Sulfonates as Terminal Electron Acceptors for Growth of Sulfite-Reducing Bacteria (Desulfitobacterium spp.) and Sulfate-Reducing Bacteria: Effects of Inhibitors of Sulfidogenesis

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)—Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135–149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Unsaturated organic acids as terminal electron acceptors for reductase chains of anaerobic bacteria

This paper summarizes the current knowledge of unsaturated organic acids in their role as terminal electron acceptors of anaerobic bacteria. The mechanisms and enzyme systems involved in the reduction of fumarate by Escherichia coli, Wolinella succinogenes, and some species of the genus Shewanella are considered. Particular attention is given to reduction of the double bond of the unnatural compound methacrylate by the sigma-proteobacterium Geobacter sulfurreducens Am-1. Soluble periplasmic flavocytochromes c, found in bacteria of the genera Shewanella and Geobacter, are involved in the hydration of fumarate (in Shewanella species) and methacrylate (in G. sulfurreducens Am-1). In E. coli and W. succinogenes, fumarate is reduced in cytosol by membrane-bound fumarate reductases. The prospects for research into organic acid reduction at double bonds in bacteria are discussed.     

Oxygen and pyruvate as external electron acceptors for Leuconostoc spp.

L. NURAIDA, I. GRIGOLAVA, J.D. OWENS AND G. CAMPBELL-PLATT. 1992. Leuconostoc mesenteroides NCDO 518, Leuc, mesenteroides NCIB 8710, Leur. mesenteroides NCIB 8023, Leuconostoc sp. Pz 45, Leuconostoc sp. Pz 34 and Leuconostoc sp. Pz 10 were grown in a chemically defined medium with glucose and different external electron acceptors. All strains, except Leuconostoc sp. Pz 10, formed acetate under aerobic conditions. Leuconostoc Pz 10 produced only small amounts of acetate under aerobic or anaerobic conditions. This strain was the only strain not possessing NADH oxidase. All strains produced acetate from a mixture of glucose plus pyruvate. None of the bacteria used glycerol. Oxygen and pyruvate as external electron acceptors increased the cell yield on glucose.     

Growth of sulfate-reducing bacteria with sulfur as electron acceptor

In addition to three new isolates, six strains of representative species of sulfate-reducing bacteria were tested for their capacity to use elemental sulfur as an electron acceptor for growth. There was good growth and sulfide production by strain Norway 4 and the three isolates, two of which had been enriched with sulfur flower and one isolated from a culture with green sulfur bacteria. Slow but definite growth was observed with Desuflovibrio gigas. The type strains of Desulfovibrio desulfuricans, D. vulgaris, and Desulfotomaculum nigrificans as well as Desulfomonas pigra did not grow with sulfur. The four strains that grew well with sulfur flower were straight, nonsporulating rods and did not contain desulfoviridin.     

Quinones as terminal electron acceptors for anaerobic microbial oxidation of phenolic compounds

The capacity of anaerobic granular sludge for oxidizing phenoland p-cresol under anaerobic conditions was studied. Phenol and p-cresolwere completely converted to methane when bicarbonate was the only terminal electron acceptor available. When the humic model compound, anthraquinone-2,6-disulfonate, was included as an alternative electron acceptor in the cultures, the oxidation of the phenolic compounds was coupled to the reduction of the model humic compound to its corresponding hydroquinone, anthrahydroquinone-2,6-disulfonate. These results demonstrate for the first time that the anaerobic degradation of phenolic compounds can be coupled to the reduction of quinones as terminal electron acceptor.     

Oil field souring control by nitrate-reducing Sulfurospirillum spp. that outcompete sulfate-reducing bacteria for organic electron donors

Nitrate injection into oil reservoirs can prevent and remediate souring, the production of hydrogen sulfide by sulfate-reducing bacteria (SRB). Nitrate stimulates nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB) and heterotrophic nitrate-reducing bacteria (hNRB) that compete with SRB for degradable oil organics. Up-flow, packed-bed bioreactors inoculated with water produced from an oil field and injected with lactate, sulfate, and nitrate served as sources for isolating several NRB, including Sulfurospirillum and Thauera spp. The former coupled reduction of nitrate to nitrite and ammonia with oxidation of either lactate (hNRB activity) or sulfide (NR-SOB activity). Souring control in a bioreactor receiving 12.5 mM lactate and 6, 2, 0.75, or 0.013 mM sulfate always required injection of 10 mM nitrate, irrespective of the sulfate concentration. Community analysis revealed that at all but the lowest sulfate concentration (0.013 mM), significant SRB were present. At 0.013 mM sulfate, direct hNRB-mediated oxidation of lactate by nitrate appeared to be the dominant mechanism. The absence of significant SRB indicated that sulfur cycling does not occur at such low sulfate concentrations. The metabolically versatile Sulfurospirillum spp. were dominant when nitrate was present in the bioreactor. Analysis of cocultures of Desulfovibrio sp. strain Lac3, Lac6, or Lac15 and Sulfurospirillum sp. strain KW indicated its hNRB activity and ability to produce inhibitory concentrations of nitrite to be key factors for it to successfully outcompete oil field SRB.     

Some effects of uncouplers and inhibitors on growth and electron transport in rumen bacteria.

Uncouplers and inhibitors of electron transport affected growth and electron transport of rumen bacteria in various ways. Selenomonas ruminantium was not affected by inhibitor and uncoupler concentrations which affected growth and electron transport of Bacteroides ruminicola, B. succinogenes, and Butyrivibrio fibrisolvens. Inhibitors, when active, led to accumulation of reduced electron carriers before the site of action, but differences were found among organisms in the site of action of these inhibitors. Uncouplers reduced the glucose molar growth yields (Ygluc) of B. ruminicola, B. succinogenes, and B. fibrisolvens compared with those obtained without uncouplers. The extent of Ygluc reduction accompanying inhibitor exposure reflected electron transport chain structure. S. ruminantium appeared to obtain its adenosine 5'-triphosphate from substrate-level processes only. The other organisms studied appeared to obtain adenosine 5'-triphosphate both from substrate-level processes and from electron transport but differed in the amount of adenosine 5'-triphosphate obtained from glucose catabolism and in the proportions of adenosine 5'-triphosphate obtained from substrate-level reactions and electron transport.     

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.     

Hydrogen as an electron donor for sulfate-reducing bacteria in slurries of salt marsh sediment

Experiments with a Warburg respirometer showed that a sediment slurry consumed hydrogen from a hydrogen atmosphere, and this consumption was not due to the activity of methanogenic bacteria. The hydrogren uptake was inhibited by the addition of 20 mM molybdate. Further experiments with sediment slurry held in conical flasks under an atmosphere of nitrogen showed that hydrogen accumulated in the headspace when bacterial sulfate reduction was inhibited either by the addition of 20 mM molybdate or by low (<5 mM) sulfate concentrations in the slurry. Methanogenesis was stimulated in the presence of a hydrogen atmosphere or by the addition of 20 mM molybdate. The results confirmed that hydrogren was an important electron donor for sulfate-reducing bacteria present in the sediment. The stimulation of methanogenesis by molybdate could be explained in part by a competition for hydrogen between sulfate-reducing bacteria and hydrogen-metabolizing methanogenic bacteria, but competition for another common substrate, possibly acetate, could also be significant.     

Formation of tellurium nanocrystals during anaerobic growth of bacteria that use Te oxyanions as respiratory electron acceptors

Certain toxic elements support the metabolism of diverse prokaryotes by serving as respiratory electron acceptors for growth. Here, we demonstrate that two anaerobes previously shown to be capable of respiring oxyanions of selenium also achieve growth by reduction of either tellurate [Te(VI)] or tellurite [Te(IV)] to elemental tellurium [Te(0)]. This reduction achieves a sizeable stable-Te-isotopic fractionation (isotopic enrichment factor [epsilon] = -0.4 to -1.0 per ml per atomic mass unit) and results in the formation of unique crystalline Te(0) nanoarchitectures as end products. The Te(0) crystals occur internally within but mainly externally from the cells, and each microorganism forms a distinctly different structure. Those formed by Bacillus selenitireducens initially are nanorods ( approximately 10-nm diameter by 200-nm length), which cluster together, forming larger ( approximately 1,000-nm) rosettes composed of numerous individual shards ( approximately 100-nm width by 1,000-nm length). In contrast, Sulfurospirillum barnesii forms extremely small, irregularly shaped nanospheres (diameter < 50 nm) that coalesce into larger composite aggregates. Energy-dispersive X-ray spectroscopy and selected area electron diffraction indicate that both biominerals are composed entirely of Te and are crystalline, while Raman spectroscopy confirms that they are in the elemental state. These Te biominerals have specific spectral signatures (UV-visible light, Raman) that also provide clues to their internal structures. The use of microorganisms to generate Te nanomaterials may be an alternative for bench-scale syntheses. Additionally, they may also generate products with unique properties unattainable by conventional physical/chemical methods.     

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

Anaerobic mineralization of toluene by enriched sediments with quinones and humus as terminal electron acceptors

The anaerobic microbial oxidation of toluene to CO(2) coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [(13)C]toluene were recovered as (13)CO(2) in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [(13)C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.     

Physiological response of Desulfurispirillum indicum S5 to arsenate and nitrate as terminal electron acceptors

The ability of anaerobic prokaryotes to employ different terminal electron acceptors for respiration enables these organisms to flourish in subsurface ecosystems. Desulfurispirillum indicum strain S5 is an obligate anaerobic bacterium that is able to grow by respiring a range of different electron acceptors, including arsenate and nitrate. Here, we examined the growth, electron acceptor utilization, and gene expression of D. indicum growing under arsenate and nitrate-reducing conditions. Consistent with thermodynamic predictions, the experimental results showed that the reduction of nitrate to ammonium yielded higher cell densities than the reduction of arsenate to arsenite. However, D. indicum grew considerably faster by respiration on arsenate compared with nitrate, with doubling times of 4.3 ± 0.2 h and 19.2 ± 2.0 h, respectively. Desulfurispirillum indicum growing on both electron acceptors exhibited the preferential utilization of arsenate before nitrate. The expression of the arsenate reductase gene arrA was up-regulated approximately 100-fold during arsenate reduction, as determined by qRT-PCR. Conversely, the nitrate reductase genes narG and napA were not differentially regulated under the conditions tested. The results of this study suggest that physiology, rather than thermodynamics, controls the growth rates and hierarchy of electron acceptor utilization in D. indicum.     

Microcalorimetric studies of the growth of sulfate-reducing bacteria: energetics of Desulfovibrio vulgaris growth.

The metabolism of Desulfovibrio vulgaris Hildenborough grown on medium containing lactate or pyruvate plus a high concentration of sulfate (36 mM) was studied. Molecular growth yields were 6.7 +/- 1.3 and 10.1 +/- 1.7 g/mol for lactate and pyruvate, respectively. Under conditions in which the energy source was the sole growth-limiting factor, we observed the formation of 0.5 mol of hydrogen per mol of lactate and 0.1 mol of hydrogen per mol of pyruvate. The determination of metabolic end products revealed that D. vulgaris produced, in addition to normal end products (acetic acid, carbon dioxide, hydrogen sulfide) and molecular hydrogen, 2 and 5% of ethanol per mol of lactate and pyruvate, respectively. Power-time curves of growth of D. vulgaris on lactate and pyruvate were obtained, by the microcalorimetric Tian-Calvet apparatus. The enthalpies (delta Hmet) associated with the oxidation of these substrates and calculated from growth thermograms were -36.36 +/- 5 and -70.22 +/- 3 kJ/mol of lactate and pyruvate, respectively. These experimental values were in agreement with the homologous values assessed from the theoretical equations of D. vulgaris metabolism of both lactate and pyruvate. The hydrogen production by this sulfate reducer constitutes an efficient regulatory system of electrons, from energy source through the pathway of sulfate reduction. This hydrogen value may thus facilitate interactions between this strain and other environmental microflora, especially metagenic bacteria.     

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol g _VSS ^?1 day^?1 compared to lactate (32 µmol g _VSS ^?1 day^?1), acetate (26 µmol g _VSS ^?1 day^?1) and methane (4.7 µmol g _VSS ^?1 day^?1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane.     

Amino-terminal amino acid sequences of electron transfer proteins from Gram-negative bacteria as indicators of their cellular localization: the sulfate-reducing bacteria

Abstract Data are presented that all known periplasmic redox proteins from the sulfate reducing bacteria included in the genus, Desulfovibrio have aminoterminal (N-terminal) amino-acid sequences commonly found in other Gram-negative bacteria and are indicative of recognition sites for signal peptides. In contrast, none of the cytoplasmic redox proteins exhibited these unique N-terminal amino-acid sequences. It is proposed that the N-terminal amino-acid residues of a given protein can be used as an indicator of its cellular localization within the bacterial cell.     

Trimethylamine oxide: a terminal electron acceptor in anaerobic respiration of bacteria.

Trimethylamine oxide (TMAO) stimulated both the anaerobic growth rate and the growth yield of Proteus NTHC 153. The molar growth yield from glucose and pyruvate in tryptone/yeast extract medium doubled in the presence of TMAO, and the organism grew anaerobically on the non-fermentable substrates L-lactate and formate when TMAO was added to the medium. We conclude that TMAO stimulated growth by serving as a terminal electron acceptor in an oxidative phosphorylation process.