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ABSTRACT Microorganisms that persist in fractured shale reservoirs cause several problems including secreting foul gases and forming biofilms. Current biocontrol measures often fail due to limited knowledge of their in situ activities. The plasma membrane protects the cell, mediates many of its critical functions, and responds to intracellular cues and ecological perturbations through physicochemical modifications. As such, it provides valuable insight into the physiological adaptation of microorganisms in disturbed environmental systems. Here, we (i) demonstrate how changes in salinity and hydraulic retention time (HRT) influence the plasma membrane intact polar lipid (IPL) chemistry of model bacterium, Halanaerobium congolense WG10, and mixed microbial consortia enriched from shale-produced fluids and (ii) elucidate adjustments in membrane IPL chemistry during biofilm growth relative to planktonic cells. We incubated H. congolense WG10 in chemostats under three salinities (7%, 13%, and 20% NaCl), operated under three HRTs (19.2, 24, and 48 h), and in drip flow biofilm reactors under the same salinity gradients. Also, mixed microbial consortia in produced fluids were enriched in triplicate chemostat vessels under three HRTs (19.2, 24, and 72 h) and biofilm reactors. Lipids were analyzed by ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that phosphatidylglycerols, cardiolipins, and phosphatidylethanolamines were predominantly enriched in planktonic H. congolense WG10 cells grown at hypersalinity (20%) compared to optimum (13%). In addition, several zwitterionic phosphatidylcholines and phosphatidylethanolamines were higher in abundance during biofilm growth. These observations suggest that microbial adaptation and biofilm formation in fractured shale are enabled by strategic plasma membrane IPL chemistry adjustments. IMPORTANCE Microorganisms inadvertently introduced into the shale reservoir during fracturing face multiple stressors including brine-level salinities and starvation. However, some anaerobic halotolerant bacteria adapt and persist for long periods of time. They produce hydrogen sulfide, which sours the reservoir and corrodes engineering infrastructure. In addition, they form biofilms on rock matrices, which decrease shale permeability and clog fracture networks. These reduce well productivity and increase extraction costs. Under stress, microbes remodel their plasma membrane to optimize its roles in protection and mediating cellular processes such as signaling, transport, and energy metabolism. Hence, by observing changes in the membrane lipidome of model shale bacteria, Halanaerobium congolense WG10, and mixed consortia enriched from produced fluids under varying subsurface conditions and growth modes, we provide insight that advances our knowledge of the fractured shale biosystem. We also offer data-driven recommendations for improving biocontrol efficacy and the efficiency of energy recovery from unconventional formations.

A search for the microorganisms responsible for the anaerobic degradation of osmoprotectants in soda lakes resulted in the isolation of a novel halophilic and alkalitolerant strain, designated Z-7514T. The cells were Gram-stain-negative and non-endospore-forming rods. Optimal growth occurs at 1.6–2.1 M Na+, pH 8.0–8.5, and 31–35 °C. The strain utilized mainly sugars, low molecular polyols, and ethanolamine as well. The G+C content of the genomic DNA of strain Z-7514T was 33.3 mol%. Phylogenetic and phylogenomic analyses revealed that strain Z-7514T belongs to the genus Halanaerobium. On the basis of phenotypic properties and the dDDH and ANI values with close validly published species, it was proposed to evolve strain Z-7514T within the genus Halanaerobium into novel species, for which the name Halanaerobium polyolivorans sp. nov. was proposed. The type strain was Z-7514T (=KCTC 25405T = VKM B-3577T). For species of the genus Halanaerobium, the utilization of ethylene glycol, propylene glycol, and ethanolamine were shown for the first time. The anaerobic degradation of glycols and ethanolamine by strain Z-7514T may represent a novel metabiotic pathway within the alkaliphilic microbial community. Based on a detailed genomic analysis, the main pathways of catabolism of most of the used substrates have been identified. [ABSTRACT FROM AUTHOR]

Bacteria remodel their plasma membrane lipidome to maintain key biophysical attributes in response to ecological disturbances. For Halanaerobium and other anaerobic halotolerant taxa that persist in hydraulically fractured deep subsurface shale reservoirs, salinity, and hydraulic retention time (HRT) are important perturbants of cell membrane structure, yet their effects remain poorly understood. Membrane-linked activities underlie in situ microbial growth kinetics and physiologies which drive biogeochemical reactions in engineered subsurface systems. Hence, we used gas chromatography-mass spectrometry (GC-MS) to investigate the effects of salinity and HRT on the phospholipid fatty acid composition of H. congolense WG10 and mixed enrichment cultures from hydraulically fractured shale wells. We also coupled acyl chain remodeling to membrane mechanics by measuring bilayer elasticity using atomic force microscopy (AFM). For these experiments, cultures were grown in a chemostat vessel operated in continuous flow mode under strict anoxia and constant stirring. Our findings show that salinity and HRT induce significant changes in membrane fatty acid chemistry of H. congolense WG10 in distinct and complementary ways. Notably, under nonoptimal salt concentrations (7% and 20% NaCl), H. congolense WG10 elevates the portion of polyunsaturated fatty acids (PUFAs) in its membrane, and this results in an apparent increase in fluidity (homeoviscous adaptation principle) and thickness. Double bond index (DBI) and mean chain length (MCL) were used as proxies for membrane fluidity and thickness, respectively. These results provide new insight into our understanding of how environmental and engineered factors might disrupt the physical and biogeochemical equilibria of fractured shale by inducing physiologically relevant changes in the membrane fatty acid chemistry of persistent microbial taxa. [ABSTRACT FROM AUTHOR]

Bacteria remodel their plasma membrane lipidome to maintain key biophysical attributes in response to ecological disturbances. For Halanaerobium and other anaerobic halotolerant taxa that persist in hydraulically fractured deep subsurface shale reservoirs, salinity, and hydraulic retention time (HRT) are important perturbants of cell membrane structure, yet their effects remain poorly understood. Membrane-linked activities underlie in situ microbial growth kinetics and physiologies which drive biogeochemical reactions in engineered subsurface systems. Hence, we used gas chromatography–mass spectrometry (GC–MS) to investigate the effects of salinity and HRT on the phospholipid fatty acid composition of H. congolense WG10 and mixed enrichment cultures from hydraulically fractured shale wells. We also coupled acyl chain remodeling to membrane mechanics by measuring bilayer elasticity using atomic force microscopy (AFM). For these experiments, cultures were grown in a chemostat vessel operated in continuous flow mode under strict anoxia and constant stirring. Our findings show that salinity and HRT induce significant changes in membrane fatty acid chemistry of H. congolense WG10 in distinct and complementary ways. Notably, under nonoptimal salt concentrations (7% and 20% NaCl), H. congolense WG10 elevates the portion of polyunsaturated fatty acids (PUFAs) in its membrane, and this results in an apparent increase in fluidity (homeoviscous adaptation principle) and thickness. Double bond index (DBI) and mean chain length (MCL) were used as proxies for membrane fluidity and thickness, respectively. These results provide new insight into our understanding of how environmental and engineered factors might disrupt the physical and biogeochemical equilibria of fractured shale by inducing physiologically relevant changes in the membrane fatty acid chemistry of persistent microbial taxa.GRAPHICAL ABSTRACTSalinity significantly alters membrane bilayer fluidity and thickness in Halanaerobium congolense WG10.

A search for the microorganisms responsible for the anaerobic degradation of osmoprotectants in soda lakes resulted in the isolation of a novel halophilic and alkalitolerant strain, designated Z-7514T. The cells were Gram-stain-negative and non-endospore-forming rods. Optimal growth occurs at 1.6–2.1 M Na+, pH 8.0–8.5, and 31–35 °C. The strain utilized mainly sugars, low molecular polyols, and ethanolamine as well. The G+C content of the genomic DNA of strain Z-7514T was 33.3 mol%. Phylogenetic and phylogenomic analyses revealed that strain Z-7514T belongs to the genus Halanaerobium. On the basis of phenotypic properties and the dDDH and ANI values with close validly published species, it was proposed to evolve strain Z-7514T within the genus Halanaerobium into novel species, for which the name Halanaerobium polyolivorans sp. nov. was proposed. The type strain was Z-7514T (=KCTC 25405T = VKM B-3577T). For species of the genus Halanaerobium, the utilization of ethylene glycol, propylene glycol, and ethanolamine were shown for the first time. The anaerobic degradation of glycols and ethanolamine by strain Z-7514T may represent a novel metabiotic pathway within the alkaliphilic microbial community. Based on a detailed genomic analysis, the main pathways of catabolism of most of the used substrates have been identified.

Subsurface lineages of microorganisms are often questioned for their autochthonous nature. We hypothesize that sporulation is critical to the delivery and ubiquity of Halanaerobium congolense in global subsurface hydrocarbon reservoirs, forming spores, or spore-like structures in order to survive surface conditions, and thriving in subsurface reservoir conditions. Genomic analysis of H. congolense revealed the presence of various genes essential for sporulation, many of which are homologous to those involved in various stages of sporulation in clostridia and bacilli. Transcriptomics revealed transcription of sporulation genes in H. congolense grown in sub-optimal conditions. This is further supported by microscopy evidence of spore-like structures. [ABSTRACT FROM AUTHOR]

Produced waters from hydraulically fractured shale formations give insight into the microbial ecology and biogeochemical conditions down-well. This study explores the potential for sulfide production by persistent microorganisms recovered from produced water samples collected from the Marcellus shale formation. Hydrogen sulfide is highly toxic and corrosive, and can lead to the formation of “sour gas” which is costly to refine. Furthermore, microbial colonization of hydraulically fractured shale could result in formation plugging and a reduction in well productivity. It is vital to assess the potential for sulfide production in persistent microbial taxa, especially when considering the trend of reusing produced waters as input fluids, potentially enriching for problematic microorganisms. Using most probable number (MPN) counts and 16S rRNA gene sequencing, multiple viable strains of bacteria were identified from stored produced waters, mostly belonging to the Genus Halanaerobium, that were capable of growth via fermentation, and produced sulfide when supplied with thiosulfate. No sulfate-reducing bacteria (SRB) were detected through culturing, despite the detection of relatively low numbers of sulfate-reducing lineages by high-throughput 16S rRNA gene sequencing. These results demonstrate that sulfidogenic produced water populations remain viable for years post production and, if left unchecked, have the potential to lead to natural gas souring during shale gas extraction.

Produced waters from hydraulically fractured shale formations give insight into the microbial ecology and biogeochemical conditions down-well. This study explores the potential for sulfide production by persistent microorganisms recovered from produced water samples collected from the Marcellus shale formation. Hydrogen sulfide is highly toxic and corrosive, and can lead to the formation of "sour gas" which is costly to refine. Furthermore, microbial colonization of hydraulically fractured shale could result in formation plugging and a reduction in well productivity. It is vital to assess the potential for sulfide production in persistent microbial taxa, especially when considering the trend of reusing produced waters as input fluids, potentially enriching for problematic microorganisms. Using most probable number (MPN) counts and 16S rRNA gene sequencing, multiple viable strains of bacteria were identified from stored produced waters, mostly belonging to the Genus Halanaerobium , that were capable of growth via fermentation, and produced sulfide when supplied with thiosulfate. No sulfate-reducing bacteria (SRB) were detected through culturing, despite the detection of relatively low numbers of sulfate-reducing lineages by high-throughput 16S rRNA gene sequencing. These results demonstrate that sulfidogenic produced water populations remain viable for years post production and, if left unchecked, have the potential to lead to natural gas souring during shale gas extraction. [ABSTRACT FROM AUTHOR]

The Bakken Shale has become one of the United States' most important oil and gas producing regions. This study examined the microbiology and geochemical characteristics of Bakken region produced water from 17 well sites sampled from the three-phase separator and produced water holding tank over a 6-month time frame. Produced water samples had high total dissolved solids (220 000–350 000 mg/L) and low dissolved organic carbon concentrations (41–132 mg/L). Microbial abundances varied between 101 and 104 16S rRNA gene copies/mL, approximately four orders of magnitude below those observed for produced waters from other hydraulic fracturing regions. The most abundant bacterial orders found in produced water samples were Bacillales, Halanaerobiales and Pseudomonadales, consistent with observations from other unconventional resource plays. Our observations suggest temporal community structuring, as produced waters sampled early in our sampling period were dominated by Halanaerobiales, and produced waters sampled at the remaining winter sampling time points were characterized by high relative abundances of Bacillales and Pseudomonadales. Data from this study extends the current available knowledge of the microbiology and chemistry associated with produced water from the Bakken region and provides insights into microbial community dynamics in hypersaline subsurface fluids. [ABSTRACT FROM AUTHOR]

ABSTRACT Hydraulic fracturing of black shale formations has greatly increased United States oil and natural gas recovery. However, the accumulation of biomass in subsurface reservoirs and pipelines is detrimental because of possible well souring, microbially induced corrosion, and pore clogging. Temporal sampling of produced fluids from a well in the Utica Shale revealed the dominance of Halanaerobium strains within the in situ microbial community and the potential for these microorganisms to catalyze thiosulfate-dependent sulfidogenesis. From these field data, we investigated biogenic sulfide production catalyzed by a Halanaerobium strain isolated from the produced fluids using proteogenomics and laboratory growth experiments. Analysis of Halanaerobium isolate genomes and reconstructed genomes from metagenomic data sets revealed the conserved presence of rhodanese-like proteins and anaerobic sulfite reductase complexes capable of converting thiosulfate to sulfide. Shotgun proteomics measurements using a Halanaerobium isolate verified that these proteins were more abundant when thiosulfate was present in the growth medium, and culture-based assays identified thiosulfate-dependent sulfide production by the same isolate. Increased production of sulfide and organic acids during the stationary growth phase suggests that fermentative Halanaerobium uses thiosulfate to remove excess reductant. These findings emphasize the potential detrimental effects that could arise from thiosulfate-reducing microorganisms in hydraulically fractured shales, which are undetected by current industry-wide corrosion diagnostics. IMPORTANCE Although thousands of wells in deep shale formations across the United States have been hydraulically fractured for oil and gas recovery, the impact of microbial metabolism within these environments is poorly understood. Our research demonstrates that dominant microbial populations in these subsurface ecosystems contain the conserved capacity for the reduction of thiosulfate to sulfide and that this process is likely occurring in the environment. Sulfide generation (also known as “souring”) is considered deleterious in the oil and gas industry because of both toxicity issues and impacts on corrosion of the subsurface infrastructure. Critically, the capacity for sulfide generation via reduction of sulfate was not detected in our data sets. Given that current industry wellhead tests for sulfidogenesis target canonical sulfate-reducing microorganisms, these data suggest that new approaches to the detection of sulfide-producing microorganisms may be necessary.

This study revealed for the first time the dynamic changes of the bacterial community during the fermentation of traditional Chinese fish sauce (TCFS) using high-throughput sequencing. In the early phase of TCFS fermentation, Shewanella (approximately 90%) within Proteobacteria was the dominant bacteria. Then, Halanaerobium (3%−86%) within Firmicutes rapidly replaced Shewanella as the dominant genus until the 12th month. Lactococcus (3.31%) and Bacillus (45.56%) belonging to Firmicutes were detected abundantly in the 3rd and 9th months after fermentation, respectively. In the late phase (12−15 months), Tetragenococcus within Firmicutes replaced Halanaerobium as the most dominant bacteria (29.54%). Many other genera including Pseudomonas, Psychrobacter, Tissierella, Carnobacterium and Gallicola were abundantly present in the 15th month after fermentation. Furthermore, the relationships between the bacterial community and major functional substances of TCFS, including amino nitrogen (AAN), free amino acids (FAAs), total soluble nitrogen (TSN), and trimethylamine (TMA), were investigated by partial least squares regression (PLSR). Tetragenococcus was positively correlated with the formation of TMA, while Halanaerobium showed the opposite result, suggesting that Tetragenococcus might be a good starter for TCFS fermentation. These results contribute to our knowledge about bacterial participation in the process of TCFS fermentation and will help improve the quality of fermented seafood.

Microbial activity in the produced water from hydraulically fractured oil and gas wells may potentially interfere with hydrocarbon production and cause damage to the well and surface infrastructure via corrosion, sulfide release, and fouling. In this study, we surveyed the microbial abundance and community structure of produced water sampled from 42 Marcellus Shale wells in southwestern Pennsylvania (well age ranged from 150 to 1,846 days) to better understand the microbial diversity of produced water. We sequenced the V4 region of the 16S rRNA gene to assess taxonomy and utilized quantitative PCR (qPCR) to evaluate the microbial abundance across all 42 produced water samples. Bacteria of the order Halanaerobiales were found to be the most abundant organisms in the majority of the produced water samples, emphasizing their previously suggested role in hydraulic fracturing-related microbial activity. Statistical analyses identified correlations between well age and biocide formulation and the microbial community, in particular, the relative abundance of Halanaerobiales. We further investigated the role of members of the order Halanaerobiales in produced water by reconstructing and annotating a Halanaerobium draft genome (named MDAL1), using shotgun metagenomic sequencing and metagenomic binning. The recovered draft genome was found to be closely related to the species H. congolense, an oil field isolate, and Halanaerobium sp. strain T82-1, also recovered from hydraulic fracturing produced water. Reconstruction of metabolic pathways revealed Halanaerobium sp. strain MDAL1 to have the potential for acid production, thiosulfate reduction, and biofilm formation, suggesting it to have the ability to contribute to corrosion, souring, and biofouling events in the hydraulic fracturing infrastructure. IMPORTANCE: There are an estimated 15,000 unconventional gas wells in the Marcellus Shale region, each generating up to 8,000 liters of hypersaline produced water per day throughout its lifetime (K. Gregory, R. Vidic, and D. Dzombak, Elements 7:181-186, 2011, https://doi.org/10.2113/gselements.7.3.181; J. Arthur, B. Bohm, and M. Layne, Gulf Coast Assoc Geol Soc Trans 59:49-59, 2009; https://www.marcellusgas.org/index.php). Microbial activity in produced waters could lead to issues with corrosion, fouling, and souring, potentially interfering with hydraulic fracturing operations. Previous studies have found microorganisms contributing to corrosion, fouling, and souring to be abundant across produced water samples from hydraulically fractured wells; however, these findings were based on a limited number of samples and well sites. In this study, we investigated the microbial community structure in produced water samples from 42 unconventional Marcellus Shale wells, confirming the dominance of the genus Halanaerobium in produced water and its metabolic potential for acid and sulfide production and biofilm formation. [ABSTRACT FROM AUTHOR]

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O32-, SO42-, and HS-). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure. [ABSTRACT FROM AUTHOR]

Abstract: Raw glycerol is a tempting substrate for fermentations, but contains impurities that can be inhibitory for organisms. In this study, raw glycerol tolerance and contamination risk of pure bacterial culture at hypersaline process conditions were evaluated. The inhibitory effect of raw glycerol was similar on a halophilic (Halanaerobium saccharolyticum) and a non-halophilic (Clostridium butyricum) bacterium implying the inhibition originating from methanol or other impurities rather than salt. The hypersaline process conditions decreased efficiently contaminations and no growth of contaminants was observed at and above 125 g/l NaCl. Halophilic H2 and 1,3-PD production from raw glycerol were studied separately as 1-stage processes and jointly as 2-stage process in non-sterile conditions. Non-sterile conditions were successfully applied and the highest production yields obtained were 3.0 mol H2/mol glycerol and 0.66 mol 1,3-PD/mol glycerol (1-stage processes), whereas the highest cumulative production was 74 mmol H2/l culture and 31 mmol 1,3-PD/l culture (2-stage process). [Copyright &y& Elsevier]

Abstract: An upflow anaerobic packed bed reactor was operated continuously with synthetic saline wastewater at different initial COD concentrations (COD0 =1900–6300mg/L), salt concentrations (0–5%, w/v) and hydraulic retention times (θ H =11–30h) to investigate the effect of those operating parameters on COD removal from saline synthetic wastewater. Anaerobic salt tolerant bacteria, Halanaerobium lacusrosei, were used as dominant microbial culture in the process. The percent COD removal reached up to 94% at COD0 =1900mg/L, 19h hydraulic retention time and 3% salt concentration. No substrate inhibition effect was observed at high feed CODs. Increasing hydraulic retention time from 11h to 30h resulted in a substantial improvement in the COD removal from 60% to 84% at around COD0 =3400mg/L and 3% salt concentration. Salt inhibition effect on COD utilization was observed at above 3% salt concentration. Modified Stover–Kincannon model was applied to the experimental data to determine the biokinetic coefficients. Saturation value constant, and maximum utilization rate constant of Stover–Kincannon model for COD were determined as K B =5.3g/Lday, U max =7.05g/Lday, respectively. [Copyright &y& Elsevier]

Abstract: The recently described anaerobic moderately halophilic bacterium Halanaerobium congolense has been shown to reduce thiosulfate and sulfur—but not sulfate—into sulfide. When cultivated in the presence of thiosulfate as terminal electron acceptor, H. congolense possesses a highly active thiosulfate:cyanide sulfur-transferase activity (rhodanese-like enzyme). A gene library of H. congolense (DSM 11287T) was constructed, and a 3.1-kb Sau3A DNA that encompassed a thiosulfate:cyanide sulfur-transferase-encoding gene was isolated in Escherichia coli. This fragment contains 2 orfs, which were separately subcloned in E. coli. The 900-bp gene encoding the rhodanese-like protein was named rdlA. RdlA differs from other known rhodanese-like proteins by having two potential catalytic sites, one N-terminal and one C-terminal, both harboring a cysteine. The two putative active sites are preceded by a highly-conserved region of unknown function. Closely related genes were also characterized in other thiosulfate-reducing non-sulfate-reducing anaerobes belonging to phylogenetically distant microorganisms, thus suggesting that RdlA is of importance in the mechanism of thiosulfate reduction by numerous members of the domain Bacteria. [Copyright &y& Elsevier]

While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions. Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin. Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance. However, some species show a specific requirement for chloride. In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent. In this organism the expression of a large number of proteins is chloride regulated. Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride. A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber. It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms. [ABSTRACT FROM AUTHOR]

• Microbial Enhanced Oil Recovery with Halanerobium sp. was performed in sandpacks. • Sucrose consumption with 10 % carbonate was 8 times higher compared to pure quartz. • 1 % of carbonate and sulfide maintained microbial growth and led to 21.6 % more oil. • Superficial mineral dissolution by microbially produced acids was confirmed by XPS. • Acid neutralization capacity of rock is proposed as a screening criterion for MEOR. Microbial enhanced oil recovery (MEOR) is an economically attractive tertiary recovery technique and fermentative bacteria are frequently suggested for MEOR, partly because microbially produced organic acids have the potential for rock matrix dissolution. In this study, the metabolic activity and the community shift of a diverse microbiome of a high-salinity oilfield upon supplying MEOR nutrients was investigated in dynamic sandpacks set-up with and without crude oil using pure quartz sand and two types of reservoir rock. Geochemical characterization of the porous media included specific surface area (SSA), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). During the experiments, substrate and metabolites, incremental oil and differential pressure were monitored and the microbial community shift was investigated via Illumina sequencing. Fermentative Halanaerobiales outcompeted other microbes and led to an incremental oil recovery of 24.5 ± 9.6 % OOIP in reservoir rock. Microbial-induced dissolution of surface minerals was indicated by a decrease in SSA and surface-bound ferrous iron and concluded to be an important MEOR mechanism. Fermentation of sucrose was primarily limited by an insufficient acid neutralization capacity (ANC), but a carbonate content of 12% sustainably buffered the pH in a favorable growth range. Even minor amounts of other non-inert minerals (1% pyrite and calcite) facilitated microbial growth significantly, resulting in six-fold higher acetate production rates compared to quartz sand. Besides emphasizing the relevance of accessory minerals in MEOR, our results imply that the ANC could serve as potential screening parameter for predicting the performance of fermentation - based MEOR in the field. [ABSTRACT FROM AUTHOR]

As a dynamic screening tool for a high-salinity oilfield (186 g/L), anaerobic sandpacks were established to simulate Microbial Enhanced Oil Recovery (MEOR) under defined laboratory conditions. Glass beads, quartz sand or crushed reservoir rock were used to produce porous media which varied in permeability, wettability, homogeneity and geochemistry. In total, 14 sandpacks were flooded with oil and inoculated with indigenous fermentative bacteria of the order Halanaerobiales. After waterflooding, these were treated with nutrients in different injection scenarios during which incremental oil recovery, permeability, microbial activity and produced metabolites were measured. Our results indicate that the efficiency of MEOR is dependent on the type of porous medium used: Both glass beads and outcrop quartz sand were found to be no suitable analogue to reservoir material because not all potential MEOR effects were accounted for. MEOR was least efficient in quartz sandpacks with a recovery factor of 7.0 ± 1.7% with respect to the original oil in place (IRF OOIP), attributed mainly to fluid-fluid interactions. In sandpacks with reservoir rock, wettability alteration, matrix dissolution and bioplugging were additional MEOR mechanisms and resulted in an incremental recovery which was almost three-fold higher compared to pure quartz sandpacks (IRF OOIP = 23.2 ± 6.4%). Bioplugging was not detected in sandpacks with a permeability of 8–10 D, although cell retention was observed. Mean pore sizes of these sandpacks were calculated to be in the range of 100 μm, thus considered to be too large to allow for significant plugging. Our findings support the use of MEOR as potential tertiary recovery method but also emphasize the importance of carefully designing laboratory experiments. We argue that porous medium properties such as permeability, pore size, wettability and mineralogy play a crucial role during dynamic MEOR feasibility studies, because they directly influence incremental recovery. • Bacteria of the genus Halanaerobium were tested for MEOR in dynamic and anaerobic sandpacks at 180 g*L-1 salinity • 14 experiments with four different porous media (glass beads, quartz sand and two reservoir rock samples) were conducted • Incremental recovery was three-fold higher in reservoir rock sandpacks (IRF OOIP = 23.2 ± 6.4 %) compared to pure quartz sand • Pure outcrop quartz sand did not meet requirements for all potential MEOR effects to occur • A wettability change from oil- towards water-wet was identified as one of the main MEOR effects [ABSTRACT FROM AUTHOR]

This study revealed for the first time the dynamic changes of the bacterial community during the fermentation of traditional Chinese fish sauce (TCFS) using high-throughput sequencing. In the early phase of TCFS fermentation, Shewanella (approximately 90%) within Proteobacteria was the dominant bacteria. Then, Halanaerobium (3%–86%) within Firmicutes rapidly replaced Shewanella as the dominant genus until the 12th month. Lactococcus (3.31%) and Bacillus (45.56%) belonging to Firmicutes were detected abundantly in the 3rd and 9th months after fermentation, respectively. In the late phase (12–15 months), Tetragenococcus within Firmicutes replaced Halanaerobium as the most dominant bacteria (29.54%). Many other genera including Pseudomonas, Psychrobacter, Tissierella, Carnobacterium and Gallicola were abundantly present in the 15th month after fermentation. Furthermore, the relationships between the bacterial community and major functional substances of TCFS, including amino nitrogen (AAN), free amino acids (FAAs), total soluble nitrogen (TSN), and trimethylamine (TMA), were investigated by partial least squares regression (PLSR). Tetragenococcus was positively correlated with the formation of TMA, while Halanaerobium showed the opposite result, suggesting that Tetragenococcus might be a good starter for TCFS fermentation. These results contribute to our knowledge about bacterial participation in the process of TCFS fermentation and will help improve the quality of fermented seafood. [ABSTRACT FROM AUTHOR]

Bacterial Halanaerobium strains become the dominant persisting microbial community member in produced fluids across geographically distinct hydraulically fractured shales. Halanaerobium is believed to be inadvertently introduced into this environment during the drilling and fracturing process and must therefore tolerate large changes in pressure, temperature, and salinity. Here, we used a Halanaerobium strain isolated from a natural gas well in the Utica Point Pleasant formation to investigate metabolic and physiological responses to growth under high-pressure subsurface conditions. Laboratory incubations confirmed the ability of Halanaerobium congolense strain WG8 to grow under pressures representative of deep shale formations (21 to 48 MPa). Under these conditions, broad metabolic and physiological shifts were identified, including higher abundances of proteins associated with the production of extracellular polymeric substances. Confocal laser scanning microscopy indicated that extracellular polymeric substance (EPS) production was associated with greater cell aggregation when biomass was cultured at high pressure. Changes in Halanaerobium central carbon metabolism under the same conditions were inferred from nuclear magnetic resonance (NMR) and gas chromatography measurements, revealing large per-cell increases in production of ethanol, acetate, and propanol and cessation of hydrogen production. These metabolic shifts were associated with carbon flux through 1,2-propanediol in response to slower fluxes of carbon through stage 3 of glycolysis. Together, these results reveal the potential for bioclogging and corrosion (via organic acid fermentation products) associated with persistent Halanaerobium growth in deep, hydraulically fractured shale ecosystems, and offer new insights into cellular mechanisms that enable these strains to dominate deep-shale microbiomes. IMPORTANCE The hydraulic fracturing of deep-shale formations for hydrocarbon recovery accounts for approximately 60% of U.S. natural gas production. Microbial activity associated with this process is generally considered deleterious due to issues associated with sulfide production, microbially induced corrosion, and bioclogging in the subsurface. Here we demonstrate that a representative Halanaerobium species, frequently the dominant microbial taxon in hydraulically fractured shales, responds to pressures characteristic of the deep subsurface by shifting its metabolism to generate more corrosive organic acids and produce more polymeric substances that cause "clumping" of biomass. While the potential for increased corrosion of steel infrastructure and clogging of pores and fractures in the subsurface may significantly impact hydrocarbon recovery, these data also offer new insights for microbial control in these ecosystems., (Copyright © 2019 Booker et al.)