Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO₂. Rates up to 0.4 μmol min⁻¹ mg of wet cells⁻¹ were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO₂ reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min⁻¹ mg of protein⁻¹ (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO₂ to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO₂ do not possess a CO-oxidizing system.     

Fermentation of fumarate and L-malate by Clostridium formicoaceticum.

The fermentation of fumarate and L-malate by Clostridium formicoaceticum was investigated. Growing and nongrowing cells degraded fumarate by dismutation to succinate, acetate, and CO2; on the other hand, only small amounts of succinate were detected when the organism was grown on L-malate. This dicarboxylic acid was mainly converted to acetate and CO2. The fermentation balances were modified if bicarbonate or formate were present in the medium. When C. formicoaceticum was grown in the presence of both dicarboxylic acids, fumarate was consumed before L-malate. The latter was mainly converted to acetate, whereas fumarate was fermented to acetate and succinate. Molar growth yields were determined to be 6 g of dry weight per mol of fumarate and 8 g of dry weight per mol of L-malate fermented.     

Kinetics of Homoacetic Fermentation of Lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converted lactate to acetate at mesophilic temperatures (30 to 42°C) and at pHs between 6.6 and 9.6. The production of acetate was found to be growth associated. Approximately 0.96 g of acetic acid and 0.066 g of cells were formed from each gram of lactic acid consumed at 37°C. The concentration of the substrate (lactate) had little or no effect on the growth rate; however, the fermentation was inhibited by acetic acid. The bacterium grew at an optimal pH of 7.6 and an optimal temperature of 37°C. Small amounts of bicarbonate were stimulatory to bacterial growth. Bacterial growth was enhanced, however, by the use of higher concentrations of bicarbonate in the media, only because higher buffer capacities were obtained and proper medium pH could be maintained for growth. Based on its ability to convert lactate to acetate, this homoacetic bacterium may be important in the anaerobic methanogenic process when lactate is a major intermediary metabolite.     

Fermentation of Glucose, Fructose, and Xylose by Clostridium thermoaceticum: Effect of Metals on Growth Yield, Enzymes, and the Synthesis of Acetate from CO2

Clostridium thermoaceticum ferments xylose, fructose, and glucose with acetate as the only product. In fermentations with mixtures of the sugars, xylose is first fermented, then fructose, and last, glucose. Fructose inhibits the fermentation of glucose, and this inhibition appears to be due to a repression of the synthesis of an enzyme needed for glucose utilization. Addition of metals to the culture medium increases the cell yield drastically from about 7 to 18 g per liter, and Y(glucose) values between 40 and 50 are obtained. According to the postulated pathways of the fermentation of glucose and synthesis of acetate from CO(2) by C. thermoaceticum, 3 mol of ATP are available as energy for growth. Thus a Y(adenosine 5'-triphosphate) of 13 to 16 is obtained. Because the normal Y(ATP) value is 10.5, this could mean that an additional source of ATP is available by an unknown mechanism. The addition of metals also increases the nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase activity, the overall reaction ((14)CO(2) --> acetate), and the incorporation of the methyl group of 5-methyltetrahydrofolate into acetate. These reactions are catalyzed very efficiently by cells harvested in early growth, whereas cells obtained at the end of a fermentation have very low formate dehydrogenase activity and capacity to incorporate CO(2) into acetate. The following enzymes involved in the synthesis of acetate from CO(2) and in the metabolism of pyruvate are present in extracts of C. thermoaceticum: 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, phosphate acetyltransferase, and acetate kinase. These enzymes are not or are very little affected by the addition of metals to the growth medium.The amount of corrinoids in cells from early growth is low, whereas it is high in cells harvested late in growth. The opposite is found for the activity of delta-aminolevulinate dehydratase, which is high at the beginning of growth and low at the end.     

Methanogenesis from lactate by a co-culture of Clostridium formicoaceticum and Methanosarcina mazei

Summary A co-culture of Clostridium formicoaceticum and Methanosarcina mazei converted lactate to methane and carbon dioxide at mesophilic temperatures and pH values near 7.0. Lactate was first converted to acetate by the homoacetogen, and then to CH₄ and CO₂ by the methanogen, with the second reaction as the rate-limiting step. The methane yield was about 1.45 mol/mol lactate. These two organisms formed a mutualistic association and may be useful together with the homolactic bacterium Stretococcus lactis to convert lactose to methane. Offprint requests to: S. T. Yang     

Effects of Acetate and Butyrate During Glycerol Fermentation by Clostridium butyricum

The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied. At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth. The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL⁻¹ had distinct effects on the metabolism and growth of Clostridium butyricum. Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production. In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production. It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production. The effects of acetate and butyrate highlight the metabolic flexibility of Cl. butyricum CNCM 1211 during glycerol fermentation. Received: 2 January 2001 / Accepted: 6 February 2001     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.     

Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or l-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2–3.5 U per mg of protein. Reduced benzyl viologen, FMNH₂ and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates.The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. These results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - O.D optical density - DTE dithioerythritol     

On a reversible molybdenum-containing aldehyde oxidoreductase from Clostridium formicoaceticum

Clostridium formicoaceticum grown in the presence of 1 mM molybdate and about 1.5×10^-5 mM tungsten (present in the 5 g yeast extract/l of the growth medium) forms two reversible aldehyde oxidoreductases in an activity ratio of about 45:55. The fraction of 45% does not bind to the octyl-Sepharose column, whereas the 55% aldehyde oxidoreductase binds to this column. From cells grown on a synthetic medium without the addition of tungstate only about 2% of the aldehyde oxidoreductase of the crude extract binds to octyl-Sepharose. The enzyme not binding to octyl-Sepharose has been purified as judged by electrophoresis. It is pure after about 50 fold enrichment. According to SDS gel electrophoresis the enzyme consists of identical 100 kD subunits. Based on gel chromatography it seems to be a trimer. Per subunit 0.6 molybdenum, 7 iron, 6.6 acid labile sulphur, about 0.1 pterin-6-carboxylic and <0.05 tungsten have been found. The first 13 amino acids from the amino end show no similarity with the W-containing aldehyde oxidoreductase from the same bacterium. With reduced tetramethylviologen (E₀=–550 mV) the new molybdenum containing enzyme can reduce various aliphatic and aromatic acids to aldehydes. The pH optimum is at 6.0. For the dehydrogenation of butyraldehyde a rather broad pH region from pH 6 to 10 shows almost no variation of rate. From 15 different aldehydes acetaldehyde exhibits the highest rate. The K_m value for butanal is 0.002 and for propionate 7.0 mM. Compared with the tungsten enzyme the molybdenum enzyme is only moderately oxygen-sensitive.Abbreviations AOR aldehyde oxidoreductase - BV benzylviologen - MV methylviologen - NH₂CO-MV 1,1-carbamoylmethylviologen - TMV 1,1,2,2-tetramethylviologen     

Presence of Cytochrome and Menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum

Cytochrome b and menaquinone have been demonstrated in the homoacetate-fermenting Clostridium formicoaceticum and Clostridium thermoaceticum.     

Kinetics and mathematical modeling of homoacetic fermentation of lactate by Clostridium formicoaceticum

Fermentation kinetics of Clostridium formicoaceticum grown on lactate at pH 7.0 and 35 degrees C was studied. Acetate was the only fermentation product and its production was growth associated. The growth of this bacterium was insensitive to the lactate concentrations studied, but was inhibited by acetic acid. A Monod-type expression with product inhibition similar to the noncompetitive inhibition of enzyme kinetics was used to model the batch fermentation. An integrated equation was developed and used to help estimating the kinetic parameters in the model. This mathematical model can be used to simulate the homoacetic fermentation of lactate by C. formicoaceticum at pH 7.0 and 35 degrees C.     

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose. Received: 14 May 1998 / Accepted: 7 August 1998     

Effects of Carbon Dioxide on Growth and Maltose Fermentation by Bacteroides amylophilus

The requirement of carbon dioxide for growth of Bacteroides amylophilus is quantitatively similar to that of certain other rumen bacteria. Carbon dioxide could be replaced by bicarbonate, but not by formate or certain amino acids. Label from ¹⁴CO₂ was incorporated into the succinate produced during maltose fermentation by B. amylophilus, and during glucose fermentation by B. ruminicola, and during cellobiose fermentation by B. succinogenes. All of the incorporated label could be associated with the carboxyl function of the molecule. The depression in radioactivity per micromole of carbon in the succinate formed from the fermentation of uniformly labeled ¹⁴C-maltose by B. amylophilus was greater than would be expected if all of the succinate formed was produced via a direct CO₂ fixation pathway(s) involving phosphoenolpyruvate or pyruvate; the radioactivity per micromole of carbon suggests that as much as 60% of the total succinate results from a pathway(s) involving direct CO₂ fixation. Maltose fermentation by B. amylophilus was dependent upon CO₂ concentration, but CO₂ concentration could not be shown to influence either the fermentation end-product ratios or the proportion of total succinate formed attributable to CO₂ fixation.     

Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum

Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.     

Purification and some properties of the tungsten-containing carboxylic acid reductase from Clostridium formicoaceticum.

Judged by properties observed during the purification and based on the sequence of the first 25 amino acids, the enzyme from Clostridium formicoaceticum catalysing the reversible reduction of non-activated carboxylic acids to aldehydes at the expense of reduced viologens, is astonishingly different from that found by us in C. thermoaceticum. According to native and SDS gel electrophoresis the reductase is nearly homogeneous after only 26-fold purification. The specificity for various substrates and artificial electron carriers is also broad, but V of the purified aldehyde dehydrogenase activity (54 U/mg enzyme for butanal) is about 1 order of magnitude lower than that of the enzyme from C. thermoaceticum. The reductase is a dimer of two identical subunits with an Mr of 67,000 each. Increased enzyme concentrations seem to lead to higher oligomers. Per dimer 11 +/- 1 iron, 16 +/- 1 acid labile sulphur, 1.4 tungsten and after permanganate oxidation 1.6 mol pterin-6-carboxylic acid have been found.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.