Biodiversity and Degradation Potential of Oil-Degrading Bacteria Isolated from Deep-Sea Sediments of South Mid-Atlantic Ridge

Marine Pollution Bulletin xxx (2015) xxx–xxx

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Marine Pollution Bulletin

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Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge ⇑ Xiangxing Gao a,1, Wei Gao a,b,1, Zhisong Cui a, Bin Han a, Peihua Yang c, Chengjun Sun a, Li Zheng a, a Marine Ecology Research Center, First Institute of Oceanography, State Oceanic Administration of China, Qingdao, China b College of Marine life, Ocean University of China, Qingdao, China c College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China article info abstract

Article history: The indigenous oil-degrading bacterial consortia MARA and MARB were enriched from the deep-sea sed- Received 17 February 2015 iments of South Mid-Atlantic Ridge (MAR) with crude oil as sole carbon and energy sources. Biodiversity Revised 28 May 2015 and community analyses showed that members of a-Proteobacteria were the key players in consortium Accepted 29 May 2015 MARA, whereas those of c-Proteobacteria were the key players in consortium MARB, which were studied Available online xxxx by MiSeq sequencing method. Gravimetric method estimated the oil degradation rates of MARA and MARB to be 63.4% and 85.8%, respectively, after 20 d. Eleven cultivable oil degraders with different mor- Keywords: phologies were isolated. These strains were identiﬁed as Alcanivorax, Bacillus, Dietzia, Erythrobacter, Mid-Atlantic Ridge Marinobacter, Nitratireductor, and Oceanicola based on 16S rRNA gene sequences. Three strains belonging Crude oil biodegradation Microbial consortium to Dietzia exhibited the highest oil degradation capability. Results indicated that the intrinsic biodegra- Metagenome dation capacity of oil contaminants by indigenous microbial communities exists in South MAR sediments. Sediments Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction indigenous oil-degrading bacteria would be the most feasible mea- sure because of the extreme environment (e.g., depth and temper- Petroleum contamination is a widespread environmental prob- ature) (Hazen et al., 2010). Thus, the in situ oil degradation capacity lem. Annually, large amounts of petroleum hydrocarbons spread of indigenous microbial communities would (or be obligated to) into the marine environment from both natural (Head et al., play a signiﬁcant role in bioremediation of deep-sea oil contamina- 2006) and anthropogenic sources (Hassanshahian et al., 2012). tion (Hazen et al., 2010). Schwarz et al. (1974) reported that This phenomenon causes great concern to marine environmental deep-sea bacteria from Atlantic Ocean sediment can utilize hydro- safety among coastal areas (Hassanshahian et al., 2014) and pelagic carbons and grow under in situ pressure. Cui et al. (2008) and Shao areas (Hazen et al., 2010). As an instinct response of nature itself, et al. (2010) presented the responses of bacterial community from bioremediation by marine oil-degrading microorganisms is inves- the Mid-Atlantic Ridge (MAR) sediments to polycyclic aromatic tigated worldwide. Oil-degrading bacteria comprise at least 60 hydrocarbons (PAHs) by DGGE on laboratory scale. However, infor- genera of aerobic bacteria and 5 genera of anaerobic bacteria mation regarding indigenous bacterial communities that execute (Prince, 2005), namely. Alcanivorax, Marinobacter, Rhodococcus oil degradation in the MAR sediments remains limited. (Hassanshahian et al., 2012), Pseudomonas (Zhang et al., 2011), Knowledge about the active bacterial communities in biodegrada- and Acinetobacter (Sakai et al., 1994). Their biodegradation was tion of petroleum hydrocarbon is even less. successful to naturally eliminate oil pollutants (Cappello et al., In this study, oil-degrading consortia were enriched from the 2007a). South MAR sediments with crude oil as the sole sources of carbon The oil spill in the Deepwater Horizon in the Gulf of Mexico (Lu and energy. This research aims to (i) reveal the predominant taxa et al., 2011) resulted in large amount of oil depositing into the of the microbial communities engaged in oil degradation and esti- deep-sea environment. Oil plumes were detected at depths of mate their oil-degrading potential on laboratory scale to reveal the approximately 1000–1300 m (Hazen et al., 2010), as gaseous or biodegradation capacity of crude oil in deep-sea environment, as non-gaseous components (Kessler et al., 2011). Biodegradation by well as (ii) investigate the biodegradability of crude oil by isolated predominant strains of the consortia, thereby understanding the

⇑ Corresponding author. microbial biodiversity and degradation potential at the deep-sea E-mail address: Zhengli@ﬁo.org.cn (L. Zheng). sediments of South MAR. 1 Both authors contributed equally. http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 0025-326X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 2 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

2. Materials and methods 2.4. Phylogenetic analysis of the isolates

2.1. Sampling Purified strains were cultured in M8 medium at 25 °C for 3 d. Bacterial pellets were obtained by centrifugation at 12,000 rpm Sediment samples were collected using a multi-core sampler for 3 min. Genomic DNA was then prepared with an Axygen DNA during the 26th Da-Yang Ke Kao in 2012. Two sites of the South extraction kit (Axygen, USA) in accordance with the manufacturer’s MAR (Fig. 1) were defined as site A (15°100S; 13°210W) and site B protocol. The 16S rRNA gene sequences were amplified on a ther- (18°290S; 12°420W) with depths of 3032 m and 2152 m, respec- mal cycler (TaKaRa, Japan) as previously described (Cui et al., tively. Samples were wrapped in tinfoil and preserved at 4 °C until 2008). The PCR products were sequenced by Invitrogen use. (Invitrogen, Shanghai, China) using 16S rRNA sequencing primer sets 27F and 1492r. Subsequently, the 16S rRNA gene sequences were aligned by BLAST and then utilized to construct the phyloge- 2.2. Quantification of total petroleum hydrocarbons in the sediments netic tree through the methods described by Shao et al. (2010).

Total petroleum hydrocarbons (TPHs) in the sediments were 2.5. Community structure analysis of the two culturable oil-degrading extracted and determined using GC–MS. The procedures were per- consortia formed in triplicate as previously described (Cui et al., 2008). Grain sizes of the sediments were measured by sieving techniques 2.5.1. DNA extraction, PCR amplification, and MiSeq sequencing (Dell’Anno et al., 2012). Genomic DNA was extracted by CTAB/NaCl method as described by Rochelle (2001). The V4 + V5 region of the 16S rRNA gene was amplified using a primer sets 515F (50-GTGCCAGCMGCCGCGG-30) 2.3. Enrichment of oil-degrading bacterial consortia and isolation of oil and 907R (50-CCGTCAATTCMTTTRAGTTT-30). PCR was performed degraders in a 20 lL reaction system containing 4 lLof5 FastPfu Buffer, 2 L of dNTPs (2.5 mM), 0.8 L of each primer (5 M), 0.4 Lof Sediment (2 g) from each site was added to an Erlenmeyer flask l l l l FastPfu polymerase, 0.2 L of BSA, and 10 ng of template DNA. containing 100 mL of ONR7a medium (Dyksterhouse et al., 1995) l Distill water was added for the remaining of the reaction systems. with 1% (w/v) sterilized crude oil as the sole carbon and energy The PCR program was conducted as follows: 95 °C for 2 min, fol- source. The crude oil was sterilized by autoclave at 115 °C for lowed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 20 min. The flask without sediment addition was used as negative 55 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C control. Each treatment was performed in triplicate. The flasks for 10 min. PCR was performed on an ABI GeneAmpÒ 9700 thermal were incubated on a rotary shaker at 25 °C and 115 rpm for cycler. PCR products were purified using a DNA gel extraction kit 2 weeks under aerobic conditions. Enriched cultures were then (Axygen, USA). The concentration of PCR products was quantified transferred to the same fresh medium with 2% inoculum and culti- by QuantiFluor™-ST blue fluorescence quantitative system in vated under the same conditions. After the enrichments were accordance with the manufacturer’s protocol. The purified PCR repeated thrice, two oil-degrading consortia, namely, MARA and products were sequenced by the Illumina MiSeq sequencing sys- MARB, were obtained. The culture (100 lL) was then serially tem (Majorbio, Shanghai, China). diluted and plated on M8 agar plates (Cui et al., 2008). All plates were incubated at 25 °C for 7 d. Bacteria with different morphologies were streaked on fresh M8 plates for purification. The two 2.5.2. Statistical analysis consortia and all the isolates were stored with 20% glycerol in a Raw sequence reads were processed using the Trimmomatic cryogenic vial at 80 °C for further analyses. software (version 0.32) as follows: (i) <50 bp reads were filtered

Western Africa

The gulf of Guina

Brazil site A (15°10′S; 13°21′W)

site B (18°29′S; 12°42′W)

South Atlantic Ocean

Fig. 1. Sampling sites of the South Mid-Atlantic Ridge.

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx 3

Table 1 Bacterial diversity and richness estimate of the two culturable oil-degrading consortia.

Sample ID Reads 0.97 Similarity OTU ACE Chao Shannon Simpson Coverage MARA 28714 58 91.2 77.43 1.49 0.37 99.94 (75.39, 121.39) (64.06, 120.32) (1.47, 1.50) (0.37, 0.38) MARB 27747 60 73.46 75.17 0.99 0.64 99.95 (64.72, 98.39) (64.30, 113.53) (0.97, 1.01) (0.64, 0.65) to abandon the base pair with quality value of <20; (ii) overlapped grain size analysis showed that both of the sediments were com- V4 + V5 region gene sequences were generated on the basis of the posed of slit clay with pale gray in color. sequences of barcodes and primers in different reads; and (iii) the chimera, barcodes and primers were all removed. Effective 3.2. Biodiversity and community structure analyses of the two sequences were then clustered by Usearch software (version 7.1) culturable bacterial consortia to operational taxonomic units (OTUs) at 97% similarity level. The taxonomic identification of each OTU was classified phyloge- Comparative analysis of the two culturable oil-degrading con- netically to different taxonomic levels with the reference sequence sortia was conducted using diversity and richness indices, and database SILVA 119 (Quast et al., 2012) based on RDP Classifier cluster method. A total of 77,797 reads were recovered via (Wang et al., 2007) under a set confidence threshold of 70%. Illumina MiSeq sequencing analysis of 16S rRNA gene sequences Alpha-diversity indices (ACE, Chao1, Shannon and Simpson) were from the two consortia. Up to 56,461 effective reads were gener- analyzed with Mothurv (version 1.30.1) (Schloss et al., 2011)at ated after trimming as previously described, and the optimized 97% similarity level. data of the reads ranged from 395 bp to 396 bp in length. A total of 118 OTUs were obtained from the two consortia with 2.6. Hydrocarbon biodegradation analysis 97% similarity by RDP Classifier (Table 1). However, no obvious difference existed in the number of OTUs between the two consortia. The enriched oil-degrading bacterial consortia and the isolates Furthermore, the average alpha-diversity indices (ACE, Chao, were transferred into 100 mL of ONR7a medium containing 1% Shannon and Simpson) were estimated at a dissimilarity of 3%. (w/v) sterilized crude oil; the transfer was performed in triplicate The results showed that the community richness (ACE and Chao) with an inoculum density of OD630 = 0.05. The flask without any was slightly higher in MARA consortium, which was similar in con- inoculum was used as control. All the samples were incubated at fidence intervals. The community diversity indices (Shannon and 25 °C on a rotator shaker (115 rpm) for 20 d. The turbidity of the Simpson) of the MARA consortium were also slightly higher than oil-degrading cultures was also determined by OD630nm to indi- that of the MARB consortium. Additionally, the coverage values rectly represent the bacterial density on an ELIASA (TECAN, of the two consortia were both >99%, indicating that the performed Switzerland). After 20 d of biodegradation, the total residual sequencing depth can reflect the composition of prokaryotic com- hydrocarbons and their derivates were extracted with 50 mL of munity at a dissimilarity of 3%. The rarefaction curves reached dichloromethane. Bacteria and water were removed by centrifuga- asymptote, indicating that the sequencing performance covered tion at 6000 rpm for 5 min. Up to 20 mL of the organic phase was most of the taxonomic structures and diversities of the community processed to determine the residual oil content via gravimetric (data not shown). All the data showed no obvious difference in the analysis as previously described (Zheng et al., 2012). Afterward, two culturable consortia between community diversity and 1 mL of the residual organic phase was dehydrated in a column richness. with 2 g of anhydrous Na2SO4 and filtered through a 0.22 lm nylon For the bacterial community structure of the two consortia, tax- membrane (JINTENG, Tianjin, China). The filtrate was evaporated onomic distributions of the microbial OTUs were shown at differ- to dry under a stream of nitrogen at ambient temperature. The ent levels based on phylogenetic analysis. At the phylum level residual was redissolved in chromatography-grade n-hexane con- (Fig. 2A), Proteobacteria was the prominent phylum in both consor- taining N-Tetracosane-D50 and P-Terphenyl-D14 as internal stan- tium MARA and MARB, accounting for 97.1% and 95.9% of the total 1 dards (10 lgmL each). The samples were separated by a 6890A effective reads, respectively. The second dominant phylum was gas chromatograph (Agilent Technologies) in a HP-5 MS capillary Actinobacteria (2.5% and 2.9%, correspondingly). Moreover, column (30 m 0.25 mm, 0.22 lm). Hydrocarbons were analyzed Deinococcus-Thermus (0.31%), Bacteroidetes (0.03%), Firmicutes by a 5973 mass spectrometer equipped with a quadrupole axis (0.007%), and others (0.0035%) existed in consortium MARA, detector. The residual hydrocarbons were quantified using the pro- whereas Planctomycetes (0.97%) and Bacteroidetes (0.20%) appeared tocols described by Cui et al. (2013). in consortium MARB. At the class level, an obvious variation between the OTUs of the 2.7. Nucleotide sequence accession numbers two consortia was observed (Fig. 2B). a-Proteobacteria was the most dominant class in consortium MARA, accounting for 79.11%. The partial 16S rRNA gene sequences of the strains are available The second dominant class was c-Proteobacteria (17.45%), whereas in the GenBank nucleotide sequence database and the accession b- and d-Proteobacteria were relatively little (0.53% and 0.0035%). numbers are listed in Table 2. Additionally, Actinobacteria was the third dominant class (2.5%), and Deinococci (0.31%) was also detected. In the consortium 3. Results MARB, the most dominant class was c-Proteobacteria, accounting for 92.07%. This rate is much higher than the abundance of 3.1. TPHs in sediments a-Proteobacteria (3%). No obvious difference existed in the abundance of b- and d-Proteobacteria between the two consortia, which The concentration of the detected TPHs in the sediment at site A both exhibited low abundance (<0.7%). Moreover, Actinobacteria was 1082 ± 5 ng g1 dry weight. No obvious difference was noted accounted for 2.9% in the consortium MARB. Both Phycisphaerae from site B sediment (985 ± 10 ng g1 dry weight). Additionally, (0.97%) and Cytophagia (0.15%) were also detected in MARB.

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 4 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

A, Bacterial phyla levels

MARA MARB Proteobacteria Actinobacteria Planctomycetes Bacteroidetes Deinococcus-Thermus Firmicutes others

B, Bacterial class levels Gammaproteobacteria MARA MARB Alphaproteobacteria Deltaproteobacteria Betaproteobacteria Actinobacteria Phycisphaerae Deinococci Cytophagia others

C, Bacterial genus levels MARA Marinobacter MARB Alcanivorax Dietzia Rhodobacteraceae unclassified Gammaproteobacteria unclassified Rhodobium Erythrobacter Amorphus Peredibacter Marinicella others

D, Others MARA MARB

Unclassified Unclassified

Bauldia Georgenia Vibrio Delftia Truepera Pseudomonas Muricauda Fulvivirga Roseobacter Arcobacter Celeribacter Halomonas Hyphomonas Desulfotalea Nocardioides Streptomyces Sphingobium Psychrobacter Sandaracinus Parvularcula Sulfitobacte Microbacterium Pelagibacterium Sphingomonas Pseudospirillum Brevundimonas Hyphomicrobium Ochrobactrum Nitratireductor Propionibacterium Lactobacillus WinogradskyellaĂ Pseudoalteromonas Escherichia-Shigella Stenotrophomonas unclassified

Fig. 2. Biodiversity and community structure of the two oil-degrading consortia MARA and MARB.

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx 5

Table 2 16S rRNA gene analysis of the isolated strains in the two oil-degrading consortia.

Strains (GenBank accession no.) Most closely related type strain (GenBank accession no.) Similarity (%) Size (bp) A1 (KP265727) Dietzia maris DSM 43672T (NR118596) 100 885 A2 (KP265728) Dietzia maris DSM 43672T (NR118596) 99.19 859 A3 (KP265729) Dietzia maris DSM 43672T (NR118596) 99.88 834 A4 (KP265730) Bacillus siamensis KCTC 13613T (AJVF01000043) 99.88 871 A5 (KP265731) Alcanivorax venustensis ISO4T (AF328762) 99.55 863 A6 (KP265732) Erythrobacter citreus RE35F-1T (AF118020) 99.64 837 B1 (KP265733) Marinobacter lipolyticus SM19T (ASAD01000031) 98.84 861 B3 (KP265735) Dietzia maris DSM 43672T (NR118596) 99.04 866 B4 (KP265736) Oceanicola nanhaiensis DSM 18065T (JHZF01000008) 99.88 851 B5 (KP265737) Erythrobacter ﬂavus SW-46T (AF500004) 99.07 852 B6 (KP265738) Nitratireductor aquibiodomus JCM 21793T (BAMP01000145) 100 860

Strains initial with A before the ﬁgures were obtained from the consortium MARA, and initial with B were obtained from the consortium MARB.

Oceanicola nanhaiensis DSM 18065T (JHZF010000) 100 B4 (KP265736) 99 Nitratireductor aquibiodomus JCM 21793T (BAMP010001) Alpha-proteobacteria 100 99 B6 (KP265738) T 100 Erythrobacter citreus RE35F-1 (AF118020) A6 (KP265732) T 100 Erythrobacter flavus SW-46 (AF500004) 100 99 B5 (KP265737)

T 100 Alcanivorax venustensis ISO4 (AF328762) A5 (KP265731) Gamma-proteobacteria T 100 Marinobacter lipolyticus SM19 (ASAD010000)

100 B1 (KP265733) Bacillus siamensis KCTC 13613T (AJVF010000) 100 Firmicutes A4 (KP265730)

A3 T Dietzia maris DSM 43672 ˄NR118596˅ 100 A1 (KP265727) Actinobacteria 74

38 A2 (KP265728) 84 B3 (KP265735)

0.05

Fig. 3. Phylogenetic tree of the oil-degrading strains isolated from the MAR deep-sea sediments. The phylogenetic tree was constructed based on 16S rRNA gene fragments (approximately 860 bp). The type strains and their GenBank accession numbers are shown in boldface type. The numbers at the branch nodes are bootstrap values based on 1000 re-samplings for maximum likelihood. Scale bar equals approximately 5% nucleotide divergence.

Table 3 Crude oil degradation rates by consortium MARA and MARB. (Mean ± SD).

Treatment % of oil-degradation % of total n-alkanes % of total PAHs %/(C17/Pr) %/(C18/Ph) Growth (OD630nm) MARA 63.4a ± 3.4 66.6a ± 5.0 68.8b ± 4.5 52.3a ± 8.7 56.6a ± 3.1 0.78 ± 0.02 MARB 85.5b ± 0.8 82.5a ± 2.9 91.2a ± 4.8 67.9a ± 6.4 66.9a ± 4.8 0.91 ± 0.05 NC 19.4C ± 1.2 7.3B ± 0.72 11.6C ± 2.0 5.0B ± 10.9 6.1B ± 3.5 0.10 ± 0.01

The different letters of superscript lowercase indicates the signiﬁcant difference with level at p < 0.05, while the different letters of superscript uppercase at p < 0.01.

At the genus level, the difference between the two consortia namely, Rhodobacteraceae (57.8%), Erythrobacter (18%), was more remarkable (Fig. 2C). Up to 67 genera were generated c-Proteobacteria unclassiﬁed (14%), Rhodobium (2.8%), and in the two consortia. Among these genera, 28 were shared by Dietzia (2.2%). Four genera were major members of the consor- both consortia. The top 10 genera in each consortium were listed, tium MARB, namely, Marinobacter (80.5%), Alcanivorax (7.4%), with the genera of relatively lower abundance pooled into others. Dietzia (2.9%), and Amorphus (1.2%). Five genera accounted for over 95% in the consortium MARA,

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 6 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

3.3. Isolation and identiﬁcation of cultivable strains from the two A Napthalene Fluorene Chrysene consortia Control dibenzothiophene Phenanthrene Abundance Eleven strains with different morphologies were isolated from the two consortia with oil-degrading capacity (Table 2). Analysis of 16S rRNA gene sequences indicated that these strains belong to eight species in seven genera within the phyla of Proteobacteria, Firmicutes, and Actinobacteria. These strains were dominant in both consortia, as detected by Illumina MiSeq sequencing analysis (Fig. 2). The most closely related types of these strains are listed in Table 2. Strain B1 might be a new species MARA because it demonstrates a low similarity to Marinobacter lipolyticus Abundance SM19T (98.8%). Moreover, four strains of genus Dietzia were isolated from the two consortia. As shown in the phylogenetic tree (Fig. 3), Erythrobacter and Dietzia strains existed in both consortia preferentially. Proteobacteria strains comprised the largest branch (6/11), followed by Actinobacteria (4/11). Notably, four strains of Dietzia exhibited three colors in morphology, with pale yellow for strain A1 and A3, red for strain A2, and orange for strain B3. MARB

3.4. Biodegradability of crude oil by the two consortia

After 20 d, the oil content was degraded to 63% and 85% by con- Abundance sortium MARA and MARB, respectively, accompanied by consider- able increase in bacterial biomass (OD630nm)(Table 3). The results indicated that the two consortia exhibited excellent oil-degrading capacity. The consortium MARB exhibited higher degradation rates (85%) than the consortium MARA (63%) (Table 3). Furthermore, GC–MS analysis showed that both n-alkanes and aromatic com- pounds can be steadily utilized by the two consortia to different degrees. The consortium MARB degraded 82.5% of n-alkanes and 91.2% of PAHs in 20 d, which were much better than those of the B C22 consortium MARA (66.6% and 68.8%). Additionally, hopane within C21 C23 Control C20 C25 oil was used as a conserved internal biomarker (Zakaria et al., C19 C24 C18

2000). The oil-degrading effect, based on the evaluation of Abundance C17 C26 C17/pristane and C18/phytane, was remarkably enhanced by the C16 C27 two consortia compared with abiotic samples (p < 0.01). C15 C29 C28 As shown in the GC–MS chromatogram (Fig. 4), both consortia C30 can utilize a wide range of oil components, including aromatic C14 C31 hydrocarbons and n-alkanes, compared with abiotic samples. The C13 C32 C33 C12 consortium MARB exhibited better degradation of the petroleum hydrocarbons than the consortium MARA, as most peaks of PAHs and n-alkane fractions of the crude oil significantly declined MARA (Fig. 4A and B). In detail, two- to four-ring PAHs, including naph- Abundance thalene, fluorene, dibenzothiophene, phenanthrene and chrysene, were noted in the figures with various degrees of utilization. The two consortia preferentially employed two- to three-ring PAHs compared with four-ring PAH chrysene. With regard to the n-alkane utilization, the two consortia exhibited better degradation for alkanes with medium-length chains (C12–C21) (>70% of consortium MARA and >80% of consortium MARB) than long chains (C25–C39) (<50% of consortium MARA and <70% of consortium MARB MARB). Abundance 3.5. Degradability of crude oil by the isolated strains

The oil-degrading capacity of the isolated strains was evaluated by incubation experiment with crude oil as carbon source for 20 d in triplicate. Among the 11 strains, the cultures of A1, A2, A3, and B3 showed obvious growth on day 3, whereas the growth of the other strains occurred on day 5 (Fig. 5). Compared with the abiotic samples, 48–88% of crude oil was degraded by different strains. Fig. 4. Abundance of petroleum hydrocarbons after 20 d0 biodegradation by Five strains (A1, A3, A5, B1, and B3) can degrade >60% of crude consortium MARA and MARB. (A) the chromatogram of aromatic hydrocarbons; (B) the chromatogram of n-alkanes.

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100 Oil degradation rate reported that c- and a-Proteobacteria were the key members in OD630nm beach sands of the Gulf of Mexico responding to the Deepwater 1.2 Horizon oil spill, with Alcanivorax, Marinobacter, and 80 Rhodobacteraceae being the dominant taxa. Members of Marinobacter (Head et al., 2006) and Rhodobacteraceae (Gutierrez 0.8 et al., 2011) were also associated with the degradation of crude 60 oil components. c-Proteobacteria had been reported as 630nm hydrocarbon-utilizing bacteria in deep-sea oil biodegradation pro- OD cess (Hazen et al., 2010). Consistent with the previous work, our 40 0.4

Degradation rate/% research showed that the above members were also dominant in the two consortia from South MAR sediments, whereas most of them were uncultivable and unclassified. However, the two con- 20 0.0 sortia differed significantly in their community structure, with a-Proteobacteria and c-Proteobacteria being the dominant mem- A1 A2 A3 A4 A5 A6 B1 B3 B4 B5 B6 NC bers in MARA and MARB, respectively. Given the differences in Strains oil-degrading capacity, members of c-Proteobacteria are potential Fig. 5. Oil degrading-capacity and bacterial growth of the isolated strains. key players in biodegradation of oil contaminants in the marine environment. The genus Dietzia is one of the most interesting taxa found in the two consortia. This genus is broadly distributed in nature, such oil, which is better than other strains (about 50%) (Fig. 5). Notably, as in mud of the Mariana Trench (Takami et al., 1997), and strain B3 exhibited the highest oil-degradation rates among the 11 deep-sea sediments (Colquhoun et al., 1998). As a multifunctional strains (approximately 90%), followed by A1 (80%) and A3 (75%). bioresource, strains of Dietzia have been applied in many areas Additionally, analysis showed that the Pearson correlation coeffi- including therapeutic biotreatments, biosurfactants, and biodegra- cient (Zhang et al., 2010) was 0.943 (p < 0.01), indicating that the dation of petroleum hydrocarbons (Gharibzahedi et al., 2014). In biodegradation rates of crude oil were positively related to bacte- the present study, four Dietzia strains exhibited significant rial biomass (OD630nm)(Fig. 5). oil-degrading capacity (50–88%). These strains showed a broad Degradation profile of the petroleum hydrocarbons by each petroleum hydrocarbon degradation profile, including C11–C39 strain was analyzed by GC–MS. The degradation rates of each n-alkanes and PAHs. Notably, the abundance of this genus in both n-alkane (C11–C39) in crude oil by the 11 strains were calculated consortia is relatively high. This finding indicated that Dietzia may (Table S1). The results showed that a remarkable increase (20– play an important role in oil biodegradation in the environment. 90%) in n-alkane degradation was achieved by the isolated strains Other strains in the consortia, including Alcanivorax, Bacillus, compared with the abiotic samples. Strains A1, A3, and B3 exhib- Erythrobacter, Marinobacter, Nitratireductor, and Oceanicola also ited higher degradation rates of n-alkanes (C11–C39) than the showed oil-degrading capacity. The interactions between microor- other strains. Strains A4 and B6 exhibited the lowest n-alkane ganisms in the consortia deserve further investigation. degradation rates. GC–MS analyses also showed that two-, three-, and four-ring PAH derivatives were steadily degraded by the isolated strains 5. Conclusion (Table S2). Among the 11 strains, B3 exhibited the optimum capacity of PAH utilization (>90%), followed by strains A1, A3, B1, and B5, Strains of c- and a-Proteobacteria (Marinobacter, Alcanivorax, with a total PAH degradation rate of >60%. Furthermore, no obvious Erythrobacter and Rhodobacteraceae) were detected as the key difference existed in the degradation rates of fluorene, dibenzoth- degraders of petroleum hydrocarbons in two deep-sea sediments iophene and phenanthrene by each strain. from the South MAR. A significant coupling relationship existed between the bacterial community structures and the oil-degrading capacity. Members of c-Proteobacteria were superior 4. Discussion to other degraders in crude oil degradation. Different strains of Dietzia were isolated and reconfirmed to demonstrate significant Petroleum contaminants can enter the deep ocean through nat- capability for crude oil degradation. ural seepage or adherence to materials heavier than water (Schwarz et al., 1974). Hydrocarbons were also thought to be pro- Acknowledgements duced by both abiotic and biogenic processes in vent systems (Foustoukos and Seyfried, 2004), which had been found at the This research was supported by the National Natural Science South MAR. Cui et al. (2008) and Shao et al. (2010) detected the Foundation of China (41406127 and 41476103), the National existence of PAHs in deep-sea sediments with a total concentration Natural Science Foundation of China, Basic Scientific Fund for 1 of 260 and 445 ng g dry weight, respectively. In the current National Public Research Institutes of China (2015T05), the 1 study, the TPHs were detected as 1082 and 895 ng g dry weight National Natural Science Foundation of China-Shandong Joint from the MAR sediments, indicating an occurrence of higher con- Funded Project (U1406403), National Key Basic Research centration of TPHs at the South MAR sites. Two oil-degrading con- Development Program (973 Program) sub-project sortia were obtained from the sediments, and 11 crude-oil (2015CB755904) and the 2012 Taishan Scholar Award. degrading bacteria were further isolated and characterized. To our knowledge, this research is the first to report about oil-degrading bacterial consortia from South MAR environments Appendix A. Supplementary material on a laboratory scale. Various hydrocarbon-utilizing bacteria in the marine environ- Supplementary data associated with this article can be found, in ment were isolated (Cappello et al., 2007a; Kasai et al., 2002) with the online version, at http://dx.doi.org/10.1016/j.marpolbul.2015. crude oil as the sources of carbon and energy. Kostka et al. (2011) 05.065.

Please cite this article in press as: Gao, X., et al. Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge. Mar. Pollut. Bull. (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.05.065 8 X. Gao et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

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