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.

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.

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.

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.

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.

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.

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.

Microbial diversity and ecotoxicity of sediments 3 years after the Jiaozhou Bay oil spill.

In 2013, the "Qingdao oil pipeline explosion" released an estimated 2000 tons of oil into the environment. Sediment samples were collected from ten sites in Jiaozhou Bay and Shilaoren Beach to evaluate the influence of the spilled oil on the benthic environment 3 years after the oil spill accident. The compositions of oil, bacterial diversity and biotoxicity were examined in this study. The results showed that the concentration of total petroleum hydrocarbons (TPHs) peaked near the oil leak point and gradually decreased along the coastline, ranging from 21.5 to 133.2 µg/g. The distribution of polycyclic aromatic hydrocarbons (PAHs) was correlated with TPH, and naphthalenes were dominant in the 20 detected PAHs. The bacterial diversities in seriously polluted and slightly polluted sediments were completely different. As degrading bacteria, Alcanivorax and Lutibacter were the main genera at the oil-polluted sites. The analysis of biotoxicity by the luminescent bacteria method showed great differences among the polluted sites, the control site in Jiaozhou Bay, and the non-polluted site outside of Jiaozhou Bay. The biotoxicity also peaked at the site near the oil leak point. These results indicate that the oil spill that occurred 3 years ago still affects the environment and impacts the bacterial communities in the sediments.

Application of a bacterial whole cell biosensor for the rapid detection of cytotoxicity in heavy metal contaminated seawater.

A toxicity biosensor Acinetobacter baylyi Tox2 was constructed with the host strain A. baylyi ADP1 harboring a new and medium-copy-number plasmid pWH1274_lux, and was applied to detect the cytotoxicity of heavy metal contaminated seawater. The gene cassette luxCDABE was controlled by constitutively expressed promoter Ptet on pWH1274_lux and the bioluminescence intensity of the biosensor reduces in proportional to the concentrations of toxic compounds. A. baylyi Tox2 exhibits tolerance to salinity, hence it is applicable to seawater samples. A. baylyi Tox2 and Mugilogobius chulae were exposed to different concentrations of heavy metals (Hg2+, Zn2+, Cu2+, and Cd2+) in artificial seawater for performance comparison and Pearson correlation analysis showed a significant correlation (p < 0.01) between A. baylyi Tox2 toxicity detection and the fish (M. chulae) exposure test. This suggests that the performance of A. baylyi Tox2 is comparable to the conventional fish toxicity test in terms of cytotoxicity detection of heavy metal contaminated seawater. Furthermore, A. baylyi Tox2 was used to evaluate cytotoxicity of field-collected seawater samples. The results indicate that there was a significant correlation between the luminescence inhibition ratio (IR) of A. baylyi Tox2 and heavy metal concentrations detected by ICP-MS in the samples. Two seawater samples, which contained a high concentration of total heavy metals, exhibited stronger cytotoxicity than the samples containing low concentrations of heavy metals. In conclusion, A. baylyi Tox2 can be used as an alternative tool to aquatic animals for the evaluation of the cytotoxicity of heavy metal contamination in the marine environment.

Genome Sequence of a Marine Alkane Degrader, Alcanivorax sp. Strain 97CO-6.

Alcanivorax sp. strain 97CO-6 was isolated from a crude oil-consuming bacterial consortium, enriched from Yellow Sea sediments from China. Here, we present the draft genome of strain 97CO-6, which contains 3,253,423 bp, with a G+C content of 54.53%, as well as 2,931 protein-coding genes and 42 tRNAs.

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.

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).

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.

Draft Genome Sequence of Rhodobacteraceae Strain PD-2, an Algicidal Bacterium with a Quorum-Sensing System, Isolated from the Marine Microalga Prorocentrum donghaiense.

Rhodobacteraceae strain PD-2 was isolated from the marine microalga Prorocentrum donghaiense. It has algicidal activity toward its host and could produce N-acylhomoserine lactone signals. Here, we present the draft genome of strain PD-2, which contains 5,227,214 bp with an average GC content of 66.19%. There were 4,864 encoding gene sequences and two clusters of luxI and luxR homologues identified.