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.     

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.     

Isolation and physiological characterisation of Thiobacillus thyasiris sp. nov., a novel marine facultative autotroph and the putative symbiont of Thyasira flexuosa

A novel facultatively chemolithoautotropic Thiobacillus, isolated from the gill tissue of the marine bivalve Thyasira flexuosa, is described. It is believed to be the symbiont from this animal, providing the animal with carbon fixed by the Calvin cycle. The organism grows lithoautotrophically on thiosulphate, tetrathionate and elemental sulphur, which are oxidised to sulphate. It oxidizes sulphide, thiosulphate, trithionate, tetrathionate and hexathionate, but not thiocyanate. Kinetic constants for these substrates are presented. In autotrophic batch culture it produces yields that are among the lowest reported for thiosulphate or tetrathionate as energy substrates (1.25 and 2.5 g cell-carbon per mol substrate, respectively). Autotrophic cultures contain ribulose bisphosphate carboxylase and excreted 20% of their fixed carbon into the medium during growth. Mixotrophic growth on acetate and thiosulphate resulted in partial repression of the carboxylase. The organism is slightly halophilic and markedly halotolerant, showing optimum growth at about pH 7.5 and maximum growth rate at 37° C. It contains ubiquinone Q-10 and its DNA contains 52 mol % G+C. These characteristics distinguish it from any other Thiobacillus or Thiomicrospira species previously described. The organism is formally described and named as Thiobacillus thyasiris.     

Isolation and characterisation of Thiobacillus halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from a Western Australian hypersaline lake

The isolation of a novel obligately chemolithotrophic, halophilic and extremely halotolerant Thiobacillus from a hypersaline lake is described. Attempts to demonstrate sulphur- and ferrous iron-oxidizing chemolithotrophs in neighbouring hypersaline lakes were unsuccessful. The organism isolated differs from any other Thiobacillus species previously described and is formally named as Thiobacillus halophilus. It possesses ribulose bisphosphate carboxylase and grows chemolithoautotrophically on thiosulphate, tetrathionate and sulphur, oxidising them to sulphate. Kinetic constants for oxidation of sulphide, thiosulphate, trithionate and tetrathionate are presented. The organism is obligately halophilic, growing best with 0.8–1.0 M NaCl, and tolerating up to 4 M NaCl. Optimum growth was obtained at about 30° C and pH 7.0–7.3. It contains ubiquinone Q-8 and its DNA contains 45 mol % G+C. Organisms of this type might contribute significantly to the autotrophic fixation of carbon dioxide in some hypersaline extreme environments of the kind described.     

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.     

Isolation and characterization of Thiobacillus hydrothermalis sp. nov., a mesophilic obligately chemolithotrophic bacterium isolated from a deep-sea hydrothermal vent in Fiji Basin

A novel obligately chemolithotrophic Thiobacillus species isolated from a deep-sea hydrothermal vent is described. This organism grows lithoautotrophically on thiosulphate, tetrathionate, sulphide and sulphur which are oxidized to sulphate. The isolate is slightly halophilic and markedly halotolerant, showing optimum growth at pH 7.5 and at 35°C. The G+C content of the DNA is 67.1 mol%. The 16S rRNA sequence is distinct from any other Thiobacilli sequences. Phylogenetic analysis shows the organism to be a representative of the -group of proteobacteria and a specific relative of Thiobacillus neapolitanus. The ubiquinone is ubiquinone-8. These characters distinguish the isolate from any other Thiobacillus or Thiomicrospira species previously reported and is a new species described as Thiobacillus hydrothermalis. The type strain is isolate R3, DSM7121.     

Isolation and physiological characterisation of Thiobacillus aquaesulis sp. nov., a novel facultatively autotrophic moderate thermophile

A moderately thermophilic, facultatively chemolithoautotrophic thiobacillus isolated from a thermal sulphur spring is described. It differs from all other species currently known to be in culture. It grows lithoautotrophically on thiosulphate, trithionate or tetrathionate, which are oxidized to sulphate. Batch cultures on thiosulphate do not produce tetrathionate, but do precipitate elemental sulphur during growth. In autotrophic chemostat cultures the organism produces yields on thiosulphate, trithionate and tetrathionate that are among the highest observed for a Thiobacillus. Autotrophic cultures contain ribulose bisphosphate carboxylase. Heterotrophic growth has been observed only on complex media such as yeast extract and nutrient broth. It is capable of autotrophic growth and denitrification under anaerobic conditions with thiosulphate and nitrate. It grows between 30 to 55° C, and pH 7 to 9, with best growth at about 43°C and pH 7.6. It contains ubiquinone Q-8, and its DNA contains 65.7 mol% G+C. The organism is formally described and named as Thiobacillus aquaesulis.Now the Department of Biological Sciences     

Autotrophic growth and inorganic sulphur compound oxidation by Sulfolobus sp. in chemostat culture

Sulfolobus strain LM was grown in tetrathionate and thiosulphate-limited continuous culture. CO₂ limitation resulted in a decrease of the steady-state biomass and an increase in the specific rate of thiosulphate oxidation so that substrate did not accumulate in the medium. The initial step in thiosulphate utilization appeared to be its conversion to tetrathionate. The affinity for tetrathionate oxidation appeared to increase with prolonged continuous culture giving an apparent K _m of about 6 M tetrathionate, a higher affinity than for thiosulphate oxidation and in the same range as values observed with acidophilic, sulphur-oxidizing eubacteria.     

Chemolithotrophic potential of a Hyphomicrobium species,capable of growth on methylated sulphur compounds

The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Y_sulphide and Y_thiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time.Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H₂O₂, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.Abbreviations DMSO dimethyl sulphoxide - DMS dimethyl sulphide - MM methyl mercaptan - TMAO trimethylamine N-oxide - D dilution rate - GSH redticed glutathione - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - PES phenazine ethosulphate - RubPCase ribulose 1,5-bisphosphate carboxylase - PEPCase phosphoenol pyruvate carboxylase - Wurster's blue (TMPD) N,N,N,N-tetramethyl-p-phenylenediamine     

Tetrathionate Disproportionation by Thiomonas intermedia K12

Thiomonas intermedia K12, a moderately acidophilic bacterium, which oxidises sulphur compounds, – exhibited the capability to use tetrathionate under oxic and anoxic conditions. Whereas under oxic conditions, the reduced sulphur tetrathionate compound was oxidised, under anoxic conditions, the organism disproportionated the compound. In both cases, trithionate and sulphate were produced but in different amounts. The results of the tetrathionate degradation experiments under oxic conditions pointed towards a cyclic degradation process with a transient formation of trithionate and sulphate as the final products, similar to the mechanism described for acidophilic sulphur compound oxidising bacteria. The results of the tetrathionate degradation experiments under anoxic conditions hinted to a partial reduction of tetrathionate to thiosulphate and a fractional oxidation to trithionate and sulphate. 4 M tetrathionate were converted to 6 M thiosulphate, 1 M trithionate, 1 M sulphate, and 8 M protons. The ΔG₀' of this reaction was found to be –16.1 kJ per mol tetrathionate degraded. Additionally, Thiomonas intermedia K12 grew under anoxic conditions with tetrathionate as the sole energy source. The cell numbers increased from 10⁵ as the start value to 10⁷/mL at the end. Organic compounds, excluding traces of yeast extract, did not enhance growth. Therefore, it is proposed that tetrathionate disproportionation is a novel lithotrophic metabolism, which allowed Thiomonas intermedia K12 to survive changing conditions of oxygen supply in sulphur‐compound‐rich environments and even to grow during this reaction. The extensive sulphur compound analysis was carried out by ion‐pair chromatography.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

Thiobacillus strain Q,a chemolithoheterotrophic sulphur bacterium

The physiological properties of an organism isolated from a selective chemostat enrichment using acetate and thiosulphate as the limiting substrates, provisionally called Thiobacillus Q, were investigated. Although the organism made up 85% of the community in the enrichment culture, its expected chemolithotrophic nature was not apparent in batch experiments. The growth yield was not enhanced by the addition of thiosulphate to an acetate containing mineral medium, even though up to 50% of the thiosulphate was oxidized. Under acetate limitation in the chemostat, there was a linear increase in yield with thiosulphate addition up to a concentration of 7 mM. Higher thiosulphate concentrations resulted in loss of thiosulphate oxidizing capacity and a decrease in the biomass to the level obtained with acetate alone. This loss may be due to the presence of inhibitory (50–100 M) levels of sulphite which is probably produced as an intermediate of the biological thiosulphate oxidation. Experiments with sulphide showed that Thiobacillus Q could also use it as an additional energy source. The complete lack of autotrophic growth, both in batch and chemostat experiments, together with the absence of even very low amounts of the key enzymes of the Calvin cycle demonstrated that this organism is a typical chemolithoheterotroph. Although this organism has provisionally been placed in the genus Thiobacillus, standard taxonomic procedures showed a close relationship with Pseudomonas alcaligenes. This study stresses the importance of quantitative chemostat studies in establishing the role of inorganic oxidations in energy metabolism and in the understanding of the role of heterotrophic sulphur oxidation in natural environments.     

Light-driven reduction of oxygen as a method for studying electron transport in the green photosynthetic bacterium Chlorobium limicola

The use of O₂ uptake as a valid assay for non-cyclic photosynthetic electron flow in membranes from Chlorobium limicola is discussed. It is recommended that methyl viologen, catalase and superoxide dismutase should be added to the experimental medium. The addition of methyl viologen more than doubled the rate of O₂ uptake observed on illumination with 1 mM sulphide as donor. Superoxide dismutation was shown to be efficient under the experimental conditions by means of standard additions of potassium superoxide dissolved in dimethylsulphoxide. The highest rates of light stimulated O₂ uptake were obtained with sulphide as electron donor, and approached 50 mol O₂ · h^-1 · mg bacteriochlorophyll c ^-1 with 0.2 mM sulphide. The presence of 5 mM 2-mercaptoethanol or 3 mM sulphite as electron donor led to lower light stimulated rates of O₂ uptake, while 5 mM thiosulphate had little effect. The rates were insensitive to uncoupler. The light stimulated O₂ uptake with 0.2 mM sulphide as donor was 20–30% inhibited by 10 M antimycin A and 50 M cyanide.Abbreviations APS Adenosine 5-phosphosulphate - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - MeV methyl viologen - P-840 the photoreactive bacteriochlorophyll     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

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.     

Cell yield and bioenergetics of Thiomicrospira denitrificans compared with Thiobacillus denitrificans

From cell yields of Thiomicrospira denitrificans grown in the chemostat at different growth rates under anaerobic conditions a value of 1.4mm S₂O _inf3 ^sup= per g dry wt and per h could be calculated for maintenance energy requirements, and of 5.65 g dry wt per mole S₂O _inf3 ^sup= for the true growth yield.Cell yields of Thiomicrospira denitrificans appeared to be almost half of those of Thiobacillus denitrificans. Though in Thiobacillus denitrificans at D=0.03 h^-1 under anaerobic conditions a value was found of 11.60 g dry wt per mole of thiosulphate used for energetic purposes, a value of 5.72 g dry wt per mole of thiosulphate was found under comparable conditions in Thiomicrospira denitrificans. Under aerobic conditions at D=0.03 h^-1 values of 18.54 g dry wt per mole of thiosulphate were found in Thiobacillus denitrificans whereas Thiomicrospira denitrificans yielded only 9.38 g dry wt per mole of thiosulphate.As in Thiobacillus denitrificans anaerobic cell yields on sulphide were comparable to those on thiosulphate.Calculations have been made which indicate that the biosynthetic efficiency of Thiomicrospira denitrificans is lower than that of Thiobacillus denitrificans. This can only partly be explained by the absence of adenosine-phosphosulphate (APS) reductase.     

Purification and characterization of a periplasmic thiosulfate dehydrogenase from the obligately autotrophicThiobacillus sp. W5

A periplasmic thiosulfate dehydrogenase (EC 1.8.2.2) was purified to homogeneity from the neutrophilic, obligately chemolithoautotrophicThiobacillus sp. W5. A five-step procedure resulted in an approximately 2,300-fold purification. The purified protein had a molecular mass of 120±3 kDa, as determined by gel filtration. It is probably a tetramer containing two different subunits with molecular masses of 33±1 kDa and 27±0.5 kDa, as determined by SDS-PAGE. UV/visible spectroscopy revealed that the enzyme contained haemc; haem staining showed that both subunits contained haemc. A haemc content of 4 mol per mol of enzyme was calculated using the pyridine haemochrome test. The pH optimum of the enzyme was 5.5 At pH 7.5, the K_m and V_max were 120±10 M and 1,160±30 U mg^-1, respectively. The absence of 2-heptyl-4-hydroquinoline-N-oxide (HQNO) inhibition for the oxidation of thiosulfate by whole cells suggested that the electrons enter the respiratory chain at the level of cytochromec. Comparison with thiosulfate dehydrogenases from otherThiobacillus species showed that the enzyme was structurally similar to the thiosulfate dehydrogenase of the acidophilic, facultatively chemolithoautotrophicThiobacillus acidophilus, but not to the thiosulfate dehydrogenases published for the obligately chemolithoautotrophicThiobacillus tepidarius andThiobacillus thioparus.Abbreviations BV Benzyl viologen - DCPIP 2,6-Dichloroindophenol - HQNO 2-Heptyl-4-hydroquinoline-N-oxide - NEM N-ethylmaleimide - PES Phenazine ethosulfate - PMS Phenazine methosulfate     

The effect of carbon dioxide on the growth kinetics of fructose-limited chemostat cultures of Acetobacterium woodii DSM 1030

The growth of the anaerobic acetogenic bacterium Acetobacterium woodii DSM 1030 was investigated in fructose-limited chemostat cultures. A defined medium was developed which contained fructose, mineral salts, cysteine · HCl and Ca pantothenate (1 mg · 1^–1) supplied in a vitamin supplement. Growth at high dilution rates was dependent on the presence of CO₂ in the gas phase. The ^max was found to be 0.16 h^–1 and the fructose maintenance requirement was 0.1 to 0.13 mmol fructose · (g dry wt)^–1 · h^–1. A growth yield of 61 g dry wt · (mol fructose)^–1, corrected for the cell maintenance requirement and for incorporation of fructose carbon into cell biomass, was determined from the fructose consumption. A corresponding growth yield of 69 g dry wt · (mol fructose)^–1 was calculated from the acetate production assuming that fructose fermentation was homoacetogenic. A Y_ATP of 12.2 to 13.8 g dry wt · (mol ATP)^–1 was calculated from these growth yields using a value of 5 mol ATP · (mol fructose)^–1 as an estimate of the amount of ATP synthesised from fructose fermentation. The addition of yeast extract (0.5 g · 1^–1) to the medium did not influence the ^max or cell yield. After prolonged growth under fructose-limited conditions the requirement of the culture for CO₂ in the gas phase was reduced.Abbreviations YE yeast extract - IC inorganic carbon - D fermenter dilution rate : h^–1 - M_X maintenance requirement for X: mmol X · (g dry wt)^–1 · h^–1 - X may be fructose (Fruct), fructose consumed in energy metabolism (Fruct [E]), acetate (Ac) - ATP CO₂, NH _inf4 ^sup+ or Pi - qX specific rate of utilisation or consumption of X: mmol X · (g dry wt)^–1 · h^–1 - V fermenter volume: litre - rC · Cell, fermenter cell carbon production: mmol C · h^–1 - Y_X yield of cells on X: g dry wt · (mol X)^–1 - Y _infx ^supmax the yield corrected for cell maintenance: g dry wt · (mol X)^–1 - S_ATP stoichiometry of ATP synthesis from fructose: mol ATP · (mol frucose)^–1 - x cell concentration: g dry wt · 1^–1 - specific growth rate : h^–1 - ^max maximum specific growth rate: h^–1     

Utilization of 35S-Thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans

Differentially labelled ³⁵S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated K _m value of 0.5 mM for ³⁵S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound ³⁵S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-³⁵S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-³⁵S]thiosulphate or ³⁵S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.Abbreviation APS adenosine-5-sulphatophosphate     

Mechanisms of sulphide tolerance in the peanut worm,Sipunculus nudus (Sipunculidae) and in the lugworm,Arenicola marina (Polychaeta)

Summary The peanut worm Sipunculus nudus and the lugworm Arenicola marina are inhabitants of intertidal flats. Both species may be exposed to H₂S within their habitat. Sulphide concentrations in the vicinity of A. marina burrows are as high as 340 mol · 1^-1, whereas the pore water in sipuncle areas contains much lower sulphide levels of 13 mol · 1^-1 at most. During in vivo sulphide incubations, H₂S increases within the coelomic fluid of both species. In S. nudus the concentration of total sulphide after 8 h is about 40% of that of the incubation medium containing 200 and 1000 mol · 1^-1, respectively, which is partly due to the acidification of the coelomic fluid by 0.2 pH units during anaerobiosis. After 8 h, the sulphide concentration in A. marina was only 15% of that in the incubation medium containing 1000 mol · 1^-1. When oxygen is available, both species oxidize sulphide to thiosulphate, but in A. marina this capability is more pronounced than in S. nudus. If sulphide is not completely oxidized internally both intertidal worms switch to an anaerobic metabolism as indicated by the accumulation of opines and succinate.Abbreviations ATP adenosine triphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - HPLC high-performance liquid chromatography - wwt wet weight