Diego Ahumada<sup>1</sup> , Claudia Muñoz-Villagrán1,3, Fabián Cornejo<sup>1</sup> , Esteban Vargas <sup>4</sup> , Mauricio Latorre5,6,7,8, Eduardo Morales <sup>9</sup> , Claudio Vásquez <sup>1</sup> and Felipe Arenas <sup>1</sup> \*

#### Edited by:

Edgardo Donati, National University of La Plata, Argentina

#### Reviewed by:

M. Oves, King Abdulaziz University, Saudi Arabia Ana Isabel Pelaez, Universidad de Oviedo Mieres, Spain

\*Correspondence:

Felipe Arenas felipe.arenass@usach.cl

†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

> Received: 12 December 2017 Accepted: 24 April 2018 Published: 15 May 2018

#### Citation:

Figueroa M, Fernandez V, Arenas-Salinas M, Ahumada D, Muñoz-Villagrán C, Cornejo F, Vargas E, Latorre M, Morales E, Vásquez C and Arenas F (2018) Synthesis and Antibacterial Activity of Metal(loid) Nanostructures by Environmental Multi-Metal(loid) Resistant Bacteria and Metal(loid)-Reducing Flavoproteins. Front. Microbiol. 9:959. doi: 10.3389/fmicb.2018.00959 <sup>1</sup> Laboratorio Microbiología Molecular, Departamento de Biología, Facultad de Química y Biología, Universidad de Santiago de Chile, Santiago, Chile, <sup>2</sup> Centro de Bioinformática y Simulación Molecular, Universidad de Talca, Talca, Chile, <sup>3</sup> Departamento de Ciencias Básicas, Facultad de Ciencia, Universidad Santo Tomas, Sede Santiago, Chile, <sup>4</sup> Center for the Development of Nanoscience and Nanotechnology, Santiago, Chile, <sup>5</sup> Mathomics, Centro de Modelamiento Matemático, Universidad de Chile, Beauchef, Santiago, Chile, <sup>6</sup> Fondap-Center of Genome Regulation, Facultad de Ciencias, Universidad de Chile, Santiago, Chile, <sup>7</sup> Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, Santiago, Chile, 8 Instituto de Ciencias de la Ingeniería, Universidad de O'Higgins, Rancagua, Chile, <sup>9</sup> uBiome, San Francisco, CA, United States

Microbes are suitable candidates to recover and decontaminate different environments from soluble metal ions, either via reduction or precipitation to generate insoluble, non-toxic derivatives. In general, microorganisms reduce toxic metal ions generating nanostructures (NS), which display great applicability in biotechnological processes. Since the molecular bases of bacterial reduction are still unknown, the search for new -environmentally safe and less expensive- methods to synthesize NS have made biological systems attractive candidates. Here, 47 microorganisms isolated from a number of environmental samples were analyzed for their tolerance or sensitivity to 19 metal(loid)s. Ten of them were highly tolerant to some of them and were assessed for their ability to reduce these toxicants in vitro. All isolates were analyzed by 16S rRNA gene sequencing, fatty acids composition, biochemical tests and electron microscopy. Results showed that they belong to the Enterobacter, Staphylococcus, Acinetobacter, and Exiguobacterium genera. Most strains displayed metal(loid)-reducing activity using either NADH or NADPH as cofactor. While Acinetobacter schindleri showed the highest tellurite (TeO2<sup>−</sup> 3 ) and tetrachloro aurate (AuCl<sup>−</sup> 4 ) reducing activity, Staphylococcus sciuri and Exiguobacterium acetylicum exhibited selenite (SeO2<sup>−</sup> 3 ) and silver (Ag+) reducing activity, respectively. Based on these results, we used these bacteria to synthetize, in vivo and in vitro Te, Se, Au, and Ag-containing nanostructures. On the other hand, we also used purified E. cloacae glutathione reductase to synthesize in vitro Te-, Ag-, and Se-containing NS, whose morphology, size, composition, and chemical composition were evaluated. Finally, we assessed the putative anti-bacterial activity exhibited by the in vitro synthesized NS: Te-containing NS were more effective than Au-NS in inhibiting Escherichia coli and Listeria monocytogenes growth. Aerobically synthesized

**84**

TeNS using MF09 crude extracts showed MICs of 45- and 66- µg/ml for E. coli and L. monocytogenes, respectively. Similar MIC values (40 and 82µg/ml, respectively) were observed for TeNS generated using crude extracts from gorA-overexpressing E. coli. In turn, AuNS MICs for E. coli and L. monocytogenes were 64- and 68- µg/ml, respectively.

Keywords: metal, metalloid, reduction, resistance, environmental bacteria, flavoprotein, nanostructure, bioremediation

#### INTRODUCTION

More than half of the elements in the Periodic Table are represented by metals and metalloids. Whereas some of them are essential for different cellular processes, including electron transfer, enzyme catalysis, and stabilization of protein structure, others such as Ag+, Hg+, SeO2<sup>−</sup> 4 , and TeO2<sup>−</sup> 4 are highly toxic even at very low concentrations (Pérez et al., 2007; Zannoni et al., 2008; Lemire et al., 2013). Thus, organisms like bacteria must maintain a strict balance of the intracellular concentration of these metal(loid)s. Under aerobic conditions the toxicity of compounds containing these elements is mainly related to the production of reactive oxygen species (ROS), which can attack key cell components including polyunsaturated fatty acids, proteins, and nucleic acids (Borsetti et al., 2005; Tremaroli et al., 2007; Vrionis et al., 2015). Additionally, protein carbonylation (Contreras and Vásquez, 2010), [4Fe-4S] cluster dismantling of iron-sulfur proteins (Calderón et al., 2009), membrane lipid peroxidation (Lemire et al., 2013; Pradenas et al., 2013), blocking of cysteine and methionine residues (Stolz and Oremland, 1999), increased cell permeability, and the interference of metabolic pathways have also been observed (Carotti et al., 2000).

Given their great biotechnological potential, we focused our interest in characterizing NS containing silver, gold or the metalloids tellurium and selenium. Selenium can be found in the environment as a solid [Se (0), Se<sup>0</sup> ], forming part of seleniumcontaining amino acids, and as the selenium oxyanions selenate [Se (VI), SeO2<sup>−</sup> 4 ] and the most toxic, selenite [Se (IV), SeO2<sup>−</sup> 3 ] (Stolz and Oremland, 1999). Conversely to selenium, tellurium (Te) has no known biological role to date (Ba et al., 2010) and given its metallic characteristics, it is found in various redox states, mainly elemental tellurium [Te (0), Te<sup>0</sup> ], telluride [Te (II), Te2−], tellurite [Te (IV), TeO2<sup>−</sup> 3 ], and tellurate [Te (VI), TeO2<sup>−</sup> 4 ], both oxyanions being highly toxic to most organisms (Nies and Silver, 1995; Taylor, 1999). In turn, silver is mainly found in the 1+ oxidation state [Ag (I)], and less commonly as [Ag (II)], [Ag (III)], or metallic silver (Ag<sup>0</sup> ) (Burriel et al., 2006). Finally, gold is found as metallic gold (Au<sup>0</sup> ) as well as in six other oxidation states, 1+ [Au (I)] and 3+ [Au (III)] being the most commonly found in nature (Nies and Grass, 2009).

To date, a number of genetic and biochemical mechanisms that microbial systems use to cope with the toxicity of defined metal(loid)s are relatively well known; however, a strategy providing universal resistance to all these toxicants has not been reported so far. In general, one of the most common strategies employed by microbes to tolerate high metal(loid) concentrations, is their chemical modification to less-toxic forms (Silver and Phung, 2005). For instance, this kind of resistance mechanisms have been observed in Rhodobacter capsulatus (Turner et al., 2011), Geobacillus sp. (Correa-Llantén et al., 2013), and Shewanella oneidensis (Klonowska et al., 2005) which reduce Te (IV), Au (III) and Se (IV), respectively.

Metal(loid) reduction by microbial systems could be of great help in decontaminating soluble metal ions from soils, forming insoluble, less-toxic derivatives that often generate metal arrangements at the nanoscale (Narayanan and Sakthivel, 2010; Thakkar et al., 2010), also known as nanostructures (NS) exhibiting one or more dimensions <100 nm. Gold (Correa-Llantén et al., 2013), selenium (Song et al., 2017), silver (Gurunathan et al., 2009), tellurium (Arenas-Salinas et al., 2016), and uranium (Suresh, 2012) NS have been reported, opening a great interest because of their potential application in the field of biotechnology.

A number of flavoproteins able of metal ion reduction to less toxic, insoluble forms have been described, including (i) mercury reductase (MerA) (Silver and Phung, 2005), (ii) glutathione reductase (Au3<sup>+</sup> and TeO2<sup>−</sup> 3 reduction, Pugin et al., 2014), (iii) fumarate reductase (selenite reduction, Song et al., 2017), (iv) thioredoxin reductase and alkyl hydroperoxide reductase that reduce tellurite (Arenas-Salinas et al., 2016) and (v) nitrate reductase, able to reduce tellurite as well as silver (Kumar et al., 2007).

Because the optical, mechanical, and fluorescent properties of metal(loid)-containing NS are different from those exhibited by matter at larger scales (Suresh, 2012), NS display a wide range of applications in physics, chemistry, electronics, optics, materials science, biomedicine, and agriculture (Thakkar et al., 2010). The fast development of nanotechnology has provided several kinds of materials for biomedical applications, including cancer detection therapies (Rao, 2008), and antibacterial treatments (Amstad et al., 2011; Azam et al., 2012). In fact, the use of metallic NS displaying bactericidal activity has been reported (Sharma et al., 2009; Li et al., 2011; Cui et al., 2012; Xiu et al., 2012; Dizaj et al., 2014; Pugin et al., 2014; Oves et al., 2017). In other studies, gold-, tellurium-, copper -, silver-, zinc-, and titanium-containing NS proved to be effective as antibacterials against Escherichia coli (Cioffi and Rai, 2012).

Summarizing, since NS production by chemical methods often involve high temperature, anaerobic conditions and toxic reagents that in the end affect the clinical application of nanostructures, the generation and characterization of new metallic NS by biological systems is important either for basic as well as for applied research. In this general context, the search for new environmentally friendly and inexpensive methods for metal(loid)-containing NS generation has made biological systems desirable candidates for both in vivo and in vitro NS production, either using whole microorganisms or purified enzymes.

## MATERIALS AND METHODS

### Isolation and Selection of Metal(loid)-Resistant Bacteria

Twenty-two samples were collected from two different Chilean regions from which bacterial isolates were obtained: (i) north (Blanca, Chaxa and Céjar lagoons, El Tatío geyser, Death Valley) and (ii) mid-south (Maule lagoon, El Teniente copper mine). A sample from Uyuni Salar, Bolivia, was also included in this study. In turn, isolates MF02 and MF05 were kindly provided by Dr. Juan Carlos Tantaléan, Universidad San Luis Gonzaga, Ica, Perú.

Four different culture media were individually inoculated with each sample and incubated at 25 or 37◦C for 24 h: LB (Sambrook and Russell, 2001); R2A (0.5 g/L yeast extract, 0.5 g/L peptone, 0.5 g/L casamino acids, 0.5 g/L glucose, 0.5 g/L soluble starch, 0.3 g/L sodium pyruvate, 0.3 g/L K2HPO4, and 0.5 g/L MgSO<sup>4</sup> adjusted to pH 7.2); Acidiphilium (2 g/L (NH4)2SO4, 0.1 g/L KCl, 0.5 g/L K2HPO4, 0.5 g/L MgSO4, 0.3 g/L yeast extract and 1 g/L D-glucose adjusted to pH 3.0); and Aciduric thermophilic Bacillus strains (ATB) (0.2 g/L (NH4)2SO4, 0.5 g/L MgSO4, 0.25 g/L CaCl2, 3 g/L KH2PO4, 1 g/L yeast extract, 1 g/L tryptone, 1 g/L glucose and 1 g/L starch adjusted to pH 4.3). Pure cultures were obtained after sequential dilutions and successive passages using appropriate solid media.

Purified colonies were seeded on LB-agar plates that contained different concentrations of the following compounds: K2TeO<sup>3</sup> (0.04–0.4 mM), K2CrO<sup>4</sup> (1–10 mM), CdCl<sup>2</sup> (3–5 mM), CoCl<sup>2</sup> (3–5 mM), CuSO<sup>4</sup> (3–5 mM), AgNO<sup>3</sup> (1–5 mM), Al(SO4)<sup>3</sup> (5 mM), FeCl<sup>3</sup> (5–10 mM), PbCl<sup>2</sup> (3 mM), UO2(CH3COO)<sup>2</sup> (2–10 mM), ZnSO<sup>4</sup> (2–5 mM), NiSO<sup>4</sup> (2–5 mM), MnSO<sup>4</sup> (10– 20 mM), HgCl<sup>2</sup> (0.02–0.1 mM), Na2SeO<sup>3</sup> (10–50 mM), FeCl<sup>2</sup> (5–10 mM), NaAsO<sup>2</sup> (5–10 mM), Na2HAsO<sup>4</sup> (10–50 mM), or HAuCl<sup>4</sup> (0.02–1 mM). Selection was for bacterial growth at the highest concentrations and when applicable, also by observing metal(loid) reduction. Strains MF01, MF03, MF04, and MF08 were from Maule lagoon, MF06 from Blanca lagoon (Atacama, Chile), MF07 from Death Valley and MF09 from El Teniente copper mine. Isolate MF10 was from Uyuni Salar, Bolivia.

#### Growth, Identification, and Characterization of the Environmental Strains

Cells were grown in LB medium at 37◦C; E. coli BL21 carrying the pET 101/gorA vector was grown in ampicillin (0.1 mg/ml)-supplemented LB medium. This gorA-expressing plasmid was constructed as follows: the gorA gene was amplified from the environmental strain MF01 using primers CACCATGACTAAGCATTATGACTACATCGCA (Gforward) and ACGCATGGTGACAAATTCTTCG (Greverse). PCR products were inserted into the vector Champion TM pET101 Directional TOPO Expression (Invitrogen <sup>R</sup> ). Correct gene insertion was checked by PCR using primers pETForward (ATGCGTCCGGCGTAGAGG) and pETReverse (GCTAGTTATTGCTCAGCGGTGG). Their identity was confirmed by DNA sequencing. The expression of the cloned gene was induced with 1 mM IPTG for 16 h. GorA was purified by Ni-affinity chromatography (HisTrap HP, GE Healthcare).

Physiological characterization of the isolates was carried out using standard biochemical assays (API ZYM and API 20E; BioMérieux Inc.), which were conducted according to the manufacturer's instructions. Morphological analyses were initially done by Gram staining and in the case of selected strains, also by scanning electron microscopy (SEM) as described by Arenas et al. (2014a). Briefly, samples were visualized and analyzed by low voltage electron microscopy using a LVEM5 Benchtop equipment (Center for Bioinformatics and Molecular Simulation).

Bacterial identification was accomplished by sequencing the 16S rRNA gene using the universal primer 8F (forward) and 1492R (reverse). GeneBank accession numbers for the 16S rRNA nucleotide sequences are: MG461635 (MF01), MG459178 (MF02), MG461634 (MF03), MG461632 (MF04), MG461629 (MF05), MG461631 (MF06), MG461630 (MF07), MG461633 (MF08), MG461636 (MF09), and MG459165 (MF10).

Phylogenetic trees were constructed using the MEGA 6.06 program applying the neighbor-joining method (Saitou and Nei, 1987), while distances were calculated with bootstrapping (100) applying the Tamura-Nei method (Tamura et al., 2004). Bacterial identity was also assessed by analyzing the fatty acid composition at the DSMZ Institute (Braunschweig, Germany).

Determination of minimal inhibitory concentrations (MIC) was carried out using serial dilutions (1: 2) of sterile solutions of K2TeO3, AgNO3, HAuCl4, K2CrO4, CdCl2, NaAsO2, CuSO<sup>4</sup> and Na2SeO<sup>3</sup> in 1 ml of LB medium in 48-well plates. Subsequently, 10 µl of cultures grown in LB medium up to OD<sup>600</sup> ∼0.6 were added to each well and incubation proceeded with constant shaking at the optimal growth temperature of the particular isolate. MICs were determined after 24 h of incubation. For growth curve construction, overnight cultures were diluted 1:100 with fresh LB medium and incubated with shaking up to OD<sup>600</sup> ∼ 0.6. Then, 10 µL were added to 1 mL of fresh LB medium containing 4µM K2TeO3, 15µM AgNO<sup>3</sup> or HAuCl4. Bacterial growth was monitored at 600 nm every 30 min for 14 h in a TECAN Infinite M200 Pro multimode plate reader.

## Determination of Enzymatic Activity

Metal(loid)-reducing activity was determined in a final volume of 200 µl which contained 50 mM Tris-HCl buffer pH 8.0 (or 9.0), 50 mM potassium phosphate pH 7.0, 1 mM NADH (or NADPH), 1 mM β-mercaptoethanol and the crude extract; metal(loid)s tested for reduction were TeO2<sup>−</sup> 3 (1 mM), SeO2<sup>−</sup> 3 (1 mM), AgNO<sup>3</sup> (0.2 mM) and HAuCl<sup>4</sup> (1 mM). Reactions were monitored in a TECAN Infinite M200 Pro multimode plate reader at the wavelength that the metal(loid) absorbs in its elemental state: 500 nm (Te; Pugin et al., 2014), 400 nm (Se; Song et al., 2017), 424 nm (Ag; Kumar et al., 2007), and 540 nm (Au; Correa-Llantén et al., 2013). The optimal buffer and best nicotinamide cofactor were determined for each crude extract; an enzyme unit (U) was defined as the amount of enzyme required to increase the absorbance by 0.001 units/min at the respective wavelength. Metal(loid)-reducing activity was also assessed by native polyacrylamide gel electrophoresis (native PAGE). Briefly, proteins in crude extracts were fractionated in 10% gels; after the run, activity was revealed by submerging the gel in the appropriate buffer containing the metal(loid) and a mixture of 1 mM NADH/NADPH as electron donor.

## In vivo and in vitro Synthesis of Nanostructures

The isolates selected for synthesizing NS in vivo were grown to exponential phase (OD600∼0.5) and treated with ¼ of the metal(loid) MIC (TeO2<sup>−</sup> 3 , SeO2<sup>−</sup> 3 , Ag<sup>+</sup> or AuCl<sup>−</sup> 4 ), incubated for 4 h and then centrifuged for 10 min at 9,000 g. The bacterial pellet was sent for thin sectioning and transmission electron microscopy (TEM) analysis (Advanced Microscopy Unit (AMU), at Pontificia Universidad Católica de Chile).

Formation of NS in vitro was carried out using 150µg/ml of purified recombinant glutathione reductase (GorA) from E. cloacae (isolate MF01). Additionally, crude extracts that contained GorA overexpressed for 2 h were used with reduction buffer (50 mM potassium phosphate pH 7.0, 1 mM NADPH, 1 mM <sup>β</sup>-mercaptoethanol and 0.15 mM TeO2<sup>−</sup> 3 or 1 mM SeO2<sup>−</sup> 3 ). In addition, crude extracts (200µg/ml of protein per reaction) from strains of interest were used for the production of NS by incubation with 1 mM Ag+, AuCl4, TeO2<sup>−</sup> 3 or 10 mM SeO2<sup>−</sup> 3 for 16 h in reduction buffer under optimal pH conditions and the preferred nicotinamide cofactor.

#### NS Characterization

A drop containing in vitro generated NS was placed on a copper grid and NS morphology was analyzed by TEM using a Hitachi Transmission Electron Microscope HT7700. Their chemical composition was determined through X-ray energy dispersion spectroscopy (EDS) using a SEM Zeiss EVO MA 10, EDS Penta FET Precision X-act Oxford Instruments. Both studies were conducted at the Center for the Development of Nanoscience and Nanotechnology–CEDENNA, USACH. In turn, the hydrodynamic diameter of the metallic nanostructures was assessed by dynamic light scattering (DLS) using a Marlvern Zetasizer Nano ZS equipment at room temperature (25◦C) as described by Arenas-Salinas et al. (2016). Final values were calculated based on three independent measurements of twenty repetitions. Furthermore, metal(loid)s were quantified using total reflection X-ray fluorescence spectrophotometry (TXRF). Briefly, in vitro-synthesized NS were centrifuged at 13,000 × g for 90 min and the sediment was suspended in 200 µl of 65% HNO3. Samples were heated at 60◦C ∼2 h and analyzed in a TXRF Bruker S2 PICOFOX Spectrometer at Bioinformatics and Gene Expression laboratory of the Institute of Nutrition and Food Technology (INTA, University of Chile).

#### NS Antibacterial Activity

A suspension of in vitro synthesized NS was used to challenge Escherichia coli and Listeria monocytogenes. The NS were dialyzed against 50 mM Tris-HCl buffer (pH 8.0 or 9.0) or 50 mM potassium phosphate (pH 7.0), accordingly to the buffer used to generate them. Dialysis was for 4 h at room temperature with constant shaking. The dialyzed material was maintained at 4◦C until further analysis. Concentrations of these metallic NS were calculated by determining the dry weight which was expressed in µg/ml. MIC determinations of the dialyzed NS and growth curves of E. coli BW25113 and L. monocytogenes were carried out as described in section Growth, Identification, and Characterization of the Environmental Strains.

## Data Analysis

Plots and statistical analyses were carried out using the GraphPad Prism 6.0 (GraphPad Software, Inc.). Analysis of variance (ANOVA) and t-test were used considering p < 0.05. The statistical significance was indicated as follows: <sup>∗</sup> , for values where p < 0.05, ∗∗, for values where p < 0.01, ∗∗∗, for values where p < 0.001 and ∗∗∗∗, for values where p < 0.000; ns, no significant difference.

## RESULTS

#### Isolation, Identification, and Characterization of Metal(loid) Resistant Bacteria

A total of 47 bacterial strains isolated from environmental samples were assessed for metal(loid) resistance. Cells were routinely grown in LB or LB-agar plates at 37◦C and exposed to different concentrations of 19 different, defined metal(loid)s. None of them grew in Cd2+-, Al3+-, or Mn2+ containing LB medium nor in R2A, Acidiphilium, or ATB culture media. **Table S1** shows the ability to grow in the presence of the indicated metal(loid)s; when reduced, some of them (e.g., Te, Se, Au, and Ag) showed a characteristic color that was monitored spectrophotometrically. In turn, while strain MF47 was the most sensitive to all metal(loid)s tested, strains MF45 and MF46 tolerated only Pb2<sup>+</sup> and Te4+, respectively.

Ten strains (MF01-MF10), including cocci, bacilli and shortbacilli, from which 21 and 26 were Gram negative and Gram positive, respectively (**Table S2**), were selected for further studies because of their ability to grow in the presence of 15, 12, 11, 11, 11, 9, 13, 9, 13, and 9 different metal(loid)s and/or to reduce them (**Table S1**). They belong to the Enterobacter (MF01 and MF04), Staphylococcus (MF02, MF07, and MF10), Acinetobacter (MF05 and MF09), and Exiguobacterium (MF03, MF06, and MF08) genera, as determined by 16S rRNA gene sequencing (**Figure S1**, **Table S2**). Their fine morphology was analyzed by scanning electron microscopy (**Figure 1**): MF02, MF07, and MF10 were coccoid, MF01, MF03, MF04, MF06, and MF08 showed bacillar forms and MF09 and MF05 were cocobacilli. Their physiological characteristics, as determined by biochemical tests, are listed in **Table 1**. Excepting MF03, all strains used citrate as carbon source, but none produced indole or hydrogen sulfide. All strains were oxidase negative and catalase positive and while MF05, MF06, MF07, MF08, MF09, and MF10 did not oxidize sugars at all, MF01 oxidized almost all sugars assayed but inositol.

## Metal(loid) Resistance and Reduction by the Environmental Strains

Minimal inhibitory concentrations of K2TeO3, AgNO3, HAuCl4, K2CrO4, CdCl2, NaAsO2, CuSO4, and Na2SeO<sup>3</sup> were determined under aerobic growth conditions for the strains listed in **Table S2**. MF02 and MF04 were the most resistant and sensitive to tellurite, respectively. All strains exhibited similar resistance levels to HAuCl4, CdCl2, AgNO3, and CuSO4, while MF01 and MF03 were the most sensitive to NaAsO2. Differences up to 500-fold in the MIC of K2CrO<sup>4</sup> were observed, MF02 and MF08 tolerating the highest concentration (400 mM). Finally, while MF01 and MF02 grew in the presence of Na2SeO<sup>3</sup> up to 500 mM, MF10 was the most sensitive isolate (3.125 mM). Then, the effect of sublethal concentrations of TeO2<sup>−</sup> 3 , AuCl<sup>−</sup> 4 and Ag<sup>+</sup> on bacterial growth was assessed (**Figure 2**). Although an extended lag phase was observed, cells regained growth between ∼4 and ∼7 h after TABLE 1 | Physiological characterization of metal(loid)-resistant strains.


+, positive; −, negative. Strains: (1) MF01, (2) MF02, (3) MF03, (4) MF04, (5) MF05, (6) MF06, (7) MF07, (8) MF08, (9) MF09, and (10) MF10.

exposure to the metalloid. Growth of MF01, MF04, and MF07 was completely abolished in the presence of tellurite.

On the other hand, crude cell extracts were assayed to assess tellurium (IV)-, selenium (IV)-, gold (III)-, and silver (I) reducing activity in the presence of NADH or NADPH and in the pH range 7–9 (**Figure 3** and **Figure S2**). The reduction of three metal(loid)s generated a change of the solution's color: black, purple and yellow-orange in the case of tellurite (**Figure S2A**), AuCl<sup>−</sup> 4 (**Figure S2B**) and Ag<sup>+</sup> (**Figure S2C**), respectively. While MF09 was the best tellurium (IV) reducer (pH 8-9 with NADH as cofactor, **Figure 3A**), selenium (IV) was better reduced by MF02 at pH 7.0 using NADPH as cofactor (**Figure 3B**). In turn, MF03 exhibited the best Ag (I)-reducing activity at pH 7.0 in the presence of NADH (**Figure 3C**). Finally, a robust gold-reducing activity was observed in MF09 at pH 8.0 with NADH as the enzyme cofactor (**Figure 3D**).

To look for proteins which could be responsible of metal(loid) reduction, tellurite- and gold- reducing activities were assessed in situ by polyacrylamide gel electrophoresis under non-denaturing conditions (**Figures S3A,B**). Reduction bands generated by MF01 extracts were excised from the gel and fractionated by SDS-PAGE (**Figure S3C**). Twelve bands were identified by MALDI-TOF (**Figure S3D**) which included the enzyme glutathione reductase (GorA), belonging to the flavoprotein family. The ability of GorA from the Antarctic strain Pseudomonas sp. BNF22 to reduce TeO2<sup>−</sup> 3 had been observed previously in our laboratory (Pugin et al., 2014). However, its ability to reduce other metal(loid)s and/or its efficiency has not yet been determined, which prompted us to focus on this phenomenon. The gorA gene was amplified from the E. cloacae genome and cloned into the pET/101 expression vector to generate the pET/gorA recombinant plasmid (**Figures S3E,F**). A kinetic induction assay showed a ∼50 kDa protein band that accumulated over time (**Figure S3G**). The enzyme was purified and assayed for tellurium (IV), selenium (IV), gold (III), silver (I), and copper (II) reduction, measured in the presence of NADH or NADPH and in the pH range 6–9. GorA reduced optimally TeO2<sup>−</sup> 3 , SeO2<sup>−</sup> 3 and Ag<sup>+</sup> at pH 8.0, 6–7 and 7, respectively (**Figure S3H**).

#### Synthesis and Characterization of Nanostructures

Ultrathin sections of selected strains that were exposed to sublethal concentrations (¼ of the MIC) of defined metal(loid)s were analyzed by transmission electron microscopy (**Figure 4**). Intracellular, evenly distributed long electron-dense

rods, representing most probably tellurium nanostructures (TeNS) were observed in tellurite-exposed MF09 (**Figure 4A**). In turn, SeNS generated by MF02 appeared as spherical particles located near the cell membrane (**Figure 4B**). In both cases, alterations in size, morphology and cell division were observed (not shown). No silver-containing particles were generated by MF03 under the same conditions (**Figure 4C**). Finally, small, circular and evenly distributed gold particles were seen when MF09 was treated with sub lethal gold concentrations (**Figure 4D**).

On the other hand, in vitro generation of NS by bacterial crude extracts lasted ∼16 h, irrespective of the strain or metal(loid) tested. NS morphology, size and chemical composition were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS) and total reflection X-ray fluorescence spectrophotometry (TRXF), respectively. TeNS synthetized using MF09 extracts showed heterogeneous morphology with an average diameter ∼10 nm (**Figure 5A**, left panel) and a hydrodynamic diameter ∼9.36 nm (polydispersity index PDI 0.446), in agreement with TEM results (**Figure 5A**, middle panel). The chemical composition was 23.2% tellurium, 55.8% carbon and low amounts of chlorine, potassium, and phosphorus (**Figure 5A**, right panel). Moreover, TXRF analysis indicated that the tellurium concentration in TeNS was 78.66 ± 7.51µg/ml. In the case of the SeNS generated with crude extracts from MF02, nanoparticles showed a spherical morphology with regular edges—nanospheres—and a size of ∼100 nm (**Figure 5B**, middle panel); they showed a gaussian distribution (DLS) with a maximum at 105.1 nm (PDI 0.124) (**Figure 5B**, middle panel). Selenium content in SeNS was 83.9%, while TRXF showed that Se concentration was 158.98 ± 16,4µg/ml (**Figure 5B**, right panel). On the other hand, AgNS generated using crude extracts from MF03 showed a size of ∼10 nm, spherical-like shape with irregular edges and an electron-dense core (**Figure 5C**, left panel). These AgNS also showed a gaussian distribution with a maximum centered at 68.46 nm (PDI 0.43) (**Figure 5B**, middle panel), a size larger than that assessed by TEM; AgNS contained mostly carbon and to a lesser extent silver (9.84 ± 1.05µg/ml, representing 20.7%) (**Figure 5C**, right panel). The same MF09 crude extract used to prepare TeNS

was utilized to generate AuNS, whose morphology was not easy to determine because of the presence of organic material surrounding the particles (**Figure 5D**, left panel). Nevertheless, these NS were rather spherical with a size ∼50 nm. AuNS showed a size distribution with a maximum at 82.35 nm (**Figure 5D,** middle panel). These nanoparticles contained 21.2% gold and other elements in lower amounts such as calcium, magnesium, chlorine, sulfur, and phosphorus (**Figure 5A**, right panel). TXRF analysis indicated that AuNS exhibited a gold concentration of 77.23 ± 12.61µg/ml.

Finally, crude extracts from GorA-overproducing E. coli as well as purified GorA were used to synthesize in vitro SeNS and TeNS (**Figure 6**): (i) both, crude extracts and purified protein were able to generate spherical SeNS with a size of ∼50 nm and irregular edges (TEM). DLS analysis indicated that GorA-synthesized NS are prone to aggregation with a size distribution of 74.75 nm (PDI 0.159) (**Figure 6A**); conversely NS obtained using crude extracts did not aggregate and showed a size of 39.09 nm (PDI 0.456) (**Figure 6C**). SeNS from crude extracts showed a higher amount of selenium (131.65 ± 16.46µg/ml, representing 29.9%) than GorA-SeNS (164.93 ± 24.7µg/ml, representing 26.6%) (**Figures 6A,C**); (ii) in turn, GorA-TeNS showed a rather stick-like morphology (**Figure 6B**), while nanoparticles synthetized using crude extracts exhibited irregular, non-homogenous morphology, with some of them showing triangular shapes (**Figure 6D**). Size distribution (DLS) of GorA- and crude extract-TeNS was 35.11 nm (PDI 0.32) and 31 nm (PDI 0.63), respectively (**Figures 6B,D**). TeNS from crude extracts showed a higher amount of tellurium (127.36 ± 15.26µg/ml, representing 10%) than GorA-TeNS

(157.6 ± 25.3µg/ml, representing 8.2%) (**Figure 6B,D**). Both crude extracts and purified protein showed the presence of other elements (more abundant in the GorA solution) such as potassium, sodium, sulfur, and phosphorus, among others.

#### NS Antibacterial Activity

The minimal inhibitory concentration of in vitro synthesized NS was determined and along with the construction of growth curves, were used to assess their putative antibacterial effects. Aerobically synthesized TeNS using MF09 crude extracts showed MICs of 45- and 66- µg/ml for E. coli and L. monocytogenes, respectively. Similar MIC values (40 and 82µg/ml, respectively) were observed for TeNS generated using GorA-overproducing E. coli crude extracts. In turn, AuNS MICs for E. coli and L. monocytogenes were 64- and 68- µg/ml, respectively. At the tested concentrations, both SeNS and AgNS did not inhibit E. coli or L. monocytogenes growth. However, Te- and Au-containing NS inhibited completely the growth of E. coli and L. monocytogenes (**Figure S4**).

## DISCUSSION

Environmental bacteria survive in the presence of toxic agents such as metal(loid)s because they have evolved specific mechanism to cope with them (Summers and Silver, 1978; Bollag et al., 1994). Specifically, bacterial metal(loid)-reduction has gained attention among researchers since naturally reducingor genetically modified- bacteria are widely used in the field of bioremediation (Stephen and Macnaughtont, 1999; Das et al., 2016) and biotechnology to generate nano-scaled structures exhibiting biomedical, electronic and pharmacological applications, among others (Suresh, 2012).

In this line, environmental bacteria from different Chilean regions were isolated, identified and characterized. Such regions exhibit a combination of many extreme factors like high salinity, desiccation, high and low temperatures, volcanic intervention, etc., most of them responsible for generating oxidative stress in organisms. Since metal(loid)s are also able to induce oxidative stress (Nies, 1999; Imlay, 2003), it was inferred that those places from where the samples were taken could host metal(loid)-resistant bacteria. The 10 most resistant to- and

(B) were generated using purified GorA, SeNS (C) and TeNS (D) were generated using crude extracts of E. coli overexpressing the gorA gene. Numbers are the average of 3 independent trials ± SD.

able of metal(loid)s reduction bacteria were chosen for further analyses because of their potential biotechnological applicability.

Their resistance level to 8 metal(loid)s was evaluated under aerobic conditions (**Table S2**). In this situation, most of these toxicants exert -at least in part- toxicity because of ROS production (mainly superoxide), causing the inactivation of certain enzymes and metabolic pathways, modification of nucleic acids and lipid peroxidation of the cell membrane (Ahearn et al., 1995; Calderón et al., 2009; Contreras and Vásquez, 2010; Lemire et al., 2013). High levels of tellurite resistance (4 mM) were observed for some isolates, while tellurite MIC did not exceed 0.004 mM for sensitive bacteria such as E. coli (Taylor, 1999). In the case of chromate and selenite, resistance levels observed for some isolates were up to ∼500- (MF02 and MF08) and ∼160-fold (MF01 and MF02) higher than that of other isolates, respectively. Interestingly, phylogenetically related strains (MF01 and MF04, MF09 and MF05, MF07 and MF10) exhibited different levels of resistance, probably because of the different environmental conditions where they thrive. Growth curves also showed that the isolates are differentially affected by these toxicants (**Figure 3**).

When reduced to their elemental state, tellurite, selenite, auric tetrachloride and silver generate colored, insoluble NS (**Figure S2**), which could serve as markers for putative biotechnological applications including medicine, electronics, pharmacy and engineering, among others (Ba et al., 2010; Suresh, 2012; Oves et al., 2013). Most strains exhibited metal(loid) reducing ability (**Figure 3**, **Figure S2**), whose optimal reduction condition -pH range and enzyme cofactor among others- was determined. MF09 showed maximal tellurite reduction at pH 8 and 9 using NADH as electron donor (**Figure 3A**). This could be explained because, in general, redox centers of proteins exhibiting tellurite reductase activity contain reactive vicinal cysteines that are very sensitive to pH (Arenas et al., 2014b). Deprotonation of thiol groups of these cysteines generates the thiolate anion (-S−, pKa ∼8.0) which is highly reactive (Vlamis-Gardikas, 2008). This high pKa implies that most cysteine thiols are protonated at pH 7.0 and hence, inactive.

Similar results were observed during AuCl<sup>−</sup> 4 reduction by MF09 at pH 8.0 in the presence of NADH (**Figure 3D**). In turn, optimal selenite and silver reduction by MF03 and MF02, respectively, occurred at pH 7.0. Particularly, while Se<sup>0</sup> is slowly oxidized to SeO2<sup>−</sup> 4 in alkaline conditions (Kamal, 1994), SeO2<sup>−</sup> 3 is reduced at more acidic pH values, thus favoring the formation elemental Se. On the other hand, pH influences metal(loid) speciation thus causing the formation of complexes and the deprotonation/protonation of functional groups that may participate in the catalytic process (Panda and Deepa, 2011). Also, pH can result in precipitation of defined ions (Atkins and Jones, 2006). For instance, Ag<sup>+</sup> precipitates as silver hydroxide in alkaline conditions and is spontaneously transformed into the more stable, insoluble, silver oxide (Ag2O) (Burriel et al., 2006).

Next, the in vivo generation of tellurium-, selenium-, silver-, and gold-containing NS was evaluated using three of the environmental strains under study (**Figure 4**). TeNS generated by MF09 exhibited an elongated morphology, a situation that has been previously reported in Rhodobacter capsulatus (Turner et al., 2011). On the other hand, MF02-generated SeNS showed spherical morphology and a size >100 nm (**Figure 4B**). Actually, SeNS produced by most microorganisms exhibit spherical morphology and in some cases, transformation from spheres to nanowires do occur (Mane et al., 2015). In turn, MF03 did not form AgNS (**Figure 4C**), probably because Ag<sup>+</sup> interacts with- and precipitate with a large number of intra- or extracellular anions (thus decreasing its availability to form NS) (Atkins and Jones, 2006); such is the case when PO3<sup>−</sup> 4 present in the culture medium form stable compounds such as Ag3PO<sup>4</sup> (Xiu et al., 2011). MF09- generated AuNS were observed as small intracellular deposits evenly distributed in the cell (**Figure 4D**). It general, AuNS synthesis is directly dependent on the microorganism and pH of the medium. In this line, marine yeasts form large-sized AuNPs at acidic pH values, while it reduced at alkaline pHs; conversely, fungi belonging to the Verticillium genus form larger AuNPs at alkaline pH values (Dhillon et al., 2012).

It should be noted that the molecular basis of the in vivo metal(loid)-reducing activity is very hard to determine accurately since a large number of factors that are present in the cell context may affect and/or influence this activity; this makes almost impossible to correlate directly in vitro activity and the generation of metal(loid) deposits in vivo and variables such as culture medium, toxicant exposure time, temperature, oxygen availability, among others, should be explored further.

The ability of crude extracts to generate metal(loid) containing NS was used to characterize NS synthesis in vitro, including their morphology, size, composition and metal(loid)concentration (**Figure 5**). All the formed structures exhibited sizes <100 nm and hence, fall in the definition of nanostructures, i.e., at least one dimension does not exceed 100 nm (Suresh, 2012). MF09 crude extracts formed TeNS that were smaller in size than AuNS (**Figures 5A,D**). An important factor that affects NS morphology and size is related to the formation of the NS itself, where NS nucleation and growth are limiting steps for these NS characteristics (Xiong and Lu, 2015). Both TeNS and AuNS contain a high proportion of the respective metal(loid), 22.3 and 21.2%, respectively, in addition to other elements such as carbon. In particular, TeNS exhibited small sizes and poorly defined morphologies. Unfortunately, the presence of organic material hampered the correct visualization of AuNS. This did not affect SeNS observation, which showed a rather spherical morphology with poorly defined edges, and contained as much as 83.9% Se (**Figure 5B**). This result is consistent with the high Se concentration determined by TXRF (158.98 ± 16.4µg/ml). Different was the case of AgNS, which contained lower metal abundance (**Figure 5C**) and is probably due to the presence of phosphate in the reaction buffer (see above).

Lastly, NS were also generated using purified GorA from E. cloacae (>92% pure) as well as crude extracts of a recombinant E. coli overexpressing the E. cloacae gorA gene (**Figure 6**). Given that it is a relatively new procedure, not much information is available in the literature. This flavoprotein, which was identified and characterized in this work, exhibits the ability to efficiently reduce SeO2<sup>−</sup> 3 and TeO2<sup>−</sup> 3 (**Figure S3H**). TeNS synthesized using crude extracts showed a rather triangular shape, while those generated by the purified protein were slightly larger size, irregular and exhibited a more spherical morphology (**Figures 6B,D**). As mentioned, difference in size and morphology could be attributed to the influence of the cellular environment on the metal(loid)-reducing efficiency.

Intracellular tellurite reduction could occur also because the participation of reductants such as gluthatione (GSH) and other cytosolic thiols (painter-type reactions) whose reducing ability would promote Te<sup>0</sup> formation (Chasteen et al., 2009). Given that GorA is responsible for appropriate GSH levels, it is tempting to speculate that TeNS could be formed more efficiently when it is overproduced. This situation could explain why TeNS generated by crude extracts of GorA-overproducing E. coli are larger than those generated by the purified protein. No differences were observed regarding the Te content in TeNS. Actually, elemental analyses showed that Te was present in lower amount regarding other elements such as carbon, oxygen and phosphorus. Based on the stoichiometry of Te and O (oxygen percent is at least twice that of Te) in TeNS generated under aerobic conditions either by GorA or crude extracts, it could be inferred that in NS tellurium is present as tellurium dioxide (TeO2). Nevertheless, additional experimental evidence is required to state it definitely. It has been observed lately that GorA-generated TeNS remain attached to the enzyme, probably through covalent bonding with the active site catalytic cysteine residues (Pugin et al., 2014). Unlike TeNS, SeNS generated using crude extracts of GorA-overproducing cells were smaller than those produced by purified GorA (**Figures 6A,C**). This could be explained by some kind of electron leak, thus making selenite reduction less efficient (Zare et al., 2013; Mane et al., 2015). However, Se concentration was about the same in SeNS synthesized in both conditions.

Given that antibiotic-resistant, pathogenic bacteria have increased almost exponentially worldwide, the development of tools from biotechnological origin for biomedical applications is mandatory (Yacoby and Benhar, 2008). In this work, the antibacterial activity of the in vitro-generated NS was tested against E. coli and L. monocytogenes. TeNS made by crude extracts of GorA-overproducing E. coli were the most toxic for E. coli and L. monocytogenes. Similar results were observed with MF09-generated AuNS and TeNS (**Figure S4**). The effect of NS on E. coli and L. monocytogenes growth was also assessed by constructing growth curves. Results from **Figure S4** show that in general all NS inhibited completely the bacterial growth.

Toxic effects of NS are generally related to the specific particle. Thus, AuNS have been widely used in therapeutic as well as diagnostic procedures, and exhibit antibacterial activity against Salmonella sp., Bacillus subtilis, E. coli and Pseudomonas aeruginosa (Das et al., 2009). In particular, chemically-generated AuNS seem to exert their toxicity in two ways: (i) changing membrane potential and inhibiting ATP synthase activity, which results in a general turn down of metabolism and (ii) inhibiting the binding of tRNAs to the ribosome, thus generating a global collapse of biological processes. Other NS are toxic because the release of metal ions, as in the case of some AgNS; in fact, Ag<sup>+</sup> release causes damage to the cell membrane, inactivates proteins and inhibits DNA replication (Chaloupka et al., 2010; Xiu et al., 2012).

Our results show that in general all tested NS were more toxic for E. coli as compared to L. monocytogenes, which, as a Gram positive organism, possess a cell wall that probably retards or prevents the entry of NS. In this context, no information on the mechanism of NS uptake is available in the literature. Unlike TeNS and AuNS, in vitro-synthesized SeNS and AgNS did not inhibit bacterial growth (not shown). Results with SeNS could be explained because selenium is an essential element for microorganisms (Lemire et al., 2013), and probably higher Se amounts in NS would be needed to generate toxicity. It has been also shown that SeNS inhibits both growth and proliferation of cancer cells in culture (Selenius et al., 2010). On the other hand and although AgNS have been reported as the most toxic NS (Rai et al., 2009; Lin et al., 2012), they did not exhibit toxicity for the tested bacteria.

Finally, it seems very interesting that the same enzyme can reduce two metalloids possessing different chemical characteristics, as for instance, their reactivity with certain enzymes (Rigobello et al., 2011). We trust that the results of this work will help to define the molecular basis governing the biological synthesis of many potentially useful nanostructures.

#### CONCLUSION

Ten bacteria exhibiting resistance to various metals and metalloids were isolated and characterized from environmental samples. Whole cells as well as crude extracts derived from them were successfully used for the biological synthesis of TeNS, SeNS, AuNS, and AgNS, which exhibited different, specific characteristics. In particular, E. cloacae was used as model to look for toxicant-reducing enzymes. In this context, it was found that purified E. cloacae GorA reduced efficiently TeO2<sup>−</sup> 3 and SeO2<sup>−</sup> 3 . Finally, especially interesting were TeNS and AuNS, which exhibited antibacterial properties and inhibited E. coli and L. monocytogenes growth.

#### AUTHOR CONTRIBUTIONS

MF, VF, MA-S, CM-V, FC, EM, DA, CV, and FA: Conceived and designed the experiments; MF, VF, EV, ML, DA, FC, MA-S, and CM-V: Performed the experiments; MF, VF, DA, MA-S, EM, ML, EV CM-V, CV, and FA: Analyzed the data; MA-S, CV, ML, and FA: Contributed reagents, materials, analysis tools; CV and FA: Wrote the paper.

## FUNDING

This work received financial support from FONDECYT (Fondo Nacional de Ciencia y Tecnología) Iniciación en la Investigación #11140334 (FA), #11150679 (ML), and Regular #1160051 (CV), Support from DICYT (Dirección de Investigación en Ciencia y Tecnología, Universidad de Santiago de Chile), Basal FB0807 (CEDENNA) (EV) and Universidad de Talca (Fondo De Proyectos De Investigación Para Investigadores Iniciales) (MA-S) is also acknowledged.

#### ACKNOWLEDGMENTS

We would like to thank Natalia Valdes from the Universidad de Santiago de Chile, Facultad de Química y Biología for her support in bioinformatics analysis. Also Yerko Argandoña from the Universidad de Talca, for support in electron microscopy. In addition, to Dr. Rivas-Pardo and Mrs. Paulina Ramirez of Columbia University for his critical review of the manuscript. This paper is dedicated in memory of my daughter María Ignacia Arenas Monterey, our little angel.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00959/full#supplementary-material

Figure S1 | Phylogenetic trees based on the 16S rRNA gene sequence. Trees were constructed using the neighbor-joining method as described in Methods. (A) Exiguobacterium, (B) Enterobacter, (C) Acinetobacter, and (D) Staphylococcus. The numbers on the nodes represent the percentage of bootstrap (1,000 repetitions).

Figure S2 | in vitro metal(loid)- reduction by crude extracts of the indicated strains. The reduction of TeO2<sup>−</sup> 3 (A), AuCl<sup>−</sup> 4 (B), and Ag+(C) was performed as described in Methods. The activity was measured at different pH values and monitored for 24 h. Strains analyzed: (1) MF01, (2) MF02, (3) MF03, (4) MF04, (5) MF05, (6) MF06, (7) MF07, (8) MF08, (9) MF09, (10) MF10.

Figure S3 | Identification, cloning, and purification of GorA. Tellurite (A) and gold (B) reductase activity in situ. Crude extracts from each strain [MF01 (1), MF02 (2), MF03 (3), MF04 (4), MF05 (5), MF06 (6), MF07 (7), MF08 (8), MF09 (9), MF10(10)] were fractionated by NATIVE-PAGE and revealed as TeO2<sup>−</sup> 3 or AuCl<sup>−</sup> 4 activity as described in Methods. (C) SDS-PAGE of the reduction bands of TeO2<sup>−</sup> 3 and AuCl<sup>−</sup> 4 from MF01 extracts; Lanes 1, 2, and 5: molecular weight standards (BioRad, #1610305). Lanes 3 and 4: fractionation of proteins present in the reduction band of TeO2<sup>−</sup> 3 and AuCl<sup>−</sup> 4 , respectively. (D) Identification of protein bands by MALDI-TOF. (E) Agarose gel electrophoresis of PCR-amplified gorA; Lane 1, molecular size standards (1 kb DNA ladder, Promega). Lane 2, amplification of gorA. (F) Agarose gel electrophoresis of gorA amplification by PCR. Lane 1, molecular size standards (1 kb DNA ladder, Invitrogen); lane 2, colony PCR amplification of gorA; lane 3, directional cloning of gorA assessed by PCR using the primers Gforward and pETReverse described in section Growth, identification and characterization of the environmental strains; (G) Induction kinetics and purification of GorA; SDS-PAGE of crude extracts of E. coli overexpressing gorA and the purified enzyme; ST, SDS-PAGE molecular weight standards (low range). Lanes 1-6, induction kinetics for GorA at 0, 1, 2, 3, 4, and 16 h, respectively. Lane 7, purified recombinant GorA. (H) GorA

metal(loid)-reductase activity (U/mg protein), showing the dependence on the pH and cofactor.

Figure S4 | Growth curves of E. coli and L. monocytogenes exposed to NS. Effect of TeNS synthesized by crude extracts of cells overexpressing the E. cloacae gorA gene in E. coli (A) and L. monocytogenes (B). Effect of TeNS synthesized using crude extracts of MF09 in E. coli (C) and L. monocytogenes

#### REFERENCES


(D). Effect of AuNS synthesized by crude extracts of MF09 in E. coli (E) and L. monocytogenes (F). Black and blue lines represent the growth of control and NS-treated cells, respectively.

Table S1 | Metal(loid)-resistant strains.

Table S2 | MICs for the metal(loid)-resistant strains.


biofabricated from chromium reducing novel OS4 strain of Stenotrophomonas maltophilia. PLoS ONE 8:e59140. doi: 10.1371/journal.pone.0059140


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Figueroa, Fernandez, Arenas-Salinas, Ahumada, Muñoz-Villagrán, Cornejo, Vargas, Latorre, Morales, Vásquez and Arenas. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB

Blas Blázquez† , Manuel Carmona and Eduardo Díaz\*

Department of Microbial and Plant Biotechnology, Centro de Investigaciones Biológicas–Consejo Superior de Investigaciones Científicas, Madrid, Spain

#### Edited by:

Kian Mau Goh, Universiti Teknologi Malaysia, Malaysia

#### Reviewed by:

Maximino Manzanera, University of Granada, Spain Victor Ladero, Instituto de Productos Lácteos de Asturias (CSIC), Spain

> \*Correspondence: Eduardo Díaz ediaz@cib.csic.es

#### †Present address:

Blas Blázquez, Systems Biology Programme, Centro Nacional de Biotecnología–Consejo Superior de Investigaciones Científicas, Madrid, Spain

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

> Received: 08 January 2018 Accepted: 06 March 2018 Published: 22 March 2018

#### Citation:

Blázquez B, Carmona M and Díaz E (2018) Transcriptional Regulation of the Peripheral Pathway for the Anaerobic Catabolism of Toluene and m-Xylene in Azoarcus sp. CIB. Front. Microbiol. 9:506. doi: 10.3389/fmicb.2018.00506 Alkylbenzenes, such as toluene and m-xylene, are an important class of contaminant hydrocarbons that are widespread and tend to accumulate in subsurface anoxic environments. The peripheral pathway for the anaerobic oxidation of toluene in bacteria consists of an initial activation catalyzed by a benzylsuccinate synthase (encoded by bss genes), and a subsequent modified β-oxidation of benzylsuccinate to benzoyl-CoA and succinyl-CoA (encoded by bbs genes). We have shown here that the bss and bbs genes, which are located within an integrative and conjugative element, are essential for anaerobic degradation of toluene but also for m-xylene oxidation in the denitrifying beta-proteobacterium Azoarcus sp. CIB. New insights into the transcriptional organization and regulation of a complete gene cluster for anaerobic catabolism of toluene/m-xylene in a single bacterial strain are presented. The bss and bbs genes are transcriptionally coupled into two large convergent catabolic operons driven by the PbssD and PbbsA promoters, respectively, whose expression is inducible when cells grow anaerobically in toluene or m-xylene. An adjacent tdiSR operon driven by the PtdiS promoter encodes a putative two-component regulatory system. TdiR behaves as a transcriptional activator of the PbssD, PbbsA, and PtdiS promoters, being benzylsuccinate/(3-methyl)benzylsuccinate, rather than toluene/m-xylene, the inducers that may trigger the TdiS-mediated activation of TdiR. In addition to the TdiSR-based specific control, the expression of the bss and bbs genes in Azoarcus sp. CIB is under an overimposed regulation that depends on certain environmental factors, such as the presence/absence of oxygen or the availability of preferred carbon sources (catabolite repression). This work paves the way for future strategies toward the reliable assessment of microbial activity in toluene/m-xylene contaminated environments.

Keywords: toluene, m-xylene, Azoarcus, anaerobic degradation, tdiSR, catabolite repression, biomarker

#### INTRODUCTION

Aromatic compounds such as alkylbenzenes, e.g., toluene, xylenes, ethylbenzene and benzene, are an important class of contaminants that are prominently placed amongst the US Agency for Toxic Substances and Disease Registry's list of priority pollutants because of their carcinogenic and/or neurotoxic effects to humans. Since alkylbenzenes are relatively soluble and mobile in water, they are widespread and tend to accumulate in hostile environments, e.g., subsurface

anoxic environments, where they are of major concern to groundwater quality and ecosystem health. Therefore, the anaerobic degradation of alkylbenzenes is an important practical aspect of bioremediation and becomes crucial for the biogeochemical cycles (Kummel et al., 2013; Rabus et al., 2016; Lueders, 2017). Despite the aerobic degradation of alkylbenzenes has been extensively studied, the microbial catabolism of these aromatic hydrocarbons under anaerobic conditions is much less known, and novel pathways and enzymes have been recently described, some of which are of potential biotechnological interest (Rabus et al., 2016; Lueders, 2017). Toluene has been used widely as a model compound for studying anaerobic alkylbenzene degradation. Toluene can be degraded coupled to anaerobic respiration with nitrate, sulfate, iron (III), manganese (IV) or carbonate serving as terminal electron acceptors (Foght, 2008; Carmona et al., 2009; Rabus et al., 2016). Geobacter metallireducens GS-15 was the first pure bacterial culture described to be able to degrade toluene in anaerobic conditions (Lovley et al., 1989). Several isolates capable of anaerobic toluene degradation have been described since then, including both facultative anaerobes, e.g., beta-proteobacteria of the Azoarcus, "Aromatoleum," Thauera, Georgfuchsia, Herminiimonas genera, and alpha-proteobacteria of the Magnetospirillum genus, and obligate anaerobes, e.g., delta-proteobacteria of the Geobacter, Desulfobacula and Desulfobacterium genera, and some clostridial strains (Wöhlbrand et al., 2013; Bozinovski et al., 2014; Kim et al., 2014; Strijkstra et al., 2014; Rabus et al., 2016; Lueders, 2017; Meyer-Cifuentes et al., 2017). Most of the studies on anaerobic toluene degradation have been made on the denitrifying bacteria Thauera aromatica K172 and T1 strains, Azoarcus sp. strain T and "Aromatoleum aromaticum" EbN1 strain (Coschigano and Young, 1997; Leuthner et al., 1998; Krieger et al., 1999; Achong et al., 2001; Hermuth et al., 2002; Leutwein and Heider, 2002; Chakraborty and Coates, 2004; Coschigano and Bishop, 2004; Kube et al., 2004; Kühner et al., 2005).

The first step in the anaerobic catabolism of toluene is the addition of the methyl group of toluene to fumarate to form (R) benzylsuccinate by the strictly anoxic benzylsuccinate synthase (BSS), a glycyl radical enzyme (**Figure 1A**). The genes bssA, bssB and bssC code for the α (BssA), β (BssB) and γ (BssC) subunits of the heterohexameric (αβγ)<sup>2</sup> BSS (Leuthner et al., 1998; Krieger et al., 2001; Bhandare et al., 2006; Funk et al., 2014; Heider et al., 2016). Conversion to the active, radical-containing form of BSS depends on an activating enzyme (BssD) that belongs to the family of S-adenosyl-methionine radical enzymes and that is encoded by a gene (bssD) which is closely associated with the bssABC genes constituting the bssDCAB operon of toluenedegrading bacteria (**Figure 2**) (Selmer et al., 2005; Heider et al., 2016). The bss operons contain two additional conserved genes, bssE and bssF (**Figure 2**), whose functions are still unknown. Whereas BssE is an essential protein for toluene degradation (Bhandare et al., 2006), the role of BssF in the anaerobic oxidation of toluene has not yet been explored. Moreover, in beta-proteobacteria the bss cluster contains a set of additional genes, i.e., the bssGIJKL genes (**Figure 2**), whose function is so far unknown. Despite the primary structure of the gene products is conserved in the bss clusters from different denitrifying bacteria, the transcriptional organization of the bss clusters differs between species and even between strains of the same species (Coschigano, 2000; Achong et al., 2001; Kube et al., 2004).

Further anaerobic degradation of toluene consists of a modified beta-oxidation of (R)-benzylsuccinate to benzoyl-CoA and succinyl-CoA (**Figure 1A**). All enzymes of this pathway are encoded by the bbs gene cluster (**Figure 2**) (Leuthner and Heider, 2000; Leutwein and Heider, 2001, 2002; Kube et al., 2004). Like in the bss gene cluster, the bbs cluster contains genes, e.g., bbsI and bbsJ, of unknown function (Carmona et al., 2009). Although the gene organization of the bbsABCDEFJGH cluster is usually conserved except in some delta-proteobacteria (**Figure 2**) (Kube et al., 2004; Butler et al., 2007; Chaurasia et al., 2015), the transcriptional organization of a bbs cluster has not been studied so far in any bacteria.

Upstream of the bss cluster in most denitrifying bacteria there are two genes, tdiS and tdiR (toluene-degradation inducer) (**Figure 2**) that were proposed to encode the sensor histidine kinase and the cognate response regulator, respectively, of a two-component regulatory system likely involved in the transcriptional control of the catabolic bss and bbs genes (Coschigano, 2000; Achong et al., 2001; Hermuth et al., 2002; Kube et al., 2004; Kühner et al., 2005). Extracts of Escherichia coli cells that expressed the tdiR gene from T. aromatica K172 were able to retard the migration of a DNA probe that contained the bssD promoter, supporting the notion that TdiR likely behaves as a transcriptional regulator of the bss genes (Leuthner and Heider, 1998). Nevertheless, a clear experimental demonstration that TdiS and TdiR control the expression of both bss and bbs genes in beta-proteobacteria is still missing.

Unlike toluene degradation, anaerobic degradation of xylenes (meta-, ortho-, and para-xylene) has been much less studied. The first reaction of the m-xylene degradation pathway is performed by the BSS enzyme that catalyzes the addition of fumarate to the methyl group to form (3-methyl)benzylsuccinate (**Figure 1A**) (Krieger et al., 1999; Verfürth et al., 2004). The (3 methyl)benzylsuccinate was also detected as a key intermediate of the fumarate addition pathway in xylene-degrading sulfatereducing cultures (Morasch et al., 2004; Herrmann et al., 2009). On the other hand, it has been proposed that the bbs gene products might be responsible of the β-oxidation of (3 methyl)benzylsuccinate to 3-methylbenzoyl-CoA and succinyl-CoA in a similar way than in the catabolism of toluene (**Figure 1A**) (Krieger et al., 1999; Bozinovski et al., 2014), although there is not yet a genetic demonstration that bbs genes are responsible for anaerobic m-xylene degradation.

Azoarcus sp. CIB is facultative anaerobic bacterium able to grow aerobically in toluene and anaerobically (nitrate-reducing) in toluene and m-xylene (López-Barragán et al., 2004). Moreover, Azoarcus sp. CIB also shows an endophitic lifestyle (Fernández et al., 2014) and is able to resist some metals and metalloids (Fernández-Llamosas et al., 2016). The strain CIB has been used previously to decipher the transcriptional organization and regulation of several gene clusters involved in the anaerobic degradation of aromatic compounds and in the resistance strategies to survive in the presence of high concentrations of these contaminants (López-Barragán et al., 2004; Blázquez et al.,

2008; Carmona et al., 2009; Valderrama et al., 2012, 2014; Juárez et al., 2013; Juárez et al., 2015; Martín-Moldes et al., 2016; Zamarro et al., 2016). An in silico analysis of the Azoarcus sp. CIB genome sequence revealed the presence of a chromosomal region within the integrative and conjugative ICEXTD element coding for proteins with significant amino acid sequence identity with Bss and Bbs proteins in closely related bacteria from the Azoarcus, "Aromatoleum" and Thauera genera (Martín-Moldes et al., 2015; Zamarro et al., 2016). This sequence comparison analysis allowed us to propose a similar biochemical pathway for the anaerobic conversion of toluene and m-xylene to benzoyl-CoA and 3-methylbenzoyl-CoA, respectively, in strain CIB (**Figure 1**). Further degradation of benzoyl-CoA and 3-methylbenzoyl-CoA in Azoarcus sp. CIB proceeds via the central bzd (stands for benzoate degradation) and mbd (stands for methylbenzoate degradation) pathways, respectively (López-Barragán et al., 2004; Juárez et al., 2013). In this work we have used Azoarcus sp. CIB to study for the first time the transcriptional organization and regulation of the complete bss-bbs cluster in a facultative anaerobe. Our results reveal that both the bss and bbs genes are essential for the anaerobic degradation of toluene and m-xylene.

#### MATERIALS AND METHODS

#### Bacterial Strains, Plasmids and Growth Conditions

The E. coli and Azoarcus strains, as well as the plasmids used in this study, are detailed in **Table 1**. E. coli strains were grown at 37◦C in Lysogeny Broth (LB) mediun (Bertani, 1951). Azoarcus sp. strain CIB and its derivatives were grown anaerobically under nitrate reducing conditions (10 mM nitrate) at 30◦C in MC medium as previously described (López-Barragán et al., 2004). Aromatic hydrocarbons such as toluene, xylenes, styrene were supplied at 250 mM in an inert carrier phase of 2,2,4,4,6,8,8 heptamethylnonan (HMN). Benzylsuccinate was added at 4 mM to the culture medium as inducer. Organic acids such as succinate or pyruvate were added at 0.2% (w/v). Azoarcus sp. CIB cells were also cultivated aerobically in MC medium in the absence of nitrate. When using toluene aerobically as sole carbon source, it was added directly to the culture medium at 1 mM. Where appropriate, antibiotics were added to the culture medium at the following concentrations: ampicillin, 100 µg/ml; gentamicin, 10 µg/ml; kanamycin, 50 µg/ml. Growth was determined by measuring the optical density at 600 nm (OD600) in a Shimadzu UV-260 spectrophotometer.

## Molecular Biology Techniques

Standard molecular biology techniques were performed as previously described (Sambrook and Russell, 2001). DNA fragments were purified with Gene-Clean Turbo (BIO101 Systems); plasmids and PCR products were purified with a High Pure Plasmid and PCR Product Purifications kits (Roche), respectively. Oligonucleotides were supplied by Sigma Co. All cloned inserts and DNA fragments were confirmed by DNA sequencing with fluorescently labeled dideoxynucleotide terminators (Sanger et al., 1977) and AmpliTaq FS DNA polymerase (Applied Biosystems) in an ABI Prism 377 automated DNA sequencer (Applied Biosystems). Transformation of E. coli cells was carried out by using the RbCl method or by

electroporation (Gene Pulser, Bio-Rad) (Sambrook and Russell, 2001). Plasmids were transferred from E. coli S17-1λpir (donor strain) into Azoarcus sp. recipient strains by biparental filter mating as described previously (López-Barragán et al., 2004).

#### Construction of Azoarcus sp. CIBdtdiR, Azoarcus sp. CIBdtdiS, Azoarcus sp. CIBdbssD, Azoarcus sp. CIBdbssF, Azoarcus sp. CIBdbssJ, and Azoarcus sp. CIBdbbsB Strains

For insertional disruption of the genes through single homologous recombination, an internal region of each gene was PCR-amplified, cloned into plasmid pGEM-T Easy, and subcloned into the pK18mob suicide vector (**Table 1**). For gene disruption of the tdiR gene, a 0.4-kb internal fragment was PCRamplified using primers tdiRint5 and tdiR131.5 (Supplementary Table S1), and cloned in plasmid pGEM-tdiR (**Table 1**). The pGEM-tdiR plasmid was XbaI/PstI double-digested and the 0.4-kb fragment was then subcloned giving rise to the suicide plasmid pK18mobtdiR (**Table 1**). For gene disruption of the tdiS gene, a 0.6-kb internal fragment was PCR amplified using primers 5TdiS and 3TdiS (Supplementary Table S1), and cloned in plasmid pGEM-tdiS (**Table 1**). The pGEM-tdiS plasmid was EcoRI digested and the 623-bp fragment was then subcloned producing plasmid pK18mobtdiS (**Table 1**). The tdiS mutant allows expression of the downstream tdiR gene by transcription from the promoter of the kanamycin resistance gene present in the suicide plasmid. For gene disruption of the bssD gene, a 0.6-kb internal fragment was PCR amplified using primers 5BssD and 3BssD (Supplementary Table S1), and cloned in plasmid pGEM-bssD (**Table 1**). The pGEM-bssD plasmid was PstI/BamHI double-digested and the 593-bp fragment was then subcloned into pK18mob producing plasmid pK18mobbssD (**Table 1**). For gene disruption of the bssF gene, a 0.4-kb internal fragment was PCR-amplified using primers 5BssF and 3BssF (Supplementary Table S1) and cloned in plasmid pGEM-bssF (**Table 1**). The pGEM-bssF plasmid was HindIII/XbaI doubledigested and the 451-bp fragment was then subcloned giving rise to the suicide plasmid pK18mobbssF (**Table 1**). For gene disruption of the bssJ gene, a 0.3-kb internal fragment was PCR amplified using primers 5BssJ and 3BssJ (Supplementary Table S1) and cloned in plasmid pGEM-bssJ (**Table 1**). The TABLE 1 | Bacterial strains and plasmids used in this work.

fmicb-09-00506 March 20, 2018 Time: 16:29 # 5


<sup>a</sup>The abbreviations used are: T+, anaerobic growth in toluene; X+, anaerobic growth in m-xylene; T−, lack of anaerobic growth in toluene; X−, lack of anaerobic growth in m-xylene; Ap<sup>r</sup> , ampicillin-resistant; Gm<sup>r</sup> , gentamicin-resistant; Km<sup>r</sup> , kanamycin-resistant; Sm<sup>r</sup> , streptomycin-resistant.

pGEM-bssJ plasmid was PstI/SalI double-digested and the 347-bp fragment was then subcloned producing plasmid pK18mobbssJ (**Table 1**). For gene disruption of the bbsB gene, a 0.5-kb internal fragment was PCR amplified using primers 5BbsB and 3BbsB, and cloned in plasmid pGEM-bbsB (**Table 1**). The pGEM-bbsB plasmid was PstI/BamHI double-digested and the 512-bp fragment was then subcloned producing plasmid pK18mobbbsB (**Table 1**). The derivative pK18mob plasmids were then transferred from E. coli S17-1λpir (donor strain) into Azoarcus sp. CIB (recipient strain) by biparental filter mating as previously described (de Lorenzo and Timmis, 1994; López-Barragán et al., 2004). Exconjugant Azoarcus sp. CIB mutant strains harboring the disrupted genes by insertion of the corresponding suicide plasmids, were isolated aerobically on kanamycin-containing MC medium lacking nitrate and containing 0.2% citrate as the sole carbon source for counter-selection of donor cells. The mutant strains were analyzed by PCR to confirm the disruption of the target genes.

#### Construction of lacZ Translational Fusions

To construct plasmid pSJPbbsA that harbors the PbbsA::lacZ translational fusion, a 356-bp XbaI/PstI DNA fragment containing the bbsA (AzCIB\_4526)-orfB (AzCIB\_4527) intergenic region was PCR-amplified from Azoarcus sp. CIB chromosomal DNA by using oligonucleotides 5PbbsA and 3PbbsA (5<sup>0</sup> -AACTG CAGGACATGACGCCTCCGCAGCATTTG-3<sup>0</sup> (Supplementary Table S1), and then cloned into the XbaI/PstI double-digested pSJ3 plasmid (**Table 1**). Plasmid pBBRPbbsA was constructed by subcloning a 3.5-kb EcoRI/HindIII fragment harboring the PbbsA::lacZ translational fusion from pSJPbbsA into the EcoRI/HindIII double-digested broad-host range plasmid pBBR5T (**Table 1**). To construct plasmid pBBRPbssD that

carries the PbssD::lacZ fusion, a 335-bp SpeI/BamHI DNA fragment containing the tdiS-bssD intergenic region was PCRamplified from Azoarcus sp. CIB chromosomal DNA by using oligonucleotides 5PtdiS and 3PbssD (Supplementary Table S1), and then cloned into the SpeI/BamHI double-digested pBBRPbbsA (**Table 1**). To construct plasmid pSJ3PtdiS that carries the PtdiS::lacZ translational fusion, a 314-bp BamHI fragment containing the bssD-tdiS intergenic region was PCR-amplified from Azoarcus sp. CIB chromosomal DNA by using oligonucleotides 3PbssD and PtdiS3'(Supplementary Table S1), and then cloned into the BamHI digested pSJ3 plasmid (**Table 1**). Plasmid pBBRPtdiS was constructed by subcloning a 3.5-kb EcoRI/HindIII DNA fragment harboring the PtdiS::lacZ fusion from pSJ3PtdiS into the EcoRI/HindIII double-digested broad-host range plasmid pBBR5T (**Table 1**).

#### RNA Extraction and Reverse Transcription-PCR Experiments

Azoarcus sp. CIB cells grown in MC medium containing the appropriate carbon sources were harvested at the midexponential phase of growth and stored at −80◦C. Azoarcus sp. CIBdbbsB and CIBdbssD strains grown in kanamycin-containing MC medium with pyruvate plus toluene were harvested at the stationary phase of growth and stored at −80◦C. Pellets were thawed, and cells were lysed in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) containing 50 mg ml−<sup>1</sup> lysozyme. Total RNA was extracted using the RNeasy mini kit (Qiagen), including a DNase treatment according to the manufacturer instructions (Ambion), precipitated with ethanol, washed, and resuspended in RNase-free water. The concentration and purity of the RNA samples were measured by using a ND1000 Spectrophotometer (Nanodrop Technologies) according to the manufacturer's protocols. Synthesis of total cDNA was carried out with 20 µl of reverse transcription reactions containing 400 ng of RNA, 0.5 mM concentrations of each dNTP, 200 U of SuperScript II reverse transcriptase (Invitrogen), and 5 µM concentrations of random hexamers as primers in the buffer recommended by the manufacturer. Samples were initially heated at 65◦C for 5 min then incubated at 42◦C for 2 h, and the reactions were terminated by incubation at 70◦C for 15 min. In standard RT-PCR reactions, the cDNA was amplified with 1 U of AmpliTaq DNA polymerase (Biotools) and 0.5 µM concentrations of the corresponding primer pairs (oligonucleotides 1–20; Supplementary Table S1). Samples were initially denatured by heating at 94◦C for 3 min. A 30-cycle amplification program was followed (94◦C for 40 s, 60◦C for 40 s and 72◦C for 60 s). Control reactions in which reverse transcriptase was omitted from the reaction mixture ensured that DNA products resulted from the amplification of cDNA rather than from DNA contamination. The dnaE gene encoding the α-subunit of DNA polymerase III was used to provide an internal control cDNA that was amplified with oligonucleotides 5 <sup>0</sup>POLIIIHK/30POLIIIHK (Supplementary Table S1). The expression of the internal control was shown to be constant across all samples analyzed.

## β-Galactosidase Assays

The β-galactosidase activities from promoter-lacZ reporter fusions were measured with permeabilized cells when cultures reached mid-exponential or stationary phase of growth, as described (Miller, 1972).

#### Sequence Data Analyses

Nucleotide sequence analyses were done at the National Center for Biotechnology Information (NCBI) server<sup>1</sup> . The amino acid sequences of the open reading frames were compared with those present in databases using the TBLASTN algorithm (Altschul et al., 1990) at the NCBI server<sup>2</sup> . Pairwise and multiple protein sequence alignments were made with the ClustalW program (Thompson et al., 1994) at the EMBL-EBI server<sup>3</sup> . Phylogenetic analysis of the different proteins was carried out according to the Kimura two-parameter method (Kimura, 1980), and a tree was reconstructed using the neighbor-joining method (Saitou and Nei, 1987) of the PHYLIP program (Felsenstein, 1993) at the TreeTop-GeneBee server<sup>4</sup> and represented using TreeView X 0.5.1 (Glasgow University).

#### RESULTS AND DISCUSSION

#### The bss-bbs Cluster Is Involved in the Anaerobic Degradation of Toluene and m-Xylene in Azoarcus sp. CIB

As indicated in the Introduction, the in silico analysis of the genome sequence of Azoarcus sp. CIB revealed a gene cluster of 26 open reading frames most of which encode orthologous of the Bss and Bbs proteins involved in the peripheral pathway for the anaerobic degradation of toluene in several bacteria. This cluster is located in the close vicinity of the mbd genes encoding the 3-methylbenzoyl-CoA anaerobic central pathway (Juárez et al., 2013), strongly suggesting that it constitutes the bss-bbs cluster for the anaerobic degradation of toluene and m-xylene in strain CIB (Zamarro et al., 2016) (**Figure 1B**).

Since the toluene peripheral pathway was shown to be inducible when bacteria grow in the presence of toluene (Coschigano, 2000; Achong et al., 2001; Hermuth et al., 2002; Kube et al., 2004; Kühner et al., 2005), we checked whether the expression of the bss-bbs genes was inducible when Azoarcus sp. CIB cells were grown anaerobically on toluene or m-xylene with respect to the use of succinate or benzoate as sole carbon source. The analysis of the RT-PCR amplification products revealed that the expression of the bssA and bbsA genes is induced when the cells grow in the presence of toluene or m-xylene as sole carbon sources (**Figures 3A,B**), suggesting their participation in the anaerobic degradation of both aromatic hydrocarbons.

Usually the transcription of anaerobic degradation pathways is regulated by the presence of oxygen in the culture medium

<sup>1</sup>http://www.ncbi.nlm.nih.gov

<sup>2</sup>http://blast.ncbi.nlm.nih.gov

<sup>3</sup>http://www.ebi.ac.uk/Tools/msa/clustalw2/

<sup>4</sup>http://www.genebee.msu.su/services/phtree\_reduced.html

FIGURE 3 | The expression of the bss, bbs and tdi genes is inducible in Azoarcus sp. CIB. Agarose gel electrophoresis of the RT-PCR products. Gene expression was monitored by RT-PCR as described in Section "Materials and Methods" with the primer pairs bssA5new/bssA3new, bbsA331.3/bbsA5new and tdiRint5/tdiSF.3 (Supplementary Table S1) that amplify a bssA gene fragment (A,D), a bbsA gene fragment (B,E), or the tdiS-tdiR intergenic region (C), respectively. (A–C) Azoarcus sp. CIB cells were grown anaerobically until mid-exponential phase by using 0.2% succinate (S), 250 mM of toluene (T) or 250 mM m-xylene (X) as sole carbon source. (D,E) Azoarcus sp. CIB cells were grown under anaerobic (–O2) or aerobic (+O2) conditions using toluene as only carbon source. Lanes M, molecular size markers (HaeIII-digested 8X174 DNA); numbers indicate the sizes of the markers (in bp).

FIGURE 4 | Transcriptional organization of the tdi-bss-bbs genes: (A) Schematic representation of the tdi-bss-bbs gene cluster from Azoarcus sp. CIB. The regulatory tdi genes are indicated by black arrows; the catabolic bss and bbs genes are indicated by gray and white arrows, respectively. The PtdiS, PbssD and PbbsA promoters are represented by bent arrows. The intergenic regions whose expression was checked by RT-PCR are marked by the oligonucleotides (thin arrows) used in the analysis (1–20; see Supplementary Table S1). (B) Agarose gel electrophoresis of RT-PCR products. RT-PCRs from Azoarcus sp. CIB cells grown under denitrifying conditions on 250 mM toluene (lanes T) or 250 mM m-xylene (lanes X) were performed as described in Section "Materials and Methods." The numbers correspond to each primer pair that amplifies each of the intergenic regions indicated in (A). Lanes M, molecular size markers (HaeIII-digested 8X174 DNA); numbers indicate the sizes of the markers (in bp). (C) RT-PCRs from Azoarcus sp. CIB, Azoarcus sp. CIBdbssD and Azoarcus sp. CIBdbbsB cells anaerobically grown until stationary phase in 0.2% pyruvate plus 250 mM toluene (lanes T) by using the oligonucleotide pairs 11/12 and 13/14 (Supplementary Table S1). Lanes C, PCRs performed with the same primer pairs and with genomic DNA as a positive control. Lanes M, molecular size markers (HaeIII-digested 8X174 DNA); numbers indicate the sizes of the markers (in bp).

FIGURE 5 | TdiR is a transcriptional activator of the PbssD, PbbsA and PtdiS promoters. Cells of Azoarcus sp. CIB (A) or Azoarcus sp. CIBdtdiR (B) containing plasmids pBBRPbssD, pBBRPbbsA, or pBBRPtdiS that express the PbssD::lacZ, PbbsA::lacZ, or PtdiS::lacZ translational fusions, respectively, were grown anaerobically on 0.2% pyruvate (–) or 0.2% pyruvate plus toluene (+) until stationary growth phase. β-galactosidase activity values were determined as detailed in Section "Materials and Methods." Error bars represent standard deviation of three different experiments, and asterisks mark the results that are statistically significant (unpaired t-test; ∗∗∗P-value < 0.001, <sup>∗</sup>P-value 0.01–0.05).

m-xylene (mX), or benzylsuccinate (Bs). β-galactosidase activity values were determined as detailed in Section "Materials and Methods." Error bars represent standard deviation of three different experiments, and asterisks mark the results that are statistically significant (unpaired t-test; ∗∗∗P-value < 0.001).

(Durante-Rodríguez et al., 2006; Carmona et al., 2009; Juárez et al., 2013). To determine if the bss-bbs cluster from Azoarcus sp. CIB is under oxygen control, gene expression studies were performed with cells grown with toluene as sole carbon source under aerobic or anaerobic conditions. RT-PCR experiments showed that the bssA and bbsA genes are expressed only under anoxic conditions (**Figures 3D,E**). A similar oxygen-dependent expression of the bss genes was reported in Magnetospirillum sp. TS-6 (Shinoda et al., 2005), and it appears to be consistent with the fact that BSS is a strictly anaerobic enzyme (Beller and Spormann, 1998; Leuthner et al., 1998; Heider et al., 2016). Nevertheless, the oxygen-dependent regulation of the anaerobic toluene degradation pathway may differ from one strain to another since the bss genes from Thauera sp. DNT-1 were shown to be transcribed both in aerobic and anaerobic conditions (Shinoda et al., 2004).

To confirm that the bss-bbs cluster described above was responsible for the anaerobic degradation of toluene and m-xylene in Azoarcus sp. CIB, we constructed CIB mutant strains with insertional disruptions within some of the bss and bbs genes

(A) Phylogenetic tree built from the multiple amino acid sequence alignment of the sensor histidine kinases TdiS from Azoarcus sp. CIB (ABK15651), TutC1 from T. aromatica T1 (AAD12187), Tcs2 from "A. aromaticum" sp. EbN1 (YP\_158339), StyS from Pseudomonas sp. Y2 (CAA03998), TutC from P. mendocina (AAL13332), TodS from P. putida DOT-T1 (AAC45438), TmoS from P. mendocina (AAL13333), Tcs1 from A. aromaticum sp. EbN1 (YP\_158337), BphS from Rhodococcus sp. RHA1 (BAC75411) and BpdS from Rhodococcus sp. M5 (AAB52543) using the program CLUSTALW and visualized with the TreeView software application. The aerobic TodS-like and anaerobic TdiS-like families are circled by red and blue lines, respectively, and a scheme of their different domain architecture and primary structure is shown at the bottom. PAS, HK, and REC correspond to the PAS sensor, autokinase and receiver domains, respectively. H and D indicate the presence of key phosphorylatable histidine and aspartic acid residues at the HK and REC domains, respectively. (B) Phylogenetic tree built from the multiple amino acid sequence alignment of the regulatory protein TdiR from Azoarcus sp. CIB (ABK15650), TutB1 from T. aromatica T1 (AAD12186), Tcr2 from "A. aromaticum" sp. EbN1 (YP\_158340), TutB from T. aromatica T1 (AAD12186), StyR from Pseudomonas sp. Y2 (CAA03999), TodT from P. putida DOT-T1 (CAB43736), TmoT from P. mendocina (AAL13333), Tcr1 from A. aromaticum sp. EbN1 (YP\_158338), BphT from Rhodococcus sp. RHA1 (BAC75412) and BpdT from Rhodococcus sp. M5 (AAB52544) using the program CLUSTALW and visualized with the TreeView software application. The aerobic TodT-like and anaerobic TdiR-like families are circled by red and blue lines, respectively, and a scheme of their similar domain architecture but different primary structure is shown at the bottom. REC and HTH correspond to the receiver and helix-turn-helix DNA-binding domains, respectively. D indicates the presence of key phosphorylatable aspartic acid residue at the REC domain. The bars represent one inferred amino acid substitution per ten amino acids.

that are orthologous to those that have been reported or suggested to be essential for toluene degradation in other bacteria. Thus, we constructed the Azoarcus sp. CIBdbssD, and CIBdbbsB mutant strains harboring the bssD and bbsB disrupted genes, respectively (**Table 1**). As expected, none of the two mutant strains was able to use toluene as the sole carbon and energy source under anaerobic conditions (**Table 1**). Interestingly, none of the mutant strains was also able to use anaerobically m-xylene as carbon source (**Table 1**). Although inactivation of the bssA gene in Azoarcus sp. strain T was shown previously to prevent anaerobic growth on m-xylene (Achong et al., 2001), and (3-methyl)benzylsuccinate was proposed to be converted into 3-methylbenzoyl-CoA via a modified β-oxidation pathway (**Figure 1A**) (Krieger et al., 1999), a genetic demonstration that the bbs genes were indeed needed for the anaerobic growth on m-xylene was still lacking. In fact, some studies had suggested the existence of separate Bbs isoenzymes in toluene and m-xylene catabolism (Leutwein and Heider, 2002). However, our results reveal that both the bss and bbs genes are essential for the anaerobic degradation of toluene and m-xylene in Azoarcus sp. CIB.

So far, only the bssDCABE genes have been shown to be essential for the anaerobic conversion of toluene to benzylsuccinate (Leuthner et al., 1998; Krieger et al., 2001; Bhandare et al., 2006; Funk et al., 2014; Heider et al., 2016). However, the bss cluster contains additional genes of unknown function that are conserved in all toluene degraders, e.g., the bssF gene, or that are restricted to bss clusters from beta-proteobacteria, e.g., the bssGIJKL genes (**Figure 2**) (Kube et al., 2004; Carmona et al., 2009; Kim et al., 2014). To gain further knowledge on whether these additional bss genes might be essential for the initial reaction of the toluene/m-xylene degradation pathway, we constructed Azoarcus sp. CIB mutant strains with disruptional insertions within the bssF and bssJ genes. Whereas the strain Azoarcus sp. CIBdbssF was unable to grow anaerobically in toluene or m-xylene as sole carbon sources, the strain Azoarcus sp. CIBdbssJ could use these two aromatic hydrocarbons as growth substrates (**Table 1**). Therefore, these results reveal that inactivation of bssF prevents the anaerobic growth on toluene and m-xylene, and suggest that this gene and/or any of the downstream co-transcribed genes, i.e., bssG,

orfXY, bssI (see below), are needed for the synthesis of a functional BSS enzyme. The BssF protein (580 amino acids) does not show significant similarity to other proteins of known function, which precludes so far any prediction of its functional role in the BSS activity. On the other hand, although the BssJ protein (307 amino acids) appears to be not essential for the BSS activity, we cannot rule out its involvement in toluene degradation by, for instance, mediating in the stress response caused by toxic aromatic hydrocarbons (Trautwein et al., 2008).

## Transcriptional Organization of the bss-bbs Gene Cluster

The organization of the genes within the bss-bbs cluster in Azoarcus sp. CIB is very similar to that observed in other betaproteobacteria (**Figure 2**). The bss and bbs genes are arranged in two opposite orientations and most of the genes are separated by very short distances suggesting that they constitute two convergent operons (Zamarro et al., 2016) (**Figure 4A**). The existence of the bssDCABEFGH catabolic operon has been described in "A. aromaticum" EbN1 (Kube et al., 2004), but it is not known whether these genes are cotranscribed with the bssIJKLM genes. On the contrary, whereas the bss cluster from Azoarcus sp. strain T contains a bssDCABE operon and a transcriptional initiation site upstream of bssF (Achong et al., 2001), the tutE (bssD) gene of T. aromatica T1 constitutes an operon different to that formed by tutFDGH (bssCABE) (Coschigano, 2000). On the other hand, the transcriptional organization of the bbs cluster has not been elucidated so far in any bacteria.

Since a transcriptional analysis of the whole gene cluster for anaerobic toluene and m-xylene degradation in bacteria has not been reported so far, we aimed to validate the presumed transcriptional organization of the bss-bbs genes in strain CIB. To this end, we performed RT-PCR experiments using total RNA harvested from Azoarcus sp. CIB cells grown anaerobically in toluene or m-xylene as sole carbon sources, and different primer sets that amplify bssD-bssC (3/4), bssB-bssE (5/6), bssE-bssF (7/8), bssG-orfX-orfY-bssI (9/10), bssJ-bssK (11/12), tolR-bbsI (13/14), bbsI-bbsH (15/16), bbsH-bbsG (17/18) and bbsJ-bbsF (19/20) gene fragments (**Figure 4A**). The results obtained (**Figure 4B**) strongly suggest that the bssD-L and bbsA-tol genes are cotranscribed and, therefore, constitute two separate convergent operons. To confirm that the bss and bbs genes constitute two separate operons, we performed gene expression studies in the Azoarcus sp. CIBdbssD and CIBdbbsB mutant strains that harbor insertional disruptions within the bssD and bbsB genes, respectively (**Table 1**). To this end, the two mutant strains were grown anaerobically in the presence of toluene, and RT-PCR analyses revealed the lack of expression of the bss and bbs genes (**Figure 4C**), hence suggesting that the mutations caused polar effects on the expression of the genes located dowsntream of the insertion site and, therefore, that these genes are cotranscribed. Therefore, all these data taken together suggest that in Azoarcus sp. CIB the bssD-L and bbsA-tol genes are arranged as two separate convergent operons. A comparative analysis of the upstream regions of the bssD and bbsA genes in different Azoarcus, Aromatoleum, Thauera, and Herminiimonas strains confirmed the existence of the previously proposed PbssD and PbbsA promoters, respectively (Supplementary Figure S1). PbssD

and PbbsA contain potential −10/−35 sequences recognized by the RNA polymerase as well as the transcription initiation sites which have been mapped for bssD in T. aromatica (Coschigano, 2000; Hermuth et al., 2002; Kube et al., 2004) and Azoarcus sp. strain T (Achong et al., 2001), and for bbsA in T. aromatica K172 (Leuthner and Heider, 2000). Similar PbssD and PbbsA promoter regions driving the expression of the bss and bbs operons were identified in Azoarcus sp. CIB (**Figure 4A** and Supplementary Figure S1). Although an internal promoter or RNA processing event was reported upstream of bssC in some Thauera strains (Coschigano, 2000; Hermuth et al., 2002), the predicted stemloop structure resembling a RNase processing site (Hermuth et al., 2002) is not observed in the bss operon of Azoarcus sp. CIB.

A couple of conserved genes, i.e., tdiS (tutC1) and tdiR (tutB1), are located upstream of the bss genes in most anaerobic toluene degraders from the beta-proteobacteria group (**Figure 2**). These two genes were proposed to code for a two-component regulatory system that regulates transcription of the catabolic bss genes (Coschigano, 2000; Achong et al., 2001; Hermuth et al., 2002; Kube et al., 2004; Kühner et al., 2005). A couple of orthologous tdiSR genes are also divergently transcribed with respect to the bss genes in Azoarcus sp. CIB (**Figures 1B**, **2**, **4A**). RT-PCR studies revealed that tdiS and tdiR are co-transcribed (**Figure 4B**) and, therefore, constitute an operon controlled by the PtdiS promoter in Azoarcus sp. CIB (**Figure 4A**). Moreover, the tdiSR operon appears to be slightly inducible when the cells grow anaerobically in toluene or m-xylene (**Figure 3C**), thus suggesting a common regulatory mechanism for the catabolic (bss, bbs) and predicted regulatory (tdiSR) genes in strain CIB.

All these results taken together constitute the first transcriptional analysis of the whole gene cluster responsible for the anaerobic peripheral pathway for toluene and m-xylene degradation in a single bacterial strain. In Azoarcus sp. CIB these genes are organized in two catabolic operons, bssDCABEFGIJJKL and bbsABCDEFJGHtol, and a putative regulatory operon, tdiSR, that are inducible when cells grow in toluene and m-xylene.

#### tdiSR Control the Expression of the bss-bbs Genes in Azoarcus sp. CIB

To confirm the implication of the tdiSR genes in the anaerobic degradation of toluene and m-xylene in Azoarcus sp. CIB, we constructed two mutant strains, Azoarcus sp. CIBdtdiS and Azoarcus sp. CIBdtdiR, that harbor disruptional insertions within the tdiS (allows expression of tdiR from the kanamycin resistance gene, see Supplementary Figure S2) and tdiR genes, respectively. The two mutant strains were unable to grow using toluene or m-xylene as sole carbon sources (**Table 1**), thus suggesting that the tdiSR genes are involved in the anoxic degradation of toluene/m-xylene and they are likely behaving as an activator system of the PbssD and/or PbbsA promoters.

To study further the role of TdiSR in the activation of the PbssD and PbbsA promoters in Azoarcus sp. CIB, we constructed plasmids pBBRPbssD and pBBRPbbsA that contain the PbssD::lacZ and PbbsA::lacZ translational fusions, respectively (**Table 1**). The Azoarcus sp. CIB strains containing plasmid pBBRPbssD or pBBRPbbsA were grown anaerobically in the absence or presence of toluene or m-xylene, and β-galactosidase assays were performed at the end of the growth curve. As shown in **Figure 5A**, the presence of toluene increased the activity of the PbssD and PbbsA promoters by more than fivefold with respect to the activity observed in the absence of toluene (similar results were obtained with m-xylene, data not shown). These results are in agreement with the observed induction of the bss and bbs genes when Azoarcus sp. CIB grows anaerobically in toluene (**Figures 3A,B**). Moreover, the activity of the PbssD promoter appears to be significantly higher than that of the PbbsA promoter both in the absence or presence of toluene (**Figure 5A**). We then analyzed the activity of the PbssD and PbbsA promoters in the Azoarcus sp. CIBdtdiR mutant strain harboring the pBBRPbssD or pBBRPbbsA plasmids. In contrast to the wild-type strain, the Azoarcus sp. CIBdtdiR (pBBRPbbsA) strain did not show β-galactosidase activity even in the presence of toluene (**Figure 5B**), which strongly suggests that TdiR acts as an essential activator of the PbbsA promoter. On the other hand, the Azoarcus sp. CIBdtdiR (pBBRPbssD) strain revealed that the activity of the PbssD promoter region in the absence of toluene did not increase as much as in the wild-type strain in the presence of toluene (**Figure 5B**), suggesting that although TdiR is needed for a full induction of bss genes, there is some TdiR-independent activation of PbssD when the cells are in the presence of toluene.

Since the expression of the tdiSR genes is also induced when Azoarcus sp. CIB grows in the presence of toluene (**Figure 3C**), we checked whether the tdiR gene product could also act as a transcriptional activator of its own PtdiS promoter. To accomplish this, we constructed plasmid pBBRPtdiS, that contains the PtdiS::lacZ translational fusion (**Table 1**), and monitored the β-galactosidase activity in Azoarcus sp. CIB (pBBRPtdiS) and Azoarcus sp. CIBdtdiR (pBBRPtdiS) strains grown anaerobically in the absence or presence of toluene. Toluene increased the activity of the PtdiS promoter by more than twofold with respect to the activity observed in the absence of toluene in the wild-type but not in the tdiR mutant strain (**Figure 5**).

All these results taken together reveal for the first time that the tdiSR genes, which are highly conserved in the anaerobic toluene degradation clusters of beta-proteobacteria, encode an activator system of the promoters that drive expression of the bss and bbs catabolic genes and of that controlling transcription of the tdi regulatory genes in the presence of toluene. Whereas the expression of the bbs genes is strictly dependent on TdiR, the induction of the bss genes appears to be controlled by TdiR and some additional factors that respond to the presence of toluene in Azoarcus sp. CIB, and whose characterization requires further research.

#### Benzylsuccinate Might Be the Inducer Molecule of the TdiSR-Mediated Control

As described above, the expression of the tdi-bss-bbs genes is inducible in Azoarcus sp. CIB cells growing in the presence of toluene and m-xylene (**Figures 3**, **4**). In T. aromatica T1, benzylsuccinate or any other further intermediate in the toluene peripheral pathway (**Figure 1A**), was suggested

to be the inducer of the tut(bss) genes (Coschigano and Bishop, 2004). To determine if toluene and/or m-xylene, or an intermediate metabolite derived from the anaerobic degradation of the former, are the true inducers of the TdiSRmediated control of the peripheral pathway in Azoarcus sp. CIB, we analyzed the expression of the PbbsA::lacZ fusion in different CIB strains. We selected CIB mutants strains that lack: (i) the first enzymatic step for the conversion of toluene/m-xylene to benzylsuccinate/(3-methyl)benzylsuccinate, i.e., Azoarcus sp. CIBdbssD, which does not express the bss operon (**Figure 4C**); (ii) the modified β-oxidation pathway that converts benzylsuccinate/(3-methyl)benzylsuccinate to the central intermediates benzoyl-CoA/3-methylbenzoyl-CoA (**Figure 1**), i.e., Azoarcus sp. CIBdbbsB, which does not express the bbs operon (**Figure 4C**). Azoarcus sp. CIBdbssD (pBBRPbbsA) cells grown anaerobically on pyruvate or pyruvate supplemented with toluene or m-xylene as inducers showed very low β-galactosidase activity in all the tested conditions, thus suggesting that none of the two aromatic hydrocarbons is the real inducer of the bbs operon (**Figure 6A**). On the contrary, Azoarcus sp. CIBdbbsB (pBBRPbbsA) cells, which retain a functional bss operon and should be capable to transform toluene and m-xylene accumulating benzylsuccinate and (3 methyl)benzylsuccinate, respectively, presented β-galactosidase activity when they were cultivated anaerobically in the presence of toluene or m-xylene (**Figure 6B**). Therefore, all these data suggest that benzylsuccinate and (3-methyl)benzylsuccinate, rather than toluene and m-xylene, are the real inducers of the bbs genes. In agreement with this hypothesis, the two Azoarcus sp. CIB mutant strains grown anaerobically in the presence of benzylsuccinate showed activation of the PbbsA promoter (**Figure 6**). Interestingly, none of Azoarcus strains could use benzylsuccinate as sole carbon and energy source, suggesting a poor uptake of this compound inside the cells.

Taken together all these results reveal that benzylsuccinate is the inducer compound of the TdiSR-mediated control of the toluene peripheral pathway in Azoarcus sp. CIB. Although a similar regulatory scenario can be predicted in other toluene degrader denitrifying bacteria, such as in Thauera strains (Coschigano and Bishop, 2004), benzylsuccinate may not always behave as the inducer molecule. Thus, it was reported that "A. aromaticum" EbN1 strain grown in a mixture of pyruvate and benzylsuccinate did not induce the expression of bssA or the formation of Bss/Bbs proteins (Kühner et al., 2005). Nevertheless, the lack of induction of benzylsuccinate in strain EbN1 could be due to unefficient transport of this molecule or to putative pyruvate-dependent catabolite repression of the bss-bbs genes, as it was shown in strain CIB (see below), and therefore further studies should be carried out to confirm whether benzylsuccinate behaves also as an inducer molecule in "A. aromaticum" EbN1.

Two-component regulatory systems that consist of a sensor histidine kinase and its cognate transcriptional regulator have been shown to be involved in the control of the catabolism of aromatic hydrocarbons in several bacteria (Carmona et al., 2008). According to their primary structure and molecular architecture, the sensor histidine kinases of these two-component regulatory systems can be classified in at least three different phylogenetic groups, i.e., the TodS, TdiS and BphS groups (**Figure 7A**). Hybrid histidine kinases of the TodS family are complex enzymes that contain two sensor PAS domains, i.e., PAS1, that recognizes aromatic hydrocarbons (Busch et al., 2007), and PAS2, involved in dimerization (Koh et al., 2016), two transmitter (autokinase) domains, and a response regulator receiver domain (**Figure 7A**) (Busch et al., 2009). They are involved in the control of the genes for the aerobic degradation of toluene in Pseudomonas putida (TodS) (Busch et al., 2007), P. mendocina (TmoS) (Silva-Jiménez et al., 2012) and, probably, in T. aromatica (TutC) (Leuthner and Heider, 1998), and styrene in some Pseudomonas strains (StyS) (Velasco et al., 1998; Leoni et al., 2003). Histidine kinases of the TdiS group are shorter than TodS-like kinases. They contain two tandem PAS domains, PAS1 and PAS2, and a single transmitter domain (**Figure 7A**), and are encoded in the clusters for the anaerobic degradation of toluene in some strains of the genera Azoarcus/"Aromatoleum" (TdiS) (Achong et al., 2001); Thauera (TdiS/TutC1) (Leuthner and Heider, 1998) and Herminiimonas (**Figure 2**), as well as in the cluster for the anaerobic degradation of ethylbenzene in "A. aromaticum" sp. EbN1 (Tcs2) (Kühner et al., 2005). On the other hand, the large histidine-kinases BphS and BpdS from Rhodococcus sp. RHA1 and Rhodococcus sp. M5, respectively, are involved in the aerobic degradation of biphenyls and they harbor a N-terminal domain similar to that of serine/threonine kinases and a C-terminal histidine-kinase domain (Takeda et al., 2010). It is worth noting that the amino acid residues shown to be involved in the binding to the inducer in the PAS1 domain of TodS, i.e., Phe46, Ile74, Phe79 and Ile114, are conserved in PAS1 domains of other TodS family members but not in any of the PAS domains of TdiS-like proteins. This observation is in agreement with the fact that TodS family members recognize aromatic hydrocarbons as inducers (Busch et al., 2007; Busch et al., 2009; Koh et al., 2016) but TdiS-like histidine kinases may respond to an intermediate, i.e., a benzylsuccinate derivative, rather than to the precursor aromatic hydrocarbon.

The response regulators of the two component regulatory systems that control the catabolism of aromatic hydrocarbons show a similar modular architecture with a N-terminal receiver domain that contains the phosphoaccepting Asp residue (putative Asp58 in TdiR) and a C-terminal helix-turn-helix (HTH) DNA-binding domain of the NarL/FixJ family separated by a glutamine-rich Q linker (**Figure 7B**) (Milani et al., 2005). As with the sensor histidine kinases, three different phylogenetic groups can be identified when comparing their cognate response regulator partners, i.e., the TodT, TdiR and BphT groups (**Figure 7B**). The 3D-structure of the HTH domain of StyR shows four α helices, one of them responsible of DNA recognition with three residues (Lys175, Val176 and His179) putatively involved in the interaction with the operator region of the cognate promoter (Milani et al., 2005). The same amino acid residues are conserved in other StyR-like regulators, such as TodT, TutB and TmoT, all of which may recognize the consensus ATAAACN4GTTTAT sequence at their corresponding operator regions (Lacal et al., 2006; Silva-Jiménez et al., 2012). Interestingly, these residues are not conserved in the HTH

domains of the anaerobic TdiR-like response regulators, which were proposed to recognize a different palindromic region, GGTGTTCGCACC, that is conserved upstream of the −35 regions at the PbssD and PbbsA promoters of different anaerobic toluene degraders (Supplementary Figure S1) (Kube et al., 2004).

In T. aromatica T1 the genes tutC1 (tdiS) and tutB1 (tdiR) are separated from the tutEFDGH (bssDCABE) catabolic genes by tutB and tutC (**Figure 2**). The products of the tutB and tutC genes show high identity with TodS and TodT regulators, respectively, rather than with TdiS and TdiR regulators (**Figure 7**), thus strongly suggesting that TutBC are involved in the aerobic catabolism of toluene in T. aromatica T1 (Leuthner and Heider, 1998). However, the observation that a mutation in the tutB gene disabled the capacity to grow anaerobically in toluene (Coschigano and Young, 1997) and reduced significantly the expression of the bss genes (Coschigano and Bishop, 2004), might suggest the existence of some cross-talk in the regulation of the aerobic and anaerobic toluene degradation pathways in T. aromatica T1.

## Carbon Catabolite Repression of the Toluene/m-Xylene Peripheral Pathway in Azoarcus sp. CIB

The expression of the genes responsible of the catabolism of aromatic compounds is usually under carbon catabolite control when the cells grow in the presence of preferred carbon sources. This catabolite repression phenomenon has been extensively studied in the aerobic catabolism of aromatic compounds (Marqués et al., 1994; Cases et al., 1996; Carmona et al., 2008; Rojo, 2010), and it has also been described for the central pathways involved in the anaerobic catabolism of aromatic compounds in T. aromatica (Heider et al., 1998) and Azoarcus sp. CIB (López-Barragán et al., 2004; Carmona et al., 2009; Juárez et al., 2013; Valderrama et al., 2014). However, there were no reports about the expression of the peripheral pathways for anaerobic degradation of aromatic hydrocarbons when bacteria grow in the presence of alternative carbon sources.

To determine if the expression of the toluene/m-xylene anaerobic peripheral pathway in Azoarcus sp. CIB is under catabolite repression, we checked the activation of the PbbsA and PbssD promoters in Azoarcus sp. CIB (pBBRPbbsA) and Azoarcus sp. CIB (pBBRPbssD) cells, respectively, grown in minimal medium containing toluene plus an additional carbon source, e.g, pyruvate, glutamate, glutarate or benzoate. All the carbon sources tested produced an inhibitory effect on the induction of PbbsA and PbssD promoters, but this inhibition disappeared when the cells reached the stationary phase of growth and consumed the preferred carbon source (**Figures 8A,B**). In contrast, the induction of these promoters was observed at the exponential and stationary growth phases when the cells were grown in toluene as sole carbon source (Supplementary Figure S3). These results suggest that the bss and bbs genes are under carbon catabolite repression, but induction of these genes can be observed at the stationary growth phase as reported above (**Figures 5**, **6**). Interestingly, we also observed a pyruvate-dependent catabolite repression of the PtdiS promoter when Azoarcus sp. CIB (pBBRPtdiS) cells were grown in minimal medium containing toluene plus pyruvate, and this repression was alleviated at the stationary phase of growth (**Figure 8C**). Thus, catabolite repression of the tdiST regulatory operon might be a major reason of the observed repression by organic acids of the TdiSR-dependent expression of the bss-bbs-genes in Azoarcus sp. CIB. Although carbon catabolite repression by organic acids had been reported in the aerobic catabolism of toluene in some bacteria (Duetz et al., 1996; Ruíz et al., 2004; Busch et al., 2010), catabolite repression of the peripheral pathway for the anaerobic degradation of toluene has not been shown before.

### Evolutionary Considerations on the Toluene Peripheral Pathway

A detailed sequence comparison analysis of the organization of the bss-bbs genes in different beta-proteobacteria revealed the existence of a region that shows a high variability among strains. Thus, the bssG-bssI intergenic region contains a bssH gene encoding a putative transporter in "A. aromaticum" EbN1, but this gene is lacking in Herminiimonas sp. CN, and is substituted by orfX-orfY, two genes of unknown function, in Azoarcus sp. CIB and A. toluclasticus MF63 strains (**Figure 2**). Interestingly, this region divides the bss-bbs cluster of the CIB and MF63 strains into two different DNA fragments according to their GC content, i.e., a fragment containing the bssDCABEFG genes and whose GC content (58%) is lower that than of the fragment containing the rest of the bss (bssIJKL) and bbs genes, whose GC content (65%) matches the average GC content of the Azoarcus genome. It is worth noting that the bssDCABE genes from Azoarcus sp. T, T. aromatica T1 and Thauera sp. DNT-1 have also a GC content (<57%) lower than the average GC content (65%) described for Azoarcus/Thauera strains (Liu et al., 2013; Martín-Moldes et al., 2015). On the contrary, the GC content of the bssDCABE genes from "A. aromaticum" EbN1, T. aromatica K172 and Magnetospirillum sp. TS-6 is close to the average GC content of the corresponding species. These observations suggest that the bssDCABEFG genes from Azoarcus sp. CIB, Azoarcus sp. T, T. aromatica T1 and Thauera sp. DNT-1 have an evolutionary origin different than that of their orthologous from Magnetospirillum sp. TS-6, "A. aromaticum" EbN1 and T. aromatica K172, and most probably they come from a microorganism, such as a Hermimiimonas-related strain, with a GC content lower than that of Thauera or Azoarcus strains. Interestingly, this subgrouping of Bss orthologs has also a reflect in the subtle differences observed in their reaction mechanism when using stable isotope tools (Kummel et al., 2013).

Analysis of the flanking regions of the tdi-bss-bbs cluster in Azoarcus sp. CIB revealed the presence of full or truncated mobile genetic elements (**Figure 2**). Thus, next to the tdiSR genes there is a sequence (1istA) encoding the first amino acids of a truncated IS21 transposase. istA/istB genes encoding the two subunits of a IS21 transposase are located adjacent to the bbs genes in "A. aromaticum" EbN1 (**Figure 2**) (Kube et al., 2004). On the other hand, next to the bbs operon of strain CIB there is a set of genes (orfB-E) (**Figure 2**) that are orthologous to c1A68, c1A87, c1A90, and c1A92 genes that encode proteins of unknown function and are located

within the cluster for anaerobic degradation of ethylbenzene in "A. aromaticum" EbN1 strain (Rabus et al., 2002). These genes are also present in a similar organization next to the bbs genes in A. toluclasticus MF63 (**Figure 2**). Downstream of the orfB-E genes in strain CIB there are three additional genes encoding two complete and one truncated IS4 transposase (**Figure 2**). All these observations strongly suggest that the bss-bbs genes in Azoarcus sp. CIB and some closely related bacteria have been acquired by horizontal gene transfer events as a result of different genetic rearrangements. A different genetic organization is observed in the bss-bbs clusters from phylogenetically distant delta-proteobacteria toluene-degrading obligate anaerobes, e.g., Geobacter or Desulfobacula (**Figure 2**) (Carmona et al., 2009; Wöhlbrand et al., 2013). The different genetic organization of the toluene peripheral pathway in facultative and obligate anaerobes correlates also with the significant differences found both in the predicted regulatory genes, e.g., TetR-like and XylR-like regulators are present in obligate anaerobes versus TdiSR-like regulators present in facultative anaerobes (**Figure 2**), and in the catabolic genes, e.g., bss and genes involved in auxiliary functions for Bss and Bbs enzymes are also distinct in facultative and strict anaerobes (**Figure 2**) (Wöhlbrand et al., 2013; Bozinovski et al., 2014). In summary, despite the biochemical strategy to convert toluene into benzoyl-CoA via benzylsuccinate appears to be shared by most anaerobic toluene-degraders, the complete set of genetic determinants involved in this peripheral pathway may have arisen through different evolutionary events in facultative and obligate anaerobes.

#### CONCLUSION

Toluene and m-xylene are important contaminant hydrocarbons that tend to accumulate in subsurface anoxic environments. We have shown here that the bss and bbs genes are essential for the anaerobic degradation of toluene and m-xylene in Azoarcus sp. CIB. Moreover, we have characterized for the first time the transcriptional organization and regulation of a complete cluster encoding the peripheral pathway for the anaerobic degradation of toluene and m-xylene in bacteria. Benzylsuccinate and (3-methyl)benzylsuccinate were shown to be the inducer molecules recognized by the TdiSR two-component regulatory system that specifically controls the activation of the bss, bbs and tdi operons in Azoarcus sp. CIB. In addition to the TdiSR-mediated specific control, the expression of the bss and bbs genes in Azoarcus sp. CIB is under an overimposed regulation that depends on certain environmental factors, such as the presence/absence of oxygen or the availability of preferred carbon sources (catabolite repression). Interestingly, our results indicate that the bss and bbs operons from Azoarcus sp. CIB display differential regulation in the presence of toluene. Whereas the activity of the PbbsA promoter is strictly dependent on TdiR, the activation of the PbssD promoter is also under control of unknown factor(s) triggered by the presence of the aromatic hydrocarbons. Moreover, it is worth noting that the PbssD promoter shows a significant basal activity when the cells grow in the absence of toluene/m-xylene. This basal activity and the TdiR-independent activation of PbssD by toluene could be a strategy to increase the organism's capacity to react quickly to transient toluene availability. Thus, the presence of basal levels of BSS enzyme in the cell will facilitate the formation of some inducer intermediate (benzylsuccinate) and, therefore, the subsequent full induction of all regulatory and catabolic genes when the cells face the presence of the aromatic hydrocarbons. Our results are in agreement with recent reports which suggest that bssA transcription may occur at a basal level even in the absence of toluene, supporting the idea that detection of bssA gene transcripts alone is not sufficient to indicate toluene degradation activity in contaminated environments (Brow et al., 2013; Lünsmann et al., 2016). In this context, and for a reliable assessment of microbial activity in toluene-contaminated samples, we propose here to monitor the expression of the tightly regulated bbs genes as an alternative or complementary approach to the current methods based only on the study of bssA expression. In this sense, highly conserved nucleotide sequence regions within the bbsAB genes of different bacteria could be used to design degenerate oligonucleotides primers for amplification of a bbs gene probe. Finally, the TdiSR-PbbsA regulatory couple identified in this work might constitute also an interesting genetic tool to develop whole cell biosensors for detecting benzylsuccinate, a widely used metabolic biomarker of in situ anaerobic bioremediation of toluene-contaminated sites (Young and Phelps, 2005).

## AUTHOR CONTRIBUTIONS

BB carried out the practical work. BB, MC, and ED designed the experiments, analyzed the results, and wrote the manuscript.

## FUNDING

This work was supported by grants BIO2012-39501, BIO2016- 79736-R, and PCIN-2014-113 from the Ministry of Economy and Competitiveness of Spain and by a grant of Fundación Ramón Areces XVII CN. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

## ACKNOWLEDGMENTS

We thank A. Valencia for technical assistance and Secugen S. L. for DNA sequencing.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00506/full#supplementary-material

#### REFERENCES

fmicb-09-00506 March 20, 2018 Time: 16:29 # 15



plasmid pWW0 of Pseudomonas putida for metabolism of aromatics. J. Bacteriol. 176, 2517–2524. doi: 10.1128/jb.176.9.2517-2524.1994



**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Blázquez, Carmona and Díaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Structural and Biochemical Characterization of BdsA from Bacillus subtilis WU-S2B, a Key Enzyme in the "4S" Desulfurization Pathway

Tiantian Su<sup>1</sup>† , Jing Su1,2† , Shiheng Liu<sup>1</sup> , Conggang Zhang<sup>1</sup> , Jing He<sup>1</sup> , Yan Huang<sup>1</sup> , Sujuan Xu<sup>1</sup> \* and Lichuan Gu<sup>1</sup> \*

<sup>1</sup> State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, China, <sup>2</sup> Faculty of Light Industry, Province Key Laboratory of Microbial Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan, China

#### Edited by:

Edgardo Donati, National University of La Plata, Argentina

#### Reviewed by:

Willem J. H. Van Berkel, Wageningen University & Research, Netherlands John Joseph Kilbane, Illinois Institute of Technology, United States

#### \*Correspondence:

Sujuan Xu xu\_sujuan@sdu.edu.cn Lichuan Gu lcgu@sdu.edu.cn

†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

Received: 12 September 2017 Accepted: 30 January 2018 Published: 15 February 2018

#### Citation:

Su T, Su J, Liu S, Zhang C, He J, Huang Y, Xu S and Gu L (2018) Structural and Biochemical Characterization of BdsA from Bacillus subtilis WU-S2B, a Key Enzyme in the "4S" Desulfurization Pathway. Front. Microbiol. 9:231. doi: 10.3389/fmicb.2018.00231 Dibenzothiophene (DBT) and their derivatives, accounting for the major part of the sulfur components in crude oil, make one of the most significant pollution sources. The DBT sulfone monooxygenase BdsA, one of the key enzymes in the "4S" desulfurization pathway, catalyzes the oxidation of DBT sulfone to 2<sup>0</sup> -hydroxybiphenyl 2-sulfonic acid (HBPSi). Here, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. BdsA and the BdsA-FMN complex exist as tetramers. DBT sulfone was placed into the active site by molecular docking. Seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are found to be involved in the binding of DBT sulfone. The importance of these residues is supported by the study of the catalytic activity of the active site variants. Structural analysis and enzyme activity assay confirmed the importance of the right position and orientation of FMN and DBT sulfone, as well as the involvement of Ser139 as a nucleophile in catalysis. This work combined with our previous structure of DszC provides a systematic structural basis for the development of engineered desulfurization enzymes with higher efficiency and stability.

#### Keywords: FMN-binding, dibenzothiophene, desulfurization, monooxygenase, "4S" pathway

#### INTRODUCTION

The burning of fossil fuels causes worldwide environmental pollution because of the sulfur compounds present in fossil fuels. Hydrodesulfurization (HDS) can remove sulfur from petroleum, but heterocyclic sulfur compounds are difficult to remove completely. Thus, reducing the presence of heterocyclic sulfur compounds, such as dibenzothiophene (DBT) and alkylated DBTs, has become increasingly important. Physical and chemical methods of desulfurization are not economically viable and may cause additional environmental pollution. Therefore, biological desulfurization (BDS) for the treatment of recalcitrant organic sulfur compounds has gained attention (Monticello et al., 1985; Gray et al., 2003).

Numerous efforts have been focused on investigating biodesulfurization systems, using DBT or its alkylated derivatives as model compounds. The pathway specifically cleaving the C-S bond during metabolic desulfurization has been termed the "4S" pathway (**Figure 1**).

The genes, involved in the metabolic desulfurization of the "4S" pathway (dszABCD), were first reported in Rhodococcus sp. IGTS8 (Denome et al., 1993; Kayser et al., 1993). In the initial step of the desulfurization process, DBT is oxidized into DBT sulfone by DBT monooxygenase DszC. Then, DBT sulfone monooxygenase DszA catalyzes the oxidation of DBT sulfone into 2<sup>0</sup> -hydroxybiphenyl 2-sulfonic acid (HBPSi). Finally, HBPSi is desulfurized by the HBPSi desulfinase DszB to 2 hydroxybiphenyl (2-HBP) and sulfite (Denome et al., 1994; Gray et al., 1998). Some thermophilic desulfurizing bacteria, such as Bacillus subtilis WU-S2B, Paenibacillus, and Pseudomonas, can grow in the presence of DBT, utilizing DBT as their sole source of sulfur (Konishi et al., 1997; Ishii et al., 2000a; Kahnert and Kertesz, 2000). The DBT desulfurization enzymes, TszABCD, have been isolated from Paenibacillus (Ishii et al., 2000b). TszABCD have higher heat stability than DszABCD, but the enzymatic activities of TszABCD are lower than those of DszABCD. The corresponding genes from Bacillus subtilis WU-S2B have been identified and the enzymes (BdsABCD) characterized (Kirimura et al., 2001). Additionally, recombinant Escherichia coli cells harboring the bdsABCD genes exhibit DBT desulfurizing activity at 50◦C, and purified BdsA and BdsB have higher enzymatic activities than those of DszA and DszB (Kirimura et al., 2004; Ohshiro et al., 2005). The DNA sequences of BdsABC share high identities (52–73%) with those of DszABC and TdsABC. Multiple conserved proteins motifs have also been identified (Kilbane and Robbins, 2007). These most highly conserved regions could be used to construct improved universal primers for the PCR detection/cloning of bdsA/dszA genes in uncharacterized biodesulfurizationcompetent microbial cultures, which may help identify possible

biodesulfurization genes in genomic data and provide an aid to the annotation of genes in the desulfurization pathway.

The reactions, catalyzed by DBT desulfurization enzymes, have been characterized (Gallagher et al., 1993; Yan et al., 2000; Gray et al., 2003; Kilane, 2006). DszA and DszC belong to Group C of the family of flavin-dependent monooxygenases which contain a TIM-barrel fold and receive reduced flavin from a NAD(P)H-dependent reductase (Montersino et al., 2011; Huijbers et al., 2014). The oxidoreductase DszD supplies the NADH necessary for the activity of monooxygenases DszA and DszC. DszB is a hydrolase that participates in the last desulfurization step and hydrolyzes 2<sup>0</sup> -hydroxybiphenyl-2-sulfinic acid into 2-hydroxybiphenyl and sulfite. During preliminary characterization research of these enzymes, the results showed that DszB is the rate-limiting enzyme (Abin-Fuentes et al., 2014; Kilbane, 2016). Biological desulfurization can make greater contributions in future, but there exist some issues regarding the catalytic mechanism of key enzymes in the "4S" pathway that need to be resolved.

The structures of these key enzymes, involved in the "4S" pathway, have been studied extensively. The structure of DszB was first reported in 2006, and the reaction mechanism was further illustrated (Lee et al., 2006). The crystal structures of DszC and its complex with FMN have also been reported (Liu et al., 2014; Zhang et al., 2014; Guan et al., 2015), and the homology TdsC has also been reported (Hino et al., 2017). Recently, a liquid chromatography–mass spectrometry analysis showed that the C2 hydroperoxide of the DBT sulfone reacts with reduced flavin to form a flavin-N5-oxide intermediate in DszA, which is involved in subsequent protonation (Adak and Begley, 2016). However, without the structure of BdsA, it is unknown how this process

happens in the active site of BdsA and how the protein enzyme facilitates the reaction.

To further understand the catalytic mechanism of DBT sulfone monooxygenation, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B at the resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. Site-directed mutagenesis showed that mutations in the residues involved in catalysis or in flavin substrate-binding result in a significant loss of enzymatic activity, which provides valuable clues for elucidating the catalytic mechanism of DBT sulfone monooxygenase.

#### MATERIALS AND METHODS

#### Cloning, Protein Expression, and Purification

The bdsA gene, from Bacillus subtilis WU-S2B, was synthesized and used as the template for PCR amplification. The amplified bdsA was inserted into the NdeI and XhoI sites of a pET-15b vector (Novagen) in frame with an N-terminal His-tag; the resulting plasmid was transformed into E. coli BL21 (DE3) cells for BdsA overexpression.

Escherichia coli BL21 (DE3) cells, harboring the bdsA overexpression plasmid, were grown in Luria-Bertani (LB) medium supplemented with 100 µg/mL ampicillin. The cells were induced by 0.12 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the OD<sup>600</sup> reached 0.9, and were incubated overnight at 22◦C. Cells were subsequently harvested by centrifugation (8400 g for 15 min) and lysed by ultrasonication in lysis buffer [25 mM Tris-HCl, pH 8.0; 200 mM NaCl; 0.4 mM phenylmethylsulfonyl fluoride (PMSF)]. After centrifugation at 28,000 g for 45 min at 4◦C, the supernatant was applied to a Ni-NTA affinity column (GE Healthcare) equilibrated in the lysis buffer. The 6× His-tagged BdsA was eluted with elution buffer (25 mM Tris-HCl, pH 8.0; 100 mM NaCl; 250 mM imidazole), followed by further purification by anion exchange on a 16/10 HR Source-15Q column (GE Healthcare) with gradient buffer (Buffer A: 25 mM Tris-HCl, pH 8.0; Buffer B : 25 mM Tris-HCl, pH 8.0, 1 M NaCl), and then by size-exclusion chromatography using a gel-filtration column (Superdex 200 10/300 GL, GE Healthcare) in 10 mM Tris-HCl pH 8.0, 100 mM NaCl. Purified BdsA was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining (R250). The concentrations of purified proteins were determined using Nanodrop (A-280 nm) (Thermo Scientific).

#### Site-Directed Mutagenesis of BdsA

Site-directed mutagenesis of BdsA was conducted by two-step PCR using the wild-type pET-15b/bdsA plasmid as template. The mutant clones were confirmed by DNA sequencing (Invitrogen). The expression and purification processes were the same as that for the wild-type BdsA protein.

#### Crystallization and Data Collection

For crystallization, the purified BdsA proteins were concentrated to 10–20 mg/mL measured by Nanodrop (A-280nM). Crystals TABLE 1 | X-ray data collection and refinement statistics.


<sup>a</sup>Rmerge = 6hkl6<sup>i</sup> |Ii (hkl) − <I(hkl)>|/6hkl6<sup>i</sup> I i (hkl). where <I(hkl)> is the mean intensity of multiply recorded reflections.

The coordinates and structure factors have been deposited in the Protein Data Bank with the accession code 5XKC (apo-BdsA) and 5XKD (FMN-bound).

were first screened by sitting drop vapor diffusion using crystallization screen kits (Hampton Research) at 20◦C. After optimization, native crystals were obtained from the hanging drops by mixing equal volumes of the protein solution and reservoir solution [0.1 M sodium citrate (pH 5.5) and 35% PEG 200]. To obtain crystals of the BdsA-FMN complex, native crystals were soaked in the solution, containing 0.1 M sodium citrate (pH 5.5), 35% PEG 200 and 1 mM FMN, for 30 min. We also tried to obtain the crystals of the BdsA-FMNsubstrate complex by the same means. Unfortunately, the efforts failed.

For data collection, crystals were flash-frozen in liquid nitrogen, with 15–20% (v/v) ethylene glycol used as cryoprotectant. The X-ray diffraction data sets were collected at 100K on a beam line BL17U at the Shanghai Synchrotron Radiation Facility (Shanghai, China) equipped with an ADSC Q315r CCD-detector.

### Structure Determination and Refinement

The X-ray diffraction data were integrated and scaled using the HKL-2000 program suite (Otwinowski and Minor, 1997). The native BdsA structure was resolved by molecular replacement using Phaser from the CCP4 suit of programs (Winn et al., 2011) with LadA (PDB entry 3B9N) as the search model. Refinement was performed using the PHENIX crystallography suite (Adams et al., 2002) and the COOT interactive modelbuilding program (Emsley and Cowtan, 2004). The final R-values were Rwork = 17.47% and Rfree = 21.30% based on a subset of 5% of the reflections. The cofactor FMN was added to the complex model, based on the Fo–Fc density map of the ligand structure, and refinement was conducted in the same manner as that for apo-BdsA. The final model had a Rwork = 18.11% and Rfree = 22.11% based on a subset of 5% of the reflections.

Diffraction data collection and refinement statistics are listed in **Table 1**. The final models were checked and validated using PROCHECK (Laskowski et al., 1993), QMEAN (Benkert et al., 2008), and ProQ (Cristobal et al., 2001) model quality assessment tools, which indicated that the models were of good quality. Structure graphics were illustrated with the PyMOL molecular visualization system (Lill and Danielson, 2011). The atomic coordinates and structure factors of BdsA and the BdsA-FMN complex were deposited in the Protein Data Bank with accession codes 5XKC and 5XKD, respectively.

## Calculations of Enzyme-Substrate Molecular Docking

AutoDock 4.2 was used for the molecular docking calculations (Morris et al., 2009). The substrate coordinates were designed using the Dundee PROGRG server ( Schuttelkopf and van Aalten, 2004). The DBT sulfone docking was based on the BdsA-FMN complex structure. The ligands and BdsA were prepared using AutoDockTools, and the BdsA was designed to be rigid. Polar hydrogens and Kollman United Atom charges were added to the enzyme; Gasteiger charges were assigned; and nonpolar hydrogen atoms were merged for the ligands. Docking calculations were performed using the default settings for the genetic algorithm parameters with 25,000,000 energy evaluations per run. A composite file of all possible conformers was analyzed by AutoDock Tools. The chemical reasonableness of best results was evaluated by examining the interactions between the BdsA and best-docked conformer.

#### Enzymatic Activity Assays

The activity of BdsA was determined by measuring the amount of HBPSi using high-performance liquid chromatography (HPLC) according to a method described previously (Ohshiro et al., 1997, 1999). The enzyme reaction system for measuring the activity of BdsA contained 100 mM potassium phosphate buffer (pH 7.0), 0.25 mM DBT sulfone, 6 mM NADH,

from the protein core is located between α7 and β8, which contains four small helices (α8, α9, α10, and α11).

10 µM FMN, 20 nM Fre (flavin reductase from E. coli O157), and 2 µM BdsA, in a total volume of 1 mL. The reaction was performed with rotary shaking (2000 rpm) at 35◦C for 20 min, and stopped by the addition of 100 µL of 12 M HCl. The mixture was extracted with ethyl acetate, centrifuged at 15,000 g for 5 min, and then the supernatant was injected into an HPLC system using 20 mM KH2PO<sup>4</sup> (pH 2.5) and methanol as the mobile phase, at a ratio of 2:3.

The reaction mixture was loaded onto a C18 reversed-phase column (Venusil XBP-C18, Agela Technologies) attached to an HPLC system (LC-10AT pump, SPD-10A UV/VIS detector). An increase in absorbance at 280 nm, which indicates the amount of HBPSi, was observed. The enzymatic assays for all mutants were performed in the same manner as that for the wild-type BdsA protein.

#### RESULTS

#### Overall Structure of apo-BdsA

The crystal structure of apo-BdsA (unbound structure) from Bacillus subtilis belongs to the P21212 space group with the cell dimension a = 132.494 Å, b = 174.574 Å, and c = 85.345 Å. The asymmetric unit contains four peptide chains, designated A, B, C, and D, which form two homodimers, AB and CD (**Figure 2A**). Each BdsA monomer is composed of 453 amino acids. In the structure of BdsA, the electron densities for the N-terminus to Gln4 and Ser451 to the C-terminus are not visible, likely because these regions are flexible and disordered. In the final model of apo-BdsA, more than 96.85% of the residues were located in the favored regions of the Ramachandran plot and only 2.98% in the generous and allowed regions.

The BdsA monomer contains nine β-strands (β1–β9) and 13 α-helices (α1–α13), which fold into a triosephosphate isomerase (TIM)-barrel with five extended insertion regions (**Figure 2B**). These extended insertion regions are located in the connecting regions between β-strands and α-helices, designated as insertion segments IS1 to IS5 (**Figure 2C**). IS1 is located between β1 and α1, IS2 between β2 and α2, IS3 between β3 and α3, and IS4 between α4 and β5. According to the structure, IS1 to IS4 play important roles in the formation of the BdsA homodimer (**Figure 2C**). IS5 is located between α7 and β8, which contain four small helices (α8, α9, α10, and α11). IS5, the largest insertion region, is separated from the protein core and forms a deep groove with the protein core (**Figures 2B,C**). The deep groove is important in pro 8.

fmicb-09-00231 February 15, 2018 Time: 11:33 # 6

FMN-binding and substrate catalysis. IS5 forms the entrance for FMN and the substrate.

#### Tetramer Structure of BdsA

The crystal structure of BdsA shows that there are four molecules in an asymmetric unit (**Figure 2A**). To determine its oligomeric state in solution, we performed gel-filtration experiments using a Superdex-200 column (GE Healthcare) running in 10 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The elution volume of BdsA indicated a molecular mass of ∼220 kDa, indicating that BdsA exists as a tetramer in the solution.

Structure comparison showed that the two homodimers, AB and CD, have a high structural similarity. The root-mean-square deviation (RMSD) value for Cα is 0.165 Å. As predicted by PISA (Krissinel and Henrick, 2005), the AB dimer has a surface area of approximately 3,177 Å<sup>2</sup> , and CD has a surface area of approximately 3,217 Å<sup>2</sup> . The dimer interface is extensive and consists predominantly of hydrophobic residues, which are derived from α1, IS1, IS2, α2, α3, IS3, and IS4 (**Figure 2C**).

## Structure of the BdsA–FMN Complex

The structure of the tetrameric BdsA–FMN complex contains four FMN molecules, with each subunit binding one FMN (**Figure 3A**). The TIM-barrel structure confirms that BdsA belongs to the group C flavin-dependent monooxygenases. The binding pocket is mainly formed by IS1, IS2, IS3, and IS5 (**Figure 3B**). The overall Cα RMSD between apo-BdsA and the BdsA-FMN complex is only 0.240 Å. The most substantial structural discriminations were observed at the side chains of residues Phe56, Val137, and Phe246 (**Figure 3C**), which may facilitate the binding of FMN.

The binding of FMN to BdsA is maintained through hydrogen bonds and hydrophobic interactions. The flavin ring of FMN lies in the barrel, with its plane nearly parallel with the staves of the barrel, and its si-face exposed to the solvent. There are several hydrogen bonding interactions between BdsA and FMN: the NE2 atom of His 156 with the O3 atom of the flavin phosphate groups (2.7 Å); the OG atom of Ser139 with the N1 atom of the flavin ring (2.8 Å); backbone nitrogen of Leu230 with the phosphate group (3.1 Å); and the Arg159 with the O4 atom of the flavin ring (3.4 Å). Two residues, Tyr160 and Val137, form van der Waals contacts with FMN (**Figure 3D**).

Frontiers in Microbiology | www.frontiersin.org

and IS3.

by black dashed line), including α8, α9, α10, and α11. It is the key component of the active pocket, which controls the size of the pocket accompanied with IS1, IS2,

Based on the structural analysis, six single-point mutations of BdsA were constructed: V137A, S139A, H156A, R159A, Y160A, and L230A. The secondary structures of mutant BdsA proteins were in good agreement with the wild type protein when examined by circular dichroism spectroscopy (**Supplementary Figure S1**). The results of enzymatic activity showed that mutant proteins V137A, S139A, R159A, Y160A, and L230A completely lost their activity (**Figure 4**). The activity of H156A was decreased by approximately 50% (**Figure 4**). We also showed the superposition of the wild type and variant amino acids, which indicated that all the point mutations significantly disrupt the interactions between the side chains of the active site residues and FMN (**Supplementary Figure S2**). These results indicate that these residues play significant roles in the binding of FMN, which was consistent with the results of the structural analysis.

fmicb-09-00231 February 15, 2018 Time: 11:33 # 7

## Substrate Docking and Analysis of Mutagenesis

The substrate DBT sulfone is the intermediate product of DBT desulfurization. It is difficult to crystallize the BdsA-FMNsubstrate complex because DBT sulfone binds poorly to the oxidized form of the enzyme. To examine the binding and catalytic mechanism of DBT sulfone, we used flexible docking (Autodock 4.2) to build three-dimensional models of the BdsA-FMN-DBT sulfone complex. The docking result is illustrated in **Figure 3D**. As shown, the DBT sulfone substrate is located in the active site, on the si-face of the FMN isoalloxazine ring (**Supplementary Figure S3**). According to the results of the molecular docking analysis, seven residues (Phe12, His20, Phe56, Phe246, Val248, His316, and Val372) are involved in the binding of DBT sulfone (**Figure 3D**). The distance between flavin C4a and the carbon atom of the DBT sulfone attacked by the flavin hydroperoxide is 4.5 Å. The structure suggests that there is space between flavin C4a and the attacked atom of DBT sulfone for the formation of a (hydro) peroxyflavin intermediate (Li et al., 2008).

To illustrate the function of the substrate-binding residues, seven single-point mutants of BdsA were constructed: F12A, H20F, F56A, F246A, V248A, H316F, and V372A. The enzyme activities of these proteins were analyzed by measuring the formation of HBPSi on HPLC, and each assay was repeated four times (**Figure 4**). The results showed that mutant proteins H20F, F56A, F246A, and H316F lost more than 90% activity compared with wild-type BdsA. The activity of F12A was decreased by approximately 80%, and those of V248A and V372A were decreased by approximately 50%. These results were consistent with the results of the molecular docking calculations. Taken together, our results indicate that residues F12, H20, F56, F246, V248, H316, and V372 may play vital roles in substrate binding.

## DISCUSSION

## The Overall Structure and Active Site of BdsA Are Highly Conserved

A structure-based alignment was conducted, using the DALI program (Holm and Rosenström, 2010), against the Protein Data Bank (PDB). The similarity results showed that the scaffold of BdsA is highly conserved among all the group C flavoprotein monooxygenase superfamily. The nitrilotriacetate monooxygenase (PDB: 3sdo Z-score 48.8) is most similar to BdsA, followed by Ytnj (PDB: 1ywl, Z-score 47.6), alkane monooxygenase (Lada, PDB: 3b9n, Z-score 46.9) (Li et al., 2008), ethylenediaminetetra acetic acid (EDTA) monooxygenase (Emoa, PDB: 5dqa, Z-score 44.2) (Macdonald et al., 2016), and luciferase-like monooxygenase (PDB: 3rao, Z-score 32.9). The Cα superposition of BdsA with Ytnj, Lada, Emoa, and luciferaselike monooxygenase yielded the RMSD values of 1.178, 1.322, 1.316, and 2.364 Å, respectively. The homologs fold as TIMbarrels and are highly conserved in structural folding. However, the structural identities of the insertion segment IS5 were very low (**Figure 5**). In BdsA, IS5 ranges from Val304 to Val375, including α8, α9, α10, and α11. It is the key component of the active pocket, which, along with IS1, IS2, and IS3, controls the size of the pocket. The volume of the active pocket of BdsA, and those of homologous proteins, are calculated using CASTp server (Dundas et al., 2006). The volume of the BdsA activity

pocket is 5134.9 Å<sup>3</sup> . Nitrilotriacetate monooxygenase catalyzes the degradation of the nitrilotriacetate substrate, with the active pocket volume of 5288.4 Å<sup>3</sup> . Ytnj has an active pocket with the volume of 5030.2 Å<sup>3</sup> , but its function remains unknown. Alkane monooxygenase catalyzes the terminal hydroxylation of long-chain n-alkanes (C15–C36), and its active pocket volume is 4118.7 Å<sup>3</sup> . The luciferase-like monooxygenase-active pocket volume is 2611.1 Å<sup>3</sup> . The maximum cavity, 8158.3 Å<sup>3</sup> , is that of the EDTA monooxygenase. The figure highlighting the different binding modes of substrate are shown in **Supplementary Figure S4**. We interpreted that the structural variations may be related to the specificities of substrate-binding.

In this study, we have determined the putative residues participating in substrate binding of BdsA through molecular docking and enzyme activity assays. According to the molecular docking result, the substrate-binding site of BdsA may be located on the si-face of the isoalloxazine ring of FMN. As shown in **Figure 3D**, the DBT sulfone is in its stable form and fits well with the active pocket. According to the structure, 13 mutations (six mutations related to FMN-binding and seven mutations related to DBT-sulfone-binding) were constructed. Single mutations in these residues caused near-complete loss in the activity of BdsA. To illustrate the putative substrate-binding site, a sequence conservation analysis was performed, using BdsA homologs from different species with sequence identities from 48 to 72% (**Figure 6**). H20, F56, V137, S139, R159, Y160, F246, and H316 are all conserved in BdsA from different species. As shown in **Figure 3D**, these residues constitute a hydrophobic pocket, which fits well with the substrate DBT sulfone and can stabilize the conformation of the substrate, contributing to the formation of a C4a-(hydro) peroxyflavin intermediate on the si-face of the flavin isoalloxazine ring.

#### Mechanism of BdsA

The three-dimensional structure of BdsA is essential for elucidating the catalytic mechanism of the DBT sulfone monooxygenation. Recently, the crystal structure of BdsA was reported by Masahiko at a resolution of 2.8 Å (Okai et al., 2017), which provides architectural information and comparison with the homologous proteins of BdsA. However, the study by Masahiko did not give the structure of the complex with FMN or the substrate. In our study, we determined the crystal structure of BdsA from Bacillus subtilis WU-S2B, at a resolution of 2.2 Å, and the structure of the BdsA-FMN complex at 2.4 Å. In addition, we have also constructed a three-dimensional model of the BdsA-FMN-DBT sulfone complex by using flexible molecular docking. These structures may help in the future design of experiments aimed at elucidating the catalytic mechanism in further detail.

BdsA is a DBT sulfone monooxygenase that shares a 79% sequence identity with DszA from Rhodococcus sp. IGTS8. We reason that the catalysis of BdsA most probably exploits the same mechanism as DszA. Based on the structures we have resolved and previous works on homologous proteins, we propose a general catalytic mechanism for BdsA in sulfinic acid formation (**Figure 7**). The first step is Ser139 as an electron donor accepting the hydride from the N1 atom of the reduced flavin ring to activate FMNH2 (Massey et al., 1969; Bruice, 1984); and the second step of the reaction was a radical combination of 2 (activate FMNH2) to oxygen resulting in flavin hydroperoxide 3, which is the nucleophilicity reagent established in the flavoenzyme-mediated Baeyer–Villiger oxidation of ketones (Walsh and Chen, 1988) and the RutAcatalyzed uracil ring opening reaction (Mukherjee et al., 2010). Then, the addition of compound 3 to DBT sulfone forms the sulfone-stabilized carbanion 4. Succeedent forms 5 (FMN) and hydroperoxide 6.

BdsA play two roles in this process. First, the protein provides an environment that keeps the DBT sulfone and FMN in the correct positions with right orientation (**Figure 3D**). Second, the amino acid Ser139 functions as a nucleophile, activating FMNH2. According to the suggested mechanism of DszA (Adak and Begley, 2016), the catalytic cycle is then completed by the conversion of 6 to benzenesulfinic acid and flavin-N5-oxide intermediate to FMN. In the process, the protein only supplies the reaction environment and may not catalyze directly.

#### AUTHOR CONTRIBUTIONS

LG, SX, TS, JS, and SL designed the research. TS, JS, SL, CZ, and JH performed the experiments. TS, JS, SL, YH, SX, and LG analyzed the data and wrote the paper. All authors contributed to the editing of the manuscript.

## FUNDING

This work was supported by National Natural Science Foundation of China (Grant No. 30800167).

## ACKNOWLEDGMENTS

We thank Professor Minyong Li at School of Pharmaceutical Sciences, Shandong University for assistance with mechanism discussion. We also thank the staff at the Beamline BL17U1 of the Shanghai Synchrotron Radiation Facility for assistance with data collection.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 00231/full#supplementary-material

FIGURE S1 | The binding modes of FMN in these homology proteins.

FIGURE S2 | The CD spectra result. The wild-type and mutant proteins are shown in different colors.

FIGURE S3 | Superposition of the wild and variants amino acids of the active site. The cofactor FMN is shown as yellow sticks. Wild amino acids are shown as cyan sticks, and mutant amino acids are shown as magenta sticks.

FIGURE S4 | BdsA, 1yw1, and 3b9o are illustrated in cyan, magentas, and wheat cartoon, respectively.

## REFERENCES

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erythropolis D-1: purification and characterization. J. Ferment. Bioeng. 83, 233–237. doi: 10.1016/S0922-338X(97)80985-3


developments. Acta Crystallogr. 67(Pt 4), 235–242. doi: 10.1107/S09074449100 45749


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Su, Su, Liu, Zhang, He, Huang, Xu and Gu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fmicb-09-00231 February 15, 2018 Time: 11:33 # 11

# Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast *Blastobotrys (Arxula) adeninivorans*

Anna K. Meier <sup>1</sup> , Sebastian Worch<sup>1</sup> , Erik Böer <sup>1</sup> , Anja Hartmann<sup>1</sup> , Martin Mascher <sup>1</sup> , Marek Marzec1, 2, Uwe Scholz <sup>1</sup> , Jan Riechen<sup>1</sup> , Kim Baronian<sup>3</sup> , Frieder Schauer <sup>4</sup> , Rüdiger Bode<sup>4</sup> and Gotthard Kunze<sup>1</sup> \*

<sup>1</sup> Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany, <sup>2</sup> Department of Genetics, Faculty of Biology and Environmental Protection, University of Silesia, Katowice, Poland, <sup>3</sup> Department of Microbiology, School of Biological Sciences, University of Canterbury, Christchurch, New Zealand, <sup>4</sup> Institute of Microbiology, University of Greifswald, Greifswald, Germany

#### *Edited by:*

Rajesh K. Sani, South Dakota School of Mines and Technology, United States

#### *Reviewed by:*

Willem J. H. Van Berkel, Wageningen University & Research, Netherlands Maria Fátima Carvalho, Centro Interdisciplinar de Pesquisa Marine e Ambiental (CIIMAR), Portugal

> *\*Correspondence:* Gotthard Kunze kunzeg@ipk-gatersleben.de

#### *Specialty section:*

This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology

*Received:* 22 May 2017 *Accepted:* 31 August 2017 *Published:* 15 September 2017

#### *Citation:*

Meier AK, Worch S, Böer E, Hartmann A, Mascher M, Marzec M, Scholz U, Riechen J, Baronian K, Schauer F, Bode R and Kunze G (2017) Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans. Front. Microbiol. 8:1777. doi: 10.3389/fmicb.2017.01777 Tannins and hydroxylated aromatic acids, such as gallic acid (3,4,5-trihydroxybenzoic acid), are plant secondary metabolites which protect plants against herbivores and plant-associated microorganisms. Some microbes, such as the yeast Arxula adeninivorans are resistant to these antimicrobial substances and are able to use tannins and gallic acid as carbon sources. In this study, the Arxula gallic acid decarboxylase (Agdc1p) which degrades gallic acid to pyrogallol was characterized and its function in tannin catabolism analyzed. The enzyme has a higher affinity for gallic acid (K<sup>m</sup> −0.7 <sup>±</sup> 0.2 mM, kcat <sup>−</sup>42.0 <sup>±</sup> 8.2 s−<sup>1</sup> ) than to protocatechuic acid (3,4-dihydroxybenzoic acid) (K<sup>m</sup> <sup>−</sup>3.2 <sup>±</sup> 0.2 mM, kcat <sup>−</sup>44.0 <sup>±</sup> 3.2 s−<sup>1</sup> ). Other hydroxylated aromatic acids, such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid are not gallic acid decarboxylase substrates. A. adeninivorans G1212/YRC102-AYNI1-AGDC1, which expresses the AGDC1 gene under the control of the strong nitrate inducible AYNI1 promoter achieved a maximum gallic acid decarboxylase activity of 1064.4 U/l and 97.5 U/g of dry cell weight in yeast grown in minimal medium with nitrate as nitrogen source and glucose as carbon source. In the same medium, gallic acid decarboxylase activity was not detected for the control strain G1212/YRC102 with AGDC1 expression under the control of the endogenous promoter. Gene expression analysis showed that AGDC1 is induced by gallic acid and protocatechuic acid. In contrast to G1212/YRC102-AYNI1- AGDC1 and G1212/YRC102, A. adeninivorans G1234 [1agdc1] is not able to grow on medium with gallic acid as carbon source but can grow in presence of protocatechuic acid. This confirms that Agdc1p plays an essential role in the tannic acid catabolism and could be useful in the production of catechol and cis,cis-muconic acid. However, the protocatechuic acid catabolism via Agdc1p to catechol seems to be not the only degradation pathway.

Keywords: biodegradation*,* aromatic acids, gallic acid, antimicrobial plant substances, fungi

## INTRODUCTION

The non-conventional, non-pathogenic, imperfect, dimorphic yeast Arxula adeninivorans (syn. Blastobotrys adeninivorans) is a fungal organism with great biotechnological potential due to its wide substrate spectrum and robustness against osmotic, salt and temperature stresses (Middelhoven et al., 1984; Wartmann et al., 1995; Malak et al., 2016). It grows in harsh environments, tolerates up to 20% NaCl in the medium, endures temperatures of ≤48◦C. It is also able to use a variety of tannins including tannic acid and hydroxylated aromatic acids as carbon sources (Wartmann et al., 1995; Tag et al., 1998; Yang et al., 2000; Böer et al., 2009a). To date, this research has focused on the wildtype strain, A. adeninivorans LS3 that was isolated from wood hydrolysate in Siberia (Gienow et al., 1990). In 2014, the complete genome of A. adeninivorans LS3 was sequenced and genome analysis revealed the presence of two potential tannin acyl hydrolases (Atan1p, Atan2p) as well as gallic acid decarboxylase (Agdc1p) (Kunze et al., 2014).

Tannins are part of almost all types of plant tissues and their hydrolysis releases glucose and gallic acid. Gallic acid belongs to the hydroxylated aromatic acid compounds, which share primarily antifungal and bacteriostatic properties and are toxic for most microbes at very low concentrations (Sikkema et al., 1995). Gallic acid recently became a useful compound in pharmaceutical and chemical industries because of its antifungal, bacteriostatic, anticancer, antimelanogenic, and antioxidant properties (Badhani et al., 2015).

A large amount of gallic acid is produced in the food and agriculture industries, which has contributed to a remarkable increase of phenolic compounds, mostly toxic agents, in the environment. Unfortunately, di- and trihydroxylated aromatic acids such as gallic acid and protocatechuic acid are difficult to degrade and often accumulate in water and soil, which is resulting in additional environmental pollution (Zhang et al., 2014). Before there can be an attempt to increase the efficiency of bioremediation processes, the microorganisms and the enzymes that degrade these pollutants need to be investigated.

Many bacteria and filamentous fungi have been characterized as organisms able to degrade hydroxylated aromatic acids by enzymatic degradation. These microorganisms developed several different ways to degrade these aromatic compounds. One option is the decarboxylation of a carboxyl group attached to the aromatic ring, which is catalyzed by enzymes from the decarboxylase family (Grant and Patel, 1969; Yoshida et al., 1982; Wright, 1993; O'Donovan and Brooker, 2001; Hashidoko et al., 2002; Mukherjee and Banerjee, 2004). In most cases, these decarboxylases are encoded by inducible genes, which ensure that the synthesis of the enzymes only occurs in the presence of their substrates. However, only a few of these enzymes have been characterized to date because they exhibit high oxygen sensitivities and are relatively unstable (Yoshida et al., 1982; Nakajima et al., 1992; Zeida et al., 1998; Jiménez et al., 2013).

The general strategy of aerobic degradation of aromatic compounds is the cleavage of the aromatic ring with the degradation products being channeled into the central metabolism. Usually the compounds go through ortho or meta pathways and are completely utilized (Williams and Sayers, 1994). However, there are microorganisms which can nonoxidatively decarboxylate gallic acid but the pathway results in death of the cell because of a lack in further degradative enzymes (Nakajima et al., 1992; Zeida et al., 1998; Rodríguez et al., 2008).

The decarboxylation of gallic acid results in the production of pyrogallol. This phenol derivate is very sensitive to oxygen and is used in many industrial applications, for example as a developing agent in photography and in cosmetic products, such as in hair dying agents.

The tannin acyl hydrolase 1 (Atan1p) has been already characterized and described as the enzyme which catalyzes the first reaction step in tannin degradation by A. adeninivorans LS3 (Böer et al., 2009a). It was described as extracellular enzyme, induced by tannins or gallic acid. The transformation of gallic acid and protocatechuic acid by A. adeninivorans LS3 wildtype strain have also been investigated (Sietmann et al., 2010). However, it is still not known if the degradation pathway is common for all hydroxylated aromatic acids and the role of gallic acid decarboxylase is currently unclear.

In this study, the Arxula gallic acid decarboxylase gene, AGDC1, was identified, isolated and expressed using a strong promoter in A. adeninivorans. The recombinant enzyme was purified, characterized and in addition, its function in the yeast cell determined. Growth of the AGDC1 expression yeast strain and a ∆agdc1 gene disruption mutant on different aromatic acids was studied to elucidate the role of the gene product in A. adeninivorans.

#### MATERIALS AND METHODS

#### Strains and Culture Conditions

Escherichia coli strains XL1 blue [recA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac [F'proABlacl q Z DM15 Tn10 (Tetr)], from Invitrogen (Grand Island, NY, USA) or DH5α [F– 880dlacZ1M15 1(lacZYA-argF) U169, deoR, recA1, endA1, hsdR17 (rK−, mK+) phoA supE44, λ <sup>−</sup>, thi-1, gyrA96 relA1] were used as host strains for bacterial transformation and plasmid isolation. Both strains were cultured in Luria Bertani medium (LB - Sigma, USA) supplemented with 50 mg/l ampicillin (AppliChem, Germany) or 50 mg/l kanamycin (Roth, Germany).

Arxula adeninivorans G1212 (aleu2 ALEU2::atrp1) (Steinborn et al., 2007) and the wildtype strain A. adeninivorans LS3 (Kunze and Kunze, 1994), originally isolated from wood hydrolysate in Siberia (Russia), were used as experimental strains. Both strains are deposited in the yeast collection of the Department of Biology of the University of Greifswald (SBUG, Germany). They were cultivated in shaking flasks at 30◦C, 180 rpm either under non-selective conditions in complex medium (YEPD-yeast

**Abbreviations:** GDC, gallic acid decarboxylases; AGDC gene, gallic acid decarboxylase 1 gene from A. adeninivorans; Agdc1p, gallic acid decarboxylase 1 protein from A. adeninivorans; dcw, dry cell weight; YRC, yeast rDNA integrative expression cassette; YIC, yeast integrative expression cassette; YEPD, complex medium; YMM: yeast minimal medium; U, unit; GA, gallic acid; PA, protocatechuic acid; 3-HBA, 3-hydroxybenzoic acid; 4-HBA, 4-hydroxybenzoic acid; 2,3-DHBA, 2,3-dihydroxybenzoic acid; 2,4-DHBA, 2,4-dihydroxybenzoic acid; 2,5-DHBA, 2,5-dihydroxybenzoic acid; ORF, open reading frame.

extracts-peptone-dextrose) or in yeast minimal medium (YMM) supplemented with 43.5 mM NaNO<sup>3</sup> as nitrogen source and 20 g/l glucose as carbon source (YMM-glucose-NaNO3) (Tanaka et al., 1967; Rose et al., 1990).

## Transformation Procedures and Isolation of Nucleic Acids

Transformations of E. coli and A. adeninivorans were performed according to Böer et al. (2009a). Plasmid DNA isolation and DNA restriction were carried out as described by Wartmann et al. (2002).

### Construction of *AGDC1* Expression Plasmids and Generation of Transgenic *A. adeninivorans* Strains

For AGDC1 overexpression, the AGDC1 ORF (PRJEB19393) with a HisTag encoding region at the 3′ -end (AGDC1-6H) was amplified from genomic DNA of A. adeninivorans LS3 in a PCR reaction using primers that incorporated flanking EcoRI and NotI cleavage sites: primer AGDC1-1-6H-5′ -**GAA TTC** ATG ACT ACT TCC TAC GAG CCC TGG-3′ (primer nucleotide positions 1-20, EcoRI restriction site is in bold type and underlined); primer AGDC1-2-6H 5′ -**GCG GCC GC**T TAG TGG TGG TGA TGA TGG TGC CAG TGG AGG TCA ATC TCC T-3′ (primer nucleotide positions 674–693, HisTag encoding region underlined, NotI restriction site is in bold type and underlined). The resulting EcoRI-NotI flanked AGDC1- 6H ORF was inserted into the plasmid pBS-AYNI1-PHO5- SS between the A. adeninivorans derived AYNI1 promoter and the S. cerevisiae PHO5 terminator (Böer et al., 2009c). The AYNI1 promoter - AGDC1-6H gene - PHO5 terminator flanked by SpeI–SacII restriction sites expression modules were inserted into the plasmid Xplor2.2 to generate Xplor2.2-AYNI1- AGDC1-6H-PHO5 (Böer et al., 2009b). The resulting plasmids contained fragments of 25S rDNA, which are interrupted by the selection marker module (ALEU2 promoter-ATRP1m gene-ATRP1 terminator), the AGCD1 expression module and an E. coli resistance marker and replicator. To prepare the cassettes for yeast transformation, Xplor2.2-AYNI1-AGDC1-6H-PHO5 and the control plasmid Xplor2.2 lacking AGDC1 expression module were digested with AscI (YRC) or SbfI (YIC) to remove the E. coli sequences including the resistance marker. The resulting restriction products YRC102-AYNI1-AGDC1-6H, YIC102-AYNI1-AGDC1-6H and YRC102 (control) were used to transform A. adeninivorans G1212.

Yeast transformants were selected by tryptophan auxotrophy in YMM-glucose-NaNO3. The cells were stabilized by passaging on selective (YMM-glucose-NaNO3) and non-selective (YEPD) agar medium, to attain a high level of protein production (Klabunde et al., 2003).

## Construction of 1*agdc1* Gene Disruption Mutant

To create disruption mutants, the gene disruption cassette contained 1,019 bp up and 1,032 bp down the ORF of AGDC1 as well as the ATRP1m selection marker module was constructed. All fragments were amplified by PCR (up and down ORF overlapped fragments using chromosomal DNA of A. adeninivorans LS3 as the template and ATRP1m selection marker module using plasmid pBS-ALEU2-TRP1m as the template-Steinborn et al., 2007). Primers used for fragment amplification were created with additional 15 bp overlapping sides (**Table 1A**). This strategy allowed ligation of fragments in one step by using an In-Fusion Cloning Kit (TaKaRa Clontech, USA Inc.). The resulting construct was amplified in E. coli DH5α. Finally, the complete construct covering 1019 bp in front of the AGDC1 gene, the ATRP1m selection marker module and 1032 bp behind the AGDC1 was amplified using the primers 5′ -AAATTCTTCTACAGGAAGTCAGG-3′ and 5′ - ATTGTTATGCACTATTCGTTAACGG-3′ . The resulting 3,548 bp PCR-product was used to transform A. adeninivorans G1212 (Böer et al., 2009a).

#### Microscopic Analysis

The phenotype of A. adeninivorans G1212/YRC102, G1212/YRC102-AYNI1-AGDC1-6H and G1234 [1agdc1] was investigated. For this purpose, strains were cultivated on agar plates containing YMM-glucose-NaNO<sup>3</sup> at 30◦C for 48 h. Colony morphology was investigated using a VHX-5000 Digital Microscope (KEYENCE Deutschland GmbH, Neu-Isenburg, Germany). For detailed analysis, a FESEM S 4100 device (Hitachi High-Technologies Europe GmbH, Krefeld, Germany) was used. Fragments of agar plates containing colonies were dried in 30◦C, attached to carbon-coated aluminum sample blocks and gold-coated in an Edwards S150B sputter coater (Edwards High Vacuum Inc., Clevedon, United Kingdom).

## Assay for Determination of Gallic Acid Decarboxylase Activity

Gallic acid decarboxylase activity was assayed by following gallic acid or protocatechuic acid biotransformation during the enzymatic reaction in 50 mM potassium phosphate buffer pH 6.2. The reactions were carried out in 96-well UV-transparent microtiter plates (Greiner Bio-One GmbH) in triplicates, in a total volume of 100 µl. Reaction was started by adding 0.5 mM substrate solution to the enzyme and monitored 15 min at 40◦C, 259 nm (ε = 7100 [1/M/cm]) and 288 nm (ε = 1,570 [1/M/cm]) for gallic acic and protocatechuic acid respectively (Krumholz et al., 1987). Blank values were established by assaying using water instead of the enzyme. One Unit (1 U) of enzyme activity was defined as the amount of enzyme required to decarboxylate 1 µmol gallic acid to pyrogallol or 1 µmol protocatechuic acid to catechol per min at 40◦C, pH 6.2.

Gallic acid decarboxylase activity with 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4 dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid as substrates were measured by GC-MS. 470 µl 1 mg/ml substrate in 50 mM potassium phosphate buffer pH 6.2 and 30 µl purified Agdc1p were incubated at 40◦C for 0, 5, 15, 25, and 35 min. The blank for the reaction was assayed using boiled enzyme (15 min, 95◦C). The reactions were stopped by increasing the temperature for 95◦C for 15 min. The pH of the samples was adjusted to 2 with HCl and the compounds extracted using TABLE 1 | (A) Primers used for construction of deletion mutant strain G1234 [1agdc1]. (B) Primers used for analysis of AGDC1 expression levels.



#### (B) Primers for nested quantitative RT PCR


organic solvent MTBS (Sigma-Aldrich, USA). Extracts were lyophilized and resuspended in 400 µl pyridine and 100 µl BSTFA (Sigma-Aldrich, USA) and incubated overnight at 60◦C. GC/MS measurements were done according to method described in "Analysis of supernatant from strains growing on YMM-NaNO<sup>3</sup> supplemented with different carbon sources."

#### Agdc1-6hp Analysis

Agdc1-6hp was purified by column chromatography on HisTrap FF (1 ml) Novagen, USA) in 20 mM Tris, 0.5 mM NaCl pH 7.9, and 5 mM imidazole as binding buffer and 20 mM Tris, 0.5 mM NaCl pH 7.9, and 1 M imidazole as elution buffer. Finally, the purified protein was desalted with PD10 columns (GE Health Care Europe GmbH, Germany).

The indicative molecular mass determination of native Agdc1- 6hp was done by gel filtration using SuperdexTM 200 (Amersham Biosciences, UK). The flow rate was 1 ml/min and fractions of about 1 ml were collected for 182 min (buffer: 50 mM Tris pH 8 + 0.15 M NaCl). A calibration curve was constructed using blue dextran, ferritin, catalase, bovine albumin, RNAse A and vitamin B12 as standards.

The K<sup>m</sup> value for gallic acid and protocatechuic acid were determined as described in "Assay for determination of gallic acid activity." All measurements were done in triplicate and Michaelis-Menten and Hanes plots were constructed.

The Agdc1p concentration was determined using a Coomassie stained SDS-PAGE for the calculation of kcat (Kunze et al., 1998).

#### Protein Analysis

SDS-PAGE and Western blot analysis were performed as described by Kunze et al. (1998). The antibodies used for Western blot analysis were anti-His-tag specific primary antibody produced in rabbit (Sigma-Aldrich, USA) and secondary antibody anti-mouse IgG alkaline phosphatase conjugate produced in goat (Sigma-Aldrich, USA). The staining procedure was done by membrane incubation with NBT/BCIP substrate (Roche Diagnostics, Switzerland).

The dye-binding method of Bradford was used for protein quantification (BIO-RAD, USA), using bovine serum albumin as the standard (Bradford, 1976).

#### Quantitative Reverse Transcriptase PCR Analysis (qRT PCR)

Arxula adeninivorans G1212/YRC102 cells were grown in YMMglucose-NaNO<sup>3</sup> for 24 h at 30◦C and 180 rpm. Afterwards cells were harvested (3,220 × g at 4◦C), washed with YMM-NaNO<sup>3</sup> and resuspended in YMM-NaNO<sup>3</sup> containing 0.2% glucose or hydroxylated aromatic acids. 2 ml of culture samples were collected after 0, 4, 8, 24, and 48 h. Cells were pelleted (2,300 × g, 4◦C), and disrupted mechanically using silica beads and a Mixer Mill MM400 (RETSCH, Germany) operating for 3 min, vibrational frequency of 30/s. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany) as described by the manufacturer. RNA concentration was analyzed using NanoDrop2000c spectrophotometer (Thermo Fisher, Germany). cDNA was synthesized using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Germany) with an Oligo(dT)15V-RTA primer. For the analysis of temporal gene expression patterns a nested qRT-PCR assay was applied (Worch and Lemke, 2017). The first PCR synthesis was made with cDNA-template (10 cycles) using gene-specific primers and RTA1 primer (**Table 1B**). The PCR product was diluted 1:500 and amplified in the presence of SYBR Green fluorescent dye (Power SYBR <sup>R</sup> Green PCR Master Mix, Applied Biosystems, Foster City, CA, USA) using ABI 7900HT Fast Sequence Detection System (Applied Biosystems) with gene-specific primers 2 or 4 and RTA primer (**Table 1B**). TFCI and ALG9 were used as reference (housekeeping) genes (Rösel and Kunze, 1995; Teste et al., 2009). The calculations were done using 11ct-method (Livak and Schmittgen, 2001).

## Microarray Design and Hybridization for Gene Expression Analysis

The gene expression profiling was performed using a microarray produced by Agilent Technologies in 8 × 60 k format. The microarray was based on 6,025 annotated chromosomal sequences and 36 putative mitochondrial gene oligos and was designed using Agilent Technologies eArray software (https:// earray.chem.agilent.com; design number 035454). Depending on the sequence length of the genes, up to 10 60-mers per gene were created, resulting in a total of 56,312 A. adeninivorans specific oligos.

A. adeninivorans LS3 was cultivated overnight in YMMglucose-NaNO3. Cells were then pelleted (3,220 <sup>×</sup> g at 20◦C), and shifted to YMM-NaNO<sup>3</sup> containing a mixture of 0.5% gallic acid plus 1% glucose or 1% glucose only. After 15 min, 30 min, 2 and 5 h of cultivation at 30◦C and 180 rpm, cells were harvested and total RNA was isolated using RNeasy Mini Kit (Qiagen, Germany). The samples were labeled and the microarray hybridized according to the Agilent Technologies "One-Color Microarray-Based Gene Expression Analysis (v6.5)" instructions. The R package limma was used for microarray data analysis (Smyth, 2005). The background of raw expression data was corrected by "normexp" and normalized between arrays using "quantile." Differentially expressed genes were detected by fitting a linear model to log2-transformed data by an empirical Bayes method (Smyth, 2004). The Bonferroni method was used to correct for multiple testing.

### Analysis of Supernatant from Strains Growing on YMM-NaNO<sup>3</sup> Supplemented With Different Carbon Sources

Quantities of glucose, hydroxylated aromatic acids, as well as various phenol derivatives were measured by GC-MS (Clarus SQ 8 GC Mass Spectrometers). Samples were previously extracted and derivatized. For this purpose, 2 ml cultures were centrifuged for 10 min, 16,000 × g, 4◦C, 500 µl supernatant was adjusted to pH 2 with HCl and compounds were extracted with 1 ml MTBS (Sigma-Aldrich, USA). Extracts were lyophilized in a Freeze Dryer, Alpha 1-4 LSC plus (Christ), resuspended in 400 µl pyridine, 100 µl BSTFA (Sigma-Aldrich, USA) was added and samples were incubated overnight at 60◦C. 1 µl sample was injected into an Elite-5MS column, Length 30 m, I.D 0.25 mm, 25 µm (Elite, USA). The injection was performed with a split speed 10 ml/min. The temperature was held at 60◦C for 1 min and was then ramped up to 230◦C at 15◦C/min and held at this temperature for 11.33 min. The peak areas (triplicates) were calculated by TurboMass 6.1 using data from external quantification standards assayed four times.

## RESULTS

#### Identification of the *A. adeninivorans AGDC1* Gene Encoding Gallic Acid Decarboxylase (Agdc1p)

A putative gallic acid decarboxylase gene (ARAD1C45804g) was annotated in the genome of A. adeninivorans LS3. The gene, Arxula gallic acid decarboxylase 1 (AGDC1), is localized on chromosome Arad1C. The open reading frame is a 696 bp encoding a protein with 232 amino acids. The predicted subunit molecular mass of Agdc1p is 27.3 kDa. Protein analysis according to the SignalP (version 4.1) program (http://www.cbs.dtu.dk/ services/SignalP/) predicted the absence of a secretion signal sequence, which was corroborated by cytoplasmic localization in cell fractionation experiments (data not shown).

In Lactobacillus plantarum WCFS1 the enzyme comprises three subunits. There are several conserved domains that are fundamental for enzyme catalytic activity and structure were found in the Agdc1p sequence such as subunit B which is homologous with Lactobacillus plantarum WCFS1 gallic acid decarboxylase and putative gallic acid decarboxylase protein sequences from other lactic bacteria described by Jiménez et al. (2013) (**Figure 1A**).

A phylogenetic tree between Agdc1p and gallic acid decarboxylase from L. plantarum as well as the putative gallic acid decarboxylase protein sequences from lactic bacteria was developed using the Neighbor-Joining method. It demonstrated that the Lactobacillus sakei and Pediococcus pentosaceus putative enzymes originated from the same ancestral node, which shares a common ancestor with Agdc1p, whereas Enterococcus faecium, Streptococcus gallolyticus, Oenococcus oeni, Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus brevis form other branches with common ancestors (**Figure 1B**).

## Generation of an Agdc1-6hp Producing Yeast Strain

Arxula adeninivorans G1212 [1trp1] served as a host strain for overexpression of AGDC1 gene with a HisTag encoding sequence fused to the 3′ -end of the ORF under control of the nitrate inducible AYNI1 promoter (A. adeninivorans nitrate reductase promoter). Cassettes with the AGDC expression module (YRC102-AYNI1-AGDC1-6H, YIC102-AYNI1-AGDC1-6H-**Figure 2A**) and control (YRC102) were prepared as described in "Material and Methods." After genome integration of the cassettes, a number of selected clones (YICs and YRCs) were passaged to establish high plasmid stability. The transformants were then cultivated in YMM-glucose-NaNO<sup>3</sup> at 30◦C for 48 h. The yeast cells were harvested, disrupted and screened for gallic acid decarboxylase activity. This activity was detected in all stabilized A. adeninivorans G1212/YRC102-AYNI1- AGDC1-6H and G1212/YIC102-AYNI1-AGDC1-6H strains. However transgenic yeast strains with genome integrated YRCs achieved approximately 2.2-fold higher enzyme activity

method of Agdc1p to subunit B of selected lactic bacteria without distance corrections. An asterisk (\*) indicates positions which have a single, fully conserved residue, a colon (:) indicates conservation between groups of strongly similar properties, and a period (·) indicates conservation between groups of weakly similar properties.

than YIC transformants. In contrast, gallic acid decarboxylase activity was not detectable in the control strain A. adeninivorans G1212/YRC102 using glucose as carbon source. A. adeninivorans G1212/YRC102-AYNI1-AGDC1-6H with the highest gallic acid decarboxylase activity was used in further investigations.

AGDC1 expression and control strains A. adeninivorans G1212/YRC102-AYNI1-AGDC1-6H and G1212/YRC102 were cultivated in YMM-glucose-NaNO<sup>3</sup> at 30◦C and 180 rpm for 120 h and dry cell weight (dcw), gallic acid decarboxylase activity with gallic acid as substrate and yield [Y(P/X)] were determined each 24 h. The experiment was performed in triplicate.

All yeast strains reached a maximum dcw after 24 h of cultivation and then remained constant until the end of the experiment. In contrast gallic acid decarboxylase activity was detected in A. adeninivorans G1212/YRC102-AYNI1-AGDC1- 6H only. This strain achieved its maximum enzyme activity of 972 U/l with 94 U/g dcw after 96 h (**Figure 2B**).

#### Phenotype of Transgenic *A. adeninivorans* Strains

The deletion of gallic acid decarboxylase gene results in change of cells shape and through this, changes in morphology of colonies. The difference was visible already on the agar plate (YMM-NaNO<sup>3</sup> supplemented with 2% glucose as sole source of carbon) without magnification. The microscopic analysis showed that the deletion mutant strains were growing as mycelia at 30◦C; this phenomenon was not observed in the case of G1212/YRC102 control strain and G1212/YRC102-AYNI1- AGDC1-6H overexpression strain (**Figure 3**).

#### Purification and Characterization of Agdc1-6hp

Arxula adeninivorans G1212/YRC102-AYNI1-AGDC1-6H with HisTag encoding sequence at the 3′ -end of the AGDC1 ORF was

selected for synthesis of recombinant Agdc1-6hp. Purification was by Immobilized Metal Affinity Chromatography (IMAC). Crude extract and a fraction containing purified Agdc1-6hp were analyzed on Coomassie-stained SDS-PAGE and Western blot.

acid as substrate, calculated yield [Y(P/X)] (circles) and dry cell weight (squares). Measurements were done in triplicate.

Predicted molecular mass of Agdc1-6hp is 27.3 kDa. The denatured protein was visible at around the 25 kDa position on SDS-PAGE and Western blot in both the crude extract and the purified protein fraction (**Figure 4A**). Gallic acid decarboxylase activity was measured for crude extract and purified Agdc1-6hp. Total activity in crude extract was 1 U/mg whereas it was 5.9 U/mg in the purified fraction, corresponding to 77% of yield. The specific activity was 1 and 5.9 U/mg in crude extract and the purified Agdc1-6hp fraction respectively (**Figure 4**).

The indicative molecular mass of native Agdc1-6hp was estimated by gel filtration on a SuperdexTM 200 column (as described in "Material and methods"). The calculated molecular mass of the native enzyme was 27.1 kDa, whereas predicted molecular mass of the His-tagged Agdc1-6hp was 28.1 kDa. This data indicates that Agdc1-6hp is a monomeric protein.

In vitro study of Agdc1-6hp established the optimal conditions for enzymatic activity. The reaction mixture contained 0.5 mM gallic acid as substrate. The optimum pH was determined using the following buffers: 50 mM citrate-phosphate (pH 2.6–8), 50 mM sodium acetate (pH 3–6), 50 mM sodium citrate (pH 3.5– 6.5), 50 mM potassium phosphate buffer (pH 5.8–6.8), 50 mM sodium phosphate (pH 5.5–8), and 50 mM TRIS-HCl (pH 7–9). Agdc1-6hp was most active when reaction was carried out in 50 mM potassium phosphate buffer pH 6.2 at 40◦C. Enzyme activity of 80% was observed between pH 5.6–7.1 and at temperatures between 25 and 45◦C. Some buffer systems reduced the activity of Agdc1-6hp, for example citrate-phosphate buffer exhibited 52% activity whereas sodium-citrate buffer had only 15% of activity in comparison to potassium phosphate buffer when the reaction was carried out under optimal conditions.

A study of thermostability demonstrated that Agdc1-6hp is stable between 4 and 30◦C. After 24 h 100% of the initial activity was detected, however incubation at 35◦C resulted in loss of 60% of initial activity after 24 h. Incubation at optimum

temperature (40◦C) resulted 100% gallic acid decarboxylase activity after only 7 h of incubation, however activity decreased drastically thereafter. Temperatures above 40◦C led to a complete loss of activity after 4 h. Enzyme stored at −80◦C exhibited no loss of activity even after 1 month of storage in these conditions.

device (Scale bar: D-3 µm, E-9 µm, F-4 µm).

A number of metal ions and reagents were tested to determine which acted as inhibitors and/or cofactors. When 1 mM Ni2+, Fe2+, Fe3+, Co2+, or Cu2<sup>+</sup> were present in a reaction mixture, the enzyme was almost completely inhibited (relative activity <30%). In contrast addition of 1 mM Mg2+, Mn2+, Ca2<sup>+</sup> K + , Na2S2O3, PEG4000, PEG6000, or PEG8000 to the reaction mixture had no significant influence on gallic acid decarboxylase activity. In all cases activities were >80% of activity in control reaction mixture. The slight positive effect was detected when 1 mM EDTA, ascorbic acid or DTT were present in reaction mixture (**Table 2**).

The kinetic parameters of purified Agdc1-6hp were determined photometrically for gallic acid and protocatechuic acid. The K<sup>m</sup> of Agdc1-6hp for gallic acid is 4 times lower than for protocatechuic acid (0.7 ± 0.2 mM and 3.2 ± 0.2 mM respectively). Turnover, kcat, is 42.0 <sup>±</sup> 8.2 s−<sup>1</sup> for gallic acid and 44.0 ± 3.4 s−<sup>1</sup> for protocatechuic acid. However, the catalytic efficiency is much lower for protocatechuic acid (14.0 ± 2.0 TABLE 2 | Relative Agdc1-6hp activity [%] assayed for 0.5 mM gallic acid as substrate and presence of different additives in the reaction mixture.


[mM−<sup>1</sup> s −1 ]) than for gallic acid (57.8 ± 7.6 [mM−<sup>1</sup> s −1 ]) (Supplementary Table S1).

Five additional hydroxylated aromatic acids (3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid) with different numbers and positions of hydroxyl groups were tested to determine the in vitro substrate specificity of Agdc1-6h. The enzyme, incubated in optimal conditions for 35 min, did not catalyze the decarboxylation of these compounds.

#### Expression Analysis of *AGDC1* on Various Carbon Sources

The expression level of AGDC1 relative to the housekeeping genes TFCI and ALG9 was analyzed by nested quantitative RT-PCR. A. adeninivorans G1212/YRC102 was cultivated on YMM-NaNO<sup>3</sup> supplemented with 0.2% (w/v) of glucose or different hydroxylated aromatic acids as sole source of carbon. Samples were collected at different times and total RNA was isolated. An increase of gene transcription of AGDC1 was observed when cells were cultivated on gallic acid and protocatechuic acid. None of the other carbon sources (glucose, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,3 dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid) induced the expression of AGDC1. The highest expression level was detected after 8 h of cultivation (**Figure 5**).

In order to get information on whole transcriptome variations upon incubation of A. adeninivorans with gallic acid, microarray expression analysis was performed. The changes in genes expression of wildtype strain, A. adeninivorans LS3, cultivated in YMM-NaNO<sup>3</sup> supplemented with 1% glucose and YMM-NaNO<sup>3</sup> supplemented with 1% glucose plus 0.5% gallic acid were analyzed. Cells were harvested after 15 min, 30 min, 5 h, and 12.5 h of incubation in shaking flasks at 30◦C and 180 rpm. Total RNA was isolated and analysis of microarray data allowed recognition of the candidate genes involved in tannic acid degradation pathway (**Figure 6**). Significant upregulation was observed for gallic acid decarboxylase 1 (AGDC1) and tannase 1 (ATAN1) genes, as well as for genes annotated as putative tannase 2 (ATAN2), catechol-1,2-dioxygenase, oxalocrotonate

FIGURE 4 | Purification of Agdc1-6hp on Ni Sepharose. (A) Crude extract and purified Agdc1-6hp visualized on Coomassie-stained SDS-PAGE (12%) and Western blot, using primary antibodies anti-polyhistidine from rabbit and secondary antibodies anti-rabbit from goat. (B) Summary of the Agdc1-6hp purification.

decarboxylase, 2-oxopent-4-dienoate hydratase, 4-hydroxy-2 oxovalerate aldolase. These data suggest that additional enzymes are involved in tannic acid degradation. These enzymes convert the pyrogallol into pyruvate and acetaldehyde and make them available to the central metabolism (**Figure 6**). In addition, microarray data showed the upregulation of genes involved in β-oxidation process but not in the glyoxylate cycle, methyl citrate cycle and in the catabolism of the branched-chain amino acids valine, leucine and isoleucine (Supplementary Figures S1–S4).

#### Gallic Acid Tolerance by *A. adeninivorans*

The growth behavior of A. adeninivorans G1212/YRC102 on different gallic acid concentrations was investigated. The cells were cultivated at 30◦C, 180 rpm on YMM-NaNO<sup>3</sup> supplemented with 0.05–2% (w/v) gallic acid as sole source of carbon. Growth occurred in all concentrations and the growth profile at all concentrations was characterized by long adaptation phase. Cell growth required 48 h of cultivation (Supplementary Figure S5) to start to increase in rate. The OD600 nm values were strongly correlated to the amount of carbon sources in medium. Despite the long adaptation phase, no growth inhibition was observed. Additionally, the final optical densities, reached during cultivation on gallic acid were lower than those seen in growth on medium supplemented with an equal concentration of glucose.

#### Role of Agdc1p in *A. adeninivorans* on Metabolizing Gallic Acid

Previous investigations indicated that Agdc1p is an enzyme expressed by a gallic acid inducible gene. To analyse its in vivo role, three A. adeninivorans strains G1212/YRC102, G1212/YRC102-AYNI1-AGDC1-6H, and G1234 [∆agdc1] were studied. All strains were cultivated at 30◦C up to 146 h on YMM-NaNO<sup>3</sup> supplemented with 2% (w/v) glucose, 2% (w/v) gallic acid, or 1% (w/v) gallic acid plus 1% (w/v) glucose. Dcw, gallic acid decarboxylase activity and its yield [Y(P/X)] were determined. Deletion of AGDC1 contributed to cells death or growth inhibition when gallic acid was present in culture medium (**Figure 7**). No gallic acid decarboxylase activity was detected in crude extract of A. adeninivorans G1234 [1agdc1] in all cultivation conditions. Additional metabolite analysis showed that gallic acid remains in the culture medium during the entire cultivation. This was not detected when A. adeninivorans G1234 [∆agdc1] was cultivated in a medium with protocatechuic acid as the carbon source

(**Figure 8**). In contrast gallic acid decarboxylase was synthesized and accumulated in the control strain A. adeninivorans G1212/YRC102 when gallic acid was present in the medium. On the other hand, the overexpression strain A. adeninivorans G1212/YRC102-AYNI1-AGDC1-6H exhibited activity during cultivation on glucose and reached 1,064 U/l and 97.5 U/g between 96–146 h of cultivation. The highest enzyme activity of 1,617 U/l and 148 U/g was detected in the crude extract of A. adeninivorans G1212/YRC102-AYNI1-AGDC1-6H after cultivation on a mixture of gallic acid and glucose. In comparison, the cultivation of G1212/YRC102 in the same conditions exhibited maximal 446.7 U/l and 37.5 U/g. However, the overexpression of AGDC1 did not significantly improve the cells growth on gallic acid (**Figure 7**).

To demonstrate that the main role of gallic acid decarboxylase in A. adeninivorans is to degrade gallic acid, A. adeninivorans G1212/YRC102 and G1234 [∆agdc1] were cultivated for 48 h on YMM-NaNO<sup>3</sup> supplemented with 0.2% (w/v) of different hydroxylated aromatic acids as sole source of carbon. Samples were collected at 0, 30, and 48 h of cultivation for metabolite analysis. This investigation demonstrated that A. adeninivorans G1212/YRC102 is unable to grow on 2.3-dihydroxybenzoic acid. However, this strain was able to grow on all other carbon sources (gallic acid, protocatechuic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid) with different adaptation times. The deletion mutant was unable to grow on gallic acid and 2.3 dihydroxybenzoic acid as carbon source. In all other cases, the adaptation phase of A. adeninivorans G1234 [1agdc1] was shorter and cells grew faster compared to the control strain. Interestingly, the best growth of both strains was observed for 3-hydroxybenzoic acid and the slowest growth was with 4 hydroxybenzoic acid (**Figure 9**).

In vivo transformation of gallic acid to pyrogallol by A. adeninivorans was investigated. A. adeninivorans G1212/YRC102 and G1212/YRC102-AYNI1-AGDC1-6H were cultivated on YMM-NaNO<sup>3</sup> supplemented with 0.25% gallic acid plus 0.5% glucose as the carbon source. The metabolites were analyzed by GS-MS. Complete consumption of gallic acid occurred after 48 h cultivation of the control strain and after 96 h of the G1212/YRC102-AYNI1-AGDC1-6H strain. The maximum extracellular pyrogallol production was detected after 24 h incubation. The yield, calculated as the % of substrate converted into the product detected in the culture medium, was similar for both strains; approximately 2.7% for control strain and 2.2% for overexpression strain (**Figure 10**). Pyrogallol was only product detected during degradation of gallic acid by A. adeninivorans.

enzymes (rectangular square). Enzymes are enriched with color-coded fold change values of time resolved expression data of the respective genes. The colors represent upregulation (blue) and downregulation (red) of genes in cells shifted to a medium containing gallic acid as the carbon source compared to cells grown with glucose. Metabolites or enzymes occurring multiple times in the metabolic network are decorated with a clone marker (e.g., NAD+) [produced using VANTED–(Junker et al., 2012; Rohn et al., 2012)].

Degradation of protocatechuic acid in A. adeninivorans was also studied and the products produced during the degradation of protocatechuic acid by G1212/YRC102 and G1212/YRC102- AYNI1-AGDC1-6H were analyzed. The products detected in the culture medium during cultivation were catechol, cis,cis-muconic acid and trace amounts of 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 1,4-dihydroxybenzene and 1,3,4 benzentriol (**Figures 11A,B**).

In addition, a time course analysis of protocatechuic acid consumption (**Figures 11A,B**) by the overexpression strain A. adeninivorans G1212/YRC102-AYNI1-AGDC1-6H was conducted. The protocatechuic acid was completely degraded after 48 h and the maximum catechol concentration was detected after 24 h of cultivation. Cis,cis-muconic acid appeared even later, and the highest concentration was observed at 48 h of cultivation of G1212/YRC102 strain and after 72 h in G1212/YRC102-AYNI1-AGDC1-6H. The yield, calculated as the % of protocatechuic acid converted into the product (cis,cis-muconic acid) detected in the culture medium, was 42.5% for G1212/YRC102 and 71.2% for G1212/YRC102-AYNI1- AGDC1-6H (**Figure 11B**).

#### DISCUSSION

Decarboxylation of hydroxylated aromatic acids has been reported in bacteria and fungi. A well-known example is the non-oxidative decarboxylation of gallic acid, which leads to the synthesis of pyrogallol (Grant and Patel, 1969; Yoshida et al., 1982; Li and Wang, 2015). The yeast A. adeninivorans LS3 has been described as a microorganism able to utilize some hydroxylated aromatic acids such as tannic acid, gallic acid, protocatechuic acid, as a sole source of carbon and energy (Sietmann et al., 2010). The authors described the pathways for the degradation of gallic acid, protocatechuic acid and 4 hydroxybenzoic acid in the LS3 wildtype strain. However, the enzymes involved in the degradation of these compounds have

not been identified and characterized. The complete genome data of A. adeninivorans has been available since 2014 (Kunze et al., 2014), which has allowed detailed studies of the tannic acid degradation pathway.

For the first time, it has been demonstrated that deletion of the gene encoding gallic acid decarboxylase causes changes in the morphology of A. adeninivorans cells. This organism is a dimorphic microorganism but the exact physiological regulation of dimorphism is still unknown. The only factor known to initiate a change of cell shape from budding cells to mycelia is a temperature above 42◦C. It is not clear why the lack of this enzyme causes the change in morphology and it is not known if deletion of further enzymes involved in this pathway will have a similar effect. Gallic acid decarboxylase plays very important role in the metabolism of tannins and phenol derivatives, which are toxic for cells and the lack of this enzyme may cause cell stress and possible changes in shape. It is also possible that the enzyme is somehow connected with a signaling pathway which is controlled by temperature allowing mycelia to form at 30◦C. Finally, it is possible that the enzyme plays some other important roles which are still not known and further study is required to explain the enzymes role in the dimorphism of A. adeninivorans.

Gallic acid decarboxylase is one enzyme of this pathway that transforms gallic acid to pyrogallol. Gallic acid decarboxylases described so far are known for their instability due to a high oxygen sensitivity, and only a few have been successful purified (Zeida et al., 1998; Jiménez et al., 2013). Some gallic acid decarboxylases have been partially characterized, whereas the rest of them remain uncharacterized due to their complete loss of activity after purification (Grant and Patel, 1969; Brune and Schink, 1992; Yoshida et al., 2010). In this study gallic acid decarboxylase from A. adeninivorans fused with C-terminal HisTag region (Agdc1-6hp) has been successfully purified and characterized for the first time. Selection of the appropriate buffer and pH seems to be crucial for the storage of purified Agdc1- 6hp. The enzyme stored in crude extract at 4◦C for 5 days led to 42% loss of activity which is similar to the results obtained by Zeida et al. (1998). In 50 mM potassium phosphate buffer (pH 6) the purified enzyme retained 60% activity after 9 days at 4◦C. A decrease in enzyme activity was not observed after one-month storage at −80◦C. This suggests the possibility for long-term storage of Agdc1-6hp. The native molecular mass is approximately 27 kDa which indicates a monomeric enzyme. This is uncommon in comparison to known bacterial gallic acid decarboxylases, some of which consist of six subunits (Zeida et al., 1998; Jiménez et al., 2013) and decarboxylases from Bacillus megaterium and Clostridium hydroxybenzoicum possess a minimum two enzyme subunits (He and Wiegel, 1995, 1996; Omura et al., 1998).

Several metal ions were tested as potential cofactors for Agdc1p. Almost all metal ions had negative influence and some like Fe2<sup>+</sup> or Ni2<sup>+</sup> completely inhibited the activity. Na2S2O<sup>3</sup> used as stabilizer for gallic acid decarboxylases from Pantotea agglomerans T71 or Lactobacillus plantarum WCFS1 did not show any stabilization effect on Agdc1p. Instead, it resulted in decrease of activity up to 85% (Zeida et al., 1998; Jiménez et al., 2013). Addition of DTT and ascorbic acid to the reaction mixture exhibited 107 and 116% of relative activity respectively, compared to the control. Addition of EDTA alone led to 146% relative activity which is in contrast to the inhibitory effect of EDTA on gallic acid decarboxylase observed in P. agglomerans T71 (Zeida et al., 1998). Because the purification procedure was carried out on Ni Sepharose column, it is possible that the EDTA does not directly affect the enzyme but acts by chelating the nickel residues after purification, which could have inhibitory effects on the enzyme activity. However, it is also possible that EDTA positively affects the enzyme. In vitro investigation of kinetic and substrate specificity of Agdc1-6hp indicated that the enzyme has four times higher affinity for gallic acid than for protocatechuic acid. The K<sup>m</sup> value for gallic acid is 0.7 ± 0.2 mM and 3.2 ± 0.2 mM for protocatechuic acid. In comparison, the K<sup>m</sup> for gallic acid is 0.96 mM for gallic acid decarboxylase from P. agglomerans T71 which is unable to decarboxylate other gallic acid analogs (Zeida et al., 1998). The ability of Agdc1-6hp to decarboxylate other hydroxylated aromatic acids in vitro has not been confirmed. Most of the gallic acid decarboxylases, which have been characterized have a limited substrate spectrum (He and Wiegel, 1995, 1996; Zeida

0.2% gallic acid, protocatechuic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid) as carbon source. Measurements were made in triplicate. (C) Summarize of growth behavior of A. adeninivorans on different hydroxylated aromatic acids as sole source of carbon.

et al., 1998). The 3,4-dihydroxybenzoate decarboxylase from Enterobacter cloacae P can decarboxylate protocatechuic acid, but not gallic acid (Yoshida et al., 2010).

The cultivation of the control strain, A. adeninivorans G1212/YRC102, on YMM-glucose-NaNO<sup>3</sup> did not reveal gallic acid decarboxylase activity during the cultivation. This suggests that Agdc1p is an enzyme encoded by an inducible gene. Nested qRT-PCR assays confirmed the inducibility of the AGDC1 gene in presence of gallic acid and also protocatechuic acid. In contrast, glucose and the other selected hydroxybenzoic acid compounds were not able to induce the AGDC1 gene. The most vigorous gallic acid degrading microorganisms contain inducible GDC genes (Yoshida and Yamada, 1985; O'Donovan and Brooker, 2001; Gauri et al., 2013; Jiménez et al., 2013; Li and Wang, 2015; Reverón et al., 2015). An exception is Klebsiella aerogenes where decarboxylation of gallic acid, protocatechuic acid and 2.5 dihydroxybenzoic acid were shown to be constitutive (Grant and Patel, 1969).

A. adeninivorans tolerates as much as 2% (w/v) gallic acid however, regardless of the gallic acid concentration, the growth curves exhibit long lag phases. Since gallic acid is a weak acid, its presence results in the lowering of the pH value of the culture medium a moderate amount e.g. supplementation with 1% (w/v) gallic acid lowered the pH value to approximately 3.8. At this pH, gallic acid is undissociated and can go across the yeast cell membrane. Inside the cell, the higher pH causes the gallic acid to dissociate, which reduces the pH<sup>i</sup> to below the physiological range of the cell and thus interferes homeostasis. Both factors generate antimicrobial properties of hydroxylated aromatic acids, generating cell stress, extending the lag phase and inhibiting cell growth (Noda et al., 1982; Restaino et al., 1982; Beales, 2003; Ibraheem and Ndimba, 2013). An experiment which cultivated A. adeninivorans on a medium with a constant pH 6 (optimal for growth of A. adeninivorans) showed that cells were unable to degrade gallic acid resulting in cell death (data not showed). The overexpression of AGDC1 did not shorten the

lag phase or increase the cell growth period significantly over 120 h (**Figure 7**). Also, the activity of Agdc1p was lower and appeared later in comparison to cultivation on glucose. These results suggest a stress response at the level of gene expression generated by gallic acid. A longer log phase was also observed when A. adeninivorans was cultivated on other hydroxylated aromatic acids as sole source of carbon which indicates a similar effect on the cells (**Figure 9**).

The deletion of AGDC1 in A. adeninivorans genome results in cell death when cells were cultivated on gallic acid (**Figure 8**). This suggests that there is no other pathway able to degrade gallic acid which is supported by extracellular metabolite analysis of the control strain, A. adeninivorans G1212/YRC102, which showed only the production of pyrogallol. In the previous study Sietmann et al. (2010) reported that in A. adeninivorans, oxidative and non-oxidative decarboxylation of protocatechuic acid occur simultaneously; indeed the G1234 [1agdc1] was still able to grow and degrade protocatechuic acid. Analysis of the metabolites in culture medium showed that control strain and the overexpression strain produced catechol and cis,cis-muconic acid. Trace amounts of 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 1,4-dihydroxybenzene and 1,3,4-benzentriol were also detected. Our investigations have confirmed that the degradation of protocatechuic acid involves gallic acid decarboxylase and other enzymes.

To confirm which compounds are produced during the cultivation of A. adeninivorans on protocatechuic acid, further investigations are required. Cultivation of A. adeninivorans G1234 [1agdc1] on media containing different hydroxylated aromatic acids as sole source of carbon revealed that only cultivation on gallic acid led to cell death (**Figure 9**). This indicates that gallic acid decarboxylase is the key enzyme in the degradation of gallic acid by A. adeninivorans. Furthermore, we determined that all of the tested aromatic acids except 2,3 dihydroxybenzoic acid can serve as sole source of carbon for A. adeninivorans. However, the first step of degradation is not carried out by gallic acid decarboxylase. Considering all aspects of growth and gene expression analysis, it seems that the tannic acid degradation pathway is used exclusively for tannins and gallic acid. Protocatechuic acid can be degraded also by this pathway, but the other aromatic acids are degraded through a separate pathway(s).

Interestingly the overexpression of AGDC1 did not increase the accumulation of extracellular pyrogallol when cells were cultivated on glucose and gallic acid. The yield was approximately the same for both the control and the overexpression strain. The reason could be that most extracellular pyrogallol is accumulated in the first 24 h of cultivation. At this time, the cells are under strong stress conditions created by gallic acid. This results in low gene expression and low recombinant enzyme content. Probably the pre-adaptation of cells on low gallic acid concentration increases the yield of pyrogallol in the strain with overexpression of AGDC1. To obtain a higher yield of extracellular pyrogallol, further engineering of A. adeninivorans and optimization of the cultivation conditions are necessary.

Unexpectedly, the overexpression of gallic acid decarboxylase contributed to the accumulation of cis,cis-muconic acid. The yield increased almost by 71.2% over the control which suggests that A. adeninivorans could be an efficient synthesizer of this difficult to produce molecule. Nevertheless, further investigation and optimization are necessary to demonstrate this possibility.

The microarray data revealed the genes involved in the degradation of gallic acid. The AGDC1 and catechol-1,2 dioxyenase genes encode the first two enzymes involved in the degradation of gallic acid which cleave the aromatic ring, resulting in the production of pyruvate and acetaldehyde. This is similar to the situation in Eubacterium oxidoreducens and Pelobacter acidigallici (Krumholz et al., 1987; Brune and Schink, 1992). The highest upregulation of genes in the presence of gallic acid are found in the ß-oxidation pathway. It is still an open question whether or not transporters are involved in the transport of gallic acid into A. adeninivorans cells. Microarray data did not exhibit any potential genes which could be recognized as gallic acid transporter(s) and there is little in the literature concerning the transport of hydroxylated aromatic acids in microorganisms. Two possibilities exist, either A. adeninivorans does not have transporters, and therefore gallic acid couldn't enter the cells at pH 6 because it is in the dissociated form or there exist pH dependent transporter(s), which are not yet annotated in the genome.

In this study, physiological and transcriptome analysis was used to understand the catabolism of hydroxylated aromatic

#### REFERENCES


acids in A. adeninivorans. We confirmed that the genes encoding enzymes involved catabolic pathway of gallic acid are induced and aromatic acid substrates are the inducers. The construction of gallic acid decarboxylase disruption mutants revealed that gallic acid decarboxylase Agdc1p is the only enzyme responsible for transformation of gallic acid. However, other enzymes were demonstrated to be responsible for the transformation of other hydroxylated aromatic acids.

Gallic acid decarboxylase affinity for gallic acid, its substrate specificity and the possibility of long-term storage of the Agdc1p will be advantageous if the enzyme were to be commercialized. The enzyme may have useful industrial applications e.g. in bioremediation processes or in chemical synthesis. Further culture optimization as well as strain engineering could also contribute to more efficient conversion of protocatechuic acid to cis,cis-muconic acid which may also have industrial application.

#### AUTHOR CONTRIBUTIONS

AM and EB carried out the construction of the E. coli and A. adeninivorans strains, enzyme activities determination and Agdc1p analysis. AM prepared the quantitative reverse transcriptase PCR analysis. AM, AH, MM, US, and SW participated in microarray design, hybridization, gene annotation and gene expression analysis as well as visualization, AM and JR in GC–MS analysis. MM and AM prepared the microscopic analysis. RB and FS gave several useful suggestions. AM, KB, and GK drafted the manuscript. All authors read and approved the final manuscript.

#### ACKNOWLEDGMENTS

The research work was supported by grant from the Marie Curie Actions–Initial Training Networks (Grant No. FP7-PEOPLE-2013-ITN).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01777/full#supplementary-material

the yeast Arxula adeninivorans. Appl. Microbiol. Biotechnol. 84, 583–594. doi: 10.1007/s00253-009-2167-5


in Azotobacter sp. SSB81. J. Appl. Microbiol. 114, 84-95. doi: 10.1111/jam. 12030


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Meier, Worch, Böer, Hartmann, Mascher, Marzec, Scholz, Riechen, Baronian, Schauer, Bode and Kunze. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives

Himadri Bose and Tulasi Satyanarayana\*

*Department of Microbiology, University of Delhi, New Delhi, India*

All the leading cities in the world are slowly becoming inhospitable for human life with global warming playing havoc with the living conditions. Biomineralization of carbon dioxide using carbonic anhydrase (CA) is one of the most economical methods for mitigating global warming. The burning of fossil fuels results in the emission of large quantities of flue gas. The temperature of flue gas is quite high. Alkaline conditions are necessary for CaCO<sup>3</sup> precipitation in the mineralization process. In order to use CAs for biomimetic carbon sequestration, thermo-alkali-stable CAs are, therefore, essential. CAs must be stable in the presence of various flue gas contaminants too. The extreme environments on earth harbor a variety of polyextremophilic microbes that are rich sources of thermo-alkali-stable CAs. CAs are the fastest among the known enzymes, which are of six basic types with no apparent sequence homology, thus represent an elegant example of convergent evolution. The current review focuses on the utility of thermo-alkali-stable CAs in biomineralization based strategies. A variety of roles that CAs play in various living organisms, the use of CA inhibitors as drug targets and strategies for overproduction of CAs to meet the demand are also briefly discussed.

Keywords: global warming, polyextremophilic microbes, thermo-alkali-stable, carbonic anhydrase, biomineralization, CA inhibitors

#### INTRODUCTION

In the early Eighteenth century, industrial revolution took the world by storm. This led to large scale manufacture, which at that time proved to be a major economic boost world over. In the last 300 years, there has been a marked transformation in human life. Due to improved farming practices, food production increased. This along with technological advances in health, communication and transport sectors paved the way toward modern age for human civilization. This modernization led to a heavy toll on "mother nature" (Shakun et al., 2012).

The concentrations of greenhouse gases are increasing day by day mainly due to anthropogenic activities, of them about two-thirds is contributed by fossil fuels. The burning of fossil fuels results in the emission of large quantities of flue gas that contains ∼ 71% N2, 14% CO2, 1–2% hydrocarbons, carbon monoxide, NOx, and minor amounts of SO<sup>x</sup> (Perry and Gee, 1995). According to IPCC, among all the greenhouse gases, CO<sup>2</sup> is emitted most (65% from fossil fuels and 11% from forestry). Other gases such as methane (16%), nitrous oxide (6%), and fluorinated gases (2%) are emitted in smaller amounts by anthropogenic activities (IPCC, 2014). Cumulative carbon emissions from different sectors have increased by

#### Edited by:

*Rajesh K. Sani, South Dakota School of Mines and Technology, United States*

#### Reviewed by:

*Navanietha Krishnaraj Rathinam, National Institute of Technology, Durgapur, India Saurabh Dhiman, South Dakota School of Mines and Technology, United States*

> \*Correspondence: *Tulasi Satyanarayana tsnarayana@gmail.com*

#### Specialty section:

*This article was submitted to Microbiotechnology, Ecotoxicology and Bioremediation, a section of the journal Frontiers in Microbiology*

> Received: *28 April 2017* Accepted: *08 August 2017* Published: *25 August 2017*

#### Citation:

*Bose H and Satyanarayana T (2017) Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives. Front. Microbiol. 8:1615. doi: 10.3389/fmicb.2017.01615*

**145**

about 40% since 1970s. The concentrations of hazardous gases such as NOx, SOx and methane are well beyond their threshold in many cities all over the world, thus, the Air Quality Index (AQI) is declining (IPCC, 2000). CO<sup>2</sup> levels in the atmosphere have surged past the threshold of 400 ppm and it may not climb down for generations. This 400 ppm benchmark was broken first time in the recorded history last year. According to World Meteorological Organization (WMO), 2016 would be the first full year to exceed the mark (NOAA, 2016). As per the latest measurement by NOAA in April 2017, the concentration of CO<sup>2</sup> at present is 406.17 ppm (NOAA, 2017). Some of the isolated places (Arctic regions) have already breached this mark in the past few years as recorded in Mouna Loa Observatory in 2013 (IPCC, 2013). Emission Database for Global Atmospheric Research stated that global emission of CO<sup>2</sup> has increased by 48% in the last two decades (http://edgar.jrc.ec. europa.eu/ overview.php?v)<sup>1</sup> . The increase in GHG emissions has led to increase in earth's surface temperature by about 2 ◦C from pre-industrial times (IPCC, 2000). These conditions have also led to widespread natural calamities and affected the environment adversely. An increase in warm temperature extremes and decrease in cold temperature extremes have been noted in the past few years. This has also led to an impact on the precipitation patterns around the world and disturbed the water cycle. Agricultural production has been affected due to adverse climatic changes. There has been a reduction in crop yields leading to increase in food prices, food shortage and insecurity (Adams et al., 1998). These climatic hazards are affecting the lives of people round the world particularly those who are living below the poverty line. Many freshwater, marine and terrestrial species are already on the verge of extinction. A change in the distribution and interaction pattern has been observed in many freshwater and marine phytoplanktons. According to IPCC, the period from 1983 to 2012 were the warmest years (IPCC, 2014).

Air pollution causes various respiratory and cardiac diseases. Tiny particles produced by vehicular engines and industry worsen heart and brain related disorders and increase the risk of stroke. Global warming is taking the earth toward peril and it is essential to tackle this catastrophe for our survival. It is next to impossible for developing and under developed nations to control large scale CO<sup>2</sup> emissions. Nevertheless, global warming has to be mitigated. Scientific and global consensus on global warming and climate change has brought the world powers together in order to hunt for new technologies for mitigating the global warming (Kheshgi et al., 2012).

The year 2015 ushered in an era of optimism and action with Paris climate change agreement. It also marks a new era of climate change reality with record levels of high greenhouse gases. In order to tackle increasing carbon emissions, carbon trading and taxation have been implemented by various countries (Princiotta, 2007). The progress made in developing carbon capture technologies has been reviewed from time to time (Boone et al., 2013; Frost and McKenna, 2013). IPCC has also published a wholesome review on different CCS technologies providing a precious input for policy makers and researchers in developing schemes for reducing GHG emissions (IPCC, 2000, 2013, 2014). Some reviews have also outlined various holistic approaches for carbon capture and also described methods for mitigating global warming. This review discusses biomimetic approach for mitigation of global warming with a special focus on utilizing nature's own catalyst, carbonic anhydrase (CA), as the biomimetic carbon sequestering agent.

#### APPROACHES FOR MITIGATING GLOBAL WARMING

Countries round the world have initiated a number of measures to counter the alarming rise of CO<sup>2</sup> in the environment which includes use of low carbon fuels (nuclear power, natural gas etc.), increasing the use of renewal energy and applying geo-engineering approaches [afforestation and reforestation, and CO<sup>2</sup> capture, storage and utilization (CCSU)]. All these approaches have their own set of merits and demerits. It is impossible to curtail the carbon emissions by using only one of the measures. The major drawback of these measures is very high capital investments as non-conventional energy sources are associated with high cost and at times they are not user friendly as well (Faridi and Satyanarayana, 2015). Among them, CCSU can reduce the carbon emissions by 80–85% by capturing CO<sup>2</sup> directly from power plants and energy intensive processes such as cement industries, which are described in detail (Boot-Handford et al., 2014; Leung et al., 2014) in the following sections.

#### CARBON CAPTURE AND STORAGE (CCS)

## CO<sup>2</sup> Capture

CO<sup>2</sup> emissions are captured and separated from flue gas, transported via huge pipelines and stored permanently deep underground or reutilized for various applications. Various approaches have been designed for efficient CO<sup>2</sup> capture. These approaches are basically divided into three phases:


In pre-combustion CO<sup>2</sup> capture, the fuel is treated initially before combustion as in the case of coal or natural gas. Coal undergoes gasification process resulting in the formation of syngas (CO + H2). The reaction takes place in low O<sup>2</sup> environment. Further, this syngas (which is free from pollutants) undergoes water gas shift reaction to form CO<sup>2</sup> and H2O. This technique can be applied in power plants using coal as a fuel source, but it incurs an efficiency loss of 7–8%. Natural gas containing mainly methane (CH4) can be converted to syngas and rest of the procedure is same like that of coal (Olajire, 2010). In post-combustion CO<sup>2</sup> capture, carbon is captured from flue gas generated after combustion. This technique has been considered useful for the existing power plants and approved in small scale with a CO<sup>2</sup> recovery rate of 800 t/day (Wall, 2007). Oxyfuel combustion uses oxygen in place of air for combustion. This substantially reduces the NOx load in the exhaust gas. Hence

<sup>1</sup>Emission database for global atmospheric research EDGAR, European Commission. Available online at: http://edgar.jrc.ec.europa.eu/ (Accessed February 27, 2017).

the remaining gas only contains SO2, water and CO2. SO<sup>2</sup> can be easily removed by precipitation or desulphurization leading to about 80–98% recovery of CO2. This process requires large quantities of pure O<sup>2</sup> that increases overall cost of the process. The presence of a large amount of SO<sup>2</sup> in flue gas would cause corrosion problems (Buhre et al., 2005; Pfaff and Kather, 2009).

CO<sup>2</sup> separation from flue gas is carried out by various ways such as absorption [liquid sorbents such as monoethanolamine (MEA), diethanolamine (DEA) used to separate CO2], adsorption (solid adsorbent such as zeolite, activated carbon are used to bind CO<sup>2</sup> on its surface), chemical looping combustion (metal oxide is used as oxygen carrier for combustion), membrane separation (allows only CO<sup>2</sup> to pass through while trapping other components), hydrate based separation (flue gas exposed to high pressure of water, leading to formation of hydrates and CO<sup>2</sup> gets trapped within them) and cryogenic distillation (distillation at low temperature and high pressure), thereby separating the solidified CO<sup>2</sup> (Aaron and Tsouris, 2005; Knudsen et al., 2009; Bhown and Freeman, 2011).

## CO<sup>2</sup> Transport

Large pipelines are used in transport of CO<sup>2</sup> to storage sites or to facilities where it can be reutilized. Pipelines are the most efficient and viable method of CO<sup>2</sup> transport to long distances. Various factors have to be considered for the proper transport of CO2. Mass/volume ratio is optimized by transporting the CO<sup>2</sup> in supercritical conditions (i.e., pressure is maintained at 72.1 atm and temperature around 32◦C; Svensson et al., 2004). Impurities in the CO<sup>2</sup> stream leads to alteration in pressure and temperature, thus, impurities must be taken care of. The formation of carbonic acid due to the presence of water leads to corrosion of the pipelines, which are mostly made of carbon steel. Large integrated network of pipelines are needed for a commercial CCS project (Aspelund et al., 2006). These large networks will help in decreasing overall length of the pipeline by 25%, thereby reducing the associated cost. The cost of transport also depends on the regional and economic conditions of a country. A cost analysis in China showed that for a mass flow of 4,000 t CO2/day, the use of ship tankers will cost 7.48 USD/tonne CO<sup>2</sup> as compared to 12.64 USD/tonne CO<sup>2</sup> for railway tankers and 7.05 USD/tonne CO<sup>2</sup> for 300 km pipelines. Periodical monitoring and assessment of the pipelines is needed for efficient and regular transport (Gao et al., 2014).

#### CO<sup>2</sup> Storage: Geological Storage

Carbon dioxide can be stored into rock pores 800 m below the surface in abandoned deep saline aquifers and oil or gas reservoirs. This is called geological carbon sequestration (Orr, 2009). The choice of a good sequestering site is always useful for long term carbon storage. Porous and permeable surfaces having non-potable ground water or the sites having sedimentary rock formations made of such chemicals (e.g., calcium) can react with CO<sup>2</sup> leading to more stable formations, which are preferable sites for storage (Benson et al., 2005). The first large scale CO<sup>2</sup> sequestration project which started in 1996 was called Sleipner, located in the North Sea where Norway's Statoil Hydro strips carbon dioxide from natural gas with amine solvents and disposed of carbon dioxide in a deep saline aquifer. CO<sup>2</sup> can be transported in high pressures into nearly depleted oil/gas wells in order to extract the residual oil and gases, while the injected CO<sup>2</sup> remains stored (enhanced oil recovery). CO<sup>2</sup> can be injected into deep coal beds to release methane which is trapped in porous structure of coal seams (CO2–enhanced coal bed mehane). Deep aquifers at a depth of 700–1,000 m below ground level contain high salinity brines, which can be used to store injected CO<sup>2</sup> captured from CCS process (Hart and Gnanendran, 2009).

## Deep Ocean Storage

Oceans are the natural sinks for CO<sup>2</sup> and it can be injected into the ocean by direct injection and ocean fertilization. Oceans can take up about 2 billion metric tons of CO<sup>2</sup> per annum, the amount of carbon that would double the load in the atmosphere and would increase the concentration in the deep ocean by only 2%. At depths of more than 3 km, CO<sup>2</sup> gets liquefied and can easily sink to the sea floor due to its higher density than the adjoining seawater and can be stored there for a long time. Beside these, three other mechanisms have also been proposed for CO<sup>2</sup> storage. Physical trapping of CO<sup>2</sup> using immobilization in a gaseous or supercritical phase in geological formations causes its immobilization that leads to either static trapping in structural traps or residual gas trapping in porous structure. Chemical trapping in formation fluids (water/hydrocarbon) either by ionic trapping or by dissolution, where CO<sup>2</sup> can react chemically with minerals after dissolution to form mineral trappings or it can get adsorbed on mineral surface (adsorption trapping). Hydrodynamic trapping through the upward migration of CO<sup>2</sup> at very slow speed results in its trapping into the intermediate layers. In this manner, large quantities of CO<sup>2</sup> can be stored (Benson et al., 2005). Polymer microcapsules composed of liquid carbonate cores, which have high surface area for controlled uptake of CO2, have been utilized for large scale carbon capture process. Higher adsorption of carbon dioxide was achieved and these microcapsules were found to be stable under typical industrial operating conditions (Vericella et al., 2015).

## Demerits of CCS Technology

Although, CCS approaches are quite efficient, they still have certain implications. First of all the cost of the overall process is quite high. It has been estimated that CCS will lead to a rise in power tariff by about 10% in US alone. It also requires a large amount of energy as it consumes 25 percent of the power plant's output capacity. The use of MEA in capture methods may lead to corrosion, evaporative losses, generation of toxic degradation products, and may require significant energy to remove CO<sup>2</sup> during sorbent regeneration (Nielsen et al., 2012; Reynolds et al., 2012; Da Silva and Booth, 2013). There are some other disadvantages associated with the post-combustion amine capture method. The equipment will be very large as compared to the small size of a coal-fired power plant. Large volumes of solvents are needed. Regeneration of solvent by heating produces toxic byproducts, which should be scrubbed and eliminated. This

process also utilizes large volumes of water (Herzog, 1998). In this approach, the energy utilization increases by about 15–25%. During transport and storage of CO2, there is a risk of leakage. Direct injection of CO<sup>2</sup> will lead to the acidification of deep ocean (Dutreuil et al., 2009) which may cause disastrous effects on marine ecosystem. That's why only geological storage of CO<sup>2</sup> into deep saline aquifers and unused mines is somewhat acceptable (Shahbazi and Nasab, 2016). The CCS technology is still in pilot scale and a large scale commercialization is yet to be undertaken. Way back in 2010, Tsouris et al. (2010) pointed high costs associated with CCS technology and urged the world to direct its energies on alternative energy technology. It is also true that in near future it will be very difficult to move away from fossil fuels as energy source. Biomineralization of CO<sup>2</sup> using metal oxides (MgO and CaO) with the help of ubiquitous biocatalyst carbonic anhydrase (CA) provides a cost effective and environmentally benign solution for mitigation of global warming, which will be discussed in detail in the ensuing sections.

#### Mineralization of Atmospheric Carbon

This process mimics the mineralization process occurring in nature which is responsible for the presence of huge amounts of limestone on the surface of Earth. This is called silicate weathering. It traps the atmospheric carbon by reacting with large limestone rocks such as wollastonite (CaSiO3), serpentine (Mg3Si2O5(OH)4) and olivine (Mg2SiO4) (Huijgen et al., 2007; Santos et al., 2007). This process occurs in both salt and fresh waters as CO<sup>2</sup> gets dissolved in water easily and there exists equilibrium between CO2, HCO<sup>−</sup> 3 , and CO2<sup>−</sup> 3 . The set of reactions involved in CO<sup>2</sup> mineralization is outlined below (Farrell, 2011):

Gaseous CO<sup>2</sup> dissolves quickly in water and produces a loosely hydrated aqueous form (1).

$$\text{CO}\_2\text{ (g)} \rightarrow \text{CO}\_2\text{ (aq)}\tag{1}$$

Then carbonic acid is formed when aqueous CO<sup>2</sup> reacts with water (2).

$$\rm{CO\_2\ (aq)} + \rm{OH^-} \rightarrow \rm{H\_2CO\_3} \tag{2}$$

In the 2nd step, carbonic acid breaks down into carbonate and bicarbonate ions [(3), (4)]

$$\text{H}\_2\text{CO}\_3 \rightarrow \text{wHCO}\_3^- + \text{H}^+ \tag{3}$$

$$\rm{HCO\_3^- + OH^- \to \rm CO\_3^{2-} + H\_2O} \tag{4}$$

The presence of metal ions such as Ca2+, Mg2+, and Fe2<sup>+</sup> drives the precipitation of carbonate into mineral carbonates as depicted below (5):

$$\begin{array}{l} \text{CO}\_{3}^{2-} + \text{Ca}^{2+} \rightarrow \text{CaCO}\_{3} \downarrow \text{ (calcite)}\\ \text{CO}\_{3}^{2-} + \text{Mg}^{2+} \rightarrow \text{MgCO}\_{3} \downarrow \text{ (magnesite)}\\ \text{CO}\_{3}^{2-} + \text{Ca}^{2+} + \text{Mg}^{2+} \rightarrow \text{CaMg(CO}\_{3}\text{)}\_{2} \downarrow \text{ (dolomite)}\\ \text{CO}\_{3}^{2-} + \text{Fe}^{2+} \rightarrow \text{FeCO}\_{3} \downarrow \text{ (siderite)} \end{array}$$

This process is pH dependent. At pH below 8.0, reaction 2 becomes insignificant as OH<sup>−</sup> ions are absent. Between pH 8.0 and 10.0, both the reactions (2 and 3) occur, and above pH 10, reaction 2 occurs mainly. Due to abundant supply of OH<sup>−</sup> at alkaline pH, mainly HCO<sup>−</sup> 3 (bicarbonate) and CO<sup>−</sup> 3 form leading to CaCO<sup>3</sup> precipitation. Also at acidic pH, the solubility of carbonate increases. In order to increase carbonate precipitation, it is necessary to make the environment alkaline. Mineral carbonation is being studied at length for its utility in biomineralization of CO<sup>2</sup> from flue gas. Some pilot scale studies have already been undertaken to demonstrate the viability of the process (Reddy et al., 2010).

This technique has several advantages over other sequestration based approaches:


Despite being very effective, it has certain limitations. The process is very slow in ambient conditions (Haywood et al., 2001). According to the study of kinetics of calcite precipitation by Dreybodt et al. (1997), except at high pH, the formation of HCO<sup>−</sup> 3 (bicarbonate) is the rate limiting step. Equilibrium constants for reactions (2) and (3) are 2.6 × 10−<sup>3</sup> and 1.7 × 10−<sup>4</sup> , respectively (Mirjafari et al., 2007). The rate of reaction (3) and (4) is being virtually diffusion controlled and very rapid. If CO<sup>2</sup> hydration rate could be enhanced in some way or the other, then maximal amount of anthropogenic CO<sup>2</sup> can be converted into mineral carbonates. As it is said that the "Nature has solution to every problem," we are endowed with a natural solution to the climate change problem in the form of carbonic anhydrase (CA). The CAs can speed up the entire mineralization process by catalyzing the hydration of dissolved CO<sup>2</sup> into bicarbonate i.e., the reaction 2 at a faster rate (biomineralization). The addition of dolomite and K-feldspar to the soil can further enhance carbon sequestration in soil (Xiao et al., 2016). Use of CA as a potential biocatalyst has caught the attention of many researchers and much work has been done on exploring the possibilities of using this "Nature's own catalyst" for CCS (Farrell, 2011; Alvizo et al., 2014). Zinc(II) cyclen, which is a mimic of the enzyme carbonic anhydrase, was evaluated for its utilization in carbon capture process in rigorous conditions as that in industries and it was shown to be inhibited by bicarbonate accumulation (Floyd et al., 2013). There are some CA variants which can minimize bicarbonate inhibition by protecting the active site with a hydrophobic pocket. Hence, it is worthwhile to search for natural CA enzymes which can circumvent bicarbonate inhibition. Power et al. (2016) have successfully demonstrated the utility and efficiency of bovine carbonic anhydrase (BCA) and CO2-rich gas streams in the carbonation rate of brucite [Mg(OH)2], which is a highly reactive mineral. Carbonation was affected by decrement in CO<sup>2</sup> supply. In the following sections, the role of CA in CCS has been described in greater detail.

#### Carbonic Anhydrase: Vital Cog in the Wheel of Life

Carbonic anhydrases catalyze CO<sup>2</sup> hydration and HCO<sup>−</sup> 3 dehydration, in almost all organisms. It (EC No. 4.2.1.1) is a zinc metalloenzyme which is used as a catalyst in living systems for the conversion of carbon dioxide to bicarbonates and vice-versa. It was the first zinc metalloenzyme to be discovered in living systems (Smith and Ferry, 2000). Zinc ion complex facilitates carbon dioxide hydration activity. In most of the organisms, CAs are required for rapid processes, particularly transport processes. For example, it is required for the removal of CO<sup>2</sup> from lungs and for synthesis of eye secretions. CAs maintain optimum level of CO<sup>2</sup> and HCO<sup>−</sup> 3 in the body as they are utilized as substrate for many enzymatic reactions. It maintains acid—base balance in blood and helps in maintaining its physiological pH and also actively participates in ion transport and respiration. Mutations in CA genes can lead to osteoporosis and mental retardation. Carbonic anhydrase II (hCAII) is present in relatively high concentrations in red blood cells (Berg et al., 2002).

Initially the presence of the enzyme was found in the animal kingdom but later on CAs showed their signatures in all three living domains (Kaur et al., 2012; Di Fiore et al., 2015). This enzyme is either found intracellularly in cytoplasm or secreted outside (extracellular) associated with the periplasm and an essential component for survival of nearly all life forms. CAs are one of the fastest among the enzymes known having kcat in the order of 10<sup>6</sup> s −1 and kcat/K<sup>m</sup> in the order of 108M−<sup>1</sup> s −1 . An "anhydrase" is defined as an enzyme that catalyzes the removal of a water molecule from a compound, and so it is this "reverse" reaction that gives carbonic anhydrase its name, because it removes a water molecule from carbonic acid (Smith et al., 1999). In plants, β—CAs have a role in photosynthesis in chloroplasts by raising the concentration of CO<sup>2</sup> to enhance the carboxylation rate of ribulose 1, 5-bisphosphate carboxylase (RuBisCO) (Smith and Ferry, 2000). It functions in three modes: conversion of CO<sup>2</sup> to bicarbonate (to be utilized by RuBisCO in C<sup>4</sup> plants), conversion of bicarbonate into CO<sup>2</sup> [for fixation by phosphoenol pyruvate carboxylase (PEPC)] and also aids in facilitated diffusion by rapid equilibration between CO<sup>2</sup> and HCO<sup>−</sup> 3 . It also provides bicarbonate, which is required for the metabolism in plants (Monti et al., 2013). Recently β–CA has been shown to play a role in the perception of salicylic acid in Arabidopsi thaliana, suggesting its requirement in defense response (Medina-Puche et al., 2017). Besides higher organisms, CAs are also required in lower organisms. In some of the heterotrophs such as Propionibacterium, CAs help in CO<sup>2</sup> reduction during glycerol fermentation that results in the formation of oxaloacetate (Wood et al., 1941). In cyanobacteria and microalgae, CAs are involved in the CO<sup>2</sup> concentrating mechanism (CCM), which helps the cells to photosynthesize in the absence of inorganic carbon and also due to decline in levels of CO<sup>2</sup> in their surrounding environment (Badger and Price, 2003). CCM helps in maintenance of CO<sup>2</sup> levels around the RuBisCo active centers thereby improving the efficiency of Calvin cycle. CAs have also been reported from facultative anaerobes such as Rhodospirillum rubrum (Gill et al., 1984). CAs supply the cellular transporters with HCO<sup>−</sup> 3 by converting CO<sup>2</sup> penetrating the cells into HCO<sup>−</sup> 3 . Extracellular CA in alkaliphilic cyanobacteria plays a role in their survival in high alkaline conditions in alkaline soda lakes (Soltes-Rak et al., 1997; So et al., 1998; Kupriyanova et al., 2007, 2011). In Microcoleus and Rhabdoderma, CA doesn't allow CO<sup>2</sup> to leak out from the cell by converting it into bicarbonate, thus preserving the intracellular C<sup>i</sup> pool for photoautotrophic assimilation. In Rhabdoderma, out of two CAs present, one is bound to photosystem II (PSII) of thylakoid membranes. It participates in the light photosynthetic reactions, regulating operation of the water oxidizing complex via its protection against excess of protons, similar to luminal CA of microalgae and higher plants (Shutova et al., 2008). CA in cyanobacterial thylakoid membranes supplies CO<sup>2</sup> for photosynthesis in cyanobacteria. CA helps in the formation of oxaloacetic acid from carboxylase and phosphoenolpyruvate (PEP) carboxylase by providing bicarbonate which is utilized by pyruvate (Norici et al., 2002). This oxaloacetate is used for the synthesis of aspartate family of amino acids. Lysine production increases in elevated CO<sup>2</sup> conditions owing to the action CA and PEP carboxylase (Puri and Satyanarayana, 2010). CA in carboxysomal shell of chemolithoautotrophic cyanobacterium Halothiobacillus neapolitanus (CsoS3) supplies the active sites of RuBisCO with high concentrations of CO<sup>2</sup> necessary for RuBisCO activity and efficient carbon fixation (So et al., 2004). β–CA present in Escherichia coli is a major player in the cyanate degradation pathway of the organism. This type of CA has also been shown to play a role in pathogenesis of some bacteria such as Salmonella typhimurium (Valdivia and Falkow, 1997). In methanogens, CA plays an active role in acetate metabolism by converting the excess carbon dioxide produced into bicarbonates. It also helps in the conversion of acetate into methane. γ–class homologs of the CamH subclass are found in mitochondria, where it might have a role in the carbon transport system to increase the efficiency of photosynthetic carbon dioxide fixation (Tripp and Ferry, 2000). Hence CA and its classes play an important role in metabolic functions of all life forms. Life without CA is virtually out of question. CA levels are also correlated with aging (Cabiscol and Levine, 1995). A variety of diseases have been associated with such oxidative damage, which includes Parkinson's disease, diabetes, rheumatoid arthritis and alzheimer's disease. Classes of CA have been briefly described in the next section.

#### Types of CA and Mechanism of Action

There are basically six types of CAs discovered till now, namely α, β, γ, δ, ζ , and η. They don't have any specific sequence similarity, hence representing a classical case of convergent evolution (Lane and Morel, 2000; Smith and Ferry, 2000; Lapointe et al., 2008; Del Prete et al., 2014). Characteristic features of these classes are briefly described in **Table 1**.

Despite their structural differences, CAs have similar action mechanism. This mechanism has been widely studied in α–CAs. It is a two–step ping-pong reaction that catalyzes the reversible hydration/dehydration of CO<sup>2</sup> into bicarbonate and a proton (Silverman, 1982). A histidine residue present near the water molecule accepts a proton (H+) which gets released from the water molecule. It leaves only hydroxide ion attached to zinc ion. The active site has specific pockets for binding CO2, where it gets bound to the hydroxide ion. Further, nucleophilic attack on the carbonyl group by the zinc-bound hydroxide takes place that results in HCO<sup>−</sup> 3 formation. The enzyme is then regenerated and the bicarbonate ion is released (Lindskog and Coleman, 1973; Silverman and Lindskog, 1988).

$$\text{EZnOH}^- + \text{CO}\_2 + \text{H}\_2\text{O} \longleftrightarrow \text{EZnH}\_2\text{O} + \text{HCO}\_3^- \tag{1}$$

$$\text{EZnH}\_2\text{O} + \text{PSR} \longleftrightarrow \text{EZnOH}^- + \text{PSR} - \text{H}^+ \tag{2a}$$

$$\text{PSR}-\text{H}^+ + \text{B} \longleftrightarrow \text{PSR} + \text{B} - \text{H}^+ \tag{2b}$$

#### CA Specific Inhibitors and their Biological Relevance

CA abnormality leads to several diseases such as edema, glaucoma, hypertension, epilepsy, and cancer (Supuran, 2008). Pharmaceuticals that suppress the activity of carbonic anhydrase are classified as carbonic anhydrase inhibitors. Their clinical use has been established as diuretics, anti-glaucoma agents and antiepileptics in the management of gastric and duodenal ulcers, mountain sickness, neurological disorders, or osteoporosis (Supuran et al., 2003). Among human CA isoforms, CAIX and CAXII have already been identified as potent drug targets and molecular markers for the treatment of various types of cancer and tumors. Both show upregulated expression in cancerous cells as compared to normal cells. The two isoforms are required for maintaining intracellular pH of tumor cells. The inhibition of these two CAs is, therefore, important for cancer treatment. Several workers and companies have developed several ureido and sulfonamide based pharmaceuticals for cancer treatment which inhibit CAIX and CAXII (Cabiscol and Levine, 1995; Lomelino and McKenna, 2016).

#### Sulfonamide, Sulfamate, and Sulfamide Inhibitors

Sulfonamides (R–NH2SO2) constitute an important group of classical drugs which have been known for their pharmaceutical and CA inhibitory properties. The most effective are the heterocyclic sulfonamides (acetazolamide, methazolamide, ethoxzolamide). Acetazolamide is one of the well**-**known inhibitors of carbonic anhydrase and has rendered its effectiveness for the in vivo inhibition of intracellular CAs (Teicher et al., 1992). It is used in the treatment of glaucoma, epilepsy (rarely), idiopathic intracranial hypertension, and altitude sickness. Similarly other sulfanamide inhibitors such as dorzolamide and brinzolamide are used in the treatment


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of glaucoma as well. Near about 20 CA inhibitors have been granted FDA approval and they are being put to wide clinical usage (More et al., 1946; Sugrue, 2000; Frost and McKenna, 2013; Wulf and Matuszewski, 2013; Supuran, 2016a). Sulfamic acid (NH3SO3) and sulfamide (NH2SO2NH2) are the simplest CA inhibitors containing the –NH2SO<sup>2</sup> moiety. However, the sulfonamides are less effective against some of the β–CAs like Cab and also some of the γ–CAs (Zimmerman et al., 2004) and fungal MG-CA where they inhibit in millimolar/micromolar concentrations. Sulfamic acid and sulfamide are the weakest with inhibition constants (Ki) in the millimolar range. Interestingly, a completely different inhibition profile was observed against Zn-Cam and MG-CA, where sulfamic acid and sulfananilamide were shown to be more potent than sulfonamide inhibitors. Over the years various other inhibitors of CAs (benzenesulfonamides, arylbenzenesulfonamides, tetrafluorobenzenesulfonamides, 4-aminoethylbenzenesulfonamide) have been synthesized/discovered and their potential inhibitory properties have been evaluated (Pastoreková and Pastorek, 2004; Pala et al., 2014). Two CAs (LpCA1 and LpCA2) from Legionella pneumophila, a pathogenic bacterium, also get inhibited by sulfanamide inhibitors (sulfonylated aromatic sulphonamides, acetazolamide, ethoxzolamide, methazolamide, and dichlorophenamide). They can serve as a promising drug target for this pathogen (Supuran, 2016b). MGM Institute of Health Sciences has developed cerium oxide nanoparticles which are plant based that exhibit CA inhibitory and antioxidant properties (Lomelino and McKenna, 2016). An electrochemical enzyme inhibition biosensor, based on CA entrapped in a carbon paste electrode using carbon black nanoparticles and solid paraffin, was developed that measures sulfanilamide inhibition of CA (Bourais et al., 2017). This biosensor can even detect the sub-micromolar levels of sulfanilamide and the detection limit was 0.4µm (Bourais et al., 2017).

#### Novel Inhibitors

Coumarins, phenols, polyamines, fullerenes, boronic acid and their substituted derivatives have been effective against animal CAs (Innocenti et al., 2009b; Carta et al., 2012; Supuran, 2013). Their efficacy against microbial CAs, however, must be investigated. Carta et al. (2012) recently introduced dithiocarbamates (DTCs) as a new class of Zn-binding CA inhibitors, which can interact with the nearby amino acid residues for effective binding. Indeed these promising inhibitors have been effective even in sub-nanomolar concentrations. Nitroimidazoles decrease the pH of the hypoxic cancer cells, thereby helping in the uptake of chemotherapeutic agents. Scientists are also developing CA inhibitory antibodies for cancer treatment (Lomelino and McKenna, 2016). Incorporation of sugar moieties into sulfanilamide has resulted in sharp improvement in their solubility and effectiveness. The inhibition constants for ribose and galactose sulfanilamides against bsCA1 were reduced to 8.9 and 9.2 nM as compared to 2,500 nM for the unsubstituted sulfanilamide. In contrast to other sulfonamides and sulfanilamides, glycosylsulfanilamides have a good balance between its hydro- and liposolubility, therefore, it can easily penetrate through membranes to affect the growth of microorganisms (Supuran, 2015). Mete et al. (2016) synthesized a series of new thienyl-substituted pyrazoline benzenesulfonamides and showed their effective inhibition against hCAI and hCAII.

### Thermo-Alkalistable Carbonic Anhydrases

Advances in recombinant DNA technology have enabled us to modify/change a protein's structure and function as per the demand. As already stated, utilization of CAs in biomineralization of CO<sup>2</sup> requires the enzyme to be alkalistable as the mineralization is favored at alkaline pH (Farrell, 2011) and the temperature of flue gas is too high (around 140◦C), which is cooled to about 60◦C for post-combustion CO<sup>2</sup> capture. Hence, the enzymes needed for carbon sequestration must be thermostable. Several efforts have been made to modify the mesophilic CAs in order to make them thermo-alkali-stable. CO<sup>2</sup> Solutions Inc. has developed a thermally optimized CA by genetic engineering which is stable at 90◦C for 24 h (CO<sup>2</sup> Solutions Inc., 2012). Fisher et al. (2012) modified the surface amino acid residues of hCAII (which are far away from active site) to increase the thermostability by 6◦C. Some of the amino acid residues present at the surface (Tyr7, Leu224, Leu100, Leu240, Asn67, and Asn62) were replaced by Phe, Ser, His, Pro, Gln, and Leu, respectively. Directed evolution technique was utilized in order to develop a thermostable CA from β–CA from Desulfovibrio vulgaris. It can even retain activity at 107◦C. The enzyme was stable in the presence of primary flue gas contaminats such as NOx and SOx as well as in the presence of 4.2 M concentration of N-methyl-diethanolamine (MDEA) at 50◦C for about 14 weeks. The enzyme retains 40% residual activity at alkaline pH (11.8). This highly stable CA has been efficiently used for biomineralization of CO<sup>2</sup> at high temperature (87◦C) (Alvizo et al., 2014). A whole cell bacterial catalyst was generated from the CA of Neisseria gonorrhoeae (ngCA) by engineering it in such a way that it is secreted in the periplasm of E. coli. This whole cell catalyst was also found stable at low pH. It might be possible to sequester CO<sup>2</sup> more efficiently using whole cell enzyme systems even at a pH below the pKa of HCO3<sup>−</sup> or CO2<sup>−</sup> 3 , thereby reducing the cost of maintaining it at elevated pH (Jo et al., 2013). The modified CA thus generated by these strategies can aid in efficiently capturing CO2, but adds to the cost. It is worthwhile to search for enzymes that are stable at two/more extreme conditions. The use of thermo-alkali-stable CA from polyextrmophiles would simplify the process. Jo et al. (2016) engineered the de-novo sulfide bond of ngCA which resulted in enhancement in both kinetic and thermodynamic stabilities. The major reason for this enhancement is the loss of conformational entropy of the unfolded state, thereby increasing rigidity.

Recent advances in technology have enabled biologists to reach the extreme maxima of earth, sea and sky for exploring their diversity. Extremophiles have been isolated from a wide array of extreme environments like deep sea vents, hot springs, upper troposphere and stratosphere, outer space and others (Wilson and Brimble, 2009). There are also reports on the isolation of polyextremophiles from mines and industries (Onstott et al., 2003; Bhojiya and Joshi, 2012). Their characterization and applications have added new dimension to applied biology. Polyextremophiles are known to produce a variety of useful products (Coker, 2016)**.** Metagenomics and data mining studies have revealed the presence of α, β, γ CA genes in microbes from stressed environments. β– CA from Methanobacterium thermoautotrophicum (CabCA) and γ–CA from Methanosarcina thermophila (CamCA) were the first known CAs from extremophiles (Alber and Ferry, 1994; Smith and Ferry, 2000). Two novel and highly thermo-alkalistable α–CAs (SazCA and Ssp CA) have been discovered from thermophilic archaea Sulfurihydrogenibium azorense and Sulfurihydrogenibium yellowstonense. YO3AOP1, respectively. The former being one of the fastest known CAs till date (kcat/ <sup>K</sup><sup>m</sup> value of 3.5 <sup>×</sup> <sup>10</sup>8M−<sup>1</sup> s −1 ) (De Luca et al., 2013). SSp CA is even active after 3 h incubation at 70◦C. SazCA is also highly thermostable having 53 days and 8 days of halflife at 40◦ and 70◦C, respectively. It retains carbon dioxide hydration activity even after incubation at 80 and 90◦C for several hours (Russo et al., 2013). Both these enzymes are alkalistable (active at pH 9.6) and stable in the presence of flue gas contaminants such as NO2−, NO3−, and SO2<sup>−</sup> 4 (Vullo et al., 2012; De Luca et al., 2013). Two highly thermophilic bacteria isolated from hydrothermal vent ecosystems Persephonella marina EX-H1 (PMCA) and Thermovibrio ammonificans produced highly thermostable α–CA. The CA of T. ammonificans (TaCA) was more stable than SazCA and SspCA. This enzyme (taCA) showed thermo-stimulating properties (activity of taCA was elevated after the high temperature incubation (Jo et al., 2014). Faridi and Satyanarayana (2016a) reported a moderately thermostable and highly alkalistable α–CA(BhCA) from polyextremophilic bacterium Bacillus halodurans. It has a unique property of sulfate stimulation (its activity enhanced in the presence of sulfate ions). This property can be exploited for CCS as the flue gas contains SOx as one of the contaminants. BhCA did not get denatured in the presence of EDTA (Faridi and Satyanarayana, 2016b). Bose and Satyanarayana (2016) reported a moderately thermostable and alkalistable γ–CA from Aeribacillus pallidus, a polyextremophilic bacterium. Both ApCA and BhCA were stable in presence presence of bicarbonate (0.1M). Psychrobacter sp. SHUES1 isolated from frozen alkaline samples from Shanghai (China) produced carbonic anhydrase and urease which are important in microbially induced carbonate precipitation (MICP; Li et al., 2016). List of some of the thermoalkali-stable CAs which have been characterized in the last few years have been highlighted in **Table 2** with their characteristics.

Crystal structures of three of the most thermo-alkali-stable CAs were solved and their analysis revealed the reasons of their higher thermostabilities. As per the crystallographic structure, SspCA was found to have increased structural compactness. It contained a large number of charged residues on the protein surface with a greater number of ionic networks. These might be the key factors involved in the higher thermostability of this enzyme with respect to its mesophilic homologs. It has a fold which is characterized by a 10—stranded centrally placed β–sheet, which is surrounded by several helices and β– strands. A deep conical cavity that extends from the center to the protein surface harbors the active site. Several polar and hydrophobic interactions play active role in stabilization of SspCA. SazCA has a similar structure to that of SSpCA with minor differences. SspCA has Glu2 and Gln207 residues which are substituted with His2 and His207 in SazCA. This substitution is responsible for higher SazCA catalytic activity. The crystallographic structures of both SazCA and SspCA confirmed the dimeric nature of the enzymes (Di Fiore et al., 2013; De Simone et al., 2015). Crystallographic structure analysis of TaCA revealed it to be similar to the structure of other previously known bacterial homologs (Huang et al., 1998; Di Fiore et al., 2013; De Simone et al., 2015), but having entirely novel oligomeric pattern. Indeed TaCA forms a tetramer that comprises two dimers, which are structurally similar to that of SazCA and SspCA. The two dimers are joined together by two intermolecular disulfide bridges and by inter-subunit ionic interactions (James et al., 2014). This tetrameric state may be a possible reason for the enhanced thermostability of TaCA. Tahirov et al. (1998) recorded that thermostable enzymes have higher degree of oligomerization than mesophilic enzymes. It is also worthwhile to mention that the two conserved cysteine residues, Cys202 and Cys47, are reduced partially in SspCA and SazCA due to insufficiently oxidative expression conditions. It is suggested that the presence of two cysteine residues in TaCA leads to its increased stability (James et al., 2014). These structural analyses of thermostable CAs not only provide structural insights of the enzymes but also aid in their modification, so that they can be efficiently utilized for biomineralization.

#### Utilization of Thermo-alkali-stable CAs in Biomineralization

Several strategies are being developed for utilization of thermo-alkali-stable CAs in mitigating global warming. CO<sup>2</sup> Solutions Company has developed a CA based reactor for capturing CO<sup>2</sup> from different CO<sup>2</sup> intensive industries (http:// www.co\_2solutions.com/en/the-process). This process requires immobilized CA. Utilization of thermo-alkali-stable CAs for carbon capture requires a series of bioreactors which will have direct supply of flue gas. It is directly supplied to a bioreactor containing the immobilized enzyme. This enzyme in aqueous condition will hydrate CO<sup>2</sup> present in flue gas and releases HCO<sup>−</sup> 3 and H+. Alvizo et al. (2014) developed a process which utilizes a highly thermostable β–CA engineered from the β–CA of Desulfovibrio vulgaris. In this process, the set up consists of absorber column where carbon dioxide gets absorbed into MDEA; CA present in the column generates proton and bicarbonate. The residual flue gas is released and the rest (bicarbonate+amine+CA) is transferred to the next column maintained at 87◦C, where CO<sup>2</sup> is again regenerated, accompanied by regeneration of the solvent. Pure CO<sup>2</sup> is later reutilized and the solvent returns back to the initial absorber column for the next cycle. The rate of CO<sup>2</sup> absorption increases by about 25-fold in the catalyzed reaction as compared to the non-catalyzed reaction. Normal conditions for steadystate experiments were 2 l per minute of solvent, 180 l per minute of flue gas at 25◦C and absorber and desorber temperature at 87◦C. All enzyme–solvent mixtures were made


using normal tap water without any further treatment (Alvizo et al., 2014). CAs from Caminibacter mediatlanticus (CmCA) and Sulfurihydrogenibium yellowstonense YO3AOP1 (SspCA), both belonging to α–class, have been used to capture CO<sup>2</sup> from flue gas (Daigle and Fradette, 2014; Rossi, 2014). SspCA was characterized as prospective biocatalyst for CO<sup>2</sup> capture as it has regenerative absorption ability in alkaline conditions. Its prolonged half life (53 days at 40◦C and 8 days at 70◦C) making it one of the most suitable candidates for CCS. Ssp CA was, therefore, tested for its biomimetic carbon sequestration (Russo et al., 2013). Ssp CA immobilized in polyurethane foam (PU-SspCA) has been used in designing a bioreactor to mimic CO<sup>2</sup> capture process in industries (Migliardini et al., 2014). The immobilized CA (PU-SspCA) showed exceptional thermostability for very long duration even at 70◦C and was highly stable at 100◦C even after 48 h (Capasso et al., 2012). Heat stable carbonic anhydrases from M. thermophila and Caminibacter sp. have been used in bioreactors for efficient CO<sup>2</sup> removal from flue gases. Pyrococcus horikoshii and M. thermophila secreted γ–CA which was used in developing γ– CA nanoassemblies. These assemblies were developed by joining the single entitites to make multiple linked interactions with the surface of the reactor. Biotinylation sites were created by specifically mutating some of the residues to cysteines. Firm nanostructures were thus created by cross-linking biotinylatedγ–CAs with streptavidin tetramers (Salemme and Weber, 2014).

There are basically three different crystal phases for calcium carbonate (CaCO3), viz. calcite (rhombic), aragonite (needle like) and vaterite (spherical)]. Calcite phase is thermodynamically the most stable phase, while the vaterite is the metastable phase which is more soluble (Favre et al., 2009). When PMCA and TaCA (both α–CAs) were utilized for biomineralization; it resulted in the formation of stable calcite (Jo et al., 2014). α–CA from Dunaliella sp. produced 8.9 mg of calcite per 100 µg (172 U/mg) of enzyme in presence of 10 mM Ca2<sup>+</sup> (Kanth et al., 2012). Another thermo-alkali-stable γ– CA (ApCA) from polyextremophilic bacterium A. pallidus was used for biomineralization and well faceted rhombohedral calcite crystals were observed when viewed under scanning electron microscopy (SEM; **Figure 1**). The partially purified enzyme caused precipitation of 42.5 mg CO2<sup>−</sup> <sup>3</sup> mg−<sup>1</sup> protein (Bose and Satyanarayana, 2016). Optimization of the process parameters led to an increase in carbonate precipitation up to 200 mg CO2<sup>−</sup> <sup>3</sup> mg−<sup>1</sup> protein (Bose and Satyanarayana, 2017).

When a highly alkalistable and moderately thermostable α–CA (BhCA) from Bacillus halodurans was used for biomineralization, well defined calcite crystals were observed (Faridi and Satyanarayana, 2016a). ApCA and BhCA were also utilized for testing their efficacy in CO<sup>2</sup> sequestration from flue gas using vehicular exhaust (**Figure 2**). Vehicular exhaust has a comparable composition as that of flue gas emitted from the thermal power plants (∼71% N2, 14% CO2, 1–2%

formed in the control. (B) Rhombohedral and well faceted calcite crystals were formed in the presence of ApCA (adopted from Bose and Satyanarayana, 2016).

hydrocarbons, carbon monoxide, NOx, and minor amount of SO2; Perry and Gee, 1995). Both the enzymes were useful in sequestering CO<sup>2</sup> from vehicular exhaust, hence making them efficient candidates for biomineralization (Faridi and Satyanarayana, 2016b; **Figure 3**). Biomineralization process for ApCA and BhCA was performed at 37◦C in a 75 mL total reaction volume. CA (0.05 mg) was dispersed in 15 mL Tris buffer containing 0.9 g of CaCl2.2H2O in Tris (2.52 g) buffer according to a protocol used by Mirjafari et al. (2007). To initiate the mineralization process, 60 mL of CO<sup>2</sup> solution was added to the enzyme mix. The reaction was performed for 10 min at 37◦C. In an interesting study, the effect of CA from Bacillus mucilaginosus on carbonate formation and wollastonite dissolution were explored under variable CO<sup>2</sup> conditions. Real time PCR was used to analyze the correlation between CA gene expression, sufficiency or deficiency of calcium and CO<sup>2</sup> concentration. The findings reaffirmed the belief that CO<sup>2</sup> concentration is not related to effects of CA. Further, they also showed that microbial CA has some role in silicate weathering (Xiao et al., 2014). Some of the other alkalistable CAs have also shown efficacy in carbon sequestration. Another α–CA from Serratia sp., which was alkalistable in nature, was successful in generating calcite crystals (Srivastava et al., 2015). Some of the mesophilic CAs from some other microorganisms such as Pseudomonas fragi (27.33 mg CO2<sup>−</sup> 3 /mg of protein), Bacillus pumillus (25.43 CO2<sup>−</sup> 3 /mg of protein), M. lylae (24.02 CO2<sup>−</sup> 3 /HCO3−/mg of protein) (Prabhu et al., 2011) and Citrobacter freundii SW3 (Ramanan et al., 2009) [225 mg CO32−/mg of protein] also produced calcite crystals. Rhodobacter sphaeroides was genetically modified to harbor a surface displayed CA with inducible expression of phosphoenolpyruvate carboxylase led to very high CO<sup>2</sup> reduction efficiency and the production of several organic compounds (carotenoids, polyhydroxybutyrate, malic acid, succinic acid; Park et al., 2017).

#### Production and Over Expression of Carbonic Anhydrase Genes by Molecular Approaches

One of the key factors that determine the cost effectiveness of any biotechnological application is the availability and cost of the biocatalysts. Microorganisms are well known to produce CAs for use in CCS technologies. Researchers have also displayed the ability of many prokaryotic species such as planktonic Bacillus mojavensis cells for microbially enhanced CCS by mineral trapping and solubility trapping (Mitchell et al., 2010). The production levels of CAs are, however, quite low for industrial use. Hence the production parameters must be optimized. Statistical approaches such as Response surface methodology (RSM) and two factorial designs are some of the well-known methods for optimizing production of biocatalysts (Kumar and Satyanarayana, 2014). The production of carbonic anhydrase is growth associated; hence, optimization of growth parameters leads to high enzyme titres. Optimization of growth conditions by mono-factor tests with blank control and orthogonal design methodology resulted in 7-fold increase in CA titres in B. mucilaginosus K02 (Zhang et al., 2011). Bose and Satyanarayana (2016) reported about 4-fold increment in enzyme titres on optimization of growth parameters. In both the studies, the CA production was found to be synchronous to that of the growth of the bacterium. The CA production by the wild type strain becomes too cumbersome. The production of CA from thermoalkaliphilic microorganisms is difficult as it is not easy to provide accurate conditions [anaerobic, high temperature, pH (high/low) etc.] for their growth and enzyme production. Many archaeal life forms producing bioactive compounds need specialized growth conditions (Alber and Ferry, 1994; Mesbah and Wiegel, 2012). The advent of genome mining, metagenomics and bioinformatics approaches have enabled to use molecular biological tools for cloning and over expression of industrially relevant enzymes in prokaryotic (E. coli, Bacillus subtilis) and eukaryotic (Pichia

pastoris) hosts. Downstream processing becomes easy as the enzymes have various protein tags (e.g., His, GST) for easy purification of the recombinant proteins (Swartz, 2001). Since late 1990s, the prokaryotic CA genes from different strata have been cloned and the protein was over expressed in E. coli for analytical and application purposes. The first CA gene cloned from prokaryotes was α–CA (NCA) from N. gonorrhoeae, which encoded a protein of 28 kDa (Chirica et al., 1997 ˘ ). This enzyme was also expressed heterologously in E. coli as a periplasmic protein in order to use it as a whole cell biocatalyst (Jo et al., 2013). The yield of ∼106 mg L <sup>−</sup><sup>1</sup> with a specific activity of 3090 U mg−<sup>1</sup> in the pure protein was achieved (Kim et al., 2012).

Since then many other microbial CAs have been cloned and expressed in prokaryotic hosts. Many researchers have already cloned and over expressed all the CA-encoding genes from E. coli (can, cynT, cynT2, caiE, pay, and yrdA) and studied their expression profiles in response to different growth conditions (Fujita et al., 1994; Cronk et al., 2001; Merlin et al., 2003). CA genes from thermophilic bacteria such as Thermovibrio ammonificans, M. thermobacterium, M. thermophila, Sulfurihydrogenibium, B. halodurans and others have been cloned and over expressed in E. coli to assess their efficacy in biomineralization. These CAs have been shown to be useful in sequestering CO<sup>2</sup> from flue gas (Capasso et al., 2012; Faridi and Satyanarayana, 2016a). The role of CAs in virulence/pathogenesis have been studied. They play an active role in metabolism during pathogenesis in many bacterial and fungal pathogens such as Vibrio cholerae, Helicobacter pylori, Candida albicans, Aspergillus fumigatus, and others. List of CAs from some of the pathogens have been summarized in **Table 3**.

FIGURE 3 | Mineralization of vehicular exhaust gas CO2 using BhCA in the presence of Ca2+. Test reaction containing CA showed efficient mineralization of CO2 as compared to the control (adopted from Faridi and Satyanarayana, 2016a).

One of the major problems with cloning of genes in heterologous hosts is obtaining the active protein in soluble



form, as the recombinant proteins are aggregated as inclusion bodies, which need to be solubilised (Clark, 2001). Generally optimization of IPTG concentration, growth temperature (after induction) and incubation time brings the protein into the soluble form (Prasad et al., 2011). Techniques such as sizeexclusion chromatography (Li et al., 2004), dialysis and dilution (Marston, 1986; Tsumoto et al., 2003; Umetsu et al., 2003), zeolite absorbing systems (Nara et al., 2009), reversed micelle systems (Sakono et al., 2004), microfluidic chips (Jahn et al., 2007) and the natural GroEL—GroES chaperone system (Zhi et al., 1992) could be used in soluble expression of recombinant proteins. Other problems include codon bias and lack of post translational machinery in E. coli (Gustafsson et al., 2004; Sahdev et al., 2008). The latter can be overcome by cloning the gene of interest in eukaryotic hosts such as Saccharomyces cerevisiae and P. pastoris. In this case, protein is expressed extracellularly, hence, we are saved from the energy intensive step of breaking the cells in disruptors or sonicators. Codon optimization also results in faster translation rates. α–CA from Dunaliella species was codon optimized and effectively expressed in E. coli, which was used for biosequestration of CO<sup>2</sup> (Kanth et al., 2012). A synthetic α–CA (HC-aCA) from Hahella chejuensis was cloned and expressed (Ki et al., 2013). The codon-optimized carbonic anhydrase gene of thermophilic Persephonella marina EX-H1 (PMCA) was expressed and characterized. The removal of the signal peptide resulted in 5-fold enhancement of CA (Kanth et al., 2014).

## Role of Metagenomics in CCS Technologies

The advent of metagenomics has permitted discovery of numerous microorganisms from extreme environments, which could be of biotechnological interest. Metagenomics analysis has revealed the presence of CA encoding genes in the Indian Ocean viral metagenome (Williamson et al., 2012). Jones et al. (2012) showed the existence of RuBisCo and CA gene clusters in the Acidithiobacillus, an extremely acidophilic sulfur-oxidizing biofilm by community genome analysis. Microarray data of a metagenome of acid mine drainage also showed the presence of CA encoding genes along with the RuBisCo gene clusters (Guo et al., 2013). Three gene copies were identified which encoded CA in the metagenome of the marine ammonium oxidizing bacteria (van de Vossenberg et al., 2003). The metagenomes of the serpentinite-hosted Lost City hydrothermal field and Mid-Atlantic Ridge also indicated the presence of CA encoding genes. These metagenomes were found to be similar to the genome of Thiomicrospira crunogena XCL-2, an isolate from a basalt-hosted hydrothermal vent in the Pacific Ocean (Brazelton and Baross, 2010). Microorganisms exhibit great potential as bioindicators to detect leakages from CO<sup>2</sup> storage projects and for that metagenomics becomes handy. Metagenomics and high throughput screen (HTS) methods can also be utilized for studying the effect of environmental changes on microbial communities at the CCS sites (Caporaso et al., 2012; Håvelsrud et al., 2012, 2013; Howe et al., 2014). Information about amplicons and metagenomes helps in establishing a CCS monitoring approach, which could even be useful in the detection of CO<sup>2</sup> leaks (Noble et al., 2012). The use of HTS has already been found to be effective in evaluating the response of in situ bacterial populations to increased CO2, and matching community shifts to metabolic potential (Mu et al., 2014). The metagenomic approach along with appropriate bioinformatics tools makes the system competent. Metagenomics can thus be used as a biosensor for monitoring the CCS sites efficiently (Hicks et al., 2017).

## Utilization of Immobilized CA in CCSU

Immobilization strategies are necessary so that the enzyme can be recycled number of times. Their stability can be enhanced by various immobilization techniques. Immobilization has long been used as an approach for increasing the stability of mesophilic enzymes. There are several reports of immobilization of mesophilic CAs. These CAs have been proved to be more efficient than the free enzymes for CO<sup>2</sup> capture. CAs from P. fragi, Bacilllus pumilus, and Micrococcus lylae have been immobilized on chitosan and surfactant-modified silylated chitosan beads (Prabhu et al., 2009; Wanjari et al., 2011); these displayed enhanced CO<sup>2</sup> hydration capacity. Chitosan is made of glucosamine and acetyl glucosamine units. The functional groups present on chitosan are amino and hydroxyl groups which are required for enzyme immobilization. The enzyme is adsorbed on the surface of chitosan beads. Immobilization of CAs also improved their thermal stabilities, for e.g., the immobilized CA retained at least 60% of the initial activity after 90 days at 50◦C compared to about 30% for their free counterparts under the same conditions. The CAs also exhibit a high stability in the presence of inhibitors upon immobilization (Prabhu et al., 2009; Kanbar and Ozdemir, 2010). CaCO<sup>3</sup> precipitation rate doubled in a period of 5 min when pure CA from P. fragi was immobilized by adsorption on chitosan beads in comparison with the free enzyme (Wanjari et al., 2012; Yadav et al., 2012). Immobilization of CAs on several other matrices such as ordered mesoporous aluminosilicate, octa(aminophenyl)silsesquioxanefunctionalized Fe3O4/SiO<sup>2</sup> nanoparticles, silica nanoparticles and by single/multiple attachments to polymers deposited on Fe3O<sup>4</sup> particles was also attempted (Sharma et al., 2011; Rayalu et al., 2012). CA immobilized on ordered mesoporous aluminosilicate exhibited CO<sup>2</sup> sequestration efficiency of 16.14 mg of CaCO3/mg CA as compared to that of free enzyme which sequesters 33.08 mg of CaCO3/mg CA. Immobilized CA even showed enhanced stability and retained 67% of initial activity even after six cycles (Yadav et al., 2011). Kinetics of the immobilized CAs (immobilized on ordered mesoporous aluminosilicate) were studied and compared with that of the free enzyme by Yadav et al. (2010). The Km, Vmax, and kcat values of the immobilized enzyme were 0.158 mM, 2.307 µ mole min−1ml−<sup>1</sup> , and 1.9 s−<sup>1</sup> , and these for free CA were 0.876 mM, 0.936 µ mole min−1ml−<sup>1</sup> , and 2.3 s−<sup>1</sup> , respectively (Yadav et al., 2010). A high CO<sup>2</sup> sequestration and improved stability have been achieved when CA was immobilized on core-shell CA-chitosan nanoparticles (SEN-CA; Rayalu et al., 2012). Ssp CA was immobilized on solid matrix made of silica particles (silanized Sipernat <sup>R</sup> 22 particles). Enzyme-carrier covalent bonding was adopted as immobilization technique and it was observed that the enzyme stability and activity increased on immobilization. Immobilization of this CA in polyutherane foam also exhibited enhanced stability. The immobilized CA (PU-SspCA) showed exceptional thermostability for very long duration even at 70◦C. The CO<sup>2</sup> absorption capacity of PU-SspCA was verified in a three phase trickled bed bioreactor which mimics the post-combustion processes in a thermal power plant. The three-phase reactor was filled with shredded foam with PU-SspCA. The gas mixture (CO2/N2) was fed from both the sides (i.e., concurrent and countercurrent). Increasing the flow rate of water and decreasing the CO<sup>2</sup> flow rate also greatly improved CO<sup>2</sup> capture in these reactors. SspCA showed good CO<sup>2</sup> capture performance when the PU-SspCA-shredded foam was used in the bioreactor (Migliardini et al., 2014). Concerted efforts are needed to develop CA immobilization techniques that allow reuse of the enzyme 100–500 times with sustained activity. Immobilization can also lead to unwanted release of enzyme on surface of the reactor. In order to overcome this problem, some novel immobilization techniques have been developed using γ– CA from P. horikoshii and M. thermophila (Salemme and Weber, 2014). The immobilization techniques allowed the development of γ–CA nanoassemblies (Salemme and Weber, 2014). An immobilization sequence was added at amino- or carboxyterminus which aided in proper and reversible immobilization of γ–CA to the functionalized surface. Gas-liquid membrane contractors are also emerging as potential bio reactors for using CAs for CO<sup>2</sup> capture. Iliuta and Iliuta (2017) has demonstrated a novel, cost effective and environmentally friendly approach for CO<sup>2</sup> capture by immobilizing CA in a hollow-fiber membrane bioreactor (HFMB) by multiscale steady-state model, under partially liquid-filled and gas-filled membrane pores conditions.

Nanoparticles are also widely used for immobilization of enzymes due to their unique size and physical properties. The immobilization of enzymes on nanoparticles (NPs) offers high surface area and may lead to reduction in protein unfolding, improvement in storage stability and performance (Laurent et al., 2008; Xu et al., 2014). Iron mangnetic nanoparticles (MNPs) are being synthesized with various surface modifications in order to use them for immobilizing protein/enzyme. The CA from B. pumilus TS1 was immobilized successfully on chitosan stabilized iron MNPs (Yadav et al., 2012). Silanization of iron MNPs is being widely used for surface modifications of iron MNPs. It is also very easy and cost effective technique, which can be carried out simply in aqueous or organic media at moderate temperatures (Xu et al., 2014). Faridi et al. (2017) showed that even after 22 cycles of reuse, recombinant α–CA (rBhCA) from B. halodurans TSLV1 immobilized on silanized iron MNPs (rBhCA-Si-MNPs) lost only 50% of activity. Nickel nanoparticles have been successfully used as a direct catalyst for CO<sup>2</sup> hydration reaction for assessing their application in CO<sup>2</sup> mineralization (Bhaduri et al., 2015).

#### Other Applications of Carbonic Anhydrases

Apart from their utility in CCSU, the CAs have some other applications too as discussed below:

#### Carbonic Anhydrase in Formation of Bioconcrete

Biomineralization by bacteria facilitates the development of bioconcrete, wherein calcium carbonate is formed by the metabolic activity of microorganisms, which involves a series of complex reactions directed mainly by urease and carbonic anhydrase enzymes (Castro et al., 2016). The activity of urease, CA, concentration of calcium and pH are very important in bioconcrete formation (Achal and Mukherjee, 2015).

#### Artificial Lungs

This equipment helps to overcome respiratory problems. The major drawback of this technique is the insufficient transfer of CO<sup>2</sup> per square inch through the polymeric hollow fiber membranes (HFM). CAs can be utilized by immobilizing on HFM in order to increase the rate of CO<sup>2</sup> transfer. CAs can, therefore, be used in developing small artificial lungs, which could be efficiently utilized within the human body (Kaar et al., 2007).

#### Biosensors

HCA II based biosensors have been employed to check the toxic effects of zinc on marine life (Thompson and Jones, 1993; Rout and Das, 2003; Muyssen et al., 2006). Many researchers are attempting to develop biosensor variants for other transition metals (Thompson et al., 2000, 2002; Frederickson et al., 2006; Bozym et al., 2008; Wang et al., 2011; McCranor et al., 2012).

### Pharmalogical Considerations

CAs can be incorporated into stimuli-triggered drug delivery systems which utilize CO2, bicarbonate or pH changes as signaling molecules. This can improve the efficiency of these antidote based delivery systems (Satav et al., 2010).

#### Blood Substitutes

The current blood substitutes have inadequate CO<sup>2</sup> removal rate, which leads to coma or death. CAs can be utilized along with catalase (CAT) and superoxide dismutase (SOD into the PolySFHb substitute (PolySFHb-SOD-CAT-CA) in order to overcome this limitation (Bian et al., 2011).

#### CONCLUSIONS AND FUTURE PERSPECTIVES

CAs are a class of enzymes which are essential for the survival of living beings. This enzyme not only helps in our metabolic activities but also aids in the protection of Mother Nature. Due to anthropogenic activities, the nature is getting destroyed day by day. Global warming is having its toll on the climate and weather. Carbonic anhydrase can aid in tackling the future catastrophes due to global warming. Unfortunately the CCS and biomineralization techniques are either in the lab stage or pilot plant stage. Researchers are constantly attempting to address a few of the key issues related to this technology, thus, it has become quite difficult to generate a public consensus on CCS technology. The technology needs to be ameliorated. We are also not able to bring down the carbon emissions as fossil fuels are (also will be in near future) the main energy source which drives today's world. Although much is known about CAs, the current metadata reveals the presence of many CA genes in the extreme realms of earth which are waiting to be discovered. These are mostly from unculturable microbes. Bioinformatics tools have been useful in identifying different CA encoding genes. Once identified, they can be easily cloned and expressed in different microbial hosts for studying their novel properties and utility in CO<sup>2</sup> biomineralization. Currently the unexplored data is so vast that we may discover a few novel classes of CAs in addition to the known six classes in future. CAs from polyextremophilic microbial sources have already been tried for CCS related strategies. Enzyme immobilization techniques permit the repeated use of the enzyme and designing continuously operating reactors. We need to improve the CA immobilization technology so that it becomes cost effective and readily accepted by people. Further research efforts are called for developing highly efficient and robust CAs, efficient immobilization techniques and designing continuously operating reactors for cost effective biomimetic carbon sequestration.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

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#### ACKNOWLEDGMENTS

We wish to thank University Grants Commission, Govt. of India, New Delhi for awarding Faculty Fellowship to one of us (TS) while writing this review.


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer SD and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Bose and Satyanarayana. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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