Conditions In Chemostat Cultures Research Articles

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

ABSTRACT Most of the current industrial processes for L-leucine production are based on fermentation, usually in fed-batch operation mode. Although the culture technology has advanced in recent decades, the process still has significant drawbacks. To solve these problems, we investigated the effects of chemostat culture conditions on the production of L-leucine by Corynebacterium glutamicum CP. The dilution rate, the nitrogen source, and the carbon–nitrogen ratio of the medium were optimized. With the addition of ammonium acetate to the chemostat medium, the initial C/N ratio was adjusted to 57.6, and the L-leucine titer reached the highest level at the optimal dilution rate of 0.04 h−1. Compared with fed-batch culture, the L-leucine titer was reduced (from 53.0 to 24.8 g L−1), but the yield from glucose was increased by 10.0% (from 0.30 to 0.33 mol mol−1) and productivity was increased by 58.3% (from 1.2 to 1.9 g L−1 h−1). Moreover, the titer of the by-product L-alanine was significantly reduced (from 8.9 to 0.8 g L−1). In addition, gene expression levels and activity of key enzymes in the synthesis of L-leucine and L-alanine were analyzed to explain the difference of production performance between chemostat culture and fed-batch culture. The results indicate that chemostat culture has great potential to increase the industrial production of L-leucine compared to current fed-batch approaches.

Resolving the contributions of the membrane-bound and periplasmic nitrate reductase systems to nitric oxide and nitrous oxide production in Salmonella enterica serovar Typhimurium

The production of cytotoxic nitric oxide (NO) and conversion into the neuropharmacological agent and potent greenhouse gas nitrous oxide (N₂O) is linked with anoxic nitrate catabolism by Salmonella enterica serovar Typhimurium. Salmonella can synthesize two types of nitrate reductase: a membrane-bound form (Nar) and a periplasmic form (Nap). Nitrate catabolism was studied under nitrate-rich and nitrate-limited conditions in chemostat cultures following transition from oxic to anoxic conditions. Intracellular NO production was reported qualitatively by assessing transcription of the NO-regulated genes encoding flavohaemoglobin (Hmp), flavorubredoxin (NorV) and hybrid cluster protein (Hcp). A more quantitative analysis of the extent of NO formation was gained by measuring production of N₂O, the end-product of anoxic NO-detoxification. Under nitrate-rich conditions, the nar, nap, hmp, norV and hcp genes were all induced following transition from the oxic to anoxic state, and 20% of nitrate consumed in steady-state was released as N₂O when nitrite had accumulated to millimolar levels. The kinetics of nitrate consumption, nitrite accumulation and N₂O production were similar to those of wild-type in nitrate-sufficient cultures of a nap mutant. In contrast, in a narG mutant, the steady-state rate of N₂O production was ~30-fold lower than that of the wild-type. Under nitrate-limited conditions, nap, but not nar, was up-regulated following transition from oxic to anoxic metabolism and very little N₂O production was observed. Thus a combination of nitrate-sufficiency, nitrite accumulation and an active Nar-type nitrate reductase leads to NO and thence N₂O production, and this can account for up to 20% of the nitrate catabolized.

Nitrous oxide production in soil isolates of nitrate‐ammonifying bacteria

Here we provide the first demonstration of the potential for N2 O production by soil-isolated nitrate-ammonifying bacteria under different C and N availabilities, building on characterizations informed from model strains. The potential for soil-isolated Bacillus sp. and Citrobacter sp. to reduce NO3 (-) , and produce NH4 (+) , NO2 (-) and N2 O was examined in batch and continuous (chemostat) cultures under different C-to-NO3 (-) ratios, NO3 (-) -limiting (5 mM) and NO3 (-) -sufficient (22 mM) conditions. C-to-NO3 (-) ratio had a major influence on the products of nitrate ammonification, with NO2 (-) , rather than NH4 (+) , being the major product at low C-to-NO3 (-) ratios in batch cultures. N2 O production was maximum and accompanied by high NO2 (-) production under C-limitation/NO3 -sufficiency conditions in chemostat cultures. In media with lower C-to-NO3 -N ratios (5- and 10-to-1) up to 2.7% or 5.0% of NO3 (-) was reduced to N2 O by Bacillus sp. and Citrobacter sp., respectively, but these reduction efficiencies were only 0.1% or 0.7% at higher C-to-NO3 (-) ratios (25- and 50-to-1). As the highest N2 O production did not occur under the same C-to-NO3 (-) conditions as highest NH4 (+) production we suggest that a re-evaluation may be necessary of the environmental conditions under which nitrate ammonification contributes to N2 O emission from soil.

Severe Zinc Depletion of Escherichia coli: ROLES FOR HIGH AFFINITY ZINC BINDING BY ZinT, ZINC TRANSPORT AND ZINC-INDEPENDENT PROTEINS

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The Effect of Temperature on the Proteome of Recombinant Pichia pastoris

The impact of environmental factors on the productivity of yeast cells is poorly investigated so far. Therefore, it is a major concern to improve the understanding of cellular physiology of microbial protein production hosts, including the methylotrophic yeast Pichia pastoris. Two-Dimensional Fluorescence Difference Gel electrophoresis and protein identification via mass spectrometry were applied to analyze the impact of cultivation temperature on the physiology of a heterologous protein secreting P. pastoris strain. Furthermore, specific productivity was monitored and fluxes through the central carbon metabolism were calculated. Chemostat culture conditions were applied to assess the adaption to different growth temperatures (20, 25, 30 degrees C) at steady-state conditions. Many important cellular processes, including the central carbon metabolism, stress response and protein folding are affected by changing the growth temperature. A 3-fold increased specific productivity at lower cultivation temperature for an antibody Fab fragment was accompanied by a reduced flux through the TCA-cycle, reduced levels of proteins involved in oxidative stress response and lower cellular levels of molecular chaperones. These data indicate that folding stress is generally decreased at lower cultivation temperatures, enabling more efficient heterologous protein secretion in P. pastoris host cells.

The influence of reduced oxygen availability on pathogenicity and gene expression in Mycobacterium tuberculosis

We investigated how Mycobacterium tuberculosis responded to a reduced oxygen tension in terms of its pathogenicity and gene expression by growing cells under either aerobic or low-oxygen conditions in chemostat culture. The chemostat enabled us to control and vary the oxygen tension independently of other environmental parameters, so that true cause-and-effect relationships of reduced oxygen availability could be established. Cells grown under low oxygen were more pathogenic for guinea pigs than those grown aerobically. The effect of reduced oxygen on global gene expression was determined using DNA microarray. Spearman rank correlation confirmed that microarray expression profiles were highly reproducible between repeat cultures. Using microarray analysis we have identified genes that respond to a low-oxygen environment without the influence of other parameters such as nutrient depletion. Some of these genes appear to be involved in the biosynthesis of cell wall precursors and their induction may have contributed to increased infectivity in the guinea pig. This study has shown that a combination of chemostat culture and microarray presents a biologically robust and statistically reliable experimental approach for studying the effect of relevant and specific environmental stimuli on mycobacterial virulence and gene expression.

Tailored Synthesis of Poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) inRalstonia eutrophaDSM 428

AbstractRalstonia eutrophawas grown in a chemostat at a dilution rate ofD= 0.1 ± 0.01 h−1under the conditions of simultaneous limitation by carbon (butyric and/or valeric acid) and nitrogen (ammonium). In order to achieve a physiological state with a high cellular polyhydroxyalkanoate (PHA) content, the ratio of total carbon to nitrogen of the feed medium was kept constant at 17 mol C/molN and only the ratio of butyric to valeric acid was varied in the carbon feed. When only butyric acid was fed, poly([R]‐3‐hydroxybutyrate) (PHB) was isolated. For substrate mixtures consisting of butyric and valeric acid, and valeric acid alone, theco‐polymer poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) was incorporated with a maximum content of (R)‐3‐hydroxyvalerate of 62 mol%. The melting temperature of PHA decreased from 178 °C for PHB to about 80 °C for PHA with the highest HV content. The molecular weight (Mw) of the polymer was between 0.9 and 1.2 × 106with a polydispersity of 3 ± 0.3. The polymer composition in the cells was a non‐linear function of the substrate mixture. It was concluded that dual (C, N) limited growth conditions in chemostat cultures could be used to tailor the composition of PHB/HV in an accurate way, which is not possible when using N limited batch cultures.

The Genome-wide Transcriptional Responses of Saccharomyces cerevisiae Grown on Glucose in Aerobic Chemostat Cultures Limited for Carbon, Nitrogen, Phosphorus, or Sulfur

Profiles of genome-wide transcriptional events for a given environmental condition can be of importance in the diagnosis of poorly defined environments. To identify clusters of genes constituting such diagnostic profiles, we characterized the specific transcriptional responses of Saccharomyces cerevisiae to growth limitation by carbon, nitrogen, phosphorus, or sulfur. Microarray experiments were performed using cells growing in steady-state conditions in chemostat cultures at the same dilution rate. This enabled us to study the effects of one particular limitation while other growth parameters (pH, temperature, dissolved oxygen tension) remained constant. Furthermore, the composition of the media fed to the cultures was altered so that the concentrations of excess nutrients were comparable between experimental conditions. In total, 1881 transcripts (31% of the annotated genome) were significantly changed between at least two growth conditions. Of those, 484 were significantly higher or lower in one limitation only. The functional annotations of these genes indicated cellular metabolism was altered to meet the growth requirements for nutrient-limited growth. Furthermore, we identified responses for several active transcription factors with a role in nutrient assimilation. Finally, 51 genes were identified that showed 10-fold higher or lower expression in a single condition only. The transcription of these genes can be used as indicators for the characterization of nutrient-limited growth conditions and provide information for metabolic engineering strategies.

Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the methylotrophic yeast Candida boidinii.

The methylotrophic yeast Candida boidinii exhibits formaldehyde dehydrogenase activity (FLD, EC 1.2.1.1) during growth on methanol as a sole carbon source. The structural gene, FLD1, was cloned from a genomic library of C. boidinii. The 1263 bp FLD1 gene contained a 123 bp intron and its exon encoded a gene product of 380 amino acids, whose predicted amino acid sequence showed high similarity to the sequences of FLDs from other organisms. The FLD1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fld1Delta strain could not grow on methanol as a carbon source under methanol-limited chemostat culture conditions, even with low dilution rates (D<0.05 x h(-1)), whereas a strain with a disruption in the gene for formate dehydrogenase (FDH; another NADH-generating dehydrogenase involved in the formaldehyde oxidation pathway) could survive. These results indicated that FLD, but not FDH, is essential for growth of C. boidinii on methanol.

Cell competence and membrane protein production in perturbed cultures of B. subtilis

The work presents a comparison of three different cultivation methods used to stimulate competence in Bacillus subtilis cells. The level of cell competence achieved under forced perturbations in cyclic fed-batch culture, was compared to competence achieved under steady state conditions in chemostat culture and under transient conditions in batch culture. The percentage of competent cells found using cyclic fed-batch culture was highest with a maximum value of 4.9% transformed cells obtained. Transient conditions achieved in batch culture resulted in a maximum value of 3.9% transformed cells. In chemostat culture, at steady state, a maximum value of 2.9% transformed cells was reached. Cyclic fed-batch culture was shown to induce a greater number membrane proteins in comparison to the membrane proteins produced under both batch and chemostat culture conditions.

Growth kinetics of suspended microbial cells: from single-substrate-controlled growth to mixed-substrate kinetics.

Growth kinetics, i.e., the relationship between specific growth rate and the concentration of a substrate, is one of the basic tools in microbiology. However, despite more than half a century of research, many fundamental questions about the validity and application of growth kinetics as observed in the laboratory to environmental growth conditions are still unanswered. For pure cultures growing with single substrates, enormous inconsistencies exist in the growth kinetic data reported. The low quality of experimental data has so far hampered the comparison and validation of the different growth models proposed, and only recently have data collected from nutrient-controlled chemostat cultures allowed us to compare different kinetic models on a statistical basis. The problems are mainly due to (i) the analytical difficulty in measuring substrates at growth-controlling concentrations and (ii) the fact that during a kinetic experiment, particularly in batch systems, microorganisms alter their kinetic properties because of adaptation to the changing environment. For example, for Escherichia coli growing with glucose, a physiological long-term adaptation results in a change in KS for glucose from some 5 mg liter-1 to ca. 30 microg liter-1. The data suggest that a dilemma exists, namely, that either "intrinsic" KS (under substrate-controlled conditions in chemostat culture) or micromax (under substrate-excess conditions in batch culture) can be measured but both cannot be determined at the same time. The above-described conventional growth kinetics derived from single-substrate-controlled laboratory experiments have invariably been used for describing both growth and substrate utilization in ecosystems. However, in nature, microbial cells are exposed to a wide spectrum of potential substrates, many of which they utilize simultaneously (in particular carbon sources). The kinetic data available to date for growth of pure cultures in carbon-controlled continuous culture with defined mixtures of two or more carbon sources (including pollutants) clearly demonstrate that simultaneous utilization results in lowered residual steady-state concentrations of all substrates. This should result in a competitive advantage of a cell capable of mixed-substrate growth because it can grow much faster at low substrate concentrations than one would expect from single-substrate kinetics. Additionally, the relevance of the kinetic principles obtained from defined culture systems with single, mixed, or multicomponent substrates to the kinetics of pollutant degradation as it occurs in the presence of alternative carbon sources in complex environmental systems is discussed. The presented overview indicates that many of the environmentally relevant apects in growth kinetics are still waiting to be discovered, established, and exploited.

Use of the inlet gas composition to control the respiratory quotient in microaerobic bioprocesses

Different aspects of the estimation and control of the respiratory quotient (RQ) have been investigated for the purpose of providing well characterized microaerobic, or oxygen limited, conditions in chemostat cultures. The RQ was controlled by changing the oxygen concentration in the inlet gas. The standard, steady-state equation for calculating the RQ in this case could not be used for dynamic RQ control. However, the dynamic characteristics of the equation could be considerably improved, by applying a first-order exponential filter to the inlet gas composition. Substituting the momentaneous inlet oxygen concentration for the filtered concentration in the standard gas balance equations resulted in favourable dynamic characteristics and high steady-state accuracy. Computer simulations showed that the sensitivity of the RQ to noise and bias in the oxygen analyses was acceptable for precision values typically achieved with mass spectrometry. A PID controller, and an adaptive pole placement controller, have been tried for the control of the RQ, calculated by the filtering procedure, during microaerobic ethanol production with Saccharomyces cerevisiae CBS 8066. The PID controller could not be used with constant control parameters, because the sensitivity of the RQ to the inlet oxygen concentration differed between different RQ setpoints. When the controller gain was adjusted for each RQ setpoint, the PID controller worked very well. The adaptive pole placement controller also worked well at all interesting operating conditions. In situ NAD(P)H fluorescence measurements indicated that the intracellular redox state is correlated to the RQ for RQ values in the range 10–30.

A Growth Yield Model for Substrate-Sufficient Continuous Culture of Microorganisms

It is generally observed that the growth yields are greater under substrate-limited conditions than under substrate-sufficient conditions in chemostat cultures. However, the Pirt maintenance theory could not correctly explain the effect of excessive substrate on the growth yield. The excess substrate should cause serious uncoupling between anabolism and catabolism leading to energy spilling. Based on a balance on substrate reaction, the total quantity of substrate consumed by microorganisms is taken to be the sums of the quantity of substrate utilization for growth, the quantity of substrate utilization for maintenance and that due to energy spilling. A kinetic model is proposed to describe the dependence of the observed growth yield on residual substrate concentration for substrate-sufficient culture. It is demonstrated that the observed growth yield decreases with increasing the residual substrate concentration. This model describes the literature data satisfactorily.

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.

Dihydrofolate reductase synthesis in continuous culture using a methotrexate-resistant Escherichia coli

A methotrexate-resistant strain of Escherichia coli Type I produced exceptionally high levels of the enzyme dihydrofolate reductase (EC 1.5.1.3) in 6 h of batch fermentation. The culture had markedly improved performance under chemostat culture conditions in terms of enzyme yield and output. Temperature, pH, dilution rates, and nutrient composition were optimized under chemostat culture conditions. A maximum enzyme yield of 10,360 U l −1 and a specific activity of 22.84 U mg −1 were obtained under chemostat conditions at pH 7.0, temperature 37°C, and dilution rate 0.2 h −1, using media containing 1.0 and 0.6% (w/v) dextrose and yeast extract, respectively. The culture's performance and enzyme yields in chemostat and the feasibility of large-scale production are discussed.

Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures ofEnterococcus faecalisNCTC 775

Enterococcus faecalis was grown under anaerobic conditions in chemostat cultures on energy sources with different degress of reduction (i.e. mannitol, glucose, pyruvate) at various culture pH values. Intracellular NADH/NAD ratios were measured and were found to be influenced both by the nature of the energy source and by the culture pH value. Highest ratios were found with mannitol as energy source and with high culture pH values. A role for the redox potential of the NADH/NAD couple as a regulatory effector is suggested by a correlation of the redox potential with the in vivo distribution of the carbon flux between pyruvate formate lyase and the pyruvate dehydrogenase complex.

Effect of the energy source on the NADH/NAD ratio and on pyruvate catabolism in anaerobic chemostat cultures of Enterococcus faecalis NCTC 775

Enterococcus faecalis was grown under anaerobic conditions in chemostat cultures on energy sources with different degress of reduction (i.e. mannitol, glucose, pyruvate) at various culture pH values. Intracellular NADH/NAD ratios were measured and were found to be influenced both by the nature of the energy source and by the culture pH value. Highest ratios were found with mannitol as energy source and with high culture pH values. A role for the redox potential of the NADH/NAD couple as a regulatory effector is suggested by a correlation of the redox potential with the in vivo distribution of the carbon flux between pyruvate formate lyase and the pyruvate dehydrogenase complex.