Marine Pollution Bulletin xxx (2015) xxx–xxx Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul 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 lLof5Â 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 morpholo- gies 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 À80 °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.