Influence of CO2-HCO3− Levels and pH on Growth, Succinate Production, and Enzyme Activities of Anaerobiospirillum succiniciproducens

Growth and succinate versus lactate production from glucose by Anaerobiospirillum succiniciproducens was regulated by the level of available carbon dioxide and culture pH. At pH 7.2, the generation time was almost doubled and extensive amounts of lactate were formed in comparison with growth at pH 6.2. The succinate yield and the yield of ATP per mole of glucose were significantly enhanced under excess-CO₂-HCO₃⁻ growth conditions and suggest that there exists a threshold level of CO₂ for enhanced succinate production in A. succiniciproducens. Glucose was metabolized via the Embden-Meyerhof-Parnas route, and phosphoenolpyruvate carboxykinase levels increased while lactate dehydrogenase and alcohol dehydrogenase levels decreased under excess-CO₂-HCO₃⁻ growth conditions. Kinetic analysis of succinate and lactate formation in continuous culture indicated that the growth rate-linked production rate coefficient (K) cells was much higher for succinate (7.2 versus 1.0 g/g of cells per h) while the non-growth-rate-related formation rate coefficient (K′) was higher for lactate (1.1 versus 0.3 g/g of cells per h). The data indicate that A. succiniciproducens, unlike other succinate-producing anaerobes which also form propionate, can grow rapidly and form high final yields of succinate at pH 6.2 and with excess CO₂-HCO₃⁻ as a consequence of regulating electron sink metabolism.     

Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic

pH affected significantly the growth and the glucose fermentation pattern of Propionibacterium microaerophilum. In neutral conditions (pH 6.5-7.5), growth and glucose fermentation rate (qs) were optimum producing propionate, acetate, CO(2), and formate [which together represented 90% (wt/wt) of the end products], and lactate representing only 10% (wt/wt) of the end products. In acidic conditions, propionate, acetate, and CO(2) represented nearly 100% (wt/wt) of the fermentation end products, whereas in alkaline conditions, a shift of glucose catabolism toward formate and lactate was observed, lactate representing 50% (wt/wt) of the fermentation end products. The energy cellular yields ( Y(X/ATP)), calculated (i) by taking into account extra ATP synthesized through the reduction of fumarate into succinate, was 6.1-7.2 g mol(-1). When this extra ATP was omitted, it was 11.9-13.1 g mol(-1). The comparison of these values with those of Y(X/ATP) in P. acidipropionici and other anaerobic bacteria suggested that P. microaerophilum could not synthesize ATP through the reduction of fumarate into succinate and therefore differed metabolically from P. acidipropionici.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production

Succinic acid is a four-carbon dicarboxylic acid produced as one of the fermentation products of anaerobic metabolism. Based on the complete genome sequence of a capnophilic succinic acid-producing rumen bacterium, Mannheimia succiniciproducens, gene knockout studies were carried out to understand its anaerobic fermentative metabolism and consequently to develop a metabolically engineered strain capable of producing succinic acid without by-product formation. Among three different CO2-fixing metabolic reactions catalyzed by phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, and malic enzyme, PEP carboxykinase was the most important for the anaerobic growth of M. succiniciproducens and succinic acid production. Oxaloacetate formed by carboxylation of PEP was found to be converted to succinic acid by three sequential reactions catalyzed by malate dehydrogenase, fumarase, and fumarate reductase. Major metabolic pathways leading to by-product formation were successfully removed by disrupting the ldhA, pflB, pta, and ackA genes. This metabolically engineered LPK7 strain was able to produce 13.4 g/liter of succinic acid from 20 g/liter glucose with little or no formation of acetic, formic, and lactic acids, resulting in a succinic acid yield of 0.97 mol succinic acid per mol glucose. Fed-batch culture of M. succiniciproducens LPK7 with intermittent glucose feeding allowed the production of 52.4 g/liter of succinic acid, with a succinic acid yield of 1.16 mol succinic acid per mol glucose and a succinic acid productivity of 1.8 g/liter/h, which should be useful for industrial production of succinic acid.     

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.     

Changes in metabolism of the rumen bacterium Streptococcus bovis H13/1 resulting from alteration in dilution rate and glucose supply per unit time

Streptococcus bovis H13/1 was grown in a glucose-limited chemostat. A concomitant increase in dilution rate and glucose supply per unit time caused both an increase in lactate production per mole of glucose fermented and a linear increase in growth yield over the dilution rate range 0.052 to 0.141/h. When the dilution rate was increased with no change in glucose supply per unit time there was a reduction in lactate production and an increase in that of acetate and ethanol coinciding with a non-linear increase in growth yield. YMaxglu = 38.6 and a maintenance coefficient, ms = 0.290 mmol/l glucose/g cells/h were calculated. The results also suggested an interaction between the formate and CO2 pools.     

Effect of methotrexate (MTX) on NAD(P)+ dehydrogenases of HeLa cells: malic enzyme, 2-oxoglutarate and isocitrate dehydrogenases

The effects of methotrexate (MTX) on oxygen uptake by permeabilized HeLa cells were evaluated. MTX did not inhibit state III respiration when the oxidizable substrate was succinate, but when the substrates were 2-oxoglutarate or isocitrate the respiration decreased about 50 per cent at 1·0 mM concentration of the drug. This effect was explained by inhibition of 2-oxoglutarate and isocitrate dehydrogenases by MTX. No effect was observed on succinate dehydrogenase. An evaluation of the effects of MTX on malic enzyme activity as measured by pyruvate plus lactate production in intact cells supplied with malate showed a decrease of about 40 per cent in metabolite production using 0·4 mM MTX. HeLa cell malic enzyme, as observed for other tumour cells, is compartmentalized in mitochondria and cytosol, and is another example of a dehydrogenase inhibited by MTX. © 1997 John Wiley & Sons, Ltd.     

Toward homosuccinate fermentation: metabolic engineering of Corynebacterium glutamicum for anaerobic production of succinate from glucose and formate

Previous studies have demonstrated the capability of Corynebacterium glutamicum for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δcat Δpqo Δpta-ackA ΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase gene pyc(P458S) into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the fdh gene from Mycobacterium vaccae, coding for an NAD(+)-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene gapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gap accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength.

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dissolved CO2 levels on the growth of Mannheimia succiniciproducens and succinic acid production

A capnophilic rumen bacterium Mannheimia succiniciproducens produces succinic acid as a major fermentation end product under CO(2)-rich anaerobic condition. Since succinic acid is produced by carboxylation of C3 compounds during the fermentation, intracellular CO(2) availability is important for efficient succinic acid formation. Here, we investigated the metabolic responses of M. succiniciproducens to the different dissolved CO(2) concentrations (0-260 mM). Cell growth was severely suppressed when the dissolved CO(2) concentration was below 8.74 mM. On the other hand, cell growth and succinic acid production increased proportionally as the dissolved CO(2) concentration increased from 8.74 to 141 mM. The yields of biomass and succinic acid on glucose obtained at the dissolved CO(2) concentration of 141 mM were 1.49 and 1.52 times higher, respectively, than those obtained at the dissolved CO(2) concentration of 8.74 mM. It was also found that the additional CO(2) source provided in the form of NaHCO(3), MgCO(3), or CaCO(3) had positive effects on cell growth and succinic acid production. However, growth inhibition was observed when excessive bicarbonate salts were added. By the comparison of the activities of key enzymes, it was found that PEP carboxylation by PEP carboxykinase (PckA) is the most important for succinic acid production as well as the growth of M. succiniciproducens by providing additional ATP.     

Regulation of carbon and electron flow in Propionispira arboris: relationship of catabolic enzyme levels to carbon substrates fermented during propionate formation via the methylmalonyl coenzyme A pathway.

A detailed study of the glucose fermentation pathway and the modulation of catabolic oxidoreductase activities by energy sources (i.e., glucose versus lactate or fumarate) in Propionispira arboris was performed. 14C radiotracer data show the CO2 produced from pyruvate oxidation comes exclusively from the C-3 and C-4 positions of glucose. Significant specific activities of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were detected, which substantiates the utilization of the Embden-Meyerhoff-Parnas path for glucose metabolism. The methylmalonyl coenzyme A pathway for pyruvate reduction to propionate was established by detection of significant activities (greater than 16 nmol/min per mg of protein) of methylmalonyl coenzyme A transcarboxylase, malate dehydrogenase, and fumarate reductase in cell-free extracts and by 13C nuclear magnetic resonance spectroscopic demonstration of randomization of label from [2-13C]pyruvate into positions 2 and 3 of propionate. The specific activity of pyruvate-ferredoxin oxidoreductase, malate dehydrogenase, fumarate reductase, and transcarboxylase varied significantly in cells grown on different energy sources. D-Lactate dehydrogenase (non-NADH linked) was present in cells of P. arboris grown on lactate but not in cells grown on glucose or fumarate. These results indicate that growth substrates regulate synthesis of enzymes specific for the methylmalonyl coenzyme A path and initial substrate transformation.     

pH and base counterion affect succinate production in dual-phase <Emphasis Type="Italic">Escherichia coli</Emphasis> fermentations

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO₂. A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using ¹³C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)₂ was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)₂, the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Glucose catabolism by Spirochaeta thermophila RI 19.B1.

Spirochaeta thermophila RI 19.B1 (DSM 6192) fermented glucose to lactate, acetate, CO2, and H2 with concomitant formation of cell material. The cell dry mass yield was 20.0 g/mol of glucose. From the fermentation balance data and knowledge of the fermentation pathway, a YATP of 9.22 g of dry mass per mol of ATP was calculated for pH-uncontrolled batch-culture growth on glucose in a mineral medium. Measurement of enzyme activities in glucose-grown cells revealed that glucose was taken up by a permease and then subjected to ATP-dependent phosphorylation by a hexokinase. Glucose-6-phosphate was further metabolized to pyruvate through the Embden-Meyerhof-Parnas pathway. The phosphoryl donor for phosphofructokinase activity was PPi rather than ATP. This was also found for the type strain of S. thermophila, Z-1203 (DSM 6578). PPi was probably formed by pyrophosphoroclastic cleavage of ATP, with recovery of the resultant AMP by the activity of adenylate kinase. All other measured kinase activities utilized ATP as the phosphoryl donor. Pyruvate was further metabolized to acetyl coenzyme A with concomitant production of H2 and CO2 by pyruvate synthase. Lactate was also produced from pyruvate by a fructose-1,6-diphosphate-insensitive lactate dehydrogenase. Evidence was obtained for the transfer of reducing equivalents from the glycolytic pathway to hydrogenase to produce H2. No formate dehydrogenase or significant ethanol-producing enzyme activities were detected.     

Evidence for cytochrome involvement in fumarate reduction and adenosine 5'-triphosphate synthesis by Bacteroides fragilis grown in the presence of hemin.

Growth of Bacteroides fragilis subsp. fragilis on glucose was very much stimulated by the addition of hemin (2 mg/liter) to the medium. The generation time decreased from 8 to 2 h, and the molar growth yield increased from YM = 17.9 to YM = 47 g (dry weight) of cells per mol of glucose. In the absence of hemin, glucose was fermented to fumarate, lactate, and acetate. The cells did not contain detectable amounts of cytochromes or fumarate reductase. In the presence of hemin, the major products of fermentation were succinate, propionate, and acetate. A b-type cytochrome, possibly a c-type cytochrome, and a very active fumarate reductase were present in the cells. It is concluded from these results that hemin is required by B. fragilis to synthesize a functional fumarate reductase and that the hemin-dependent, enormous increase of the growth yield may be due to adenosine 5'-triphosphate production during reduction of fumarate to succinate.     

A new process for the continuous production of succinic acid from glucose at high yield, titer, and productivity

A novel three stages continuous fermentation process for the bioproduction of succinic acid at high concentration, productivity and yield using A. succiniciproducens was developed. This process combined an integrated membrane-bioreactor-electrodialysis system. An energetic characterization of A. succiniciproducens during anaerobic cultured in a cell recycle bioreactor was done first. The very low value of Y(ATP) obtained suggests that an ATP dependent mechanism of succinate export is present in A. succiniciproducens. Under the best culture conditions, biomass concentration and succinate volumetric productivity reach values of 42 g/L and 14.8 g/L.h. These values are respectively 28 and 20 times higher compared to batch cultures done in our laboratory. To limit end-products inhibition on growth, a mono-polar electrodialysis pilot was secondly coupled to the cell recycle bioreactor. This system allowed to continuously remove succinate and acetate from the permeate and recycle an organic acids depleted solution in the reactor. The integrated membrane-bioreactor-electrodialysis process produced a five times concentrated succinate solution (83 g/L) compared to the cell recycle reactor system, at a high average succinate yield of 1.35 mol/mol and a slightly lower volumetric productivity of 10.4 g/L.h. The process combined maximal production yield to high productivity and titer and could be economically viable for the development of a biological route for succinic acid production.     

Homolactate fermentation by metabolically engineered Escherichia coli strains

We report the homofermentative production of lactate in Escherichia coli strains containing mutations in the aceEF, pfl, poxB, and pps genes, which encode the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, and phosphoenolpyruvate synthase, respectively. The process uses a defined medium and two distinct fermentation phases: aerobic growth to an optical density of about 30, followed by nongrowth, anaerobic production. Strain YYC202 (aceEF pfl poxB pps) generated 90 g/liter lactate in 16 h during the anaerobic phase (with a yield of 0.95 g/g and a productivity of 5.6 g/liter . h). Ca(OH)(2) was found to be superior to NaOH for pH control, and interestingly, significant succinate also accumulated (over 7 g/liter) despite the use of N(2) for maintaining anaerobic conditions. Strain ALS961 (YYC202 ppc) prevented succinate accumulation, but growth was very poor. Strain ALS974 (YYC202 frdABCD) reduced succinate formation by 70% to less than 3 g/liter. (13)C nuclear magnetic resonance analysis using uniformly labeled acetate demonstrated that succinate formation by ALS974 was biochemically derived from acetate in the medium. The absence of uniformly labeled succinate, however, demonstrated that glyoxylate did not reenter the tricarboxylic acid cycle via oxaloacetate. By minimizing the residual acetate at the time that the production phase commenced, the process with ALS974 achieved 138 g/liter lactate (1.55 M, 97% of the carbon products), with a yield of 0.99 g/g and a productivity of 6.3 g/liter . h during the anaerobic phase.     

Fermenter growth of Streptococcus agalactiae and large-scale production of CAMP factor

Streptococcus agalactiae (group B) was grown in Todd-Hewitt broth (36.4 g l-1, pH 7.8) in a Braun Fermenter (type B20) to investigate the conditions of optimal bacterial growth and maximal production of CAMP factor. The influence of different gas atmospheres (air, N2, CO2, and gas mixtures) on growth, CAMP production and chain length of S. agalactiae was studied. The organisms grew best in the presence of 2% (w/v) glucose, at pH 6.2, with a constant flow of CO2. The number of diplococci and monococci under these conditions reached almost 80% of the total population.     

Glucose and carbon dioxide metabolism by Succinivibrio dextrinosolvens.

Growth rates and culture conditions affect the molar yields of catabolic end products and cells of Succinivibrio dextrinosolvens growing on glucose. When growth in chemostats occurred, a trend toward decreased succinate and acetate formation, increased lactate formation, and a higher yield of cells correlated with an increase in the growth rate. End product and cellular yields on defined medium indicate a high maintenance requirement for S. dextrinosolvens and are consistent with energy conservation steps during the formation of acetate and succinate. Simultaneous carbon dioxide consumption and production were determined from batch studies with NaH14CO3, and the amounts were used to calculate a fermentation balance. These data also indicated that CO2 consumption lags behind CO2 production early in the growth phase, becoming equivalent to it toward stationary phase. Significantly more CO2 was fixed by S. dextrinosolvens when the organism was cultured in chemostats sparged with CO2. Formate is in part derived from free CO2 in the medium, as shown by 13C nuclear magnetic resonance studies, and may be sensitive to CO2 availability. Nuclear magnetic resonance data are consistent with the carboxylation of a C3 intermediate of the Embden-Meyerhof-Parnas pathway of glycolysis to a C4 compound to eventually form succinate.