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.     

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.     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Oxidation of Elemental Sulfur to Sulfite by Thiobacillus thiooxidans Cells

Thiobacillus thiooxidans cells oxidized elemental sulfur to sulfite, with 1 mol of O₂ consumption per mol of sulfur oxidized to sulfite, when the oxidation of sulfite was inhibited with 2-n-heptyl-4-hydroxyquinoline N-oxide.     

Oxidation of Ferrous Iron and Elemental Sulfur by Thiobacillus ferrooxidans

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.     

Bacterial Disproportionation of Elemental Sulfur Coupled to Chemical Reduction of Iron or Manganese

A new chemolithotrophic bacterial metabolism was discovered in anaerobic marine enrichment cultures. Cultures in defined medium with elemental sulfur (S⁰) and amorphous ferric hydroxide (FeOOH) as sole substrates showed intense formation of sulfate. Furthermore, precipitation of ferrous sulfide and pyrite was observed. The transformations were accompanied by growth of slightly curved, rod-shaped bacteria. The quantification of the products revealed that S⁰ was microbially disproportionated to sulfate and sulfide, as follows: 4S⁰ + 4H₂O → SO₄^2- + 3H₂S + 2H⁺. Subsequent chemical reactions between the formed sulfide and the added FeOOH led to the observed precipitation of iron sulfides. Sulfate and iron sulfides were also produced when FeOOH was replaced by FeCO₃. Further enrichment with manganese oxide, MnO₂, instead of FeOOH yielded stable cultures which formed sulfate during concomitant reduction of MnO₂ to Mn²⁺. Growth of small rod-shaped bacteria was observed. When incubated without MnO₂, the culture did not grow but produced small amounts of SO₄^2- and H₂S at a ratio of 1:3, indicating again a disproportionation of S⁰. The observed microbial disproportionation of S⁰ only proceeds significantly in the presence of sulfide-scavenging agents such as iron and manganese compounds. The population density of bacteria capable of S⁰ disproportionation in the presence of FeOOH or MnO₂ was high, > 10⁴ cm^-3 in coastal sediments. The metabolism offers an explanation for recent observations of anaerobic sulfide oxidation to sulfate in anoxic sediments.     

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.     

Growth of Thiobacillus ferrooxidans on Elemental Sulfur

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day⁻¹, both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.     

Effect of Various Ions, pH, and Osmotic Pressure on Oxidation of Elemental Sulfur by Thiobacillus thiooxidans

The oxidation of elemental sulfur by Thiobacillus thiooxidans was studied at pH 2.3, 4.5, and 7.0 in the presence of different concentrations of various anions (sulfate, phosphate, chloride, nitrate, and fluoride) and cations (potassium, sodium, lithium, rubidium, and cesium). The results agree with the expected response of this acidophilic bacterium to charge neutralization of colloids by ions, pH-dependent membrane permeability of ions, and osmotic pressure.     

Sulfur Chemistry in Bacterial Leaching of Pyrite

In the case of pyrite bioleaching by Leptospirillum ferrooxidans, an organism without sulfur-oxidizing capacity, besides the production of tetra- and pentathionate, a considerable accumulation of elemental sulfur occurred. A similar result was obtained for chemical oxidation assays with acidic, sterile iron(III) ion-containing solutions. In the case of Thiobacillus ferrooxidans, only slight amounts of elemental sulfur were detectable because of the organism's capacity to oxidize sulfur compounds. In the course of oxidative, chemical pyrite degradation under alkaline conditions, the accumulation of tetrathionate, trithionate, and thiosulfate occurred. The data indicate that thiosulfate, trithionate, tetrathionate, and disulfane-monosulfonic acid are key intermediate sulfur compounds in oxidative pyrite degradation. A novel (cyclic) leaching mechanism is proposed which basically is indirect.     

Anaerobic Elemental Sulfur Reduction by Fungus Fusarium oxysporum

Reduction of inorganic sulfur compounds by the fungus Fusarium oxysporum was examined. When transferred from a normoxic to an anoxic environment, F. oxysporum reduced elemental sulfur to hydrogen sulfide (H₂S). This reaction accompanied fungal growth and oxidation of the carbon source (ethanol) to acetate. Over 2-fold more of H₂S than of acetate was produced, which is the theoretical correlation for the oxidation of ethanol to acetate. NADH-dependent sulfur reductase (SR) activity was detected in cell-free extracts of the H₂S-producing fungus, and was found to be up-regulated under the anaerobic conditions. On the other hands both O₂ consumption by the cells and cytochrome c oxidase activity by the crude mitochondrial fractions decreased. These results indicate that H₂S production involving SR was due to a novel dissimilation mechanism of F. oxysporum, and that the fungus adapts to anaerobic conditions by replacing the energy-producing mechanism of O₂ respiration with sulfur reduction.     

Involvement of Intermediate Sulfur Species in Biological Reduction of Elemental Sulfur under Acidic,Hydrothermal Conditions

The thermoacidophile and obligate elemental sulfur (S₈⁰)-reducing anaerobe Acidilobus sulfurireducens 18D70 does not associate with bulk solid-phase sulfur during S₈⁰-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H₂S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81°C. The production of polysulfide is likely due to the abiotic reaction between S₈⁰ and the biologically produced H₂S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S₈⁰ was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S₈⁰ can serve as terminal electron acceptors (TEA) supporting the growth of A. sulfurireducens, total sulfide concentration and cell density were monitored in batch cultures with S₈⁰ provided as a solid phase in the medium or with S₈⁰ sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S₈⁰ sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S₈⁰ provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S₈⁰ particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S₈⁰ particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for A. sulfurireducens.     

Reduction of Cupric Ions with Elemental Sulfur by Thiobacillus ferrooxidans

In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu²⁺) was reduced enzymatically with elemental sulfur (S⁰) by washed intact cells of Thiobacillus ferrooxidans AP19-3 to give cuprous ion (Cu⁺). The rate of Cu²⁺ reduction was proportional to the concentrations of S⁰ and Cu²⁺ added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70°C. The activity of Cu²⁺ reduction with S⁰ by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as α,α′-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S⁰ with Fe³⁺ or Mo⁶⁺ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu²⁺ by S⁰, and the Michaelis constant of SFORase for Cu²⁺ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe³⁺ and Mo⁶⁺ but also Cu²⁺.     

Investigation of Elemental Sulfur Speciation Transformation Mediated by Acidithiobacillus ferrooxidans

The speciation transformation of elemental sulfur mediated by the leaching bacterium Acidithiobacillus ferrooxidans was investigated using an integrated approach including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray absorption near edge spectroscopy (XANES). Our results showed that when grown on elemental sulfur powder, At. ferrooxidans ATCC23270 cells were first attached to sulfur particles and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to polysulfides. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested that these globules consisted of elemental sulfur bound to thiol groups of protein. Huan He and Cheng-Gui Zhang made equal contributions to this paper.     

Precision and Variability in Bacterial Temperature Sensing

In Escherichia coli, the ratio of the two most abundant chemoreceptors, Tar/Tsr, has become the focus of much attention in bacterial taxis studies. This ratio has been shown to change under various growth conditions and to determine the response of the bacteria to the environment. Here, we present a study that makes a quantitative link between the ratio Tar/Tsr and the favored temperature of the cell in a temperature gradient and in various chemical environments. From the steady-state density-profile of bacteria with one dominant thermo-sensor, Tar or Tsr, we deduce the response function of each receptor to temperature changes. Using the response functions of both receptors, we determine the relationship between the favored temperature of wild-type bacteria with mixed clusters of receptors and the receptor ratio. Our model is based on the assumption that the behavior of a wild-type bacterium in a temperature gradient is determined by a linear combination of the independent responses of the two receptors, factored by the receptor’s relative abundance in the bacterium. This is confirmed by comparing our model predictions with measurements of the steady-state density-profile of several bacterial populations in a temperature gradient. Our results reveal that the density-profile of wild-type bacteria can be accurately described by measuring the distribution of the ratio Tar/Tsr in the population, which is then used to divide the population into groups with distinct Tar/Tsr values, whose behavior can be described in terms of independent Gaussian distributions. Each of these Gaussians is centered about the favored temperature of the subpopulation, which is determined by the receptor ratio, and has a width defined by the temperature-dependent speed and persistence time.     

Mechanism of Bacterial Pyrite Oxidation

The oxidation by Ferrobacillus ferrooxidans of untreated pyrite (FeS(2)) as well as HCl-pretreated pyrite (from which most of the acid-soluble iron species were removed) was studied manometrically. Oxygen uptake was linear during bacterial oxidation of untreated pyrite, whereas with HCl-pretreated pyrite both a decrease in oxygen uptake at 2 hr and nonlinear oxygen consumption were observed. Ferric sulfate added to HCl-pretreated pyrite restored approximately two-thirds of the decrease in total bacterial oxygen uptake and caused oxygen uptake to revert to nearly linear kinetics. Ferric sulfate also oxidized pyrite in the absence of bacteria and O(2); recovery of ferric and ferrous ions was in excellent agreement with the reaction Fe(2)(SO(4))(3) + FeS(2) = 3FeSO(4) + 2S, but the elemental sulfur produced was negligible. Neither H(2)S nor S(2)O(3) (2-) was a product of the reaction. It is probable that two mechanisms of bacterial pyrite oxidation operate concurrently: the direct contact mechanism which requires physical contact between bacteria and pyrite particles for biological pyrite oxidation, and the indirect contact mechanism according to which the bacteria oxidize ferrous ions to the ferric state, thereby regenerating the ferric ions required for chemical oxidation of pyrite.     

Comparison of NTA and Elemental Sulfur as Potential Soil Amendments in Phytoremediation

The effect of NTA (nitrilotriacetic acid) and elemental sulfur (S), two soil amendments suggested for the enhancement of metal phytoavailability in phytoextraction, on heavy metal uptake by Nicotiana tabacum (tobacco) and Zea mays (maize) were studied and compared in two Zn-, Cu-, Cd -, and Pb-contaminated soils from northern Switzerland. Experiments were performed in the greenhouse with topsoil (0 to 20 cm) material from two locations, Dornach and Rafz. The Dornach soil was calcareous and had been contaminated by dust emissions from a nearby brass metal smelter. The Rafz soil, free of carbonates, had been polluted by former sewage sludge application. Soil amendments with S increased the solubility (NaNO₃ extraction) of Zn and Cd about 10-fold in Dornach soil and up to 30-fold in Rafz soil after 55 days. Zn and Cd removal by N. tabacum and Z. mays, however, increased only about 5.5- and 2.5-fold in these treatments in Rafz soil, respectively, while in the Dornach soil only a slight increase for Cd was found. Repeated NTA application increased soluble Zn, Cu, and Cd about 100-, 20-, and 19-fold in the Dornach soil and 13-, 4-, and 2-fold in the Rafz soil shortly after application. Soluble Pb was increased by NTA up to 50-fold in Rafz soil. After 90 days soluble heavy metal concentrations were only slightly elevated in both soils. Again, however, Zn, Cd, and Cu removal by N. tabacum and Z. mays increased only about 1.5- to 2.5-fold in the two soils, whereas Pb removal by N. tabacum increased about fivefold in the Rafz soil as a result of NTA application     

Inorganic Sulfur Oxidation by Aureobasidium pullulans

Aureobasidium pullulans (de Bary) Arnaud isolated from the phylloplane of sycamore exposed to heavy atmospheric pollution oxidized S⁰ to S₂O₃²⁻, S₄O₆²⁻, and SO₄²⁻ in vitro. The intermediates S₂O₃²⁻ and S₄O₆²⁻ were also oxidized to SO₄²⁻. Cell-free extracts of A. pullulans also oxidized reduced forms of S, the oxidation increasing linearly with increasing protein concentration, showing that the process is enzymatic. The possible role of fungi in S oxidation in soils is discussed.