Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent α-glucosidase

The gene for the α-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hy-drolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritimaα-glucosidase has the unusual property of requiring NAD⁺ and Mn²⁺ for activity. Co²⁺ and Ni²⁺ also activated AglA, albeit less efficiently than Mn²⁺. T. maritima AglA represents the first example of a maltodextrin-degrading α-glucosidase with NAD⁺ and Mn²⁺ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or β-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD⁺ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of d-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90°C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50°C and 70°C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h. Received: October 6, 1999 / Accepted: February 9, 2000     

Demonstration of hydrogenase electrode operation in a bioreactor

This work describes the first step towards combination of the bioreactor with a starch-degrading microbial consortium and hydrogenase electrode (HE) in one unit for electricity generation. For this purpose, the bioreactor for microbial fermentation was designed with a set of electrodes (pH-sensor, Ag|AgCl reference electrode, Pt-electrode, and HE) inside the bioreactor. Potentials of all electrodes and H₂ accumulation were monitored in the system under the precise pH control. Results obtained with the hydrogen-producing microbial consortium indicated that HE generates the potential equal to the H₂|2H⁺ equilibrium potential. Furthermore, HE was able to catalyze the current generation (200 μA) by consuming H₂ gas produced in the microbial consortium from starch. After 220 h of operation, HE retained at least 81% of the initial activity. Calculations of carbon balance indicated that fermentation products were similar in microbial cells without HE and with HE generating the current due to H₂ consumption.     

Glucose fermentation to acetate,CO2 and H2 in the anaerobic hyperthermophilic eubacterium Thermotoga maritima: involvement of the Embden-Meyerhof pathway

The hyperthermophilic anaerobic eubacterium Thermotoga maritima was grown on glucose as carbon and energy source. During growth 1 mol glucose was fermented to 2 mol acetate, 2 mol CO₂ and 4 mol H₂. The molar growth yicld on glucose (Y_glucose) was about 45 g cell dry mass/mol glucose. In the presence of elemental sulfur growing cultures of T. maritima converted 1 mol glucose to 2 mol acetate, 2 mol CO₂ about 0.5 mol H₂ and about 3.5 mol H₂S. Y_glucose was about 45 g/mol. Cell extracts contained all enzymes of the Embden-Meyerhof pathway: hexokinase (0.29 U/mg, 50°C), glucose-6-phosphate isomerase (0.56 U/mg, 50°C), phosphofructokinase (0.19 U/mg, 50° C), fructose-1,6-bisphosphate aldolase (0.033 U/mg, 50°C), triosephosphate isomerase (6.3 U/mg, 50°C), glyceraldehyde-3-phosphate dehydrogenase (NAD⁺ reducing: 0.63 U/mg, 50°C), phosphoglycerate kinase (3.7 U/mg, 50°C), phosphoglycerate mutase (0.4 U/mg, 50°C); enolase (4 U/mg, 80°C), pyruvate kinase (0.05 U/mg, 50°C). Furthermore, cell extracts contained pyruvate: ferredoxin oxidoreductasee (0.43 U/mg, 60°C); NADH: ferredoxin oxidoreductase (benzylviologen reduction: 0.46 U/mg, 80°C); hydrogenase (benzylviologen reduction: 15 U/mg, 80°C), phosphate acetyltransferase (0.13 U/mg, 80°C), acetate kinase (1.2 U/mg, 55°C), lactate dehydrogenase (0.16 U/mg, 80°C) and pyruvate carboxylase (0.02 U/mg, 50°C). The findings indicate that the hyperthermophilic eubacterium T. maritima ferments sugars (glucose) to acetate, CO₂ and H₂ involving the Embden-Meyerhof pathway, phosphate acetyltransferase and acetate kinase. Thus, the organism differs from the hyperthermophilic archaeon Pyrococcus furiosus which ferments sugars to acetate, CO₂ and H₂ involving a modified non-phosphorylated Entner-Doudoroff pathway and acetyl-CoA synthetase (ADP forming).     

Effect of Supplemental Electron Donors on the Microbial Reduction of Fe(III), Sulfate, and CO2 in Coal Mining–Impacted Freshwater Lake Sediments

Abstract In acidic mining-impacted lake sediments, the microbial reduction of Fe(III) is the dominant electron-accepting process, whereas the reduction of sulfate seems to be restricted to a narrow sediment zone of elevated pH and lower amounts of total and reactive iron. To evaluate the microbial heterogeneity and the commensal interactions of the microbial community, the flow of supplemental carbon and reductant was evaluated in four different zones of the sediment in anoxic microcosms at the in situ temperature of 12°C. Substrate consumption, product formation, and the potential to reduce Fe(III) and sulfate were similar with both upper and lower sediment zones. In the upper acidic iron-rich sediment zone, the rate of Fe(II) formation 204 nmol ml⁻¹ d⁻¹ was enhanced to 833 nmol ml⁻¹ d⁻¹ and 462 nmol ml⁻¹ d⁻¹ by supplemental glucose and H₂, respectively. Supplemental lactate and acetate were not consumed under acidic conditions and decreased the rate of Fe(II) formation to 130 nmol ml⁻¹ d⁻¹ and 52 nmol ml⁻¹ d⁻¹, respectively. When the pH of the upper sediment increased above pH 5, acetate-dependent reduction of sulfate was initiated even though the pool of Fe(III) was not depleted. In deeper sediment zones with elevated pH, the rapid consumption of acetate was always coincident to a decrease in the concentration of sulfate and soluble Fe(II), indicating the formation of Fe(II) sulfides. Although the reduction of Fe(III) was still an ongoing process in deeper sediment zones, the formation of Fe(II) was only slightly enhanced by the consumption of glucose or cellobiose, but not by H₂ or acetate. H₂-utilizing acetogens seemed to be involved in the consumption of H₂. These collective results indicated (i) that the reduction of Fe(III) predominated over the reduction of sulfate as long as the sediment remained acidic and carbon-limited, and (ii) that the sulfate-reducing microbiota in this heterogeneous sediment were better adapted to the geochemical gradients present than were other neutrophilic dissimilatory Fe(III) reducers. Received: 17 February 2000; Accepted: 22 June 2000; Online Publication: 28 August 2000     

Production and Starch Saccharification by a Thermostable and Neutral Glucoamylase of a Thermophilic Mould Thermomucor Indicae-Seudaticae

The present investigation was aimed at producing a thermostable and neutral glucoamylase (amyloglucosidase, EC 3.2.1.3) by a thermophilic mould, Thermomucor indicae-seudaticae in submerged cultivation and testing its applicability in starch saccharification. Parametric optimization resulted in the secretion of 30,000 U/l of glucoamylase in a synthetic medium (5% soluble starch, 0.1% yeast extract, 0.05% K₂HPO₄ and 0.01% MgSO₄· 7H₂O) using 5 × 10⁶ spores/50 ml of a 3-day-old inoculum at 40 °C and 250 rev/min in shake flasks in 48 h. The enzyme secretion was not affected to any significant extent by the tested additives and detergents. A 1.7-fold increase in glucoamylase secretion was attained when T. indicae-seudaticae was grown in a laboratory fermenter. The enzyme alone catalysed the hydrolysis of soluble starch to an extent of 65%. A prior treatment of starch with thermostable α-amylase and amylopullulanase, followed by glucoamylase, resulted in a greater extent of hydrolysis, 79 and 91%, respectively.     

Characterization of microbial souring in Berea‐sand porous medium with a North Sea oil field inoculum

Microbial souring (H₂S production) in porous medium was investigated in an anaerobic upflow porous medium reactor at 60°C using produced waters obtained from the North Sea Ninian oilfield as the inoculum. Multiple carbon sources commonly found in oil field waters (formate, acetate, propionate, iso‐ and n‐butyrates) with inorganic sulfate as the electron acceptor were used as the substrates. Stoichiometry and the rate of souring in the reactor column were calculated. A large proportion of H₂S was trapped in the column as FeS and possibly as a gas phase. Concentration gradients for the substrates (organic acids and sulfate) and H₂S were generated along the column. At steady state, the highest volumetric substrate consumption and H₂S production were found at the front part (inlet) of the reactor column. The average volumetric sulfate reduction rate after H₂S production had stabilized was calculated to be 203 ± 51 mg sulfate‐S.l^‐1.d^‐1. Comparison of the results with the authors’ previous work on the Alaska Kuparuk oilfield waters indicates that the two different microbial inocula (produced waters) exhibited the same experimental trends (rates and location) for souring in the experimental reactor system. This indicates that abiotic factors, as well as microbial parameters, may play an important role for microbial souring in the system.     

Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii

Summary The hydrolysis and fermentation of cellulose (Avicel) by continuous cultures of Ruminococcus albus strain 7 and Methanobrevibacter smithii strain PS were studied. Cellulose destruction ranged from ca. 22% to 71% for 0.25 to 2.27 days solids retention time, respectively. The cellulose hydrolysis rate constant (k) was 1.3 days^–1. Concentrations of soluble reducing sugars were low, showing that cellulose hydrolysis was the rate-limiting step of cellulose fermentation. The estimated methane-based molar growth yield for M. smithii was 2.8 g mol^–1. Its maximum specific growth rate was ca. 4 days^–1. The dissolved H₂ half-saturation constant (K _s ) for methanogenesis was ca. 1 M. The final products of the co-culture were primarily acetate, CH₄ and CO₂ and low levels of ethanol and H₂. The co-culture produced more H₂ (used for reduction of CO₂ to CH₄) and acetate than a monoculture of R. albus. These differences coulb be accounted for by the lower production of ethanol, confirming to the theory of interspecies H₂ transfer. Offprint requests to: M. J. Wolin     

Hydrogen production by the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis> part I: effects of sulfured nutriments,with thiosulfate as model,on hydrogen production and growth

Background

Thermotoga maritima and T. neapolitana are hyperthermophile bacteria chosen by many research teams to produce bio-hydrogen because of their potential to ferment a wide variety of sugars with the highest theoretical H₂/glucose yields. However, to develop economically sustainable bio-processes, the culture medium formulation remained to be optimized. The main aim of this study was to quantify accurately and specifically the effect of thiosulfate, used as sulfured nutriment model, on T. maritima growth, yields and productivities of hydrogen. The results were obtained from batch cultures, performed into a bioreactor, carefully controlled, and specifically designed to prevent the back-inhibition by hydrogen.

Results

Among sulfured nutriments tested, thiosulfate, cysteine, and sulfide were found to be the most efficient to stimulate T. maritima growth and hydrogen production. In particular, under our experimental conditions (glucose 60 mmol L^?1 and yeast extract 1 g L^?1), the cellular growth was limited by thiosulfate concentrations lower than 0.06 mmol L^?1. Under these conditions, the cellular yield on thiosulfate (Y X/Thio) could be determined at 3617 mg mmol^?1. In addition, it has been shown that the limitations of T. maritima growth by thiosulfate lead to metabolic stress marked by a significant metabolic shift of glucose towards the production of extracellular polysaccharides (EPS). Finally, it has been estimated that the presence of thiosulfate in the T. maritima culture medium significantly increased the cellular and hydrogen productivities by a factor 6 without detectable sulfide production.

Conclusions

The stimulant effects of thiosulfate at very low concentrations on T. maritima growth have forced us to reconsider its role in this species and more probably also in all thiosulfato-reducer hyperthermophiles. Henceforth, thiosulfate should be considered in T. maritima as (1) an essential sulfur source for cellular materials when it is present at low concentrations (about 0.3 mmol g^?1 of cells), and (2) as both sulfur source and detoxifying agent for H₂ when thiosulfate is present at higher concentrations and, when, simultaneously, the pH₂ is high. Finally, to improve the hydrogen production in bio-processes using Thermotoga species, it should be recommended to incorporate thiosulfate in the culture medium.

     

The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature,oxygen, and pH on hydrogen production

The hyperthermophilic bacterium, Thermotoga neapolitana, has potential for use in biological hydrogen (H₂) production. The objectives of this study were to (1) determine the fermentation stoichiometry of Thermotoga neapolitana and examine H₂ production at various growth temperatures, (2) investigate the effect of oxygen (O₂) on H₂ production, and (3) determine the cause of glucose consumption inhibition. Batch fermentation experiments were conducted at temperatures of 60, 65, 70, 77, and 85°C to determine product yield coefficients and volumetric productivity rates. Yield coefficients did not show significant changes with respect to growth temperature and the rate of H₂ production reached maximum levels in both the 77°C and 85°C experiments. The fermentation stoichiometry for T. neapolitana at 85°C was 3.8 mol H₂, 2 mol CO₂, 1.8 mol acetate, and 0.1 mol lactate produced per mol of glucose consumed. Under microaerobic conditions H₂ production did not increase when compared to anaerobic conditions, which supports other evidence in the literature that T. neapolitana does not produce H₂ through microaerobic metabolism. Glucose consumption was inhibited by a decrease in pH. When pH was adjusted with buffer addition cultures completely consumed available glucose. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

In Situ Cultivation of Subsurface Microorganisms in a Deep Mafic Sill: Implications for SLiMEs

An in situ culturing device was incubated within a flowing borehole in a mafic sill at 1.474 km depth in Evander Au mine, South Africa. The device was designed to enrich methanogenic, Fe^{3 +}-reducing and SO₄ ^{2 ?}-reducing microorganisms using acetate, formate, methanol, Fe^{3 +}-citrate and SO₄ ^{2 ?} enriched agar and sand cartridges. At the end of the 33 day incubation geochemical analyses detected elevated H₂, acetate, CH₄ and Fe concentrations and depleted SO₄ ^{2 ?} concentrations. 16S rDNA sequences and PLFA analyses revealed that the microbial community composition of the substrate-bearing cartridges were distinct from that of the original borehole water and the non-substrate-bearing control cartridge. 16S rDNA and dissimilatory sulfite reductase, dsrAB, gene sequences indicated the device successfully targeted SO₄ ^{2 ?} reducing bacteria (SRB), which were not detected in the original borehole water. 16S rDNA sequences also revealed a shift in the microbial community from one relying on H₂ based methanogenesis to one suggestive of H₂ based acetogenesis supporting aceticlastic methanogenesis and SO₄ ^{2 ?} reduction compatible with the subsurface lithoautotrophic hypothesis.     

Thermodynamic and kinetic analysis of the H2 threshold for Methanobacterium bryantii M.o.H

H₂ thresholds, concentrations below which H₂ consumption by a microbial group stops, have been associated with microbial respiratory processes such as dechlorination, denitrification, sulfate reduction, and methanogenesis. Researchers have proposed that observed H₂ thresholds occur when the available Gibbs free energy is minimal (ΔG ≈ 0) for a specific respiratory reaction. Others suggest that microbial kinetics also may play a role in controlling the thresholds. Here, we comprehensively evaluate H₂ thresholds in light of microbial thermodynamic and kinetic principles. We show that a thermodynamic H₂ threshold for Methanobacterium bryantii M.o.H. is not controlled by ΔG for methane production from H₂ + HCO₃⁻. We repeatedly attain a H₂ threshold near 0.4 nM, with a range of 0.2–1 nM, and ΔG for methanogenesis from H₂ + HCO₃⁻ is positive, +5 to +7 kJ/mol-H_2, at the threshold in most cases. We postulate that the H₂ threshold is controlled by a separate reaction other than methane production. The electrons from H₂ oxidation are transferred to an electron sink that is a solid-phase component of the cells. We also show that a kinetic threshold (S _min) occurs at a theoretically computed H₂ concentration of about 2400 nM at which biomass growth shifts from positive to negative.     

Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract

The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD⁺, Mn²⁺) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

Permeability and reactivity of Thermotoga maritima in latex bimodal blend coatings at 80°C: a model high temperature biocatalytic coating

Thermostable polymers cast as thin, porous coatings or membranes may be useful for concentrating and stabilizing hyperthermophilic microorganisms as biocatalysts. Hydrogel matricies can be unstable above 65°C. Therefore a 55-m thick, two layer (cell coat + polymer top coat) bimodal, adhesive latex coating of partially coalesced polystyrene particles was investigated at 80°C using Thermotoga maritima as a model hyperthermophile. Coating permeability (pore structure) was critical for maintaining T. maritima viability. The permeability of bimodal coatings generated from 0.8 v/v of a suspension of non-film-forming 800 nm polystyrene particles with high glass transition temperature (T_g= 94°C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (T_g –5°C, 40.9% total solids) with 0.3 g sucrose/g latex was measured in a KNO₃ diffusion cell. Diffusivity ratio remained above 0.04 (D_eff/D) when incubated at 80°C in artificial seawater (ASW) for 5 days. KNO₃ permeability was corroborated by cryogenic-SEM images of the pore structure. In contrast, the permeability of a mono-dispersed acrylate/vinyl acetate latex Rovace SF091 (T_g~10°C) rapidly decreased and became impermeable after 2 days incubation in ASW at 80°C. Thermotoga maritima were entrapped in these coatings at a cell density of 49 g cell wet weight/liter of coating volume, 25-fold higher than the density in liquid culture. Viable T. maritima were released from single-layer coatings at 80°C but accurate measurement of the percentage of viable entrapped cells by plate counting was not successful. Metabolic activity could be measured in bilayer coatings by utilization of glucose and maltose, which was identical for latex-entrapped and suspended cells. Starch was hydrolyzed for 200 h by latex-entrapped cells due to the slow diffusion of starch through the polymer top coat compared to only 24 h by suspended T. maritima. The observed reactivity and stability of these coatings was surprising since cryo-SEM images suggested that the smaller low T_g polyacrylate/styrene particles preferentially bound to the T. maritima toga-sheath during coat formation. This model system may be useful for concentrating, entrapment and stabilization of metabolically active hyperthermophiles at 80°C.     

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (10¹²/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (10¹¹/g dry weight), but low numbers of hydrolytic-fermentative bacteria (10⁹/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H₂-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H₂, formate, acetate and butyrate but they were involved in propionate degradation.Correspondence to: M. K. Jain     

Hydrogen production by the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis> Part II: modeling and experimental approaches for hydrogen production

Background

Thermotoga maritima is a hyperthermophilic bacterium known to produce hydrogen from a large variety of substrates. The aim of the present study is to propose a mathematical model incorporating kinetics of growth, consumption of substrates, product formations, and inhibition by hydrogen in order to predict hydrogen production depending on defined culture conditions.

Results

Our mathematical model, incorporating data concerning growth, substrates, and products, was developed to predict hydrogen production from batch fermentations of the hyperthermophilic bacterium, T. maritima. It includes the inhibition by hydrogen and the liquid-to-gas mass transfer of H₂, CO₂, and H₂S. Most kinetic parameters of the model were obtained from batch experiments without any fitting. The mathematical model is adequate for glucose, yeast extract, and thiosulfate concentrations ranging from 2.5 to 20 mmol/L, 0.2–0.5 g/L, or 0.01–0.06 mmol/L, respectively, corresponding to one of these compounds being the growth-limiting factor of T. maritima. When glucose, yeast extract, and thiosulfate concentrations are all higher than these ranges, the model overestimates all the variables. In the window of the model validity, predictions of the model show that the combination of both variables (increase in limiting factor concentration and in inlet gas stream) leads up to a twofold increase of the maximum H₂-specific productivity with the lowest inhibition.

Conclusions

A mathematical model predicting H₂ production in T. maritima was successfully designed and confirmed in this study. However, it shows the limit of validity of such mathematical models. Their limit of applicability must take into account the range of validity in which the parameters were established.

     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

Dissimilatory Iron [Fe(III)] Reduction by a Novel Fermentative,Piezophilic Bacterium Anoxybacter fermentans DY22613T Isolated from East Pacific Rise Hydrothermal Sulfides

Dissimilatory iron-reducing microorganisms play an important role in the biogeochemical cycle of iron and influence iron mineral formation and transformation. However, studies on microbial iron-reducing processes in deep-sea hydrothermal fields are limited. A novel piezophilic, thermophilic, anaerobic, fermentative iron-reducing bacteria of class Clostridia, named Anoxybacter fermentans DY22613^T, was isolated from East Pacific Rise hydrothermal sulfides. In this report, we examined its cell growth, fermentative metabolites, and biomineralization coupled with dissimilatory iron reduction. Both soluble ferric citrate (FC) and solid amorphous Fe(III) oxyhydroxide (FO) could promote cell growth of this strain, accompanied by increased peptone consumption. More acetate, butyrate, and CO₂ were produced than without adding FO or FC in the media. The highest yield of H₂ was observed in the Fe(III)-absent control. Coupled to fermentation, magnetite particles, and iron-sulfur complexes were respectively formed by the strain during FO and FC reduction. Under experimental conditions mimicking the pressure prevailing at the deep-sea habitat of DY22613^T (20?MPa), Fe(III)-reduction rates were enhanced resulting in relatively larger magnetite nanoparticles with more crystal faces. These results implied that the potential role of A. fermentans DY22613^T in situ in deep-sea hydrothermal sediments is coupling iron reduction and mineral transformation to fermentation of biomolecules. This bacterium likely contributes to the complex biogeochemical iron cycling in deep-sea hydrothermal fields.     

Oxygen Reduction Reaction Affected by Sulfate-Reducing Bacteria: Different Roles of Bacterial Cells and Metabolites

Sulfate-reducing bacteria (SRB) were found to be capable of tolerating a certain amount of oxygen (O₂), but how they affect oxygen reduction reaction (ORR) has not been clear. The present work investigated the impact of SRB on ORR in 3.5 wt% sodium chloride solution with the cyclic voltammetry method. The addition of SRB culture solution hampered both the reduction of O₂ to superoxide (O ₂ ^·? ) and hydrogen peroxide (H₂O₂) to water (H₂O), and the influence of SRB metabolites was much larger than that of bacterial cells. Sulfide and extracellular polymeric substances (EPS), typical inorganic and organic metabolic products, had great impact on ORR. Sulfide played an important role in the decrease of cathodic current for H₂O₂ reduction due to its hydrolysis and chemical reaction activity with H₂O₂. EPS were sticky, easy to adsorb on the electrode surface and abundant in functional groups, which hindered the transformation of O₂ into O ₂ ^·? and favored the reduction of H₂O₂ to H₂O.     

A Cluster of Thermotoga neapolitana Genes Involved in the Degradation of Starch and Maltodextrins: the Molecular Structure of the Locus

A 5451-bp genome fragment of the hyperthermophilic anaerobic eubacterium Thermotoga neapolitana has been cloned and sequenced. The fragment contains one truncated and three complete open reading frames highly homologous to the starch/maltodextrin utilization gene cluster from T. maritima whose genome sequence is known. The incomplete product of the first frame is highly homologous to MalG, the Escherichia coli protein of starch and maltodextrin transport. The product of the second frame, AglB, is highly homologous to cyclomaltodextrinase with the -glucosidase activity TMG belonging to family 13 of glycosyl hydrolases (GH13). The product of the third frame, AglA, is homologous to the T. maritima cofactor-dependent -glucosidase from the GH4 family. The two enzymes form a separate branch on the phylogenetic tree of the family. The AglA and AglB proteins supplement each other in substrate specificity and can ensure complete hydrolysis to glucose of cyclic and linear maltodextrins, the intermediate products of starch degradation. The product of the fourth reading frame has sequence similarity with the riboflavin-specific deaminase RibD from T. maritima. The homologous locus of this bacterium, between the aglA and ribD genes, has five open reading frames missing in T. neapolitana. The nucleotide sequences of two frames are homologous to transposase genes. The deletion size is 2.9 kb.