Bacterial community analysis of biofilm on API 5LX carbon steel in an oil reservoir environment.

This study aimed to characterize the biofilm microbial community that causes corrosion of API 5LX carbon steel. API 5LX carbon steel coupons were incubated with raw produced water collected from two oil reservoir stations or filter-sterilized produced water. Biofilm 16S rRNA amplicon sequencing revealed that the bacterial community present in the biofilm was dominated by Proteobacteria, including Marinobacter hydrocarbonoclaustics and Marinobacter alkaliphilus. Electrochemical analysis such as impedance and polarization results indicated that Proteobacteria biofilm accelerated corrosion by ~ twofold (2.1 ± 0.61 mm/years) or ~ fourfold (~ 3.7 ± 0.42 mm/years) when compared to the control treatment (0.95 ± 0.1 mm/years). Scanning electron and atomic force microscopy revealed the presence of a thick biofilm and pitting corrosion. X-ray diffraction revealed higher amounts of the corrosion products Fe2O3, γ-FeOOH, and α-FeOOH, and confirmed that the microbial biofilm strongly oxidized the iron and contributed to the acceleration of corrosion of carbon metal API 5LX.

Activation of Protein Expression in Electroactive Biofilms.

Microbes that form biofilms on electrodes and generate electrical current responses could be integrated into devices to perform sensing, conduct signals, or act as living microprocessors. A challenge in working with these species is the ability to visualize biofilm formation and protein expression in real-time while also measuring current, which is not possible with typical bio-electrochemical reactors. Here, we present a three-dimensional-printed flow cell for simultaneous electrochemistry and fluorescence imaging. Current-producing biofilms of Marinobacter atlanticus constitutively expressing green fluorescent protein were grown on the flow cell working electrode. Increasing current corresponded with increasing surface coverage and was comparable to biofilms grown in typical stirred-batch reactors. An isopropyl ß-d-1-thiogalactopyranoside (IPTG) inducible system driving yellow fluorescent protein was used to assess the spatiotemporal activation of protein expression within the biofilm at different stages of growth and induction dynamics. The response time ranged from 30 min to 5 h, depending on the conditions. These data demonstrate that the electrochemical flow cell can evaluate the performance of an electrically active environmental bacterium under conditions relevant for development as a living electronic sensor.

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench.

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45 °C (optimum 37 °C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1 MPa (megapascal) with tolerance up to 70 MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3 mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

The extracellular matrix of the oleolytic biofilms of Marinobacter hydrocarbonoclasticus comprises cytoplasmic proteins and T2SS effectors that promote growth on hydrocarbons and lipids.

The assimilation of the nearly water insoluble substrates hydrocarbons and lipids by bacteria entails specific adaptations such as the formation of oleolytic biofilms. The present article reports that the extracellular matrix of an oleolytic biofilm formed by Marinobacter hydrocarbonoclasticus at n-hexadecane-water interfaces is largely composed of proteins typically cytoplasmic such as translation factors and chaperones, and a lesser amount of proteins of unknown function that are predicted extra-cytoplasmic. Matrix proteins appear to form a structured film on hydrophobic interfaces and were found mandatory for the development of biofilms on lipids, alkanes and polystyrene. Exo-proteins secreted through the type-2 secretion system (T2SS) were shown to be essential for the formation of oleolytic biofilms on both alkanes and triglycerides. The T2SS effector involved in biofilm formation on triglycerides was identified as a lipase. In the case of biofilm formation on n-hexadecane, the T2SS effector is likely involved in the mass transfer, capture or transport of alkanes. We propose that M. hydrocarbonoclasticus uses cytoplasmic proteins released by cell lysis to form a proteinaceous matrix and dedicated proteins secreted through the T2SS to act specifically in the assimilation pathways of hydrophobic substrates.

Reconstructing metabolic pathways of hydrocarbon-degrading bacteria from the Deepwater Horizon oil spill.

The Deepwater Horizon blowout in the Gulf of Mexico in 2010, one of the largest marine oil spills(1), changed bacterial communities in the water column and sediment as they responded to complex hydrocarbon mixtures(2-4). Shifts in community composition have been correlated to the microbial degradation and use of hydrocarbons(2,5,6), but the full genetic potential and taxon-specific metabolisms of bacterial hydrocarbon degraders remain unresolved. Here, we have reconstructed draft genomes of marine bacteria enriched from sea surface and deep plume waters of the spill that assimilate alkane and polycyclic aromatic hydrocarbons during stable-isotope probing experiments, and we identify genes of hydrocarbon degradation pathways. Alkane degradation genes were ubiquitous in the assembled genomes. Marinobacter was enriched with n-hexadecane, and uncultured Alpha- and Gammaproteobacteria populations were enriched in the polycyclic-aromatic-hydrocarbon-degrading communities and contained a broad gene set for degrading phenanthrene and naphthalene. The repertoire of polycyclic aromatic hydrocarbon use varied among different bacterial taxa and the combined capabilities of the microbial community exceeded those of its individual components, indicating that the degradation of complex hydrocarbon mixtures requires the non-redundant capabilities of a complex oil-degrading community.

Marinobacter shengliensis sp. nov., a moderately halophilic bacterium isolated from oil-contaminated saline soil.

Two moderately halophilic strains, designated SL013A34A2(T) and SL013A24A, were isolated from oil-contaminated saline soil from Shengli Oilfield, eastern China. Cells were found to be Gram-staining negative, aerobic, rod-shaped with a single polar flagellum. The isolates were found to grow at 10-40 °C (optimum 35 °C), pH 6.0-9.0 (optimum pH 8.0), and NaCl concentrations of 0.5-18.0 % (w/v) (optimum 3.0-6.0 NaCl). The 16S rRNA gene sequence analysis indicated that the isolates belong to the genus Marinobacter. Strain SL013A34A2(T) shares the highest 16S rRNA gene sequence similarities with strain SL013A24A (99.3 %), followed by M. hydrocarbonoclasticus CGMCC 1.7683(T) (97.8 %), M. vinifirmus CGMCC 1.7265(T) (97.8 %), and M. excellens KMM 3809(T) (97.4 %), respectively, but low similarities (93.8-96.4 %) with type strains of the other numbers of genus Marinobacter. DNA-DNA relatedness values of strain SL013A34A2(T) with strains SL013A24A, M. hydrocarbonoclasticus CGMCC 1.7683(T), M. vinifirmus CGMCC 1.7265(T) and M. excellens KMM 3809(T) were 88.7, 29.2, 33.4 and 29.4 %, respectively. The major fatty acids of strain SL013A34A2(T) were identified as C18:1 ω9c, C16:0, C12:03-OH, C12:0, C16:1 ω9c and 10-methyl C18:0. The major respiratory quinone of strain SL013A34A2(T) was found to be ubiquinone-9, and its predominant polar lipids were identified as diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and unidentified glycolipid. The genomic DNA G + C content was found to be 56.1 mol %. Based on the phenotypic, genetic and chemotaxonomic characteristics, these two isolates are representatives of a novel species of the genus Marinobacter, for which the name Marinobacter shengliensis sp. nov. is proposed. The type strain is SL013A34A2(T)(=LMG 27740(T) = CGMCC 1.12758(T)).

The marine bacterium Marinobacter hydrocarbonoclasticus SP17 degrades a wide range of lipids and hydrocarbons through the formation of oleolytic biofilms with distinct gene expression profiles.

Hydrophobic organic compounds (mainly lipids and hydrocarbons) represent a significant part of the organic matter in marine waters, and their degradation has an important impact in the carbon fluxes within oceans. However, because they are nearly insoluble in the water phase, their degradation by microorganisms occurs at the interface with water and thus requires specific adaptations such as biofilm formation. We show that Marinobacter hydrocarbonoclasticus SP17 develops biofilms, referred to as oleolytic biofilms, on a large variety of hydrophobic substrates, including hydrocarbons, fatty alcohols, fatty acids, triglycerides, and wax esters. Microarray analysis revealed that biofilm growth on n-hexadecane or triolein involved distinct genetic responses, together with a core of common genes that might concern general mechanisms of biofilm formation. Biofilm growth on triolein modulated the expression of hundreds of genes in comparison with n-hexadecane. The processes related to primary metabolism and genetic information processing were downregulated. Most of the genes that were overexpressed on triolein had unknown functions. Surprisingly, their genome localization was restricted to a few regions identified as putative genomic islands or mobile elements. These results are discussed with regard to the adaptive responses triggered by M. hydrocarbonoclasticus SP17 to occupy a specific niche in marine ecosystems.

The study of marine corrosion of copper alloys in chlorinated condenser cooling circuits: the role of microbiological components.

The present paper reports the on-line monitoring of corrosion behavior of the CuNi 70:30 and Al brass alloys exposed to seawater and complementary offline microbiological analyses. An electrochemical equipment with sensors specifically set for industrial application and suitable to estimate the corrosion (by linear polarization resistance technique), the biofilm growth (by the BIOX electrochemical probe), the chlorination treatment and other physical-chemical parameters of the water has been used for the on-line monitoring. In order to identify and better characterize the bacteria community present on copper alloys, tube samples were collected after a long period (1year) and short period (2days) of exposition to treated natural seawater (TNSW) and natural seawater (NSW). From the collected samples, molecular techniques such as DNA extraction, polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and identification by sequencing were performed to better characterize and identify the microbial biodiversity present in the samples. The monitoring data confirmed the significant role played by biofouling deposition against the passivity of these Cu alloys in seawater and the positive influence of antifouling treatments based on low level dosages. Molecular analysis indicated biodiversity with the presence of Marinobacter, Alteromonas and Pseudomonas species.

Type 2 quorum sensing monitoring, inhibition and biofilm formation in marine microrganisms.

The quorum sensing (QS) dependent behaviour of micro-organisms, in particular expression of virulence genes, biofilm formation and dispersal, have provided impetus for investigating practical approaches to interfere with microbial QS. This study tests Halomonas pacifica and Marinobacter hydrocarbonoclasticus, two halophilic marine micro-organism, for their AI-2 dependent QS signalling and the effect of two well-known quorum-sensing inhibitors (QSIs), patulin and penicillic acid, on biofilm formation. We report, for the first time, the successful amplification of a putative luxS gene in H. pacifica using degenerated primers and AI-2 dependent QS as well as inhibition using QSIs. Penicillic acid had a strong inhibitory effect on AI-2 induction of H. pacifica at non-growth inhibitory concentrations, while patulin has an adverse effect only at the highest concentration (25 µM). QSIs effect on biofilm forming capability was isolate specific, with maximum inhibition at 25 µM of patulin in H. pacifica. In M. hydrocarbonoclasticus, no adverse effects were noted at any tested concentration of either QSIs. Detection of bioluminescence and the presence of a putative luxS gene provide biochemical and genetic evidence for the production of a signalling molecule(s) which is the essential first step in characterizing H. pacifica QS. This study highlights the importance of AI-2 dependent QS in a marine setting, not previously reported. It further suggests that QSI compounds must be selected in the specific system in which they are to function, and they cannot easily be transferred from one QS system to another.

Kinetics analysis of a salt-tolerant perchlorate-reducing bacterium: effects of sodium, magnesium, and nitrate.

Salt-tolerant perchlorate-reducing bacteria can be used to regenerate ion-exchange brines or resins exhausted with perchlorate. A salt-tolerant perchlorate-reducing Marinobacter vinifirmus strain P4B1 was recently purified. This study determined the effects of Na(+) and Mg(2+) concentrations on the perchlorate reduction rate of P4B1. The results showed that strain P4B1 could utilize perchlorate and grow in the presence of 1.8% to 10.2% NaCl. Lower NaCl concentrations allowed faster perchlorate reduction. The addition of Mg(2+) to the culture showed significant effects on perchlorate reduction when perchlorate was the sole electron acceptor. A molar Mg(2+)/Na(+) ratio of ∼0.11 optimized perchlorate degradation and cell growth. When perchlorate and nitrate were both present, nitrate reduction did not start significantly until perchlorate was below 100 mg/L. Tests with washed cell suspensions indicated that strain P4B1 had both perchlorate and nitrate reduction enzymes. When the culture was exposed to both perchlorate and nitrate, the nitrate reduction enzyme activity was low. The maximum specific substrate utilization rate (Vm) and the half saturation coefficient (KS) for P4B1 (30 g/L NaCl) determined in this study were 0.049 ± 0.003 mg ClO4(-)/mg VSS-h and 18 ± 4 mg ClO4(-)/L, respectively.

[Isolation and identification of Marinobacter adhaerens HY-3 and its allelopathy on Skeletonema costatum].

UNLABELLED: The allelopathy between bacteria and algae is a very complicated physical and ecological phenomenon. A marine bacterium was isolated from the water of a shrimp and crab mix-culturing pond. By 16S rRNA analysis, it was identified as Marinobacter adhaerens HY-3. Skeletonema costatum, a common dominant species of red-tide microorganism in China, was chosen as the other research object. The allelopathic effect of Marinobacter adhaerens HY- 3 on S. costatum was studied. Using the growth mass of Skeletonema costatum and the content of chlorophyll a as the parameters, the effects of HY-3 on the growth and photosynthesis of Skeletonema costatum were studied after co-cultivation and addition of extracellular metabolites of HY-3. The results showed that the growth of S. costatum was inhibited when the concentration of the strain HY-3 was above 10(4), and the growth mass of the 10(4), 10(6) and 10(8) HY-3 group was 70%, 23% and 22% of the control group respectively on the 10th day, with the content of chlorophyll being 88%, 62% and 60% of the control group, respectively. Therefore, the suppression increased with increasing concentration of HY-3. However, addition of extracellular metabolites of HY-3 had no effect on the growth of S. costatum. CONCLUSIONS: M. adhaerens HY-3 had certain allelopathy on S. costatum and affected its growth and photosynthesis. Moreover, interaction between M. adhaerens HY-3 and S. costatum was achieved by their direct contact and the extracellular metabolites did not contain allelopathy factors.

An electrically reversible switchable surface to control and study early bacterial adhesion dynamics in real-time.

Bacterial adhesion can be controlled by applying electrical potentials to surfaces incorporating well-spaced negatively charged 11-mercaptoundecanoic acids. When combined with electrochemical surface plasmon resonance, these dynamic surfaces become powerful for monitoring and analysing the passage between reversible and non-reversible cell adhesion, opening new opportunities to advance our understanding of cell adhesion processes.

Marinobacter nanhaiticus sp. nov., polycyclic aromatic hydrocarbon-degrading bacterium isolated from the sediment of the South China Sea.

A Gram-negative, rod-shaped, slightly halophilic and facultatively anaerobic bacterium, designated strain D15-8W(T), was isolated from the sediment of the South China Sea. Growth was found to occur optimally at 25 °C, between pH 7.0 and 8.0 and with 1-5 % (w/v) NaCl. The strain was observed to utilize a variety of organic substrates and polycyclic aromatic hydrocarbons as sole carbon sources. The G+C content of the genomic DNA was determined to be 58.7 %. The predominant respiratory quinone was found to be Q-9. The significant fatty acids were determined to be C(16:0), C(16:1) ω9c, C(18:1) ω9c, C(12:0) and C(14:0) 3OH. Analysis of 16S rRNA gene sequences showed that strain D15-8W(T) fits within the phylogenetic cluster of the genus Marinobacter and is most closely related to Marinobacter segnicrescens CGMCC 1.6489(T), Marinobacter bryozoorum DSM 15401(T), Marinobacter lacisalsi CECT 7297(T) and Marinobacter daqiaonensis CGMCC1.9167(T). The DNA-DNA hybridization values between strain D15-8W(T) and the type strains of the most closely related species were 42.3 % (CGMCC 1.6489(T)), 39.8 % (DSM 15401(T)), 37.3 % (CECT 7297(T)) and 35.2 % (CGMCC1.9167(T)). The results of this polyphasic study indicate that strain D15-8W(T) represents a novel species of the genus Marinobacter, for which the name Marinobacter nanhaiticus sp. nov. is proposed. The type strain is D15-8W(T) (=CGMCC 1.11019(T)=KCTC 23749(T)).

Chemotaxis of Marinobacter adhaerens and its impact on attachment to the diatom Thalassiosira weissflogii.

Alga-bacterium interactions are crucial for aggregate formation and carbon cycling in aquatic systems. To understand the initiation of these interactions, we investigated bacterial chemotaxis within a bilateral model system. Marinobacter adhaerens HP15 has been demonstrated to attach to the diatom Thalassiosira weissflogii and induce transparent exopolymeric particle and aggregate formation. M. adhaerens possesses one polar flagellum and is highly motile. Bacterial cells were attracted to diatom cells, as demonstrated by addition of diatom cell homogenate or diatom culture supernatant to soft agar, suggesting that chemotaxis might be important for the interaction of M. adhaerens with diatoms. Three distinct chemotaxis-associated gene clusters were identified in the genome sequence of M. adhaerens, with the clusters showing significant sequence similarities to those of Pseudomonas aeruginosa PAO1. Mutations in the genes cheA, cheB, chpA, and chpB, which encode histidine kinases and methylesterases and which are putatively involved in either flagellum-associated chemotaxis or pilus-mediated twitching motility, were generated and mutants with the mutations were phenotypically analyzed. ΔcheA and ΔcheB mutants were found to be swimming deficient, and all four mutants were impaired in biofilm formation on abiotic surfaces. Comparison of the HP15 wild type and its chemotaxis mutants in cocultures with the diatom revealed that the fraction of bacteria attaching to the diatom decreased significantly for mutants in comparison to that for the wild type. Our results highlight the importance of M. adhaerens chemotaxis in initiation of its interaction with the diatom. In-depth knowledge of these basic processes in interspecies interactions is pivotal to obtain a systematic understanding of organic matter flux and nutrient cycling in marine ecosystems.

Marinobacter adhaerens sp. nov., isolated from marine aggregates formed with the diatom Thalassiosira weissflogii.

A Gram-negative, motile, rod-shaped bacterial strain, HP15(T), was isolated from aggregates taken from surface waters of the German Wadden Sea (German Bight). Of 82 marine isolates, HP15(T) was chosen for further study because of its high potential to induce production of transparent exopolymeric particles and aggregate formation while interacting with the diatom Thalassiosira weissflogii. HP15(T) grew optimally at 34-38 °C and pH 7.0-8.5, and was able to tolerate salt concentrations of 0.5-20% (w/v) NaCl. HP15(T) was characterized chemotaxonomically by possessing ubiquinone-9 as the major respiratory lipoquinone, as well as C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The DNA G+C content of strain HP15(T) was 56.9 mol%. The closest relative based on 16S rRNA gene sequence analysis was the type strain of Marinobacter flavimaris, with 99% similarity. Whole-genome relatedness values of HP15(T) to the type strains of M. flavimaris, Marinobacter salsuginis, Marinobacter lipolyticus and Marinobacter algicola were less than 70%, as determined by DNA-DNA hybridization. On the basis of phenotypic and chemotaxonomic properties as well as phylogenetic analyses, the isolate represents a novel species, Marinobacter adhaerens sp. nov.; the type strain is HP15(T) (=DSM 23420(T)=CIP 110141(T)).

Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells.

Diatom aggregation is substantial for organic carbon flux from the photic zone to deeper waters. Many heterotrophic bacteria ubiquitously found in diverse marine environments interact with marine algae and thus impact organic matter and energy cycling in the ocean. In particular, Marinobacter adhaerens HP15 induces aggregate formation while interacting with the diatom, Thalassiosira weissflogii. To study this effect at the molecular level, a genetic tool system was developed for strain HP15. The antibiotic susceptibility spectrum of this organism was determined and electroporation and conjugation protocols were established. Among various plasmids of different incompatibility groups, only two were shown to replicate in M. adhaerens. 1.4×10(-3) transconjugants per recipient were obtained for a broad-host-range vector. Electroporation efficiency corresponded to 1.1×10(5)CFU per µg of DNA. Transposon and gene-specific mutageneses were conducted for flagellum biosynthetic genes. Mutant phenotypes were confirmed by swimming assay and microscopy. Successful expression of two reporter genes in strain HP15 revealed useful tools for gene expression analyses, which will allow studying diverse bacteria-algae interactions at the molecular level and hence to gain a mechanistic understanding of micro-scale processes underlying ocean basin-scale processes. This study is the first report for the genetic manipulation of a Marinobacter species which specifically interacts with marine diatoms and serves as model to additionally analyze various previously reported Marinobacter-algae interactions in depth.

[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.

Cells dispersed from Marinobacter hydrocarbonoclasticus SP17 biofilm exhibit a specific protein profile associated with a higher ability to reinitiate biofilm development at the hexadecane-water interface.

Biofilm formation by marine hydrocarbonoclastic bacteria is commonly observed and has been recognized as an important mechanism for the biodegradation of hydrocarbons. In order to colonize new oil-water interfaces, surface-attached communities of hydrocarbonoclastic bacteria must release cells into the environment. Here we explored the physiology of cells freshly dispersed from a biofilm of Marinobacter hydrocarbonoclasticus developing at the hexadecane-water interface, by combining proteomic and physiological approaches. The comparison of the dispersed cells' proteome with those of biofilm, logarithmic- and stationary-phase planktonic cells indicated that dispersed cells had lost most of the biofilm phenotype and expressed a specific proteome. Two proteins involved in cell envelope maturation, DsbA and CtpA, were exclusively detected in dispersed cells, suggesting a reshaping of the cell envelopes during biofilm dispersal. Furthermore, dispersed cells exhibited a higher affinity for hexadecane and initiated more rapidly biofilm formation on hexadecane than the reference planktonic cells. Interestingly, storage wax esters were rapidly degraded in dispersed cells, suggesting that their observed physiological properties may rely on reserve mobilization. Thus, by promoting oil surface colonization, cells emigrating from the biofilm could contribute to the success of marine hydrocarbonoclastic bacteria in polluted environments.

Marinobacter oulmenensis sp. nov., a moderately halophilic bacterium isolated from brine of a salt concentrator.

A Gram-negative, aerobic, moderately halophilic bacterium, designated Set74(T), was isolated from brine of a salt concentrator at Ain Oulmene, Algeria. The strain grew optimally at 37-40 °C, at pH 6.5-7.0 and with 5-7.5 % (w/v) NaCl and used various organic compounds as sole carbon, nitrogen and energy sources. Ubiquinone 9 (Q-9) was the major lipoquinone. The main cellular fatty acids were C16:0, C18:1ω9c, summed feature 7 (ECL 18.846; C19:0 cyclo ω10c and/or C19:1ω6c), C12:0 3-OH, C16:1ω9c, C18:0 and C12:0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 57.4 mol%. The 16S rRNA gene sequence analysis indicated that strain Set74(T) was a member of the genus Marinobacter. The closest relatives of strain Set74(T) were Marinobacter santoriniensis NKSG1(T) (97.5 % 16S rRNA gene sequence similarity) and Marinobacter koreensis DD-M3(T) (97.4 %). DNA-DNA relatedness between strain Set74(T) and M. santoriniensis DSM 21262(T) and M. koreensis DSM 17924(T) was 45 and 37 %, respectively. On the basis of the phenotypic, chemotaxonomic and phylogenetic features, a novel species, Marinobacter oulmenensis sp. nov., is proposed. The type strain is Set74(T) ( = CECT 7499(T)  = DSM 22359(T)).

Hydrostatic pressure affects membrane and storage lipid compositions of the piezotolerant hydrocarbon-degrading Marinobacter hydrocarbonoclasticus strain #5.

A new piezotolerant alkane-degrading bacterium (Marinobacter hydrocarbonoclasticus strain #5) was isolated from deep (3475 m) Mediterranean seawater and grown at atmospheric pressure (0.1 MPa) and at 35 MPa with hexadecane as sole source of carbon and energy. Modification of the hydrostatic pressure influenced neither the growth rate nor the amount of degraded hexadecane (approximately 90%) during 13 days of incubation. However, the lipid composition of the cells sharply differed under both pressure conditions. At 0.1 MPa, M. hydrocarbonoclasticus #5 biosynthesized large amounts ( approximately 62% of the total cellular lipids) of hexadecane-derived wax esters (WEs), which accumulated in the cells under the form of individual lipid bodies. Intracellular WEs were also synthesized at 35 MPa, but their proportion was half that at 0.1 MPa. This lower WE content at high pressure was balanced by an increase in the total cellular phospholipid content. The chemical composition of WEs formed under both pressure conditions also strongly differed. Saturated WEs were preferentially formed at 0.1 MPa whereas diunsaturated WEs dominated at 35 MPa. This increase of the unsaturation ratio of WEs resembled the one classically observed for bacterial membrane lipid homeostasis. Remarkably, the unsaturation ratio of membrane fatty acids of M. hydrocarbonoclasticus grown at 35 MPa was only slightly higher than at 0.1 MPa. Overall, the results suggest that intracellular WEs and phospholipids play complementary roles in the physiological adaptation of strain #5 to different hydrostatic pressures.