Marinicella rhabdoformis sp. nov., isolated from coastal sediment.

A Gram-stain-negative, rod-shaped, facultative anaerobic bacterium, designated strain 3539T, was isolated from coastal sediment of Weihai, PR China. Optimal growth occurred at 28 °C, pH 7.5-8.0 and in the presence of 3.0 % (w/v) NaCl. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 3539T formed a robust clade with members of the genus Marinicella and was closely related to Marinicella litoralis JCM 16154T, Marinicella sediminis F2T and Marinicella pacifica sw153T with 97.7, 96.2 and 95.4 % sequence similarity, respectively. The average amino acid identity, percentage of conserved proteins, average nucleotide identity and digital DNA-DNA hybridization values between strain 3539T and M. litoralis JCM 16154T were 64.9, 68.3, 72.8 and 18.9 %, respectively. The genomic DNA G+C content of strain 3539T was 42.0 mol%. The dominant respiratory quinone was ubiquinone-8, and the major fatty acids were iso-C15 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). The polar lipids of strain 3539T consisted of phosphatidyldimethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, one unidentified aminophospholipid, one unidentified lipid and three unidentified phospholipids. Based on the combination of phylogenetic, phenotypic and chemotaxonomic data, strain 3539T is considered to represent a novel species within the genus Marinicella in he family Alcanivoracaceae, for which the name Marinicella rhabdoformis sp. nov. is proposed. The type strain of the new species is 3539T (=KCTC 72414T=MCCC 1H00388T).

Alcanivorax profundi sp. nov., isolated from deep seawater of the Mariana Trench.

A Gram-stain-negative, rod-shaped, non-motile, strictly aerobic strain, designated as MTEO17T, was isolated from a 1000 m deep seawater sample of the Mariana Trench. Growth was observed at 10-45 °C (optimum, 37 °C), in the presence of 0.0-12.0 % NaCl (w/v; optimum, 3.0 %) and at pH 6.0-10.0 (optimum, pH 7.0-8.0). Phylogenetic analysis, based on the 16S rRNA gene sequence, revealed that strain MTEO17T belonged to the genus Alcanivorax and showed the highest sequence similarity of 97.9 % to Alcanivorax nanhaiticus MCCC 1A05629T. The estimated average nucleotide identity and DNA-DNA hybridization values between strain MTEO17T and A. nanhaiticus MCCC 1A05629T were 78.98 and 23.80 %, respectively. The significant dominant fatty acids were C16 : 0, summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The polar lipids comprised two phosphatidylethanolamines, one phosphatidylglycerol, one unidentified phospholipid and four unidentified polar lipids. The DNA G+C content of strain MTEO17T was 57.5 %. On the basis of the polyphasic evidence, strain MTEO17T is proposed to represent a novel species of the genus Alcanivorax, for which the name Alcanivorax profundi sp. nov. is proposed. The type strain is MTEO17T (=KCTC 52694T=MCCC 1K03252T).

Marinicella sediminis sp. nov., isolated from marine sediment.

A novel heterotrophic, Gram-stain-negative, aerobic, rod-shaped, pale yellow, non-motile and non-spore-forming bacterium, designated strain F2T, was isolated from marine sediment collected from the Weihai coast, Shandong Province, PR China. Optimal growth occurred at 33 °C (range, 10-37 °C), with 3.0-4.0 % (w/v) NaCl (1.0-8.0 %) and at pH 7.5-8.0 (pH 6.5-9.0). Q-8 was the sole respiratory quinone. The major polar lipids of strain F2T were phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and two unidentified polar lipids. The major cellular fatty acid in strain F2T was iso-C15 : 0. The genomic DNA G+C content of the strain was 48.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that strain F2T is most closely related to Marinicella litoralis JCM 16154T (97.5 %) and Marinicella pacifica sw153T (96.0 %). Based on the results of our polyphasic analysis, we conclude that strain F2T represents a novel species of the genus Marinicella, for which the name Marinicella sediminis sp. nov. is proposed. The type strain of the new species is F2T (=KCTC 42953T=MCCC 1H00149T).

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)).

Kangiella profundi sp. nov., isolated from deep-sea sediment.

A taxonomic study employing a polyphasic approach was carried out on strain FT102(T), which was isolated from a deep-sea sediment sample collected in the south-west Indian Ocean at a depth of 2784 m. The strain was Gram-stain-negative, non-motile, rod-shaped and non-spore-forming. It grew optimally at 37-42 °C, pH 6.5-8.5 and in the presence of 1-4% (w/v) NaCl. Phylogenetic analysis of 16S rRNA gene sequences confirmed the separation of the novel strain from recognized members of the genus Kangiella that are available in public databases. Strain FT102(T) exhibited 95.5-98.6% 16S rRNA gene sequence similarity to the type strains of the eight recognized species of the genus Kangiella. The chemotaxonomically characteristic fatty acid iso-C15:0 and ubiquinone Q-8 were also detected. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The DNA G + C content of strain FT102(T) was 45.0 mol%. The mean DNA-DNA relatedness values between strain FT102(T) and the type strains of Kangiella aquimarina and Kangiella koreensis were 47.3% and 13.7%, respectively. The combined results of phylogenetic, physiological and chemotaxonomic studies indicated that strain FT102(T) was affiliated with the genus Kangiella but differed from the recognized species of the genus Kangiella. Therefore, strain FT102T represents a novel species of the genus Kangiella, for which the name Kangiella profundi sp. nov. is proposed. The type strain is FT102(T) ( = CGMCC 1.12959(T) = KCTC 42297(T) = JCM 30232(T)).

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.

[Microbial community structure analysis of unexploited oil and gas fields by PCR-DGGE].

Microbial communities of different depths (30, 60, 100, 150, 200cm) from the unexploited oilfield, gas field and control area were studied by PCR-DGGE and sequencing methods. The objectives of this study were to understand the microbial distribution in the regions of unexploited oil and gas fields, and to investigate the potential microbial indicators of oil and gas resources. The results showed that the Dice coefficients between different depths were very low (26-69.9). The microbial communities in the soil of 150 cm and 200 cm depth had greater richness (S > or = 19), diversity (H > or = 2.69) and evenness (E > or = 0. 90). The results of sequencing demonstrated that the bands from oilfield were mainly grouped into alpha-Proteobacteria, gamma-Proteobacteria, Actinobacteria, Acidobacteria with the predominance of gamma-Proteobacteria (75%). Most of the bands were related to oil-associated and hydrocarbon degrading bacteria, such as Methylophaga and Alcanivorax. While the gas field had alpha, beta, gamma, delta-Proteobacteria and Bacteroidetes, and gamma-Proteobacteria accounted for only 24%. More strains showed relativity to methanotrophs, such as Methylocystaceae. Thus, 150 cm and 200 cm were more suitable as the oil-gas exploration sampling depth. Methylocystaceae may act as potential indicators for gas resources, Methylophaga and Alcanivorax for oil.

Kangiella taiwanensis sp. nov. and Kangiella marina sp. nov., marine bacteria isolated from shallow coastal water.

Two Gram-negative, heterotrophic, aerobic, marine bacteria, designated strains KT1(T) and KM1(T), were isolated from seawater samples collected from the shallow coastal regions of northern Taiwan. Cells grown in broth cultures were non-flagellated rods. NaCl was required for growth. Optimal growth occurred with 2-5 % NaCl, at 25-30 °C and at pH 8. They grew aerobically and were not capable of anaerobic growth by fermenting D-glucose or other carbohydrates. Q-8 was the only isoprenoid quinone. The major polar lipid detected in strain KT1(T) was phosphatidylmonomethylethanolamine, whereas those detected in KM1(T) were phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and an unidentified phospholipid. Cellular fatty acids were nearly all iso-branched, with iso-C(15 : 0) as the most abundant component (54.6-57.2 % of the total). Strains KT1(T) and KM1(T) had DNA G+C contents of 43.9 and 46.3 mol%, respectively. The two strains shared 98.1 % 16S rRNA gene sequence similarity; levels of similarity with the type strains of species of the genus Kangiella were 95.6-98.4 %. Data from the present taxonomic study conducted using a polyphasic approach revealed that the isolates could be classified as representatives of two novel species of the genus Kangiella, for which the names Kangiella taiwanensis sp. nov. (type strain KT1(T) = BCRC 80330(T) = JCM 17727(T)) and Kangiella marina sp. nov. (type strain KM1(T) = BCRC 80329(T) = JCM 17728(T)) are proposed.

Transcriptional profiling of the marine oil-degrading bacterium Alcanivorax borkumensis during growth on n-alkanes.

The marine oil-degrading bacterium Alcanivorax borkumensis SK2 has attracted significant interest due to its hydrocarbonoclastic lifestyle, its alkane-centered metabolism, and for playing an important ecological role in cleaning up marine oil spills. In this study, we used microarray technology to characterize the transcriptional responses of A. borkumensis to n-hexadecane exposure as opposed to pyruvate, which led to the identification of a total of 220 differentially expressed genes, with 109 genes being upregulated and 111 genes being downregulated. Among the genes upregulated on alkanes are systems predicted to be involved in the terminal oxidation of alkanes, biofilm formation, signal transduction, and regulation.

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.

Populations of heavy fuel oil-degrading marine microbial community in presence of oil sorbent materials.

AIMS: To investigate the feasibility of applying sorbent material X-Oil in marine oil spill mitigation and to survey the interactions of oil, bacteria and sorbent. METHODS AND RESULTS: In a series of microcosms, 25 different treatments including nutrient amendment, bioaugmentation with Alcanivorax borkumensis and application of sorbent were tested. Microbial community dynamics were analysed by DNA fingerprinting methods, RISA and DGGE. Results of this study showed that the microbial communities in microcosms with highly active biodegradation were strongly selected in favour of A. borkumensis. Oxygen consumption measurements in microcosms and gas chromatography of oil samples indicated the fast and intense depletion of linear alkanes as well as high oxygen consumption within 1 week followed by consequent slower degradation of branched and polyaromatic hydrocarbons. CONCLUSION: Under given conditions, A. borkumensis was an essential organism for biodegradation, dominating the biofilm microbial community formation and was the reason of emulsification. SIGNIFICANCE AND IMPACT OF THE STUDY: This study strongly emphasizes the pivotal importance of A. borkumensis as an essential organism in the initial steps of marine hydrocarbon degradation. Interaction with the sorbent material X-Oil proved to be neutral to beneficial for biodegradation and also promoted the growth of yet unknown micro-organisms.

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.

Alcanivorax balearicus sp. nov., isolated from Lake Martel.

A bacterial strain designated MACL04(T) was isolated from Lake Martel, a subterraneous saline lake in Mallorca (Spain). The complete 16S rRNA gene sequence of this strain showed nearly 100 % similarity to that of Alcanivorax dieselolei B-5(T). Despite this high similarity, strain MACL04(T) showed phenotypic, chemotaxonomic and molecular differences with respect to A. dieselolei, indicating that strain MACL04(T) represents a separate species. Cells of strain MACL04(T) were motile by means of a single polar or subpolar flagellum and colonies formed on media containing 1 % (v/v) Tween 20 were opaque and mucoid, with blue-green iridescence. The generation time of strain MACL04(T) in this medium was approximately half that of A. dieselolei B-5(T) and strain MACL04(T) did not produce lipases after incubation for 5 days. Strain MACL04(T) did not require NaCl for growth and grew in the presence of up to 15 % (w/v) NaCl. The strain was able to use alkanes as a sole carbon source; however, glucose could also be used, albeit weakly, as a carbon source. Several amino acids and organic acids were used as carbon sources. Strain MACL04(T) produced acid in media containing pyruvate as the sole carbon source. The major fatty acids were C(19 : 0) cyclo omega8c and C(16 : 0). The fatty acid C(16 : 1)omega8c, present in strain MACL04(T), was not detected in the recognized Alcanivorax species. The sequences of the large and short 16S-23S intergenic spacer regions showed similarities of 97.2 and 98.8 % (ungapped) with respect to A. dieselolei B-5(T). Partial sequences of gyrB and alkb genes showed 94.0 % similarity between strain MACL04(T) and A. dieselolei B-5(T). The G+C content of strain MACL04(T) was 62.8 mol%. The data from this polyphasic study indicate that strain MACL04(T) represents a novel species of the genus Alcanivorax, for which the name Alcanivorax balearicus sp. nov. is proposed. The type strain is MACL04(T) (=LMG 22508(T)=CECT 5683(T)).

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.