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.

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.

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.

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.

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.

Periodically spilled-oil input as a trigger to stimulate the development of hydrocarbon-degrading consortia in a beach ecosystem.

In this study, time-series samples were taken from a gravel beach to ascertain whether a periodic oil input induced by tidal action at the early stage of an oil spill can be a trigger to stimulate the development of hydrocarbon-degrading bacteria under natural in situ attenuation. High-throughput sequencing shows that the microbial community in beach sediments is characterized by the enrichment of hydrocarbon-degrading bacteria, including Alcanivorax, Dietzia, and Marinobacter. Accompanying the periodic floating-oil input, dynamic successions of microbial communities and corresponding fluctuations in functional genes (alkB and RDH) are clearly indicated in a time sequence, which keeps pace with the ongoing biodegradation of the spilled oil. The microbial succession that accompanies tidal action could benefit from the enhanced exchange of oxygen and nutrients; however, regular inputs of floating oil can be a trigger to stimulate an in situ "seed bank" of hydrocarbon-degrading bacteria. This leads to the continued blooming of hydrocarbon-degrading consortia in beach ecosystems. The results provide new insights into the beach microbial community structure and function in response to oil spills.

A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples.

Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013. The chromium(VI) contaminated soils were pretreated by water extraction, and directly exposed to the bioreporter in two phases: aqueous soil extraction (water phase) and soil supernatant (solid phase). The results indicated that both extractable and soil particle fixed chromium(VI) were bioavailable to the bioreporter, and the solid-phase contact bioreporter assay provided a more precise evaluation of soil genotoxicity. For crude oil contaminated seawater, the response of the bioreporter clearly illustrated the spatial and time change in genotoxicity surrounding the spill site, suggesting that the crude oil degradation process decreased the genotoxic risk to ecosystem. In addition, the performance of the bioreporter was simulated by a modified cross-regulation gene expression model, which quantitatively described the DNA damage response of the E. coli bioreporter. Accordingly, the bioluminescent response of the bioreporter was calculated as the mitomycin C equivalent, enabling quantitative comparison of genotoxicities between different environmental samples. This bioreporter assay provides a rapid and sensitive screening tool for direct genotoxicity assessment of environmental samples.

Biodiversity and degradation potential of oil-degrading bacteria isolated from deep-sea sediments of South Mid-Atlantic Ridge.

The indigenous oil-degrading bacterial consortia MARA and MARB were enriched from the deep-sea sediments of South Mid-Atlantic Ridge (MAR) with crude oil as sole carbon and energy sources. Biodiversity and community analyses showed that members of α-Proteobacteria were the key players in consortium MARA, whereas those of γ-Proteobacteria were the key players in consortium MARB, which were studied by MiSeq sequencing method. Gravimetric method estimated the oil degradation rates of MARA and MARB to be 63.4% and 85.8%, respectively, after 20d. Eleven cultivable oil degraders with different morphologies were isolated. These strains were identified as Alcanivorax, Bacillus, Dietzia, Erythrobacter, Marinobacter, Nitratireductor, and Oceanicola based on 16S rRNA gene sequences. Three strains belonging to Dietzia exhibited the highest oil degradation capability. Results indicated that the intrinsic biodegradation capacity of oil contaminants by indigenous microbial communities exists in South MAR sediments.

[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.

[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.

[Isolation, identification and diversity analysis of petroleum-degrading bacteria in Shengli Oil Field wetland soil].

The petroleum-degrading bacteria in Shengli Oil Field wetland soil were isolated and identified by traditional experiment methods, and their diversity was analyzed by PCR-DGGE (denaturing gradient gel electrophoresis). A total of thirteen petroleum-degrading bacterial strains were isolated, among which, six strains were found to have the ability of degrading the majority of C12-C26 petroleum hydrocarbon, with a degradation rate of > 90%. These petroleum degraders were phylogeneticly identified as the members of Halomonas, Alcanivorax, and Marinobacter, which were all belonged to gamma-proteobacteria. The uncultured predominant bacteria in Shengli Oil Field wetland soil were of Sulfurovum, Gillisia and Arcobacter. Among the predominant bacteria, gamma-proteobacteria accounted for a larger proportion, followed by alpha-proteobactiria, epsilon-proteobactiria, Actinobacteria, and Flavobacteria.