Oxidation of Inorganic Sulfur Compounds by Obligately Organotrophic Bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria which are most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

PRODUCTS OF THE OXIDATION OF THIOSULFATE BY BACTERIA IN MINERAL MEDIA

Various cultures (previously described), which oxidize thiosulfate in mineral media have been studied in an attempt to determine the products of oxidation. The transformation of sodium thiosulfate by Cultures B, T, and K yields sodium tetrathionate and sodium hydroxide; secondary chemical reactions result in the accumulation of some tri- and pentathionates, sulfate, and elemental sulfur. As a result of the initial reaction, the pH increases; the secondary reactions cause a drop in pH after this initial rise. The primary reaction yields much less energy than the reactions effected by autotrophic bacteria. No significant amounts of assimilated organic carbon were detected in media supporting representatives of these cultures. It is concluded that they are heterotrophic bacteria. Th. novellus oxidizes sodium thiosulfate to sodium sulfate and sulfuric acid; the pH drops progressively with growth and oxidation. Carbon assimilation typical of autotrophic bacteria was detected; the ratio of sulfate-sulfur formed to carbon assimilated was 56:1. It is calculated that 5.1 per cent of the energy yielded by the oxidation of thiosulfate is accounted for in the organic cell substance synthesized from inorganic materials. This organism is a facultative autotroph. The products of oxidation of sodium thiosulfate by Th. thioparus are sodium sulfate, sulfuric acid, and elemental sulfur; the ratio of sulfate sulfur to elemental sulfur is 3 to 2. The pH decreases during growth and oxidation. The elemental sulfur is produced by the primary reaction and is not a product of secondary chemical changes. The bacterium synthesizes organic compounds from mineral substances during growth. The ratio of thiosulfate-sulfur oxidized to carbon assimilated was 125:1, with 4.7 per cent of the energy of oxidation recovered as organic cell substance. This bacterium is a strict autotroph.     

Microbial thiocyanate utilization under highly alkaline conditions

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO-) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a "cyanate pathway" in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.     

Inorganic sulfur oxidizing system in green sulfur bacteria

Green sulfur bacteria use various reduced sulfur compounds such as sulfide, elemental sulfur, and thiosulfate as electron donors for photoautotrophic growth. This article briefly summarizes what is known about the inorganic sulfur oxidizing systems of these bacteria with emphasis on the biochemical aspects. Enzymes that oxidize sulfide in green sulfur bacteria are membrane-bound sulfide-quinone oxidoreductase, periplasmic (sometimes membrane-bound) flavocytochrome c sulfide dehydrogenase, and monomeric flavocytochrome c (SoxF). Some green sulfur bacteria oxidize thiosulfate by the multienzyme system called either the TOMES (thiosulfate oxidizing multi-enzyme system) or Sox (sulfur oxidizing system) composed of the three periplasmic proteins: SoxB, SoxYZ, and SoxAXK with a soluble small molecule cytochrome c as the electron acceptor. The oxidation of sulfide and thiosulfate by these enzymes in vitro is assumed to yield two electrons and result in the transfer of a sulfur atom to persulfides, which are subsequently transformed to elemental sulfur. The elemental sulfur is temporarily stored in the form of globules attached to the extracellular surface of the outer membranes. The oxidation pathway of elemental sulfur to sulfate is currently unclear, although the participation of several proteins including those of the dissimilatory sulfite reductase system etc. is suggested from comparative genomic analyses.     

Reduced sulfur compound oxidation by Thiobacillus caldus.

The oxidation of reduced inorganic sulfur compounds was studied by using resting cells of the moderate thermophile Thiobacillus caldus strain KU. The oxygen consumption rate and total oxygen consumed were determined for the reduced sulfur compounds thiosulfate, tetrathionate, sulfur, sulfide, and sulfite in the absence and in the presence of inhibitors and uncouplers. The uncouplers 2,4-dinitrophenol and carbonyl cyanide m-chlorophenyl-hydrazone had no affect on the oxidation of thiosulfate, suggesting that thiosulfate is metabolized periplasmically. In contrast, the uncouplers completely inhibited the oxidation of tetrathionate, sulfide, sulfur, and sulfite, indicating that these compounds are metabolized in the cytoplasm of T. caldus KU. N-Ethylmaleimide inhibited the oxidation of tetrathionate and thiosulfate at the stage of elemental sulfur, while 2-heptyl-4-hydroxyquinoline-N-oxide stopped the oxidation of thiosulfate, tetrathionate, and elemental sulfur at the stage of sulfite. The following intermediates in the oxidation of the sulfur compounds were found by using uncouplers and inhibitors: thiosulfate was oxidized to tetrathionate, elemental sulfur was formed during the oxidation of tetrathionate and sulfide, and sulfite was found as an intermediate of tetrathionate and sulfur metabolism. On the basis of these data we propose a model for the metabolism of the reduced inorganic sulfur compounds by T. caldus KU.     

Chemolithoautotrophic oxidation of thiosulfate, tetrathionate and thiocyanate by a novel rhizobacterium belonging to the genus Paracoccus

Two tropical leguminous-rhizospheric strains, SST and JT 001, phylogenetically closest to Paracoccus thiocyanatus and Paracoccus pantotrophus, respectively, were isolated on reduced sulfur compounds as sole energy and electron sources. While SST had versatile chemolithotrophic abilities to oxidize thiosulfate, tetrathionate, thiocyanate, sulfide and elemental sulfur, JT 001 could oxidize thiosulfate, soluble sulfide, elemental sulfur and a relatively lesser amount of tetrathionate. Positive hybridization signals were detected for JT 001 but not SST, when their genomic DNAs were probed with DIG-labeled sulfur oxidation genes amplified from the chemolithotrophic alphaproteobacterium Pseudaminobacter salicylatoxidans KCT001. Though the new isolate SST exhibited high 16S rRNA gene sequence similarity with the monotypic species P. thiocyanatus, it was found to be considerably distinct from the latter in terms of phenotypic and chemotaxonomic characteristics. Polyphasic systematic analysis, however, confirmed that JT 001 was a strain of P. pantotrophus.     

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.     

Microbial Thiocyanate Utilization under Highly Alkaline Conditions

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS⁻) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO⁻) to ammonia and CO₂. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.     

H<Subscript>2</Subscript>S biotreatment with sulfide-oxidizing heterotrophic bacteria

Many industrial activities produce H₂S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H₂S to thiosulfate and sulfite. These bacteria may also potentially be used in H₂S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H₂S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min^?1 g^?1 of cell dry weight at pH 7.4, sufficient for H₂S biotreatment. The immobilized bacteria removed H₂S as efficiently as the bacteria in suspension, and the inclusion of Fe₃O₄ nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H₂S by Fe₃O₄. Thus, heterotrophic bacteria may be used for H₂S biotreatment under aerobic conditions.     

Roseinatronobacter thiooxidans gen. nov., sp. nov., a new alkaliphilic aerobic bacteriochlorophylla—containing bacterium isolated from a soda lake

Several samples of microbial mat obtained from soda lakes of the Kunkurskaya steppe (Chita region) 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 bacteriochlorophylla. Pigments were synthesized only at high concentrations of nitrogen-containing organic compounds (peptone or yeast extract). The production of bacteriochlorophylla 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 α-3 subgroup of Proteobacteria and to constitute a distinct branch located between nonsulfur purple bacteriaRhodobacter andRhodovulum. Based on the unique phenotypic properties and the results of phylogenetic analysis, the alkaliphilic isolate ALG 1 was assigned to a new genus and speciesRoseinatronobacter thiooxidans with the type strain DSM-13087     

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H₂, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O₂ as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O₂ concentrations or ceased after repeated O₂ additions. The amounts of O₂ consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O₂ completely to CO₂. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O₂ probably required a reductive first step, since it was obtained only with energized intact cells.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - APS adenosine phosphosulfate or adenylyl sulfate     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

Sulfur cycling in Catenococcus thiocyclus

Abstract: Since its isolation from marine volcanic areas, Catenococcus thiocyclus has been known to be able to oxidize thiosulfate to tetrathionate, but the benefits gained from the reaction were unknown. The energy to be gained from such a reaction is so small (1 electron per mol of thiosulfate, compared with 8 electrons if the thiosulfate is oxidized to sulfate) that it seemed unlikely to be a useful metabolic reaction. However, continuous culture experiments have now revealed that C. thiocyclus is able to gain metabolically useful energy from this oxidation (biomass yields increased by approximately 20% after the addition of 7.75 mM thiosulfate to medium containing 20 mM acetate) by combining it with the chemical reduction of the tetrathionate by sulfide. The enzymes for thiosulfate oxidation appear to be constitutive. Moreover, with a suitable primary energy source (e.g. glucose), C. thiocyclus can reduce sulfur (S°) to sulfide and Fe³⁺ to Fe²⁺. A chemical reaction then generates FeS. Such reactions may have important implications for the sulfur cycle at oxic:anoxic interfaces in marine and freshwater systems.     

Thiosulfate stimulation of microbial dark assimilation of carbon dioxide in shallow marine waters

The effect of thiosulfate on dark assimilation of carbon dioxide in shallow marine environments was investigated in order to explain the recent discovery of bacterial thiosulfate oxidation in aerobic, open ocean seawater. The results demonstrate that the potential exists for microbial thiosulfate oxidation to increase both dark assimilation of carbon dioxide and the utilization of organic compounds in the sea. Thiosulfate-stimulated microbial activity may be caused not only by chemoautotrophic sulfur bacteria, but also by heterotrophic species which oxidize thiosulfate to tetrathionate. Measurements of dark assimilation of carbon dioxide made at different incubation times indicate that great care must be taken both in experimental procedure and in interpretation of results obtained with the dark assimilation technique.     

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     

Colourless sulfur bacteria and their role in the sulfur cycle

Summary The bacteria belonging to the families of the Thiobacteriaceae, Beggiatoaceae and Achromatiaceae are commonly called the colourless sulfur bacteria. While their ability to oxidize reduced inorganic sulfur compounds has clearly been established, it is still not known whether all these organisms can derive metabolically useful energy from these oxidations. During the last decades research has mainly focussed on the genus Thiobacillus. Bacteria belonging to this genus can oxidize a variety of reduced inorganic sulfur compounds and detailed information is available on the biochemistry and physiology of these energy-yielding reactions. The thiobacilli, most of which can synthesize all cell material from CO₂, possess a well-regulated metabolic machinery with high biosynthetic capacities, which is essentially similar to that of other procaryotic organisms. Although the qualitative role of colourless sulfur bacteria in the sulfur cycle is well documented, quantitative data are virtually absent. Activities of colourless sulfur bacteria in nature must be related to direct and indirect parameters, such as: the rate of oxidation of (S³⁵) sulfur compounds, the rate of C¹⁴O₂-fixation, the rate of acid production and numbers and growth rates of the bacteria. However, chemical reactions and similar activities of heterotrophic organisms mask the activities of the colourless sulfur bacteria to various extents, depending on the condition of the natural environment. This interference is minimal in regions where high temperature and/or low pH allow the development of a dominant population of colourless sulfur bacteria, such as hot acid sulfur springs, sulfide ores, sulfur deposits and some acid soils. The oxidation of inorganic sulfur compounds is carried out by a spectrum of sulfur-oxidizing organisms which includes: 1) obligately chemolithotrophic organisms 2) mixotrophs 3) chemolithotrophic heterotrophs 4) heterotrophs which do not gain energy from the oxidation of sulfur compounds but benefit in other ways from this reaction, and 5) heterotrophs which do not benefit from the oxidation of sulfur compounds. The spectrum is completed by a hypothetical group of heterotrophic organisms, which may have a symbiotic relationship with thiobacilli and related bacteria. Such heterotrophs may stimulate the growth of colourless sulfur bacteria and thereby contribute to the oxidation of sulfur compounds. Future research should focus in the first place on obtaining and studying pure cultures of many of the colourless sulfur bacteria. In the second place, studies on the physiological and ecological aspects of mixed cultures of colourless sulfur bacteria and heterotrophs may add to a better understanding of the role of the colourless sulfur bacteria in the sulfur cycle. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974.     

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.     

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.     

Homologs from sulfur oxidation (Sox) and methanol dehydrogenation (Xox) enzyme systems collaborate to give rise to a novel pathway of chemolithotrophic tetrathionate oxidation

The SoxXAYZB(CD)₂‐mediated pathway of bacterial sulfur‐chemolithotrophy explains the oxidation of thiosulfate, sulfide, sulfur and sulfite but not tetrathionate. Advenella kashmirensis, which oxidizes tetrathionate to sulfate, besides forming it as an intermediate during thiosulfate oxidation, possesses a soxCDYZAXOB operon. Knock‐out mutations proved that only SoxBCD is involved in A. kashmirensis tetrathionate oxidation, whereas thiosulfate‐to‐tetrathionate conversion is Sox independent. Expression of two glutathione metabolism‐related proteins increased under chemolithotrophic conditions, as compared to the chemoorganotrophic one. Substrate‐dependent oxygen consumption pattern of whole cells, and sulfur‐oxidizing enzyme activities of cell‐free extracts, measured in the presence/absence of thiol inhibitors/glutathione, corroborated glutathione involvement in tetrathionate oxidation. Furthermore, proteome analyses detected a sulfite:acceptor oxidoreductase (SorAB) exclusively under chemolithotrophic conditions, while expression of a methanol dehydrogenase (XoxF) homolog, subsequently named thiol dehydrotransferase (ThdT), was found to increase 3‐ and 10‐fold during thiosulfate‐to‐tetrathionate conversion and tetrathionate oxidation respectively. A thdT knock‐out mutant did not oxidize tetrathionate but converted half of the supplied 40 mM S‐thiosulfate to tetrathionate. Knock‐out of another thiosulfate dehydrogenase (tsdA) gene proved that both ThdT and TsdA individually converted ～ 20 mM S‐thiosulfate to tetrathionate. The overexpressed and isolated ThdT protein exhibited PQQ‐dependent thiosulfate dehydrogenation, whereas its PQQ‐independent thiol transfer activity involving tetrathionate and glutathione potentially produced a glutathione:sulfodisulfane adduct and sulfite. SoxBCD and SorAB were hypothesized to oxidize the aforesaid adduct and sulfite respectively.     

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.