biochemical reaction mechanisms in sulfur oxidation by chemosynthetic bacteria

Summary Aspects of the biochemistry of the oxidation of inorganic sulfur compounds are discussed in thiobacilli but chiefly inThiobacillus denitrificans. Almost all of the thiobacilli (e.g. T. denitrificans, T. neapolitanus, T. novellus, andThiobacillus A ₂) were capable of producing approximately 7.5 moles of sulfuric acid aerobically from 3.75 moles of thiosulfate per gram of cellular protein per hr. By far the most prolific producer of sulfuric acid (or sulfates) from the anaerobic thiosulfate oxidation with nitrates wasT. denitrificans which was capable of producing 15 moles of sulfates from 7.5 moles of thiosulfate with concomitant reduction of 12 moles of nitrate resulting in the evolution of 6 moles of nitrogen gas/g protein/hr. The oxidation of sulfide was mediated by the flavo-protein system and cytochromes ofb, c, o, anda-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. The aerobic thiosulfate oxidation on the other hand involved cytochromec : O₂ oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate byT. denitrificans, however, was severely inhibited by the flavoprotein inhibitors because of the splitting of the thiosulfate molecule into the sulfide and sulfite moieties produced by the thiosulfate-reductase. Accumulation of tetrathionate and to a small extent trithionate and pentathionate occurred during anaerobic growth ofT. denitrificans. These polythionates were subsequently oxidized to sulfate with the concomitant reduction of nitrate to N₂. Intact cell suspensions catalyzed the complete oxidation of sulfide, thiosulfate, tetrathionate, and sulfite to sulfate with the stoichiometric reduction of nitrate, nitrite, nitric oxide, and nitrous oxide to nitrogen gas thus indicating that NO₂ ⁻, NO, and N₂O are the possible intermediates in the denitrification of nitrate. This process was mediated by the cytochrome electron transport chain and was sensitive to the electron transfer inhibitors. The oxidation of sulfite involved cytochrome-linked sulfite oxidase as well as the APS-reductase pathways. The latter was absent inT. novellus andThiobacillus A ₂. In all of the thiobacilli the inner as well as the outer sulfur atoms of thiosulfate were oxidized at approximately the same rate by intact cells. The sulfide oxidation occurred in two stages: (a) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (b) a slower oxidation reaction stage catalyzed by the cellfree extracts, probably involving polysulfides. InT. novellus andT. neapolitanus the oxidation of inorganic sulfur compounds is coupled to energy generation through oxidative phosphorylation, however, the reduction of pyridine nucleotides by sulfur compounds involved an energy-linked reversal of electron transfer. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974. Summary already inserted on p. 189 of the present volume.     

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.     

极端嗜酸性专性化能自养硫细菌有机质代谢的研究进展

极端嗜酸性专性化能自养硫细菌具有独特的生理特性, 在农业、细菌冶金、含硫废水处理以及环境保护等方面发挥着重要作用, 但这类细菌在其特殊能源缺乏时不能代谢有机质, 生长缓慢, 代时长, 细胞得率低, 限制了它在实际生产中的应用效率。对其进行遗传改造, 构建能够利用有机质快速生长的基因工程菌, 将为这类细菌的工业化应用提供一条可行的途径。主要对极端嗜酸性专性化能自养硫细菌有机质代谢的研究进展进行了综述, 其中包括有机化合物的抑制作用、有机化合物的有限利用、中心代谢途径及物质的转运等, 还包括专性化能自养硫细菌有机质代谢遗传改造研究的最新进展。     

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.     

The organosulphur cycle: aerobic and anaerobic processes leading to turnover of C1-sulphur compounds

Abstract The processes generating and transforming those C₁-organosulphur compounds which are major intermediates in the biogeochemical cycling of sulphur are summarised. The biological and chemical interconversions of the methylated sulphides, methane sulphonate, carbon disulphide and carbonyl sulphide are significantly influenced by diverse microorganisms, including autotrophic thiobacilli, methylotrophs, methanogens and sulphate-reducing bacteria. The major biogenic sulphur gas is dimethyl sulphide. A major atmospheric photochemical oxidation product from this is methane sulphonate, which can contribute to the acidity of rain. New data and ideas on the microbiological fate of methane sulphonate and of natural and anthropogenic carbon disulphide are presented.     

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.     

Thermodynamic aspects of energy conservation by chemolithotrophic sulfur bacteria in relation to the sulfur oxidation pathways

The free-energy data on which assessments of the autotrophic growth efficiencies of chemolithotrophic bacteria are commonly based have been reevaluated and new values have been calculated. It has been concluded that many earlier calculations are in error and that many values previously reported in the literature are overestimates of efficiency. A problem posed by the chemolithotrophic sulfur-oxidizing bacteria is the elucidation of the mechanism by which elemental sulfur and the sulfane-sulfur (-S-) of the thionic acids are converted to sulfite. Even after decades of studies on sulfur oxidation by these bacteria, this problem has not been fully resolved although it is widely thought that conversion of sulfur to sulfite is brought about by an oxygenase. The biochemically feasible mechanisms by which sulfur and “sulfane” oxidation to sulfite might occur are reviewed. The possible insight afforded by chemical thermodynamics into the most likely mechanisms for oxidation to sulfate in relation to the efficiency of energy conservation is discussed. Energetic calculations and growth yield data indicate that the energy-yielding oxidation of sulfur and “sulfane” to sulfite, either coupled to energy-conserving electron transport or catalyzed by an oxygenase, could explain divergent growth yields among different sulfur-chemolithotrophs. Received: 30 October 1998 / Accepted: 25 January 1999     

Enzymes of Carbohydrate Metabolism in Thiobacillus species

A study was made of enzymes of carbohydrate metabolism in representative thiobacilli grown with and without glucose. The data show that Thiobacillus perometabolis possesses an inducible Entner-Doudoroff pathway and is thus similar to T. intermedius and T. ferrooxidans. T. novellus lacks this pathway. Instead, a non-cyclic pentose phosphate pathway along with the Krebs cycle is apparently the major route of glucose dissimilation in this organism. Glucose does not support or stimulate the growth of strains of T. neapolitanus, T. thioparus, and T. thiooxidans examined, nor does its presence in the growth medium greatly influence their enzymatic constitution. These obligately chemolithotrophic thiobacilli do not possess the Entner-Doudoroff pathway. Their nicotinamide adenine dinucleotide (NAD)-linked isocitrate dehydrogenase activity predominates over their nicotinamide adenine dinucleotide phosphate (NADP)-linked activity; the converse is true for the other thiobacilli. The data suggest that NAD-linked isocitrate dehydrogenase activity in thiobacilli is involved in biosynthetic reactions.     

Biochemical Studies on Accelerated Treatment of Thiocyanate by Activated Sludge using Growth Factors such as Pyruvate

S ummary . Micro-organisms in activated sludge oxidizing thiocyanate behave in a similar way to thiobacilli. Organic compounds such as pyruvate stimulate thiocyanate oxidation and in so doing become incorporated into cellular material; such metabolites are not oxidized to produce energy. The presence of thiocyanate in batch cultures accelerated pyruvate uptake. The carbon of thiocyanate is recovered mainly as CO₂; carbonate becomes incorporated into cellular protein, but less so in sludges previously treated with pyruvate. TCA cycle enzymes are not altered appreciably after pyruvate is added to activated sludge.     

Importance of Hydrogen Sulfide, Thiosulfate, and Methylmercaptan for Growth of Thiobacilli during Simulation of Concrete Corrosion

Biogenic sulfuric acid corrosion of concrete surfaces caused by thiobacilli was reproduced in simulation experiments. At 9 months after inoculation with thiobacilli, concrete blocks were severely corroded. The sulfur compounds hydrogen sulfide, thiosulfate, and methylmercaptan were tested for their corrosive action. With hydrogen sulfide, severe corrosion was noted. The flora was dominated by Thiobacillus thiooxidans. Thiosulfate led to medium corrosion and a dominance of Thiobacillus neapolitanus and Thiobacillus intermedius. Methylmercaptan resulted in negligible corrosion. A flora of heterotrophs and fungi grew on the blocks. This result implies that methylmercaptan cannot be degraded by thiobacilli.     

Molecular genetic studies on the thiobacilli and the development of improved biomining bacteria

Acidophilic autotrophic thiobacilli, which are able to oxidize metal and solubilize sulphide ores, are used industrially to leach metals from mineral ores. Genetic manipulation of the thiobacilli has the potential for the production of leaching bacteria with desirable characteristics for industry. In this review we examine the development of genetic systems in the thiobacilli and the present status of molecular genetics in the group.     

铜蓝精矿的生物浸出

采用两种嗜酸硫杆菌(嗜酸氧化亚铁硫杆菌和喜温硫杆菌)对铜蓝进行生物浸出,实验在有或没有4 g/L硫酸亚铁pH 2．0、150转/分、35℃的三角瓶中进行。实验结果表明:用两种菌混合浸出的铜几乎等于嗜酸氧化亚铁硫杆菌单独浸出的铜;另外,亚铁的加入能提高铜的浸出。     

Chemolithotrophic ATP synthesis and NAD(P) reduction in Thiobacillus tepidarius and T. versutus

Thiosulphate-dependent reduction of NAD and NADP in intact cells of Thiobacillus tepidarius and T. versutus was severely inhibited or abolished by FCCP at concentrations that did not affect ATP synthesis over the same time scale. Thiosulphate-dependent ATP synthesis in T. tepidarius was abolished by the ATPase inhibitor DCCD, which did not affect NAD or NADP reduction at the concentrations tested. These results indicate that energy-dependent NAD(P) reduction using reversal of electron transfer from cytochrome b or c in thiobacilli is directly driven by the p generated by thiosulphate oxidation, and does not require the participation of ATP. While NAD(P) reduction and ATP synthesis are thus both effected by sulphur compound oxidation, there is no obligatory link between them.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCCD dicyclohexylcarbodiimide - DNP 2,4-dinitrophenol - G-6-P glucose 6-phosphate - FCCP carbonylcyanide p-fluoromethoxyphenylhydrazone     

Metabolism of organic acids by Thiobacillus neapolitanus

Summary A comparative study has been made of the metabolism in several strains of Thiobacillus neapolitanus of formate, acetate, propionate, butyrate, valerate and pyruvate. Conflicting reports in the literature concerning the mechanism of pyruvate assimilation in thiobacilli have been resolved. Pyruvate undergoes decarboxylation to yield acetyl coenzyme A, which is converted to glutamate, proline and arginine via reactions of the incomplete Krebs' cycle of this organism. Pyruvate is converted also to alanine, valine, isoleucine, leucine and lysine by mechanisms like those in heterotrophs. No aspartate is formed from the C-3 of pyruvate. Removal of the C-1 of pyruvate yields carbon dioxide, which is refixed into all cell constituents. Formate is not produced by this scission reaction, as formate itself is incorporated almost exclusively into purines. Aspartate can be synthesized by the activities of phosphoenolpyruvate carboxylase and oxaloacetate-glutamate transamination. Carbon from propionate is converted principally to lipids, although some amino acid production occurs with the same distinctive labelling pattern as is found after acetate assimilation by T. neapolitanus strains C and X. Butyrate and valerate also showed some distinctive patterns of incorporation into cell constituents. Fluoropyruvate and fluoropropionate inhibited the growth of T. neapolitanus and the mechanisms of this poisoning are discussed.Generally these compounds contributed only small proportions of the total cell carbon and tended to be converted to limited numbers of cell components. The thiobacilli thus tend to conserve carbon from these compounds and not to degrade them to carbon dioxide on a large scale when growing in an otherwise autotrophic medium.     

The energy landscape for ubihydroquinone oxidation at the Q(o) site of the bc(1) complex in Rhodobacter sphaeroides

Activation energies for partial reactions involved in oxidation of quinol by the bc(1) complex were independent of pH in the range 5. 5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH(2). Separation to products after the first electron transfer and movement of semiquinone formed in the Q(o) site would allow rapid electron transfer to heme b(L). This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation.     

Biochemical Basis of Obligate Autotrophy in Blue-Green Algae and Thiobacilli

Differential rates of incorporation of sugars, organic acids, and amino acids during autotrophic growth of several blue-green algae and thiobacilli have been determined. In obligate autotrophs (both blue-green algae and thiobacilli), exogenously furnished organic compounds make a very small contribution to cellular carbon; acetate, the most readily incorporated compound of those studied, contributes about 10% of newly synthesized cellular carbon. In Thiobacillus intermedius, a facultative chemoautotroph, acetate contributes over 40% of newly synthesized cellular carbon, and succinate and glutamate almost 90%. In the obligate autotrophs, carbon from pyruvate, acetate, and glutamate is incorporated into restricted groups of cellular amino acids, and the patterns of incorporation in all five organisms are essentially identical. These patterns suggest that the tricarboxylic acid cycle is blocked at the level of alpha-ketoglutarate oxidation. Enzymatic analyses confirmed the absence of alpha-ketoglutarate dehydrogenase in the obligate autotrophs, and also revealed that they lacked reduced nicotinamide adenine dinucleotide oxidase, and had extremely low levels of malic and succinic dehydrogenase. These enzymatic deficiencies were not manifested by the two facultative chemoautotrophs examined. On the basis of the data obtained, an interpretation of obligate autotrophy in both physiological and evolutionary terms has been developed.     

Growth of sulfate-reducing bacteria with solid-phase electron acceptors

Hannebachite (CaSO3 x 0.5H2O), gypsum (CaSO4 x 2H2O), anglesite (PbSO4), and barite (BaSO4) were tested as electron acceptors for sulfate-reducing bacteria with lactate as the electron donor. Hannebachite and gypsum are commonly associated with flue gas desulfurization products, and anglesite is a weathering product found in lead mines. Barite was included as the most insoluble sulfate. Growth of sulfate-reducing bacteria was monitored by protein and sulfide (dissolved H2S and HS-) measurements. Biogenic sulfide formation occurred with all four solid phases, and protein data confirmed that bacteria grew under these electron acceptor conditions. Sulfide formation from gypsum was almost comparable in rate and quantity to that produced from soluble sulfate salt (Na2SO4); hannebachite reduction to sulfide was not as fast. Anglesite as the electron acceptor was also reduced to sulfide in the solution phase and galena (PbS) was detected in solids retrieved from spent cultures. Barite as the electron acceptor supported the least amount of growth and H2S formation. The results demonstrate that low-solubility crystalline phases can be biologically reactive under reducing conditions. Furthermore, the results demonstrate that galena precipitation through sulfide production by sulfate-reducing bacteria serves as a lead enrichment mechanism, thereby also alleviating the potential toxicity of lead. In view of the role of acidophilic thiobacilli in the oxidation of sulfides, the present work accentuates the role of anaerobic and aerobic microbes in the biogeochemical cycling of solid-phase sulfates and sulfides.     

Organic Carbon Utilization by Resting Cells of Thiosulfate-Oxidizing Marine Heterotrophs

Two thiosulfate-oxidizing marine heterotrophs, strains 12W and 16B, were tested for utilization of [¹⁴C]glucose and [¹⁴C]acetate, respectively, in the presence or absence of thiosulfate. Thiosulfate oxidation caused an increase in organic carbon incorporation and a corresponding decrease in respiration at pH 6.5, near the optimum pH for thiosulfate oxidation and thiosulfate-stimulated growth in these bacteria. The amount of glucose or acetate metabolized remained virtually unaffected by thiosulfate oxidation. The metabolic shift in carbon utilization was diminished by increasing the initial pH to 8.0. The results indicate that marine heterotrophs 12W and 16B exhibit a type of mixotrophic metabolism which differs from that observed in the thiobacilli.     

Simultaneous sewage sludge digestion and metal leaching — effect of temperature

The effect of temperature on the simultaneous sewage sludge digestion and metal leaching process was studied in laboratory bioreactors of 20 l working volume. The results thus obtained showed that the process can be employed efficiently for metal solubilization, elimination of indicator microorganisms and sewage sludge stabilization at temperatures between 10°C and 30°C. Rates of pH reduction, sulfur oxidation, growth of thiobacilli, elimination of indicator microorganisms and solids degradation were found to decrease with temperature. Low metal solubilization efficiency was observed at 10°C; however, metals were solubilized to below the recommended level. The solubilization of organic matter and nutritive elements (N, P and K) was not significantly affected by the variation in temperature. The fertilizer value of sludge after leaching and digestion did not change significantly and remained the same irrespective of temperature. *** DIRECT SUPPORT *** AG903078 00005     

Structural insights into the mechanism of amine oxidation by monoamine oxidases A and B

Due to their pharmacological importance in the oxidation of amine neurotransmitters, the membrane-bound flavoenzymes monoamine oxidase A and monoamine oxidase B have attracted numerous investigations and, as a result, two different mechanisms; the single electron transfer and the polar nucleophilic mechanisms, have been proposed to describe their catalytic mechanisms. This review compiles the recently available structural data on both enzymes with available mechanistic data as well as current NMR data on flavin systems to provide an integration of the approaches. These conclusions support the proposal that a polar nucleophilic mechanism for amine oxidation is the most consistent mechanistic scheme as compared with the single electron transfer mechanism.