Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.     

Biogeochemical Importance of the Bacterial Community in Uranium Waste Deposited at Key Lake,Northern Saskatchewan

The long-term stability of immobilized elements of concern in uranium tailings deposited in the Deilmann Tailings Management Facility (DTMF), northern Saskatchewan, is dependent upon maintenance of highly oxic conditions within the tailings mass. The main objective of this study was to investigate the effect of stimulating microbial activity on the redox potential and state of ferrihydrite, which are considered to be the primary controlling condition and mineral phase, respectively, within the tailings. To determine the potential for biologically mediated decreases in redox potential and ferrihydrite reduction, a series of microcosm assays were performed. Non-sterile material from the tailings–water interface of the DTMF site was inoculated with indigenous flora previously isolated from the tailings material and enriched with a carbon source (50 ppm trypticase soy broth) and incubated under continuous-flow or intermittent-flow conditions, and compared with an uninoculated, no-carbon control that received continuous flow. Highly reducing conditions with redox potentials of less than ?300 mV were detected after 2 days of incubation within the carbon-enriched tailings of microcosms receiving continuous flow, and less than ?280 mV after 11 days of incubation within carbon-enriched tailings in microcosms receiving intermittent flow. The lowest recorded Eh value (?545 mV) was recorded after 14 days in a carbon-enriched microcosm receiving intermittent flow. In contrast, the redox conditions in the control microcosm never dropped below ?93 mV; thus, it was clear that microbial activity and available carbon drove the Eh conditions to become highly reducing. The occurrence of low redox conditions was concomitant with the bulk chemical detection of Fe (II) in the effluent of treated microcosms. Sites of microbial ferrihydrite reduction were also detected using scanning transmission X-ray microscopy where Fe (II) species were observed in close proximity with bacterial cells. Analysis of the microbial diversity present within the microcosms confirmed that microbes indigenous to the DTMF system have the potential to generate conditions suitable for the proliferation of sulfate and iron reducing bacteria, such as Desulfosporosinus, which was detected by high-throughput 16S rRNA gene sequencing.     

Anaerobic Methane Oxidation and Sulfate Reduction in Bacterial Mats on Coral-Like Carbonate Structures in the Black Sea

A detailed study of the processes of anaerobic methane oxidation and sulfate reduction in the bacterial mats occurring on coral-like carbonate structures in the region of methane seeps in the Black Sea, as well as of the phenotypic diversity of sulfate-reducing bacteria developing in this zone, has been performed. The use of the radioisotopic method shows the microbial mat structure to be heterogeneous. The peak activity of the two processes was revealed when a mixture of the upper (dark) and underlying (intensely pink) layers was introduced into an incubation flask, which confirms the suggestion that methanotrophic archaea and sulfate-reducing bacteria closely interact in the process of anaerobic methane oxidation. Direct correlation between the rate of anaerobic methane oxidation and the methane and electron acceptor concentrations in the medium has been experimentally demonstrated. Several enrichment and two pure cultures of sulfate-reducing bacteria have been obtained from the near-bottom water and bacterial mats. Both strains were found to completely oxidize the substrates to CO₂ and H₂S. The bacteria grow at temperatures ranging from −1 to 18 (24)°C, with an optimum in the 10–18°C range, and require the presence of 1.5–2.5% NaCl and 0.07–0.2% MgCl 2⋅6H₂O. Regarding the aggregate of their phenotypic characteristics (cell morphology, spectrum of growth substrates, the capacity for complete oxidation), the microorganisms isolated have no analogues among the psychrophilic sulfate-reducing bacteria already described. The results obtained demonstrate the wide distribution of psychrophilic sulfate-reducing bacteria in the near-bottom water and bacterial mats covering the coral-like carbonate structures occurring in the region of methane seeps in the Black Sea, as well as the considerable catabolic potential of this physiological group of psychrophilic anaerobes in deep-sea habitats__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 420–429.Original Russian Text Copyright © 2005 by Pimenov, Ivanova.     

Nutrient Amendments of Inorganic Fertiliser and Oil Palm Empty Fruit Bunch and Their Influence on Bacterial Species Dominance and Degradation of the Associated Crude Oil Constituents

The effects of inorganic commercial fertiliser (N:P:K = 8:8:1) and oil palm empty fruit bunch (EFB) as nutrient amendments for crude oil degradation and microbial population shift by a microbial consortium [Pseudomonas sp. (UKMP-14T), Acinetobacter sp. (UKMP-12T), Trichoderma sp. (TriUKMP-1M and TriUKMP-2M)] were assessed. The bacterial populations present during crude oil degradation were analysed by spread plate method and 16S rRNA sequences, whereas the presence of fungi was assessed by growth on potato dextrose agar. Crude oil degradation analysed using gas chromatography-flame ionisation detection showed total petroleum hydrocarbon reduced between 70 and 100%, depending on the type of amendments compared to control (≈55%) after 30 days of incubation. Nutrient amendments using NPK fertiliser or EFB were found to influence the domination of different bacterial species, which in turn preferentially utilised different hydrocarbons. This study suggested different nutrient amendments could be used to preferentially select bacteria to degrade different components of crude oil, particularly pertaining to the recalcitrant phytane. This information is very useful for application of in situ bioremediation of soil hydrocarbon contamination.     

Upgrading the efficiency of dissimilatory sulfate reduction by Desulfovibrio desulfuricans via adjustment of the COD/SO4 ratio

The potential for upgrading the microbiological reduction of sulfates and for decreasing the organic pollution levels in industrial waste-water by the adjustment of the COD/SO4 ratio was investigated. The experiments involved waste-water samples coming from industrial pig farming, baker's yeast production and organic dye manufacture. The results show that in the presence of Desulfovibrio desulfuricans both the objectives can be achieved by abating the disproportion between the content of sulfates and that of organic substances.     

铁还原菌降解石油烃的研究进展

铁还原菌是指能够利用细胞外Fe(III)作为末端电子受体,通过氧化有机物将Fe(III)还原为Fe(II)微生物的总称。铁还原作用广泛存在于土壤、河流、海洋、地表含水层以及高温高压的地下深部油藏。在厌氧或兼性厌氧条件下,Fe(III)还原耦合有机物的降解,对铁、碳元素的生物地球化学循环具有重要意义。本文介绍了铁还原菌的多样性和铁还原作用机理,综述了铁还原菌在石油烃降解方面的研究进展。此外,还总结了铁还原菌在生物修复中的潜在作用,并对未来的研究方向进行了展望。     

Organic Carbon Degradation in Arctic Marine Sediments,Svalbard: A Comparison of Initial and Terminal Steps

Degradation of marine organic matter under anoxic conditions involves microbial communities working in concert to remineralize complex substrates to CO ₂ . In order to investigate the coupling between the initial and terminal steps of this sequence in permanently cold sediments, rates of extracellular enzymatic hydrolysis and sulfate reduction were measured in parallel cores collected from 5 fjords on the west and northwest coast of Svalbard, in the high Arctic. Inventories of total dissolved carbohydrates were also measured in order to evaluate their potential role in carbon turnover. Polysaccharide hydrolysis rates exhibited substrate-related and, to a lesser extent, depth-related differences (p < 0.0001); laminarin hydrolysis was consistently most rapid at nearly all depths and sites, and fucoidan hydrolysis was least rapid. Although there was a high degree of variability in parallel cores, sulfate reduction rates also exhibited statistically significant depth-and station-related differences. A comparison with data from previous investigations in Svalbard sediments suggests that this variability is linked to substrate availability rather than to organism distribution. Total dissolved carbohydrate concentrations were comparable to those measured in more temperate sediments, and likely comprise a considerable fraction of porewater dissolved organic carbon. A comparison of dissolved carbohydrate inventories with hydrolysis and sulfate reduction rates suggests that the turnover of carbon through the dissolved pool occurs quite rapidly, on the order of a few days to weeks. The transformation of particulate to dissolved organic matter must also be sufficiently rapid to maintain the measured rates of terminal remineralization.     

Anaerobic oxidation of alkanes by newly isolated denitrifying bacteria

The capacity of denitrifying bacteria for anaerobic utilization of saturated hydrocarbons (alkanes) was investigated with n-alkanes of various chain lengths and with crude oil in enrichment cultures containing nitrate as electron acceptor. Three distinct types of denitrifying bacteria were isolated in pure culture. A strain (HxN1) with oval-shaped, nonmotile cells originated from a denitrifying enrichment culture with crude oil and was isolated with n-hexane (C₆H₁₄). Another strain (OcN1) with slender, rod-shaped, motile cells was isolated from an enrichment culture with n-octane (C₈H₁₈). A third strain (HdN1) with oval, somewhat pleomorphic, partly motile cells originated from an enrichment culture with aliphatic mineral oil and was isolated with n-hexadecane (C₁₆H₃₄). Cells of hexane-utilizing strain HxN1 grew homogeneously in the growth medium and did not adhere to the alkane phase, in contrast to the two other strains. Quantification of substrate consumption and cell growth revealed the capacity for complete oxidation of alkanes under strictly anoxic conditions, with nitrate being reduced to dinitrogen. Received: 3 August / Accepted: 6 October 1999     

Anaerobic mineralization of pentachlorophenol (PCP) by combining PCP-dechlorinating and phenol-degrading cultures

The dechlorination and mineralization of pentachlorophenol (PCP) was investigated by simultaneously or sequentially combining two different anaerobic microbial populations, a PCP-dechlorinating culture capable of the reductive dechlorination of PCP to phenol and phenol- degrading cultures able to mineralize phenol under sulfate- or iron-reducing conditions. In the simultaneously combined mixture, PCP (about 35 microM) was mostly dechlorinated to phenol after incubation for 17 days under sulfate-reducing conditions or for 22 days under iron-reducing conditions. Thereafter, the complete removal of phenol occurred within 40 days under both conditions. In the sequentially combined mixture, most of the phenol, the end product of PCP dechlorination, was degraded within 12 days of inoculation with the phenol degrader, without a lag phase, under both sulfate- and iron-reducing conditions. In a radioactivity experiment, [14C-U]-PCP was mineralized to 14CO2 and 14CH4 by the combined anaerobic microbial activities. Analysis of electron donor and acceptor utilization and of the production and consumption of H2, CO2, and CH4 suggested that the dechlorinating and degrading microorganisms compete with other microorganisms to perform PCP dechlorination and part of the phenol degradation in complex anoxic environments in the presence of electron donors and acceptors. The presence of a small amount of autoclaved soil slurry in the medium was possibly another advantageous factor in the successful dechlorination and mineralization of PCP by the combined mixtures. This anaerobic-anaerobic combination technology holds great promise as a cost-effective strategy for complete PCP bioremediation in situ.     

Formation of Siderite in Microbial Microcosms Derived from a Marine Sediment

Abstract

Exceptionally well-preserved fossils are frequently encased by carbonate concretions. The initial steps of their formation in marine and freshwater sediments are induced by microbial activity. The role of the involved microbial communities, however, is not well understood. In this study, siderite (FeCO₃) formation in microbial microcosms is observed, with various fatty acyl compounds (lipids, surfactants) as substrates and Wadden Sea sediment samples as inocula. In actively growing microcosms, sulfate-reducing bacteria (the genus Desulfofrigus in particular) dominate the microbial community and submicroscopic siderite precipitates on bacterial cell surfaces were identified. We suggest that these biologically induced mineralization processes may, in the natural environment, initiate the formation of large concretions under suboxic conditions in coastal sediments.     

球形红细菌厌氧降解邻二氯苯及其机理研究

研究分析光合细菌球形红细菌在厌氧光照条件下降解邻二氯苯的条件和机理。结果表明, 在厌氧光照条件下球形红细菌的最佳生长和对邻二氯苯的最佳降解条件为：pH 7.0, 温度为30℃, 接种量10％。在最佳条件下, 邻二氯苯的去除率可达90％以上; 其降解中间产物主要有氯苯、4-羟基苯甲酸; 根据降解产物的分析, 推断球形红细菌降解邻二氯苯的机理主要是按照先脱掉一个氯原子生成氯苯, 然后氯苯进一步脱氯并通过4-羟基苯甲酸的代谢途径开环进行。     

Biodegradation of Petroleum Tar by Pseudomonas Spp. From Oil Field of Assam,India

ABSTRACT

Petroleum tar produced during the processing of crude oil is one of the earth's major pollutants. The potential of certain soil bacteria in the biodegradation of petroleum tar was assessed to develop an active indigenous bacterial consortium for bioremediation of petroleum tar–polluted sites of Assam, India. In vitro enrichment cultures of five Pseudomonas spp. were found to metabolize petroleum tar. The Fourier transform infrared (FTIR) analyses of the enrichment cultures revealed the presence of the functional groups, viz., –OH, –CHO, C?O, and –COOH, which provided evidence for the biodegradation of petroleum tar. Further, gas chromatography–flame ionization detection (GC-FID) analyses revealed complete degradation of low-molecular-weight hydrocarbons, and the subsequent appearance of some additional peaks reflected the formation of intermediate metabolites during the degradation of petroleum tar. A mixed culture with 0.1% Tween 80 as a surfactant exhibited almost complete degradation in contrast to the degradation by the mixed culture without Tween 80. This confirmed the effect of a surfactant for acceleration of the biodegradation process of petroleum tar.     

球形红细菌厌氧降解邻二氯苯及其机理研究

研究分析光合细菌球形红细菌在厌氧光照条件下降解邻二氯苯的条件和机理.结果表明,在厌氧光照条件下球形红细菌的最佳生长和对邻二氯苯的最佳降解条件为:pH 7.0,温度为30℃,接种量10%.在最佳条件下,邻二氯苯的去除率可达90%以上;其降解中间产物主要有氯苯、4-羟基苯甲酸;根据降解产物的分析,推断球形红细菌降解邻二氯苯的机理主要是按照先脱掉一个氯原子生成氯苯,然后氯苯进一步脱氯并通过4-羟基苯甲酸的代谢途径开环进行.     

Degradation of Linseed Oil Vapors by Soil Bacteria in Trickling Biofilters

Oxidation products of linseed oil were produced by impinging a stream of air onto the surface of pure linseed oil and injecting the vapor-laden air into soil percolation columns to enrich the population of bacteria capable of degrading linseed oil vapors. As the populations of bacteria increased, the linseed oil vapors were consumed by these organisms, and the air that emerged from the columns was free of linseed oil contaminants. Five different kinds of bacteria capable of growing on the linseed oil oxidation products as sole source of carbon and energy were found and isolated in pure culture. Chromatographic analyses showed that individual organisms removed specific components of the vapor at specific rates, but none was able to remove them all within a 30-day period of time. When the five were grown together and presented the linseed oil vapor, all vapor constituents were utilized, and the rate of utilization was greater than that seen when the isolates were tested in pure culture. This indicated that the five organisms operated as a bacterial consortium in the degradation of linseed oil vapors. Trickling biofilters prepared from pregrown populations of the five organisms challenged with linseed oil vapors were able to remove all volatile constituents found in linseed oil vapor. Bioremediation of the air was complete and it was accomplished in a single pass of the air through the filter.

This work shows that bacteria found in the soil are capable of degrading linseed oil vapors and that they can be grown in the laboratory and used successfully in bench scale trickling biofilters.     

Regulation of geochemical activity of microorganisms in a petroleum reservoir by injection of H<Subscript>2</Subscript>O<Subscript>2</Subscript> or water-air mixture

In the course of pilot trials of biotechnologies for the enhancement of oil recovery in formation waters of the Gangxi bed of the Dagang oil field (China), microbiological processes were investigated. The biotechnologies are based on injection into the petroleum reservoir of different oxygen sources (H₂O₂ solution or a water-air mixture) with nitrogen and phosphorus salts. The injection of water-air mixture with nitrogen and phosphorus salts resulted in an increase in the number of aerobic and anaerobic organotrophic bacteria, rates of sulfate reduction and methanogenesis in formation water and also the content of CO₂ (from 4.8–12 to 15–23.2%) and methane (from 86–88 to 91.8%) in the gas. The preferential consumption of isotopically light bicarbonate by methanogens resulted in a higher content of the light ¹²C in methane; the δ¹³C/CH₄ value changed from ?45.1…?48.3 to ?50.7…?59.3‰. At the same time, mineral carbonates of the formation water became isotopically heavier; the δ¹³C/Σcarbonates value increased from 3.4…4.0 to 5.4…9.6‰. Growth of hydrocarbon-oxidizing bacteria was accompanied by production of biosurfactants and decreased interfacial tension of formation water. Injection of H₂O₂ solution resulted in the activation of aerobic processes and in suppression of both sulfate reduction and methanogenesis. Methane content in the gas decreased from 86–88 to 75.7–79.8%, probably due to its consumption by methanotrophs. Due to consumption of isotopically light methane, the residual methane carbon became heavier, with the δ¹³C/CH₄ values from ?39.0 to ?44.3‰. At the same time, mineral carbonates of the formation water became isotopically considerably lighter; the δ¹³C/Σcarbonates value decreased from 5.4…9.6 to ?1.4…2.7‰. The additional amount of oil recovered during the trial of both variants of biotechnological treatment was 3819 t.     

Analysis of bacterial diversity in acidic pond water and compost after treatment of artificial acid mine drainage for metal removal

The microbial population of a sludge amended leaf compost material utilized for treatment of artificial acid mine drainage was studied by culture-independent molecular methods. Iron-rich and sulfurous wastewater (artificial acid mine drainage) was circulated through a column bioreactor for 16 months. After 12 months the column was inoculated with a mixed culture from an acidic pond receiving acid mine drainage from a tailings impoundment at a decommissioned site in Kristineberg, North Sweden. Hydrogen sulfide odor and the formation of black precipitates indicated that sulfate-reduction occurred in the column. 16S rDNA gene analysis by denaturing gradient gel electrophoresis, cloning, and sequencing as well as fluorescent in situ hybridization confirmed the presence of microorganisms closely related to sulfate-reducing bacteria and microorganisms from the genera Pseudoxanthmonas, Dechlorosoma, Desulfovibrio, Agrobacterium, Methylocapsa, Rhodococcus, Sulfobacillus, and some unidentified bacteria. Sulfate-reducing bacteria were found in the column bioreactor 2 weeks after inoculation, but not thereafter. This suggests they were in low abundance, even though sulfate remediation rates were significant. Instead, the population contained species similar to those previously found to utilize humic substances released from the compost material.     

Evidence of the activity of dissimilatory sulfate-reducing prokaryotes in nonsulfidogenic tropical mobile muds

In spite of the nonsulfidic conditions and abundant reactive iron(III) commonly found in mobile tropical deltaic muds, genes encoding dissimilatory sulfite reductase (dsr) were successfully amplified from the upper approximately 1 m of coastal deposits sampled along French Guiana and in the Gulf of Papua. The dsr sequences retrieved were highly diverse, were generally represented in both study regions and fell into six large phylogenetic groupings: Deltaproteobacteria, Thermodesulfovibrio groups, Firmicutes and three groups without known cultured representatives. The spatial and temporal distribution of dsr sequences strongly supports the contention that the sulfate-reducing prokaryote communities in mobile mud environments are cosmopolitan and stable over a period of years. The decrease in the (35)SO(4) (2-) tracer demonstrates that, despite abundant reactive sedimentary iron(III) ( approximately 350-400 mumol g(-1)), the sulfate-reducing prokaryotes present are active, with the highest levels of sulfide being generated in the upper zones of the cores (0-30 cm). Both the time course of the (35)S-sulfide tracer activity and the lack of reduced sulfur in sediments demonstrate virtually complete anaerobic loss of solid phase sulfides. We propose a pathway of organic matter oxidation involving at least 5-25% of the remineralized carbon, wherein sulfide produced by sulfate-reducing prokaryotes is cyclically oxidized biotically or abiotically by metal oxides.     

Effect on the Medium Salinity on Oil Degradation by Nocardioform Bacteria

Oil degradation by cultures of Rhodococcus erythropolisand Dietzia mariswas found to depend on the NaCl concentration in the medium. Optimal utilization of turbine oil by R. erythropolisand D. mariswas observed at 0.5 and 2 to 5% NaCl concentration, respectively. Mineral oil and a mixture of paraffins (C₁₄–C₁₈) were utilized within a broader range of the medium salinity. As shown by fluorescent microscopy, D. mariscolonies formed on the oil drop surface, whereas R. erythropoliscells penetrated the drops. The strains studied may populate various ecological niches in oil-containing ecosystems. They are promising for the development of microbial preparations for cleaning the environment from oil pollution.     

Temperature-dependent differences in community structure of bacteria involved in degradation of petroleum hydrocarbons under sulfate-reducing conditions

Aim: The aim of this study was to characterize the microbial community involved in anaerobic degradation of petroleum hydrocarbon under low‐ and moderate‐temperature conditions. Methods and Results: Sulfate‐reducing enrichment cultures growing on crude oil and p‐xylene were established at low and moderate temperatures. Bacterial community structures of the cultures were characterized by 16S rRNA gene‐based analysis and organisms responsible for degradation of p‐xylene were investigated by analysis of the bamA gene, involved in anaerobic degradation of aromatic compounds. The PCR‐denaturing gradient gel electrophoresis analysis indicated significant differences in microbial community structures among the cultures, depending on the temperatures of incubation. Difference depending on the temperatures was also observed in the cloning analysis of the bamA gene performed on the p‐xylene‐degrading enrichment cultures. Majority of clones detected in the culture of moderate temperature were related to Desulfosarcina ovata, whereas more diverse bamA gene sequences were obtained from the culture incubated at low temperature. Conclusions: Temperature‐dependent differences in microbial community were demonstrated by the analyses of two genes. It was suggested that sulfate‐reducing bacteria of phylogenetically different groups might be involved in the degradation of petroleum hydrocarbons in different temperature environments. Significance and Impact of the Study: This study is the first report of p‐xylene‐degrading sulfate‐reducing enrichment culture at low temperature. The results of the experiments at low temperature were distinctly different from those reported in previous studies performed at moderate temperatures.     

印度洋表层海水石油降解菌的多样性分析

【目的】为了研究印度洋石油降解菌多样性,并获得新的石油降解菌。【方法】本研究通过印度洋表层海水样品采集、以柴油与原油1:1混合物作为碳源,从中富集、分离筛选石油降解菌,并通过PCR-DGGE对13个站点富集菌群的菌群结构进行分析。【结果】通过形态观察、生理生化反应和16SrRNA分析,共得到共29个属的51株不同的细菌,它们主要是属于α亚群和γ亚群。其中,Alcanivorax属(占18%),Novosphingobium属(占10%)和Marinobacter(占6%)Thalassospira(占6%)为主要的优势菌。通过生态多样性分析表明,Shannon-Winner指数(H)为4.57968,说明其具有较高的多样性;均匀度指数(E)为0.8664771,表示其分布比较均匀。单菌实验表明,49株具有石油降解能力其中,Sinomonas,Knoellia,Mesoflavibacter等属的细菌为首次发现有降解能力。DGGE分析表明Alcanivorax属的细菌是印度洋表层海水中的重要石油降解菌。【结论】本研究首次揭示了印度洋表层海水中石油降解菌的多样性,并获取了若干在海洋石油污染生物修复中具有应用前景的降解菌。