Isolation and characterization of alkaliphilic, chemolithoautotrophic, sulphur-oxidizing bacteria

Alkaliphilic sulphur-oxidizing bacteria were isolated from samples from alkaline environments including soda soil and soda lakes. Two isolates, currently known as strains AL 2 and AL 3, were characterized. They grew over a pH range 8.0–10.4 with an optimum at 9.5–9.8. Both strains could oxidize thiosulphate, sulphide, polysulphide, elemental sulphur and tetrathionate. Strain AL 3 more actively oxidized thiosulphate and sulphide, while isolate AL 2 had higher activity with elemental sulphur and tetrathionate. Isolate AL 2 was also able to oxidize trithionate. The pH optimum for thiosulphate and sulphide oxidation was between 9–10. Some activity remained at pH 11, but was negligible at pH 7. Metabolism of tetrathionate by isolate AL 2 involved initial anaerobic hydrolysis to form sulphur, thiosulphate and sulphate in a sequence similar to that in other colourless sulphur-oxidizing bacteria. Sulphate was produced by both strains. During batch growth on thiosulphate, elemental sulphur and sulphite transiently accumulated in cultures of isolates AL 2 and AL 3, respectively. At lower pH values, both strains accumulated sulphur during sulphide and thiosulphate oxidation. Both strains contained ribulose bisphosphate carboxylase. Thiosulphate oxidation in isolate AL 3 appeared to be sodium ion-dependent. Isolate AL 2 differed from AL 3 by its high GC mol % value (65.5 and 49.5, respectively), sulphur deposition in its periplasm, the absence of carboxysomes, lower sulphur-oxidizing capacity, growth kinetics (lower growth rate and higher growth yield) and cytochrome composition.     

Oxidation of reduced sulphur compounds by intact cells of Thiobacillus acidophilus

Oxidation of reduced sulphur compounds by Thiobacillus acidophilus was studied with cell suspensions from heterotrophic and mixotrophic chemostat cultures. Maximum substrate-dependent oxygen uptake rates and affinities observed with cell suspensions from mixotrophic cultures were higher than with heterotrophically grown cells. ph Optima for oxidation of sulphur compounds fell within the pH range for growth (pH 2–5), except for sulphite oxidation (optimum at pH 5.5). During oxidation of sulphide by cell suspensions, intermediary sulphur was formed. Tetrathionate was formed as an intermediate during aerobic incubation with thiosulphate and trithionate. Whether or not sulphite is an inter-mediate during sulphur compound oxidation by T. acidophilus remains unclear. Experiments with anaerobic cell suspensions of T. acidophilus revealed that trithionate metabolism was initiated by a hydrolytic cleavage yielding thiosulphate and sulphate. A hydrolytic cleavage was also implicated in the metabolism of tetrathionate. After anaerobic incubation of T. acidophilus with tetrathionate, the substrate was completely converted to equimolar amounts of thiosulphate, sulphur and sulphate. Sulphide- and sulphite oxidation were partly inhibited by the protonophore uncouplers 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) and by the sulfhydryl-binding agent N-ethylmaleimide (NEM). Oxidation of elemental sulphur was completely inhibited by these compounds. Oxidation of thiosulphate, tetrathionate and trithionate was only slightly affected. The possible localization of the different enzyme systems involved in sulphur compound oxidation by T. acidophilus is discussed.     

Correlation of viable cell counts,metabolic activity of sulphur-oxidizing bacteria and chemical parameters of marine sediments

Viable counts of aerobic and anaerobic chemotrophic sulphur-oxidizers as well as phototrophic sulphur bacteria were determined in sediment samples taken from two different areas along the Baltic Sea shore which were known to regularly develop sulphidic conditions. Depth profiles of bacterial cell counts were correlated with concentration profiles of chloride, sulphate, sulphide, nitrate and phosphate in the pore water of these sediments and with potential activities of nitrate reduction, thiosulphate transformation and sulphate formation. The data revealed a complex multilayered structure within the sediments. Sulphide was released into the water from sediments of both sampling areas, but it was found that light and the availability of oxygen significantly reduced this amount. In the highly reduced sediment at Hiddensee, the highest numbers of phototrophic and chemotrophic sulphur-oxidizers were found near the sediment surface. Therefore, it was concluded that the combined action of both groups of bacteria most efficiently oxidizes reduced sulphur compounds in the top layers of the sediments. Nitrate may replace oxygen as final electron acceptor and will support oxidation of sulphide, in particular when oxygen and light are limiting.     

Corrosion of Pembina crude oil pipeline

Summary Corrosion failure of the Pembina pipeline system of North Central Alberta, Canada, was frequent and was associated with constant bacterial load and sulphide in the crude oil and produced water. The bacterial load included a variety of anaerobic and aerobic/facultative bacteria which acted in concert to produce sulphide, giving rise to a cascade of sulphide generation.A total of 256 isolates from the crude oil were tested for ability to reduce oxidized sulphur compounds to sulphide. Five groups of bacteria, (A-E), based on this ability to reduce sulphur compounds, existed in the crude oil system. Group A reduced sulphur compounds with oxidation states +6; and lower, Group B reduced oxidation state +4 and below; Group C, oxidation states +2 and lower. Group D reduced only oxidation state 0 (elemental Sulphur), while Group E could reduce no sulphur compound to sulphide. It was found that a ceiling on the reductive capability of each bacterial group was set by the oxidation state of the sulphur compounds. The result is a synergistic relationship whereby intermediate products of reductive activities of each group form the substrate for subsequent action by other groups until sulphide is produced.     

Novel domain packing in the crystal structure of a thiosulphate-oxidizing enzyme

A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains. The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide. The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold. The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.     

Growth of Thiocapsa roseopersicina purple sulfur bacteria in darkness under anaerobic conditions]

The phototrophic sulphur bacterium. Thiocapsa roseopersicina, strain BBS, was grown under anaerobic conditions in the darkness on the medium containing glucose and thiosulphate or molecular sulphur. The assimilation of glucose is accompanied by the accumulation of small amounts of pyruvate in the medium, and the uptake of thiosulphate or molecular sulphur leads to the formation of sulphates and hydrogen sulphide.     

Reduction of tetrathionate,trithionate and thiosulphate,and oxidation of sulphide in Proteus mirabilis

The reductase catalyzing the reduction of tetrathionate and thiosulphate in Proteus mirabilis is also concerned with the reduction of trithionate and the oxidation of sulphide. Tetrathionate is reduced to thiosulphate, thiosulphate to sulphite and sulphide, and trithionate is reduced to thiosulphate plus sulphite. The oxidation of sulphide in cell-free extracts proceeds most likely to polysulphanes or to elemental sulphur, depending on the conditions. The kinetics of the reduction of tetrathionate imply a simultaneous interaction of tetrathionate and thiosulphate on the reductase molecule. The reduction of tetrathionate is activated by thiosulphate causing a non-linear progress of this reaction. On the other hand the reduction of thiosulphate is completely blocked until tetrathionate has been depleted. The order of reduction in a mixture of thiosulphate and trithionate is imputed by the enzymatic constants of the reductase for both substrates. Therefore in cell-free extracts thiosulphate is reduced prior to trithionate and afterwards, when thiosulphate has been exhausted, trithionate and the produced thiosulphate are reduced simultaneously. Fast growing cells, however, reduce trithionate first since their intracellular redox potential is insulfficiently low to permit the reduction of any thiosulphate.     

Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells ofChromatium okenii

Growth experiments and short term experiments in a stirred cuvette showed thatChromatium okenii strain Ostrau is not able to oxidize any reduced sulphur compounds except sulphide and elementary sulphur; thiosulphate, sulphite, and thioglycolate can not be utilized as reducing agents for photosynthesis. The cells are not able to use H₂; hydrogenase could not be demonstrated. In the dark, sulphide is formed from intracellular sulphur and the carbon content of the cells decreases. Growth and turnover of sulphur compounds was followed in the light in the presence and absence of acetate as a second carbon source. Sulphide oxidation depends on the presence of CO₂ and on light intensity, i.e. sulphur metabolism is governed by the photosynthetic activity of the cells.     

Chemolithoutotrophic growth of Thiothrix ramosa

Thiothrix has been shown for the first time to be able to grow chemolithoautotrophically with thiosulphate or carbon disulphide as sole energy substrate. Thiosulphate served as the growth-limiting substrate for Thiothrix ramosa in chemostat culture. Maximum growth yield (Y_max) from yields at growth rates between 0.029–0.075 h^-1 was 4.0 g protein/mol thiosulphate oxidized. The key enzyme of the Calvin cycle, ribulose 1,5-bisphosphate carboxylase, was present in these cells, as were rhodanese, adenylyl sulphate (APS) reductase and sulphur-oxidizing enzyme. Thiosulphate-grown cells oxidized thiosulphate, sulphide, tetrathionate and carbon disulphide. Oxidation kinetics for sulphide, thiosulphate and tetrathionate were biphasic: oxygen consumption during the fast first phase of oxidation indicated oxidation of sulphide, and the sulphane moieties of thiosulphate and tetrathionate, to elemental sulphur, before further oxidation to sulphate. Kinetic constants for these four substrates were determined. T. ramosa also grew mixotrophically in batch culture on lactate with a number of organic sulphur compounds: carbon disulphide, methanethiol and diethyl sulphide. Substituted thiophenes were also used as sole substrates. The metabolic versatility of T. ramosa is thus much greater than previously realised.     

The Types and Distribution of Thiobacilli in Biological Systems Treating Carbonization Effluents

Summary: An extensive investigation of 30 strains of bacteria which oxidize inorganic sulphur compounds led to the recognition of three major groups. A study of the occurrence of these groups in biological effluent systems suggested that the organisms generally believed to be responsible for the oxidation of thiosulphate and thiocyanate, the autotrophic thiobacilli, were absent in many instances. It is suggested that in these instances heterotrophic organisms, which are found throughout all the systems, may be responsible for the destruction of the sulphur compounds. A heterotrophic organism which destroys thiocyanate, but not thiosulphate, has been isolated.     

The bacteria of the sulphur cycle

This paper concentrates on the bacteria involved in the reductions and oxidations of inorganic sulphur compounds under anaerobic conditions. The genera of the dissimilatory sulphate-reducing bacteria known today are discussed with respect to their different capacities to decompose and oxidize various products of fermentative degradations of organic matter. The utilization of molecular hydrogen and formate by sulphate reducers shifts fermentations towards the energetically more favourable formation of acetate. Since acetate amounts to about two-thirds of the degradation products of organic matter, the complete anaerobic oxidation of acetate by several genera of the sulphate-reducing bacteria is an important function for terminal oxidation in sulphate-sufficient environments. The results of pure culture studies agree well with ecological investigations of several authors who showed the significance of sulphate reduction for the complete oxidation of organic matter in anaerobic marine habitats. In the dissimilatory sulphur-reducing bacteria of the genus Desulfuromonas the oxidation of acetate is linked to the reduction of elemental sulphur. Major characteristics of the anaerobic, sulphide-oxidizing phototrophic green and purple sulphur bacteria as well as of some facultative anoxygenic cyanobacteria, are given. By the formation of elemental sulphur and sulphate, these bacteria establish sulphur cycles with the sulphide-forming bacteria. In view of the morphological diversity of the sulphate-reducing bacteria and question of possible evolutionary relations to phototrophic sulphur bacteria is raised.     

The isolation ofChromatium Okenii and its behaviour in different media

Summary An enrichment culture ofChromatium Okenii, obtained from mud of a ditch at Delft, was used for isolation of this species. The bacteria of the strain, isolated by making 5 successive shake cultures in an agar medium were, however, less than half the size ofChr. Okenii when cultivated under the same conditions as the bacteria in the original culture.By variation of the culture conditions for the pure strain, it appeared that in a mineral medium containing fairly high concentrations of sodium thiosulphate and fairly low concentrations of sodium malate, bacteria developed of nearly the same size as present in the enrichment culture. This supports the view ofWinogradsky that media of this type offer the most natural conditions to purple sulphur bacteria.It is, therefore, evident thatChr. Okenii is a highly pleomorphic species, and the consequences hereof for the differentiation of species within the genusChromatium were discussed.     

Effect of industrial immissions with high sulphur dioxide content on thiobacilli and oxidative activity of spruce forest soils towards inorganic sulphur compounds

Effect of industrial immissions with high sulphur dioxide content on the upper horizons of spruce forest soils in NW Bohemia was investigated. The content of sulphates, oxidative activity towards sulphide, elemental sulphur, thiosulphate and sulphite, concentration and species representation of thiobacilli in horizons F, H and A in regions highly affected by immissions (two localities) and in regions relatively less influenced (three localities) were followed. In the affected areas the sulphur content in the soil was higher, the species representation of thiobacilli was similar and their concentration was higher, the ability of the soil to oxidize thiosulphate was inhibited and oxidation of elemental sulphur was stimulated. The oxidation of sulphide and sulphite was not significantly affected by the immissions. Changes caused by immissions could be observed only in horizons F and H and did not involve horizons A.     

The effect of a duckweed cover on sulphide volatilisation from waste stabilisation ponds

The effects of a duckweed (DW) cover on the surface of waste stabilisation ponds on sulphide emissions were studied in a laboratory scale set-up of an anaerobic pond-reactor, followed by two algae (A) pond-reactors and two duckweed (Lemna gibba) pond-reactors. The concentrations of various S-components were measured at different depth in the reactors, while sulphide emissions were measured at the surface. Presence of a duckweed cover on the anaerobic pond-reactor resulted in a reduction of 99% in sulphide emission. In algae pond-reactors, sulphide emissions were negligible through chemical and biological conversion of sulphide. In the duckweed pond-reactors, colourless sulphur bacteria (Beggiatoa sp.) were observed on the duckweed roots. Batch tests showed that both micro-biological and possibly chemical oxidation occurred in a typical duckweed pond environment. The duckweed cover reduced H₂S volatilisation via two mechanisms, by forming a physical barrier and by providing attachment area for sulphide oxidising bacteria.     

Isolation and characterization of sulphate-reducing bacteria <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> from Vajreshwari thermal springs in Maharashtra,India

Sulphate-reducing bacteria (SRB) in the thermal springs of Vajreshwari were investigated with combined microbiological and molecular approaches. A sulphate-reducing bacteria medium containing lactate was used for enrichment and isolation, which yielded Gram negative, rod shaped, anaerobic, non-spore forming and motile bacteria capable of reducing sulphate to sulphide. These grew at temperatures ranging from 25 to 55 °C and could use pyruvate, lactate and ethanol as electron donors. Desulfoviridin was detected in all the isolates. The partial 16S rRNA and dissimilatory sulphite reductase (DSR) gene sequences of five representative isolates revealed that the strains belonged to the sulphur reducing bacterial species Desulfovibrio vulgaris.     

Sulphur metabolism in Thiorhodaceae. II. stoichiometric relationship of CO2 fixation to oxidation of hydrogen sulphide and intracellular sulphur inChromatium okenii

Stoichiometry of sulphide and intracellular sulphur oxidation in connection with CO₂ fixation was studied inChromatium okenii. The equipment used was a special stirred cuvette with a rapid-sampling arrangement, which allowed short-time experiments with illuminated bacterial suspensions under anaerobic conditions. Turnover of the sulphur compounds is controlled by a linear CO₂ fixation rate which amounts to 0.069µmoles of CO₂/min mg of cell protein at light saturation. Van Niel's equations for bacterial photosynthesis could be confirmed for short periods under the condition that sulphate is produced during increase of intracellular sulphur; i.e., oxidation of sulphide and of intracellular sulphur do not occur consecutively but simultaneously. The full oxidation rate of intracellular sulphur starts after complete consumption of sulphide. The time during which sulphide is oxidized to intracellular sulphur amounts to 1/3–1/4 of the time necessary for the complete quantitative oxidation of the sulphide to sulphate.     

Isolation of some new sulphur bacteria from activated sludge

E mtiazi , G., H abibi , M.H. & S etareh , M. 1990. Isolation of some new sulphur bacteria from activated sludge. Journal of Applied Bacteriology 69 , 864–870.
During studies on bulking of activated sludge some new sulphur micro-organisms, which were able to grow aerobically and anaerobically on reduced sulphur compounds, were isolated on thiosulphate agar. These were capable of autotrophic and heterotrophic growth on a wide range of substrates. In view of their ability to oxidize reduced sulphur compounds, and because one of them was an oxidase- and catalase-positive, Gram-negative, motile coccus (0.36–0.48 μm) it was named Thiosphaera persica . The second one was an oxidase- and catalase-positive, Gram-negative, motile rod (1.32–1.80 μm) and was named Thiobacillus persica . The third one was oxidase-negative, catalase-positive, motile, Gram-negative and polymorphic and was named Sulphobacter polymorpha .     

Some factors influencing production of sulphate by oxidation of elemental sulphur and thiosulphate in upper horizons of spruce forest soils

Some factors influencing the oxidative activity of upper horizons of spruce forest soils (a mixture of fermentative and humus layers) toward intermediates of the oxidative part of the sulphur cycle were investigated. Preincubation of the soil with added cysteine, sulphide, elemental sulphur or thiosulphate was found to stimulate enzyme systems oxidating any of these compounds. Sulphite and sulphate were ineffective in this respect. The oxidation of elemental sulphur was stimulated by CaCO3, technical urea and high doses of superphosphate and potassium sulphate. It was inhibited by KH2PO4, pure urea, 40 % potassium salt, ammonium nitrate with calcium carbonate and the fertilizer NPK I. It proceeded at the highest rate at approximately 60 % capillary capacity (61 % of mass water content). Oxidation of thiosulphate was stimulated by KH2PO4, pure urea, superphosphate, potassium sulphate and only slightly by the fertilizer NPK I. It was inhibited by CaCO3, 40 % potassium salt and only slightly by ammonium nitrate with calcium carbonate. Potassium chloride, glucose and technical urea were without effect. The oxidation proceeded at the highest rate at 35 % maximal capillary capacity (48 % mass water content).     

Effect of some sulphur compounds on soil microflora of spruce rhizosphere

The effect of a long-term application of sulphite, thiosulphate and sodium sulphate on the soil microflora and spruce seedlings was investigated in a pot experiment. Sulphur compounds decreased the concentration of bacteria, including thiobacilli, increased the concentration of microscopic fungi and sulphate-reducing bacteria; they inhibited respiration, nitrification and oxidation of thiosulphate, stimulated ammonification and oxidation of elemental sulphur. In certain cases the spruce rhizosphere exhibited just the opposite effect. In the rhizosphere the sulphate-reducing bacteria were suppressed together with thiobacilli, whose unit oxidative activity increased substantially. Growth of seedlings was inhibited by sulphite and stimulated by thiosulphate and sulphate. Sulphite, the effects of which were similar to those of sulphur dioxide immissions, was the most effective compound. In regions influenced by immissions the soil is apparently intoxicated by the absorbed sulphite.