Influence of dilution rate on the acidogenic phase products distribution during two-phase lactose anaerobiosis

Acidogenic fermentation of lactose was carried out in a continuous stirred reactor with a mixed anaerobic culture. From the variation of the reactor products with pH and dilution rate two possible carbon flow schemes were proposed for the reaction. In both schemes the carbon flow from pyruvate to butyrate and lactate was assumed to occur in parallel. A change in gas composition and in product concentrations at dilution rates between 0.1 and 0.15 h(-1) for pH levels between 4.5 and 6.0 was ascribed to a shift in microbial population. To clarify the mechanism radiotracer tests were made using [U-(14)C]-butyrate, [2-(14)C]-propionate and [U-(14)C]-lactate to determine the path of carbon flow during acidogenesis of lactose using a mixed culture. At a dilution rate between 0.1 and 0.15 h(-1) and pH from 4.5 to 6.0 a rise in the lactate concentration in the product was shown to be due to a microbial population shift which disabled the conversion of lactate to other intermediary metabolites. It was also found that the flow of carbon from pyruvate to butyrate and lactate occurred by parallel pathways. Also, in the presence of hydrogen reducing methanogens, lactate was almost completely converted to acetate and not propionate. Butyrate was found to be converted to acetate at a slow rate as long as hydrogen reducing methanogens were present. The role played by propionibacteria in this lactose acidogenic eocosystem was minor. From the carbon flow model it can be concluded that lactate is the most suitable marker for optimizing an acidogenic reactor in a two-phase biomethanation process.     

Benzoate fermentation by the anaerobic bacterium Syntrophus aciditrophicus in the absence of hydrogen-using microorganisms.

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (DeltaG') of -9.2 kJ/mol was reached (-4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO(2), and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than -20 kJ/mol, the postulated minimum free energy value for substrate metabolism.     

Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H(2)-consuming methanogens was <-20 kJ mol of CH(4)(-1) and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >-10 kJ mol of propionate(-1). Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0. 07 day(-1) and produced maximum biomass yields of 2.6 g mol of propionate(-1) and 7.6 g mol of CH(4)(-1) for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP(-1). However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h(-1) mol of biomass C(-1)) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Pure-culture growth of fermentative bacteria, facilitated by H2 removal: bioenergetics and H2 production

We used an H2-purging culture vessel to replace an H2-consuming syntrophic partner, allowing the growth of pure cultures of Syntrophothermus lipocalidus on butyrate and Aminobacterium colombiense on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (DeltaG) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H2 production changed such that DeltaG remained nearly constant for each organism (-11.1 +/- 1.4 kJ mol butyrate(-1) for S. lipocalidus and -58.2 +/- 1.0 kJ mol alanine(-1) for A. colombiense). The cellular maintenance energy, determined from the DeltaG value and the hydrogen production rate at the point where the cell number was constant, was 4.6 x 10(-13) kJ cell(-1) day(-1) for S. lipocalidus at 55 degrees C and 6.2 x 10(-13) kJ cell(-1) day(-1) for A. colombiense at 37 degrees C. S. lipocalidus, in particular, seems adapted to thrive under conditions of low energy availability.     

Application of response surface methodology for optimization of acidogenesis of cattail by rumen cultures

Acidogenesis of cattail using rumen cultures was carried out to produce volatile fatty acids (VFA) in this study. The influences of pH and substrate concentration on cattail degradation, VFA yield and microbial growth were investigated by using response surface methodology (RSM). Experimental results showed that a low substrate concentration and pH of 6.9 were optimal for acidogenesis of cattail. The highest cattail degradation efficiency, VFA yield and microbial yield were 75.9%, 0.41 g/g VS and 0.110 g/g VS, respectively. Further experiments confirmed that the main VFA in the acidogenesis of cattail were acetate, propionate and butyrate, while i-butyrate, valerate and i-valerate were also produced at low levels. The results suggested that acidogenesis using rumen cultures is a promising method for cattail disposal.     

Response of a biohydrogen-producing reactor to the substrate shift from sucrose to lactose

The response of an upflow acidogenic granule-based reactor to the substrate shift from sucrose to lactose was investigated in this study. Experimental results show that it took 60h for the reactor to completely degrade the new substrate. Hydrogen production performance, in terms of H(2) partial pressure, H(2) production rate and H(2) yield, was affected. Acetate, propionate, butyrate, valerate, caporate, ethanol and propanol were present in the reactor effluent, and their distribution changed significantly after the substrate shift. As the substrate was changed, the caproate- and ethanol-type fermentation was weakened, while the propionate-type fermentation was strengthened. Throughout the experiment, the butyrate-type fermentation played an important role. The H(2) yield had a close correlation with both propionate and B/A (butyrate/acetate) in this substrate shift process.     

Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge

Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.     

Benzoate Fermentation by the Anaerobic Bacterium Syntrophus aciditrophicus in the Absence of Hydrogen-Using Microorganisms

The anaerobic bacterium Syntrophus aciditrophicus metabolized benzoate in pure culture in the absence of hydrogen-utilizing partners or terminal electron acceptors. The pure culture of S. aciditrophicus produced approximately 0.5 mol of cyclohexane carboxylate and 1.5 mol of acetate per mol of benzoate, while a coculture of S. aciditrophicus with the hydrogen-using methanogen Methanospirillum hungatei produced 3 mol of acetate and 0.75 mol of methane per mol of benzoate. The growth yield of the S. aciditrophicus pure culture was 6.9 g (dry weight) per mol of benzoate metabolized, whereas the growth yield of the S. aciditrophicus-M. hungatei coculture was 11.8 g (dry weight) per mol of benzoate. Cyclohexane carboxylate was metabolized by S. aciditrophicus only in a coculture with a hydrogen user and was not metabolized by S. aciditrophicus pure cultures. Cyclohex-1-ene carboxylate was incompletely degraded by S. aciditrophicus pure cultures until a free energy change (ΔG′) of −9.2 kJ/mol was reached (−4.7 kJ/mol for the hydrogen-producing reaction). Cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at micromolar levels during growth of an S. aciditrophicus pure culture with benzoate. High hydrogen (10.1 kPa) and acetate (60 mM) levels inhibited benzoate metabolism by S. aciditrophicus pure cultures. These results suggest that benzoate fermentation by S. aciditrophicus in the absence of hydrogen users proceeds via a dismutation reaction in which the reducing equivalents produced during oxidation of one benzoate molecule to acetate and carbon dioxide are used to reduce another benzoate molecule to cyclohexane carboxylate, which is not metabolized further. Benzoate fermentation to acetate, CO₂, and cyclohexane carboxylate is thermodynamically favorable and can proceed at free energy values more positive than −20 kJ/mol, the postulated minimum free energy value for substrate metabolism.     

Comparative thermodynamic analysis of DNA--protein interactions using surface plasmon resonance and fluorescence correlation spectroscopy

We report a kinetic and thermodynamic analysis of interactions between ssDNA and replication protein A (RPA) using surface plasmon resonance (SPR) and fluorescence correlation spectroscopy (FCS) at variable temperature. The two methods yield different values for the Gibbs free energy but nearly the same value for the reaction enthalpy of ssDNA-RPA complex formation. The Gibbs free energy was determined by SPR and FCS to be -62.6 and -54.7 kJ/mol, respectively. The values for the reaction enthalpy are -64.4 and -66.5 kJ/mol. It is concluded that the difference in Gibbs free energy measured by the two methods is due to different reaction entropies. The entropic contribution to the free energy at 25 degrees C is -1.8 kJ/mol for SPR and -11.8 kJ/mol for FCS. In SPR, the reaction is restricted to two dimensions because of immobilization of the DNA molecules to the sensor surface. In contrast, FCS is able to follow complex formation without spatial restrictions. In consequence, the reaction entropy determined from SPR experiments is lower than for FCS experiments.     

Kinetic and isothermal studies on liquid-phase adsorption of 2,4-dichlorophenol by palm pith carbon

Adsorption studies were conducted to study the removal of 2,4-dichlorophenol (2,4-DCP) from aqueous solution on palm pith carbon under varying experimental conditions such as agitation time, adsorbent dose, pH and temperature. Higher 2,4-DCP was removed with decrease in the initial concentration of 2,4-DCP and increase in amount of adsorbent used. Kinetic study showed that the adsorption of 2,4-DCP on palm pith carbon was a gradual process. Adsorption capacities were 19.16 mg/g for the particle size of 250-500 microm. The equilibrium time was 60 and 80 min for 10 and 20 mg/L and 100 min for both 30 and 40 mg/L phenol concentrations, respectively. Acidic pH was favourable for the adsorption of 2,4-DCP. Studies on pH effect and desorption showed that chemisorption seemed to play a major role in the adsorption process. Thermodynamic study showed that adsorption of 2,4-DCP on palm pith carbon was more favoured. The change in entropy (DeltaS0) and heat of adsorption (DeltaH0) of palm pith carbon was estimated as 30.72 J/mol/k and 7.16 kJ/mol, respectively. The high positive value of change in Gibbs free energy indicated the feasible and spontaneous adsorption of 2,4-DCP on palm pith carbon. The results indicated that palm pith carbon was an attractive candidate for removing phenols from wastewater.     

Energetics of Syntrophic Propionate Oxidation in Defined Batch and Chemostat Cocultures

Propionate consumption was studied in syntrophic batch and chemostat cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. The Gibbs free energy available for the H₂-consuming methanogens was <−20 kJ mol of CH₄⁻¹ and thus allowed the synthesis of 1/3 mol of ATP per reaction. The Gibbs free energy available for the propionate oxidizer, on the other hand, was usually >−10 kJ mol of propionate⁻¹. Nevertheless, the syntrophic coculture grew in the chemostat at steady-state rates of 0.04 to 0.07 day⁻¹ and produced maximum biomass yields of 2.6 g mol of propionate⁻¹ and 7.6 g mol of CH₄⁻¹ for S. fumaroxidans and M. hungatei, respectively. The energy efficiency for syntrophic growth of S. fumaroxidans, i.e., the biomass produced per unit of available Gibbs free energy was comparable to a theoretical growth yield of 5 to 12 g mol of ATP⁻¹. However, a lower growth efficiency was observed when sulfate served as an additional electron acceptor, suggesting inefficient energy conservation in the presence of sulfate. The maintenance Gibbs free energy determined from the maintenance coefficient of syntrophically grown S. fumaroxidans was surprisingly low (0.14 kJ h⁻¹ mol of biomass C⁻¹) compared to the theoretical value. On the other hand, the Gibbs free-energy dissipation per mole of biomass C produced was much higher than expected. We conclude that the small Gibbs free energy available in many methanogenic environments is sufficient for syntrophic propionate oxidizers to survive on a Gibbs free energy that is much lower than that theoretically predicted.     

Biological production of hydrogen from cellulose by natural anaerobic microflora

The capability of natural anaerobic microflora to produce hydrogen was examined with artificial wastewater containing cellulose. The microflora in sludge compost was found to produce a significant amount of hydrogen (2.4 mol/mol-hexose). Among the fermentation products other than hydrogen and carbon dioxide, the lower fatty acids, mainly acetate and butyrate, constituted more than approximately 90% of the total soluble metabolites.     

Effect of substrate and feed antibiotics on in vitro production of volatile fatty acids and methane in caecal contents of chickens

An attempt was done to identify some factors influencing the caecal fermentation pattern in poultry. Experiments were conducted to study effects of carbohydrate substrates (feed components and supplements) and antibiotics on the formation of volatile fatty acids (VFA) and methane in in vitro incubations of the caecal contents of 7-week-old chickens. Stoichiometry of fermentation differed in cultures with different carbohydrates. Fermentation pattern characterized by high propionate and low acetate production was found in cultures with lactose (0.447 and 0.376 mol/1 mol of VFA produced, respectively) and, to a lesser extent, also in cultures with raffinose. Acetate was the predominant metabolite of starch, pectin and xylan (0.727, 0.773 and 0.685 mol/1 mol of VFA produced, respectively). Fermentation of inulin resulted in high proportion of butyrate, 0.221 mol/1 mol of VFA. Other polysaccharides produced only 0.060–0.111 mol of butyrate per 1 mol of VFA. Oligosaccharides (lactose, raffinose) were fermented more rapidly than polysaccharides. Fermentation of inulin yielded more VFA than fermentation of starch, pectin and xylan. No production of VFA from carboxymethylcellulose was observed. On average, 11 mols of VFA were produced per mol of methane. Lasalocid significantly increased molar proportion of propionate, which is potentially beneficial from the point of view of salmonellae control. The magnitude of improvement, however, was small. Other feed antibiotics tested (avoparcin, bacitracin, lincomycin, spiramycin, tylosin, virginiamycin) produced only non-significant or marginal fermentation shifts. Formation of valerate, isoacids and methane was not significantly influenced by the substrate or by antibiotic treatment.     

Detection of luciferase gene sequences in nonluminescent bacteria from the Chesapeake Bay

Washed excised roots of rice (Oryza sativa) produced H(2), CH(4) and fatty acids (millimolar concentrations of acetate, propionate, butyrate; micromolar concentrations of isovalerate, valerate) when incubated under anoxic conditions. Surface sterilization of the root material resulted in the inactivation of the production of CH(4), a strong reduction of the production of fatty acids and a transient (75 h) but complete inhibition of the production of H(2). Radioactive bicarbonate was incorporated into CH(4), acetate, propionate and butyrate. About 20-40% of the fatty acid carbon originated from CO(2) reduction. In the presence of phosphate, CH(4) was exclusively produced from H(2)/CO(2), since phosphate selectively inhibited acetoclastic methanogenesis. Acetoclastic methanogenesis was also selectively inhibited by methyl fluoride, while chloroform or 2-bromoethane sulfonate inhibited CH(4) production completely. Production of CH(4), acetate, propionate and butyrate from H(2)/CO(2) was always exergonic with Gibbs free energies <-20 kJ mol(-1) product. Chloroform inhibited the production of acetate and the incorporation of radioactive CO(2) into acetate. Simultaneously, H(2) was no longer consumed and accumulated, indicating that acetate was produced from H(2)/CO(2). Chloroform also resulted in increased production of propionate and butyrate whose formation from CO(2) became more exergonic upon addition of chloroform. Nevertheless, the incorporation of radioactive CO(2) into propionate and butyrate was inhibited by chloroform. The accumulation of propionate and butyrate in the presence of chloroform probably occurred by fermentation of organic matter, rather than by reduction of acetate and CO(2). [U-(14)C]Glucose was indeed converted to acetate, propionate, butyrate, CO(2) and CH(4). Radioactive acetate, CO(2) and CH(4) were also products of the degradation of [U-(14)C]cellulose and [U-(14)C]xylose. Addition of chloroform and methyl fluoride did not affect the product spectrum of [U-(14)C]glucose degradation. The application of combinations of selective inhibitors may be useful to elucidate anaerobic metabolic pathways in mixed microbial cultures and natural microbial communities.     

Purification, kinetic and thermodynamic studies of a new ribonuclease from a mutant of Aspergillus niger

Ribonuclease was purified from Aspergillus niger SA-13-20 to homogeneity level by using (NH(4))(2)SO(4) precipitation, DEAE-cellulose anion-exchange chromatography, ultrafiltration and Sephacryl HR-200 chromatography. The molecular weight and isoelectric point of the enzyme was 40.1kDa and 5.3, respectively. The pH- and temperature-dependent kinetic parameters were determined. The RNase showed the strongest affinity with RNA as the substrate, and the highest catalytic efficiency for hydrolysis of the substrate at pH 3.5 and 65 degrees C. It exhibited Michaelis-Menten Kinetics with k(cat) of 118.1s(-1) and K(m) of 57.0 microg ml(-1), respectively. Thermodynamic parameters for catalysis and thermal denaturation were also determined. Activation energy (E(a)) for catalysis of A. niger SA-13-20 RNase was 50.31 kJ mol(-1) and free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation for catalysis of the enzyme at 65 degrees C were 69.76, 47.50 and -65.83 Jmol(-1)K(-1), respectively. Activation energy (E(a,d)) for denaturation of the enzyme was 200.53 kJ mol(-1) and free energy (DeltaG(d)(#)), enthalpy (DeltaH(d)(#)) and entropy (DeltaS(d)(#)) of activation for denaturation of the enzyme at 45 degrees C were 79.18 kJ mol(-1), 197.88 and 373.09 Jmol(-1)K(-1), respectively.     

Hydrolysis and acidogenic fermentation of gelatin under anaerobic conditions in a laboratory scale upflow reactor

Summary Gelatin was dissolved in a mineral salts medium for growth under carbon limitation and fed to a mixed population of bacteria in a lab-scale upflow reactor for hydrolysis and acidogenic fermentation under anaerobic conditions, at pH=7 and 30°C.With the shortest applied liquid retention time (30 min), 84% of the protein was hydrolysed. The majority (85%) of the hydrolysed protein was fermented. The ammonia concentration in the reactor was about 1,400 mg N·l^-1.The fermentation products were mainly acetate, propionate, and valerate. Iso-butyrate, butyrate, and iso-valerate were formed to a limited extent. Gas production was very low and consisted of carbon dioxide and methane. The product composition was independent of the retention time applied. The sludge formed was slimy. No granules were formed, however a hold-up factor of 46 could be obtained.     

Intrinsic fermentation kinetics of lactose in acidogenic biofilms

The intrinsic fermentation kinetics of lactose in acidogenic biofilms were investigated in situ in a continuous flow fermentor at 35 degrees C and pH 4.6. The external and internal mass transfer resistances to lactose molecules from bulk solution to inside the biofilms were experimentally minimized or eliminated in a thin biofilm and recycled medium. In a chemically defined culture medium, the immobilized acidogens converted lactose mainly to acetate and butyrate; the minor products included ethanol. propionate, lactate, and hydrogen. The utilization rate of lactose, as a function of lactose concentration in the fermentor, can be described by a Michaelis-Menten equation, as can the formation rates of acetate, butyrate, and ethanol. The production rates of propionate and lactate had a liner relationship with lactose concentration under the experimental conditions. The low pH (4.6) of culture medium could depress the formation of propionate, and intermediate which is most difficulty digested by acetogenic bacteria located in the second fermentor in a two-phase process. Production rate of acetate quickly reached a constant, and additional utilization of lactose produced more butyrate and other minor products. (c) 1993 John Wiley & Sons, Inc.     

Effects of hydrogen and acetate on benzene mineralisation under sulphate-reducing conditions

Syntrophic mineralisation of benzene, as recently proposed for a sulphate-reducing enrichment culture, was tested in product inhibition experiments with acetate and hydrogen, both putative intermediates of anaerobic benzene fermentation. Using [(13)C(6)]-benzene enabled tracking the inhibition of benzene mineralisation sensitively by analysis of (13)CO(2). In noninhibited cultures, hydrogen was detected at partial pressures of 2.4 × 10(-6) ± 1.5 × 10(-6) atm. Acetate was detected at concentrations of 17 ± 2 μM. Spiking with 0.1 atm hydrogen produced a transient inhibitory effect on (13)CO(2) formation. In cultures spiked with higher amounts of hydrogen, benzene mineralisation did not restart after hydrogen consumption, possibly due to the toxic effects of the sulphide produced. An inhibitory effect was also observed when acetate was added to the cultures (0.3, 3.5 and 30 mM). Benzene mineralisation resumed after acetate was degraded to concentrations found in noninhibited cultures, indicating that acetate is another key intermediate in anaerobic benzene mineralisation. Although benzene mineralisation by a single sulphate reducer cannot be ruled out, our results strongly point to an involvement of syntrophic interactions in the process. Thermodynamic calculations revealed that, under in situ conditions, benzene fermentation to hydrogen and acetate yielded a free energy change of ΔG'=-83.1 ± 5.6 kJ mol(-1). Benzene mineralisation ceased when ΔG' values declined below -61.3 ± 5.3 kJ mol(-1) in the presence of acetate, indicating that ATP-consuming reactions are involved in the pathway.     

Microcalorimetric studies of the growth of sulfate-reducing bacteria: energetics of Desulfovibrio vulgaris growth.

The metabolism of Desulfovibrio vulgaris Hildenborough grown on medium containing lactate or pyruvate plus a high concentration of sulfate (36 mM) was studied. Molecular growth yields were 6.7 +/- 1.3 and 10.1 +/- 1.7 g/mol for lactate and pyruvate, respectively. Under conditions in which the energy source was the sole growth-limiting factor, we observed the formation of 0.5 mol of hydrogen per mol of lactate and 0.1 mol of hydrogen per mol of pyruvate. The determination of metabolic end products revealed that D. vulgaris produced, in addition to normal end products (acetic acid, carbon dioxide, hydrogen sulfide) and molecular hydrogen, 2 and 5% of ethanol per mol of lactate and pyruvate, respectively. Power-time curves of growth of D. vulgaris on lactate and pyruvate were obtained, by the microcalorimetric Tian-Calvet apparatus. The enthalpies (delta Hmet) associated with the oxidation of these substrates and calculated from growth thermograms were -36.36 +/- 5 and -70.22 +/- 3 kJ/mol of lactate and pyruvate, respectively. These experimental values were in agreement with the homologous values assessed from the theoretical equations of D. vulgaris metabolism of both lactate and pyruvate. The hydrogen production by this sulfate reducer constitutes an efficient regulatory system of electrons, from energy source through the pathway of sulfate reduction. This hydrogen value may thus facilitate interactions between this strain and other environmental microflora, especially metagenic bacteria.     

Reductive unfolding of serum albumins uncovered by Raman spectroscopy

The reductive unfolding of bovine serum albumin (BSA) and human serum albumin (HSA) induced by dithiothreitol (DTT) is investigated using Raman spectroscopy. The resolution of the S-S Raman band into both protein and oxidized DTT contributions provides a reliable basis for directly monitoring the S-S bridge exchange reaction. The related changes in the protein secondary structure are identified by analyzing the protein amide I Raman band. For the reduction of one S-S bridge of BSA, a mean Gibbs free energy of -7 kJ mol(-1) is derived by studying the reaction equilibrium. The corresponding value for the HSA S-S bridge reduction is -2 kJ mol(-1). The reaction kinetics observed via the S-S or amide I Raman bands are identical giving a reaction rate constant of (1.02 +/- 0.11) M(-1) s(-1) for BSA. The contribution of the conformational Gibbs free energy to the overall Gibbs free energy of reaction is further estimated by combining experimental data with ab initio calculations.