Dehalococcoides as a Potential Biomarker Evidence for Uncharacterized Organohalides in Environmental Samples

The massive production and improper disposal of organohalides resulted in worldwide contamination in soil and water. However, their environmental survey based on chromatographic methods was hindered by challenges in testing the extremely wide variety of organohalides. Dehalococcoides as obligate organohalide-respiring bacteria exclusively use organohalides as electron acceptors to support their growth, of which the presence could be coupled with organohalides and, therefore, could be employed as a biomarker of the organohalide pollution. In this study, Dehalococcoides was screened in various samples of bioreactors and subsurface environments, showing the wide distribution of Dehalococcoides in sludge and sediment. Further laboratory cultivation confirmed the dechlorination activities of those Dehalococcoides. Among those samples, Dehalococcoides accounting for 1.8% of the total microbial community was found in an anaerobic granular sludge sample collected from a full-scale bioreactor treating petroleum wastewater. Experimental evidence suggested that the influent wastewater in the bioreactor contained bromomethane which support the growth of Dehalococcoides. This study demonstrated that Dehalococcoides could be employed as a promising biomarker to test the present of organohalides in wastestreams or other environmental samples.

The massive production and improper disposal of organohalides resulted in worldwide contamination in soil and water. However, their environmental survey based on chromatographic methods was hindered by challenges in testing the extremely wide variety of organohalides. Dehalococcoides as obligate organohalide-respiring bacteria exclusively use organohalides as electron acceptors to support their growth, of which the presence could be coupled with organohalides and, therefore, could be employed as a biomarker of the organohalide pollution. In this study, Dehalococcoides was screened in various samples of bioreactors and subsurface environments, showing the wide distribution of Dehalococcoides in sludge and sediment. Further laboratory cultivation confirmed the dechlorination activities of those Dehalococcoides. Among those samples, Dehalococcoides accounting for 1.8% of the total microbial community was found in an anaerobic granular sludge sample collected from a full-scale bioreactor treating petroleum wastewater. Experimental evidence suggested that the influent wastewater in the bioreactor contained bromomethane which support the growth of Dehalococcoides. This study demonstrated that Dehalococcoides could be employed as a promising biomarker to test the present of organohalides in wastestreams or other environmental samples.

INTRODUCTION
Organohalide compounds are a giant group of halogen-substituted hydrocarbons produced in large quantities as solvents, plastics, pesticides, and chemical intermediates for industrial and agricultural uses (Stringer and Johnston, 2001;Jugder et al., 2016). The improper handling and disposal of harmful halogenated compounds resulted in their worldwide contamination in soil and water as well as bioaccumulation through food webs, posing threat to both human health and the environment (Stringer and Johnston, 2001;Lu et al., 2017). Due to the side effects on biota, 69 out of the 126 EPA Priority Pollutants are organohalide compounds (United States Environmental Protection Agency, 2013). However, detection and monitoring of their environmental transport and fate using chromatographybased methods were limited due to the extremely wide variety of organohalide compounds (Stringer and Johnston, 2001).
In this study, we first screened Dehalococcoides in sludge and sediment samples collected from various anaerobic bioreactors for industrial wastewater treatment and contaminated blackodorous urban rivers. Further source-tracking together with laboratory cultivation confirmed which organohalide compounds supported the growth of Dehalococcoides. These results opened up opportunities employing Dehalococcoides as a biomarker to track unknown sources of organohalide compounds in wastewater and environmental samples.

Microbial Cultures Setup and Transfer
Sludge and sediment samples collected from bioreactors and black-odorous urban rivers were employed as inoculum for culture setup (Table 1). These samples were acquired directly by filling sterile 50 ml plastic Falcon tubes that were capped and transported to the laboratory at an ambient temperature. To control exposure of the samples to oxygen, Falcon tubes were sealed with Parafilm, and microcosm setup was performed in anaerobic chamber soon after their arrivals. For granular sludge, it was smashed into floc-form sludge before inoculation. Defined anaerobic mineral medium in 160 ml serum bottles for microbial cultivation was prepared as described (He et al., 2003;Wang and He, 2013a), which contains salts, trace elements and vitamins. L-cysteine and Na 2 S·9H 2 O (0.2 mM each) were added to the medium to achieved reduced conditions. The bottles were sealed with black butyl rubber septa and secured with aluminum crimp caps. The organohalide-fed cultures were transferred in 100 ml medium supplemented with 10 mM lactate, 10 mM 2-bromoethanesulphonate (BES, to inhibit methanogen growth), and 1 mM PCE or 10 ppm chloromethane. The control cultures without organohalide-amendment were transferred in the same mineral medium. Unless stated otherwise, cultures were incubated at 30 • C in the dark without shaking. All the experiments were set up in duplicates.

Analytical Techniques
Headspace samples of chloroethenes (i.e., PCE, TCE, cis-DCE, trans-DCE, VC and ethane)and chloromethane were injected manually with a glass, gastight, luer lock syringe (Hamilton, Reno, NV, United States) into a gas chromatography (GC) 7890N equipped with a flame ionization detector (Agilent, Wilmington, DE, United States) and a GS-GasPro column (30 m × 0.32 mm; Agilent, Wilmington, DE, United States) as described (Wang and He, 2013b). The standards compounds (with analytical pure or above) were purchased from Sigma-Aldrich.

Data Deposition
Raw Illumina Miseq sequencing reads were deposited into NCBI Sequence Read Archive (SRA) with accession no. SRP112682.

Screening of Obligate Organohalide-Respiring Dehalococcoides in Anaerobic Sludge and Sediment Samples
Dehalococcoides as an obligate dehalogenating bacterial group can only utilize organohalides as electron acceptors to support their growth (Löffler et al., 2013). In this study, sediment and sludge samples from black-odorous urban rivers and anaerobic bioreactors, respectively, were selected to screen the presence of Dehalococcoides (Table 1). PCR amplification with Dehalococcoides genus-specific primers, FpDHC1/RpDHC1377 (Hendrickson et al., 2002), showed the positive detection of Dehalococcoides in all urban river sediment samples, as well as in a granular sludge sample collected from a full-scale mesophilic UASB reactor treating petrochemical wastewater ( Table 1).
To profile microbial communities of those Dehalococcoidescontaining environmental samples, Miseq 16S rRNA gene sequencing was performed, showed the very different microbial community structure in samples between Dehalococcoidescontaining granular sludge and urban river sediments (Figure 1). In granular sludge collected from the UASB reactor, acidogenic populations, Syntrophorhabdus (of Syntrophorhabdaceae) and Syntrophus, formed syntrophic interactions with methanogenic Methanosaeta and Methanosarcinaceae (Figure 1). Surprisingly, the obligate organohalide-respiring Dehalococcoides presented abundant in the full-scale UASB reactor, accounting for 1.83% of the total microbial community, comparable with the relative abundance of Dehalococcoides in enrichment cultures dechlorinating PCBs (Wang and He, 2013a) and PCE (Lee et al., 2015). The presence of abundant obligate organohaliderespiring Dehalococcoides implied that the TA-wastewater contained uncharacterized organohalide compound(s). In the UASB reactor, acetate and H 2 generated from degradation of aromatic compounds in petrochemical wastewater by Syntrophorhabdus, Syntrophus and other syntrophs, together with low redox potential and the uncharacterized organohalide compounds, provide ideal growth niches for the fastidious Dehalococcoides. No other obligate dechlorinating bacteria, e.g., Dehalogenimonas and Dehalobacter, were found in the granular sludge sample. In a control sample collected from a lab-scale anaerobic sludge digester without organohalide amendment, no known dechlorinating bacteria can be detected (Figure 1). The highly similar microbial community structures of the three black-odorous river sediments, distinguish themselves from the community compositions of the granular sludge, especially the predominant lineages of Chloroflexi (i.e., Longilinea, GCA004, WCHB1-05 and Anaerolinaceae) and Proteobacteria (i.e., Syntrophobacter and Dechloromonas) (Şimsir et al., 2017) (Figure 1). Dehalococcoides were shown the appearance in the microbial community, on which indicate the potential of organohalides' contamination.

Dechlorination Activities in Dehalococcoides-Containing Cultures
To further evaluate the dechlorination activities, perchloroethene (PCE) was spiked into microcosms established with those Dehalococcoides-containing sediment and sludge samples. After around 2 months' incubation, PCE dechlorination activities were observed in all three microcosms with the river sediment inocula (data not shown). Subsequent consecutive culture transfers of the three microcosms generated three active cultures which reductively dechlorinate PCE into vinyl chloride (VC) or ethene (Figure 2). No dechlorination activity was observed in the control microcosm established with digester sludge (Figure 2D).
In contrast to PCE dechlorination in sediments of the three black-odorous urban rivers, microcosms inoculated with the Dehalococcoides-containing granular sludge showed negative PCE-dechlorination activity. To identify potential organohalides to support the growth of Dehalococcoides in the granular sludge, organohalide pollution in the petrochemical wastewater as influent of the UASB reactor was evaluated. The petrochemical wastewater was generated from a AMOCO process that oxidize para-xylene to terephthalic-acid, using a homogeneous catalyst of cobalt and manganese together with bromide as a promoter, in which bromomethane was generated as a byproduct (Tomás et al., 2013). Due to difficulties in obtaining bromomethane, dehalogenation activity test was performed with chloromethane as a homolog alternative to bromomethane. In chloromethanefed culture, over 70% chloromethane was dechlorinated within 8 days (Figure 3). No obvious dechlorination activity was observed in abiotic control.

Dehalococcoides in the Granular Sludge
The partial 16S rRNA gene sequences (∼400 bp) generated from Miseq sequencing of V4-V5 hypervariable regions were unable to differentiate Dehalococcoides between Cornell and Victoria subgroups. Therefore, Dehalococcoides genus-specific primers (i.e., FpDHC1/RpDHC1377) were utilized to generate longer 16S  To provide insight into the spatial distribution of Dehalococcoides in the granular sludge, FISH was conducted with Dehalococcoides-specific, bacterial and archaeal oligonucleotide probes (Amann et al., 1995;Yang and Zeyer, 2003). FISH analysis showed the scattered distribution of Dehalococcoides inside granules, closely colonized with other bacteria (Figure 4B) but separated from archaea ( Figure 4C). Degradation of aromatic compounds by fermentative bacteria is thermodynamically restricted and will become endergonic ( G > 0) as metabolic byproducts (e.g., acetate and H 2 ) accumulate in the biosystem. Similar with methanogenic archaea, Dehalococcoides might form syntrophic interactions with aromatic compound degrading acidogens in the granular sludge: the degradation of aromatic compounds by Syntrophorhabdus and other syntrophs provide acetate as carbon source and H 2 as electron donor for the halorespiration of Dehalococcoides; correspondingly, Dehalococcoides help maintain acetate and H 2 at low concentration in the biosystem and 'pull' degradation of aromatic compounds toward completion through consuming metabolic byproducts generated by acidogenic bacteria. The close colonization of Dehalococcoides with syntrophic bacteria could facilitate the interspecies transfer of H 2 (Mao et al., 2015).

DISCUSSION
Thus far, it remains challenging to detect organohalide compounds in wastewater and environmental samples based on chromatography methods due to their extremely wide variety, e.g., PCBs are a family of 209 structurally similar congeners (Chu and Hong, 2004;Elder et al., 2008). Bromomethane, similar with many other organohalide compounds produced as intermediate or byproducts in chemical synthesis processes, was FIGURE 4 | (A) Phylogenetic tree of Dehalococcoides identified in TA-degrading anaerobic granular sludge. Phylogenetic tree was calculated by neighbor-joining method using MEGA4 (Tamura et al., 2007). FISH analysis revealed the space distribution of (B) bacteria (red) and Dehalococcoides (yellow), and (C) archaea (red) and Dehalococcoides (green). a noteless synthesis byproduct in the petrochemical wastewater generated from terephthalic acid industry (Tomás et al., 2013). In this study, we reported the abundant presence of obligate organohalide-respiring Dehalococcoides in a full-scale UASB reactor for petrochemical wastewater treatment, and further cultivation experiments suggested the possible contamination of bromomethane in the petrochemical wastewater. Recent studies showed experimental evidences of biosynthesis of aromatic organohalides in nature, which might explain the detection of Dehalococcoides in the three black-odorous urban rivers (Agarwal et al., 2014;El Gamal et al., 2016;Şimsir et al., 2017). Also, Dehalococcoides was detected in various environmental samples contaminated with organohalides, including sludge/sediment collected from anaerobic digesters (Smith et al., 2015) and hyporheic zone of a wastewater treatment plant (WWTP)-impacted eutrophic river (Atashgahi et al., 2015). Therefore, Dehalococcoides might be a promising biomarker, complementing current chromatography-based methods, to test organohalide compounds in wastewater and environmental samples.
The UASB reactors provided ideal ecological niches for the growth of Dehalococcoides which further formed syntrophic interactions, as methanogens in syntrophic methanogenic communities (Stams and Plugge, 2009), with aromaticcompound degrading bacteria to overcome the thermodynamic limit through consuming acetate and H 2 . To our knowledge, this is the first report of the strictly organohalide-respiring Dehalococcoides present abundantly in a full-scale bioreactor for industrial wastewater treatment. In previous studies, Dehalococcoides was documented in various lab-scale bioreactors, including membrane biofilm reactors (Chung et al., 2008), UASB reactor (Hwu and Lu, 2008) and anaerobic biotrickling filter (Popat and Deshusses, 2009). The presence of Dehalococcoides in high abundance in both full-and lab-scale bioreactors showed the feasibility of removing toxic and persistent organohalides from various industrial wastewaters in anaerobic bioreactors through employing the microbial reductive dehalogenation process.

AUTHOR CONTRIBUTIONS
SW and DL conceived the idea. QL and LY performed the experiments and data analysis. SW, QY, TL, and ZH provided materials. QL, LY, and SW wrote the manuscript with inputs from all authors. All authors read and approved the final manuscript.