Anerobic degradation of 1,2-propanediol by a new Desulfovibrio strain and D. alcoholovorans

A sulfate-reducing bacterium, strain HDv, was isolated from the anoxic soil of a ricefield using lactate as electron donor. Cells were gram-negative, motile, nonsporulating curved rods, with single polar flagella. Substrates were incompletely oxidized to acetate and included glycerol, 1,2-and 1,3-propanediol. Sulfate, sulfite, thiosulfate, elemental sulfur, fumarate, maleate, and malate were utilized as electron acceptors. Pyruvate, fumarate, maleate, malate and dihydroxyacetone were fermented. Desulfoviridin and c-type cytochromes were present. The DNA base composition was 66.6 ± 0.3 mol% G+C. The isolate was identified as a Desulfovibrio sp.; its metabolic properties were somewhat different from those of previously described Desulfovibrio species. Comparative biochemical study of 1,2-propanediol dissimilation by the new isolate and Desulfovibrio alcoholovorans showed that NAD-dependent dehydrogenases play a key role in the catabolism of this substrate. The hypothetical pathways of 1,2-propanediol degradation by Desulfovibrio spp. are presented.     

Characterization of Desulfovibrio fructosovorans sp. nov.

Desulfovibrio strain JJ isolated from estuarine sediment differed from all other described Desulfovibrio species by the ability to degrade fructose. The oxidation was incomplete, leading to acetate production. Fructose, malate and fumarate were fermented mainly to succinate and acetate in the absence of an external electron acceptor. The pH and temperature optima for growth were 7.0 and 35° C respectively. Strain JJ was motile by means of a single polar flagellum. The DNA base composition was 64.13% G+C. Cytochrome c ₃ and desulfoviridin were present. These characteristics established the isolate as a new species of the genus Desulfovibrio, and the name Desulfovibrio fructosovorans is proposed.     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Methanogenesis from furfural by defined mixed cultures

Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO₂. In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural. Received: 15 August 2001 / Accepted: 20 September 2001     

Metabolism of 2,4,6-trinitrotoluene (TNT) by Desulfovibrio sp. (B strain)

A sulfate-reducing bacterium, Desulfovibrio sp. (B strain) isolated from an anaerobic reactor treating furfural-containing waste-water was studied for its ability to metabolize trinitrotoluene (TNT). The result showed that this isolate could transform 100 ppm TNT within 7 to 10 days of incubation at 37°C, when grown with 30 mm pyruvate as the primary carbon source and 20 mm sulfate as electron acceptor. Under these conditions, the main intermediate produced was 2,4-diamino-6-nitrotoluene. Under culture conditions where TNT served as the sole source of nitrogen for growth with pyruvate as electron donor and sulfate as electron acceptor, TNT was first converted to 2,4-diamino-6-nitrotoluene within 10 days of incubation. This intermediate was further converted to toluene by a reductive deamination process via triaminotoluene. Apart from pyruvate, various other carbon sources such as ethanol, lactate, formate and H₂ + CO₂ were also studied as potential electron donors for TNT metabolism. The rate of TNT biotransformation by Desulfovibrio sp. (B strain) was compared with other sulfate-reducing bacteria and the results were evaluated. This new strain may be useful in decontaminating TNT-contaminated soil and water under anaerobic conditions in conjunction with toluene-degrading denitrifiers (Pseudomonas spp.) or toluene-degrading sulfate reducers in a mixed culture system. Correspondence to: R. Boopathy     

A sulfate-reducing bacterium from the oxic layer of a microbial mat from Solar Lake (Sinai), Desulfovibrio oxyclinae sp. nov.

In an investigation on the oxygen tolerance of sulfate-reducing bacteria, a strain was isolated from a 10⁷-fold dilution of the upper 3-mm layer of a hypersaline cyanobacterial mat (transferred from Solar Lake, Sinai). The isolate, designated P1B, appeared to be well-adapted to the varying concentrations of oxygen and sulfide that occur in this environment. In the presence of oxygen strain P1B respired aerobically with the highest rates [260 nmol O₂ min^–1 (mg protein)^–1] found so far among marine sulfate-reducing bacteria. Besides H₂ and lactate, even sulfide or sulfite could be oxidized with oxygen. The sulfur compounds were completely oxidized to sulfate. Under anoxic conditions, it grew with sulfate, sulfite, or thiosulfate as the electron acceptor using H₂, lactate, pyruvate, ethanol, propanol, or butanol as the electron donor. Furthermore, in the absence of electron donors the isolate grew by disproportionation of sulfite or thiosulfate to sulfate and sulfide. The highest respiration rates with oxygen were obtained with H₂ at low oxygen concentrations. Aerobic growth of homogeneous suspensions was not obtained. Additions of 1% oxygen to the gas phase of a continuous culture resulted in the formation of cell clumps wherein the cells remained viable for at least 200 h. It is concluded that strain P1B is oxygen-tolerant but does not carry out sulfate reduction in the presence of oxygen under the conditions tested. Analysis of the 16S rDNA sequence indicated that strain P1B belongs to the genus Desulfovibrio, with Desulfovibrio halophilus as its closest relative. Based on physiological properties strain P1B could not be assigned to this species. Therefore, a new species, Desulfovibrio oxyclinae, is proposed. Received: 7 August 1996 / Accepted: 29 January 1997     

Eubacterium acidaminophilum sp. nov., a versatile amino acid-degrading anaerobe producing or utilizing H2 or formate

An obligately anaerobic, rod-shaped bacterium was isolated on alanine in co-culture with H₂-scavenging Desulfovibrio and obtained in pure culture with glycine as sole fermentation substrate. The isolated strain, al-2, was motile by a polar to subpolar flagellum and stained Gram-positive. The guanine plus cytosine content of the DNA was 44.0 mol%. Strain al-2 grew in defined, reduced glycine media supplemented with biotin. The pure culture fermented 4 mol glycine to 3 mol acetate, 4 mol ammonia and 2 mol CO₂. Under optimum conditions (34°C, pH 7.3), the doubling time on glycine was 60 min and the molar growth yield 7.6 g cell dry mass. Serine was fermented to acetate, ethanol, CO₂, H₂ and ammonia. In addition, betaine, sarcosine or creatine served as substrates for growth and acetate production if H₂, formate or e.g. valine were added as H-donors. In pure culture on alanine under N₂, strain al-2 grew very poorly and produced H₂ up to a partial pressure of 3.6 kPa (0.035 atm). Desulfovibrio species, Methanospirillum hungatei and Acetobacterium woodii served as H₂-scavengers that allowed good syntrophic growth on alanine. The co-cultures also grew on aspartate, leucine, valine or malate. Alanine and aspartate were stoichiometrically degraded to acetate and ammonia, whereas the reducing equivalents were recovered as H₂S, CH₄ or newly synthetized acetate, respectively. Growth of strain al-2 in co-culture with the hydrogenase-negative, formate-utilizing Desulfovibrio baarsii indicated that a syntrophy was also possible by interspecies formate transfer. Growth on glycine, or on betaine, sarcosine or creatine (plus H-donors) depended strictly on the addition of selenite (0.1 M); selenite was not required for fermentation of serine, or for degradation of alanine, aspartate or valine by the co-cultures. Cell-free extracts of glycine-grown cells contained active glycine reductase, glycine decarboxylase and reversible methyl viologen-dependent formate dehydrogenase in addition to the other enzymes necessary for an oxidation to CO₂. In all reactions NADP was the preferred H-carrier. Both formate and glycine could be synthesized from bicarbonate. Serine-grown cells did not contain serine hydroxymethyl transferase but serine dehydratase and other enzymes commonly involved in pyruvate metabolism to acetate, CO₂ and H₂. The enzymes involved in glycine metabolism were repressed during growth on serine. By its morphology and physiology, strain al-2 did not resemble described amino acid-degrading species. Therefore, the new isolate is proposed as type strain of a new species, Eubacterium acidaminophilum.     

Desulfovibrio brasiliensis sp. nov., a moderate halophilic sulfate-reducing bacterium from Lagoa Vermelha (Brazil) mediating dolomite formation

A novel halotolerant sulfate-reducing bacterium, Desulfovibrio brasiliensis strain LVform1, was isolated from sediments of a dolomite-forming hypersaline coastal lagoon, Lagoa Vermelha, in the state of Rio de Janeiro, Brazil. The cells are vibrio-shaped and 0.30 to 0.45 m by 1.0 to 3.5 m in size. These bacteria mediate the precipitation of dolomite [CaMg(CO₃)₂] in culture experiments. The strain was identified as a member of the genus Desulfovibrio in the -subclass of the Proteobacteria on the basis of its 16S rRNA gene sequence, its physiological and morphological properties. Strain LVform1 is obligate sodium-dependent and grows at NaCl concentrations of up to 15%. The 16S rRNA sequence revealed that this strain is closely related to Desulfovibrio halophilus (96.2% similarity) and to Desulfovibrio oxyclinae (96.8% similarity), which were both isolated from Solar Lake, a hypersaline coastal lake in the Sinai, Egypt. Strain LVform1 is barotolerant, growing under pressures of up to 370 bar (37 MPa). We propose strain LVform1 to be the type strain of a novel species of the genus Desulfovibrio, Desulfovibrio brasiliensis (type strain LVform1 = DSMZ No. 15816 and JCM No. 12178). The GenBank/EMBL accession number for the 16S rDNA sequence of strain LVform1 is AJ544687.     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Classification of secondary alcohol-utilizing methanogens including a new thermophilic isolate

A thermophilic coccoid methanogenic bacterium, strain TCI, that grew optimally around 55° C was isolated with 2-propanol as hydrogen donor for methanogenesis from CO₂. H₂, formate or 2-butanol were used in addition. Each secondary alcohol was oxidized to its ketone. Growth occurred in defined freshwater as well as salt (2% NaCl, w/v) medium. Acetate was required as carbon source, and 4-aminobenzoate and biotin as growth factors. A need for molybdate or alternatively tungstate was shown.Strain TCI was further characterized together with two formerly isolated mesophilic secondary alcohol-utilizing methanogens, the coccoid strain CV and the spirilloid strain SK. The guanine plus cytosine content of the DNA of the three strains was 55,47, and 39 mol%, respectively. Determination of the molecular weights of the methylreductase subunits and sequencing of ribosomal 16S RNA of strains TCI and CV revealed close relationships to the genus Methanogenium. The new isolate TCI is classified as a strain of the existing species, Methanogenium thermophilum (thermophilicum). For strain CV, that uses ethanol or 1-propanol in addition, a classification as new species, Methanogenium organophilum, is proposed. Strain SK is affiliated with the existing species, Methanospirillum hungatei. The ability to use secondary alcohols was also tested with described species of methanogens. Growth with secondary alcohols was observed with Methanogenium marisnigri, Methanospirillum hungatei strain GP1 and Methanobacterium bryantii, but not with Methanospirillum strains JF1 and M1h, Methanosarcina barkeri, Methanococcus species or thermophilic strains or species other than the new isolate TCI.     

A novel isolate of Desulfovibrio sp. with enhanced ability to reduce Cr(VI)

Kinetic analysis of the reduction of Cr(VI) by resting cell suspensions of Desulfovibrio vulgaris ATCC 29579 and a new isolate, Desulfovibrio sp. (`Oz7') was studied using lactate as the electron donor at 30 °C. The apparent K <sub>m</sub> (K <sub>m app</sub>) and V <sub>max</sub> with respect to Cr(VI) reduction was compared for both strains. Desulfovibio sp. `Oz7' had a K _{m app} of 90 M (threefold lower than that of D. vulgaris ATCC 29579) and a V _max of 120 nmol h^–1 mg^–1 biomass dry wt (approx. 30% lower than for the reference strain). The potential of the new isolate for bioremediation of Cr(VI) wastewaters is discussed.     

Isolation and characterization of a thermophilic benzoate-degrading,sulfate-reducing bacterium,Desulfotomaculum thermobenzoicum sp. nov.

A new thermophilic sulfate-reducing bacterium, strain TSB, that was spore-forming, rod-shaped, slightly motile and gram-positive, was isolated from a butyrate-containing enrichment culture inoculated with sludge of a thermophilic methane fermentation reactor. This isolate could oxidize benzoate completely. Strain TSB also oxidized some fatty acids and alcohols. SO _inf4 ^sup2- , SO _inf3 ^sup2- , S₂O _inf3 ^sup2- and NO _inf3 ^sup- were utilized as electron acceptors. With pyruvate or lactate the isolate grew without an external electron acceptor and produced acetate. The optimum temperature for growth was 62°C. The G+C content of DNA was 52.8 mol%. This isolate is described as a new species, Desulfotomaculum thermobenzoicum.     

Adhesion of anaerobic microorganisms to solid surfaces and the effect of sequential attachment on adhesion characteristics

The attachment of three anaerobic microorganisms, Desulfomonile tiedjei, Syntrophomonas wolfei, and Desulfovibrio sp. strain G11, was investigated to determine if the presence of one species could influence the adhesion of another species to glass surfaces. The results indicated that the numbers and distribution of attached cells of one species could be influenced considerably by the presence of another species and the order in which the test species were exposed to the surface. D. tiedjei was found to detach readily from surfaces when it was not the primary colonizer. The attachment of Desulfovibrio G11 as the primary colonizer appeared to be stabilized by exposure to another test species. Under certain experimental conditions the test organisms formed close associations with each other on the surfaces. These findings demonstrate that the characteristics of anaerobic community biofilms can be determined by both the adhesion characteristics of the individual species and the interactions among those microorganisms.     

Growth of a Strictly Anaerobic Bacterium on Furfural (2-Furaldehyde)

A strictly anaerobic bacterium was isolated from a continuous fermentor culture which converted the organic constituents of sulfite evaporator condensate to methane and carbon dioxide. Furfural is one of the major components of this condensate. This furfural isolate could degrade furfural as the sole source of carbon and energy in a defined mineral-vitamin-sulfate medium. Acetic acid was the major fermentation product. This organism could also use ethanol, lactate, pyruvate, or fumarate and contained cytochrome c₃ and desulfoviridin. Except for furfural degradation, the characteristics of the furfural isolate were remarkably similar to those of the sulfate reducer Desulfovibrio gigas. The furfural isolate has been tentatively identified as Desulfovibrio sp. strain F-1.     

Isolation and characterization of a furfural degrading sulfate-reducing bacterium from an anaerobic digester

A sulfate-reducing bacterium (SRB) was isolated from a continuous anaerobic digester, which converted the furfural-containing wastewater to methane and CO₂. This SRB isolate could use furfural, furfuryl alcohol, and 2-furoic acid as sole source of carbon and energy in a defined mineral sulfate medium. Acetic acid was the major end product of furfural degradation. This organism also used wide varieties of other carbon sources, including ethanol, pyruvate, lactate, succinate, propanol, formate, and malate. The SRB isolate contained the electron carrier desulfoviridin. It used SO₄, NO₃, and thiosulfate as electron acceptors. This isolate used ammonium chloride, nitrate and glutamate as nitrogen source. The characteristics of the SRB isolate were closely similar toDesulfovibrio sp.     

Utilization of l-alanine as carbon and nitrogen source by Deusulfovibrio HL21

Desulfovibrio HL21 is unable to grow with amino acids as energy substrates. Alanine, serine, aspartate and to some extent glutamate were used as carbon and nitrogen sources in the presence of hydrogen as the energy substrate. Dense cell suspensions converted alanine stoichiometrically to acetate, NH ₄ ⁺ and presumably HCO ₃ ^- , but at a very low rate. Desulfovibrio HL21 cells grown with alanine as carbon and nitrogen source contained increased levels of NAD(P)-dependent l-alanine dehydrogenase as compared to cells grown with NH₄Cl as nitrogen source. Unfavourable kinetic properties of this alanine dehydrogenase, repression of the synthesis of the enzyme by NH ₄ ⁺ and a low rate of NADH oxidation all have a negative effect on the rate of degradation of alanine and may partly explain the inability of the strain to grow with alanine as an energy substrate.     

Isolation and characterization of a new spore-forming sulfate-reducing bacterium growing by complete oxidation of catechol

A new mesophilic sulfate-reducing bacterium, strain Groll, was isolated from a benzoate enrichment culture inoculated with black mud from a freshwater ditch. The isolate was a spore-forming, rod-shaped, motile, gram-positive bacterium. This isolate was able of complete oxidation of several aromatic compounds including phenol, catechol, benzoate, p-and m-cresol, benzyl alcohol and vanillate. With hydrogen and carbon dioxide, formate or O-methylated aromatic compounds, autotrophic growth during sulfate reduction or homoacetogenesis was demonstrated. Lactate was not used as a substrate. SO _inf4 ^sup2- , SO _inf3 ^sup2- , and S₂O _inf3 ^sup2- were utilized as electron acceptors. Although strain Groll originated from a freshwater habitat, salt concentrations of up to 30 g·l^-1 were tolerated. The optimum temperature for growth was 35–37°C. The G+C content of DNA was 42.1 mol%. This isolate is described as a new species of the genus Desulfotomaculum.     

Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a <Emphasis Type="Italic">Desulfovibrio</Emphasis> species under anaerobic conditions

Past handling practices associated with the manufacturing and processing of the high explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has resulted in extensive environmental contamination. In-situ biodegradation is a promising technology for remediating RDX contaminated sites but often relies on the addition of a cosubstrate. A sulfate-reducing bacterium isolated from an RDX-degrading enrichment culture was studied for its ability to grow on RDX as a sole source of carbon and nitrogen and for its ability to mineralize RDX in the absence of a cosubstrate. The results showed the isolate degraded 140 μM RDX in 63 days when grown on RDX as a carbon source. Biomass within the carbon limited culture increased 9-fold compared to the RDX unamended controls. When the isolate was incubated with RDX as sole source of nitrogen it degraded 160 μM RDX in 41 days and exhibited a 4-fold increase in biomass compared to RDX unamended controls. Radiolabeled studies under carbon limiting conditions with ¹⁴C-hexahydro-1,3,5-trinitro-1,3,5-triazine confirmed mineralization of the cyclic nitramine. After 60 days incubation 26% of the radiolabel was recovered as ¹⁴CO₂, while in the control bottles less than 1% of the radiolabel was recovered as ¹⁴CO₂. Additionally, ~2% of the radiolabeled carbon was found to be associated with the biomass. The 16S rDNA gene was sequenced and identified the isolate as a novel species of Desulfovibrio, having a 95.1% sequence similarity to Desulfovibrio desulfuricans. This is the first known anaerobic bacterium capable of mineralizing RDX when using it as a carbon and energy source for growth.     

Isolation and characterization of Desulfovibrio senezii sp. nov., a halotolerant sulfate reducer from a solar saltern and phylogenetic confirmation of Desulfovibrio fructosovorans as a new species

A new halotolerant Desulfovibrio, strain CVL^T (T = type strain), was isolated from a solar saltern in California. The curved, gram-negative, nonsporeforming cells (0.3 × 1.0–1.3 μm) occurred singly, in pairs, or in chains, were motile by a single polar flagellum and tolerated up to 12.5% NaCl. Strain CVL^T had a generation time of 60 min when grown in lactate-yeast extract medium under optimal conditions (37°C, pH 7.6, 2.5% NaCl). It used lactate, pyruvate, cysteine, or H₂/CO₂ + acetate as electron donors, and sulfate, sulfite, thiosulfate, or fumarate as electron acceptors. Elemental sulfur, nitrate, or oxygen were not used. Sulfite and thiosulfate were disproportionated to sulfate and sulfide. The G+C content of the DNA was 62 mol%. Phylogenetic analysis revealed that Desulfovibrio fructosovorans was the nearest relative. Strain CVL^T is clearly different from other Desulfovibrio species, and is designated Desulfovibrio senezii sp. nov. (DSM 8436). Received: 27 February 1998 / Accepted: 15 June 1998     

Analysis of the Anaerobic Microbial Community Capable of Degrading p-Toluene Sulfonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.