Desulfovibrio tunisiensis sp. nov., a novel weakly halotolerant, sulfate-reducing bacterium isolated from exhaust water of a Tunisian oil refinery.

A novel weakly halotolerant, sulfate-reducing bacterium, designated strain RB22(T), was isolated from exhaust water of a Tunisian oil refinery. Cells of strain RB22(T) were Gram-negative, motile, vibrio-shaped or sigmoid and non-spore-forming, and occurred singly or in chains. Strain RB22(T) grew between 15 and 45 degrees C (optimum, 37 degrees C) and at pH 4.5 to 9 (optimum, pH 7). NaCl was not required for growth, but the strain tolerated high NaCl concentrations (up to 70 g l(-1)) with an optimum of 40 g l(-1). Sulfate, thiosulfate, sulfite and elemental sulfur served as electron acceptors, but not fumarate. Nitrate and nitrite were not reduced. Strain RB22(T) utilized lactate, formate, fumarate, succinate, glycerol, H(2)+CO(2) and methanol as substrates. The DNA G+C content was found to be 59.6 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the isolate was a member of the genus Desulfovibrio, with no close relatives at the species level (16S rRNA gene sequence similarity of less than 95 %). Strain RB22(T) exhibited levels of 16S rRNA gene sequence similarity of 94.6 and 94.12 % to the type strains of the closely related species Desulfovibrio aespoeensis and Desulfovibrio dechloracetivorans, respectively. On the basis of genotypic and phylogenetic characteristics, and significant phenotypic differences, we suggest that strain RB22(T) represents a novel species, for which the name Desulfovibrio tunisiensis sp. nov. is proposed. The type strain is RB22(T) (=NCIMB 14400(T)=JCM 15076(T)=DSM 19275(T)).

Desulfatiferula olefinivorans gen. nov., sp. nov., a long-chain n-alkene-degrading, sulfate-reducing bacterium.

A novel anaerobic, long-chain alkene-degrading, sulfate-reducing bacterium, strain LM2801T, was isolated from brackish sediment of a wastewater decantation facility of an oil refinery (Berre lagoon, France). Cells of strain LM2801T were Gram-negative, motile, slightly curved or vibrioid rods. Its optimum growth conditions were 30-36 degrees C, 6-10 g NaCl l(-1) and pH 7.5. Strain LM2801T incompletely oxidized long-chain alkenes (from C14 to C23) and fatty acids (C14 to C24). The DNA G+C content was 45.5 mol%. Sequence analyses of the 16S rRNA and dsrAB genes indicated that the strain was a member of the family Desulfobacteraceae within the Deltaproteobacteria. This novel isolate possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus. Therefore, strain LM2801T is described as a member of a new genus, Desulfatiferula gen. nov., of which Desulfatiferula olefinivorans sp. nov. is the type species. The type strain of Desulfatiferula olefinivorans is LM2801T (=DSM 18843T=JCM 14469T).

Bioremediation of coal contaminated soil under sulfate-reducing condition.

The objective of this study was to investigate the biodegradation of coal-derived hydrocarbons, especially high molecular weight (HMW) components, under anaerobic conditions. For this purpose biodegradation experiments were performed, using specifically designed soil column bioreactors. For the experiment, coal-contaminated soil was prepared, which contains high molecular weight hydrocarbons at high concentration (approx. 55.5 mgC g-drysoil(-1)). The experiment was carried out in two different conditions: sulfate reducing (SR) condition (SO4(2-) = 10 mmol l(-1) in the liquid medium) and control condition (SO4(2-)<0.5 mmol l(-1)). Although no degradation was observed under the control condition, the resin fraction decreased to half (from 6,541 to 3,386 mgC g-soil(-1)) under SR condition, with the concomitant increase of two PAHs (phenanthrene and fluoranthene, 9 and 2.5 times, respectively). From these results, we could conclude that high molecular hydrocarbons were biodegradable and transformed to low molecular weight PAHs under the sulfate-reducing condition. Since these PAHs are known to be biologically degraded under aerobic condition, a serial combination of anaerobic (sulfate reducing) and then aerobic bioremediations could be effective and useful for the soil pollution by petroleum and/or coal derived hydrocarbons.

Uranium immobilization by sulfate-reducing biofilms.

Hexavalent uranium [U(VI)] was immobilized using biofilms of the sulfate-reducing bacterium (SRB) Desulfovibrio desulfuricans G20. The biofilms were grown in flat-plate continuous-flow reactors using lactate as the electron donor and sulfate as the electron acceptor. U(VI)was continuously fed into the reactor for 32 weeks at a concentration of 126 microM. During this time, the soluble U(VI) was removed (between 88 and 96% of feed) from solution and immobilized in the biofilms. The dynamics of U immobilization in the sulfate-reducing biofilms were quantified by estimating: (1) microbial activity in the SRB biofilm, defined as the hydrogen sulfide (H2S) production rate and estimated from the H2S concentration profiles measured using microelectrodes across the biofilms; (2) concentration of dissolved U in the solution; and (3) the mass of U precipitated in the biofilm. Results suggest that U was immobilized in the biofilms as a result of two processes: (1) enzymatically and (2) chemically, by reacting with microbially generated H2S. Visual inspection showed that the dissolved sulfide species reacted with U(VI) to produce a black precipitate. Synchrotron-based U L3-edge X-ray absorption near edge structure (XANES) spectroscopy analysis of U precipitated abiotically by sodium sulfide indicated that U(VI) had been reduced to U(IV). Selected-area electron diffraction pattern and crystallographic analysis of transmission electron microscope lattice-fringe images confirmed the structure of precipitated U as being that of uraninite.

Optimization of metal sulphide precipitation in fluidized-bed treatment of acidic wastewater.

Sulphate-reducing biofilm and suspension processes were studied for treatment of synthetic wastewater containing sulphate, zinc and iron. With lactate supplemented wastewater with 170-230mg/l Zn and 58mg/l Fe, the following precipitation rates were obtained: 250 and 350mg/l d for Zn in fluidized-bed (FBR) and upflow anaerobic sludge blanket reactors, respectively, and 80mg/l d for Fe in both reactors with hydraulic retention time of 16h. The effluent Zn and Fe concentrations remained at less than 0.1 mg/l. The alkalinity produced in lactate oxidation increased the initial pH of 2.5-3, resulting in effluent pH of 7.5-8.5. The highest sulphate reduction rate was over 2000 mg/l d. In terms of sulphate reduction, hydrogen sulphide production and effluent alkalinity, the start-up of the FBR with the 10% fluidization rate was superior to the FBRs with 20-30% fluidization rates. With increased loading rates, high recycling rate became an advantage. After process failure caused by intentional overloading, the sulphate reduction partially recovered within 2 weeks. Metal precipitates in the reactors were predominantly FeS2, ZnS and FeS. The metal mass balance was as follows: 73-86% of Zn and Fe accumulated into the reactors and water level adjustors, 14-23% of the metals were washed out as precipitates and 0.05-0.15% remained as soluble metals. Biomass yield in the sulphate-reducing processes was 0.039-0.054g dry biomass (VS or VSS) per g of lactate oxidized or 0.035-0.074g dry biomass per g of sulphate reduced. The results of this work demonstrate that the lactate supplemented sulphate-reducing processes precipitated the metals as sulphides and neutralized the acidity of the synthetic wastewater.

Anaerobic, sulfate-dependent degradation of polycyclic aromatic hydrocarbons in petroleum-contaminated harbor sediment.

It has previously been demonstrated that [14C]-labeled polycyclic aromatic hydrocarbons (PAHs) can be oxidized to 14CO2 in anoxic, PAH-contaminated, marine harbor sediments in which sulfate reduction is the terminal electron-accepting process. However, it has not previously been determined whether this degradation of [14C]-PAHs accurately reflects the degradation of the in situ pools of contaminant PAHs. In coal tar-contaminated sediments from Boston Harbor, [14C]-naphthalene was readily oxidized to 14CO2, but, after 95 d of incubation under anaerobic conditions, there was no significant decrease in the detectable pool of in situ naphthalene in these sediments. Therefore, to better evaluate the anaerobic biodegradation of the in situ PAH pools, the concentrations of these contaminants were monitored for ca. 1 year during which the sediments were incubated under conditions that mimicked those found in situ. There was loss of all of the PAHs that were monitored (2-5 ring congeners), including high molecular weight PAHs, such as benzo[a]pyrene, that have not previously been shown to be degraded under anaerobic conditions. There was no significant change in the PAH levels in the sediments amended with molybdate to inhibit sulfate-reducing bacteria or in sediments in which all microorganisms had been killed with glutaraldehyde. In some instances, over half of the detectable pools of in situ 2-3 ring PAHs were degraded. In general, the smaller PAHs were degraded more rapidly than the larger PAHs. A distinct exception in the Boston Harbor sediment was naphthalene which was degraded very slowly at a rate comparable to the larger PAHs. In a similar in situ-like study of fuel-contaminated sediments from Liepaja Harbor, Latvia, there was no decline in PAH levels in samples that were sulfate-depleted. However, when the Latvia sediments were supplemented with sufficient sodium sulfate or gypsum to elevate pore water levels of sulfate to approximately 14-25 mM there was a 90% decline in the naphthalene and a 60% decline in the 2-methylnaphthalene pool within 90 days. These studies demonstrate for the first time that degradation by anaerobic microorganisms can significantly impact the in situ pools of PAHs in petroleum-contaminated, anoxic, sulfate-reducing harbor sediments and suggest that the self-purification capacity of contaminated harbor sediments is greater than previously considered.

Consequences of linseed oil spills in salt marsh sediments.

In a simulated spill in a salt marsh, linseed oil penetrated rapidly into the sediments at a rate of 10(-7) cm2 s(-1). The oil concentration remained unchanged for the first month after the spill, but 60% of the oil disappeared from the top 30 cm after a further month. The oil adsorbed to and accumulated in the muddy sediments (top 15 cm) leading to decreased sediment permeability, pH, Eh, abundance of plant roots and infauna and to the establishment of anoxic conditions. These changes accompanied transformations in the original fatty acid composition of the linseed oil, mainly associated with a decrease in 18 : 3omega3, an increase in the other fatty acids and the presence of 'new' fatty acids. A rapid increase in the abundance of heterotrophic aerobic and anaerobic bacteria and aerobic oil degrading bacteria, suggested that these micro-organisms degraded the oil. The role of the bacteria in oil degradation was confirmed in laboratory experiments where the fatty acids composition of the linseed oil underwent identical transformations to those obtained in the field. The degradation of linseed oil appears to be a sequential process initiated by aerobic and/or anaerobic bacteria and continued by sulphate reducing bacteria, which were unable to degrade the raw oil.

Desulfotomaculum halophilum sp. nov., a halophilic sulfate-reducing bacterium isolated from oil production facilities.

A halophilic endospore-forming, sulfate-reducing bacterium was isolated from an oilfield brine in France. The strain, designated SEBR 3139, was composed of long, straight to curved rods. It grew in 1-14% NaCl with an optimum at 6%. On the basis of morphological, physiological and phylogenetical characteristics, strain SEBR 3139 should be classified in the genus Desulfotomaculum. However, it is sufficiently different from the hitherto described Desulfotomaculum species to be considered as a new species. Strain SEBR 3139T (= DSM 11559T) represents the first moderate halophilic species of the genus Desulfotomaculum. The name Desulfotomaculum halophilum sp. nov. is proposed.

Desulfobulbus rhabdoformis sp. nov., a sulfate reducer from a water-oil separation system.

A mesophilic, Gram-negative, rod-shaped, marine, propionate-oxidizing sulfate reducer (strain M16T) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 31 degrees C, pH 6.8-7.2 and 1.5-2.0% (w/v) NaCl and 0.1-0.3% (w/v) MgCl2.6H2O in the medium. The growth yield with sulfate was 4.6 g cell biomass (mol propionate oxidized)-1. Strain M16T is nutritionally related to members of the genus Desulfobulbus, but differs in that it has no vitamin requirement and is able to utilize fumarate and malate as carbon and energy sources. Hydrogenase activity measured as hydrogen uptake was mainly membrane-bound and varied with the growth substrate. Highest activity [28 mumol min-1 (mg protein)-1] was found in cells grown with hydrogen and lowest [50 nmol min-1 (mg protein)-1] in cells grown with propionate as electron donors for sulfate reduction. Desulforubidin, menaquinone-5(H2) and cytochromes of the c- and b-type were present. The fatty acid pattern was similar to that found for Desulfobulbus propionicus. The DNA base composition was 50.6 mol% G + C. Strain M16T is equidistantly related to D. propionicus and Desulfobulbus elongatus with 96.1% 16S rDNA similarity. On the basis of differences in genotypic, phenotypic and immunological characteristics, strain M16T (= DSM 8777T) is proposed as the type strain of a new species, Desulfobulbus rhabdoformis.