Use of Acetate, Propionate, and Butyrate for Reduction of Nitrate and Sulfate and Methanogenesis in Microcosms and Bioreactors Simulating an Oil Reservoir.

Acetate, propionate, and butyrate (volatile fatty acids [VFA]) occur in oil field waters and are frequently used for microbial growth of oil field consortia. We determined the kinetics of use of these VFA components (3 mM each) by an anaerobic oil field consortium in microcosms containing 2 mM sulfate and 0, 4, 6, 8, or 13 mM nitrate. Nitrate was reduced first, with a preference for acetate and propionate. Sulfate reduction then proceeded with propionate (but not butyrate) as the electron donor, whereas the fermentation of butyrate (but not propionate) was associated with methanogenesis. Microbial community analyses indicated that Paracoccus and Thauera (Paracoccus-Thauera), Desulfobulbus, and Syntrophomonas-Methanobacterium were the dominant taxa whose members catalyzed these three processes. Most-probable-number assays showed the presence of up to 107/ml of propionate-oxidizing sulfate-reducing bacteria (SRB) in waters from the Medicine Hat Glauconitic C field. Bioreactors with the same concentrations of sulfate and VFA responded similarly to increasing concentrations of injected nitrate as observed in the microcosms: sulfide formation was prevented by adding approximately 80% of the nitrate dose needed to completely oxidize VFA to CO2 in both. Thus, this work has demonstrated that simple time-dependent observations of the use of acetate, propionate, and butyrate for nitrate reduction, sulfate reduction, and methanogenesis in microcosms are a good proxy for these processes in bioreactors, monitoring of which is more complex.IMPORTANCE Oil field volatile fatty acids acetate, propionate, and butyrate were specifically used for nitrate reduction, sulfate reduction, and methanogenic fermentation. Time-dependent analyses of microcosms served as a good proxy for these processes in a bioreactor, mimicking a sulfide-producing (souring) oil reservoir: 80% of the nitrate dose required to oxidize volatile fatty acids to CO2 was needed to prevent souring in both. Our data also suggest that propionate is a good substrate to enumerate oil field SRB.

Roles of Thermophiles and Fungi in Bitumen Degradation in Mostly Cold Oil Sands Outcrops.

Oil sands are surface exposed in river valley outcrops in northeastern Alberta, where flat slabs (tablets) of weathered, bitumen-saturated sandstone can be retrieved from outcrop cliffs or from riverbeds. Although the average yearly surface temperature of this region is low (0.7°C), we found that the temperatures of the exposed surfaces of outcrop cliffs reached 55 to 60°C on sunny summer days, with daily maxima being 27 to 31°C. Analysis of the cooccurrence of taxa derived from pyrosequencing of 16S/18S rRNA genes indicated that an aerobic microbial network of fungi and hydrocarbon-, methane-, or acetate-oxidizing heterotrophic bacteria was present in all cliff tablets. Metagenomic analyses indicated an elevated presence of fungal cytochrome P450 monooxygenases in these samples. This network was distinct from the heterotrophic community found in riverbeds, which included fewer fungi. A subset of cliff tablets had a network of anaerobic and/or thermophilic taxa, including methanogens, Firmicutes, and Thermotogae, in the center. Long-term aerobic incubation of outcrop samples at 55°C gave a thermophilic microbial community. Analysis of residual bitumen with a Fourier transform ion cyclotron resonance mass spectrometer indicated that aerobic degradation proceeded at 55°C but not at 4°C. Little anaerobic degradation was observed. These results indicate that bitumen degradation on outcrop surfaces is a largely aerobic process with a minor anaerobic contribution and is catalyzed by a consortium of bacteria and fungi. Bitumen degradation is stimulated by periodic high temperatures on outcrop cliffs, which cause significant decreases in bitumen viscosity.

Souring in low-temperature surface facilities of two high-temperature Argentinian oil fields.

Produced waters from the Barrancas and Chihuido de la Salina (CHLS) fields in Argentina had higher concentrations of sulfate than were found in the injection waters, suggesting that the formation waters in these reservoirs had a high sulfate concentration and that sulfate-reducing bacteria were inactive downhole. Incubation of produced waters with produced oil gave rapid reduction of sulfate to sulfide (souring) at 37 °C, some at 60 °C, but none at 80 °C. Alkylbenzenes and alkanes served as electron donor, especially in incubations with CHLS oil. Dilution with water to decrease the ionic strength or addition of inorganic phosphate did not increase souring at 37 or 60 °C. These results indicate that souring in these reservoirs is limited by the reservoir temperature (80 °C for the Barrancas and 65-70 °C for the CHLS field) and that souring may accelerate in surface facilities where the oil-water mixture cools. As a result, significant sulfide concentrations are present in these surface facilities. The activity and presence of chemolithotrophic Gammaproteobacteria of the genus Thiomicrospira, which represented 85% of the microbial community in a water plant in the Barrancas field, indicated reoxidation of sulfide and sulfur to sulfate. The presence of these bacteria offers potential for souring control by microbial oxidation in aboveground facilities, provided that formation of corrosive sulfur can be avoided.

Comparison of microbial communities involved in souring and corrosion in offshore and onshore oil production facilities in Nigeria.

Samples were obtained from the Obigbo field, located onshore in the Niger delta, Nigeria, from which oil is produced by injection of low-sulfate groundwater, as well as from the offshore Bonga field from which oil is produced by injection of high-sulfate (2,200 ppm) seawater, amended with 45 ppm of calcium nitrate to limit reservoir souring. Despite low concentrations of sulfate (0-7 ppm) and nitrate (0 ppm), sulfate-reducing bacteria (SRB) and heterotrophic nitrate-reducing bacteria (NRB) were present in samples from the Obigbo field. Biologically active deposits (BADs), scraped from corrosion-failed sections of a water- and of an oil-transporting pipeline (both Obigbo), had high counts of SRB and high sulfate and ferrous iron concentrations. Analysis of microbial community composition by pyrosequencing indicated anaerobic, methanogenic hydrocarbon degradation to be a dominant process in all samples from the Obigbo field, including the BADs. Samples from the Bonga field also had significant activity of SRB, as well as of heterotrophic and of sulfide-oxidizing NRB. Microbial community analysis indicated high proportions of potentially thermophilic NRB and near-absence of microbes active in methanogenic hydrocarbon degradation. Anaerobic incubation of Bonga samples with steel coupons gave moderate general corrosion rates of 0.045-0.049 mm/year, whereas near-zero general corrosion rates (0.001-0.002 mm/year) were observed with Obigbo water samples. Hence, methanogens may contribute to corrosion at Obigbo, but the low general corrosion rates cannot explain the reasons for pipeline failures in the Niger delta. A focus of future work should be on understanding the role of BADs in enhancing under-deposit pitting corrosion.

Sphingomonas ginsengisoli sp. nov. and Sphingomonas sediminicola sp. nov.

Two novel bacteria, designated strains Gsoil 634(T) and Dae 20(T), were isolated in South Korea from soil of a ginseng field and freshwater sediment, respectively and were characterized by a polyphasic approach to clarify their taxonomic positions. Phylogenetic analysis based on 16S rRNA gene sequences indicated that, although they probably represented two distinct species (indicated by a sequence similarity of 96.6 %), both strain Gsoil 634(T) and strain Dae 20(T) belonged to the genus Sphingomonas and were most closely related to 'Sphingomonas humi' PB323 (97.8 % and 96.7 % sequence similarity, respectively), Sphingomonas kaistensis PB56(T) (96.8 % and 96.7 %), Sphingomonas astaxanthinifaciens TDMA-17(T) (96.6 % and 95.4 %) and Sphingomonas jaspsi TDMA-16(T) (95.6 % and 95.8 %). For both novel strains, the major ubiquinone was Q-10, the major polyamine was homospermidine, the major cellular fatty acids included summed feature 7 (C(18 : 1)ω7c, C(18 : 1)ω9t and/or C(18 : 1)ω12t), C(17 : 1)ω6c and C(16 : 0), and the polar lipids included sphingoglycolipid. These chemotaxonomic data supported the affiliation of both strains to the genus Sphingomonas. However, the DNA-DNA relatedness value between strain Gsoil 634(T) and 'Sphingomonas humi' PB323(T) was 31 %. Moreover, the results of physiological and biochemical tests allowed the phenotypic differentiation of strains Gsoil 634(T) and Dae 20(T) from established members of the genus Sphingomonas. Based on these data, the two isolates represent two novel species in the genus Sphingomonas, for which the names Sphingomonas ginsengisoli sp. nov. (type strain Gsoil 634(T) = KCTC 12630(T) = DSM 18094(T) = LMG 23739(T)) and Sphingomonas sediminicola sp. nov. (type strain Dae 20(T)  = KCTC 12629(T) = DSM 18106(T) = LMG 23592(T)) are proposed.

Microbial community and potential functional gene diversity involved in anaerobic hydrocarbon degradation and methanogenesis in an oil sands tailings pond.

Oil sands tailings ponds harbor large amounts of tailings resulting from surface mining of bitumen and consist of water, sand, clays, residual bitumen, and hydrocarbon diluent. Oxygen ingress in these ponds is limited to the surface layers, causing most hydrocarbon degradation to be catalyzed by anaerobic, methanogenic microbial communities. This causes the evolution of large volumes of methane of up to 10(4) m(3)/day. A pyrosequencing survey of 16S rRNA amplicons from 10 samples obtained from different depths indicated the presence of a wide variety of taxa involved in anaerobic hydrocarbon degradation and methanogenesis, including the phyla Proteobacteria, Euryarchaeota, Firmicutes, Actinobacteria, Chloroflexi, and Bacteroidetes. Metagenomic sequencing of DNA isolated from one of these samples indicated a more diverse community than indicated by the 16S rRNA amplicon survey. Both methods indicated the same major phyla to be present. The metagenomic dataset indicated the presence of genes involved in the three stages of anaerobic aromatic hydrocarbon degradation, including genes for enzymes of the peripheral (upper), the central (lower), and the methanogenesis pathways. Upper pathway genes showed broad phylogenetic affiliation (Proteobacteria, Firmicutes, and Actinobacteria), whereas lower pathway genes were mostly affiliated with the Deltaproteobacteria. Genes for both hydrogenotrophic and acetotrophic methanogenesis were also found. The wide variety of taxa involved in initial hydrocarbon degradation through upper pathways may reflect the variety of residual bitumen and diluent components present in the tailings pond.

Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

[Corynebacterium aquaticum infection: one case report and literature review].

OBJECTIVE: To study the diagnosis and treatment of Corynebacterium aquaticum infection. METHODS: A retrospective analysis of one case of Corynebacterium aquaticum infection and literature review were conducted. RESULTS: A 39-year old male patient was admitted because of cough, sputum production, fever and right chest pain for 10 days. Broad-spectrum antibiotic therapy had been given in another hospital, but the patient's condition had deteriorated.Nuclear magnetic resonance-guided percutancous transthoracic needle aspiration and lung tissue, pleural fluid and blood culture were performed after admission to our hospital. Corynebacterium aquaticum was grown from the lung tissue, the blood and the pleural effusion. Therefore the diagnosis of Corynebacterium aquaticum pneumonia complicated with pyothorax and septicemia was confirmed. After draining of pleural pus and intravenous vancomycin therapy, the patient recovered and was discharged from hospital. After literature search, we did not find reports on Corynebacterium aquaticum pneumonia complicated with pyothorax and septicemia. CONCLUSIONS: Corynebacterium aquaticum pneumonia complicated with pyothorax and septicemia is rare. The diagnosis could be confirmed by bacterial culture of lung tissue, pleural fluid and blood. Thoracic cavity draining and intravenous vancomycin are effective therapies for the disease.

Characterization of a novel ginsenoside-hydrolyzing α-L-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T.

The gene encoding an α-L-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-[α-L-arabinofuranosyl-(1-6)-ß-D-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[ß-D-glucopyranosyl-(1-2)-ß-D-glucopyranosyl]-20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054(T), and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K (m) values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V (max) values of 27.1 ± 1.7 and 49.6 ± 4.1 µmol min(-1) mg(-1) of protein for p-nitrophenyl-α-L-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-L-arabinofuranosidase that can transform ginsenoside Rc to Rd.

Phycicoccus ginsenosidimutans sp. nov., isolated from soil of a ginseng field.

A Gram-positive, non-motile, non-spore-forming, aerobic, coccoid-shaped bacterium, designated BXN5-13(T), was isolated from the soil of a ginseng field from Baekdu Mountain in Jilin district, China. Strain BXN5-13(T) grew optimally at 30 °C and pH 6.5-7.5 with 0-2  % (w/v) NaCl. Strain BXN5-13(T) had ß-glucosidase activity that was connected with ginsenoside-converting ability, so that it was able to convert ginsenoside Rb(1) to ginsenoside F2. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic relatives of strain BXN5-13(T) were Phycicoccus aerophilus 5516T-20(T) (98.4  % 16S rRNA gene sequence similarity), P. bigeumensis MSL-03(T) (98.3  %), P. dokdonensis DS-8(T) (97.9  %) and P. jejuensis KSW2-15(T) (96.9  %). Lower sequence similarity (<97.0  %) was found with the type strains of other recognized species of the family Intrasporangiaceae. The predominant quinone was MK-8(H4). The major fatty acids (>10  %) were iso-C15:0, C17:0, anteiso-C15:0 and iso-C16:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and phosphatidylinositol. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The chemotaxonomic data and the high genomic DNA G+C content of strain BXN5-13(T) (70.8l %) supported its affiliation with the genus Phycicoccus. DNA-DNA relatedness between strain BXN5-13(T) and its closest phylogenetic neighbours was below 16  %. Strain BXN5-13(T) represents a novel species within the genus Phycicoccus, for which the name Phycicoccus ginsenosidimutans sp. nov. is proposed. The type strain is BXN5-13(T) (=KCTC 19419(T)=DSM 21006(T)=LMG 24462(T)).

Rhodanobacter panaciterrae sp. nov., a bacterium with ginsenoside-converting activity isolated from soil of a ginseng field.

A novel gammaproteobacterium, designated LnR5-47(T), was isolated from soil of a ginseng field in Liaoning province, China. The isolate was a Gram-negative, aerobic, non-motile, non-spore-forming rod. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain LnR5-47(T) belonged to the genus Rhodanobacter. The isolate was most closely related to Rhodanobacter ginsengisoli GR17-7(T), Rhodanobacter terrae GP18-1(T), Dyella ginsengisoli Gsoil 3046(T), Rhodanobacter soli DCY45(T), Dyella soli JS12-10(T) and Dyella japonica IAM 15069(T) (98.0, 97.9, 97.7, 97.3, 97.2 and 97.1% 16S rRNA gene sequence similarity, respectively). Chemotaxonomic data (Q-8 as the predominant ubiquinone, and iso-C(16:0), iso-C(17:1)ω9c and iso-C(15:0) as the major fatty acids) also supported the affiliation of strain LnR5-47(T) with the genus Rhodanobacter. However, DNA-DNA relatedness between strain LnR5-47(T) and its closest phylogenetic neighbours was <25.8%. Moreover, physiological and biochemical tests phenotypically differentiated the isolate from other members of the genus Rhodanobacter. Therefore, strain LnR5-47(T) represents a novel species, for which the name Rhodanobacter panaciterrae sp. nov. is proposed; the type strain is LnR5-47(T) (=KACC 12826(T)=KCTC 22232(T)=LMG 24460(T)).

Solirubrobacter ginsenosidimutans sp. nov., isolated from soil of a ginseng field.

A Gram-reaction-positive, non-motile, non-spore-forming, aerobic rod, designated BXN5-15(T), was isolated from the soil of a ginseng field on Baekdu Mountain in China. Strain BXN5-15(T) grew optimally at 30 °C and pH 6.0-7.0 in the absence of NaCl on R2A agar. Strain BXN5-15(T) displayed ß-glucosidase activity, which allowed it to transform ginsenoside Rb(1) (one of the dominant active components of ginseng) via Rd to minor ginsenoside F(2). On the basis of 16S rRNA gene sequence analysis, strain BXN5-15(T) was shown to belong to the genus Solirubrobacter. The closest phylogenetic relatives were Solirubrobacter soli Gsoil 355(T) (98.4 % 16S rRNA gene sequence similarity) and Solirubrobacter pauli B33D1(T) (96.4 %). Lower sequence similarities (<96.0 %) were found with all of the other recognized members of the order Solirubrobacterales. The predominant quinone was MK-7(H(4)). The major fatty acids (>10 %) were C(18 : 1)ω9c, iso-C(16 : 0) and C(18 : 3)ω6,9,12c. The G+C content of the genomic DNA was 70.6 mol%. DNA-DNA relatedness between strain BXN5-15(T) and S. soli KCTC 12628(T) was 23.3 %. On the basis of genotypic, phenotypic and chemotaxonomic data, strain BXN5-15(T) represents a novel species within the genus Solirubrobacter, for which the name Solirubrobacter ginsenosidimutans sp. nov. is proposed. The type strain is BXN5-15(T) ( = KACC 20671(T) = LMG 24459(T)).

Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV.

A new beta-glucosidase from a novel strain of Terrabacter ginsenosidimutans (Gsoil 3082(T)) obtained from the soil of a ginseng farm was characterized, and the gene, bgpA (1,947 bp), was cloned in Escherichia coli. The enzyme catalyzed the conversion of ginsenoside Rb1 {3-O-[beta-D-glucopyranosyl-(1-2)-beta-D-glucopyranosyl]-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol} to the more pharmacologically active rare ginsenosides gypenoside XVII {3-O-beta-D-glucopyranosyl-20-O-[beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, gypenoside LXXV {20-O-[beta-v-glucopyranosyl-(1-6)-beta-D-glucopyranosyl]-20(S)-protopanaxadiol}, and C-K [20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol]. A BLAST search of the bgpA sequence revealed significant homology to family 3 glycoside hydrolases. Expressed in E. coli, beta-glucosidase had apparent K(m) values of 4.2 +/- 0.8 and 0.14 +/- 0.05 mM and V(max) values of 100.6 +/- 17.1 and 329 +/- 31 micromol x min(-1) x mg of protein(-1) against p-nitrophenyl-beta-D-glucopyranoside and Rb1, respectively. The enzyme catalyzed the hydrolysis of the two glucose moieties attached to the C-3 position of ginsenoside Rb1, and the outer glucose attached to the C-20 position at pH 7.0 and 37 degrees C. These cleavages occurred in a defined order, with the outer glucose of C-3 cleaved first, followed by the inner glucose of C-3, and finally the outer glucose of C-20. These results indicated that BgpA selectively and sequentially converts ginsenoside Rb1 to the rare ginsenosides gypenoside XVII, gypenoside LXXV, and then C-K. Herein is the first report of the cloning and characterization of a novel ginsenoside-transforming beta-glucosidase of the glycoside hydrolase family 3.

Baekduia soli gen. nov., sp. nov., a novel bacterium isolated from the soil of Baekdu Mountain and proposal of a novel family name, Baekduiaceae fam. nov.

A taxonomic study was conducted on BR7-21T, a bacterial strain isolated from the soil of a ginseng field in Baekdu Mountain. Comparative studies of the 16S rRNA gene sequence showed that the isolate was most closely related to Conexibacter woesei DSM 14684T, Solirubrobacter pauli ATCC BAA-492T, Patulibacter minatonensis JCM 12834T, with 93.8%, 92.4%, and 91.5% sequence similarity, respectively; each genus represented a family in the order Solirubrobacterales. Strain BR7-21T was Gram-reaction positive, non-spore forming, aerobic, non-motile, and short rod-shaped. It grew well on half-strength R2A medium. The G + C content of the genomic DNA was 73.9%. It contained meso-diaminopimelic acid in the cell wall and the major menaquinones were MK-7(H4) and MK-8(H4). The major fatty acids were summarized as (C16:1ω7c/iso-C15:0 2-OH), iso-C16:0, and C17:0 cyclo. On the basis of polyphasic evidence, it was proposed that strain BR7-21T should be placed in a new genus and species, for which the name Baekduia soli gen. nov., sp. nov. was proposed with the type strain BR7-21T (= KCTC 22257T = LMG 24797T). The family Baekduiaceae fam. nov. is proposed to encompass the genus Baekduia gen. nov.

Chitinophaga soli sp. nov. and Chitinophaga terrae sp. nov., isolated from soil of a ginseng field in Pocheon Province, Korea.

Two novel strains of the Cytophaga-Flexibacter-Bacteroides (CFB) group, designated Gsoil 219" and Gsoil 2381, were isolated from soil of a ginseng field of Pocheon Province in Korea. Both strains were Gram-negative, aerobic, nonmotile, nonspore-forming, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both isolates belong to the genus Chitinophaga but were clearly separated from established species of this genus. The sequence similarities between strain Gsoil 219T and type strains of the established species and between strain Gsoil 238T and type strains of the established species ranged from 91.4 to 94.7% and 91.6 to 94.2%, respectively. Phenotypic and chemotaxonomic data (major menaquinone, MK-7; major fatty acids, iso-C15:0 and C(16:1) omega5c; major hydroxy fatty acid, iso-C(17:0) 3-OH; major polyamine, homospermidine) supported the affiliation of both strains Gsoil 219T and Gsoil 238T to the genus Chitinophaga. Furthermore, the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of both strains from the other validated Chitinophaga species. Therefore, the two isolates represent two novel species, for which the name Chitinophaga soli sp. nov. (type strain, Gsoil 219T=KCTC 12650T=DSM 18093T) and Chitinophaga terrae sp. nov. (type strain, Gsoil 238T=KCTC 12651T=DSM 18078T) are proposed.

Biocide-mediated corrosion of coiled tubing.

Coiled tubing corrosion was investigated for 16 field water samples (S5 to S20) from a Canadian shale gas field. Weight loss corrosion rates of carbon steel beads incubated with these field water samples averaged 0.2 mm/yr, but injection water sample S19 had 1.25±0.07 mm/yr. S19 had a most probable number of zero acid-producing bacteria and incubation of S19 with carbon steel beads or coupons did not lead to big changes in microbial community composition. In contrast other field water samples had most probable numbers of APB of 102/mL to 107/mL and incubation of these field water samples with carbon steel beads or coupons often gave large changes in microbial community composition. HPLC analysis indicated that all field water samples had elevated concentrations of bromide (average 1.6 mM), which may be derived from bronopol, which was used as a biocide. S19 had the highest bromide concentration (4.2 mM) and was the only water sample with a high concentration of active bronopol (13.8 mM, 2760 ppm). Corrosion rates increased linearly with bronopol concentration, as determined by weight loss of carbon steel beads, for experiments with S19, with filtered S19 and with bronopol dissolved in defined medium. This indicated that the high corrosion rate found for S19 was due to its high bronopol concentration. The corrosion rate of coiled tubing coupons also increased linearly with bronopol concentration as determined by electrochemical methods. Profilometry measurements also showed formation of multiple pits on the surface of coiled tubing coupon with an average pit depth of 60 µm after 1 week of incubation with 1 mM bronopol. At the recommended dosage of 100 ppm the corrosiveness of bronopol towards carbon steel beads was modest (0.011 mm/yr). Higher concentrations, resulting if biocide is added repeatedly as commonly done in shale gas operations, are more corrosive and should be avoided. Overdosing may be avoided by assaying the presence of residual biocide by HPLC, rather than by assaying the presence of residual surviving bacteria.

Gram-Scale Production of Ginsenoside F1 Using a Recombinant Bacterial ß-Glucosidase.

Naturally occurring ginsenoside F1 (20-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol) is rare. Here, we produced gram-scale quantities of ginsenoside F1 from a crude protopanaxatriol saponin mixture comprised mainly of Re and Rg1 through enzyme-mediated biotransformation using recombinant ß-glucosidase (BgpA) cloned from a soil bacterium, Terrabacter ginsenosidimutans Gsoil 3082T. In a systematic step-by-step process, the concentrations of substrate, enzyme, and NaCl were determined for maximal production of F1. At an optimized NaCl concentration of 200 mM, the protopanaxatriol saponin mixture (25 mg/ml) was incubated with recombinant BgpA (20 mg/ml) for 3 days in a 2.4 L reaction. Following octadecylsilyl silica gel column chromatography, 9.6 g of F1 was obtained from 60 g of substrate mixture at 95% purity, as assessed by chromatography. These results represent the first report of gramscale F1 production via recombinant enzyme-mediated biotransformation.

Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite.

Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S(0)) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe(0)). Overall this accelerates reaction of of S(0) and Fe(0) to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function.

Ramlibacter ginsenosidimutans sp. nov., with ginsenoside-converting activity.

A novel beta-proteobacterium, designated BXN5-27(T), was isolated from soil of a ginseng field of Baekdu Mountain in China, and was characterized using a polyphasic approach. The strain was Gram-staining-negative, aerobic, motile, non-spore-forming, and rod shaped. Strain BXN5-27(T) exhibited beta-glucosidase activity that was responsible for its ability to transform ginsenoside Rb1 (one of the dominant active components of ginseng) to compound Rd. Phylogenetic analysis based on 16S rRNA gene sequences showed that this strain belonged to the family Comamonadaceae; it was most closely related to Ramlibacter henchirensis TMB834(T) and Ramlibacter tataouinensis TTB310(T) (96.4% and 96.3% similarity, respectively). The G+C content of the genomic DNA was 68.1%. The major menaquinone was Q-8. The major fatty acids were C16:0, summed feature 4 (comprising C16:1 omega7c and/or iso-C15:0 2OH), and C17:0 cyclo. Genomic and chemotaxonomic data supported the affiliation of strain BXN5-27(T) to the genus Ramlibacter. However, physiological and biochemical tests differentiated it phenotypically from the other established species of Ramlibacter. Therefore, the isolate represents a novel species, for which the name Ramlibacter ginsenosidimutans sp. nov. is proposed, with the type strain being BXN5-27(T) (= DSM 23480(T) = LMG 24525(T) = KCTC 22276(T)).

Kinetics of a cloned special ginsenosidase hydrolyzing 3-O-glucoside of multi-protopanaxadiol-type ginsenosides, named ginsenosidase type III.

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-ß-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-ß-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.