Variability in Pseudomonas aeruginosa Lipopolysaccharide Expression during Crude Oil Degradation

Bacterial utilization of crude oil components, such as the n-alkanes, requires complex cell surface adaptation to allow adherence to oil. To better understand microbial cell surface adaptation to growth on crude oil, the cell surface characteristics of two Pseudomonas aeruginosa strains, U1 and U3, both isolated from the same crude oil-degrading microbial community enriched on Bonny Light crude oil (BLC), were compared. Analysis of growth rates demonstrated an increased lag time for U1 cells compared to U3 cells. Amendment with EDTA inhibited U1 and U3 growth and degradation of the n-alkane component of BLC, suggesting a link between cell surface structure and crude oil degradation. U1 cells demonstrated a smooth-to-rough colony morphology transition when grown on BLC, while U3 cells exhibited rough colony morphology at the outset. Combining high-resolution atomic force microscopy of the cell surface and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracted lipopolysaccharides (LPS), we demonstrate that isolates grown on BLC have reduced O-antigen expression compared with that of glucose-grown cells. The loss of O-antigen resulted in shorter LPS molecules, increased cell surface hydrophobicity, and increased n-alkane degradation.     

Biological souring of crude oil under anaerobic conditions

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil–water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring.     

Effect of Four Dispersants on Biodegradation and Growth of Bacteria on Crude Oil

Four chemical dispersants, Corexit 8666, Gamlen Sea Clean, G. H. Woods Degreaser-Formula 11470, and Sugee 2 were examined singly and in individual combinations with Arabian Crude Oil (1:1 ratio) at 10 and 25 C for their effects on the growth of bacteria indigenous to local marine waters, the bacterial population composition, and biodegradation of crude oil; in addition, their emulsifying capacities, at approximately 24 C, were determined. None of the dispersants used alone were toxic even at relatively high concentrations (1.25%), although Gamlen Sea Clean and G. H. Woods Degreaser-Formula 11470 did cause an increase in the lag phase which was more pronounced at 10 than at 25 C; addition of the crude oil reduced the lag phase increase. All of the dispersants used alone supported good growth of microorganisms, but qualitative population shifts were caused by the dispersant-oil combinations. The degrees of degradation of the n-alkane fraction of the crude oil varied depending upon the dispersant used. Under these test conditions, only Sugee 2, which had the poorest emulsifying capacity, promoted n-alkane degradation compared with the values obtained by using the crude oil alone.     

Effect of pyocyanin on a crude-oil-degrading microbial community

Pseudomonas aeruginosa is an n-alkane degrader that is frequently isolated from petroleum-contaminated sites and produces factors that enhance its competitiveness and survival in many environments. In this study, one such factor, pyocyanin, has been detected in an oil-degrading culture containing P. aeruginosa and is a redox-active compound capable of inhibiting microbial growth. To examine the effects of pyocyanin further, an oil-degrading culture was grown with and without 9.5 microM pyocyanin and microbial community structure and oil degradation were monitored for 50 days. Denaturing gradient gel electrophoresis (DGGE) analysis of cultures revealed a decrease in the microbial community diversity in the pyocyanin-amended cultures compared to that of the unamended cultures. Two members of the microbial community in pure culture exhibited intermediate and high sensitivities to pyocyanin corresponding to intermediate and low levels of activity for the antioxidant enzymes catalase and superoxide dismutase, respectively. Another member of the community that remained constant in the DGGE gels over the 50-day culture incubation period exhibited no sensitivity to pyocyanin, corresponding to a high level of catalase and superoxide dismutase when examined in pure culture. Pyocyanin also affected the overall degradation of the crude oil. At 50 days, the culture without pyocyanin had decreased polycyclic aromatic hydrocarbons compared to the pyocyanin-amended culture, with a specific reduction in the degradation of dibenzothiophenes, naphthalenes, and C(29) and C(30) hopanes. This study demonstrated that pyocyanin influenced the diversity of the microbial community and suggests the importance of understanding how interspecies interactions influence the degradation capability of a microbial community.     

Is the CvaA* protein, encoded within the colicin V export gene cvaA, required for colicin V transport?

Abstract We investigated the expression of important Actinobacillus pleuropneumoniae surface polysaccharides, namely, capsular polysaccharides (CPS) and lipopolysaccharides (LPS), after growth under iron-restricted conditions. Iron restriction did not seem to affect the production of CPS, as determined by labelling with a monoclonal antibody (mAb) against the serotype l K-antigen and flow cytometry analysis, and also as determined by electron microscopy. SDS-PAGE revealed that the LPS profiles of these cells were also unaffected by iron restriction. Using flow cytometry analysis, however, we observed that binding of mAb against serotype 1 O-antigen was altered in cells of A. pleuropneumoniae serotype l reference strain (4074) grown under iron-restricted conditions. This strain exhibited two subpopulations with distinct patterns of reactivity with the mAb against the O-antigen. When strain 4074 was grown under iron-restricted conditions, a shift from one cell subpopulation (moderately fluorescent) to another cell subpopulation (highly fluorescent, thus binding more antibodies) was observed. Our results indicate that growth of A. pleuropneumoniae serotype l under iron-restricted conditions did not seem to affect CPS production, but might alter, at least for the reference strain, the expression of LPS.     

Hydrocarbon assimilation by Candida lipolytica: Formation of a biosurfactant; Effects on respiratory activity and growth

Summary Candida lipolytica is shown to produce an extracellular polymer with emulsifying properties when grown on n-tetradecane or a mixture of linear hydrocarbons. A device for biosurfactant isolation is presented. The polymers recovered from the fermentation broth were found to be complex molecules with a protein, a lipid and a carbohydrate moiety. Their surface active properties suggest a possible role in hydrocarbon uptake by cells. Effectively, the addition of crude polymer resulted in an enhancement of respiration rate which was dependent on n-alkane concentration with glucose grown cells. Likewise in batch culture, maximum growth rate, cell productivity and yield were increased by the presence of the biosurfactant.     

高效石油降解菌群的构建及对芳香烃化合物萘的协同降解性能

【背景】石油作为一类混杂有机化合物,一旦产生污染就会对人类和环境造成严重的危害。【目的】从新疆石油污染土壤中分离筛选石油降解菌,为石油污染土壤的生物修复提供数据支持及技术参考。【方法】以石油为唯一碳源,通过富集培养、筛选分离得到123株单菌,根据菌落形态挑选出30个不同形态菌株,通过16S rRNA基因序列确定其种属,构建系统发育树;通过原油降解实验筛选出高效石油降解菌,以芳香烃的标志化合物萘为唯一碳源筛选出高效降解菌株,并分别筛选可降解水杨酸、邻苯二酚的菌株。【结果】分离筛选出5株高效石油降解菌,降解率高于85%;萘、水杨酸和邻苯二酚降解菌株各获得一株,将3种菌株按照1:1:1的接种比例对萘进行降解,萘的降解率从单菌60.74%提升到89.40%,菌株间的分工协作可以提高有机物的降解效率。【结论】筛选得到的菌株丰富了石油降解微生物菌种库,不同微生物菌株之间的分工协作为石油污染物的降解提供了新思路,为进一步研究石油污染治理提供参考。     

超低渗油藏微生物吞吐技术的矿场试验

【目的】通过对渭北低渗油藏内源微生物的研究,考察分离纯化的内源解烃菌产生表面活性剂和降解原油的能力、岩心驱替增油效率,同时验证其在超低渗油田单井吞吐矿场实验的应用效果,探讨微生物采油技术在超低渗油田提高采收率的工艺和可行性。【方法】采集超低渗油藏的油水样,应用油平板进行产表面活性剂解烃菌的分离,通过生理生化特性和16S r RNA基因序列分析对菌株进行种属鉴定,评价其油藏环境适应性,利用内源-外源功能微生物复配体系进行原油降解,在填砂管和岩心物模上进行驱油实验,将优化好的微生物复配体系应用于现场实施单井吞吐工艺的实验。【结果】从渭北某区块超低渗油藏的原油样品中分离得到一株铜绿假单胞菌(Pseudomonas aeruginosa),命名为WB-001。该菌株可使发酵液的表面张力降至29.04 m N/m,使渭北原油蜡质含量降至8.48%。填砂管实验表明WB-001与外源枯草芽胞杆菌OPUS-HOB-001(Bacillus subtilis)复配后,驱油效率较单纯水驱提高了9.72%;岩心驱替实验较水驱提高12.54%。微生物单井吞吐措施后,平均日产油由措施前的0.42 t增加到0.89 t,累计增油44.47 t;原油降粘率为11.70%,降凝率为9.41%,采出水表面张力降低幅度为18.93%。【结论】通过详细的室内评估和成功的矿场实验,证明微生物采油技术在超低渗油藏有一定的应用可行性,并为后续规模化应用提供了理论基础和物质基础,为超低渗油田的高效精细开发探索一条新的途径。     

Variable expression of O-antigen and the role of lipopolysaccharide as an adhesin in Aeromonas sobria

Abstract On initial isolation of Aeromonas sobria 3767 from a diarrhoeal stool specimen, two colony types were obtained: opaque (3767O) and translucent (3767T). Strain 3767O consistently produced lipopolysaccharide (LPS) core and O-antigen side chain, detectable by SDS-PAGE and by Western blotting with an O-antigen-specific monoclonal antibody. Strain 3767T produced LPS core but the amount of O-antigen was dependent on factors including growth medium and bacterial growth phase. Strain 3767T exhibited significantly lower levels of adhesion to HEp-2 cells than 3767O and this correlated with the level of LPS expression, with the greatest reduction (61%) at stationary phase when no LPS was detectable. The results implicate LPS as an adhesin for A. sobria 3767.     

Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces

There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 +/- 1 h) and similar morphology as cells grown in cell culture flasks (32 +/- 1 h), indicating good biocompatibility. However, more than 200 genes showed different expression levels in cells grown on SU-8 treated with HNO3-CAN compared to cells grown in cell culture flasks. This shows that gene expression profiling is a simple and precise method for determining differences in cells grown on different surfaces that are otherwise difficult to find using conventional methods. It is particularly noteworthy that no correlation was found between surface hydrophobicity and biocompatibility.     

Effect of dispersing oil phase on the biodegradability of a solid alkane dissolved in non-biodegradable oil

Acinetobacter sp. CR was grown on a model oil, which consisted of an inert oil matrix of pristane with n-heneicosane dissolved in it as the sole carbon source, in a stirred-tank bioreactor. This bacterium takes up substrates from the oil phase by direct contact with the oil phase. A previously established mathematical model was applied to reveal the effect of agitation conditions on the growth and n-alkane degradation kinetics of the bacterium. Higher impeller speed resulted in both lower microbial growth and lower n-alkane degradation rate of the bacterium, although it increased the specific surface area of the oil, which was measured by a previously developed device. This result was due to the decreased number of cells adhering to the oil surface, i.e., intense agitation inhibited the adhesion of cells to the oil surface. The addition of a surfactant below a critical micelle concentration (CMC) inhibited the degradation of n-heneicosane dissolved in pristane, although the biodegradability of the substrate recovered gradually with the increase in the dose of surfactant over CMC. The results suggest that efforts to increase the specific surface area of the oil phase have the undesirable result of inhibiting oil degradation when the dominant microbial degraders take up substrates in oil by direct contact with the oil. Electronic Publication     

Acid-induced expression of an LPS-associated gene in Helicobacter pylori

To investigate urease-independent mechanisms by which Helicobacter pylori resists acid stress, subtractive RNA hybridization was used to identify H. pylori genes whose expression is induced after exposure to acid pH. This approach led to the isolation of a gene that encoded a predicted 34.8 kDa protein (WbcJ), which was homologous to known bacterial O-antigen biosynthesis proteins involved in the conversion of GDP-mannose to GDP-fucose. An isogenic wbcJ null mutant strain failed to express O-antigen and Lewis X or Lewis Y determinants and was more sensitive to acid stress than was the wild-type strain. Qualitative differences in LPS profiles were observed in H. pylori cells grown at pH 5 compared with pH 7, which suggests that H. pylori may alter its LPS structure in response to acidic pH. This may be an important adaptation facilitating H. pylori colonization of the acidic gastric environment.     

Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.     

Integration of biosorption and biodegradation in a fed-batch mode for the enhanced crude oil remediation

Microbial bioremediation of oil-contaminated sites is still a challenge due to the slower rate and susceptibility of microbes to a higher concentration of oil. The poor bioavailability, hydrophobicity, and non-polar nature of oil slow down microbial biodegradation. In this study, biodegradation of crude oil is performed in fed-batch mode using an oil-degrader Pseudomonas aeruginosa to address the issue of substrate toxicity. The slower biodegradation was integrated with faster biosorption for effective oil remediation. Highly fibrous and porous sugarcane bagasse was surface modified with hydrophobic octyl groups to improve the surface-oil interactions. The microbe showed 2 folds enhanced oil degradation in the fed-batch study, which was further increased by 1·5 folds in the integrated biosorption coupled biodegradation approach. The biosorption-assisted biodegradation approach supported the microbial growth to 2 folds higher than the fed-batch study without biosorbent. The analysis of biosurfactant production indicated the 3 folds higher concentration in fed-batch modes as compared to batch study. In the integrated strategy, the concentration of contaminant (oil) reduces to quite a tolerable level to microbes, which improved effective metabolism and thus overall biodegradation. This study puts forward a promising strategy for improved degradation of hazardous hydrophobic contaminants in a sustainable, economic and eco-friendly manner.     

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag⁺/Cl^? colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔH_ITC upon interacting with free Ag⁺ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag⁺ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag⁺/Cl^? colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag⁺/Cl^? colloids. Biomineralization of Ag⁺/Cl^? colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.     

The influence of Matrigel or growth factor reduced Matrigel on human intervertebral disc cell growth and proliferation

Matrigel (reconstituted basement membrane extract) is a potent inducer of cell growth and differentiation in vitro. This study examined phenotypic variation and proliferative responses of human annular intervertebral disc cells in vitro in Matrigel and Growth Factor Reduced Matrigel (GFR-Matrigel). Cells from age- and gender-matched control subjects and patients with degenerative disc disease were grown either on the surface of, or suspended within, either matrices. Disc cells grew well on top of both matrices with cells spontaneously forming cell projections. Cells grown within either matrix migrated within the gel to form colonies. Increased colony formation within the matrices was seen with young control and patient cells (p < 0.05). Old and young control and patient cells showed increased proliferation within GFR-Matrigel compared to Matrigel. When grown on the matrix surface, young patient and control donor cells showed increased proliferation on GFR-Matrigel compared to Matrigel. Cellular proliferation was significantly greater inside a 3-dimensional environment than a two-dimensional surface monolayer environment. Disc cells had increased proliferation when grown in or on GFR-Matrigel compared to Matrigel. These studies serve as a baseline for subsequent investigations regarding effects of cytokines on disc cells and increase our knowledge of the influence of extracellular matrices on disc cell proliferation.     

The O:34-antigen lipopolysaccharide as an adhesin in Aeromonas hydrophila

Abstract We compared the ability of different Aeromonas hydrophila strains from serogroup O:34 grown at different temperatures to adhere to Hep-2 cells. We found a high level of adhesion when the strains were grown at 20 °C but not when they were grown at 37 °C. We previously described that these strains were able to form the O-antigen lipopolysaccharide when they grow at low temperature but not at high temperature. We also obtained by transposon mutagenesis mutants only devoid of the O-antigen lipopolysaccharide ( rfb mutants), and they showed significantly lower levels of adhesion to Hep-2 cells than the smooth strains. All these results prompted us to conclude that the O-antigen LPS, in these strains, is an important adhesin.     

Mouse polyomavirus large T antigen inhibits cell growth and alters cell and colony morphology in Saccharomyces cerevisiae

The gene for mouse polyomavirus large tumor (LT) antigen, a potent oncoprotein, was expressed in Saccharomyces cerevisiae from the inducible GAL1 promoter. Substantial cell growth inhibition as well as colony and cell morphology changes dependent on cyclic adenosine monophosphate (cAMP) were observed. In contrast to cell and colony morphology alterations, the growth inhibition appeared to be transient, thus indicating the existence of an active adaptation of yeast cells to the LT antigen presence.     

Effect of the tropical grass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf on microbial population and activity in petroleum-contaminated soil

The effect of the tropical pasture grass Brachiaria brizantha on numbers of bacteria, fungi and degraders of alkanes, aromatics, cycloalkanes and crude oil in petroleum hydrocarbon contaminated and uncontaminated savannah soil was evaluated. Substrate induced soil respiration and soil pH were compared between planted and unplanted soil. B. brizantha had a mostly increasing effect on microbial numbers. As an exception, growth of bacteria was not or negatively affected. Microbial respiration and pH were always lower in planted than in unplanted soil. Low pH may result from enhanced oil degradation in planted soil leading to an accumulation of organic acids. A comparable stimulation of crude oil degraders and fungi in planted soil points to the importance of fungi. Since they tolerate lower pH values than bacteria, they are considered to play a central role in oil degradation. Given that the enhancement of crude oil degradation under the influence of B. brizantha could not clearly be correlated to microbial numbers and activity, other factors like oxygen availability, plant enzymes and synergistic degradation by microbial consortia have to be considered.     

Bacteria belonging to the genus cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment

To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 10(3) cells/g of grains before crude oil was added to the tanks and increased to 3 x 10(6) cells/g of grains after crude oil was added. The number increased further after 14 days to 10(8) cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 x 10(6) cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.