Sulfide removal and elemental sulfur recycling from a sulfide-polluted medium by Allochromatium vinosum strain 21D.

Phototrophic purple sulfur bacteria oxidize sulfide to elemental sulfur, which is stored as intracellular sulfur globules. The mutant Allochromatium vinosum strain 21D, containing an inactivated dsrB gene, is unable to further oxidize intracellularly stored sulfur to sulfate. This mutant was used as a biocatalyst in a biotechnological process to eliminate sulfide from synthetic wastewater and to recycle elemental sulfur as a raw material. For this purpose, the mutant was grown in an illuminated 5-liter bioreactor (30 microE/m2/s PAR) at 30 degrees C for 61 days in anoxic phototrophic medium. The process of sulfide removal was semi-continuous and consisted of three consecutive fed-batch sections. Sulfide was repeatedly added into the bioreactor and oxidized by the cells to sulfur. In the presence of the mutant, no unwanted sulfate was produced during sulfide removal. A maximum sulfide removal rate of 49.3 microM/h, a maximum sulfide removal efficiency of 98.7%, and 60.4% sulfur recycling were achieved.     

Sulfide oxidation under chemolithoautotrophic denitrifying conditions

Chemolithoautotrophic denitrifying microorganisms oxidize reduced inorganic sulfur compounds coupled to the reduction of nitrate as an electron acceptor. These denitrifiers can be applied to the removal of nitrogen and/or sulfur contamination from wastewater, groundwater, and gaseous streams. This study investigated the physiology and kinetics of chemolithotrophic denitrification by an enrichment culture utilizing hydrogen sulfide, elemental sulfur, or thiosulfate as electron donor. Complete oxidation of sulfide to sulfate was observed when nitrate was supplemented at concentrations equal or exceeding the stoichiometric requirement. In contrast, sulfide was only partially oxidized to elemental sulfur when nitrate concentrations were limiting. Sulfide was found to inhibit chemolithotrophic sulfoxidation, decreasing rates by approximately 21-fold when the sulfide concentration increased from 2.5 to 10.0 mM, respectively. Addition of low levels of acetate (0.5 mM) enhanced denitrification and sulfate formation, suggesting that acetate was utilized as a carbon source by chemolithotrophic denitrifiers. The results of this study indicate the potential of chemolithotrophic denitrification for the removal of hydrogen sulfide. The sulfide/nitrate ratio can be used to control the fate of sulfide oxidation to either elemental sulfur or sulfate.     

Fate of elemental sulfur in an intertidal sediment

Abstract: Sediment from a tidal flat at Wedderwarden, near the mouth of the Weser estuary, northern Germany, was amended with elemental sulfur, and concentrations of metabolic end products were monitored. The production of both sulfate and sulfide was consistent with disproportionation as the most important fate of the added elemental sulfur. A population of bacteria conducting active elemental sulfur disproportionation was also enriched from the sediment. In the enrichments, containing both elemental sulfur and Fe oxides as a sulfide 'scrub', sulfide and sulfate were produced in a ratio of     , somewhat lower than the predicted ratio of     . The mismatch between predicted and observed production ratios is explained by the channelling of electrons into autotrophic or mixotrophic CO₂ fixation rather than sulfide formation. The production of organic carbon, in the correct amount to explain the observed sulfide to sulfate production ratio, was verified by organic carbon analysis. Finally, rates of sulfate reduction were identical in the elemental sulfur amended sediment, and in control sediment with no added sulfur. Hence, the heterotrophic bacterial community was completely unaffected by an active metabolism conducting elemental sulfur disproportionation.     

Oxidation of Elemental Sulfur by Fusarium solani Strain THIF01 Harboring Endobacterium Bradyrhizobium sp.

Nineteen fungal strains having an ability to oxidize elemental sulfur in mineral salts medium were isolated from deteriorated sandstones of Angkor monuments. These fungi formed clearing zone on agar medium supplemented with powder sulfur due to the dissolution of sulfur. Representative of the isolates, strain THIF01, was identified as Fusarium solani on the basis of morphological characteristics and phylogenetic analyses. PCR amplification targeting 16S rRNA gene and analyses of full 16S rRNA gene sequence indicated strain THIF01 harbors an endobacterium Bradyrhizobium sp.; however, involvement of the bacterium in the sulfur oxidation is still unclear. Strain THIF01 oxidized elemental sulfur to thiosulfate and then sulfate. Germination of the spores of strain THIF01 was observed in a liquid medium containing mineral salts supplemented with elemental sulfur (rate of germinated spores against total spores was 60.2%), and the culture pH decreased from pH 4.8 to 4.0. On the contrary, neither germination (rate of germinated spores against total spores was 1.0%) nor pH decrease was observed without the supplement of elemental sulfur. Strain THIF01 could also degrade 30 ppmv and ambient level (approximate 500 pptv) of carbonyl sulfide.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Utilization of mercapto-2-ethanol as medium reductant for determination of the metabolic response of methanogens towards inorganic sulfur compounds

Abstract Mercapto-2-ethanol was examined as a nontoxic and non-metabolizable reducing agent for growth of methanogens. It was used as a medium reductant to prove that Methanobacterium thermoautotrophicum and Methanobacterium strain ivanov grew with either sulfide or elemental sulfur as the sole source of nutrient sulfur but not with sulfate, thiosulfate, sulfite or dithionite. The later inorganic sulfur sources, except sulfate, were potent inhibitors of growth and methanogenesis at 5 mM. The practical utility of mercapto-2-ethanol as a reducing agent and the toxicity of inorganic sulfur sources on metabolic activity of the methanogens are discussed.     

Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

Sulfur metabolism enzymes in thermoacidophilus bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur:Fe(III) oxidoreductase, and sulfite:Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus strains N1 and SSO. Enzyme activity was revealed in cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.     

Optimization of biological sulfide removal in a CSTR bioreactor

In this study, biological sulfide removal from natural gas in a continuous bioreactor is investigated for estimation of the optimal operational parameters. According to the carried out reactions, sulfide can be converted to elemental sulfur, sulfate, thiosulfate, and polysulfide, of which elemental sulfur is the desired product. A mathematical model is developed and was used for investigation of the effect of various parameters on elemental sulfur selectivity. The results of the simulation show that elemental sulfur selectivity is a function of dissolved oxygen, sulfide load, pH, and concentration of bacteria. Optimal parameter values are calculated for maximum elemental sulfur selectivity by using genetic algorithm as an adaptive heuristic search. In the optimal conditions, 87.76% of sulfide loaded to the bioreactor is converted to elemental sulfur.     

Sulfur-Metabolizing Enzymes in Thermoacidophilic Bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur : Fe(III) oxidoreductase, and sulfite : Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus, strains N1 and SSO. Enzyme activity was revealed in the cells grown on medium with elemental sulfur or in the presence of various sulfide minerals and concentrates of sulfide ores. The activity of enzymes of sulfur metabolism depended little on the degree of aeration during bacterial growth.     

The production and utilization of intermediary elemental sulfur during the oxidation of reduced sulfur compounds by Thiobacillus ferrooxidans

The intermediary production of elemental sulfur during the microbial oxidation of reduced sulfur compounds has frequently been reported. Thiobacillus ferrooxidans, an acidophilic chemolithoautotroph, was found to produce an insoluble sulfur compound, primarily elemental sulfur, during the oxidation of thiosulfate, trithionate, tetrathionate and sulfide. This was confirmed by light and electron microscopy. Sulfur was produced from sulfide by an oxidative step, while the production from tetrathionate was initiated by a hydrolytic step, probably followed by a series of chemical reactions. The oxidation of intermediary sulfur was severely inhibited by sulfhydryl-binding reagents such as N-ethylmaleimide, by the addition of uncouplers or after freezing and thawing of the cells, which probably damaged the cell membrane. The mechanisms behind these inhibitions have not yet been clarified. Finally, it was observed that elemental sulfur oxidation by whole cells depended on the medium composition. The absence of sulfate or selenate reduced the sulfur oxidation rate.Non-standard abbreviations NEM N-ethylmaleimide - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Sulfide utilization by purple nonsulfur bacteria

Summary The purple nonsulfur bacteria Rhodospirillum rubrum SMG 107, Rhodopseudomonas capsulata SMG 155, Rps. sphaeroides SMG 158 and Rps. palustris SMG 124 were tested for a possible utilization of sulfide. The first three strains were found to oxidize sulfide to extracellular elemental sulfur only, whereas Rps. palustris SMG 124 converted sulfide into sulfate without intermediate accumulation of elemental sulfur. Growth ceased at lower sulfide concentrations than usually found with purple sulfur bacteria. In consequence of the low sulfide tolerance information on the specific growth rates obtainable with sulfide as photosynthetic electron donor could not be provided by cultivation in batch cultures. Sulfide-limited chemostat cultures of Rps. capsulata SMG 155 showed that the maximum specific growth rate was close to 0.14 h^-1 (doubling time 5 h). Sulfide was converted into extracellular elemental sulfur at all dilution rates tested. The maximum specific growth rate of Rps. palustris SMG 124 was found to be much lower (less than 0.03 h^-1). Sulfate was the only product of the conversion of sulfide.These data show that at least some purple nonsulfur bacteria may play a role in the dissimilatory sulfur cycle in nature. Taxonomic implications of our results are discussed.Abbreviation SMG Sammlung für Mikroorganismen, Göttingen     

Characterization of Enrichment Cultures Involved in the Carbon,Sulfur and Iron Biogeochemical Cycles in French Guiana Mobile Muds

The fluidized sediment ecosystem off French Guiana is characterized by active physical reworking, diversity of electron acceptors and highly variable redox regime. It is well studied geochemically but little is known about specific microorganisms involved in its biogeochemistry. Based on the biogeochemical profiles and rate kinetics, several possible biotically mediated pathways of the carbon, sulfur and iron cycles were hypothesized. Enrichment studies were set up with a goal to culture microorganisms responsible for these pathways. Stable microbial consortia potentially capable of the following chemolithoautotrophic types were enriched from the environment and characterized: elemental sulfur/thiosulfate disproportionators, thiosulfate-oxidizing ferrihydrite and nitrate reducers, sulfide/ferrous sulfide oxidizers coupled with nitrate and microaerophilic iron oxidizers. Attempts to generate several enrichments (anoxic ammonia oxidation, and sulfide oxidizers with ferric iron or manganese oxide) were not successful. Heterotrophic sulfate and elemental sulfur reduction bacteria are prominent and dominate reductive sulfur transformations. We hypothesize that carbon dioxide fixation coupled with synthesis of organic matter happens mostly via sulfur disproportionation and sulfur species oxidation with iron oxidation playing a minor role.     

A combined pathway of sulfur compound disproportionation in Desulfovibrio desulfuricans

The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry. During disproportionation, ³²S-thiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in ³⁴S. The sulfate formed was isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that undergo further disproportionation to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur and sulfite. It is concluded that disproportionation of thiosulfate, sulfite and elemental sulfur includes a combined pathway.     

34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria.

Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris. D. vulgaris and C. vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D. vulgaris), oxidation of sulfide to elemental sulfur (C. vibrioforme), and oxidation of sulfur to sulfate (C. vibrioforme). In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%). Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.     

Roseinatronobacter thiooxidans Gen. Nov., sp. Nov., a new alkaliphilic aerobic bacteriochlorophyll-alpha-containing bacteria from a soda lake

Several samples of microbial mat obtained from soda lakes of the Kunkurskaya steppe (Chita oblast) abundantly populated by purple bacteria were screened for the presence of heterotrophic alkaliphiles capable of oxidizing sulfur compounds to sulfate. This capacity was found in only one pigmented strain, ALG 1, isolated on medium with acetate and thiosulfate at pH 10. The strain was found to be a strictly aerobic and obligately heterotrophic alkaliphile. Growth on medium with acetate was possible within a narrow pH range from 8.5 to 10.4. The strain formed a reddish orange carotenoid and bacteriochlorophyll a. Pigments were synthesized only at high concentrations of nitrogen-containing organic compounds (peptone or yeast extract). The production of bacteriochlorophyll a was maximal under microaerobic conditions in darkness. Strain ALG 1 could oxidize sulfide, thiosulfate, sulfite, and elemental sulfur to sulfate. In heterotrophically growing culture (pH 10), thiosulfate was not oxidized until the late logarithmic phase. The sulfur-oxidizing activity was maximal at the most alkaline pH values. The notable increase in the efficiency of organic carbon utilization observed in the presence of thiosulfate suggested that the bacterium was a sulfur-oxidizing lithoheterotroph. The phylogenetic analysis of the 16S rRNA gene showed strain ALG 1 to be a member of the alpha-3 subgroup of proteobacteria and to constitute a distinct branch located between nonsulfur purple bacteria Rhodobacter and Rhodovulum. Based on the unique phenotypic properties and the results of phylogenetic analysis, the alkaliphilic isolate ALG 1 was assigned to a new genus and species Roseinatronobacter thiooxidans with the type strain DSZM-13087.     

34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria

Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris. D. vulgaris and C. vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D. vulgaris), oxidation of sulfide to elemental sulfur (C. vibrioforme), and oxidation of sulfur to sulfate (C. vibrioforme). In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%). Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Nutritional studies with Pseudomonas aeruginosa grown on inorganic sulfur sources.

Pseudomonas aeruginosa was grown on a succinate-basal salts medium supplemented with various inorganic sulfur compounds as its sole source of sulfur. The organism was able to grow on the sodium salts of sulfide, thiosulfate, tetrathionate, dithionite, metabisulfite, sulfite, or sulfate, but not on those of dithionate. Analyses of the culture media after 24 h of growth indicated accumulation of sulfate from each inorganic sulfur source except sulfate. Manometric studies with resting cells obtained by growth on each of these sulfur sources yielded net oxygen uptake for all substrates except sulfite and dithionate. Similar results were obtained with extracts from these cells by spectrophotometric techniques. Thiosulfate oxidase activity appeared to be induced by growth on sulfide, thiosulfate, or tetrathionate, with little or no activity observed when cells were grown on inorganic sulfur sources of higher oxidative states. Metabisulfite oxidase appeared to be associated with growth on all inorganic sulfur compounds. Rhodanese activity appeared to be constitutively present, and its activity, observed only in soluble fraction, seemed independent of the growth medium employed. Thiosulfate and tetrathionate oxidase activities were studied in greater detail than some of the other sulfur oxidases, and both were found to be distributed between particulate and soluble fractions.     

Carbon source utilization and accumulation of respiration-related substances by freshwater Thioploca species

Carbon source utilization of Thioploca species from freshwater and brackish lakes in Japan was investigated. Microautoradiography demonstrated that freshwater and brackish Thioploca samples assimilate acetate. In addition, vertical nitrate transportation by freshwater Thioploca was examined by measuring substances accumulated in Thioploca filaments. The filaments of Thioploca sp. from Lake Biwa, a Japanese mesotrophic lake, contained nitrate at concentrations higher than ambient by two to three orders of magnitude. They also accumulated high concentrations of sulfate and abundant elemental sulfur. The results suggest that the Thioploca-specific strategy for sulfur oxidation, migration with accumulated nitrate, is effective even in freshwater habitats of lower sulfide supply.