Methanogenic Microbial Community Composition of Oily Sludge and Its Enrichment Amended with Alkanes Incubated for Over 500 Days

Methanogenic microbial community is responsive to the availability of hydrocarbons and such information is critical for the assessment of hydrocarbon degradation in remediation and also in biologically enhanced recovery of energy from non-producing oil reserves. In this study, methanogenic enrichment cultures from oily sludge amended with n-alkanes (C₁₅-C₂₀) showed a development of active methanogenic alkanes-degrading consortium for over a total of 1000 days of incubation at 37°C. Total genomic DNAs were extracted from three types of samples, the original oily sludge (OS), the sludge after incubation for 500 days under methanogenic condition without any external carbon addition (EC), and the enrichment culture from the EC amended with n-alkanes (ET) incubated for another 500 days. The phylogenetic diversities of microbial communities of the three samples were analyzed by PCR amplification of partial 16S rRNA genes. The catabolic genes encoding benzylsuccinate synthase (bssA) and alkylsuccinate synthase (assA) were also examined by PCR amplification. These results provide important evidence in that microbial populations in an oily sludge shifted from methanogenic aromatic compounds degrading communities to potential methanogenic alkane-degrading communities when the enrichment was supplemented with n-alkanes and incubated under anaerobic conditions.     

Methanogenesis,sulfate reduction and crude oil biodegradation in hot Alaskan oilfields

Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6–807 µmol S l^?1 day^?1), but of lesser consequence in Field B (0.1–10 µmol S l^?1 day^?1). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular‐based microbial community analysis of the methanogenic oil‐utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H₂‐using family Methanobacteriaceae. The findings demonstrate that oilfield‐associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.     

Biogenic methane production in formation waters from a large gas field in the North Sea

Methanogenesis was investigated in formation waters from a North Sea oil rimmed gas accumulation containing biodegraded oil, which has not been subject to seawater injection. Activity and growth of hydrogenotrophic methanogens was measured but acetoclastic methanogenesis was not detected. Hydrogenotrophic methanogens showed activity between 40 and 80°C with a temperature optimum (ca. 70°C) consistent with in situ reservoir temperatures. They were also active over a broad salinity range, up to and consistent with the high salinity of the waters (90 g l⁻¹). These findings suggest the methanogens are indigenous to the reservoir. The conversion of H₂ and CO₂ to CH₄ in methanogenic enrichments was enhanced by the addition of inorganic nutrients and was correlated with cell growth. Addition of yeast extract also stimulated methanogenesis. Archaeal 16S rRNA gene sequences recovered from enrichment cultures were closely related to Methanothermobacter spp. which have been identified in other high-temperature petroleum reservoirs. It has recently been suggested that methanogenic oil degradation may be a major factor in the development of the world’s heavy oils and represent a significant and ongoing process in conventional deposits. Although an oil-degrading methanogenic consortium was not enriched from these samples the presence and activity of communities of fermentative bacteria and methanogenic archaea was demonstrated. Stimulation of methanogenesis by addition of nutrients suggests that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from such petroleum systems.     

Coupling hydrocarbon degradation to anaerobic respiration and mineral diagenesis: theoretical constraints

The diagenetic mineral assemblages in petroleum reservoirs control the formation fluid pH and pCO₂. Anaerobic biodegradation of petroleum is controlled by the transfer of electrons from reduced organic species to inorganic, redox sensitive, aqueous and mineral species in many cases through intermediates such as H₂ and CH₃COO^?. The terminal electron accepting reactions induce the dissolution or precipitation of the same minerals that control the ambient pH and pCO₂ in petroleum reservoirs. In this study, we develop a model for anaerobic biodegradation of petroleum that couples the production of acetate and H₂ to ‘late stage’ diagenetic reactions. The model reveals that the principal terminal electron accepting process and electron donor control the type of diagenetic reaction, and that the petroleum biodegradation rate is controlled through thermodynamic restriction by the minimum ΔG required to support a specific microbial metabolism, the fluid flux and the mineral assemblage. These relationships are illustrated by modeling coupled microbial diagenesis and biodegradation of the Gullfaks oil reservoir. The results indicate that the complete dissolution of albite by acids generated during oil biodegradation and the corresponding elevated pCO₂ seen in the Gullfaks field are best explained by methanogenic respiration coupled to hydrocarbon degradation and that the biodegradation rate is likely controlled by the pCH₄. Biodegradation of Gullfaks oil by a consortium that includes either Fe³⁺‐reducing or ‐reducing bacteria cannot explain the observed diagenetic mineral assemblage or pCO₂. For octane, biodegradation, not water washing, was the principal agent for removal at fluid velocities <20 m Myr^?1.     

Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludge with quinones as terminal electron acceptors

A quinone-respiring, enrichment culture derived from methanogenic granular sludge was phylogenetically characterized by using a combined cloning-denaturing gradient gel electrophoresis (DGGE) method, which revealed that the consortium developed was dominated by a single microorganism: 97% related, in a sequence of 1520 base pairs, to Geobacter sulfurreducens. The enrichment culture could grow with acetate, formate or H₂ when humic acids, the humic model compound, anthraquinone-2,6-disulfonate (AQDS), or chelated Fe(III) was provided as a terminal electron acceptor. The occurrence of a humic acid- or quinone-respiring microorganism in the microbial community of a wastewater treatment system suggests that this type of microorganisms may play a potential role in anaerobic bioreactors treating humus-containing wastewaters.     

Characterizing Intrinsic Bioremediation in a Petroleum Hydrocarbon-Contaminated Aquifer by Combined Chemical,Isotopic, and Biological Analyses

Chemical, isotopic, and biological parameters were evaluated over a 1-year period to characterize microbial processes associated with intrinsic bioremediation in a petroleum hydrocarbon-contaminated aquifer located in Studen, Switzerland. Chemical parameters measured included oxidants such as O₂, NO₃ ^?, and SO₄ ^2?, reduced species such as Fe²⁺ and CH₄, and dissolved inorganic carbon (DIC). Stable carbon isotope analyses of DIC were used to differentiate between different processes that contribute to DIC production. Microbial populations were identified by sequence analysis of archaeal 16S rDNA and in situ hybridization using a general DNA binding dye (DAPI) and specific probes targeting the domain Archaea (Arch915) and Bacteria (Eub338), as well as the species Methanosaeta concilii (Rotcl1) and Methanospirillum sp. (Rotcl2). Groundwater exhibited reduced conditions and elevated concentrations of DIC within the contaminated zone. Spatially distinct values of δ¹³C ranging from ?16.5l%c to ?4.44%o were found, indicating the presence of different ongoing microbial processes. Detected microbial populations (% of DAPI-stained cells) within the contaminated zone belonged to Archaea (9±2% to 31±13%) and Bacteria (13±3% to 32±13%). In wells with methanogenic activity, Methanosaeta concilii accounted for up to 26% of all DAPI-detected microorganisms. These results demonstrate that this novel combination of chemical, isotopic, and biological analysis provides valuable insights that can be used for the characterization of microbial processes in contaminated aquifers.     

Geological gas-storage shapes deep life

Around the world, several dozen deep sedimentary aquifers are being used for storage of natural gas. Ad hoc studies of the microbial ecology of some of them have suggested that sulfate reducing and methanogenic microorganisms play a key role in how these aquifers' communities function. Here, we investigate the influence of gas storage on these two metabolic groups by using high-throughput sequencing and show the importance of sulfate-reducing Desulfotomaculum and a new monophyletic methanogenic group. Aquifer microbial diversity was significantly related to the geological level. The distance to the stored natural gas affects the ratio of sulfate-reducing Firmicutes to deltaproteobacteria. In only one aquifer, the methanogenic archaea dominate the sulfate-reducers. This aquifer was used to store town gas (containing at least 50% H₂) around 50 years ago. The observed decrease of sulfates in this aquifer could be related to stimulation of subsurface sulfate-reducers. These results suggest that the composition of the microbial communities is impacted by decades old transient gas storage activity. The tremendous stability of these gas-impacted deep subsurface microbial ecosystems suggests that in situ biotic methanation projects in geological reservoirs may be sustainable over time.     

Isolation and genetic identification of PAH degrading bacteria from a microbial consortium

Polycyclic aromatic hydrocarbons (PAH; naphthalene, anthracene and phenanthrene) degrading microbial consortium C2PL05 was obtained from a sandy soil chronically exposed to petroleum products, collected from a petrochemical complex in Puertollano (Ciudad Real, Spain). The consortium C2PL05 was highly efficient degrading completely naphthalene, phenanthrene and anthracene in around 18 days of cultivation. The toxicity (Microtox™ method) generated by the PAH and by the intermediate metabolites was reduced to levels close to non-toxic in almost 40 days of cultivation. The identified bacteria from the contaminated soil belonged to γ-proteobacteria and could be include in Enterobacter and Pseudomonas genus. DGGE analysis revealed uncultured Stenotrophomonas ribotypes as a possible PAH degrader in the microbial consortium. The present work shows the potential use of these microorganisms and the total consortium for the bioremediation of PAH polluted areas since the biodegradation of these chemicals takes place along with a significant decrease in toxicity.     

The potential for hydrocarbon biodegradation and production of extracellular polymeric substances by aerobic bacteria isolated from a Brazilian petroleum reservoir

Extracellular polymeric substances (EPS) can contribute to the cellular degradation of hydrocarbons and have a huge potential for application in biotechnological processes, such as bioremediation and microbial enhanced oil recovery (MEOR). Four bacterial strains from a Brazilian petroleum reservoir were investigated for EPS production, emulsification ability and biodegradation activity when hydrocarbons were supplied as substrates for microbial growth. Two strains of Bacillus species had the highest EPS production when phenanthrene and n-octadecane were offered as carbon sources, either individually or in a mixture. While Pseudomonas sp. and Dietzia sp., the other two evaluated strains, had the highest hydrocarbon biodegradation indices, EPS production was not detected. Low EPS production may not necessarily be indicative of an absence of emulsifier activity, as indicated by the results of a surface tension reduction assay and emulsification indices for the strain of Dietzia sp. The combined results gathered in this work suggest that a microbial consortium consisting of bacteria with interdependent metabolisms could thrive in petroleum reservoirs, thus overcoming the limitations imposed on each individual species by the harsh conditions found in such environments.     

Metabolic resistance of a psychrotolerant VFA-oxidizing microbial community from an anaerobic bioreactor to changes in the cultivation temperature

A psychrotolerant microbial consortium from a low-temperature anaerobic EGSB bioreactor was grown separately on acetate, propionate, butyrate, and H₂/CO₂ at 30 and 10°C in glass flasks. In the course of the experiments, the cultivation temperature was changed at different time intervals. The initial rates of substrate utilization were higher at 30 than at 10°C. However, the microbial consortium was found to be well adapted to low temperatures; when grown at 10°C for 1.5–5 months, the rates of butyrate, propionate, and H₂/CO₂ utilization increased steadily. When grown at 30°C for 1.5–2.5 months, this consortium retained its ability to degrade VFA and H₂/CO₂ at 10°C. However, after long-term (150 days) cultivation at 10°C, its ability to utilize the substrates at 30°C decreased. In the consortium grown in the acetate-containing medium, a Methanosaeta-like methanogen was predominant; in media with propionate and butyrate, besides VFA-degrading bacteria, acetoclastic Methanosaeta-like and hydrogenotrophic Methanospirillum-like methanogenic archaea prevailed. A Methanospirillum-like strain predominated in the H₂/CO₂-containing medium. The Methanospirillum strain of this microbial community was presumably psychrotolerant. A method based on changes in the cultivation temperature is of practical interest and can be used to start up new bioreactors.     

A proteomics approach to study synergistic and antagonistic interactions of the fungal–bacterial consortium Fusarium oxysporum wild‐type MSA 35

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil‐borne plant pathogen, including non‐pathogenic F. oxysporum strains. In this study, F. oxysporum wild‐type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to γ‐proteobacteria, was analyzed by two complementary metaproteomic approaches (2‐DE combined with MALDI‐Tof/Tof MS and 1‐D PAGE combined with LC‐ESI‐MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter‐part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

The Microbial Community Structure in Petroleum-Contaminated Sediments Corresponds to Geophysical Signatures

The interdependence between geoelectrical signatures at underground petroleum plumes and the structures of subsurface microbial communities was investigated. For sediments contaminated with light non-aqueous-phase liquids, anomalous high conductivity values have been observed. Vertical changes in the geoelectrical properties of the sediments were concomitant with significant changes in the microbial community structures as determined by the construction and evaluation of 16S rRNA gene libraries. DNA sequencing of clones from four 16S rRNA gene libraries from different depths of a contaminated field site and two libraries from an uncontaminated background site revealed spatial heterogeneity in the microbial community structures. Correspondence analysis showed that the presence of distinct microbial populations, including the various hydrocarbon-degrading, syntrophic, sulfate-reducing, and dissimilatory-iron-reducing populations, was a contributing factor to the elevated geoelectrical measurements. Thus, through their growth and metabolic activities, microbial populations that have adapted to the use of petroleum as a carbon source can strongly influence their geophysical surroundings. Since changes in the geophysical properties of contaminated sediments parallel changes in the microbial community compositions, it is suggested that geoelectrical measurements can be a cost-efficient tool to guide microbiological sampling for microbial ecology studies during the monitoring of natural or engineered bioremediation processes.     

Microbial photoelectrosynthesis: Feeding purple phototrophic bacteria electricity to produce bacterial biomass

Purple phototrophic bacteria are one of the main actors in chemolithotrophic carbon fixation and, therefore, fundamental in the biogeochemical cycle. These microbes are capable of using insoluble electron donors such as ferrous minerals or even carbon-based electrodes. Carbon fixation through extracellular electron uptake places purple phototrophic bacteria in the field of microbial electrosynthesis as key carbon capturing microorganisms. In this work we demonstrate biomass production dominated by purple phototrophic bacteria with a cathode (−0.6 V vs. Ag/AgCl) as electron donor. In addition, we compared the growth and microbial population structure with ferrous iron as the electron donor. We detect interaction between the cathode and the consortium showing a midpoint potential of 0.05 V (vs. Ag/AgCl). Microbial community analyses revealed different microbial communities depending on the electron donor, indicating different metabolic interactions. Electrochemical measurements together with population analyses point to Rhodopseudomonas genus as the key genus in the extracellular electron uptake. Furthermore, the genera Azospira and Azospirillum could play a role in the photoelectrotrophic consortium.     

The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures

The interdependence between geoelectrical signatures at underground petroleum plumes and the structures of subsurface microbial communities was investigated. For sediments contaminated with light non-aqueous-phase liquids, anomalous high conductivity values have been observed. Vertical changes in the geoelectrical properties of the sediments were concomitant with significant changes in the microbial community structures as determined by the construction and evaluation of 16S rRNA gene libraries. DNA sequencing of clones from four 16S rRNA gene libraries from different depths of a contaminated field site and two libraries from an uncontaminated background site revealed spatial heterogeneity in the microbial community structures. Correspondence analysis showed that the presence of distinct microbial populations, including the various hydrocarbon-degrading, syntrophic, sulfate-reducing, and dissimilatory-iron-reducing populations, was a contributing factor to the elevated geoelectrical measurements. Thus, through their growth and metabolic activities, microbial populations that have adapted to the use of petroleum as a carbon source can strongly influence their geophysical surroundings. Since changes in the geophysical properties of contaminated sediments parallel changes in the microbial community compositions, it is suggested that geoelectrical measurements can be a cost-efficient tool to guide microbiological sampling for microbial ecology studies during the monitoring of natural or engineered bioremediation processes.     

Application of Pneumatic Fracturing to Enhance In Situ Bioremediation

A field pilot demonstration integrating pneumatic fracturing and in situ bioremediation was carried out in a gasoline-contaminated, low permeability soil formation. A pneumatic fracturing system was used to enhance subsurface air flow and transport rates, as well as to deliver soil amendments directly to the indigenous microbial populations. An in situ bioremediation zone was established and operated for a period of 50 weeks, which included periodic subsurface injections of phosphate, nitrate, and ammonium salts. Off-gas data indicated the formation of a series of aerobic, denitrifying, and methanogenic microbial degradation zones. Based on soil samples recovered from the site, 79% of soil-phase benzene, toluene, and xylenes (BTX) was removed by the integrated technology. From mass balance calculations, accounting for all physical losses, it was estimated that 85% of the total mass of BTX removed (based on mean concentration levels) was attributable to biodegradation.     

培养法和免培养法联合检测油藏环境烃降解菌和产甲烷菌群多样性

烃降解菌和产甲烷菌是油藏环境微生物生态系统中重要的功能菌群, 采用DGGE和FISH方法分析了不同油藏样品中两类菌群的多样性和产甲烷活性。DGGE结果表明, 不同水样的alkB基因多样性相差较大, 而且注水井条带明显多于采油井。FISH结果表明, 油藏水样中产甲烷菌含量明显高于烃降解菌, 且两者空间分布的位置较近; 说明油藏环境中烃降解菌和产甲烷菌结成一定的相互关系。富集培养表明, 胜利油田产出液接种物培养130 d后, 石油烃降解率达到50%以上, 产甲烷的最大速率达到1.57×10^?2 mmol/(L?d)。利用分子生物学方法分析油藏环境功能菌群的多样性, 可以为开展微生物采油技术的应用提供有用信息。     

Bioremediation of gasoline-contaminated groundwater in a pilot-scale packed-bed anaerobic reactor

This work reports on the anaerobic treatment of gasoline-contaminated groundwater in a pilot-scale horizontal-flow anaerobic immobilized biomass reactor inoculated with a methanogenic consortium. BTEX removal rates varied from 59 to 80%, with a COD removal efficiency of 95% during the 70 days of in situ trial. BTEX removal was presumably carried out by microbial syntrophic interactions, and at the observed concentrations, the interactions among the aromatic compounds may have enhanced overall biodegradation rates by allowing microbial growth instead of co-inhibiting biodegradation. There is enough evidence to support the conclusion that the pilot-scale reactor responded similarly to the lab-scale experiments previously reported for this design.     

Conversion of propionate to acetate and methane by syntrophic consortia

Summary The anaerobic degradation of propionate to acetate and methane by a defined sulfidogenic syntrophic co-culture consisting of Syntrophobacter wolinii and Desulfovibrio G11, and a new thermophilic, methanogenic consortium T13 was studied. Tracer experiments using (¹⁴C) propionate produced evidence for the generally accepted biochemical pathway involving methylmalonyl-CoA as an intermediate in the degradation of propionate. The degradation of (1-¹⁴C) propionate led exclusively to the formation of ¹⁴CO₂ by S. wolinii/D. G11 and to the formation of ¹⁴CH₄ by the methanogenic consortium T13. The conversion of either (2-¹⁴) or (3-¹⁴) propionate by S. wolinii/D. G11 resulted in uniform labelled acetate as the endproduct. The methanogenic consortium formed (U-¹⁴C) acetate from (2-¹⁴) and (3-¹⁴) propionate as an intermediary product followed by aceticlastic splitting to yield equivalent amounts of ¹⁴CO₂ and ¹⁴CH₄.