Intensification of Microbial Degradation of Crude Oil and Oil Products in the Presence of Perfluorodecalin

The possibility of using perfluorinated organic compounds for growing microorganisms and degrading xenobiotics has been demonstrated for the first time with perfluorodecalin (PFD), a gas-transporting component of the blood substitute Perftoran. This is particularly promising for intensifying microbial degradation of oil and oil products and the production of biodegrader biomass in synthetic mineral media. The addition of PFD to a mineral medium with crude oil and masut increased by 4.5–10.2 times the maximum concentrations and growth rates of all bacterial strains under study (Pseudomonas, Rhodococcus, and Bacillus genera). The degree of oil product consumption was increased 8.7–12.7 times.     

Microbial Utilization of Crude Oil

The utilization of two crude oil samples of different quality at 4 and 30 C has been studied by using pure and mixed bacterial cultures obtained by enrichment procedures. Growth, emulsification, and utilization occurred readily at both temperatures. The crude oil residue is increased in specific gravity and readily sediments out of solution. A comparison of the chemical analysis of the oils by liquid and gas-liquid chromatographic procedures before and after growth showed that the n-saturate fraction had been preferentially used. Some utilization of the aromatic fraction also occurred. Enrichments obtained with a high-quality crude oil were not as effective in utilizing a lower quality crude oil as sole carbon source as a population enriched on the low-quality crude oil.     

Microbial Degradation of Crude Oil: Factors Affecting the Dispersion in Sea Water by Mixed and Pure Cultures

By means of the enrichment culture technique, a mixed population of microorganisms was obtained which catalyzed the dispersion of crude oil in supplemented sea water. From this enrichment culture, eight pure cultures were isolated and studied. Only one of the isolates (RAG-1) brought about a significant dispersion of crude oil. RAG-1 has been tentatively characterized as a member of the genus Arthrobacter. The other seven isolates gave rise to colonies on supplemented oil agar, but were neither able to disperse oil nor to stimulate the dispersion catalyzed by RAG-1. The dispersion of crude oil by either RAG-1 or the enrichment culture was absolutely dependent on exogenous sources of nitrogen and phosphorous and completely inhibited by 10⁻²m azide. The increase in cell number of RAG-1 was directly proportional to the concentration of crude oil added to the medium over the range 0.05 to 1.0 mg/ml. Within this linear region, 1.0 mg of crude oil yielded 9 × 10⁷ cells and approximately 65% of the oil was converted into a nonbenzene extractable form. Accompanying the emulsification was a decrease in the pH from 7.6 to 5.0. Acidic conditions, however, were neither necessary nor sufficient for oil dispersion. When sea water was supplemented with 0.029 mm K₂HPO₄ and 3.8 mm (NH₄)₂SO₄ and inoculated with RAG-1, oil dispersion occurred within 1 day. This dispersion could also be brought about by the supernatant following separation of the cells from the medium. Similarly, the supernatant obtained following growth of RAG-1 on hexadecane was capable of emulsifying crude oil in 60 min.     

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.     

Involvement of Microbial Alkyl Hydroxybenzenes in the Regulation of Autolytic Degradation of Yeast Cells

A comparative study was performed of the processes of autolytic degradation of the cells of Saccharomyces cerevisiae and Schizosaccharomyces pombe under conditions simulating the phase of cell death in microbial cultures: (1) during autolysis induced by oleic acid, which is the chemical analogue of factors d₂ (autolysis autoinducer), (2) under the effect of extracellular microbial proteinases (enzymatic lysis), and (3) under the concomitant effect of the enzymes of the endogenous autolytic complex and exogenous proteinases (heterolysis). Regulatory mechanisms controlling the rate and profundity of autolysis were elucidated, relying on the stabilization of hydrolytic enzymes and enhancement of their activity in their complexes with a chemical analogue of microbial autoregulatory factors d₁, which belong to alkylhydroxybenzenes and fulfill functions of chemical chaperones. The changes in the activity of proteinases and enzymes of the autolytic complex were shown to be dependent on the concentration of the analogue at the moment of complex formation.     

油脂下脚料中残油微生物降解初步研究

以油脂降解菌Bacillus sp DE-8为出发菌,对油脂下脚料中的残油生物降解条件进行初步研究。结果表明:该菌对油脂下脚料降解条件为:起始pH值为8,接种量4%、摇床转速为150r.min-1、温度为32℃、发酵72h,该菌株对菜籽饼的降解率可达78.8%。     

海绵中石油降解微生物的分离筛选及降解特性研究

从大连湾原油污染海域生长的海绵中分离筛选到一株原油降解菌OA58,根据其生长形态、培养特征、16S rRNA序列相似性比对分析和生化指标检测,初步鉴定为脱叶链霉菌（Streptomyces exfoliatus）。同时,考察了该链霉菌对原油的降解效果,OA58能以原油为唯一碳源生长,在人工海水培养基中,14 d内对原油（初始浓度为1 g/L）的平均降解率为83%,是一株具有开发潜力的原油降解放线菌。     

Distribution of Microbial Physiologic Types in an Aquifer Contaminated by Crude Oil

Abstract We conducted a plume-scale study of the microbial ecology in the anaerobic portion of an aquifer contaminated by crude-oil compounds. The data provide insight into the patterns of ecological succession, microbial nutrient demands, and the relative importance of free-living versus attached microbial populations. The most probable number (MPN) method was used to characterize the spatial distribution of six physiologic types: aerobes, denitrifiers, iron-reducers, heterotrophic fermenters, sulfate-reducers, and methanogens. Both free-living and attached numbers were determined over a broad cross-section of the aquifer extending horizontally from the source of the plume at a nonaqueous oil body to 66 m downgradient, and vertically from above the water table to the base of the plume below the water table. Point samples from widely spaced locations were combined with three closely spaced vertical profiles to create a map of physiologic zones for a cross-section of the plume. Although some estimates suggest that less than 1% of the subsurface microbial population can be grown in laboratory cultures, the MPN results presented here provide a comprehensive qualitative picture of the microbial ecology at the plume scale. Areas in the plume that are evolving from iron-reducing to methanogenic conditions are clearly delineated and generally occupy 25–50% of the plume thickness. Lower microbial numbers below the water table compared to the unsaturated zone suggest that nutrient limitations may be important in limiting growth in the saturated zone. Finally, the data indicate that an average of 15% of the total population is suspended. Received: 28 October 1998; Accepted: 26 February 1999     

Microbial Diversity during Biodegradation of Crude Oil in Seawater from the North Sea

Microbial communities were characterized during biodegradation of immobilized oil in seawater from the Statfjord field and the German Bight in the North Sea. Seawater samples were collected at different distances from pollution sources at the two locations. A Statfjord oil was immobilized on hydrophobic synthetic Fluortex fabrics and submerged in closed flasks (no headspace) with natural or sterile seawater and incubated at 13°C for 56 days. Biodegradation of immobilized n-alkanes was measured by gas chromatography, total microbes were enumerated by epifluorescence microscopy, and culturable heterotrophic and oil-degrading microorganisms were quantified by most probable number (MPN) analysis. Polymerase chain reaction (PCR) amplification of bacterial 16S rDNA in water samples was conducted during biodegradation experiments. The amplified 16S rDNA fragments were characterized by denaturing gradient gel electrophoresis (DGGE), and by sequence analysis of cloned inserts. Biodegradation rates of alkanes in seawater collected at different distances from the pollution sources did not differ significantly (P > 0.05). Concentrations of oil-degrading microorganisms showed a temporary peak after 7 days of degradation, with a subsequent decline later in the period. DGGE analysis of 16S rRNA genes showed that community diversity decreased during the first 2–3 weeks of biodegradation, with the emergence of a few dominant bands. Cloning, restriction analysis, and sequence analysis of the 16S rDNA fragments revealed >30 different phylotypes. Abundant types during biodegradation belonged to the -Proteobacteria, in waters from both Statfjord and the German Bight. Cloning and sequencing studies indicated that the most abundant bacteria during biodegradation belonged to the family Rhodobacteraceae, with the closest relationship to the genera Sulfitobacter and Roseobacter.     

Low-Temperature Microbial Degradation of Oil Products Differing in the Extent of Condensation

Out of the 30 strains capable of oil degradation at 4–6°C, four were selected by their ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol–benzene resins) at 4–6°C. The maximum degrees of degradation of masut and ethanol–benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). These strains provide a basis for developing biodegrader preparations applicable to the bioremediation of oil-polluted sites under the conditions of a cold climate.     

原油微生物群落构成及降解菌降解特性的研究

为分离得到高效原油降解菌,直接向原油中加入营养物刺激,培养一段时间后原油乳化降解。气相色谱法、红外光谱法和紫外吸收均表明降解菌体系具有较强的降解原油烃的能力。采用细菌16S rDNA通用引物和PCR扩增等方法,构建原油降解菌体系16S rDNA克隆文库,并对所构建的文库进行分析。同时,从降解菌体系中分离得到一株降解菌,鉴定结果表明所分离的细菌为短小芽孢杆菌(Bacillus pumilus)。采用气相色谱技术对降解过程中的原油烃的变化进行分析,结果显示原油烃类组分会随着降解时间的变化而发生变化,降解菌表现出其对烷烃类物质的降解能力。     

Responses of Microbial Communities in Arctic Sea Ice After Contamination by Crude Petroleum Oil

Microbial communities associated with Arctic fjord ice polluted with petroleum oils were investigated in this study. A winter field experiment was conducted in the Van Mijen Fjord (Svalbard) from February to June 2004, in which the ice was contaminated with a North Sea paraffinic oil. Holes were drilled in the ice and oil samples frozen into the ice at the start of the experiment. Samples, including cores of both oil-contaminated and clean ice, were collected from the field site 33, 74, and 112 days after oil application. The sampled cores were separated into three sections and processed for microbiological and chemical analyses. In the oil-contaminated cores, enumerations of total prokaryotic cells by fluorescence microscopy and colony-forming units (CFU) counts of heterotrophic prokaryotes both showed stimulation of microbial growth, while concentrations of oil-degrading prokaryotes remained at similar levels in contaminated and clean ice. Analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments by denaturing gradient gel electrophoresis (DGGE) revealed that bacterial communities in oil-contaminated ice generated fewer bands than communities in clean ice, although banding patterns changed both in contaminated and clean ice during the experimental period. Microbial communities in unpolluted ice and in cores contaminated with the paraffinic oil were examined by cloning and sequence analysis. In the contaminated cores, the communities became predominated by Gammaproteobacteria related to the genera Colwellia, Marinomonas, and Glaciecola, while clean ice included more heterogeneous populations. Chemical analysis of the oil-contaminated ice cores with determinations of n-C17/Pristane and naphthalene/phenanthrene ratios indicated slow oil biodegradation in the ice, primarily in the deeper parts of the ice with low hydrocarbon concentrations.     

The Dynamic Change of Microbial Communities in Crude Oil-Contaminated Soils from Oil Fields in China

To study the biodegradability of microbial communities in crude oil contamination, crude oil-contaminated soil samples from different areas of China were collected. Using polyphasic approach, this study explored the dynamic change of the microbial communities during natural accumulation in oil field and how the constructed bioremediation systems reshape the composition of microbial communities. The abundance of oil-degrading microbes was highest when oil content was 3–8%. This oil content is potentially optimal for oil degrading bacteria proliferation. During a ～12 months natural accumulation, the quantity of oil-degrading microbes increased from 10⁵ to 10⁸ cells/g of soil. A typical sample of Liaohe (LH, oil-contaminated site near Liaohe River, Liaoning Province, China) was remediated for 50 days to investigate the dynamic change of microbial communities. The average FDA (a fluorescein diacetate approach) activities reached 0.25 abs/hr·g dry soil in the artificially enhanced repair system, 32% higher than the 0.19 abs/hr·g dry soil in natural circumstances. The abundance of oil-degrading microbes increased steadily from 0.001 to 0.068. During remediation treatment, oil content in the soil sample was reduced from 6.0% to 3.7%. GC–MS analysis indicated up to 67% utilization of C₁₀–C₂₀ normal paraffin hydrocarbons, the typical compounds that undergo microbial degradation.     

不同石油污染区微生物修复技术研究

采集辽河油田不同石油污染地区的土样 ,经富集、驯化、分离、筛选 ,得到优势石油降解菌。它们分别是假单胞菌属、黄单胞菌属、黄杆菌属、节杆菌属、动胶杆菌属 ,这些菌既有普遍性 ,同时又不乏地域特色。经系统测定其生理特性及表面活性物质对其利用多环芳烃物质的影响 ,结果表明不同石油污染区土壤的理化特性与优势细菌的生理特性相关 ;表面活性剂Tween 80可使优势细菌降解菲的速率及程度均有提高。     

原油微生物群落构成多样性及降解菌DYL-1降解原油的研究

为分离得到高效原油降解菌,直接向原油中加入营养物刺激,培养一段时间后原油乳化降解,傅里叶红外光谱显示,2957cm-1、723cm-1处吸收峰消失;2855cm-1、1377cm-1处吸收峰减弱;880cm-1以及800cm-1吸收峰几乎消失,表明原油发生降解,效果明显。同时分离得到一株降解菌,经分子鉴定为芽孢杆菌属(Bacillus sp.),红外光谱法和紫外吸收法分析表明其具有较强的降解原油烃的能力。根据传统分子生物学的方法,构建原油菌16S rDNA克隆文库,限制性酶切片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)分析了原油中的细菌多样性。结果表明此方法有效地评估了原油中的细菌群落和多样性。     

Microbial Degradation of Sterols

A process is described for the microbial degradation of cholesterol and plant sterols, to produce androsta-1, 4-diene-3, 17-dione and androst-4-ene-3, 17-dione, by two newly isolated bacteria designated Mycobacterium sp. NRRL B-3683 and Mycobacterium sp. NRRL B-3805. These myocbacteria produce substantial amounts of 17-ketonic compounds without appreciable degradation of the steroid nucleus. No ring degradation inhibitory agents are necessary. The first microbiological production of 20alpha-hydroxymethylpregna-1, 4-dien-3-one is also reported.     

Microbial Degradation of Asphalt

Organisms of the genera Pseudomonas, Chromobacterium, and Bacillus capable of degrading asphalt were isolated by enrichment cultures. The asphalt degradation by these organisms varied from 3 to 25% after incubation for 1 week. The effects of temperature, pH, and atmosphere of incubation on asphalt degradation were investigated and were shown to vary with different organisms on the same substrate.     

Microbial Degradation of Octamethylcyclotetrasiloxane

The microbial degradation of low-molecular-weight polydimethylsiloxanes was investigated through laboratory experiments. Octamethylcyclotetrasiloxane was found to be biodegraded under anaerobic conditions in composted sewage sludge, as monitored by the occurrence of the main polydimethylsiloxane degradation product, dimethylsilanediol, compared to that found in experiments with sterilized control samples.