P. Bos

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.

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.

Methanol assimilation by yeasts

For the present several processes for the production of single-cell protein are being developed. The most promising of these processes seems to be the production of yeasts on hydrocarbon substrates and industrial wastes. Production of bio-mass on natural gas or from hydrogen and carbon dioxide is carried out on a laboratory scale only. A valuable and at t ract ive new substrate for the production of microbial cells is methanol, which can be manufactured by catalytic oxidation of methane.

Purification and Partial Characterization of Thiosulfate Dehydrogenase from Thiobacillus-Acidophilus

SUMMARY: Thiosulphate dehydrogenase (EC 1.8.2.2; thiosulphate:acceptor oxidoreductase) was purified to apparent homogeneity from Thiobacillus acidophilus by a combination of ammonium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration. The enzyme catalysed the oxidation of thiosulphate (S2O2- 3) to tetrathionate (S4O2- 6) with potassium ferricyanide as an artificial electron acceptor. The molecular mass of the native enzyme, as determined by gel filtration, was 102 ± 4.2 kDa. The enzyme contained two different subunits with a molecular mass of 24 ± 0.9 and 20 ± 1.0 kDa (SDS-PAGE), respectively. Both subunits contained c 553-type haem with absorption bands at 553, 524 and 416 nm. A 77 K spectrum of purified thiosulphate dehydrogenase revealed that the absorption at 553 nm is due to different haem groups. A cytochrome content of 5.3 mole c-type haem per mole of native enzyme was calculated. The pH optimum of the purified enzyme was 3. Apart from ferricyanide, Wursters blue (the free radical of tetramethyl p-phenylenediamine) and horse heart cytochrome c could also serve as electron acceptors, though less effectively than ferricyanide. At pH 7.0, the K m for thiosulphate was 0.54 mM. The K m could not be determined at the pH optimum due to the chemical reactivity of thiosulphate at low pH values. Sulphite was a potent inhibitor of enzyme activity.

Heterotrophic Growth of Thiobacillus-Acidophilus in Batch and Chemostat Cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 μM pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Ymax and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol−1 and 0.10 mmol · g−1 · h−1, respectively. Neither the Ymax nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.

The significance of hydrocarbon assimilation in yeast identification

A large number of yeasts were screened for the ability to assimilate hydrocarbons. Not only representatives of the genusCandida, but also species from other perfect and imperfect genera are able to usen-alkanes as sole carbon and energy source. The significance of this feature in yeast systematics is discussed. In general, all strains of a species share either the ability to assimilate hydrocarbons or the failure to do so. Exceptions are found in species regarded as heterogeneous, likeCandida sake, Candida diddensii andCandida zeylanoides. In cases where the usual criteria used in identification seem to be inadequate, the simple hydrocarbon assimilation test may be useful. Also in subgrouping the generaCandida andTorulopsis the test may be of value, because some perfect genera likeHansenula, Kluyveromyces andSaccharomyces lack hydrocarbon-assimilating representatives.

Purification and characterization of a sulfite:cytochrome c oxidoreductase from Thiobacillus acidophilus

Abstract Cell-free extracts of Thiobacillus acidophilus prepared at neutral pH showed oxidation of sulfite to sulfate with ferricyanide as electron acceptor. Horse heart cytochrome c could be used as alternative electron acceptor; however, the observed activity was only 0.1% of that found for ferricyanide. The enzyme responsible for the oxidation of sulfite was purified to homogeneity. The purified enzyme was a monomer of 42 kDa and contained one haem c per monomer. Electron paramagnetic resonance (EPR) spectroscopical analysis of the sulfite:cytochrome c oxidoreductase showed the presence of molybdenum (V), only after reduction of the enzyme with sulfite. The pH optimum for the enzymatic reaction was 7.5 and the temperature optimum 40°C. Enzymatic activity was strongly reduced in the presence of the anions: chloride, phosphate and nitrate. In contrast to other enzymes involved in sulfur metabolism and previously isolated from T. acidophilus , sulfite:cytochrome c oxidoreductase activity is not stimulated by the presence of sulfate ions.

Classification of Lipomyces

The processes preceding the development of asci and the ornamentation of the ascospore wall of several strains of Lipomyces were studied by light and by electron microscopy. The polysaccharides excreted by living cells were analyzed by paperchromatography. Standard descriptions and latin diagnoses of L. kononenkoae and L. tetrasporus are presented. A key is given for the identification of the species.

ENERGETICS OF MIXOTROPHIC AND AUTOTROPHIC C1-METABOLISM BY THIOBACILLUS ACIDOPHILUS

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 μM, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.