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.

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.

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.

Distribution of oil-degrading bacteria in coastal seawater, Toyama Bay, Japan.

Oil-degrading bacteria are considered to play an important role in the biodegradation of spilled or released oil in the sea. The distribution of indigenous oil-degrading bacteria in the coastal seawater of Toyama Bay, Japan, was examined. Surface seawater samples with or without oil film in fishing port were analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of bacterial 16S rDNA. Sequence analysis revealed that several DGGE bands clearly detected only in samples with oil film corresponded to Cyanobacteria. Moreover, we cultured surface seawater samples with oil film in two different liquid culture media, a marine broth and an NSW medium; each culture contained 0.5% (w/v) C-heavy oil. Emulsification of the oil was observed at day 6 in the marine broth and day 9 in the NSW medium. Time-dependent changes of bacterial communities in those culture media were analyzed by DGGE. Interestingly, we found that Alcanivorax sp. became one of the dominant bacteria in each culture medium when emulsification of the oil began. Alcanivorax sp. is one of the well-known oil-degrading bacteria in seawater and is associated with the production of biosurfactants. These results suggest that Cyanobacteria and Alcanivorax play important roles in the bioremediation of oil-contaminated areas in Toyama Bay.

Microbial community dynamics during assays of harbour oil spill bioremediation: a microscale simulation study.

AIMS: Microcosm experiments simulating an oil spill event were performed to evaluate the response of the natural microbial community structure of Messina harbour seawater following the accidental load of petroleum. METHODS AND RESULTS: An experimental harbour seawater microcosm, supplemented with nutrients and crude oil, was monitored above 15 days in comparison with unpolluted ones (control microcosms). Bacterial cells were counted with a Live/Dead BacLight viability kit; leucine aminopeptidase, beta-glucosidase, alkaline phosphatase, lipase and esterase enzymes were measured using fluorogenic substrates. The microbial community dynamic was monitored by isolation of total RNA, RT-PCR amplification of 16S rRNA, cloning and sequencing. Oil addition stimulated an increase of the total bacterial abundance, leucine aminopeptidase and phosphatase activity rates, as well as a change in the community structure. This suggested a prompt response of micro-organisms to the load of petroleum hydrocarbons. CONCLUSIONS: The present study on the viability, specific composition and metabolic characteristics of the microbial community allows a more precise assessment of oil pollution. Both structural and functional parameters offer interesting perspectives as indicators to monitor changes caused by petroleum hydrocarbons. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of microbial structural successions at oil-polluted sites is essential for environmental bioremediation. Data obtained in microcosm studies improve our understanding of natural processes occurring during oil spills.