KWRRI Research Reports


This report describes investigations into the physiology of microorganisms that are commonly involved in the oxidation of inorganic sulfur compounds. The metabolic activities of these bacteria play a potent role in several fields of economic importance such as strip mining operations, water pollution, corrosion, metallurgy, petroleum technology and soil fertility processes.

The oxidation of inorganic sulfur compounds was studied in the chemolithotrophs Thiobacillus denitrificans, Thiobacillus A2, Thiobacillus neapolitanus, and a photolithotroph Rhodopseudomonas palustris. Cell suspensions from all of the thiobacilli catalyzed completely the oxidation of sulfide, thiosulfate, and sulfite to sulfate. The oxidation of thiosulfate in R. palustris was light-dependent and involved tetrathionate and trithionate as the major intermediates in the formation of sulfates.

Almost all of the thiobacilli (e.g. T. neapolitanus and T. denitrificans) 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 was T. 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 flavoprotein system and cytochromes of b, c, o, and a-type. This process was sensitive to flavoprotein inhibitors, antimycin A, and cyanide. Thiosulfate oxidation, on the other hand involved cytochrome c:02 oxidoreductase region of the electron transport chain and was sensitive to cyanide only. The anaerobic oxidation of thiosulfate by T. 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 of T. 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 stiochiometric 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. In addition, the APS-reductase pathway was also operative. The latter aspect was absent in T. novellus and Thiobacillus-A2. 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: (1) a cellular-membrane-associated initial and rapid oxidation reaction which was dependent upon sulfide concentration, and (2) a slower oxidation reaction stage catalyzed by the cell-free extracts, probably involving polysulfides. In T. novellus and T. 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.

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Funding Information

The work upon which this report is based was supported in part by funds provided by the United States Department of the Interior, Office of Water Resources Research, as authorized under the Water Resources Research Act of 1964.