Use of bacteria-immobilized cotton fibers to absorb and degrade crude oil

Using enrichment culture technique, two isolates that brought a significant degradation and dispersion of crude oil were obtained from contaminated sediments of the Bohai Bay, China. 16S rRNA gene sequencing and phylogenetic analysis indicated that the two bacterial strains affiliated with the genera Vibrio and Acinetobacter. Subsequently, the bacterial cells were immobilized on the surface of cotton fibers. Cotton fibers were used as crude oil sorbent as well as a biocarrier for bacteria immobilization. Among the two isolates, the marine bacteria Acinetobacter sp. HC8-3S showed a strong binding to the cotton fibers, possibly enhanced through extracellular dispersant excreted by Acinetobacter sp. HC8-3S. Both planktonic and immobilized bacteria showed relatively high biodegradation (>60%) of saturated hydrocarbons fraction of crude oil, in the pH range of 5.6–8.6. The degradation activities of planktonic and immobilized bacteria were not affected significantly when the NaCl concentration reached 70 g/L. The immobilized bacterial cells exhibited an enhanced biodegradation of crude oil. The efficiency of saturated hydrocarbons degradation by the immobilized bacterial cells increased about 30% compared to the planktonic bacterial cells.     

Self-immobilized bacterial cultures with potential for application as ready-to-use seeds for petroleum bioremediation

Two hydrocarbon-degrading bacterial isolates, an Arthrobacter sp. and a Gram-negative bacillus isolated from Kuwait oil lakes, exhibited considerable cell-surface hydrophobicity without production of exopolysaccharides in complex media. However, the bacteria produced copious amounts of exopolysaccharides in a low nutrient medium. When incubated with sawdust, Styrofoam or wheat bran, as carriers, under low nutrient conditions, stable exopolysaccharide-mediated immobilized cultures were formed. Such immobilized cultures when air-dried at room temperature survived storage for 6 weeks at 45 °C and still retained the ability to degrade hydrocarbons. Viability was retained by the immobilized Arthrobacter sp. and the Gram-negative bacterium at 45 °C storage for up to 6 and 12 months, respectively.     

Mechanisms for enhanced biodegradation of petroleum hydrocarbons by a microbe-colonized gas-liquid foam

AIMS: A microbe-colonized gas-liquid foam formulation has been previously shown to provide enhanced biodegradation capabilities in soil microcosms. The present study considers the reservoir properties of this foam and how this affects hydrocarbon degradation rates. METHODS AND RESULTS: Oxygen solubility in protein hydrolysate solutions draining from aerated and oxygenated foams was measured. The suitability of oxygenated foam to enhance the degradation of n-hexadecane in soil microcosms was assessed. Sorption of bacterial isolates at the gas-liquid interface was also investigated using a range of microscopy techniques. CONCLUSIONS: Oxygenated bioactive foam enhanced biodegradation rates by improving oxygen availability and transfer. Biodegradation of n-hexadecane was also stimulated by the protein hydrolysate used and by the inclusion of known bacterial hydrocarbon-degrading bacteria. The interaction of bacteria with the gas-liquid interface was shown to be a significant factor governing the drainage of the bacteria from the bioactive foam. SIGNIFICANCE AND IMPACT OF THE STUDY: Protein hydrolysate-based bioactive foam may be a suitable treatment technology to enhance the biodegradation of petroleum hydrocarbons in soil.     

Persistence and Biodegradation of Spilled Residual Fuel on an Estuarine Beach

The enrichment of hydrocarbon-degrading bacteria and the persistence of petroleum hydrocarbons on an estuarine beach after a spill of residual fuel oil on 11 April 1973 in Upper Narragansett Bay, R.I. was investigated. A rapid enrichment occurred during days 4 to 16 after the oil spill and a significant population of hydrocarbon-degrading bacteria was maintained in the beach sand for at least a year. The concentration of petroleum hydrocarbons in the mid-tide area declined rapidly during the bacterial enrichment period, remained fairly constant throughout the summer, and then declined to a low concentration after 1 year. An increased concentration of branched and cyclic aliphatic hydrocarbons in the low-tide sediment 128 days after the spill suggested a migration of hydrocarbons during the summer. Hydrocarbon biodegradation was apparent during the winter months at a rate of less than 1 mug of hydrocarbon per g of dry sediment per day.     

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes

In this laboratory-scale study, we examined the potential of chitin and chitosan flakes obtained from shrimp wastes as carrier material for a hydrocarbon-degrading bacterial strain. Flakes decontamination, immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated. The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms. In terms of removal percentage of crude oil after 15 days, the microcosms treated with the immobilized inoculants proved to be the most successful. The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain. It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.     

Bioassay-based screening of microorganisms that degrade dioxin using substrate-immobilized microtubes

In the current study, we attempted to develop a method for bioassay-based screening of microorganisms that degrade dioxin. However, a crucial problem encountered was that the standard dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to bacterial medium immediately disappeared from the liquid phase due to its adsorption onto polypropylene (PP) tubes. Among other aromatic hydrocarbons, adsorption onto PP tubes was also observed in beta-naphthoflavone but not in benzo[a]pyrene. Adsorption of TCDD was observed not only onto PP tubes but also onto polystyrene, glass, and PP tubes with low affinity for DNA or protein. Silanization was not effective at preventing adsorption of TCDD. TCDD immobilized onto PP tubes was recovered by organic solvents, including ethanol, methanol, and dimethyl sulfoxide (DMSO). The elution efficiency of the immobilized TCDD by DMSO was approximately 85%. Based on these findings, screening of bacteria that degrade dioxin was attempted as follows. First, TCDD was immobilized onto PP tubes. Second, bacterial suspension was added to the tubes and incubated for biodegradation of TCDD. Third, remaining, immobilized TCDD was eluted by DMSO and subjected to a reporter bioassay to evaluate the level of TCDD. Using this method, we demonstrated successful screening of bacteria that have the potential for degradation of dioxin.     

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.     

Biodegradation of crude oil across a wide range of salinities by an extremely halotolerant bacterial consortium MPD-M,immobilized onto polypropylene fibers

The bacterial consortium MPD-M, isolated from sediment associated with Colombian mangrove roots, was effective in the treatment of hydrocarbons in water with salinities varying from 0 to 180 g L(-1). Where the salinity of the culture medium surpassed 20 g L(-1), its effectiveness increased when the cells were immobilized on polypropylene fibers. Over the range of salinity evaluated, the immobilized cells significantly enhanced the biodegradation rate of crude oil compared with free-living cells, especially with increasing salinity in the culture medium. Contrary to that observed in free cell systems, the bacterial consortium MPD-M was highly stable in immobilized systems and it was not greatly affected by increments in salinity. Biodegradation was evident even at the highest salinity evaluated (180 g L(-1)), where biodegradation was between 4 and 7 times higher with immobilized cells compared to free cells. The biodegradation of pristane (PR) and phytane (PH) and of the aromatic fraction was also increased using cells immobilized on polypropylene fibers.     

Effects of nutrient and temperature on degradation of petroleum hydrocarbons in sub-Antarctic coastal seawater

In an attempt to evaluate the potential of petroleum bioremediation at high latitudes environments, microcosm studies using Antarctic coastal seawater contaminated with diesel or crude oil were conducted in Kerguelen Archipelago (49°22′S, 70°12′E). Microcosms were incubated at three different temperatures (4, 10 and 20°C). During experiments, changes observed in microbial assemblages (total direct count, heterotrophic cultivable microorganisms and hydrocarbon-degrading microorganisms) were generally similar for all incubation temperatures, but chemical data showed only some slight changes in biodegradation indices [Σ(C12–C20)/Σ(C21–C32) and C17/pristane]. The complete data set provided strong evidence of the presence of indigenous hydrocarbon-degrading bacteria in Antarctic seawater and their high potential for hydrocarbon bioremediation. The rate of oil degradation could be increased by the addition of a commercial fertilizer, but water temperature had little effects on biodegradation efficiency which is in conflict with the typical temperature-related assumption predicting 50% rate reduction when temperature is reduced by 10°C. Global warming of Antarctic seawater should not increase significantly the rate of oil biodegradation in these remote regions.     

Enhancing the biodegradation of total petroleum hydrocarbons in oily sludge by a modified bioaugmentation strategy

The effect of successive inoculation with hydrocarbon-degrading bacteria on the dynamics of petroleum hydrocarbons degradation in soil was investigated in this study. Oily sludge was used as a source of mixed hydrocarbons pollutant. Two bacterial consortia composed of alkanes and polycyclic aromatic hydrocarbon degraders were constructed from bacteria isolated from soil and oily sludge. These consortia were applied to incubated microcosms either in one dose at the onset of the incubation or in two doses at the beginning and at day 62 of the incubation period, which lasted for 198 days. During this period, carbon mineralization was evaluated by respirometry while total petroleum hydrocarbons and its fractions were gravimetrically evaluated by extraction from soil and fractionation. Dosing the bacterial consortia resulted in more than 30% increase in the overall removal of total petroleum hydrocarbons from soil. While alkane removal was only slightly improved, aromatic and asphaltic hydrocarbon fraction removal was significantly enhanced by the addition of the second consortium. Polar compounds (resins) were enriched only as a result of aromatics and asphaltene utilization. Nonetheless, their concentration declined back to the original level by the end of the incubation period.     

Biodegradation of medium chain hydrocarbons by Acinetobacter venetianus 2AW immobilized to hair-based adsorbent mats

The natural attenuation of hydrocarbons can be hindered by their rapid dispersion in the environment and limited contact with bacteria capable of oxidizing hydrocarbons. A functionalized composite material is described herein, that combines in situ immobilized alkane‐degrading bacteria with an adsorbent material that collects hydrocarbon substrates, and facilitates biodegradation by the immobilized bacterial population. Acinetobacter venetianus 2AW was isolated for its ability to utilize hydrophobic n‐alkanes (C10–C18) as the sole carbon and energy source. Growth of strain 2AW also resulted in the production of a biosurfactant that aided in the dispersion of complex mixtures of hydrophobic compounds. Effective immobilization of strain 2AW to the surface of Ottimat? adsorbent hair mats via vapor phase deposition of silica provided a stable and reproducible biocatalyst population that facilitates in situ biodegradation of n‐alkanes. Silica‐immobilized strain 2AW demonstrated ca. 85% removal of 1% (v/v) tetradecane and hexadecane within 24 h, under continuous flow conditions. The methodology for immobilizing whole bacterial cells at the surface of an adsorbent, for in situ degradation of hydrocarbons, has practical application in the bioremediation of oil in water emulsions. Published 2011 American Institute of Chemical Engineers Biotechnol Prog., 2011     

Dynamics of the hydrocarbon-degrading Cycloclasticus bacteria during mesocosm-simulated oil spills

We used catalysed reported deposition - fluorescence in situ hybridization (CARD-FISH) to analyse changes in the abundance of the bacterial groups Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes, and of hydrocarbon-degrading Cycloclasticus bacteria in mesocosms that had received polycyclic aromatic hydrocarbons (PAHs) additions. The effects of PAHs were assessed under four contrasting hydrographic conditions in the coastal upwelling system of the Rías Baixas: winter mixing, spring bloom, summer stratification and autumn upwelling. We used realistic additions of water soluble PAHs (approximately 20-30 microg l(-1) equivalent of chrysene), but during the winter period we also investigated the effect of higher PAHs concentrations (10-80 microg l(-1) chrysene) on the bacterial community using microcosms. The most significant change observed was a significant reduction (68 +/- 5%) in the relative abundance of Alphaproteobacteria. The magnitude of the response of Cycloclasticus bacteria (positive with probe CYPU829) to PAHs additions varied depending on the initial environmental conditions, and on the initial concentration of added PAHs. Our results clearly show that bacteria of the Cycloclasticus group play a major role in low molecular weight PAHs biodegradation in this planktonic ecosystem. Their response was stronger in colder waters, when their background abundance was also higher. During the warm periods, the response of Cycloclasticus was limited, possibly due to both, a lower bioavailability of PAHs caused by abiotic factors (solar radiation, temperature), and by inorganic nutrient limitation of bacterial growth.     

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...     

Bacterial Degradation and Corrosion of Naphtha in Transporting Pipeline

Five naphtha hydrocarbon-degrading bacteria including representative strains of the two classified species (Serratia marcescensAR1, Bacillus pumilusAR2, Bacillus carboniphilus AR3, Bacillus megaterium AR4, and Bacillus cereus AR5) were identified by 16S rDNA gene sequence in a naphtha-transporting pipeline. The naphtha-degrading strains were able to be involved in the corrosion process of API 5LX steel and also utilized the naphtha as the sole carbon source. The biodegradation of naphtha by the bacterial isolates was characterized by gas chromatography-mass spectrometry. Weight-loss measurement on the corrosion of API 5LX steel in the presence/absence of consortia grown in naphtha-water aqueous media was performed. The scanning electron microscope observation showed that the consortia were able to attack the steel API 5LX surface, creating localized corrosion (pit). The biodegradation of naphtha by the strains AR1, AR2, AR3, AR4, and AR5 showed biodegradation efficiency of about 76.21, 67.20, 68.78, 68.78, and 68.15, respectively. The role of degradation on corrosion has been discussed. This basic study will be useful for the development of new approaches for the detection, monitoring, and control of microbial corrosion in a petroleum product pipeline.     

不同富集方法分离多环芳烃降解菌的比较研究

多环芳烃是一类普遍存在的环境污染物。本研究探讨了普通富集法,固定化富集法以及巴斯德消毒后富集法三种途径从相同红树林土壤中分离菲降解茵的差异。通过平板培养和变性梯度凝胶电泳两种方法分析分离结果。上述方法分别获得以鞘氨醇单胞茵、分枝杆菌以及红球茵为优势菌群的群落,表明分离方法对多环芳烃降解菌多样性的研究是一种重要的影响因素。     

Alkane Biodegradation Genes from Chronically Polluted Subantarctic Coastal Sediments and Their Shifts in Response to Oil Exposure

Although sediments are the natural hydrocarbon sink in the marine environment, the ecology of hydrocarbon-degrading bacteria in sediments is poorly understood, especially in cold regions. We studied the diversity of alkane-degrading bacterial populations and their response to oil exposure in sediments of a chronically polluted Subantarctic coastal environment, by analyzing alkane monooxygenase (alkB) gene libraries. Sequences from the sediment clone libraries were affiliated with genes described in Proteobacteria and Actinobacteria, with 67?% amino acid identity in average to sequences from isolated microorganisms. The majority of the sequences were most closely related to uncultured microorganisms from cold marine sediments or soils from high latitude regions, highlighting the role of temperature in the structuring of this bacterial guild. The distribution of alkB sequences among samples of different sites and years, and selection after experimental oil exposure allowed us to identify ecologically relevant alkB genes in Subantarctic sediments, which could be used as biomarkers for alkane biodegradation in this environment. 16?S rRNA amplicon pyrosequencing indicated the abundance of several genera for which no alkB genes have yet been described (Oleispira, Thalassospira) or that have not been previously associated with oil biodegradation (Spongiibacter-formerly Melitea-, Maribius, Robiginitomaculum, Bizionia and Gillisia). These genera constitute candidates for future work involving identification of hydrocarbon biodegradation pathway genes.     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

阿特拉津降解菌株的分离、鉴定和工业废水生物处理试验

用液体无机盐培养基富集培养法和无机盐平板直接分离法, 从生产阿特拉津的农药厂的废水和污泥混合物中分离到13个能以阿特拉津为唯一氮源生长的细菌菌株。通过16S rRNA基因序列分析, 11个菌株被鉴定为Arthrobacter spp., 2个菌株被鉴定为Pseudomonas spp.。对阿特拉津降解活力最高的Arthrobacter sp. AD30和Pseudomonas sp. AD39的降解基因组成和降解特性进行了详细研究。降解基因的PCR扩增表明, AD30和AD39都含有trzN-atzBC基因, 能将有毒的阿特拉津降解成无毒的氰尿酸。降解实验表明, 向阿特拉津浓度为200 mg/L的无机盐培养基中分别接种等量的AD30、AD39和这两个菌株的混合菌液, 30°C振荡培养48 h以后, 阿特拉津去除率分别为92.5%、97.9%和99.6%, 表明混合菌的降解效果好于单菌。用AD30和AD39的混合菌液接种阿特拉津浓度为176 mg/L的工业废水, 30°C振荡培养72 h以后, 99.1%的阿特拉津被去除, 表明混合菌株在阿特拉津工业废水的生物处理中有很好的应用潜力。     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.