Oxidation of Elemental Sulfur to Thiosulfate by Streptomyces

Streptomyces sioyaensis, which produces the antibiotic siomycin, oxidizes elemental sulfur when added to the culture medium and accumulates thiosulfate in the fermented broth. The accumulated thiosulfate was isolated as the ammonium salt and was identified by melting point, IR spectrum, and paper chromatography. A variety of other streptomycetes also oxidized elemental sulfur and accumulated thiosulfate.     

Bacterial sulfur reduction in hot vents

Abstract: Elemental sulfur can be reduced through different microbial processes, including catabolically significant sulfur respiration and reduction of sulfur in the course of fermentation. Both of these processes are found in thermophilic microorganisms inhabiting continental and submarine hot vents, where elemental sulfur is one of the most common sulfur species. Among extreme thermophiles, respresented mainly by Archaea, sulfur-respiring bacteria include hydrogen-utilizing lithoautotrophs and heterotrophs, oxidizing complex organic substrates. Some marine heterotrophic sulfur-reducing Archaea were found to ferment peptides and polysaccharides, using elemental sulfur as an electron sink and thus avoiding the formation of molecular hydrogen which is highly inhibiting. Moderately thermophilic communities contain eubacterial sulfur reducers capable of lithotrophic and heterotrophic growth. Total mineralization of organic matter is carried out by a complex microbial system consisting of fermentative heterotrophs, which use elemental sulfur as an electron sink, and sulfur-respiring bacteria of the genus Desulfurella , which oxidize other fermentation products, yielding only CO_f2 and H_f2S. The most remarkable thermophilic microbial community is the thermophilic cyanobacterial mat found in the Uzon caldera, Kamchatka, which contains elemental sulfur among the layers. Organic matter produced by the thermophilic Oscillatoria is completely and rapidly mineralized by means of sulfur reduction.     

Fermentation studies with thermophilicArchaea in pure culture and in syntrophy with a thermophilic methanogen

Two heterotrophic, thermophilic, sulfur-reducing archaea were isolated from the Guaymas Basin hydrothermal vent. The fermentation of proteinaceous and carbohydrate substrates was examined at 85°C for each isolate in the presence and absence of elemental sulfur and in coculture with a thermophilic methanogen. The heterotrophic isolates differed with respect to their requirement for sulfur. Both heterotrophic isolates exhibited a mixed organic acid fermentation from proteinaceous substrates; however, acetate was the sole organic acid produced from carbohydrate fermentation. In coculture fermentations with a thermophilic methanogen, the heterotrophic isolates exhibited enhanced growth and fermentation. Interspecies hydrogen transfer and elemental sulfur-reduction may be important microbial processes in deep-sea hydrothermal vent community metabolism.     

Oxidation of Elemental Sulfur by Sulfolobus acidocaldarius

Oxidation of elemental sulfur by Sulfolobus acidocaldarius, an autotroph which grows at high temperatures and low pH, was examined by use of (35)S-labeled elemental sulfur. When cultured at pH 3.2 and 70 C, S. acidocaldarius oxidized elemental sulfur essentially quantitatively to sulfuric acid. Oxidation rate paralleled growth rate and decrease in pH of the culture medium. Elemental sulfur was not oxidized under these conditions if the culture was poisoned with formaldehyde. During the growth phase, the proportion of cells attached to the sulfur crystals increased progressively, and in the later phases of growth over 10 times more cells were attached to sulfur than were free. Doubling times for eight strains growing on elemental sulfur varied from 37 to 55 h. The organism grows much more rapidly on yeast extract than on sulfur. In a medium containing both sulfur and yeast extract, sulfur oxidation was partially inhibited, although growth was excellent.     

Utilization of different sulfur sources by propionic acid bacteria

The object of this work was to study the ability of propionic bacteria to utilize sulfur compounds having various degrees of oxidation. Propionibacterium shermanii was found to utilize sulfite, thiosulfate, sulfide and elemental sulfur, apart from sulfate, as a sulfur source. When the culture grew in a medium with elemental sulfur, sulfide was produced. The utilization of sulfate by P. shermanii had a peculiar character. In the process of the culture growth, the utilization of sulfate alternated with its release into the medium.     

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.     

Key role for sulfur in peptide metabolism and in regulation of three hydrogenases in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S⁰). Growth rates were highest on media containing peptides and S⁰, with or without maltose. Growth did not occur on the peptide medium without S⁰. S⁰ had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S⁰ with or without maltose were the same, suggesting that S⁰ is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S⁰ in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S⁰-reducing enzyme in this organism and the mechanism of the S⁰ dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.     

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     

Biochemical Studies on Streptomyces sioyaensis

Non-proliferating mycelium of Streptomyces sioyaensis was shown to form siomycin in phosphate buffer without addition of an energy source or precursors. This increase of siomycin in phosphate buffer was suppressed by glucose, acetate, l-cysteine, casamino acid, metals (Fe⁺⁺, Cu⁺⁺), various metabolic inhibitors, and antibiotics (chloramphenicol, erythromycin), whereas it was promoted by yeast extract, beef extract, l-isoleucine, Mg, etc.

The mechanism of the inhibitory effect of glucose on siomycin formation was investigated. Although glucose suppressed siomycin formation, it was the best carbon source for Streptomyces sioyaensis and vigorously metabolized to keto acids and other metabolites. Glucose suppressed siomycin formation by promoting cellular metabolism and mycelial growth. Siomycin formation was not only different from but also competitive to mycelial growth (cellular protein synthesis).     

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.     

Fermentation and Sulfur Reduction in the Mat-Building Cyanobacterium Microcoleus chthonoplastes

The mat-building cyanobacterium Microcoleus chthonoplastes carried out a mixed-acid fermentation when incubated under anoxic conditions in the dark. Endogenous storage carbohydrate was fermented to acetate, ethanol, formate, lactate, H(inf2), and CO(inf2). Cells with a low glycogen content (about 0.3 (mu)mol of glucose per mg of protein) produced acetate and ethanol in equimolar amounts. In addition to glycogen, part of the osmoprotectant, glucosyl-glycerol, was degraded. The glucose component of glucosyl-glycerol was fermented, whereas glycerol was released into the medium. Cells with a high content of glycogen (about 2 (mu)mol of glucose per mg of protein) did not utilize glucosyl-glycerol. These cells produced more acetate than ethanol. M. chthonoplastes was also capable of using elemental sulfur as the electron acceptor during fermentation, resulting in the production of sulfide. With sulfur present, acetate production increased whereas ethanol production decreased. Also, less formate was produced and the evolution of hydrogen ceased completely. In general, the carbon recoveries were satisfactory but the oxidation-reduction balances were too high. The latter could be explained by assuming the reduction of ferric iron, which is associated with the cells, mediated by the oxidation of formate. The switch from photoautotrophic to fermentative metabolism did not require de novo protein synthesis, and fermentation started immediately upon transfer to dark anoxic conditions. From the molar ratios of the fermentation products and from measurement of enzyme activities in cell extracts, we concluded that glucose derived from glycogen and glucosyl-glycerol is degraded via the Embden-Meyerhof-Parnas pathway.     

Development of a method to measure hydrogen sulfide in wine fermentation

A hydrogen sulfide (H2S) detecting tube was developed for the quantitative determination of H2S produced by yeast during laboratory scale wine fermentations. The detecting tube consisted of a small transparent plastic tube packed with an H2S-sensitive color-indicating medium. The packed medium changed color, with the color change progressing upward from the bottom of the tube, upon exposure to H2S produced by yeast during fermentation. A calibration study using a standard H2S gas showed that the length of the portion that darkened was directly related to the quantity of H2S (microg) with a high correlation coefficient (r2=0.9997). The reproducibility of the H2S detecting tubes was determined with five repetitive measurements using a standard H2S solution [5.6 microg/200 ml (28 ppb)], which resulted in a coefficient of variation of 3.6% at this level of H2S. With the sulfide detecting tubes, the production of H2S was continuously monitored and quantified from laboratory scale wine fermentations with different yeast strains and with the addition of different levels of elemental sulfur to the grape juice. This sulfide detecting tube technology may allow winemakers to quantitatively measure H2S produced under different fermentation conditions, which will eventually lead winemakers to better understand the specific factors and conditions for the excessive production of H2S during wine fermentation in a large production scale.     

Characterization of sulfur oxidation by an autotrophic sulfur oxidizer,Thiobacillus sp. ASWW-2

An autotrophic sulfur oxidizer,Thiobacillus sp. ASWW-2, was isolated from activated sludge, and its sulfur oxidation activity was characterized.Thiobacillus sp. ASWW-2 could oxidize elemental sulfur on the broad range from pH 2 to 8. When 5–50 g/L of elemental sulfur was supplemented as a substrate, the growth and sulfur oxidation activity ofThiobacillus sp. ASWW-2 was not inhibited. The specific sulfur oxidation rate of strain ASWW-2 decreased gradually until sulfate was accumulated in medium up to 10 g/L. In the range of sulfate concentration from 10 g/L to 50 g/L, the sulfur oxidation rate could keep over 2.0 g-S/g-DCW-d. It indicated thatThiobacillus sp. ASWW-2 has tolerance to high concentration of sulfate.     

Anaerobic heterotrophic dark metabolism in the cyanobacterium Oscillatoria limnetica: Sulfur respiration and lactate fermentation

The cyanobacterium Oscillatoria limnetica, capable of anoxygenic photosynthesis in the light with sulfide as electron donor can anaerobically break down its intracellular polyglucose in the dark. In the absence of elemental sulfur, the organism carries out lactate fermentation; in its presence, anaerobic respiration occurs in which sulfur is reduced to sulfide. Induction of anoxygenic photosynthesis or synthesis of new proteins is not necessary for either process. Cells adapted in the dark to sulfur reduction are capable of anoxygenic photosynthesis during a subsequent light period, unless protein synthesis has been inhibited during the dark incubation period.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP Carbonylcyanide p-trifluoromethoxyphenylhydrazone - mgat milligramatom - OD optical density     

Simultaneous heterolactic and acetate fermentation in the marine cyanobacterium Oscillatoria limosa incubated anaerobically in the dark

The marine nitrogen-fixing cyanobacterium Oscillatoria limosa, strain 23 (Oldenburg) was investigated with respect to its dark anaerobic metabolism. As soon as the cells were incubated anaerobically in the dark, they started to ferment. Glycogen was presumably degraded via the heterolactic fermentative pathway. Glycogen-glucose was degraded to equimolar amounts of lactate, ethanol and carbon dioxide. The disaccharide trehalose, which serves as an osmoprotectant in O. limosa, was also catabolized. Most probably, this compound was fermented almost exclusively to acetate. Some hydrogen was produced as well. In the presence of elemental sulfur, fermentative hydrogen production ceased and sulfide was produced instead. The presence of elemental sulfur had no effect on the amounts and ratios of the fermentation products produced.     

A gene cloning system for the siomycin producer <Emphasis Type="Italic">Streptomyces sioyaensis</Emphasis> NRRL-B5408

Streptomyces sioyaensis NRRL-B5408 produces a siomycin complex (a group of thiopeptide antibiotics structurally related to thiostrepton). Development of genetic tools for the detection of siomycin production and DNA transfer into this strain is described. The existing tipA-based reporter system for determination of siomycin production was modified to achieve its stable integration into actinomycete genomes. Various replicative plasmids (pKC1139, pKC1218E, pSOK101) as well as actinophage ϕC31- and VWB-based vectors pSET152 and pSOK804, respectively, were conjugally transferred from E. coli into the siomycin producer at a frequency ranging from 3.7 × 10⁻⁹ to 1.1 × 10⁻⁵. The transconjugants did not differ from wild type in their ability to produce siomycin. There is one attB site for each integrative plasmid. The utility of temperature sensitive replicon of pKC1139 for insertional gene inactivation in S. sioyaensis has been validated by disruption of putative nonribosomal peptide synthetase gene.     

Complete genome sequence of Desulfobulbus propionicus type strain (1pr3)

Desulfobulbus propionicus Widdel 1981 is the type species of the genus Desulfobulbus, which belongs to the family Desulfobulbaceae. The species is of interest because of its great implication in the sulfur cycle in aquatic sediments, its large substrate spectrum and a broad versatility in using various fermentation pathways. The species was the first example of a pure culture known to disproportionate elemental sulfur to sulfate and sulfide. This is the first completed genome sequence of a member of the genus Desulfobulbus and the third published genome sequence from a member of the family Desulfobulbaceae. The 3,851,869 bp long genome with its 3,351 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Nature of the sulfur-containing component and its function in Thiobacillus ferrooxidans

As was shown using various reagents (Ag+, Cd2+) and solvents (ethanol, methanol), Thiobacillus ferrooxidans cells accumulate colloidal sulfur when they grow in the medium 9K containing elemental sulfur. Colloidal sulfur is accumulated in the periplasmic space, in large, bipolarly arranged spherical structures and in simple invaginates of the cytoplasmic membrane. T. ferrooxidans cells accumulate the sulfur at a highest rate during the stationary phase of growth and can use it as a source of energy under the conditions of starvation. The factors causing sulfur accumulation in T. ferrooxidans cells are discussed.     

Glutathione reductase/glutathione is responsible for cytotoxic elemental sulfur tolerance via polysulfide shuttle in fungi

Fungi that can reduce elemental sulfur to sulfide are widely distributed, but the mechanism and physiological significance of the reaction have been poorly characterized. Here, we purified elemental sulfur-reductase (SR) and cloned its gene from the elemental sulfur-reducing fungus Fusarium oxysporum. We found that NADPH-glutathione reductase (GR) reduces elemental sulfur via glutathione as an intermediate. A loss-of-function mutant of the SR/GR gene generated less sulfide from elemental sulfur than the wild-type strain. Its growth was hypersensitive to elemental sulfur, and it accumulated higher levels of oxidized glutathione, indicating that the GR/glutathione system confers tolerance to cytotoxic elemental sulfur by reducing it to less harmful sulfide. The SR/GR reduced polysulfide as efficiently as elemental sulfur, which implies that soluble polysulfide shuttles reducing equivalents to exocellular insoluble elemental sulfur and generates sulfide. The ubiquitous distribution of the GR/glutathione system together with our findings that GR-deficient mutants derived from Saccharomyces cerevisiae and Aspergillus nidulans reduced less sulfur and that their growth was hypersensitive to elemental sulfur indicated a wide distribution of the system among fungi. These results indicate a novel biological function of the GR/glutathione system in elemental sulfur reduction, which is distinguishable from bacterial and archaeal mechanisms of glutathione- independent sulfur reduction.