Bacterial Communities Associated with Production Facilities of Two Newly Drilled Thermogenic Natural Gas Wells in the Barnett Shale (Texas, USA)

We monitored the bacterial communities in the gas–water separator and water storage tank of two newly drilled natural gas wells in the Barnett Shale in north central Texas, using a 16S rRNA gene pyrosequencing approach over a period of 6?months. Overall, the communities were composed mainly of moderately halophilic and halotolerant members of the phyla Firmicutes and Proteobacteria (classes Βeta-, Gamma-, and Epsilonproteobacteria) in both wells at all sampling times and locations. Many of the observed lineages were encountered in prior investigations of microbial communities from various fossil fluid formations and production facilities. In all of the samples, multiple H₂S-producing lineages were encountered; belonging to the sulfate- and sulfur-reducing class Deltaproteobacteria, order Clostridiales, and phylum Synergistetes, as well as the thiosulfate-reducing order Halanaerobiales. The bacterial communities from the separator and tank samples bore little resemblance to the bacterial communities in the drilling mud and hydraulic-fracture waters that were used to drill these wells, suggesting the in situ development of the unique bacterial communities in such well components was in response to the prevalent geochemical conditions present. Conversely, comparison of the bacterial communities on temporal and spatial scales suggested the establishment of a core microbial community in each sampled location. The results provide the first overview of bacterial dynamics and colonization patterns in newly drilled, thermogenic natural gas wells and highlights patterns of spatial and temporal variability observed in bacterial communities in natural gas production facilities.     

Bacterial communities associated with hydraulic fracturing fluids in thermogenic natural gas wells in North Central Texas, USA

Hydraulic fracturing is used to increase the permeability of shale gas formations and involves pumping large volumes of fluids into these formations. A portion of the frac fluid remains in the formation after the fracturing process is complete, which could potentially contribute to deleterious microbially induced processes in natural gas wells. Here, we report on the geochemical and microbiological properties of frac and flowback waters from two newly drilled natural gas wells in the Barnett Shale in North Central Texas. Most probable number studies showed that biocide treatments did not kill all the bacteria in the fracturing fluids. Pyrosequencing-based 16S rRNA diversity analyses indicated that the microbial communities in the flowback waters were less diverse and completely distinct from the communities in frac waters. These differences in frac and flowback water communities appeared to reflect changes in the geochemistry of fracturing fluids that occurred during the frac process. The flowback communities also appeared well adapted to survive biocide treatments and the anoxic conditions and high temperatures encountered in the Barnett Shale.     

Laboratory Evaluations of Factors Affecting Biodegradation of Sulfolane and Diisopropanolamine

Sulfolane and diisopropanolamine (DIPA) are used in the Sulfinol® process to remove hydrogen sulfide from sour natural gas. This process has been used in western Canada since the early 1960s, and contamination of groundwater has occurred from surface spills and from seepage from landfills and unlined process water storage ponds. Aquifer sediments from contaminated and uncontaminated areas, and muds in a wetland downgradient from the contaminated plume, were collected from a gas plant. Vigorously agitated shake-flask cultures and gently agitated 2.5-L microcosms consisting of contaminated sediment, mud and groundwater, or wetland water were used to study the biodegradation of sulfolane and DIPA. The aerobic shake-flask method showed that all five of these materials contained microbial communities that biodegraded both compounds. Microorganisms in all samples, except the uncontaminated aquifer sediment, degraded both compounds in the aerobic 2.5-L microcosms. In general, the biodegradation occurred more rapidly in the shake-flask cultures. The addition of P greatly enhanced the degradation of sulfolane and DIPA, whereas the addition of N yielded little stimulation.     

Physicochemical impacts associated with natural gas development on methanogenesis in deep sand aquifers

The Minami-Kanto gas field, where gases are dissolved in formation water, is a potential analogue for a marine gas hydrate area because both areas are characterized by the accumulation of microbial methane in marine turbidite sand layers interbedded with mud layers. This study examined the physicochemical impacts associated with natural gas production and well drilling on the methanogenic activity and composition in this gas field. Twenty-four gas-associated formation water samples were collected from confined sand aquifers through production wells. The stable isotopic compositions of methane in the gases indicated their origin to be biogenic via the carbonate reduction pathway. Consistent with this classification, methanogenic activity measurements using radiotracers, culturing experiments and molecular analysis of formation water samples indicated the predominance of hydrogenotrophic methanogenesis. The cultivation of water samples amended only with methanogenic substrates resulted in significant increases in microbial cells along with high-yield methane production, indicating the restricted availability of substrates in the aquifers. Hydrogenotrophic methanogenic activity increased with increasing natural gas production from the corresponding wells, suggesting that the flux of substrates from organic-rich mudstones to adjacent sand aquifers is enhanced by the decrease in fluid pressure in sand layers associated with natural gas/water production. The transient predominance of methylotrophic methanogens, observed for a few years after well drilling, also suggested the stimulation of the methanogens by the exposure of unutilized organic matter through well drilling. These results provide an insight into the physicochemical impacts on the methanogenic activity in biogenic gas deposits including marine gas hydrates.     

Evaluation of deep subsurface sampling procedures using serendipitous microbial contaminants as tracer organisms

Surface microbiological investigations are critically dependent on the procedures used to collect samples for study. It can be difficult to distinguish between indigenous organisms and those encountered as contaminants during the drilling process. We found that coliform bacteria contaminated drilling mud slurries. These bacteria proved useful as tracer organisms in evaluating the degree of microbial contamination accidentally encountered while drilling for subterranean samples. While these organisms were found in high numbers in both the circulating muds and in the mud reservoir, few subsurface samples harbored conforms. Subsurface slurries did not inhibit the growth of a known coliform inoculum. These results indicate that the methods used to collect and field‐process cores from Atlantic coastal plain sediments were sufficient to prevent a large degree of bacterial contamination in most samples. The microflora in drilling fluids did not quantitatively or qualitatively account for the number and diversity of bacteria in subsurface samples. We conclude that a large and viable bacterial community is present in deep regions of the terrestrial subsurface.     

Anaerobic metabolic processes in the deep terrestrial subsurface

Anaerobic microorganisms were enumerated and metabolic activities measured in deep Coastal Plain sediments sampled from three water‐bearing formations at depths down to 300 m. Aseptically obtained sediment cores harbored the potential for anaerobic biodegradation of various substrates in almost all samples. Although the sediments were not predominantly anaerobic, viable methanogens and sulfate‐reducing bacteria (SRB) were present almost throughout the depth profile. Coliform organisms were also found at various locations, but were not recoverable from drilling muds or water used to slurry the muds. The anaerobic metabolism of lactate and formate was easily detected in most samples. However, acetate and benzoate were degraded only in portions of the subsurface that harbored methanogens. The water‐saturated transmissive zones harbored the highest numbers of SRB and the potential for the widest variety of anaerobic metabolic activities. Small or negligible anaerobic microbial activity was associated with thick clay layers. The accumulation of acetate and the production of methane in samples not amended with exogenous organic matter demonstrated that some strata contained reserves of fermentable carbon and suggested that environmental factors or nutrients other than carbon were potentially limiting in situ microbial activity.     

页岩气开采与深地微生物的相互影响

页岩气是一种特殊的天然气聚集,以吸附或游离状态存在于页岩之中。页岩气资源储量丰富,约占全球天然气能源的三分之一,主要分布在中国、北美、俄罗斯等国家和地区。页岩气开采所使用的水力压裂技术会对深地微生物产生显著影响,在水力压裂的不同阶段,微生物群落组成存在明显差异。其中,产甲烷菌能够提高页岩气的产量,而产酸细菌会造成设备腐蚀,降低页岩气的回收效率。本文概述了页岩气的开采现状、开采过程以及微生物种群的变化和潜在影响,以期促进页岩气开采与深地微生物相互影响的研究,最终推动页岩气的绿色、高效开采。     

Potential for biogeochemical cycling of sulfur,iron and carbon within massive sulfide deposits below the seafloor

Seafloor massive sulfides are a potential energy source for the support of chemosynthetic ecosystems in dark, deep‐sea environments; however, little is known about microbial communities in these ecosystems, especially below the seafloor. In the present study, we performed culture‐independent molecular analyses of sub‐seafloor sulfide samples collected in the Southern Mariana Trough by drilling. The depth for the samples ranged from 0.52 m to 2.67 m below the seafloor. A combination of 16S rRNA and functional gene analyses suggested the presence of chemoautotrophs, sulfur‐oxidizers, sulfate‐reducers, iron‐oxidizers and iron‐reducers. In addition, mineralogical and thermodynamic analyses are consistent with chemosynthetic microbial communities sustained by sulfide minerals below the seafloor. Although distinct bacterial community compositions were found among the sub‐seafloor sulfide samples and hydrothermally inactive sulfide chimneys on the seafloor collected from various areas, we also found common bacterial members at species level including the sulfur‐oxidizers and sulfate‐reducers, suggesting that the common members are widely distributed within massive sulfide deposits on and below the seafloor and play a key role in the ecosystem function.     

The search for sulfate-reducing bacteria in mat samples from the lost city hydrothermal field by molecular cloning

The work is dedicated to searching for microorganisms of the domain Bacteria capable of dissimilatory sulfate reduction in the samples of microbial mats from a carbonate chimney in the Lost City hydro-thermal field. Cloning of 16S rRNA genes, the universal phylogenetic marker, and dsrAB, the functional marker for sulfate reduction, revealed phylotypes related to spore-forming Desulfotomaculum. No members of the Deltaproteobacteria, comprising the most numerous bacterial group with demonstrated capacity for dissimilatory sulfate reduction, were found. The phylogenetic position of 16S rRNA clones from the mats suggests that this microbial community is a unique consortium, where the energy flow is related to hydrogen of hydrothermal origin, while mass growth of primary produces results from utilization of sulfide formed by sulfate-and sulfur-reducing microorganisms.     

A microbiological study of an underground gas storage in the process of gas extraction

The numbers of microorganisms belonging to ecologically significant groups and the rates of terminal microbial processes of sulfate reduction and methanogenesis were determined in the liquid phase of an underground gas storage (UGS) in the period of gas extraction. The total number of microorganisms in water samples from the operation and injection wells reached 2.1 x 10(6) cells/ml. Aerobic organotrophs (including hydrocarbon- and oil-oxidizing ones) and various anaerobic microorganisms (fermenting bacteria, methanogens, acetogens, sulfate-, nitrate-, and iron-reducing bacteria) were constituent parts of the community. The radioisotopic method showed that, in all the UGS units, the terminal stages of organic matter decomposition included sulfate reduction and methanogenesis, with the maximal rate of these processes recorded in the aqueous phase of above-ground technological equipment which the gas enters from the operation wells. A comparative analysis by these parameters of different anaerobic ecotopes, including natural hydrocarbon fields, allows us to assess the rate of these processes in the UGS as high throughout the annual cycle of its operation. The data obtained indicate the existence in the UGS of a bacterial community that is unique in its diversity and metabolic capacities and able to make a certain contribution to the geochemistry of organic and inorganic compounds in the natural and technogenic ecosystem of the UGS and thus influence the industrial gas composition.     

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs,Western Siberia,Russia

The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.     

Biological souring and mitigation in oil reservoirs

Souring in oilfield systems is most commonly due to the action of sulfate-reducing prokaryotes, a diverse group of anaerobic microorganisms that respire sulfate and produce sulfide (the key souring agent) while oxidizing diverse electron donors. Such biological sulfide production is a detrimental, widespread phenomenon in the petroleum industry, occurring within oil reservoirs or in topside processing facilities, under low- and high-temperature conditions, and in onshore or offshore operations. Sulfate reducers can exist either indigenously in deep subsurface reservoirs or can be “inoculated” into a reservoir system during oilfield development (e.g., via drilling operations) or during the oil production phase. In the latter, souring most commonly occurs during water flooding, a secondary recovery strategy wherein water is injected to re-pressurize the reservoir and sweep the oil towards production wells to extend the production life of an oilfield. The water source and type of production operation can provide multiple components such as sulfate, labile carbon sources, and sulfate-reducing communities that influence whether oilfield souring occurs. Souring can be controlled by biocides, which can non-specifically suppress microbial populations, and by the addition of nitrate (and/or nitrite) that directly impacts the sulfate-reducing population by numerous competitive or inhibitory mechanisms. In this review, we report on the diversity of sulfate reducers associated with oil reservoirs, approaches for determining their presence and effects, the factors that control souring, and the approaches (along with the current understanding of their underlying mechanisms) that may be used to successfully mitigate souring in low-temperature and high-temperature oilfield operations.     

A microbiological study of an underground gas storage in the process of gas extraction

The numbers of microorganisms belonging to ecologically significant groups and the rates of terminal microbial processes of sulfate reduction and methanogenesis were determined in the liquid phase of an underground gas storage (UGS) in the period of gas extraction. The total number of microorganisms in water samples from the operation and injection wells reached 2.1 × 10⁶ cells/ml. Aerobic organotrophs (including hydrocarbon-and oil-oxidizing ones) and various anaerobic microorganisms (fermenting bacteria, methanogens, acetogens, sulfate-, nitrate-, and iron-reducing bacteria) were constituent parts of the community. The radioisotopic method showed that, in all the UGS units, the terminal stages of organic matter decomposition included sulfate reduction and methanogenesis, with the maximal rate of these processes recorded in the aqueous phase of above-ground technological equipment which the gas enters from the operation wells. A comparative analysis by these parameters of different anaerobic ecotopes, including natural hydrocarbon fields, allows us to assess the rate of these processes in the UGS as high throughout the annual cycle of its operation. The data obtained indicate the existence in the UGS of a bacterial community that is unique in its diversity and metabolic capacities and able to make a certain contribution to the geochemistry of organic and inorganic compounds in the natural and technogenic ecosystem of the UGS and thus influence the industrial gas composition.     

Microbial Community Composition in Crude Oils and Asphalts from the Kurdistan Region of Iraq

Abstract

To identify hydrocarbon-degrading microorganisms contributing to the formation of heavy oil we investigated the microbial community composition in different types of crude oils from oil-production facilities and in crude oil and asphalt from different natural seeps from the Kurdistan Region of Iraq (KRI). Crude oils from five out of six production facilities did not contain microorganisms detectable by 16S rRNA gene PCR amplicon sequencing likely reflecting a low microbial abundance in these samples. Crude oil and asphalt from the natural seeps hosted diverse microbial communities. The same phylotypes of uncultivated Deferribacteres and Thermodesulfobacteraceae were predominant community members across crude oils and asphalts from separate geographical locations. Soils surrounding seeps did not contain these phylotypes suggesting that they originate from the subsurface and although they seem commonly detected in hydrocarbon-rich environments their role in hydrocarbon-degradation is unknown. GC-MS analyses showed that mainly aromatic hydrocarbons were present in the crude oil and asphalt and that they were undergoing biodegradation - likely with sulfate and nitrate as terminal oxidants. In agreement, only bssA gene, but not assA gene-carrying microorganisms were detectable in the analyzed sampled. Overall our study identified several abundant uncultivated taxa with likely roles in transformation of nitrate, sulfate and hydrocarbons.     

生物竞争排斥对油藏微生物群落结构变化影响

【目的】海上S油田采用生物竞争排斥技术治理油藏硫化氢产出取得一定成效,本研究通过揭示该技术对油藏环境中微生物群落结构的影响,尝试研究硫化氢治理过程中产生效果差异的因素。【方法】采用高通量测序分析等方法对加入硝酸盐、亚硝酸盐等药剂后,治理效果低效井、高效井及未治理井中不同微生物群落结构变化进行分析。【结果】与低效井和未治理井相比,高效井中的反硝化菌和石油降解菌种类和丰度明显增加,其中石油降解菌增加24.14%,反硝化细菌增加5.23%;Fe²⁺、Zn²⁺等离子的存在对不同井间治理效果差异和微生物群落变化具有一定影响。【结论】海上油田硫化氢治理中,生物竞争排斥技术不仅可以明显降低硫化氢产出,同时对油藏微生物群落环境也影响显著,微生物群落结构分析可作为硫化氢治理效果评价的重要指标,为海上油田硫化氢治理工作提供技术支持。     

Microbial community differentiation between active and inactive sulfide chimneys of the Kolumbo submarine volcano,Hellenic Volcanic Arc

Over the last decades, there has been growing interest about the ecological role of hydrothermal sulfide chimneys, their microbial diversity and associated biotechnological potential. Here, we performed dual-index Illumina sequencing of bacterial and archaeal communities on active and inactive sulfide chimneys collected from the Kolumbo hydrothermal field, situated on a geodynamic convergent setting. A total of 15,701 OTUs (operational taxonomic units) were assigned to 56 bacterial and 3 archaeal phyla, 133 bacterial and 16 archaeal classes. Active chimney communities were dominated by OTUs related to thermophilic members of Epsilonproteobacteria, Aquificae and Deltaproteobacteria. Inactive chimney communities were dominated by an OTU closely related to the archaeon Nitrosopumilus sp., and by members of Gammaproteobacteria, Deltaproteobacteria, Planctomycetes and Bacteroidetes. These lineages are closely related to phylotypes typically involved in iron, sulfur, nitrogen, hydrogen and methane cycling. Overall, the inactive sulfide chimneys presented highly diverse and uniform microbial communities, in contrast to the active chimney communities, which were dominated by chemolithoautotrophic and thermophilic lineages. This study represents one of the most comprehensive investigations of microbial diversity in submarine chimneys and elucidates how the dissipation of hydrothermal activity affects the structure of microbial consortia in these extreme ecological niches.

     

Microbial Community of a Hydrothermal Mud Vent Underneath the Deep-Sea Anoxic Brine Lake Urania (Eastern Mediterranean)

The composition of a metabolically active prokaryotic community thriving in hydrothermal mud fluids of the deep-sea hypersaline anoxic Western Urania Basin was characterized using rRNA-based phylogenetic analysis of a clone library. The physiologically active prokaryotic assemblage in this extreme environment showed a great genetic diversity. Most members of the microbial community appeared to be affiliated to yet uncultured organisms from similar ecosystems, i.e., deep-sea hypersaline basins and hydrothermal vents. The bacterial clone library was dominated by phylotypes affiliated with the epsilon-Proteobacteria subdivision recognized as an ecologically significant group of bacteria inhabiting deep-sea hydrothermal environments. Almost 18% of all bacterial clones were related to delta-Proteobacteria, suggesting that sulfate reduction is one of the dominant metabolic processes occurring in warm mud fluids. The remaining bacterial phylotypes were related to alpha- and beta-Proteobacteria, Actinobacteria, Bacteroides, Deinococcus-Thermus, KB1 and OP-11 candidate divisions. Moreover, a novel monophyletic clade, deeply branched with unaffiliated 16S rDNA clones was also retrieved from deep-sea sediments and halocline of Urania Basin. Archaeal diversity was much lower and detected phylotypes included organisms affiliated exclusively with the Euryarchaeota. More than 96% of the archaeal clones belonged to the MSBL-1 candidate order recently found in hypersaline anoxic environments, such as endoevaporitic microbial mats, Mediterranean deep-sea mud volcanoes and anoxic basins. Two phylotypes, represented by single clones were related to uncultured groups DHVE-1 and ANME-1. Thus, the hydrothermal mud of hypersaline Urania Basin seems to contain new microbial diversity. The prokaryotic community was significantly different from that occurring in the upper layers of the Urania Basin since 60% of all bacterial and 40% of all archaeal phylotypes were obtained only from mud fluids. The uniqueness of the composition of the active prokaryotic community could be explained by the complex environmental conditions at the site. The interaction of oxygenated warm mud fluids with the cold hypersaline brine of the Urania Basin seems to simultaneously select for various metabolic processes, such as aerobic and anaerobic heterotrophy, sulfide- and methane-dependent chemotrophy along with anaerobic oxidation of methane, sulfate- and metal-reduction.     

The occurrence and distribution of actinomycetes in lakes of the English Lake District

Investigation of the occurrence of mesophilic actinomycetes in the lakes of the English Lake District revealed their widespread distribution in the lacustrine environment. Although only low numbers of actinomycetes occurred in the water, high numbers were recovered from all the lake muds. Total numbers of actinomycetes in the muds correlated quite well with the lakes’ productivity status. High numbers of Micro-monospora, Streptomyces and nocardioform actinomycetes were isolated from all the lakes sampled. Low numbers of Streptosporangium were isolated from all the muds but strains of Actinomadura, Actinoplanes, Dactylosporangium, Microbispora and Thermomonospora were only encountered occasionally. Micromonospora was the numerically dominant genus isolated from all the lakes sampled. This dominance was even more striking in deeper layers of mud and this was thought to reflect a more resistant spore stage in Micromonospora than in either of the other two main genera.     

Technological aspects of the microbial treatment of sulfide-rich wastewaters: A case study

Thiobacillus denitrificans has been shown to be an effective biocatalyst for the treatment of a variety of sulfide-laden waste streams including sour water, sour gases, and refinery spent-sulfidic caustics. The term 'sour' originated in the petroleum industry to describe a waste contaminated with hydrogen sulfide or salts of sulfide and bisulfide. The microbial treatment of sour waste streams resulting from the production or refining of natural gas and crude oil have been investigated in this laboratory for many years. The application of this technology to the treatment of sour wastes on a commercially useful scale has presented several technical barriers including substrate inhibition (sulfide), product inhibition (sulfate), the need for septic operation, biomass recycle and recovery, mixed waste issues, and the need for large-scale cultivation of the organism for process startup. The removal of these barriers through process improvements are discussed in terms of a case study of the full-scale treatment of sulfide-rich wastewater. The ability of T. denitrificans to deodorize and detoxify an oil-field produced water containing sulfides was evaluated under full-scale field conditions at Amoco Production Co. Salt Creek Field in Midwest, WY. More than 800 m³/d of produced water containing 100 mg/L sulfide and total dissolved solids of 4800 mg/L were successfully biotreated in an earthen pit (3000 m³) over a six-month period. Complete removal of sulfides and elimination of associated odors were observed. The system could be upset by severe hydraulic disturbances; however, the system recovered rapidly when normal influent flow rates were restored.     

Effect of environmental conditions on the distribution of functional groups of microorganisms in the Khoito-Gol mineral springs (East Sayan)

The structure and functional activity of the microbial communities formed under different environmental conditions of the Khoito-Gol mineral springs are investigated. The habitat of microorganisms in the Khoito-Gol springs is characterized by abundant hydrogen sulfide and intense circulation of sulfur with the participation of sulfate-reducing, thionic, colorless, and purple bacteria. The main terminal process of microbial destruction of organic matter is sulfate reduction.