Purification and partial characterization of a thermostable trithionate hydrolase from the acidophilic sulphur oxidizer Thiobacillus acidophilus.

Cell-free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O6(2-) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm. Trithionate hydrolase was purified to near homogeneity from cell-free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5-4.5 and a temperature optimum of 70 degrees C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 microM at 30 degrees C and 270 microM at 70 degrees C. Enzyme activity was lost after 36 days at 0 degrees C, 27 days at 70 degrees C; but after 97 days at 30 degrees C, 40% of the initial activity was still present: The enzyme activity was inhibited by mercury chloride, N-ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O6(2-) was not hydrolysed by trithionate hydrolase.     

[35S]Thiosulphate oxidation by Thiobacillus strain C

1. Thiobacillus strain C oxidized [(35)S]thiosulphate completely to sulphate. 2. During thiosulphate oxidation [(35)S]sulphate was formed more rapidly from (S.(35)SO(3))(2-) than from ((35)S.SO(3))(2-). (35)S disappeared less rapidly from thiosulphate with ((35)S.SO(3))(2-) as substrate than with (S.(35)SO(3))(2-). 3. Thiosulphate labelled in both atoms was produced during ((35)S.SO(3))(2-) oxidation, but not during (S.(35)SO(3))(2-) oxidation. 4. No (35)S was precipitated as elementary sulphur either in the presence or absence of exogenous unlabelled sulphur. 5. During [(35)S]thiosulphate oxidation, appreciable quantities of [(35)S]trithionate accumulated and later disappeared. Other polythionates did not accumulate consistently. 6. [(35)S]Trithionate was formed initially at a greater rate from (S.(35)SO(3))(2-) than from ((35)S.SO(3))(2-), but subsequently at a similar rate from each. 7. Trithionate formed from (S.(35)SO(3))(2-) was labelled only in the oxidized sulphur atoms, but that formed from ((35)S.SO(3))(2-) was labelled in both oxidized and reduced atoms. The proportion of (35)S in the oxidized atoms increased as more trithionate accumulated. 8. The results eliminate some mechanisms of trithionate formation but are consistent both with a mechanism of thiosulphate oxidation based on an initial reductive cleavage of the molecule and with a mechanism in which thiosulphate undergoes an initial oxidative reaction.     

ESR spin-trapping studies on the formation of thiosulfate and sulfide radical anions in aqueous solutions

The first spin-trapping evidence for the formation of thiosulfate (S2O3-.) and sulfide (S-.) radical anions from the reactions of hydrogen peroxide with thiosulphate and sulphide ions, respectively, was presented by electron spin resonance (ESR) spectroscopy using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS, 1a) as a spin-trap in aqueous solutions. From the facts that the short-lived radical anions, S2O3-. and S-., could be detected during the oxidation with H2O2, it is suggested that these radical anions may become one of the candidates for the toxicity of sulfide ion in the living body.     

Kinetics of denitrification using sulphur compounds: effects of S/N ratio, endogenous and exogenous compounds

The influence of different sulphur to nitrogen (S/N) ratios on the specific autotrophic denitrification activity was studied in batch experiments using thiosulphate and nitrate as substrates. Transitory accumulations of nitrite were observed for assays with S/N ratios of 3.70 and 6.67 g/g, probably due to the higher specific reduction rate of nitrate compared to that of nitrite. Nitrite was the main end product when S/N ratios of 1.16 and 2.44 g/g were tested. The effects of endogenous (NO(3)(-),NO(2)(-),S(2)O(3)(2-)and SO(4)(2-)) and exogenous compounds (acetate and NaCl) on the specific denitrifying activity of the sludge were tested. Nitrite and sulphate did exert clear inhibitory effects over the process while thiosulphate, acetate and NaCl did not have strong effects at the concentrations tested. Similar experiments also showed that sulphur was not a suitable electron donor for these microorganisms, but sulphide was used successfully.     

[35S]Thiosulphate oxidation by rat liver mitochondria in the presence of glutathione

1. Rat liver mitochondria incubated in oxygen with glutathione and [(35)S]-thiosulphate produced labelled sulphate. 2. Inner-labelled thiosulphate (S.(35)SO(3))(2-) was converted into [(35)S]sulphate more rapidly than outer-labelled thiosulphate ((35)S.SO(3))(2-). 3. Thiosulphate labelled in both sulphur atoms was formed during ((35)S.SO(3))(2-) oxidation; the outer sulphur atom before oxidation to sulphate was incorporated into the inner position. 4. A thiosulphate cycle in the metabolic pathway of sulphate formation in animal tissues is discussed.     

Dihydrolipoyl transacetylase of Escherichia coli. Formation of 8-S-acetyldihydrolipoamide

The dihydrolipoyl transacetylase component (E2) of the pyruvate dehydrogenase complex catalyzes the reaction of acetyl coenzyme A (acetyl-CoA) with dihydrolipoamide, producing coenzyme A and S-acetyldihydrolipoamide. The acetyl group is shown by experiments reported herein to be bonded to S8 in the enzymatic product. 1H NMR analysis of synthetic samples of both structural isomers of S-acetyl-S-(phenylmercurio)dihydrolipoamide enabled structural assignments to be made. Reaction of 8-S-acetyl-6-S-(phenylmercurio)dihydrolipoamide with 3-mercaptopropionic acid in chloroform produced 8-S-acetyldihydrolipoamide which contained a small amount (5%) of the 6-S isomer. Reaction of 6,8-di-S-acetyldihydrolipoamide with NH2OH produced a 4:1 mixture of 6-S-acetyldihydrolipoamide and the 8-S isomer. These compounds did not isomerize at significant rates in chloroform but rapidly isomerized to the equilibrium mixture in aqueous solution (Keq = 3.4). The second-order rate constants for the hydroxide-catalyzed isomerization were found to be kf = (1.15 +/- 0.07) X 10(6) M-1 X s-1 and kr = (3.36 +/- 0.20) X 10(5) M-1 X s-1 in the direction of the formation of the 8-S isomer. The enzymatic product was trapped by addition of phenylmercuric hydroxide within 15 s-30 min after starting the reaction. 1H NMR analysis of the products obtained at various times showed that the enzymatic product was 8-S-acetyldihydrolipoamide, which underwent progressive isomerization to the mixture of isomers within a few minutes. In the reaction of acetyl-CoA with dihydrolipoamide, the latter substrate reacts in place of enzyme-bound dihydrolipoyl moieties. Therefore, acetylation occurs at the 8-S position of bound lipoyl groups.     

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.     

Reduction of methemerythrin-anion adducts by dithionite ion

The reduction by dithionite ion (in excess) of methemerythrin-anion adducts, Hr+X-, to deoxyhemerythrin, Hr degree, has been examined at 25 degrees and pH 6.3 and 8.2. The results accord with the scheme: S2O42- in equilibrium 2SO2- rapid Hr+X- in equilibrium Hr++X- k-1, k1 Hr++SO2- leads to PRODUCT k2 with X- = Br-, HCO2-, CNO-, and F-, k2[SO2-] greater than k1[X-], and the pseudo first-order rate constant, kobs (= k-1), is independent of [X-] and [S2O42-]. Only with X- = NCS- is k2[SO2-] approximately k1[X-] and kobs = a[S2O42-]1/2 (b[NCS-] + [S2OR2-]1/2)-1. Values at pH 6.3 of k-1 (sec-1) and k1 (M-1 sec-1), obtained by anation and anion displacement reactions, are 2.3 x 10(-3), 1.6 x 10(-2) (Br-); 1.5 x 10(-3), 1.2 x 10(-2) (HCO2-); 1.3 x 10(-4), 0.52 (CNO-) and approximately 2 x 10(-4), 3.3 x 10(-3) (CN-, pH 7.0). Values of k-1 from reduction and displacement methods are in good agreement with each other. The value of k2 (1.6 x 10(5) M-1 sec-1, pH 6.3) in somewhat smaller than that for reduction of the met form of hemoproteins. There is only a small effect of pH on rates. Direct reduction of Hr+CN- does not occur, in contrast with Mb+CN-.     

Autoxidation of soluble trypsin-cleaved microsomal ferrocytochrome b5 and formation of superoxide radicals.

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 X 10(-3) +/- 0.19 X 10(-3) S-1 (mean +/- S.E.M., n =8) when measured at 30 degrees C in 10 mM-phosphate buffer, pH 7.4. At 37 degrees C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 X 10(-3) S-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 X 10(-4) M (approx. 20kPa or 150mmHg)9 During autoxidation, 0.25 mol of O2 was consumed per mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and catalase each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a tenfold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by superoxide dismutase at a concentration of 3 mug/ml. It is concluded that autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.     

Interference of thiosulfate during colorimetric analysis of hexavalent chromium using 1,5-diphenylcarbazide method

Thiosulfate (S(2)O(3)(2-)) contained in the media for autotrophic Cr (VI) reduction was found to interfere with Cr (VI) measurement following the 1,5-diphenylcarbazide (DPC) method. The interference was confirmed at several abiotic and biotic conditions, and was influenced by S(2)O(3)(2-) concentration, pH, and the media components. At neutral to alkaline pH, 500 mg/L of S(2)O(3)(2-) did not cause interference, while 4 mg/L of S(2)O(3)(2-) resulted in the interference at pH 2.0. Atomic absorption spectrophotometry could be an alternative method when the interference by S(2)O(3)(2-) is expected.     

Functional robustness and gene pools of a wastewater nitrification reactor: comparison of dispersed and intact biofilms when stressed by low oxygen and low pH

The functional robustness of biofilms in a wastewater nitrification reactor, and the gene pools therein, were investigated. Nitrosomonas and Nitrosospira spp. were present in similar amounts (cloning-sequencing of ammonia-oxidizing bacteria 16S rRNA gene), and their estimated abundance (1.1 x 10(9) cells g(-1) carrier material, based on amoA gene real-time PCR) was sufficient to explain the observed nitrification rates. The biofilm also had a diverse community of heterotrophic denitrifying bacteria (cloning-sequencing of nirK). Anammox 16S rRNA genes were detected, but not archaeal amoA. Dispersed biofilms (DB) and intact biofilms (IB) were incubated in gas-tight reactors at different pH levels (4.5 and 5.5 vs. 6.5) while monitoring O(2) depletion and concentrations of NO, N(2)O and N(2) in the headspace. Nitrification was severely reduced by suboptimal O(2) concentrations (10-100 microM) and low pH (IB was more acid tolerant than DB), but the N(2)O/NO(3)(-) product ratio of nitrification remained low (<10(-3)). The NO(2)(-) concentrations during nitrification were generally 10 times higher in DB than in IB. Transient NO and N(2)O accumulation at the onset of denitrification was 10-10(3) times higher in DB than in IB (depending on the pH). The contrasting performance of DB and IB suggests that the biofilm structure, with anoxic/micro-oxic zones, helps to stabilize functions during anoxic spells and low pH.     

X-ray structure of [Ru3 O2 (NH3)14]6+, cation of the cytological reagent Ruthenium Red

The X-ray crystal structure of [Ru3 O2 (NH3)14] (S2 O3)3 . 4H2 O, the thiosulphate salt of Ruthenium Red, has been determined. The cation contains an essentially linear N-Ru-O-Ru-O-Ru-N backbone formed from three ruthenium coordination octahedra, giving an effectively cylindrical shape to the ion. Resonance Raman spectra are consistent with retention of this structure in solution.     

Thiosulfate Oxidation and Electron Transport in Thiobacillus novellus.

Aleem, M. I. H. (Research Institute for Advanced Studies, Baltimore, Md.). Thiosulfate oxidation and electron transport in Thiobacillus novellus. J. Bacteriol. 90:95-101. 1965.-A cell-free soluble enzyme system capable of oxidizing thiosulfate was obtained from Thiobacillus novellus adapted to grow autotrophically. The enzyme systems of autotrophically grown cells brought about the transfer of electrons from thiosulfate to molecular oxygen via cytochromes of the c and a types; the reactions were catalyzed jointly by thiosulfate oxidase and thiosulfate cytochrome c reductase. The levels of both of these enzymes were markedly reduced in the heterotrophically grown organism. Cell-free extracts from the autotrophically grown T. novellus catalyzed formate oxidation and enzymatically reduced cytochrome c with formate. Both formate oxidation and cytochrome c reduction activities were abolished under heterotrophic conditions. The thiosulfate-activating enzyme S(2)O(3) (-2)-cytochrome c reductase, as well as thiosulfate oxidase, was localized chiefly in the soluble cell-free fractions, and the former enzyme was purified more than 200-fold by ammonium sulfate fractionation and calcium phosphate gel adsorption procedures. Optimal activity of the purified enzyme occurred at pH 8.0 in the presence of 1.67 x 10(-1)m S(2)O(3) (-2) and 2.5 x 10(-4)m cytochrome c. The thiosulfate oxidase operated optimally at pH 7.5 and thiosulfate concentrations of 1.33 x 10(-3) to 3.33 x 10(-2)m in the presence of added cytochrome c at a concentration of 5 x 10(-4)m. Both enzymes were markedly sensitive to cyanide and to a lesser extent to some metal-binding agents. Although a 10(-3)m concentration of p-hydroxymercuribenzoate had no effect on S(2)O(3) (-2)-cytochrome c reductase, it caused a 50% inhibition of S(2)O(3) (-2) oxidase, which was completely reversed in the presence of 10(-3)m reduced glutathione. Carbon monoxide also inhibited S(2)O(3) (-2) oxidase; the inhibition was completely reversed by light.     

Pyruvate dehydrogenase and 3-fluoropyruvate: chemical competence of 2-acetylthiamin pyrophosphate as an acetyl group donor to dihydrolipoamide

The pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex catalyzes the decomposition of 3-fluoropyruvate to CO2, fluoride anion, and acetate. Acetylthiamin pyrophosphate (acetyl-TPP) is an intermediate in this reaction. Incubation of the pyruvate dehydrogenase complex with 3-fluoro[1,2-14C]pyruvate, TPP, coenzyme A (CoASH), and either NADH or pyruvate as reducing systems leads to the formation of [14C]acetyl-CoA. In this reaction the acetyl group of acetyl-TPP is partitioned by transfer to both CoASH (87 +/- 2%) and water (13 +/- 2%). When the E1 component is incubated with 3-fluoro[1,2-14C]pyruvate, TPP, and dihydrolipoamide, [14C]acetyldihydrolipoamide is produced. The formation of [14C]acetyldihydrolipoamide was examined as a function of dihydrolipoamide concentration (0.25-16 mM). A plot of the extent of acetyl group partitioning to dihydrolipoamide as a function of 1/[dihydrolipoamide] showed 95 +/- 2% acetyl group transfer to dihydrolipoamide when dihydrolipoamide concentration was extrapolated to infinity. It is concluded that acetyl-TPP is chemically competent as an intermediate for the pyruvate dehydrogenase complex catalyzed oxidative decarboxylation of pyruvate.     

Whole animal and gill tissue oxygen uptake in the Eastern oyster, Crassostrea virginica: Effects of hypoxia, hypercapnia, air exposure, and infection with the protozoan parasite Perkinsus marinus(1)

The Eastern oyster, Crassostrea virginica, lives in shallow coastal waters and experiences many different environmental extremes including hypoxia, hypercapnia and air exposure and many oysters are infected with the protozoan parasite Perkinsus marinus. The effects of these conditions on oyster metabolism, as measured by oxygen uptake, were investigated. Mild hypercapnia had no effect on the ability of oysters to regulate oxygen uptake in hypoxic water, as measured by the B2 coefficient of oxygen regulation. The average B2 was -0.060x10(-3) (+/-0.01x10(-3) S.E.M.; n=20; low and high CO(2) treatments combined) in oysters uninfected with P. marinus and -0.056x10(-3) (+/-0.01x10(-3) S.E.M.; n=16; low and high CO(2) treatments combined) in infected oysters. There was no significant effect of light to moderate infections of P. marinus on oxygen regulation. Nor did the presence of P. marinus have an effect on the rate of oxygen uptake of whole animals in well-aerated water. In well-aerated conditions, oxygen uptake was significantly reduced by moderate hypercapnia in oysters when data from uninfected and infected oysters were combined. Mean oxygen uptake of infected oysters under hypercapnia (pCO(2)=6-8 Torr; pH 7) was 9.10 μmol O(2) g ww(-1) h(-1) +/-0.62 S.E.M. (n=9), significantly different from oxygen uptake under normocapnia (pCO(2) 相似文献   

The reaction between the superoxide anion radical and cytochrome c.

1. The superoxide anion radical (O2-) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2- and ferrocytochrome c. 2. At 20 degrees C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4-10(6) M-1. S -1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2- and the form of cytochrome c which exists above pH approximately 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2- reacts with the form present below pH 7.45 with k = 1.4-10(6) M-1 - S-1, the form above pH 7.45 with k = 3.0- 10(5) M-1 - S-1, and the form present above pH 9.2 with k = 0. 3. The reaction has an activation energy of 20 kJ mol-1 and an enthalpy of activation at 25 degrees C of 18 kJ mol-1 both above and below pH 7.45. It is suggested that O2- may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2-6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5-10(5)-5-10(6) M-1 - S-1.     

Interruption of the MnO2 oxidative process on dopamine and L-dopa by the action of S2O3(2-)

The oxidation effects of Mn2+, Mn3+ or MnO2 on dopamine can be studied in vitro and, therefore, this offers a model of the auto-oxidation process that appears naturally in neurons causing Parkinson's disease. The use of MnO, as an oxidizer in aqueous solution at pH 7 causes the oxidation of catecholamines (L-dopa, dopamine, noradrenaline and adrenaline) to melanin. However, this work shows that, in water at pH 6-7, the oxidation of catecholamines by MnO2 in the presence of sodium thiosulphate (Na2S2O3) occurs by other mechanisms. For dopamine and L-dopa, MLCT complexes were formed with bands at 312, 350 (sh), 554 (sh) nm, and an intense band at 597 nm (epsilon approximately/= 4 x 10(3) M(-1) cm(-1)) and at ca. 336, 557 (sh) nm, and an intense band at 597 nm (epsilon approximately 6 x 10(3) M(-1) cm(-1)), respectively. The latter transitions were assigned to d(pi)-->pi*-SQ. Noradrenaline and adrenaline do not form this blue complex in solution, but generate soluble oxidized compounds. The resonance Raman spectra of these complexes in solution showed bands at 950, 1006, 1258, 1378, 1508 and 1603 cm(-1) for the complex derivation of L-dopa and at 948, 1010, 1255, 1373, 1510 and 1603 cm(-1) for the dopamine-derived compound. The most intense Raman band at ca. 1378 cm(-1) was assigned to C-O stretching with major C1-C2 characteristics and indicated that dopamine and L-dopa do not occur complexed with manganese in the catecholate or quinone form, but suggests an intermediate compound such as an anionic o-semiquinone (SQ-), forming a complex such as [Mn(II)(SQ-)3]-. All enhanced Raman frequencies are characteristic of the benzenic ring without the participation of the aminic nitrogen. A mechanism is proposed for the formation of the dopamine and L-dopa complexes and a computational simulation was performed to support it.     

Isolation of a sulphate reductase-less mutant of the blue-green alga Nostoc muscorum.

The wild type Nostoc muscorum (UW strain) has yielded various physiological mutants altered in utilization of sulphate, following mutagenic treatments with N-methyl, N'-nitro N-nitrosoguanidine (NTG). One of the mutant strains designated as Sat-20 failed to grow in a medium containing sulphate (MgSO4.7 H2O). However, the mutant strain could grow when supplemented with thiosulphate (Na2S2O3.5 H2O), while methionine could fulfil the sulphur requirement only partially. On comparative reasons, the wild type as well as the mutant showed preference for thiosulphate over other sulphur sources employed.     

Bacterial sensors based on Acidithiobacillus ferrooxidans Part I. Fe2+ and S2O32- determination

An amperometric bacterial sensor with current response to Fe(2+) and S(2)O(3)(2-) ions has been designed by immobilizing an acidophilic biomass of Acidithiobacillus ferrooxidans on a multi disk flat-front oxygen probe. The bacterial layer was located between the oxygen probe and a membrane of cellulose. A filtration technique was used to yield the bacterial membranes having reproducible activity. The decrease of O(2) flow across the bacterial layer is proportional to the concentration of the dosed species. The dynamic range appeared to be linear for the Fe(2+) ions up to 2.5 mmol L(-1) with a detection limit of 9 x 10(-7) mol L(-1) and a sensitivity of 0.25 A L mol(-1). The response of the biosensor is 84 s for a determination of 2 x 10(-4) mol L(-1) Fe(2+). Optimizing the Fe(2+) determination by A. ferrooxidans sensor was carried out owing to Design of Experiments (DOE) methodology and empirical modelling. The optimal response was thus obtained for a pH of 3.4, at 35 degrees C under 290 rpm solution stirring. S(2)O(3)(2-) concentration was determined at pH 4.7, so avoiding its decomposition. The concentration range was linear up to 0.6 mmol L(-1). Sensitivity was 0.20 A L mol(-1) with a response time of 207 s for a 2 x 10(-4) mol L(-1) S(2)O(3)(2-) concentration.     

Sulphur metabolism in Thiorhodaceae. III. Storage and turnover of thiosulphate sulphur inThiocapsa floridana andChromatium species

Thiocapsa floridana strain 1711 andChromatium strains 1211 and 1611 utilize sulphide, thiosulphate, and elementary sulphur as electron donors for growth; sulphite can be used only byChromatium strain 1611. In contrast to the other strains, thiosulphate utilization inChromatium strain 1211 is inducible and not constitutive: thiosulphate is consumed only after an induction period of about 20 hours. The turnover rate of different sulphur compounds is controlled by the CO₂ fixation rate. Using differently labeled³⁵S thiosulphates in short term experiments in a special stirred cuvette, it was shown that the maximum amount of stored intracellular sulphur depends on the strain as well as on the experimental conditions like pH and thiosulphate concentration. WhileChromatium strain 1211 showed a maximum storage of only 10% from sulphane-labeled thiosulphate at pH 6.7, and of 25.7% at pH 6.2,Thiocapsa floridana accumulated 75–90% of the radioactivity into the cells at pH 6.7. While in theChromatium strains the labeling of the cells remained at a constant level until all thiosulphate was consumed, inThiocapsa floridana a defined peak of radioactivity storage was obtained, followed by a steady but 3–4 times slower rate of excretion. With sulphonelabeled thiosulphate no significant accumulation of radioactivity occurred in the cells. During dark-incubation ofThiocapsa floridana (free of intracellular sulphur) in phosphate buffer, pH 6.5, with thiosulphate a production of sulphide could be measured while sulphite was not detected; no sulphide was produced by disrupted cells under the same conditions. The results obtained withThiocapsa floridana strongly support the concept of an initial cleavage of thiosulphate. The present observations do not allow a decision concerning the enzymatic mechanism of the cleavage itself.