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.     

Effect of increased sediment sulfide concentrations on the composition of stable sulfur isotopes (δS) and sulfur accumulation in the seagrasses Zostera marina and Posidonia oceanica

The effect of increased sediment sulfide concentrations on the sulfur isotopic composition (δ³⁴S), total sulfur (TS) and elemental sulfur (S⁰) concentrations in plant tissues was studied for the two seagrasses Zostera marina (3 weeks in laboratory) and Posidonia oceanica (4 months in situ). Porewater sulfide concentrations were experimentally regulated and plants exposed to high sediment sulfide concentrations had δ³⁴S signals closer to the δ³⁴S of sulfide, whereas plants exposed to no / low sulfide concentrations had δ³⁴S signals closer to the δ³⁴S of seawater sulfate, indicating a higher sulfide invasion in plants exposed to high sulfide concentrations. The δ³⁴S varied between the plant tissues in both species with the leaves having more positive δ³⁴S signals than roots and rhizomes, indicating that sulfide was invading into the roots and moved to the other tissues through the lacunae. TS and S⁰ concentrations were higher in plants exposed to sulfide in both experiments suggesting that sulfur derived from sediment sulfide accumulated in the plants. The δ³⁴S signal in S⁰ was similar to sediment sulfide verifying that S⁰ found in the seagrasses originated from sediment sulfide. Direct comparisons of δ³⁴S in the two different seagrasses and across the treatments were not possible due to large differences in δ³⁴S of the sulfur sources. F_sulfide adjusted for these differences and may be a useful alternative, when δ³⁴S of the sulfur sources varies between study sites. There were no significant effects of sulfide exposure on plant growth and mortality in Z. marina and P. oceanica after 3 weeks and 8 weeks exposure, respectively, but P. oceanica showed indications of reduced growth and higher mortality after 16 weeks of sulfide exposure probably due to sulfide invasion/toxicity.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Reduction of bromate by biogenic sulfide produced during microbial sulfur disproportionation

Bromate (BrO₃ ⁻) is a carcinogenic contaminant formed during ozonation of waters that contain trace amounts of bromide. Previous research shows that bromate can be microbially reduced to bromide using organic (i.e. acetate, glucose, ethanol) and inorganic (H₂) electron-donating substrates. In this study, the reduction of bromate by a mixed microbial culture was investigated using elemental sulfur (S⁰) as an electron donor. In batch bioassays performed at 30°C, bromate (0.30 mM) was completely converted to bromide after 10 days and no accumulation of intermediates occurred. Bromate was also reduced in cultures supplemented with thiosulfate and hydrogen sulfide as electron donor. Our results demonstrated that S⁰-disproportionating microorganisms were responsible for the reduction of bromate in cultures spiked with S⁰ through an indirect mechanism involving microbial formation of sulfide and subsequent abiotic reduction of bromate by the biogenic sulfide. Confirmation of this mechanism is the fact that bromate was shown to undergo rapid chemical reduction by sulfide (but not S⁰ or thiosulfate) in abiotic experiments. Bromate concentrations above 0.30 mM inhibited sulfide formation by S⁰-disproportionating bacteria, leading to a decrease in the rate of bromate reduction. The results suggest that biological formation of sulfide from by S⁰ disproportionation could support the chemical removal of bromate without having to directly use sulfide as a reagent.     

Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester

During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative λ_max at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S₈ configuration. No labeled elemental sulfur was detected when the cells were grown on [³⁵S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella.     

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⁺.     

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

Massive pyrite (FeS₂) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S _n ^2?, S⁰). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe_1?x S₂) to active sulfur species (e.g., Fe_1?x S_2?y , S⁰). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S _n ^2?/S⁰ speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S _n ^2?/S⁰ surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.     

Fe(III) oxides protect fermenter–methanogen syntrophy against interruption by elemental sulfur via stiffening of Fe(II) sulfides produced by sulfur respiration

Thermosipho globiformans (rod-shaped thermophilic fermenter) and Methanocaldococcus jannaschii (coccal hyperthermophilic hydrogenotrophic methanogen) established H₂-mediated syntrophy at 68 °C, forming exopolysaccharide-based aggregates. Electron microscopy showed that the syntrophic partners connected to each other directly or via intercellular bridges made from flagella, which facilitated transfer of H₂. Elemental sulfur (S⁰) interrupted syntrophy; polysulfides abiotically formed from S⁰ intercepted electrons that were otherwise transferred to H⁺ to produce H₂, resulting in the generation of sulfide (sulfur respiration). However, Fe(III) oxides significantly reduced the interruption by S⁰, accompanied by stiffening of Fe(II) sulfides produced by the reduction of Fe(III) oxides with the sulfur respiration-generated sulfide. Sea sand replacing Fe(III) oxides failed to generate stiffening or protect the syntrophy. Several experimental results indicated that the stiffening of Fe(II) sulfides shielded the liquid from S⁰, resulting in methane production in the liquid. Field-emission scanning electron microscopy showed that the stiffened Fe(II) sulfides formed a network of spiny structures in which the microorganisms were buried. The individual fermenter rods likely produced Fe(II) sulfides on their surface and became local centers of a core of spiny structures, and the connection of these cores formed the network, which was macroscopically recognized as stiffening.     

Sulfur accumulation in eelgrass (Zostera marina) and effect of sulfur on eelgrass growth

Eelgrass (Zostera marina) was grown under exposure to high levels of sediment sulfides to examine their ability to reoxidize sulfides intruding into the plants. The plants were kept under full light (control and high sulfide level) and at 10% of light saturation (high sulfide level) for 3 weeks and growth and accumulation of elemental sulfur (S⁰) in the plants were examined. The growth rate was reduced with ∼75% in the low light treatment, whereas there was no significant difference between the rates at full light saturation. S⁰ was accumulating in the below-ground structures of the plants exposed to high sulfide concentrations with highest concentration in the youngest roots and oldest internodes. There was no accumulation of S⁰ in the leaves, suggesting that the intruding sulfides were reoxidized in the below-ground structures before reaching the leaves. The accumulation of S⁰ was higher in the roots of the low light treatment (up to two times) suggesting a larger intrusion of sulfides. These plants also appeared highly affected by the treatment with rotting meristems and increased mortality after the 3-week growth period. These results are the first to show an accumulation of sulfur compounds internally in seagrasses as a result of reoxidation of sulfides. The reoxidation is facilitated by the internal transport of oxygen and is an example of the advantage of the internal lacunae system in seagrasses.     

Isotope exchange reactions with radiolabeled sulfur compounds in anoxic seawater

The isotope exchange between³⁵S-labeled sulfur compounds of sulfate (SO₄ ^2–), elemental sulfur (S⁰), polysulfide (S_n ^2–), hydrogen sulfide (HS^–: H₂S + HS^– + S^2–), iron sulfide (FeS), and pyrite (FeS₂) was studied at pH 7.6 and 20 °C in anoxic, sterile seawater. Isotope exchange was observed between S⁰, S₂ ^2– HS^–, and FeS, but not between³⁵S labeled SO₄ ^2– or FeS₂ and the other sulfur compounds. Polysulfide mediated the isotope exchange between S⁰ and bisulfide (HS^–). The isotope exchange between S⁰ and S_n ^2–) reached 50% of equilibrium within < 2 min while exchange between S₂ ^2– and HS^– approached equilibrium within 0.5-1 h. In all the experiments HS^–, revealed a fraction exchange from 0.79 to 1.00. Isotope exchange between S^2– and FeS took place only via S₂ ^2– and/or HS^–. The isotope exchange between iron sulfide and the other sulfur compounds was not complete within 24 h as shown by a fraction exchange of 0.07–0.83. This lack of equilibrium (fraction exchange < 1) was due to the isotope exchange between dissolved compounds and surfaces of sulfur particles. The isotopic exchange reactions limit the usefulness of radiotracers in process studies of the inorganic sulfur species. Exchange reactions will also affect the stable isotope distribution among the sulfur species. The kinetics of the isotopic exchange reactions, however, depend on both pH and temperature.     

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.     

Transformation of galena to pyromorphite produces bioavailable sulfur for neutrophilic chemoautotrophy

The aqueous concentration of lead [Pb(II)] in geochemical environments is controlled by the solubility of Pb‐bearing minerals and their weathering products. In contaminated soils, a common method for in situ stabilization of Pb(II) is the addition of phosphate to convert more redox sensitive sulfide minerals into sparingly soluble pyromorphite [Pb₅(PO₄)₃X]. In this study, we conducted experimental studies to investigate the fate of reduced sulfur during the conversion of galena [PbS] to chloropyromorphite [Pb₅(PO₄)₃Cl]. Powder X‐ray diffraction analysis indicated that the reaction of phosphate with galena under oxic conditions resulted in the oxidation of sulfide and formation of elemental sulfur [S₈]. Under oxic abiotic conditions, the S₈ was retained in the solid phase, and negligible concentrations of sulfur as sulfide and thiosulfate were detected in the aqueous phase and only a small amount of sulfate. When PbS reacted in the presence of the chemoautotrophic organism Bosea sp. WAO, the S₈ in the secondary mineral was oxidized to sulfate. Strain WAO produced significantly more sulfate from the secondary S₈ than from the primary galena. Microscopic analysis of mineral–microbe aggregates on mineral‐embedded slide cultures showed that the organism was colocalized and increased in biomass over time on the secondary mineral surface supporting a microbial role. The results of this study indicate that stimulation of sulfur‐oxidizing activity may be a direct consequence of phosphate amendments to Pb(II)‐contaminated soils.     

Isolation, Characterization, and In Situ Detection of a Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under Microaerophilic Conditions

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S⁰), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 μm) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H₂S and S⁰ to SO₄²⁻ under oxic conditions.     

Succession of Internal Sulfur Cycles and Sulfur-Oxidizing Bacterial Communities in Microaerophilic Wastewater Biofilms

The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S⁰ accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H₂S was only partially oxidized to S⁰ by mainly Thiomicrospira denitirificans with NO₃⁻ as an electron acceptor, leading to significant accumulation of S⁰ in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S⁰ accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S⁰ to SO₄²⁻ under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 10⁹ cells cm⁻³) and strain SO07 (ca. 10⁸ cells cm⁻³) were found at the sulfur-rich surface (100 μm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 10⁷ to 10⁸ cells cm⁻³. Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H₂S produced by SRB to SO₄²⁻ in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.     

Growth kinetics of haloalkaliphilic,sulfur-oxidizing bacterium<Emphasis Type="Italic"> Thioalkalivibrio versutus</Emphasis> strain ALJ 15 in continuous culture

The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (K_s=6±3 M). The maintenance requirement (m_s=8±5 mmol S₂O₃²/g dry weight h^–1) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (S_n^2–) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.Communicated by W.D. Grant     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid