Electron Donor For Sulfate Reduction Research Articles

APLICAÇÃO DE REATORES ANAERÓBIOS OPERADOS EM BATELADAS SEQUENCIAIS COM FILME FIXO NO TRATAMENTO DE ÁGUA RESIDUARIA INDUSTRIAL RICA EM SULFATO

Este trabalho apresenta e discute o potencial de uso de reatores anaeróbios operados em bateladas seqüenciais com biomassa imobilizada (ASBBR), em escala piloto, no tratamento de água residuária industrial contendo elevadas concentrações de sulfato. No ASBBR, com volume total de 1,2 m3, preenchido com carvão mineral (meio suporte) foram aplicadas cargas de sulfato de 0,15 a 1,90 kg/ciclo com duração de ciclo de 48 h, correspondendo, respectivamente, às concentrações de sulfato no afluente de 0,25 a 3,0 g.L-1. O esgoto sanitário e etanol foram usados como doadores de elétrons para a redução do sulfato. As eficiências médias na redução de sulfato ficaram entre 88 e 92% nos 92 ciclos operacionais. Como pós-tratamento dos efluentes gerados pela redução de sulfato empregou-se outro reator ASBBR com volume total de 385 L e semelhante meio suporte para imobilização da biomassa. A remoção média de DQO (valor médio afluente: 1450 mg.L-1) foi de 88%, enquanto as concentrações de sulfetos totais (H2S, HS−, S2−) mantiveram-se entre a faixa de 41 e 71 mg.L-1 nos 35 ciclos operacionais de 48 h. Os resultados obtidos permitiram concluir que o uso de reatores ASBBR constitui-se em potencial alternativa tanto para a remoção de sulfatos, como no pós-tratamento dos efluentes gerados.

Desulfovibrio paquesii sp. nov., a hydrogenotrophic sulfate-reducing bacterium isolated from a synthesis-gas-fed bioreactor treating zinc- and sulfate-rich wastewater

A hydrogenotrophic, sulfate-reducing bacterium, designated strain SB1(T), was isolated from sulfidogenic sludge of a full-scale synthesis-gas-fed bioreactor used to remediate wastewater from a zinc smelter. Strain SB1(T) was found to be an abundant micro-organism in the sludge at the time of isolation. Hydrogen, formate, pyruvate, lactate, malate, fumarate, succinate, ethanol and glycerol served as electron donors for sulfate reduction. Organic substrates were incompletely oxidized to acetate. 16S rRNA gene sequence analysis showed that the closest recognized relative to strain SB1(T) was Desulfovibrio gigas DSM 1382(T) (97.5 % similarity). The G+C content of the genomic DNA of strain SB1(T) was 62.2 mol%, comparable with that of Desulfovibrio gigas DSM 1382(T) (60.2 mol%). However, the level of DNA-DNA relatedness between strain SB1(T) and Desulfovibrio gigas DSM 1382(T) was only 56.0 %, indicating that the two strains are not related at the species level. Strain SB1(T) could also be differentiated from Desulfovibrio gigas based on phenotypic characteristics, such as major cellular fatty acid composition (anteiso-C(15 : 0), iso-C(14 : 0) and C(18 : 1) cis 9) and substrate utilization. Strain SB1(T) is therefore considered to represent a novel species of the genus Desulfovibrio, for which the name Desulfovibrio paquesii sp. nov. is proposed. The type strain is SB1(T) (=DSM 16681(T)=JCM 14635(T)).

Desulfovibrio portus sp. nov., a novel sulfate-reducing bacterium in the class Deltaproteobacteria isolated from an estuarine sediment

A strictly anaerobic, mesophilic, sulfate-reducing bacterial strain (MSL79T) isolated from an estuarine sediment in the Sea of Japan of the Japanese islands was characterized phenotypically and phylogenetically. Cells were Gram-negative, motile with a polar flagellum, non-spore-forming, curved rods. Cells had desulfoviridin and c-type cytochrome. Catalase and oxidase activities were not detected. The optimum NaCl concentration for growth was 2.0% (wt/vol). The optimum temperature was 35 degrees C and the optimum pH was 6.5. Strain MSL79T utilized H2, formate, pyruvate, lactate, fumarate, malate, succinate, ethanol, propanol and butanol as electron donors for sulfate reduction. The organic electron donors were incompletely oxidized to mainly acetate. Sulfite and thiosulfate were used as electron acceptors with lactate as an electron donor. Without electron acceptors, pyruvate, fumarate and malate supported the growth. The genomic DNA G+C content was 62.1 mol%. Menaquinone MK-6(H2) was the major respiratory quinone. Major cellular fatty acids were C16:0, iso-C15:0, anteiso-C15:0, iso-C17:0, anteiso-C17:0 and iso-C17:1omega9. Phylogenetic analysis based on the 16S rRNA gene sequence as well as the alpha-subunit of dissimilatory sulfite reductase gene sequence assigned the strain to the family Desulfovibrionaceae within the class Deltaproteobacteria. The closest validly described species based on the 16S rRNA gene sequences were Desulfovibrio aespoeensis (sequence similarity; 95.0%) and Desulfovibrio profundus (94.3%). On the basis of the significant differences in the 16S rRNA gene sequences and the phenotypic characteristics between strain MSL79T and each of the most closely related species, Desulfovibrio portus sp. nov. is proposed. The type strain is MSL79T (=JCM 14722T=DSM 19338T).

Anaerobic degradation of citrate under sulfate reducing and methanogenic conditions

Citrate is an important component of metal processing effluents such as chemical mechanical planarization wastewaters of the semiconductor industry. Citrate can serve as an electron donor for sulfate reduction applied to promote the removal of metals, and it can also potentially be used by methanogens that coexist in anaerobic biofilms. The objective of this study was to evaluate the degradation of citrate with sulfate-reducing and methanogenic biofilms. During batch bioassays, the citrate, acetate, methane and sulfide concentrations were monitored. The results indicate that independent of the biofilm or incubation conditions used, citrate was rapidly fermented with specific rates ranging from 566 to 720 mg chemical oxygen demand (COD) consumed per gram volatile suspended solids per day. Acetate was found to be the main fermentation product of citrate degradation, which was later degraded completely under either methanogenic or sulfate reducing conditions. However, if either sulfate reduction or methanogenesis was infeasible due to specific inhibitors (2-bromoethane sulfonate), absence of sulfate or lack of adequate microorganisms in the biofilm, acetate accumulated to levels accounting for 90-100% of the citrate-COD consumed. Based on carbon balances measured in phosphate buffered bioassays, acetate, CO(2) and hydrogen are the main products of citrate fermentation, with a molar ratio of 2:2:1 per mol of citrate, respectively. In bicarbonate buffered bioassays, acetogenesis of H(2) and CO(2) increased the yield of acetate. The results taken as a whole suggest that in anaerobic biofilm systems, citrate is metabolized via the formation of acetate as the main metabolic intermediate prior to methanogenesis or sulfate reduction. Sulfate reducing consortia must be enriched to utilize acetate as an electron donor in order to utilize the majority of the electron-equivalents in citrate.

Competitive Oxidation of Volatile Fatty Acids by Sulfate- and Nitrate-Reducing Bacteria from an Oil Field in Argentina

Acetate, propionate, and butyrate, collectively referred to as volatile fatty acids (VFA), are considered among the most important electron donors for sulfate-reducing bacteria (SRB) and heterotrophic nitrate-reducing bacteria (hNRB) in oil fields. Samples obtained from a field in the Neuquén Basin, western Argentina, had significant activity of mesophilic SRB, hNRB, and nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). In microcosms, containing VFA (3 mM each) and excess sulfate, SRB first used propionate and butyrate for the production of acetate, which reached concentrations of up to 12 mM prior to being used as an electron donor for sulfate reduction. In contrast, hNRB used all three organic acids with similar kinetics, while reducing nitrate to nitrite and nitrogen. Transient inhibition of VFA-utilizing SRB was observed with 0.5 mM nitrite and permanent inhibition with concentrations of 1 mM or more. The addition of nitrate to medium flowing into an upflow, packed-bed bioreactor with an established VFA-oxidizing SRB consortium led to a spike of nitrite up to 3 mM. The nitrite-mediated inhibition of SRB led, in turn, to the transient accumulation of up to 13 mM of acetate. The complete utilization of nitrate and the incomplete utilization of VFA, especially propionate, and sulfate indicated that SRB remained partially inhibited. Hence, in addition to lower sulfide concentrations, an increase in the concentration of acetate in the presence of sulfate in waters produced from an oil field subjected to nitrate injection may indicate whether the treatment is successful. The microbial community composition in the bioreactor, as determined by culturing and culture-independent techniques, indicated shifts with an increasing fraction of nitrate. With VFA and sulfate, the SRB genera Desulfobotulus, Desulfotignum, and Desulfobacter as well as the sulfur-reducing Desulfuromonas and the NR-SOB Arcobacter were detected. With VFA and nitrate, Pseudomonas spp. were present. hNRB/NR-SOB from the genus Sulfurospirillum were found under all conditions.

Desulfoluna butyratoxydans gen. nov., sp. nov., a novel Gram-negative, butyrate-oxidizing, sulfate-reducing bacterium isolated from an estuarine sediment in Japan

A strictly anaerobic, mesophilic, sulfate-reducing bacterial strain, designated MSL71T, was isolated from an estuarine sediment from the Sea of Japan bordering the Japanese islands and was characterized phenotypically and phylogenetically. The cells were found to be Gram-negative, motile, non-spore-forming, slightly curved rods. Catalase and oxidase activities were not detected. The optimum NaCl concentration for growth was 2.0 % (w/v), the optimum temperature was 30 degrees C and the optimum pH was 6.3. Strain MSL71T utilized formate, butyrate, pyruvate, lactate, malate, ethanol, propanol, butanol, glycerol and H2 as electron donors for sulfate reduction. The organic electron donors used were incompletely oxidized, mainly to acetate. The strain did not use acetate, propionate, fumarate, succinate, methanol, glycine, alanine, serine, aspartate or glutamate. Sulfite and thiosulfate were used as electron acceptors with lactate as an electron donor, but fumarate was not utilized. Without electron acceptors, pyruvate and malate, but not lactate or fumarate, were fermented. The genomic DNA G+C content was 62.0 mol%. Menaquinone MK-8(H4) was the major respiratory quinone. The major cellular fatty acids were C14 : 0, C16 : 0, C16 : 1 omega 7, C18 : 1 omega 9, C18 : 1 omega 7 and C14 : 0 3-OH. A phylogenetic analysis based on the 16S rRNA gene sequence placed the strain in the class Deltaproteobacteria. The closest recognized relative of strain MSL71T was Desulfofrigus fragile (93.9 % sequence similarity) and the next closest recognized species was Desulfofrigus oceanense (93.5 %). On the basis of the significant differences in the 16S rRNA gene sequence and phenotypic characteristics between strain MSL71T and each of the related species, a novel genus and species, Desulfoluna butyratoxydans gen. nov., sp. nov., are proposed to accommodate strain MSL71T. The type strain is MSL71T (=JCM 14721T=DSM 19427T).

Remoção de sulfato de águas residuárias industriais em reator anaeróbio de leito fixo operado em bateladas sequenciais

Avaliou-se o potencial de uso reator anaeróbio operado em bateladas seqüenciais com biomassa imobilizada (ASBBR), em escala piloto, no tratamento de água residuária industrial contendo elevadas concentrações de sulfato. O ASBBR, com volume total de 1.2 m³, foi preenchido com carvão mineral como meio suporte para imobilização da biomassa (leito fixo). Foram aplicadas cargas de 0,15; 0,30; 0,65; 1,30 e 1,90 kg SO4-2/ciclo (ou batelada) com duração de 48 h, correspondendo, respectivamente, às concentrações de sulfato no afluente de 0,25; 0,50; 1,0; 2,0 e 3,0 gSO4-2.l-1. Utilizou-se etanol como doador de elétrons para a redução do sulfato. O reator foi operado à temperatura ambiente (29±8ºC), tendo sido obtidas eficiências médias na redução de sulfato entre 88 e 92% em 92 ciclos (275 dias). Os resultados obtidos permitem concluir que o uso de reatores ASBBR constitui-se em alternativa eficiente para a remoção de sulfatos de águas residuárias com características semelhantes às utilizadas neste trabalho.

Hydrolysed Cellulose Material as Sulfate Reduction Electron Donor to Treat Metal- and Sulfate Containing Waste Water

The amenability of hydrolysed cellulose material to low cost sulfate reduction electron donor was examined with fluidized bed reactor (FBR) treating synthetic mine waste water. The studied cellulose material was dried Phalaris arundinacea reed, which was acid hydrolysed (1.5 w/w % H2SO4, 7 w/w % solids) at 120oC to hydrolyse polymeric materials to biodegradable monomers. The FBR was operated at 35oC, and ethanol has previously been used as the electron donor. FBR was fed with synthetic waste water (pH 4.5) containing soluble fraction of Phalaris arundinacea hydrolysate, metals (Fe and Zn) and sulfate. The switch of the electron donor from ethanol to hydrolysate was successful. The acidic influent was neutralized in the FBR by the alkalinity produced in the oxidation of Phalaris arundinacea hydrolysate. The main oxidation product of the soluble hydrolysate was acetate, which accumulated in the FBR during overloading. The percent sulfate reduction remained in the range of 40-95 %. The highest obtained hydrogen sulfide production was 0.91 g L-1d-1 at a hydraulic retention time (HRT) of 9 h, while highest sulfate reduction was 8.4 g L-1d-1 (HRT 8 h). Iron and zinc precipitated in the FBR, and highest metal precipitation rates were 1.14 g Fe L-1 d-1 and 30 mg Zn L-1d-1 (HRT 8 h). The electron donor load was measured as soluble chemical oxygen demand (CODs), and highest CODs removal rate was 2.13 g L-1d-1 (HRT 9 h) and CODs percent oxidation 92 % (HRT 10 h). Soluble Phalaris arundinacea hydrolysate was found to be a suitable electron donor for sulfate reducing FBR and mine waste water treatment. The soluble fraction of Phalaris arundinacea hydrolysate was used very efficiently by sulfate-reducing bacteria (SRB). Additionally, batch bottle assays showed that SRB-enrichment also used solid, dried Phalaris arundinacea as electron donor for sulfate reduction (total sulfide yield 340 mg L-1 in 14 days). The results of sulfate reduction and iron precipitation are shown in figures 1 A-B.

Function of Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.

Desulfobulbus japonicus sp. nov., a novel Gram-negative propionate-oxidizing, sulfate-reducing bacterium isolated from an estuarine sediment in Japan

Two strictly anaerobic, mesophilic, sulfate-reducing bacterial strains, Pro1(T) and Pro16, were isolated from an estuarine sediment in the Sea of Japan of the Japanese islands and were characterized by phenotypic and phylogenetic methods. Strains Pro1(T) and Pro16 had almost the same physiological and chemotaxonomic characteristics. Cells of both strains were Gram-negative, motile, non-spore-forming rods. Catalase activity was not detected. The optimum NaCl concentration for growth was 3.0 % (w/v). The optimum temperature for growth was 35 degrees C and the optimum pH was 6.7. Both strains used formate, propionate, pyruvate, lactate, fumarate, malate, ethanol, propanol, butanol, glycerol, alanine, glucose, fructose and H(2) as electron donors for sulfate reduction and did not use acetate, butyrate, succinate, methanol, glycine, serine, aspartate, glutamate, cellobiose or sucrose. Organic electron donors were incompletely oxidized mainly to acetate. Both strains also used thiosulfate as an electron acceptor. Without electron acceptors, both strains fermented pyruvate and lactate. The genomic DNA G+C contents of strains Pro1(T) and Pro16 were 48.6 and 46.0 mol%, respectively. The major respiratory quinone of both strains was menaquinone MK-5(H(2)). Major cellular fatty acids of both strains were C(15 : 0), C(16 : 0), C(17 : 1)omega6 and C(18 : 1)omega7. Phylogenetic analysis based on 16S rRNA gene sequences placed both strains in the class Deltaproteobacteria. The closest recognized relative of strains Pro1(T) and Pro16 was Desulfobulbus mediterraneus with sequence similarities of 95.2 and 94.8 %, respectively. Based on phylogenetic, physiological and chemotaxonomic characteristics, strains Pro1(T) and Pro16 represent a novel species of the genus Desulfobulbus, for which the name Desulfobulbus japonicus is proposed. The type strain is Pro1(T)(=JCM 14043(T)=DSM 18378(T)) and strain Pro16 (=JCM 14044=DSM 18379) is a reference strain.

Effect of hydraulic retention time on sulfate reduction in a carbon monoxide fed thermophilic gas lift reactor

Thermophilic hydrogenogenic carbon monoxide (CO) converting microorganisms present in anaerobic sludge play a crucial role in the application of CO as electron donor for sulfate reduction. Hydrogenogenic CO conversion was investigated in a gas lift reactor (55 °C) at different hydraulic retention times (HRT). Operation at a HRT>9 h resulted in predominant consumption of CO-derived H 2 by methanogens (up to 90%) and thus in a poor sulfate reduction efficiency of less than 15%. At HRTs<4 h, consumption of the CO-derived H 2 was dominated by sulfate-reducers, i.e. up to 95% of H 2 was used for sulfate reduction. Sulfate reduction rates of 17 mmol L −1 d −1 were achieved at a HRT of 3 h, with over 87% of the H 2 produced used for sulfate reduction. Methane production, however, persisted when operating under these conditions and increasing the HRT by returning it to values >5.5 h resulted in a dominance of methanogenesis over sulfate reduction. The sulfate reduction rates were limited by the amount of CO supplied and its conversion efficiency (about 85%) at higher CO loads likely resulting from a low biomass retention.

Desulfopila aestuarii gen. nov., sp. nov., a Gram-negative, rod-like, sulfate-reducing bacterium isolated from an estuarine sediment in Japan

A strictly anaerobic, mesophilic, sulfate-reducing bacterial strain (MSL86(T)) isolated from an estuarine sediment in the Sea of Japan (around the Japanese islands) was characterized phenotypically and phylogenetically. The cells were found to be Gram-negative, motile, non-spore-forming rods. Catalase was not detected. The optimum NaCl concentration for growth was 1.0 % (w/v) and the optimum temperature was 35 degrees C. Strain MSL86(T) was slightly alkaliphilic, with optimum growth at pH 7.5-7.6. Organic electron donors were incompletely oxidized to (mainly) acetate. Strain MSL86(T) utilized formate, pyruvate, lactate, fumarate, ethanol, propanol, butanol and glycerol as electron donors for sulfate reduction and did not use acetate, propionate, butyrate, succinate, malate, methanol, glycine, alanine, serine, aspartate, glutamate or H(2). Sulfite, thiosulfate and fumarate were used as electron acceptors with lactate as an electron donor. Without electron acceptors, the strain fermented pyruvate and fumarate. The genomic DNA G+C content was 54.4 mol%. Menaquinone MK-8(H(4)) was the major respiratory quinone. The major cellular fatty acids were C(16 : 0), C(16 : 1)omega7, C(16 : 1)omega5 and C(17 : 1)omega6. A phylogenetic analysis based on the 16S rRNA gene sequence placed the strain in the class Deltaproteobacteria. The recognized bacterium most closely related to strain MSL86(T) was [Desulfobacterium] catecholicum DSM 3882(T) (sequence similarity 94.4 %), and the next most closely related recognized species were Desulfotalea psychrophila (94.2 % sequence similarity with the type strain) and Desulfotalea arctica (93.7 %). As the physiological and chemotaxonomic characteristics of MSL86(T) were distinctly different from those of any related species, a novel genus and species Desulfopila aestuarii gen. nov., sp. nov. are proposed to accommodate the strain. The type strain of Desulfopila aestuarii is MSL86(T) (=JCM 14042(T)=DSM 18488(T)).

Chemical Characterisation of Organic Electron Donors For Sulfate Reduction For Potential Use in Acid Mine Drainage Treatment

The production of acid mine drainage (AMD) containing high amounts of sulfate, heavy metals and low pH is of increasing concern. AMD is highly corrosive and results in economic and environmental problems. Organic electron donors for sulfate reduction were chemically characterised for potential use in AMD treatment. This was done in a process to develop a correlation between chemical composition and the capacity to drive sulfate reduction. Potential organic electron donors for sulfate reduction were chemically characterised in terms of dry matter content, ash content, total Kjeldahl nitrogen, lignin content, cellulose content, crude fat, crude fibre, in vitro digestibility, water-soluble carbohydrates, total non-structural carbohydrates and starch content. The chemical composition of the organic electron donors was then compared to results obtained from pilot plant studies where the organic electron donors for sulfate reduction were evaluated in terms of sulfate reduction. The chemical composition of the carbon source severely impacted its capacity to drive sulfate reduction and may be used to assist in predicting the sulfate reduction capacity of a carbon source. Organic electron donors for sulfate reduction high in protein content and low in lignin content or high in carbohydrate and crude fat content increased the capacity of a carbon source to drive sulfate reduction. The higher the fibre content of a carbon source, the lower the capacity to drive sulfate reduction. No correlation could be drawn between % dry matter, % ash content and sulfate reduction for the organic electron donors tested. Chemical characterisation can be used to assist in predicting sulfate reduction capacity of organic electron donors.

Zero valent iron as an electron‐donor for methanogenesis and sulfate reduction in anaerobic sludge

Zero valent iron (ZVI) is a reactive media commonly utilized in permeable reactive barriers (PRBs). Sulfate reducing bacteria are being considered for the immobilization of heavy metals in PRBs. The purpose of this study was to evaluate the potential of ZVI as an electron donor for sulfate reduction in natural mixed anaerobic cultures. The ability of methanogens to utilize ZVI as an electron-donor was also explored since these microorganisms often compete with sulfate reducers for common substrates. Four grades of ZVI of different particle sizes (1.120, 0.149, 0.044, and 0.010 mm diameter) were compared as electron donor in batch bioassays inoculated with anaerobic bioreactor sludge. Methanogenesis was evaluated in mineral media lacking sulfate. Sulfate reduction was evaluated in mineral media containing sulfate and the specific methanogenic inhibitor, 2-bromoethane sulfonate. ZVI contributed to significant increases in methane production and sulfate reduction-compared to endogenous substrate controls. The rates of methane formation or sulfate reduction were positively correlated with the surface area of ZVI. The highest rates of 0.310 mmol CH4 formed/mol Fe0.day and 0.804 mmol SO4(2-) reduced/mol Fe0.day were obtained with the finest grade of ZVI (0.01 mm). The results demonstrate that ZVI is readily utilized as a slow-release electron donor for methanogenesis and sulfate reduction in anaerobic sludge; and therefore, has a promising potential in bioremediation applications.

Gene expression analysis of energy metabolism mutants of Desulfovibrio vulgaris Hildenborough indicates an important role for alcohol dehydrogenase.

Comparison of the proteomes of the wild-type and Fe-only hydrogenase mutant strains of Desulfovibrio vulgaris Hildenborough, grown in lactate-sulfate (LS) medium, indicated the near absence of open reading frame 2977 (ORF2977)-coded alcohol dehydrogenase in the hyd mutant. Hybridization of labeled cDNA to a macroarray of 145 PCR-amplified D. vulgaris genes encoding proteins active in energy metabolism indicated that the adh gene was among the most highly expressed in wild-type cells grown in LS medium. Relative to the wild type, expression of the adh gene was strongly downregulated in the hyd mutant, in agreement with the proteomic data. Expression was upregulated in ethanol-grown wild-type cells. An adh mutant was constructed and found to be incapable of growth in media in which ethanol was both the carbon source and electron donor for sulfate reduction or was only the carbon source, with hydrogen serving as electron donor. The hyd mutant also grew poorly on ethanol, in agreement with its low level of adh gene expression. The adh mutant grew to a lower final cell density on LS medium than the wild type. These results, as well as the high level of expression of adh in wild-type cells on media in which lactate, pyruvate, formate, or hydrogen served as the sole electron donor for sulfate reduction, indicate that ORF2977 Adh contributes to the energy metabolism of D. vulgaris under a wide variety of metabolic conditions. A hydrogen cycling mechanism is proposed in which protons and electrons originating from cytoplasmic ethanol oxidation by ORF2977 Adh are converted to hydrogen or hydrogen equivalents, possibly by a putative H(2)-heterodisulfide oxidoreductase complex, which is then oxidized by periplasmic Fe-only hydrogenase to generate a proton gradient.

Effects of deletion of genes encoding Fe-only hydrogenase of Desulfovibrio vulgaris Hildenborough on hydrogen and lactate metabolism.

The physiological properties of a hyd mutant of Desulfovibrio vulgaris Hildenborough, lacking periplasmic Fe-only hydrogenase, have been compared with those of the wild-type strain. Fe-only hydrogenase is the main hydrogenase of D. vulgaris Hildenborough, which also has periplasmic NiFe- and NiFeSe-hydrogenases. The hyd mutant grew less well than the wild-type strain in media with sulfate as the electron acceptor and H(2) as the sole electron donor, especially at a high sulfate concentration. Although the hyd mutation had little effect on growth with lactate as the electron donor for sulfate reduction when H(2) was also present, growth in lactate- and sulfate-containing media lacking H(2) was less efficient. The hyd mutant produced, transiently, significant amounts of H(2) under these conditions, which were eventually all used for sulfate reduction. The results do not confirm the essential role proposed elsewhere for Fe-only hydrogenase as a hydrogen-producing enzyme in lactate metabolism (W. A. M. van den Berg, W. M. A. M. van Dongen, and C. Veeger, J. Bacteriol. 173:3688-3694, 1991). This role is more likely played by a membrane-bound, cytoplasmic Ech-hydrogenase homolog, which is indicated by the D. vulgaris genome sequence. The physiological role of periplasmic Fe-only hydrogenase is hydrogen uptake, both when hydrogen is and when lactate is the electron donor for sulfate reduction.

Mechanistic study of microbial control of hydrogen sulfide production in oil reservoirs.

Microbial control of biogenic production of hydrogen sulfide in oil fields was studied in a model system consisting of pure cultures of the nitrate-reducing, sulfide-oxidizing bacterium (NR-SOB) Thiomicrospira sp. strain CVO and the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6, as well as in microbial cultures enriched from produced water of a Canadian oil reservoir. The presence of nitrate at concentrations up to 20 mM had little effect on the rate of sulfate reduction by a pure culture of Lac6. Addition of CVO imposed a strong inhibition effect on production of sulfide. In the absence of added nitrate SRB we were able to overcome this effect after an extended lag phase. Simultaneous addition of CVO and nitrate stopped the production of H2S immediately. The concentration of sulfide decreased to a negligible level due to nitrate-dependent sulfide oxidation activity of CVO. This was not prevented by raising the concentration of Na-lactate, the electron donor for sulfate reduction. Similar results were obtained with enrichment cultures. Enrichments of produced water with sulfide and nitrate were dominated by CVO, whereas enrichments with sulfate and Na-lactate were dominated by SRB. Addition of an NR-SOB enrichment to an SRB enrichment inhibited the production of sulfide. Subsequent addition of sufficient nitrate caused the sulfide concentration to drop to zero. A similar response was seen in the presence of nitrate alone, although after a pronounced lag time, it was needed for emergence of a sizable CVO population. The results of the present study show that two mechanisms are involved in microbial control of biogenic sulfide production. First, addition of NR-SOB imposes an inhibition effect, possibly by increasing the environmental redox potential to levels which are inhibitory for SRB. Second, in the presence of sufficient nitrate, NR-SOB oxidize sulfide, leading to its complete removal from the environment. Successful microbial control of H2S in an oil reservoir is crucially dependent on the simultaneous presence of NR-SOB (either indigenous population or injected) and nitrate in the environment.

Thermophilic sulfate and sulfite reduction with methanol in a high rate anaerobic reactor

Thermophilic sulfite and sulfate reduction offers good prospects as part of an alternative technology to conventional off-gas desulfurization technologies. Thermophilic sulfate and sulfite reduction with methanol as the sole carbon and energy source for the sulfate reducing bacteria was studied in lab-scale Expanded Granular Sludge Bed (EGSB) reactors operated at 65 °C and pH 7.5. At a hydraulic retention time (HRT) of 4 hr, sulfite and sulfate elimination rates of up to 0.22 mol-S.l-1.day-1 (100% elimination) and 0.15 mol-S.l-1.day-1 (80% elimination), respectively, were achieved. Sulfite and sulfate reduction accounted for 85–90% of the electrons released during degradation of methanol. In addition, 10–13% and 1–2% of the consumed methanol was converted to acetate and methane, respectively. Acetate was not utilized as electron donor for sulfate reduction. Acetate production seemed to be linearly correlated to the amount of sulfite and sulfate reduced. Sulfite disproportionating activity of the sludge was demonstrated by the simultaneous appearance of sulfide and sulfate in batch tests with sulfite. However, sulfite disproportionation rates were 4 times lower than sulfate reduction rates with methanol. The results clearly demonstrate that methanol can be efficiently used as electron and carbon source to obtain high sulfite and sulfate elimination rates in thermophilic bioreactors.

Deletion of the hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough hampers hydrogen metabolism and low-redox-potential niche establishment.

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a transmembrane redox protein complex (the Hmc complex) that has been proposed to catalyze electron transport linking periplasmic hydrogen oxidation to cytoplasmic sulfate reduction. We have replaced a 5-kb DNA fragment containing most of the hmc operon by the cat gene. The resulting chloramphenicol-resistant mutant D. vulgaris H801 grows normally when lactate or pyruvate serve as electron donors for sulfate reduction. Growth with hydrogen as electron donor for sulfate reduction (acetate and CO2 as the carbon source) is impaired. These results confirm the importance of the Hmc complex in electron transport from hydrogen to sulfate. Mutant H801 is also deficient in low-redox-potential niche establishment. On plates, colony development takes 14 days longer than colony development of the wild-type strain, when the cells use hydrogen as the electron donor. This result suggests that, in addition to transmembrane electron transport from hydrogen to sulfate, the redox reactions catalyzed by the Hmc complex are crucial in establishment of the required low-redox-potential niche that allows single cells to grow into colonies.

Effects of two diamine biocides on the microbial community from an oil field

The effects of diamine (R1-NH-R2-NH2) biocides A and B on the microbial population from an oil field were investigated with reverse sample genome probing (RSGP), a technique designed to track multiple oil field bacteria in a single assay. RSGP studies of sessile microbial populations scraped from corrosion coupons obtained from biocide-treated oil field installations indicated dominance of Desulfovibrio spp. Lac6 and Eth3, or of selected heterotrophs. RSGP of planktonic production water samples indicated a broad distribution of microorganisms that changed with the addition of medium for the growth of sulfate-reducing bacteria, containing different organic acids as electron donors for sulfate reduction. Use of lactate, propionate, or acetate enriched Desulfovibrio spp., Desulfobulbus sp. Pro4, or Desulfobacter spp., respectively. Treatment of lactate- or mixed organic acid-fed planktonic populations with biocides indicated resistance of Lac6 and Eth3 to 400 ppm of biocide B and 40 ppm of biocide A. The dominance of these two Desulfovibrio spp. in many sessile field samples is, therefore, likely caused by biocide selection.Key words: Desulfovibrio, sulfate reduction, microbial corrosion, souring, biocide.