Pontiella agarivorans sp. nov., a novel marine anaerobic bacterium capable of degrading macroalgal polysaccharides and fixing nitrogen.

Marine macroalgae produce abundant and diverse polysaccharides, which contribute substantially to the organic matter exported to the deep ocean. Microbial degradation of these polysaccharides plays an important role in the turnover of macroalgal biomass. Various members of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum are degraders of polysaccharides in widespread anoxic environments. In this study, we isolated a novel anaerobic bacterial strain NLcol2T from microbial mats on the surface of marine sediments offshore Santa Barbara, CA, USA. Based on 16S ribosomal RNA (rRNA) gene and phylogenomic analyses, strain NLcol2T represents a novel species within the Pontiella genus in the Kiritimatiellota phylum (within the PVC superphylum). Strain NLcol2T is able to utilize various monosaccharides, disaccharides, and macroalgal polysaccharides such as agar and É©-carrageenan. A near-complete genome also revealed an extensive metabolic capacity for anaerobic degradation of sulfated polysaccharides, as evidenced by 202 carbohydrate-active enzymes (CAZymes) and 165 sulfatases. Additionally, its ability of nitrogen fixation was confirmed by nitrogenase activity detected during growth on nitrogen-free medium, and the presence of nitrogenases (nifDKH) encoded in the genome. Based on the physiological and genomic analyses, this strain represents a new species of bacteria that may play an important role in the degradation of macroalgal polysaccharides and with relevance to the biogeochemical cycling of carbon, sulfur, and nitrogen in marine environments. Strain NLcol2T (= DSM 113125T = MCCC 1K08672T) is proposed to be the type strain of a novel species in the Pontiella genus, and the name Pontiella agarivorans sp. nov. is proposed.IMPORTANCEGrowth and intentional burial of marine macroalgae is being considered as a carbon dioxide reduction strategy but elicits concerns as to the fate and impacts of this macroalgal carbon in the ocean. Diverse heterotrophic microbial communities in the ocean specialize in these complex polymers such as carrageenan and fucoidan, for example, members of the Kiritimatiellota phylum. However, only four type strains within the phylum have been cultivated and characterized to date, and there is limited knowledge about the metabolic capabilities and functional roles of related organisms in the environment. The new isolate strain NLcol2T expands the known substrate range of this phylum and further reveals the ability to fix nitrogen during anaerobic growth on macroalgal polysaccharides, thereby informing the issue of macroalgal carbon disposal.

Genotoxicity detection of oil-containing drill cuttings by Comet assay based on a demersal marine fish Mugilogobius chulae.

An enormous amount of oil-containing drill cuttings have been produced by the marine oil and gas industry. The environmental impacts of discharged drilling waste have been extensively studied. However, there is still an urgent need to develop alternative methods to identify the genotoxicity of untreated and treated drill waste in a timely manner before it is discharged. In this study, we developed a relatively rapid, sensitive, and accurate genotoxicity-detection method using Comet assay and the marine benthic goby Mugilogobius chulae. This goby is sensitive to a standard toxicant mitomycin C (MMC). The optimal exposure period for genotoxicity detection using M. chulae was determined. Three genotoxic indices (tail length (TL), tail DNA content (TD), and tail moment (TM)) were used to assess the effectiveness of high-temperature treatment of oil-contaminated waste. Untreated oil-containing drill cuttings exhibited the highest genotoxicity to goby cells. Genotoxicity was dramatically reduced after thermal treatment of drill cuttings at 350 °C and 500 °C. TD and TM exhibited significant correlation with the concentration of total petroleum hydrocarbons (TPHs)/total polycyclic aromatic hydrocarbons (PAHs) according to Pearson and Mantel correlation analyses (P values were <0.05). Using redundancy analysis (RDA) and variation partition analysis (VPA), the genotoxic effects of the drill cuttings were ascribed to total alkanes and specific groups of PAHs. In conclusion, this newly established biological model has the potential to be widely used to detect the genetic damage of untreated or treated oil-containing drill cuttings discharged into the marine environment.

Study on immobilization of marine oil-degrading bacteria by carrier of algae materials.

This study investigated the immobilizations with of bacteria two kinds of algal materials, Enteromorpha residue and kelp residue. The lipophilicity of them were compared by diesel absorption rates. The immobilization efficiency of Bacillus sp. E3 was measured to evaluate whether these carriers would satisfy the requirement for biodegradation of oil spills. The bacteria were immobilized through adsorption with the sterilized and non-sterilized carriers to compare the differences between the two treatments. Oil degradation rates were determined using gravimetric and GC-MS methods. Results showed the absorption rates of Enteromorpha residue and kelp residue for diesel were 411 and 273% respectively and remained approximately 105 and 120% after 2 h of erosion in simulated seawater system. After immobilized of Bacillus sp. E3, the oil degradation rates of them were higher than 65% after 21 days biodegradations. GC-MS analysis showed that two immobilizations degraded higher than 70% of the total alkane and the total PAHs, whereas the free bacteria degraded 63% of the total alkane and 66% the total PAHs. And the bacteria immobilized with the carriers degraded more HMW-alkanes and HMW-PAHs than the free bacteria. The bacteria immobilized by non-sterilized kelp residue showed a considerably higher degradation rate than that using sterilized kelp residue. A considerably higher cells absorption rate of immobilization was obtained when using kelp residue, and the preparation of immobilization was low cost and highly efficient. The experiments show the two algae materials, especially the kelp residue, present potential application in bioremediation of marine oil spills.

Marinobacter aromaticivorans sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from sea sediment.

A rod-shaped, Gram-stain-negative, slightly halotolerant bacterium, designated strain D15-8PT, was isolated from a sediment sample from the South China Sea. The strain could grow in NaCl concentrations ranging from 0.5 % to 10 % (w/v) (optimum 0.5-1.5 %), and could be cultivated at 10-40 °C (optimum 25 °C) and pH 5.5-9.5 (optimum pH 7.0-8.0). The strain was positive for catalase, oxidase, and hydrolysis of Tween 80, but negative for hydrolysis of DNA and gelatin, nitrite reduction, indole production, Voges-Proskauer reaction, and methyl red test. Strain D15-8PT could biodegrade naphthalene, phenanthrene, and anthracene. The major respiratory quinone was Q-9. The main cellular fatty acids were C12 : 0 (11.5 %), C14 : 0 3-methyl (22.0 %), C16 : 0 (19.2 %), C16 : 1ω9c (22.9 %), and C18 : 1ω9c (6.7 %). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and an unidentified phospholipid. The DNA G+C content was 56.8 mol%. Phylogenetic analyses based on 16S rRNA genes showed that strain D15-8PT was most closely related to Marinobacter maritimus JCM 12521T (98.5 % 16S rRNA gene sequence similarity), Marinobacter antarcticus CGMCC 1.10835T (98.1 %), Marinobacter lipolyticus DSM 15157T (97.1 %), and Marinobacter guineae CECT 7243T (97.0 %). Results of the gyrB gene analysis and DNA-DNA hybridization were both less than the cut-off values (90 % for gyrB gene sequence similarity and 70 % for DNA-DNA hybridization). On the basis of this taxonomic study using a polyphasic approach, strain D15-8PT represents a novel species of the genus Marinobacter, for which the name Marinobacter aromaticivorans sp. nov. is proposed. The type strain is D15-8PT ( = CGMCC 1.11015T = KCTC 23781T).

Cycloalkane degradation by an uncultivated novel genus of Gammaproteobacteria derived from China's marginal seas.

The consumption of cycloalkanes is prevalent in low-temperature marine environments, likely influenced by psychrophilic microorganisms. Despite their significance, the primary active species responsible for marine cycloalkane degradation remain largely unidentified due to cultivation challenges. In this study, we provide compelling evidence indicating that the uncultured genus C1-B045 of Gammaproteobacteria is a pivotal participant in cycloalkane decomposition within China's marginal seas. Notably, the relative abundance of C1-B045 surged from 15.9% in the methylcyclohexane (MCH)-consuming starter culture to as high as 97.5% in MCH-utilizing extinction cultures following successive dilution-to-extinction and incubation cycles. We used stable isotope probing, Raman-activated gravity-driven encapsulation, and 16 S rRNA gene sequencing to link cycloalkane-metabolizing phenotype to genotype at the single-cell level. By annotating key enzymes (e.g., alkane monooxygenase, cyclohexanone monooxygenase, and 6-hexanolactone hydrolase) involved in MCH metabolism within C1-B045's representative metagenome-assembled genome, we developed a putative MCH degradation pathway.

Marinobacter nanhaiticus sp. nov., polycyclic aromatic hydrocarbon-degrading bacterium isolated from the sediment of the South China Sea.

A Gram-negative, rod-shaped, slightly halophilic and facultatively anaerobic bacterium, designated strain D15-8W(T), was isolated from the sediment of the South China Sea. Growth was found to occur optimally at 25 °C, between pH 7.0 and 8.0 and with 1-5 % (w/v) NaCl. The strain was observed to utilize a variety of organic substrates and polycyclic aromatic hydrocarbons as sole carbon sources. The G+C content of the genomic DNA was determined to be 58.7 %. The predominant respiratory quinone was found to be Q-9. The significant fatty acids were determined to be C(16:0), C(16:1) ω9c, C(18:1) ω9c, C(12:0) and C(14:0) 3OH. Analysis of 16S rRNA gene sequences showed that strain D15-8W(T) fits within the phylogenetic cluster of the genus Marinobacter and is most closely related to Marinobacter segnicrescens CGMCC 1.6489(T), Marinobacter bryozoorum DSM 15401(T), Marinobacter lacisalsi CECT 7297(T) and Marinobacter daqiaonensis CGMCC1.9167(T). The DNA-DNA hybridization values between strain D15-8W(T) and the type strains of the most closely related species were 42.3 % (CGMCC 1.6489(T)), 39.8 % (DSM 15401(T)), 37.3 % (CECT 7297(T)) and 35.2 % (CGMCC1.9167(T)). The results of this polyphasic study indicate that strain D15-8W(T) represents a novel species of the genus Marinobacter, for which the name Marinobacter nanhaiticus sp. nov. is proposed. The type strain is D15-8W(T) (=CGMCC 1.11019(T)=KCTC 23749(T)).

Degradation potential and pathways of methylcyclohexane by bacteria derived from Antarctic surface water.

Cycloalkanes pose a tremendous environmental risk due to their high concentration in petroleum hydrocarbons and hazardous effects to organisms. Numerous studies have documented the biodegradation of acyclic alkanes and aromatic hydrocarbons. However, insufficient attention has been paid to studies on the microbial degradation of cycloalkanes, which might be closely linked to psychrophilic microbes derived from low-temperature habitats. Here we show that endemic methylcyclohexane (MCH, an abundant cycloalkane species in oil) consumers proliferated in seawater samples derived from the Antarctic surface water (AASW). The MCH-consuming bacterial communities derived from AASW exhibited a distinct species composition compared with their counterparts derived from other cold-water habitats. We also probed Colwellia and Roseovarius as the key active players in cycloalkane degradation by dilution-to-extinction-based incubation with MCH as sole source of carbon and energy. Furthermore, we propose two nearly complete MCH degradation pathways, lactone formation and aromatization, concurrently in the high-quality metagenome-assembled genomes of key MCH consumer Roseovarius. Overall, we revealed that these Antarctic microbes might have strong interactions that enhance the decomposition of more refractory hydrocarbons through complementary degradation pathways.

Occurrence and distribution of cyclic-alkane-consuming psychrophilic bacteria in the Yellow Sea and East China Sea.

Cyclic alkanes (c-alkanes) are toxic compounds that are abundant in subsurface oil reservoirs and spilled condensate; hence, their environmental risk is significant. Although numerous studies have focused on the decomposition of other compound classes, e.g., acyclic alkanes and aromatic hydrocarbons, very little is known about the biodegradation of c-alkanes in the marine environment. Here, we enriched methylcyclohexane (MCH)-degrading bacteria derived from the cold bottom water (10-20 °C) of China's marginal seas in summer and characterized the changes to the bacterial community using high-throughput amplicon sequencing. MCH-consuming bacteria failed to grow from the warmer surface water (25-29 °C) in the same geographic sites and seasons. Notably, MCH-consuming communities derived from the cold bottom water in the Yellow Sea exhibit distinct structures compared to the other treatments. Furthermore, almost all dominant species in this setting appear to be specifically adapted to deeper cold water as indicated by significantly negative correlations to temperature (P < 0.01). From these results, we proposed that the biodegradation of MCH is effectively limited to the colder waters (10-20 °C) of China's marginal seas, with uncultured psychrophiles acting as the key taxa for MCH decomposition. Overall, this study indicates key functions for uncultivated microbes in the marine environment.

[Identification and characterization of a novel hydrocarbon-degrading Marinobacter sp. PY97S].

OBJECTIVE: To identify and characterize a hydrocarbon-degrading bacterium isolated from the sediment of the Yellow Sea. METHODS: We used 16S rRNA gene sequences based phylogenetic analysis, physiological and biochemical characterization, DNA G + C content assaying, determination of cellular fatty acids, testing of carbon sources and respiratory lipoquinone and experiment of DNA-DNA relatedness. Its capability of degrading aliphatic hydrocarbons in ONR7a media supplemented with nine n-alkanes, separately, as sole source of carbon and energy was further determined. RESULTS: The Gram-negative isolate PY97S was a member of the genus Marinobacter, catalase-and oxidase-positive, and with Q-9 as its predominant respiratory lipoquinone. The similarity between its 16S rRNA gene and that of its most closely related type strain in GenBank Marinobacter koreensis DD-M3(T) was 96.93%, and their level of DNA relatedness was 46.7%. The appropriate temperature for its growth ranged from 15 degrees C to 35 degrees C with the optimum of 30 degrees C, the appropriate initial acidity from pH 6.0 to 9.5 with the optimum of pH 7.0, and the appropriate salinity (NaCl) from 0% to 10% with the optimum of 0%. It metabolized many carbohydrates and organic acids and was sensitive to diverse antibiotics including ampicillin and piperacillin. The G + C content of its genomic DNA was 48.2 mol%. The major fatty acids were 2-methyl C15:0 (29.97%), C16: 1omega7c (27.22%), C12:0 (22.22%) and C16: 1omega9c (5.73%). CONCLUSION: The isolate PY97S was identified as a petroleum hydrocarbon-degrading novel species of genus Marinobacter, holding the potential of being applied in the bioremediation of oil spill.

Screening and validation of new diagnostic ratios of dibenzothiophenes and fluorenes for identification of seriously weathered oil spills.

Fingerprinting technique is a universal method for tracing oil spill. It is usually achieved by means of diagnostic ratios (DRs) of biomarkers. In the process of severely weathering, the important components usually change greatly and the relevant diagnostic ratios may also change. Therefore, it is more difficult to trace severely weathered oil to its source. On 22 November 2013, the huge explosion of Sinopec pipeline occurred in Qingdao, China. The beaches near the explosion site were contaminated and damaged by oil spills. After the explosion, an actual weathering experiment was carried out on an oil-polluted beach. The original and weathered spilled oil samples have been collected from this site. Synchronized with actual coastal weathering, a 360-day Lab simulated weathering experiment was carried out using the sampled original oil spill samples. According to data analysis techniques including similarity, t-test method and repeatability limit analysis, 27 new diagnostic ratios of dibenzothiophenes and fluorenes in the weathered oil samples were selected and verified. 6 of them maintained good stability during both of the simulated and actual weathering process. It is recommended that these stable DRs be used for tracing the source of severely weathered oil spills to promote the efficiency and accuracy.

In Vitro Anticancer Drug Sensitivity Sensing through Single-Cell Raman Spectroscopy.

Traditional in vitro anticancer drug sensitivity testing at the population level suffers from lengthy procedures and high false positive rates. To overcome these defects, we built a confocal Raman microscopy sensing system and proposed a single-cell approach via Raman-deuterium isotope probing (Raman-DIP) as a rapid and reliable in vitro drug efficacy evaluation method. Raman-DIP detected the incorporation of deuterium into the cell, which correlated with the metabolic activity of the cell. The human non-small cell lung cancer cell line HCC827 and human breast cancer cell line MCF-7 were tested against eight different anticancer drugs. The metabolic activity of cancer cells could be detected as early as 12 h, independent of cell growth. Incubation of cells in 30% heavy water (D2O) did not show any negative effect on cell viability. Compared with traditional methods, Raman-DIP could accurately determine the drug effect, meanwhile, it could reduce the testing period from 72-144 h to 48 h. Moreover, the heterogeneity of cells responding to anticancer drugs was observed at the single-cell level. This proof-of-concept study demonstrated the potential of Raman-DIP to be a reliable tool for cancer drug discovery and drug susceptibility testing.

[Synergic effect of marine obligate hydrocarbonoclastic bacteria in oil biodegradation].

OBJECTIVE: In order to study the synergic effect of two marine obligate hydrocarbonoclastic bacteria in the oil biodegradation process. METHODS: We combined the PAHs degrader Marinobacter sp. PY97S with the oil degrader Alcanivorax sp. 22CO-6 and Alcanivorax sp. JZ9B respectively to construct oil-degrading consortia. Multiple methods including weighting method, gas chromatography-flame ionization detection, gas chromatography-mass spectrometry and thin layer chromatography-flame ionization detection were used to analyze and compare the oil degradation rates as well as the chromatographic figures of degraded oil between the pure cultures of obligate hydrocarbonoclastic bacteria and defined consortia. RESULTS: The two consortia, 22CO-6 + PY97S and JZ9B + PY97S, exhibited synergic effects in the oil biodegradation process. The degradation rates of oil by the consortia were increased from 27.81% and 83.52% to 64.03% and 86.89% compared to the pure culture of oil degrader 22CO-6 and JZ9B, respectively. The consortia could degrade aliphatic and aromatic fraction at the same time, including high molecular weight PAHs chrysene and its alkyl derivatives. CONCLUSION: There are obvious synergic effect of Alcanivorax and Marinobacter strains in the oil biodegradation process, which accelerated the oil biodegradation and decomposed thoroughly the more ecotoxic high molecular weight compounds in crude oil.

Comparative toxicity of five dispersants to test organisms at different trophic levels: Platymonas helgolandica, Ruditapes philippinarum, and Acinetobacter sp. Tox2.

Nowadays, although dispersants have been widely applied for emergency response to oil spills, they are potentially hazardous to the marine ecosystem. Therefore, it is necessary to evaluate dispersants' toxicity in a practical and integrated way before their large-scale application. Here, we compared the acute toxicity of five chemical dispersants (concentrate RS-I, conventional RS-I, HLD-501, Fuken-2, and Weipu) to three species (a microalgae Platymonas helgolandica, a mollusk Ruditapes philippinarum, and a luminescent bacterium Acinetobacter sp. Tox2) which represent different trophic levels. Our results showed that (1) conventional RS-I was slightly toxic to all the three test organisms; (2) concentrate RS-I and Weipu were slightly toxic to R. philippinarum, but were not toxic to the other two test species; (3) Fuken-2 and HLD-501 exhibited no acute toxicity to the three test organisms. Our results could provide information on toxicity data derived from multiple test organisms for the use of these five dispersants in the future.

[Predominant strains of polycyclic aromatic hydrocarbon-degrading consortia from deep sea of the Middle Atlantic Ridge].

OBJECTIVE: In order to identify the predominant strains of polycyclic aromatic hydrocarbon (PAH)-degrading consortia harboring in sea water and surface sediment collected from deep sea of the Middle Atlantic Ridge. METHODS: We employed enrichment method and spread-plate method to isolate cultivable bacteria and PAHs degraders from deep sea samples. Phylogenetic analysis was conducted by 16S rRNA gene sequencing of the bacteria. Then we analyzed the dominant bacteria in the PAHs-degrading consortia by denaturing gradient gel electrophoresis (DGGE) combined with DNA sequencing. RESULTS: Altogether 16 cultivable bacteria were obtained, including one PAHs degrader Novosphingobium sp. 4D. Phylogenetic analysis showed that strains closely related to Alcanivorax dieselolei NO1A (5/16) and Tistrella mobilis TISTR 1108T (5/16) constituted two biggest groups among the cultivable bacteria. DGGE analysis showed that strain 4L (also 4M and 4N, Alcanivorax dieselolei NO1A, 99.21%), 4D (Novosphingobium pentaromativorans US6-1(T), 97.07%) and 4B (also 4E, 4H and 4K, Tistrella mobilis TISTR 1108T, > 99%) dominated the consortium MC2D. While in consortium MC3CO, the predominant strains were strain 5C (also 5H, Alcanivorax dieselolei NO1A, > 99%), uncultivable strain represented by band 5-8 (Novosphingobium aromaticivorans DSM 12444T, 99.41%), 5J (Tistrella mobilis TISTR 1108T, 99.52%) and 5F (also 5G, Thalassospira lucentensis DSM 14000T, < 97%). CONCLUSION: We found that strains of genus Alcanivorax, Novosphingobium, Tistrella and Thalassospira were predominant bacteria of PAHs-degrading consortia in sea water and surface sediment of Middle Atlantic Ridge deep sea, with Novosphingobium spp. as their main PAHs degraders.

[Antifungal mechanism of Bacillus marinus B-9987].

OBJECTIVE: We studied the antifungal effect of the metabolite BMME-1 from Bacillus marinus B-9987, to reveal its antifungal mechanism. METHODS: The permeability of the Alternaria solani was tested by spectrophotometer after the treating the crude extracts of B-9987. The composition of cell wall and the sterol components of the fungal plasmalemma of Alternaria solani were analyzed with Infrared Spectrum and GC-MS, respectively. RESULTS: We found that the metabolites of B-9987 had strong antifungal activity with MIC50 and MFC value being 6.2mg/L and 50mg/L. The absorbance in extracellular fluid detection showed that the tegument of the fungi was impaired. The detection of glucan and chitin indicated the change in the structure of the cell wall. The absorption peak of the carbon-hydrogen bond, beta-glucosidic bond, carbon--oxygen bond was attenuated but the hydroxyl, carbonyl absorption was enhanced on the contrary. There were only one peak change in chitin chromatogram on the absorption of amide linkage comparing to the control. These changes on the structure may affect the stability of the fungal cell wall. Ergosterol was the predominant component of sterol with the proportion of 62.52 +/- 3.31% in control cells, but showed a decline during treatment with BMME-1 at a concentration of 56.36 +/- 2.52%. Accumulation of coprostanol, the precursor of ergosterol, was found in the test. CONCLUSION: From the result we can conclude that the antifungal mechanism of the crude extracts was interfering of ergosterol synthesis resulting in the change on permeability, and also mainly changed the structure of the cell wall, mainly acting on the glucan synthesis.

Complete Genome Sequence of Cycloclasticus sp. Strain PY97N, Which Includes Two Heavy Metal Resistance Genomic Islands.

We present the complete genome sequence of fluoranthene-consuming Cycloclasticus sp. strain PY97N. This strain has one circular chromosome with a G+C content of 42.06%. Moreover, two genomic islands were identified as putative conjugative elements. These genomic details are expected to inform our understanding of the remarkable catabolic capacities of organisms of the Cycloclasticus lineage.

Nanotoxicity of different sizes of graphene (G) and graphene oxide (GO) in vitro and in vivo.

Graphene family nanomaterials (GFNs) have attracted significant attention due to their unique characteristics and applications in the fields of biomedicine and nanotechnology. However, previous studies highlighted the in vitro and in vivo toxicity of GFNs with size and oxidation state differences are still elusive. Therefore, we prepared graphene (G) and graphene oxide (GO) of three different sizes (S-small, M-medium, and L-large), and characterized them using multiple surface-sensitive analytical techniques. In vitro assays using HEK 293T cells revealed that the small and large sizes of G and GO significantly reduced the cell viability and increased DNA damage, accompanying with activated reactive oxygen species (ROS) generation and induced various expressions of associated critical genetic markers. Moreover, the bacterial assays highlighted that G and GO caused strong acute toxicity on Tox2 bacteria. Effects of G were higher than GO and showed size dependent effect: L > M > S, while the medium size of GO induced mild genetic toxicity on RecA bacteria. In vivo assays revealed that exposure to G and GO caused the developmental toxicity, induced ROS generation, and activated related pathways (specifically GO) in zebrafish. Taken together, G showed stronger ability to decrease the survival rate and induce the acute toxicity, while GO showed obvious toxicity in terms of DNA damages, ROS generation, and abnormal gene expressions. Our findings highlighted that G and GO differentially induced toxicity based on their varying physical characteristics, especially sizes and oxidation state, and exposure concentrations and sensitivity of the employed in vitro and in vivo models. In short, this study provided deep insights on the negative effects of GFNs exposure.

A pyrene-degrading consortium from deep-sea sediment of the West Pacific and its key member Cycloclasticus sp. P1.

A pyrene-degrading bacterial consortium was obtained from deep-sea sediments of the Pacific Ocean. The consortium degraded many kinds of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, pyrene, acenaphthene, fluorene, anthracene, fluoranthene, 2-methylnaphthalene and 2,6-dimethylnaphthalene, but it did not grow with chrysene and benzo[alpha]pyrene. With methods of plate cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 72 bacteria belonging to 22 genera were detected from this consortium. Among the detected bacteria, the following genera frequently occurred: Flavobacterium, Cycloclasticus, Novosphingobium, Halomonas, Achromobacter, Roseovarius and Alcanivorax. The first two genera showed the strongest bands in denaturing gradient gel electrophoresis (DGGE) profiles and appeared in all PAH treatments. By now, only one isolate designated P1 was confirmed to be a pyrene degrader. It was identified to be Cycloclasticus spirillensus (100%). Although P1 can degrade pyrene independently, other bacteria, such as Novosphingobium sp. (Band 14), Halomonas sp. (Band 16) and an unidentified bacterium (Band 35), were involved in pyrene degradation in some way; they persist in the consortium in the test of dilution to extinction if only the consortium was motivated with pyrene. However, the secondary most important member Flavobacterium sp. evaded from the community at high dilutions. As a key member of the consortium, P1 distinguished itself by both cell morphology and carbon source range among the isolates of this genus. Based on intermediate analyses of pyrene degradation, P1 was supposed to take an upper pathway different from that previously reported. Together with the results of obtained genes from P1 homology with those responsible for naphthalene degradation, its degradation to pyrene is supposed to adopt another set of genes unique to presently detected. Summarily, an efficient pyrene-degrading consortium was obtained from the Pacific Ocean sediment, in which Cycloclasticus bacterium played a key role. This is the first report to exploit the diversity of pyrene-degrading bacteria in oceanic environments.

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge.

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas, Halomonas, Marinobacter, Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus, Alteromonas, Thalassospira, Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.