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.     

一株可乳化降解原油的Compostibacillus的分离及特性

【背景】通过实施多轮次微生物采油,华北油藏产出液菌浓达到了106个/mL以上,油藏内部已经形成了较稳定的微生物发酵场,从其中筛选出能够乳化降解原油的微生物,并在地面对其进行扩大培养,然后再应用到微驱油藏,以进一步提高微生物采油实施效果。【目的】筛选乳化降解原油性能良好的菌株,对其进行多相分类学鉴定和性能评价。【方法】利用原油为底物筛选乳化降解性能良好的菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等确定菌株的分类地位。通过乳化能力、降解率等方法确定菌株的原油乳化降解特性。【结果】从华北油田采集的地层水样品中分离得到一株乳化原油的菌株BLG74,经多相分类鉴定表明其是土壤堆肥芽孢杆菌(Compostibacillus humi)的新菌株,亲源性99.6%。该菌株的生长温度为30-60℃ (最适温度45℃),pH6.5-9.5(最适pH7.0),NaCl浓度0%-7%(质量体积比)。菌株BLG74在玉米浆培养基中培养,其发酵液的表面张力为56.3 mN/m,乳化力约95%,在初始原油质量浓度0.5%、温度45℃的条件下培养20d,对原油的降解率可达40.8%。【结论】菌...     

Effect of Environmental Parameters on Bacterial Degradation of Bunker C Oil, Crude Oils, and Hydrocarbons

Mixed microbial cultures, previously enriched on Bunker C fuel oil, grew on and degraded Bunker C fuel oil at temperatures ranging from 5 to 28 C. At 15 C, 41 to 85% of the benzene-soluble components of Bunker C disappeared after incubation for 7 days; at 5 C the values ranged from 21 to 52% after 14 days of incubation. A Nocardia sp. isolated from a culture enriched on Bunker C oil grew on Venezuelan crude oil, Bunker C, hexadecane, and a hydrocarbon mixture at temperatures of 5 and 15 C. The 10-C decrease in temperature resulted in an average 2.2-fold decrease in generation time of the bacteria. Gas-liquid chromatographic measurements of Venezuelan and Arabian crude oils which had been incubated with the Nocardia sp. showed significant degradation of the n-alkane portion and the chromatographically unresolved components of the oils. The concentration of elemental nitrogen required to bring about the disappearance of 1 mg of hexadecane by the Nocardia sp. was 0.5 mg. The results confirm suggestions that the rate of natural biodegradation of oil in marine temperate-to-polar zones is probably limited by low temperatures and phosphorus concentrations, but suggest that the concentrations of nitrogen occurring naturally are probably not rate-limiting factors.     

Comparison of autochthonous bacteria and commercially available cultures with respect to their effectiveness in fuel oil degradation

Summary This study examined the microbial degradation of fuel oil by nine highly adapted different commercially available mixed bacterial cultures (DBC-plus, Flow Laboratories, Meckenheim, F.R.G.) and a bacterial community from a domestic sewage sludge sample. All mixed cultures were cultivated under aerobic batch conditions shaking (110 rpm) at 20°C in a mineral base medium containing 1 or 5% (v/v) fuel oil as the sole carbon source. Percent degradation of fuel oil and the n-alkane fraction was recorded for the nine DBC-plus cultures and the mixed population of the activated sludge sample. The increase in colony counts, protein, and optical density was studied during a 31-day incubation period for DBC-plus culture A, DBC-plus culture A2 and the activated sludge sample. The activated sludge mixed culture was most effective in degrading fuel oil, but various isolated bacterial strains from this bacterial community were not able to grow on fuel oil as the sole carbon source. In contrast, the n-alkane degradation rates of the DBC-cultures were lower, but single strains from the commercially available mixed cultures were able to mineralize fuel oil hydrocarbons. Strains ofPseudomonas aeruginosa were isolated most frequently and these organisms were able to grow very rapidly on fuel oil as a complex sole carbon source. The results indicate that fuel oil degradation in domestic sewage sludge is performed by mixed populations of naturally occurring bacteria and does not depend on the application of highly adapted commercially available cultures.     

Characteristics of bioemulsifiers synthesised in crude oil media by <Emphasis Type="Italic">Halomonas eurihalina</Emphasis> and their effectiveness in the isolation of bacteria able to grow in the presence of hydrocarbons

Halomonas eurihalina strains F2-7, H28, H96, H212 and H214 were capable of producing large amounts of exopolysaccharides (EPS) in MY medium with added crude oil. The biopolymers showed lower carbohydrate and protein content than those synthesised in control medium without oil. Nevertheless, the percentages of uronic acids, acetyls and sulphates were higher. The emulsifying activity of biopolymers was measured; crude oil was the substrate most efficiently emulsified. Furthermore, all the EPS tested emulsified higher volumes of crude oil than the commercial surfactants used as controls. We have also proved the effectiveness of both Halomonas eurihalina strains and their EPS to select indigenous bacteria able to grow in the presence of polycyclic aromatic hydrocarbons (naphthalene, phenanthrene and pyrene) from waste crude oil. The majority of isolated strains belonged to the genus Bacillus.     

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.     

Hydrocarbon-Degrading Bacteria Exhibit a Species-Specific Response to Dispersed Oil while Moderating Ecotoxicity

The Deepwater Horizon blowout in April 2010 represented the largest accidental marine oil spill and the largest release of chemical dispersants into the environment to date. While dispersant application may provide numerous benefits to oil spill response efforts, the impacts of dispersants and potential synergistic effects with crude oil on individual hydrocarbon-degrading bacteria are poorly understood. In this study, two environmentally relevant species of hydrocarbon-degrading bacteria were utilized to quantify the response to Macondo crude oil and Corexit 9500A-dispersed oil in terms of bacterial growth and oil degradation potential. In addition, specific hydrocarbon compounds were quantified in the dissolved phase of the medium and linked to ecotoxicity using a U.S. Environmental Protection Agency (EPA)-approved rotifer assay. Bacterial treatment significantly and drastically reduced the toxicity associated with dispersed oil (increasing the 50% lethal concentration [LC₅₀] by 215%). The growth and crude oil degradation potential of Acinetobacter were inhibited by Corexit by 34% and 40%, respectively; conversely, Corexit significantly enhanced the growth of Alcanivorax by 10% relative to that in undispersed oil. Furthermore, both bacterial strains were shown to grow with Corexit as the sole carbon and energy source. Hydrocarbon-degrading bacterial species demonstrate a unique response to dispersed oil compared to their response to crude oil, with potentially opposing effects on toxicity. While some species have the potential to enhance the toxicity of crude oil by producing biosurfactants, the same bacteria may reduce the toxicity associated with dispersed oil through degradation or sequestration.     

Effect of Amendments on the Microbial Utilization of Oil Applied to Soil

Replicate field plots comprising a control, plus oil, plus oil and bacteria, plus oil and fertilizer (urea-phosphate; 27:27:0), and plus oil, bacteria, and fertilizer were monitored over a 308-day period for changes in bacterial and mold numbers. Changes in the chemical composition of the oil applied to the plots was followed by using chromatographic techniques. Application of fertilizer resulted in a stimulation of bacterial numbers and in the rate of utilization of n-alkane components of the saturate fraction. The application of oil-utilizing bacteria, however, resulted in only a slightly accelerated rate of utilization of n-alkane components of chain lengths C20 to C25. The isoprenoids, phytane and pristane, were still present in gas-liquid chromatography profiles after digestion of the n-alkane components of the saturate fraction. Those plots which received fertilizer showed an accelerated rate of recovery of native vegetation.     

Efficacy of intervention strategies for bioremediation of crude oil in marine systems and effects on indigenous hydrocarbonoclastic bacteria

There is little information on how different strategies for the bioremediation of marine oil spills influence the key indigenous hydrocarbon-degrading bacteria (hydrocarbonoclastic bacteria, HCB), and hence their remediation efficacy. Therefore, we have used quantitative polymerase chain reaction to analyse changes in concentrations of HCB in response to intervention strategies applied to experimental microcosms. Biostimulation with nutrients (N and P) produced no measurable increase in either biodegradation or concentration of HCB within the first 5 days, but after 15 days there was a significant increase (29%; P < 0.05) in degradation of n-alkanes, and an increase of one order of magnitude in concentration of Thalassolituus (to 10(7) cells ml(-1)). Rhamnolipid bioemulsifier additions alone had little effect on biodegradation, but, in combination with nutrient additions, provoked a significant increase: 59% (P < 0.05) more n-alkane degradation by 5 days than was achieved with nutrient additions alone. The very low Alcanivorax cell concentrations in the microcosms were hardly influenced by addition of nutrients or bioemulsifier, but strongly increased after their combined addition, reflecting the synergistic action of the two types of biostimulatory agents. Bioaugmentation with Thalassolituus positively influenced hydrocarbon degradation only during the initial 5 days and only of the n-alkane fraction. Bioaugmentation with Alcanivorax was clearly much more effective, resulting in 73% greater degradation of n-alkanes, 59% of branched alkanes, and 28% of polynuclear aromatic hydrocarbons, in the first 5 days than that obtained through nutrient addition alone (P < 0.01). Enhanced degradation due to augmentation with Alcanivorax continued throughout the 30-day period of the experiment. In addition to providing insight into the factors limiting oil biodegradation over time, and the competition and synergism between HCB, these results add weight to the use of bioaugmentation in oil pollution mitigation strategies.     

Lag period of 14CO2 evolution from dioctyl sulpho[2,3-14C]succinate in relation to adaptation of bacterium, Comamonas terrigena, to dialkyl esters of sulphosuccinate

Comamonas terrigena, strain N3H, which was isolated from soil polluted with crude oil products, degraded dioctyl sulphosuccinate, a synthetic commercial surfactant. The primary degradation of this compound, the cleavage of ester bonds between octyl groups and sulphosuccinate, lasted significantly shorter time than the subsequent breakdown of the sulphosuccinate moiety of dioctyl sulpho[2,3-(14)C]succinate. (14)CO(2) evolution had a significant shorter lag period with cells in Tris/phosphate medium, without inorganic sulphate and adapted to surfactant, than unadapted cells. The acceleration of the primary degradation by adapted cells also suggest that some enzymes involved in surfactant degradation are inducible. The bacterium may be useful for bioremediation.     

Biodegradation of petroleum hydrocarbons in soil inoculated with yeasts

Yeast species belonging to the Candida genus were added to the greyish-brown soil of the Apsheron Peninsula under laboratory conditions. The rate of CO2 production was used to estimate the degradation of crude oil, paraffin, cycloparaffin and aromatic hydrocarbons as well as their oxidized products. The rate of hydrocarbon degradation in the soil inoculated with yeast cells was shown to drop down gradually. The effective action on the process of hydrocarbon degradation depended on the special properties of an inoculated population and on the structure of a hydrocarbon. Some yeast species stimulated the degradation of various aromatic hydrocarbons and their oxidized products. Aromatic hydrocarbons were decomposed at a lower rate comparing to their oxidized products. When the soil was inoculated with C. guilliermondii populations, n-hexadecane added to the soil at a concentration of 1% was decomposed within 250-300 days. Field experiments confirmed that crude oil biodegradation was more intensive in the soil inoculated with yeast cells.     

Soybean oil-degrading bacterial cultures as a potential for control of green peach aphids (Myzus persicae)

Microorganisms capable of degrading crude oil were isolated and grown in soybean oil as a sole carbon source. The microbial cultures were used to control green peach aphids in vitro. Approximately 60% mortality of aphids was observed when the cultures were applied alone onto aphids. To examine the cultures as a pesticide formulation mixture, the cultures were combined with a low dose of the insecticide imidacloprid (one-fourth dose of recommended field-application rate) and applied onto aphids. The cultures enhanced significantly the insecticidal effectiveness of imidacloprid, which was higher than imidacloprid alone applied at the low dose. The isolated microorganisms exhibited high emulsifying index values and decreased surface tension values after being grown in soybean oil media. GC/MS analyses showed that microorganisms degraded soybean oil to fatty acids. The cultures were suggested to play the roles of wetting, spreading, and sticking agents to improve the effectiveness of imidacloprid. This is the first report on the control of aphids by using oil-degrading microbial cultures.     

Dispersants as Used in Response to the MC252-Spill Lead to Higher Mobility of Polycyclic Aromatic Hydrocarbons in Oil-Contaminated Gulf of Mexico Sand

After the explosion of the Deepwater Horizon oil rig, large volumes of crude oil were washed onto and embedded in the sandy beaches and sublittoral sands of the Northern Gulf of Mexico. Some of this oil was mechanically or chemically dispersed before reaching the shore. With a set of laboratory-column experiments we show that the addition of chemical dispersants (Corexit 9500A) increases the mobility of polycyclic aromatic hydrocarbons (PAHs) in saturated permeable sediments by up to two orders of magnitude. Distribution and concentrations of PAHs, measured in the solid phase and effluent water of the columns using GC/MS, revealed that the mobility of the PAHs depended on their hydrophobicity and was species specific also in the presence of dispersant. Deepest penetration was observed for acenaphthylene and phenanthrene. Flushing of the columns with seawater after percolation of the oiled water resulted in enhanced movement by remobilization of retained PAHs. An in-situ benthic chamber experiment demonstrated that aromatic hydrocarbons are transported into permeable sublittoral sediment, emphasizing the relevance of our laboratory column experiments in natural settings. We conclude that the addition of dispersants permits crude oil components to penetrate faster and deeper into permeable saturated sands, where anaerobic conditions may slow degradation of these compounds, thus extending the persistence of potentially harmful PAHs in the marine environment. Application of dispersants in nearshore oil spills should take into account enhanced penetration depths into saturated sands as this may entail potential threats to the groundwater.     

Genome analysis provides insights into crude oil degradation and biosurfactant production by extremely halotolerant Halomonas desertis G11 isolated from Chott El-Djerid salt-lake in Tunisian desert

Here, we report the genomic features and the bioremediation potential of Halomonas desertis G11, a new halophilic species which uses crude oil as a carbon and energy source and displays intrinsic resistance to salt stress conditions (optimum growth at 10% NaCl). G11 genome (3.96 Mb) had a mean GC content of 57.82%, 3622 coding sequences, 480 subsystems and 64 RNA genes. Annotation predicted 38 genes involved in osmotic stress including the biosynthesis of osmoprotectants glycine-betaine, ectoine and osmoregulated periplasmic glucans. Genome analysis revealed also the versatility of the strain for emulsifying crude oil and metabolizing hydrocarbons. The ability of G11 to degrade crude oil components and to secrete a glycolipid biosurfactant with satisfying properties was experimentally confirmed and validated. Our results help to explain the exceptional capacity of G11 to survive at extreme desertic conditions, and highlight the metabolic features of this organism that has biotechnological and ecological potentialities.     

High-temperature hydrocarbon biodegradation activities in Kuwaiti desert soil samples

Soil samples taken monthly from the Burgan South oil field of Kuwait for one year degraded crude oil, phenanthrene, and hexadecane. Bacteria were better degraders at high-temperature (55°C) than fungi, especially in the drier, hotter months. Depending on the period of sampling, bacteria degraded hydrocarbons in the range of 46–86% (crude oil), 42–100% (hexadecane) and 5–58% (phenanthrene). Fungi alone accounted for degradation by 20–81% (crude oil), 30–95% (hexadecane) and less than 55% (phenanthrene).     

Application of biosurfactant produced from peanut oil cake by Lactobacillus delbrueckii in biodegradation of crude oil

Lactobacillus delbrueckii cultured with peanut oil cake as the carbon source yielded 5.35 mg ml(-1) of biosurfactant production. Five sets of microcosm biodegradation experiments were carried out with crude oil as follows: set 1 - bacterial cells+crude oil, set 2 - bacterial cells+crude oil+fertilizer, set 3 - bacterial cells+crude oil+biosurfactant, set 4 - bacterial cells+crude oil+biosurfactant+fertilizer, set 5 - with no bacterial cells, fertilizer and biosurfactant (control). Maximum degradation of crude oil was observed in set 4 (75%). Interestingly, when biosurfactant and bacterial cells were used (set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in set 4 was 7% higher degradation level in microcosm experiments. It is evident from the results that biosurfactants alone is capable of promoting biodegradation to a large extent without added fertilizers.     

Isolation and characterization of Halomonas sp. strain C2SS100, a hydrocarbon-degrading bacterium under hypersaline conditions

Aims:  To isolate and characterize an efficient hydrocarbon-degrading bacterium under hypersaline conditions, from a Tunisian off-shore oil field.
Methods and Results:  Production water collected from 'Sercina' petroleum reservoir, located near the Kerkennah island, Tunisia, was used for the screening of halotolerant or halophilic bacteria able to degrade crude oil. Bacterial strain C2SS100 was isolated after enrichment on crude oil, in the presence of 100 g l⁻¹ NaCl and at 37°C. This strain was aerobic, Gram-negative, rod-shaped, motile, oxidase + and catalase +. Phenotypic characters and phylogenetic analysis based on the 16S rRNA gene of the isolate C2SS100 showed that it was related to members of the Halomonas genus. The degradation of several compounds present in crude oil was confirmed by GC–MS analysis. The use of refined petroleum products such as diesel fuel and lubricating oil as sole carbon source, under the same conditions of temperature and salinity, showed that significant amounts of these heterogenic compounds could be degraded. Strain C2SS100 was able to degrade hexadecane (C₁₆). During growth on hexadecane, cells surface hydrophobicity and emulsifying activity increased indicating the production of biosurfactant by strain C2SS100.
Conclusions:  A halotolerant bacterial strain Halomonas sp. C2SS100 was isolated from production water of an oil field, after enrichment on crude oil. This strain is able to degrade hydrocarbons efficiently. The mode of hydrocarbon uptake is realized by the production of a biosurfactant which enhances the solubility of hydrocarbons and renders them more accessible for biodegradation.
Significance and Impact of the Study:  The biodegradation potential of the Halomonas sp. strain C2SS100 gives it an advantage for possibly application on bioremediation of water, hydrocarbon-contaminated sites under high-salinity level.     

Lag period of 14CO2 evolution from dioctyl sulpho[2,3-14C]succinate in relation to adaptation of bacterium, Comamonas terrigena, to dialkyl esters of sulphosuccinate

Comamonas terrigena, strain N3H, which was isolated from soil polluted with crude oil products, degraded dioctyl sulphosuccinate, a synthetic commercial surfactant. The primary degradation of this compound, the cleavage of ester bonds between octyl groups and sulphosuccinate, lasted significantly shorter time than the subsequent breakdown of the sulphosuccinate moiety of dioctyl sulpho[2,3-¹⁴C]succinate. ¹⁴CO₂ evolution had a significant shorter lag period with cells in Tris/phosphate medium, without inorganic sulphate and adapted to surfactant, than unadapted cells. The acceleration of the primary degradation by adapted cells also suggest that some enzymes involved in surfactant degradation are inducible. The bacterium may be useful for bioremediation.     

Influence of crude oil on changes of bacterial communities in Arctic sea-ice

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is increasing due to oil exploration in Arctic offshore areas and a growing interest in using the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and gas. However, little is known about the potential impact of accidental oil spills on this environment. We investigated the impact of crude oil on microbial community composition in six different Arctic sea-ice samples incubated with crude oil at 1 degrees C in microcosms for one year. Alterations in the composition of bacterial communities were analyzed with the culture-independent molecular methods DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). DGGE, FISH and cultivation methods revealed a strong shift in community composition toward the gamma-proteobacteria in sea-ice and melt pool samples incubated with crude oil. Marinobacter spp., Shewanella spp. and Pseudomonas spp. were the predominant phylotypes in the oil-treated microcosms. The ability of indigenous sea-ice bacteria to degrade hydrocarbons at low temperature (1 degrees C) was tested using four representative strains cultivated from sea-ice enriched with crude oil. [14C]Hexadecane was degraded by the sea-ice isolates at 20-50% capacity of the mesophilic type strain Marinobacter hydrocarbonoclasticus, a known hydrocarbon degrader, incubated at 22 degrees C.     

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.