The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge

The bacteria involved in organic pollutant degradation in pelagic deep-sea environments are largely unknown. In this report, the diversity of polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was analyzed in deep-sea water on the Southwest Indian Ridge (SWIR). After enrichment with a PAH mixture (phenanthrene, anthracene, fluoranthene, and pyrene), nine bacterial consortia were obtained from depths of 3946–4746 m. While the consortia degraded all four PAHs when supplied in a mixture, when PAHs were tested individually, only phenanthrene supported growth. Thus, degradation of the PAH mixture reflected a cometabolism of anthracene, fluoranthene, and pyrene with phenanthrene. Further, both culture-dependent and independent methods revealed many new bacteria involved in PAH degradation. Specifically, the alpha and gamma subclasses of Proteobacteria were confirmed as the major groups within the communities. Additionally, Actinobacteria, the CFB group and Firmicutes were detected. Denaturing Gradient Gel Electrophoresis (DGGE) analysis showed that bacteria closely affiliated with Alcanivorax, Novosphingobium, and Rhodovulum occurred most frequently in different PAH-degrading consortia. By using general heterotrophic media, 51 bacteria were isolated from the consortia and of these 34 grew with the PAH mixture as a sole carbon source. Of these, isolates most closely related to Alterierythrobacter, Citricella, Erythrobacter, Idiomarina, Lutibacterium, Maricaulis, Marinobacter, Martelella, Pseudidiomarina, Rhodobacter, Roseovarius, Salipiger, Sphingopyxis, and Stappia were found to be PAH degraders. To the best of our knowledge, this is the first time these bacteria have been identified in this context. In summary, this report revealed significant diversity among the PAH-degrading bacteria in the deep-sea water column. These bacteria may play a role in PAH removal in deep-sea environments.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are a group of cytotoxic, mutagenic and carcinogenic compounds with two or more fused aromatic rings. They can be found in deep-sea sediments, such as those found 2.7 m beneath the bottom surface at a water depth of 3,962 m on the Middle Atlantic Ridge. This locations has a total PAH concentration ( PAHs) of 260 ∼440 ng/g dw (dry weight) Wang et al., 2008). Recently, even higher PAH concentrations were detected in the hydrothermal vent area of the South Mid-Atlantic Ridge (Huang et al., 2014). There, the PAHs ranged from 2768 to 9826 ng/g dry sediment at different sites (Huang et al., 2014). Among the PAHs detected in these reports, phenanthrene was most abundant. However, the origin and fate of PAHs in this extreme environment are not well understood.
In deep-sea water conditions, PAHs are thought to be absorbed on suspended particles and sink to the floor. As labile carbon sources in deep water are limited, PAHs in these environments could serve as valuable carbon and energy sources for bacterial growth. However, little is known about the diversity of PAH-degrading bacteria therein. In the present report, PAH degraders were examined at water depths ranging from 3946 to 4746 m on the Southwest Indian Ridge (SWIR). These bacteria displayed significant diversity and novelty, indicating the prevalence of PAH-degrading bacteria in the deep water system of the open sea.
To enrich the PAH-degrading bacteria, 400 ml water from each layer was drained into autoclaved bottles directly from the sampler. The enrichment process was initiated on board the boat at 20 • C with PAHs as the carbon and energy sources. Samples were kept for 2 months until laboratory analysis.

PAH Enrichment in the Laboratory
Polycyclic aromatic hydrocarbons dissolved in chloroform were added to flasks containing 150 ml autoclaved NH medium, with final concentrations of 100 mg/liter phenanthrene, 10 mg/liter of anthracene, 10 mg/liter fluoranthene, and 10 mg/liter pyrene. To evaporate the chloroform, flasks were shaken at 160 rpm for 2 days. Then, enrichment was initiated by adding 5.0 ml primary enriched cultures from each layer in the flasks, followed by shaking at 160 rpm in the dark at 25 • C for 20 days. For a second round of enrichment in the lab, a 2.0 ml culture of each sample was transferred into the same medium in triplicate. In addition, each PAH was tested individually for degradation in parallel to the PAH mixture treatments. At days 4, 8, 12, and 20, 2 ml cultures of treatments with obvious bacterial growth were used for community genomic DNA extraction. At day 30, the residual PAHs in each flask quantified as described below.

Bacterial Isolation from the PAH Enriched Consortia
After the samples were enriched by being transferred twice in lab, bacteria were isolated from PAH-degrading consortia by serial dilutions ranging from 10 −3 to 10 −7 . These dilutions were then spread on both M2 agar and 216L agar plates and incubated at 18 • C for ∼2 weeks. A total of 238 isolates were purified and then subjected to Rep-PCR analysis to eliminate redundant strains, as described previously (Ma et al., 2006). The isolates were then identified by 16S rRNA gene sequencing and analyzed using BLASTN at http://www.ncbi.nlm.nih.gov/BLAST. Nearly full length sequence of 16 rRNA gene was obtained by universal primer set 27f (forward) 5 -AGAGTTTGATCCTGGCTCAG-3 /and 1502r (reverse):5 -ACGGCTACCTTGTTACGACT-3 . The ability of these bacteria to degrade PAH was tested with a mixture containing phenanthrene, anthracene, fluoranthene, and pyrene.

Quantification of PAH Compounds
To assay the biodegradation capacity of the consortia, PAHs from the mixture were analyzed with Gas Chromatography Mass Spectrometry (GC-MS) after 1 month of incubation. The residual PAHs in each flask were extracted according to Guo et al. (2005). Simultaneously, 5 mg/liter fluorene was added as an internal standard of extraction. Then, degradation percentages were determined using GC-MS QP2010 (SHIMADZU) with a 30m RTS-5MS capillary column (0.25 mm inside diameter and 0.25 µm film thickness). Details of thermal program and injection conditions were according to our previous report . Selected ion monitoring (SIM) mode was used for quantification of PAH, and full-scan mode mode was used for identification of PAH with the GC-MS Postrun Analysis software (Version 2.10, SHIMADZU).

DNA Preparation
Total community DNA was collected from 2 mL culture of each sample on days 4, 8, 12, and 20 in sterile 2.0 ml Eppendorf tubes, and then immediately extracted. Genomic DNA of both consortia and isolates were extracted using the modified SDS-CTAB method (Sambrook et al., 1989).

16S rRNA PCR for DGGE Analysis
Polymerase chain reaction amplification of the V3 variable region of 16S rRNA fragments prior to Denaturing Gradient Gel Electrophoresis (DGGE) was performed as described by (Muyzer et al., 1993). To amplify the variable V3 region of bacterial 16S rDNA genes, the primer set was used: DGGEf (forward), 5 -CGCCCGCCGCGCGCGGCGGGCG GGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3 , which contained a GC clamp linked to the 5 end; DGGEr (reverse), 5 -ATTACCGCGGCTGCTGG-3 .
All PCRs were performed with a Mastercycler (Eppendorf, Hamburg, Germany). Reaction mixtures contained 1.0 U of rTaq (Takara), each primer at a concentration of 0.5 µM, and 0.6 µl of template DNA in a total volume of 50 µl. After an initial 6 min denaturation at 95 • C, a touchdown thermal profile protocol was used. The annealing temperature was decreased by 0.5 • C per cycle from 65 to 55 • C, followed by 20 additional cycles at 55 • C. Amplification was carried out with 1 min of denaturation at 95 • C, 1 min of primer annealing, and 1.0 min of primer extension at 72 • C, followed by a final extension at 72 • C for 10 min. PCRs were carried out in replicates of four using the consortium DNA, then the mixtures were combined PCR amplifications were checked on 1% (w/v) agarose gel prior to DGGE analysis.
After electrophoresis, the gel was stained with ethidium bromide (10 µg/ml) for 15 min, and images were captured using an Alpha-Imager Imaging System with AlphaEase FC image software 4.1.0 (Alpha Innotech).
Each visible band on the DGGE gels containing the PAHdegrading consortia was excised manually, and the DNA was extracted using the method described by Muyzer et al. (1993).
Using 5 µl of the extracted DNA as template, PCR was carried out to amplify target DNA for cloning. Positive PCR products were purified using the E.Z.N.A Cycle-Pure Kit (OMEGA Bio-tek, USA) and cloned into the pMD19-T Vector (Takara). After being confirmed using another DGGE gel, they were sequenced with an ABI model 3730 DNA sequencer (Invitrogen Shanghai).

Nucleotide Sequence Deposit
The 16S rRNA sequences of bacteria isolated from the nine consortia have been deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST) under accession No. EU440952∼EU441002. The DGGE band sequences determined in this study have also been submitted to the GenBank database and have been assigned accession numbers EU441003∼EU441036.

PAH Degradation by Deep Water Layers
After the initial 2 months enrichment with a PAH mixture of phenanthrene, anthracene, fluoranthene, and pyrene as the sole carbon and energy sources, PAH degradation occurred in all treatment conditions of nine layers of water samples acquired from 3946 to 4746 m. Obvious bacterial growth was observed in all the enrichment cultures with the color changing to dark brown. In the lab, the initial enrichment cultures were transferred to fresh NH medium with PAHs as the sole carbon source (with 5% inoculum). The resulting final nine consortia were numbered IR51-IR59 (IR5, representing the sampling site IR-CTD-5; 1-9, the layer number based on the water depths listed above). The growth curves of all cultures during the second round of enrichment in the lab are shown in Figure 1. Of these cultures, layer 1 (IR51, 4746 m) showed the best growth with the highest cell concentration (OD 600 = 0.459) at day 20, followed by layer 9 (IR59, 3946 m) (OD 600 = 0.400) (Figure 1).
Meanwhile, PAH degradation was further confirmed by GC-MS quantification. As shown in Figure 2, phenanthrene was almost completely degraded in all cases after 30 days incubation, while anthracene, fluoranthene, and pyrene showed degradation ranging from 69 to 99%.
In addition to phylogenic analysis, these 51 isolates were also tested for PAH-degradation. We found that 34 isolates could grow with PAHs as the sole carbon and energy sources (labeled with "+" in Figure 3). Among them, 20 bacteria belonged to Alphaproteobacteria. These 34 degraders contained some bacteria that have been previously reported, such as Bacillus (Hunter et al., 2005), Novosphingobium (Sohn et al., 2004), Lutibacterium (Chung and King, 2001), Sphingomonas (Bastiaens et al., 2000), and Marinobacter (Hedlund et al., 2001). However, many others had not been reported to be  "+" means the bacterium can grow well in the medium with PAHs as carbon sources. "(+)" means the bacterium can grow but not very well in the medium with PAHs as carbon sources. "−" means the bacterium cannot grow in the medium with PAHs as the carbon sources. Subgroups: Alpha-, Gammaproteobacteria; F, Firmicutes; A, Actinobacteria. Isolates named as PR and 2PR were isolated on agar plates of M2 agar and 216L, respectively.

Bacterial Community Composition of the PAH-Degrading Consortia
The bacterial composition of all consortia changed from the first day to the 20th day of the incubation process and also varied from layer to layer (Figure 4, Supplementary Figures S2 and  S3). For an example, the consortium of IR51 that was generated from the first layer contained at least seven major bands (Band 2 to Band 8), as shown in the DGGE profile in Figure 4. These bands belonged to the genera Alcanivorax, Novosphingobium, Bartonella, Pseudaminobacter, Rhodovulum, and Azospirillum. Two bacteria, Novosphingobium pentaromativorans US6-1 T (98.64%; Band1-3, with "1-" indicating the first layer) and Azospirillum sp. 5C (95.21%; Band1-8), became predominant at day 8. The bacterium of Band1-3 remained a predominant member throughout the time course, while the abundance of Band1-8 decreased after day 8. At day 20, Novosphingobium (Band1-3) turned out to be the most predominant member, indicating a key role in PAH degradation. In addition, Band1-2, closely related to Alcanivorax dieselolei B-5 (99.47%), was also an important member and was a major band from days 8 to 12 (Figure 4). This bacterium reoccurred as the most dominant member in the community with phenanthrene as the sole carbon and energy source (Figure 4); however, it failed to degrade any PAHs by itself.
The community structures of the other water layers (IR52-IR59) were also examined (Supplementary Figures S2 and  S3). Consistent with the results above, Alcanivorax and Novosphingobium were the most common bacteria in all the consortia. For an instance, an isolate closely related to A. dieselolei B-5 was a predominant member of almost all consortia. Bacteria of the genus Novosphingobium were also primary members of consortia from all depths and were closely related to Novosphingobium aromaticivorans, N. hassiacum, N. pentaromativorans, and Novosphingobium sp. MG36.
Interestingly, some novel species were detected as key members of the PAH-degrading consortia. For example, those with only 90.7-96.5% similarity to Arthrobacter sp. 255-8a (AY444852), as well as the bacteria with only 95.2-96.7% similarity to Rhodovulum sp. AR2002 and the bacterium with only 93.1% similarity to Rhizobiales bacterium RR47, occurred as a strong band in consortia IR53, 54, and 58. These bacteria have never been reported to be PAH degraders. Conversely, Cycloclasticus spirillensus (100.00%), the most famous PAH-degrading bacterium in marine environments (Head et al., 2006), occurred in consortia IR52, IR53, and IR55 ( Supplementary  Figures S2 and S3), but was not as important here as Novosphingobium.

Single PAH Degradation
To detect the degradation of a single PAH, phenanthrene, pyrene, anthracene, and fluoranthene were used separately as the sole carbon source. Unexpectedly, none of the nine consortia could grow with anthracene, fluoranthene, or pyrene. However, all consortia could grow with phenanthrene.

Discussion
Oil spilling into the Gulf of Mexico from the Deepwater Horizon blowout stimulated indigenous bacteria of the order Oceanospirillales in the Gammaproteobacteria, which are likely involved in alkane degradation in the deep-water column from 1099 to 1219 m (Hazen et al., 2010). However, bacteria involved in organic pollutant degradation in deep-sea environments are largely unknown, especially in the open sea (Deming, 1998). In deep-sea water columns of the open Mediterranean Sea, various types of PAHs have been found sinking with suspended particles (Bouloubassi et al., 2006). The source and fate of PAHs in deep-sea environments are poorly understood. One possible source is hydrothermal activity. A variety of organic compounds, including PAHs and long-chain hydrocarbons, were detected in the hydrothermal fluids (McCollom and Seewald, 2007). These hydrocarbons may serve as organic carbon sources that are otherwise scarce in the deep-sea environments but needed for heterotrophic bacterial growth.
Along the SWIR, hydrothermal vent fields were recently found (German et al., 1998;Tao et al., 2012). The microbial diversity of this region remains poorly characterized, and the bacterial diversity with regards to PAH degradation is unclear (Chen and Shao, 2009;Cao et al., 2014;Li et al., 2014). In this report, a variety of PAH-degrading bacteria were found in the deep water column on the Ridge at depths ∼4766 m. Among the discovered bacterial phylotypes, Proteobacteria dominated all the consortia, which was constituted of mainly Alphaproteobacteria and Gammaproteobacteria (Figures 3 and 5). Interestingly, the predominant PAH degraders obtained from the deep-sea water were significantly different from those in marine sediments. Cycloclasticus, thought to be the key PAH-degrading bacterium in marine environments, was not as important as previously reported (Dyksterhouse et al., 1995;Wang et al., 1996Wang et al., , 2008Kasai et al., 2002;Yakimov et al., 2005;McKew et al., 2007;Teira et al., 2007;Cui et al., 2008). Instead, Novosphingobium seemed to be most important genus in this report. Bacteria of this genus were also previously detected as PAH degraders in the deep-sea hydrothermal environment of the Lau Basin of the West Pacific (Dong et al., 2011). They are closely related to those detected in this report, belonging to N. indicum, which we isolated here from sample CTD-5 (Yuan et al., 2008(Yuan et al., , 2009). This bacterium can degrade many types of aromatic hydrocarbons including biphenyl, naphthalene, acenaphthene, 2-methylnaphthalene, dibenzofuran, dibenzothiophene, 2,6-dimethylnaphthalene, 4methyldibenzothiophene, phenanthrene, anthracene, chrysene, and fluoranthene (Yuan et al., 2008). However, contrary to expectations, only phenanthrene could be degraded alone, while the three other PAHs (anthracene, fluoranthene, and pyrene) could not be individually utilized by any of the consortia, as evidenced by the lack of clear bacterial growth within 1 month. We propose that phenanthrene metabolism induced the bacterial cells to utilize the three other PAHs more rapidly. This possibility is supported by a previous report, which found that pyrene degradation relies on the presence of phenanthrene in the case of the isolate Cycloclasticus sp. N3 (Geiselbrecht et al., 1998). By comparing the bacterial structure of these consortia, a trend in bacterial distribution from 20 to 820 m above the bottom can be observed. Consortia from neighboring layers were similar in bacterial structure. For example, in phenanthrene-enriched consortia, the community structures of the upper two layers were similar to each other but different from those of the other layers (Supplementary Figure S3). Some bacteria occurred only in specific consortia. For instance, the bacterium closely related to N. aromaticivorans DSM 12444 (100%) occurred from 470 to 620 m above the bottom as bands P5-4, P6-5 and P7-5, but was not found in other layers ( Table 1). The bacterium similar to Azospirillum sp. 5C (95.21%) occurred below 470 m from the bottom as bands 1-8, 3-10, 4-10, and 5-14, as did the bacterium similar to Rhodovulum sp. AR2002 (95.21%), which frequently occurred as bands 1-7, 2-11, 3-12, 5-13, P1-8 and P3-11 (Table 1). However, the strain that displayed 96.75% similarity with Rhodovulum sp. AR2002 was detected in a wider range of 20-820 m above the bottom, presenting as bands 1-6, 2-10, 5-12, 9-7, 6-10, P6-10, and P9-6 ( Table 1). Additionally, the bacterium similar to N. hassiacum W-51 (95.81%) only occurred in the first and second layers above the bottom as bands 2-4, P1-4, and P2-6, while the bacterium similar to N. pentaromativorans US6-1 (98.64%) only occurred in the bottom layer as a key member of the PAH mixture consortia. However, the bacterium similar to A. dieselolei B-5 (99.47%) frequently occurred in all layers, and the bacterium similar to Bartonella sp. AD273 (98.79%) occurred in many consortia regardless of layer. Among these bacteria, Alcanivorax, and Novosphingobium have been reported in deep-sea sediments involved in PAH degradation Wang et al., 2008;Cui and Shao, 2009). Recently, we found that PAH-degrading bacteria, including Cycloclasticus, Pseudomonas, Pseudoalteromonas, Halomonas, Marinomonas, and Dietzia, were widespread in the deep-sea sediments of the Arctic Ocean (Dong et al., 2015). In addition, Alcanivorax frequently occurred in the Arctic PAH-degrading consortia, while bacteria Novosphingobium did not.
Among the bacteria detected in this report, the bacterium similar to A. dieselolei B-5 (99.47%) was a key member of all consortia. The predominance of A. dieselolei was further confirmed by 16S rRNA gene library construction of two communities, IR58 and IR59 (data not shown). From the library, we obtained a nearly full length 16S rRNA, which showed 99.8% (1502/1505) similarity with the type strain A. dieselolei B-5. Moreover, Alcanivorax bacteria appeared in both the PAH mixture and the phenanthrene-degrading consortia. It has been demonstrated that A. dieselolei is an obligate alkane-degrading bacterium (Liu and Shao, 2005;Wang et al., 2010). However, bacteria of this genus have never been shown to be PAH degraders. Iwabuchi et al. (2002) suggested that Alcanivorax may enhance PAH degradation by utilizing the alkyl side-chains on methylated PAHs within crude oil (Iwabuchi et al., 2002). However, in this study, such alkyl side-chain-substituted PAHs were not used. Why Alcanivorax coexist with other bacteria in these communities remains an intriguing question.
Among the PAHs used, phenanthrene is probably the easiest to degrade, as the resulted PAH-degrading consortia failed to show obvious bacterial growth when using pyrene, anthracene, or fluoranthene as the sole carbon source. However, when used in form a mixture, all of them can de degraded, suggesting a cometabolism between phenanthrene and other PAHs. This phenomenon has been found in other reports (Geiselbrecht et al., 1998;Kanaly and Harayama, 2000). In the case of pure cultures, 34 of the isolates have been confirmed to grow with the PAH mixture. By now, their degradation ability on each kind of PAHs remains undetermined except one novel species of N. indicum mentioned above (Yuan et al., 2008(Yuan et al., , 2009. In respect of temperatures and hydrostatic pressures in situ that might select bacteria different to the above described, obligate psychrophilic and barophilic microbes involved in PAH degradation probably exist in the deep sea water column. Bacterial isolates in this report ever tested showed temperature growth ranges from 10 to 41 • C mostly, with optimal growth temperatures of 20 to 25 • C, such as Alcanivorax marinus, N. indicum, Oceanibaculum indicum, and Stappia indica et al., which are the novel species isolated from the deep sea water of SWIR (Lai et al., 2009a(Lai et al., ,b, 2010(Lai et al., , 2011a(Lai et al., ,b, 2013Yuan et al., 2009). They did not show an obvious growth under 4 • C within 1 week, which is higher than the temperature (about 1 • C) in situ. Probably, they actually grow slightly under temperature at 4 • C and below, or need long time incubation. Anyhow, we believe that microbes in situ would possess some taxa that are psychrophilic and barophilic, but remain uncultivated by now.
In summary, this report describes the discovery of diverse PAHs-degrading bacteria in the deep-sea water column of the SWIR at a depth of 4766 m. Bacteria affiliated with Alcanivorax, Novosphingobium, and Rhodovulum frequently occurred in different PAH-degrading consortia, and 34 isolates were confirmed to grow with our PAH mixture. These included novel PAH-degrading bacteria belonging to Alterierythrobacter, Citricella, Erythrobacter, Idiomarina, Lutibacterium, Maricaulis, Marinobacter, Martelella, Pseudidiomarina, Rhodobacter, Roseovarius, Salipiger, Sphingopyxis, and Stappia. These bacteria are thought to play a role in PAH degradation in deep-sea water. More efforts are needed to examine the diversity of extremophiles in situ of deep sea that involved in PAH degradation.

Supplementary Material
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.00853 FIGURE S1 | Sampling site IR-CTD5 (The map based on NOAA topography). The solid red circles are hydrothermal vent fields previously detected. IR-CTD5 was in the B-B' section, where plume 2 was found by anomalies (German et al., 1998). Edmond and Kairei are confirmed vent sites.
FIGURE S3 | Community structures of the PAH-degrading consortia growing with phenanthrene in 20 days. Treatments were in parallel with those in Figure S2, except using phenanthrene as a sole carbon source.