Mesopelagic microbial community dynamics in response to increasing oil and Corexit 9500 concentrations.

Marine microbial communities play an important role in biodegradation of subsurface plumes of oil that form after oil is accidentally released from a seafloor wellhead. The response of these mesopelagic microbial communities to the application of chemical dispersants following oil spills remains a debated topic. While there is evidence that contrasting results in some previous work may be due to differences in dosage between studies, the impacts of these differences on mesopelagic microbial community composition remains unconstrained. To answer this open question, we exposed a mesopelagic microbial community from the Gulf of Mexico to oil alone, three concentrations of oil dispersed with Corexit 9500, and three concentrations of Corexit 9500 alone over long periods of time. We analyzed changes in hydrocarbon chemistry, cell abundance, and microbial community composition at zero, three and six weeks. The lowest concentration of dispersed oil yielded hydrocarbon concentrations lower than oil alone and microbial community composition more similar to control seawater than any other treatments with oil or dispersant. Higher concentrations of dispersed oil resulted in higher concentrations of microbe-oil microaggregates and similar microbial composition to the oil alone treatment. The genus Colwellia was more abundant when exposed to multiple concentrations of dispersed oil, but not when exposed to dispersant alone. Conversely, the most abundant Marinobacter amplicon sequence variant (ASV) was not influenced by dispersant when oil was present and showed an inverse relationship to the summed abundance of Alcanivorax ASVs. As a whole, the data presented here show that the concentration of oil strongly impacts microbial community response, more so than the presence of dispersant, confirming the importance of the concentrations of both oil and dispersant in considering the design and interpretation of results for oil spill simulation experiments.

Genome sequence of an obligate hydrocarbonoclastic bacterium Alcanivorax marinus NMRL4 isolated from oil polluted seawater of the Arabian Sea.

Alcanivorax belongs to the hydrocarbonoclastic group of bacteria that are known for their preferential growth on alkanes and other related compounds. Here we report the genomic features of Alcanivorax marinus strain NMRL4 (=MCC 4632) isolated from oil polluted seawater of the Arabian Sea. Its 4,062,055 bp genome with 66.1% GC content encodes for 3935 coding sequences. The genome annotations of strain NMRL4 revealed the presence of multiple hydrocarbon degradation genes suggestive of its wider hydrocarbon substrate range. The strain encodes for three alkane monooxygenases, two cytochrome P450 and two flavin binding monooxygenases for degradation of short and long-chain alkanes. The genome shows capabilities for scavenging of nutrients, biofilm formation at oil-water interfaces, chemotaxis, motility and habitat specific adaptation. The genomic insights showed that the strain NMRL4 is an ideal candidate for bioremediation of pollutant petroleum hydrocarbons from the marine environment.

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.

In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes.

Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO4(3-) uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium.

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Biodegradation of crude oil by individual bacterial strains and a mixed bacterial consortium

Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. Single strains and four bacterial consortia designed by mixing the single bacterial cultures respectively in the following ratios: (Alcanivorax: Pseudomonas, 1:1), (Alcanivorax: Rhodococcus, 1:1), (Pseudomonas: Rhodococcus, 1:1), and (Alcanivorax: Pseudomonas: Rhodococcus, 1:1:1), were analyzed in order to evaluate their oil degrading capability. All experiments were carried out in microcosms systems containing seawater (with and without addition of inorganic nutrients) and crude oil (unique carbon source). Measures of total and live bacterial abundance, Card-FISH and quali-, quantitative analysis of hydrocarbons (GC-FID) were carried out in order to elucidate the co-operative action of mixed microbial populations in the process of biodegradation of crude oil. All data obtained confirmed the fundamental role of bacteria belonging to Alcanivorax genus in the degradation of linear hydrocarbons in oil polluted environments.

Alcanivorax gelatiniphagus sp. nov., a marine bacterium isolated from tidal flat sediments enriched with crude oil.

A Gram-reaction-negative, rod-shaped marine bacterium, designated MEBiC08158(T), was isolated from sediments collected from Taean County, Korea, near the Hebei Spirit tanker oil spill accident. 16S rRNA gene sequence analysis revealed that strain MEBiC08158(T) was closely related to Alcanivorax marinus R8-12(T) (99.5% similarity) but was distinguishable from other members of the genus Alcanivorax (93.7-97.1%). The DNA-DNA hybridization value between strain MEBiC08158(T) and A. marinus R8-12(T) was 58.4%. Growth of strain MEBiC08158(T) was observed at 15-43 °C (optimum 37-40 °C), at pH 6.0-9.5 (optimum pH 7.0-8.0) and with 0.5-16% NaCl (optimum 1.5-3.0%). The dominant fatty acids were C16 : 0, C19 : 0 cyclo ω8c, C12 : 0, C18 : 1ω7c, C12 : 0 3-OH and summed feature 3 (comprising C15 : 0 2-OH and/or C16 : 1ω7c). Several phenotypic characteristics differentiate strain MEBiC08158(T) from phylogenetically close members of the genus Alcanivorax. Therefore, strain MEBiC08158(T) should be classified as representing a novel species of the genus Alcanivorax, for which the name Alcanivorax gelatiniphagus sp. nov. is proposed. The type strain is MEBiC08158(T) ( = KCCM 42990(T) = JCM 18425(T)).

Genome sequence of the alkane-degrading bacterium Alcanivorax hongdengensis type strain A-11-3.

Alcanivorax hongdengensis A-11-3(T) was isolated from an oil-enriched consortium enriched from the surface seawater of Hong-Deng dock in the Straits of Malacca and Singapore. Strain A-11-3(T) can degrade n-alkane and produce a lipopeptide biosurfactant. Here we report the genome of A-11-3(T) and the genes associated with alkane degradation.

Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study.

Diversity of indigenous microbial consortia and natural occurrence of obligate hydrocarbon-degrading bacteria (OHCB) are of central importance for efficient bioremediation techniques. To investigate the microbial population dynamics and composition of oil-degrading consortia, we have established a series of identical oil-degrading mesocosms at three different locations, Bangor (Menai Straits, Irish Sea), Helgoland (North Sea) and Messina (Messina Straits, Mediterranean Sea). Changes in microbial community composition in response to oil spiking, nutrient amendment and filtration were assessed by ARISA and DGGE fingerprinting and 16Sr RNA gene library analysis. Bacterial and protozoan cell numbers were quantified by fluorescence microscopy. Very similar microbial population sizes and dynamics, together with key oil-degrading microorganisms, for example, Alcanivorax borkumensis, were observed at all three sites; however, the composition of microbial communities was largely site specific and included variability in relative abundance of OHCB. Reduction in protozoan grazing had little effect on prokaryotic cell numbers but did lead to a decrease in the percentage of A. borkumensis 16S rRNA genes detected in clone libraries. These results underline the complexity of marine oil-degrading microbial communities and cast further doubt on the feasibility of bioaugmentation practices for use in a broad range of geographical locations.

Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

[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.

Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons.

Petroleum (or crude oil) is a complex mixture of hydrocarbons. Annually, millions of tons of crude petroleum oil enter the marine environment from either natural or anthropogenic sources. Hydrocarbon-degrading bacteria (HDB) are able to assimilate and metabolize hydrocarbons present in petroleum. Crude oil pollution constitutes a temporary condition of carbon excess coupled to a limited availability of nitrogen that prompts marine oil-degrading bacteria to accumulate storage compounds. Storage lipid compounds such as polyhydroxyalkanoates (PHAs), triacylglycerols (TAGs), or wax esters (WEs) constitute the main accumulated lipophilic substances by bacteria under such unbalanced growth conditions. The importance of these compounds as end-products or precursors to produce interesting biotechnologically relevant chemicals has already been recognized. In this review, we analyze the occurrence and accumulation of lipid storage in marine hydrocarbonoclastic bacteria. We further discuss briefly the production and export of lipophilic compounds by bacteria belonging to the Alcanivorax genus, which became a model strain of an unusual group of obligate hydrocarbonoclastic bacteria (OHCB) and discuss the possibility to produce neutral lipids using A. borkumensis SK2.

Gene diversity of CYP153A and AlkB alkane hydroxylases in oil-degrading bacteria isolated from the Atlantic Ocean.

Alkane hydroxylases, including the integral-membrane non-haem iron monooxygenase (AlkB) and cytochrome P450 CYP153 family, are key enzymes in bacterial alkane oxidation. Although both genes have been detected in a number of bacteria and environments, knowledge about the diversity of these genes in marine alkane-degrading bacteria is still limited, especially in pelagic areas. In this report, 177 bacterial isolates, comprising 43 genera, were obtained from 18 oil-degrading consortia enriched from surface seawater samples collected from the Atlantic Ocean. Many isolates were confirmed to be the first oil-degraders in their affiliated genera including Brachybacterium, Idiomarina, Leifsonia, Martelella, Kordiimonas, Parvibaculum and Tistrella. Using degenerate PCR primers, alkB and CYP153A P450 genes were surveyed in these bacteria. In total, 82 P450 and 52 alkB gene fragments were obtained from 80 of the isolates. These isolates mainly belonged to Alcanivorax, Bacillus, Erythrobacter, Martelella, Parvibaculum and Salinisphaera, some of which were reported, for the first time, to encode alkane hydroxylases. Phylogenetic analysis showed that both genes were quite diverse and formed several clusters, most of which were generated from various Alcanivorax bacteria. Noticeably, some sequences, such as those from the Salinisphaera genus, were grouped into a distantly related novel cluster. Inspection of the linkage between gene and host revealed that alkB and P450 tend to coexist in Alcanivorax and Salinisphaera, while in all isolates of Parvibaculum, only P450 genes were found, but of multiple homologues. Multiple homologues of alkB mostly cooccurred in Alcanivorax isolates. Conversely, distantly related isolates contained similar or even identical sequences. In summary, various oil-degrading bacteria, which harboured diverse P450 and alkB genes, were found in the surface water of Atlantic Ocean. Our results help to show the diversity of P450 and alkB genes in prokaryotes, and to portray the geographic distribution of oil-degrading bacteria in marine environments.

Denitrifying activity and homologous enzyme analysis of Alcanivorax dieselolei strain N1203.

An Alcanivorax dieselolei strain, termed strain N1203, was isolated from the consortia of ammonia-oxidizing bacteria (AOB) combined with denitrifying bacteria from our previous study and was shown to have ability to reduce nitrate to nitrite to either nitrous oxide or molecular nitrogen. Analysis of 16S rRNA gene sequences established strain N1203 as a member of the species Alcanivorax dieselolei. In addition, the ability of strain N1203 to utilize various organic substrates as the sole carbon source, supplemented with carbohydrates, amino acids, and n-alkane compounds, was investigated, and this strain was found to have a narrow substrate range of growth such as grycerol, succinate, ethanol and n-alkane hydrocarbon. Furthermore, N1203's stepwise denitrifying activity, utilizing succinate and hexadecane as sole carbon sources, was measured. Gene fragments of nirK and qnorB genes, which are involved in denitrifying activities, were obtained, cloned and sequenced. Phylogenetic analysis for these two genes showed that both the nirK and qnorB sequences, although found in separate branches within clusters, formed subclusters branching from uncultured environmental clones. This demonstrated the typical uniqueness of these genes from any cultivated denitrifiers. Thus, strain N1203 is novel type of denitrifying bacteria that demonstrated denitrifying activities when cultivated using succinate as the sole carbon source.

Niche-specificity factors of a marine oil-degrading bacterium Alcanivorax borkumensis SK2.

Alcanivorax borkumensis strain SK2 is a cosmopolitan hydrocarbonoclastic marine bacterium, with a specialized metabolism adapted to the degradation of petroleum oil hydrocarbons. Transposon mutagenesis was used for functional genome analysis of Alcanivorax SK2 to reveal the genetic basis of other environmentally relevant phenotypes, such as biofilm formation, adaptation to UV exposure, and to growth at either low temperature or high salinity. Forty-eight relevant transposon mutants deficient in any one of these environmentally responsive functions were isolated, and the corresponding genes interrupted by the mini-Tn5 element were sequenced using inverse PCR. Several cross connections between different phenotypes (e.g. biofilm and UV stress; biofilm and UV and osmoadaptation) on signal transduction level have been revealed, pointing at complex and tightly controlled cellular interactions involving oxygen as a primary messenger and cyclic-di-GMP as a secondary messenger required for Alcanivorax responses to environmental stresses. These results provide insights into bacterial function in a complex marine environment.

Predominant growth of Alcanivorax during experiments on "oil spill bioremediation" in mesocosms.

Mesocosm experiments were performed to study the changes on bacterial community composition following oil spill in marine environment. The analysis of 16S crDNA revealed a shift in the structure of initial bacterial population that was drastically different from that one measured after 15 days. The results showed that, after 15 days, bacteria closely related to the genus Alcanivorax became the dominant group of bacterial community in petroleum-contaminated sea water nitrogen and phosphorus amended. This suggested that these bacteria played the most important role in the process of bioremediation of oil-contaminated marine environments.