Vishvesh K. Bhupathiraju

Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms.

Abstract Strain SBT is a new, strictly anaerobic, gram-negative, nonmotile, non-sporeforming, rod-shaped bacterium that degrades benzoate and certain fatty acids in syntrophic association with hydrogen/formate-using microorganisms. Strain SBT produced approximately 3 mol of acetate and 0.6 mol of methane per mol of benzoate in coculture with Methanospirillum hungatei strain JF1. Saturated fatty acids, some unsaturated fatty acids, and methyl esters of butyrate and hexanoate also supported growth of strain SBT in coculture with Desulfovibrio strain G11. Strain SBT grew in pure culture with crotonate, producing acetate, butyrate, caproate, and hydrogen. The molar growth yield was 17 ± 1 g cell dry mass per mol of crotonate. Strain SBT did not grow with fumarate, iron(III), polysulfide, or oxyanions of sulfur or nitrogen as electron acceptors with benzoate as the electron donor. The DNA base composition of strain SBT was 43.1 mol% G+C. Analysis of the 16 S rRNA gene sequence placed strain SBT in the δ-subdivision of the Proteobacteria, with sulfate-reducing bacteria. Strain SBT was most closely related to members of the genus Syntrophus. The clear phenotypic and genotypic differences between strain SBT and the two described species in the genus Syntrophus justify the formation of a new species, Syntrophus aciditrophicus.

Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new, strictly anaerobic, dissimilatory Fe(III)-reducers.

Recent studies on the diversity and ubiquity of Fe(III)-reducing organisms in different environments led to the isolation and identification of four new dissimilatory Fe(III)-reducers (strains H-2T, 172T, TACP-2T and TACP-5). All four isolates are non-motile, Gram-negative, freshwater, mesophilic, strict anaerobes with morphology identical to that of Geobacter metallireducens strain GS-15T. Analysis of the 16S rRNA sequences indicated that the new isolates belong to the genus Geobacter, in the delta-Proteobacteria. Significant differences in phenotypic characteristics, DNA-DNA homology and G+C content indicated that the four isolates represent three new species of the genus. The names Geobacter hydrogenophilus sp. nov. (strain H-2T), Geobacter chapellei sp. nov. (strain 172T) and Geobacter grbiciae sp. nov. (strains TACP-2T and TACP-5) are proposed. Geobacter hydrogenophilus and Geobacter chapellei were isolated from a petroleum-contaminated aquifer and a pristine, deep, subsurface aquifer, respectively. Geobacter grbiciae was isolated from aquatic sediments. All of the isolates can obtain energy for growth by coupling the oxidation of acetate to the reduction of Fe(III). The four isolates also coupled Fe(III) reduction to the oxidation of other simple, volatile fatty acids. In addition, Geobacter hydrogenophilus and Geobacter grbiciae were able to oxidize aromatic compounds such as benzoate, whilst Geobacter grbiciae was also able to use the monoaromatic hydrocarbon toluene.

Metabolism of Benzoate, Cyclohex-1-ene Carboxylate, and Cyclohexane Carboxylate by “Syntrophus aciditrophicus” Strain SB in Syntrophic Association with H2-Using Microorganisms

ABSTRACT The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “Syntrophus aciditrophicus” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. 13C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-13C6]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts ofRhodopseudomonas palustris grown phototrophically orThauera aromatica grown under nitrate-reducing conditions. Cocultures of “S. aciditrophicus” andMethanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “S. aciditrophicus” grown in a coculture withDesulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “S. aciditrophicus”-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “S. aciditrophicus”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.

Pretest studies for a microbially enhanced oil recovery field pilot in a hypersaline oil reservoir

Abstract The ecological and physiological factors governing microbial activity in the Southeast Vassar Vertz Sand Unit (SEWSU), Payne County, OK, an oil reservoir selected for a microbially enhanced oil recovery field pilot, were studied. Analysis of the brines from the reservoir showed that the SEWSU reservoir is a hypersaline environment rich in calcium and magnesium cations, and contains most of the inorganic nutrients required for microbial growth. Substantial amounts of sulfate and sulfide were detected in the brines, indicating a potential for sulfate reduction activity. Of the various carbohydrate‐based nutrients tested, a molasses‐ammonium nitrate nutrient mixture best stimulated the metabolism and growth of the microbial communities in the brines. Sulfide was not detected in any brine samples that received high levels of nitrate, even when additional carbon sources such as molasses were added. The addition of nitrate also resulted in shorter lag times, higher maximum turbidities, and larger press...

Haloanaerobium salsugo sp. nov., a moderately halophilic, anaerobic bacterium from a subterranean brine.

A strictly anaerobic, moderately halophilic, gram-negative bacterium was isolated from a highly saline oil field brine. The bacterium was a non-spore-forming, nonmotile rod, appearing singly, in pairs, or occasionally as long chains, and measured 0.3 to 0.4 by 2.6 to 4 microns. The bacterium had a specific requirement for NaCl and grew at NaCl concentrations of between 6 and 24%, with optimal growth at 9% NaCl. The isolate grew at temperatures of between 22 and 51 degrees C and pH values of between 5.6 and 8.0. The doubling time in a complex medium containing 10% NaCl was 9 h. Growth was inhibited by chloramphenicol, tetracycline, and penicillin but not by cycloheximide or azide. Fermentable substrates were predominantly carbohydrates. The end products of glucose fermentation were acetate, ethanol, CO2, and H2. The major components of the cellular fatty acids were C14:0, C16:0, C16:1, and C17:0 cyc acids. The DNA base composition of the isolate was 34 mol% G+C. Oligonucleotide catalog and sequence analyses of the 16S rRNA showed that strain VS-752T was most closely related to Haloanaerobium praevalens GSLT (ATCC 33744), the sole member of the genus Haloanaerobium. We propose that strain VS-752 (ATCC 51327) be established as the type strain of a new species, Haloanaerobium salsugo, in the genus Haloanaerobium.

Haloanaerobium kushneri sp. nov., an obligately halophilic, anaerobic bacterium from an oil brine

Three strains, designated VS-751T, VS-511 and VS-732, of a strictly anaerobic, moderately halophilic, Gram-negative, rod-shaped bacterium were isolated from a highly saline (15-20%) brine from an oil reservoir in central Oklahoma, USA. The optimal concentration of NaCl for growth of these three strains was 2 M (12%), and the strains also grew in the presence of an additional 1 M MgCl2. The strains were mesophilic and grew at a pH range of 6-8. Carbohydrates used by all three strains included glucose, fructose, arabinose, galactose, maltose, mannose, cellobiose, sucrose and inulin. Glucose fermentation products included ethanol, acetate, H2 and CO2, with formate produced by two of the three strains. Differences were noted among strains in the optimal temperature and pH for growth, the maximum and minimum NaCl concentration that supported growth, substrate utilization and cellular fatty acid composition. Despite the phenotypic differences among the three strains, analysis of the 16S rRNA gene sequences and DNA-DNA hybridizations showed that these three strains were members of the same genospecies which belonged to the genus Haloanaerobium. The phenotypic and genotypic characteristics of strains VS-751T, VS-511 and VS-732 are different from those of previously described species of Haloanaerobium. It is proposed that strain VS-751T (ATCC 700103T) be established as the type strain of a new species, Haloanaerobium kushneri.

Ch. R-7 Isolation and Characterization of Novel Halophilic Anaerobic Bacteria from Oil Field Brines

Abstract The development of a succesful microbially enhanced oil recovery process requires a thorough understanding of the factors affecting microbial activity in the oil reservoir. A study has, therefore been initiated to evaluate the ecological and physiological factors governing the microbial activity in the oil field brines collected from the South East Vassar Vertz Sand Unit, Payne County, Oklahoma. The brines were highliy saline containing 11 to 19% NaCl and 1 to 2% of calcium and magnesiumions. Diverse populations of anaerobic heterotrophic bacteria were present at densities of 2.4 × 10 3 to 4.6 × 10 3 MPN/ml. Of the several strains isolated, five strictly anaerobic and obligately halophilic strains were selected for further characterization. None of the isolates grew at salt concentrations less than 3% (w/v). All strains grew in a mineral salts medium containing glucose, yeast extract and casamino acids in the presence of NaCl concentrations of up to 20% (w/v), but the optimum concentration for growth was around 12%. The doubling time of the isolates ranged between 7 and 11 hours. Addition of nitrate to the growth medium did not stimulate growth of any of the isolates. However the addition of sulfite as the external electron acceptor stimulated the growth yield of the strain TTL-30. All of the isolates metabolized a variety of carbohydrate substrates but not several of the tested aromatic compounds and amino acids. Acetate, butanol and propanol were detected as the fermentative products on glucose. The change of gas phase from N 2 /CO 2 to H 2 /CO 2 stimulated the growth of strains TA and SA, but completely inhibited the growth of the strain TTL-30. Methane was never produced by any of the five strains showing that these strains are not methanogens. The ability to use a fermentable carbohydrate make these strains suitable candidates for microbial selective plugging process in hypersaline environments.

Evaluation of a microbial method to reduce hydrogen sulfide levels in a porous rock biofilm

SummaryThe efficacy of nitrate addition, with and without inoculation with a sulfide-resistant strain ofThiobacillus denitrificans (strain F), in reducing sulfide levels in an experimental system using cores and subsurface formation water from a gas storage facility was examined. The addition of nitrate (40 mM) alone to the formation water injected into core systems operated at hydraulic retention times of 3.2 and 16.7 h resulted in lower effluent sulfide concentrations, from an influent concentration of about 170–190 μM to an effluent concentration of 110 and 3 μM, respectively. A reduction in effluent nitrate concentrations in both core systems indicated the presence of indigenous nitrate-using populations. After strain F was inoculated into the core system operated at the shorter retention time, the effluent sulfide concentration decreased from 110 to 16–25 μM. The effluent sulfate concentration increased, and the effluent nitrate concentration decreased concomitant with the presence of high concentrations of denitrifying thiobacilli in the inoculated core system. The denitrifying thiobacilli detected after inoculation were presumed to be strain F since these organisms were not detected in this core system before inoculation, or in any of the samples from the uninoculated core system. These data suggest that the efficacy of the nitrate treatment may depend on the residence times of the liquids in the core system, and that inoculation with strain F was required to reduce sulfide levels to <20 μM in the core system operated at a short hydraulic retention time.

Causes and Control of Microbially Induced Souring

Abstract Petroleum reservoirs are harsh environments, but are not devoid of life. Rather, they are functional ecosystems with diverse, and metabolically active microorganisms. Thus, there is the potential for microbial sulfide production in the reservoir when environmental conditions permit. Some oil field operations may change the environmental conditions in the reservoir and inadvertently stimulate sulfide production. Waterflooding with brines high in sulfate can supply the oxidized sulfur needed for microbial sulfide production. Many oil field chemicals can be metabolized by the anaerobic populations present in the reservoir, and thus, stimulate sulfide production. One approach to control microbially induced souring is to shift the electron-accepting reaction from sulfate reduction to nitrate reduction. The ability of a sulfide-resistant strain of Thiobacillus denitrificans (strain F) to reduce sulfide levels was tested in an experimental system using cores and formation water from a gas storage facility. The addition of nitrate alone (40 mM) decreased concentrations of effluent sulfide from 170 μM to 110 μM. After inoculation with strain F, effluent sulfide concentrations decreased to between 3 and 20 μM. A large scale test of the efficacy of nitrate treatment in controlling souring was conducted in the Southeast Vassar Vertz Sand Unit, Payne Co., OK. The injection of 2.6 metric tonnes of ammonium nitrate resulted in a 56% decrease in sulfide concentrations in coproduced brine from three adjacent wells. These studies indicated that nitrate additions may be effective in controlling souring during actual field operations.

Microbially enhanced oil recovery field pilot, Payne County, Oklahoma.

Abstract A multi-well MEOR field study was performed in the Vassar Vertz Sand Unit in Oklahoma. The purpose of the field trial was to determine whether microorganisms could be used to preferentially plug high permeability zones to improve waterflood sweep efficiency. Laboratory studies determined that a nutrient system based on molasses and ammonium nitrate would induce the growth of nitrate - utilizing bacteria indigenous to the Vertz unit reservoir while limiting sulfate reduction. Tracer studies were done to determine the predominant flow pattern from the pilot injection well within the reservoir. The molasses and ammonium nitrate nutrients were injected over a four-month period. Samples were routinely analyzed for sulfate-reducing bacteria, molasses-nitrate utilizing bacteria, general fermentative bacteria, microbial metabolites, carbohydrates, sulfide, sulfate, nitrite, and nitrate. Over the period of nutrient injection, 56.2 tonnes of molasses and 18.8 tonnes of ammonium nitrate were injected into the pilot area. The results show that large-scale injection of readily metabolizable carbohydrates did not detrimentally affect the ongoing operation in the field but did result in an alteration of the existing flow patterns and reduction in transmissibility within the pilot area.