Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Systematic engineering of TCA cycle for optimal production of a four-carbon platform chemical 4-hydroxybutyric acid in Escherichia coli

To address climate change and environmental problems, it is becoming increasingly important to establish biorefineries for the production of chemicals from renewable non-food biomass. Here we report the development of Escherichia coli strains capable of overproducing a four-carbon platform chemical 4-hybroxybutyric acid (4-HB). Because 4-HB production is significantly affected by aeration level, genome-scale metabolic model-based engineering strategies were designed under aerobic and microaerobic conditions with emphasis on oxidative/reductive TCA branches and glyoxylate shunt. Several different metabolic engineering strategies were employed to develop strains suitable for fermentation both under aerobic and microaerobic conditions. It was found that microaerobic condition was more efficient than aerobic condition in achieving higher titer and productivity of 4-HB. The final engineered strain produced 103.4 g/L of 4-HB by microaerobic fed-batch fermentation using glycerol. The aeration-dependent optimization strategy of TCA cycle will be useful for developing microbial strains producing other reduced derivative chemicals of TCA cycle intermediates.     

Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli

Microbial cells possess numerous sensing/regulator systems in order to respond rapidly to environmental changes. Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. A group of global regulators, which include the one component Fnr protein and the two-component Arc system, coordinate the adaptive responses. To quantitate the contribution of Arc and FNR-dependent regulation under microaerobic conditions, the gene expression pattern of the electron transfer chain genes and the TCA cycle genes in wild-type E. coli, an arcA mutant, an fnr mutant, and a double arcA, fnr mutant, in glucose limited cultures and different oxygen concentrations was studied in chemostat cultures at steady state using QRT-PCR. It was found that the TCA cycle genes, icd, gltA, sucC, and sdhC are repressed by ArcA while Fnr has a minor or no effect on the expression of these genes under microaerobic conditions. The expression levels of the electron transfer chain genes, nuoA, ndh, and ubiE, were not significantly affected by either ArcA or Fnr regulation proteins, while a lower expression of cydA (up to 9-fold lower) and a higher expression of cyoA (up to 31-fold higher) were observed in cultures of the arcA mutant strain compared to those of the wild type. Since significantly higher NADH/NAD+ ratios were previously observed in cultures of the arcA mutant strain compared to the wild type it seems that the cytochrome o oxidase (the product of cyoABCDE) cannot efficiently support aerobic respiration when the cells are grown under microaerobic conditions.     

Stoichiometric analysis and experimental investigation of glycerol–glucose co-fermentation in Klebsiella pneumoniae under microaerobic conditions

The ratio between two substrates is an important parameter in microbial co-fermentation, such as 1,3-propanediol production from glycerol by Klebsiella pneumoniae using glucose as the cosubstrate. In this study, the glycerol–glucose cometabolism by K. pneumoniae is stoichiometrically analyzed according to energy (ATP), reducing equivalent (NADH₂) and product balances. The theoretical analysis reveals that the yield of 1,3-propanediol to glycerol under microaerobic conditions depends not only on the ratio of glucose to glycerol initially added, but also on the molar fraction of reducing equivalent oxidized completely by molecular oxygen in tricarboxylic acid (TCA) cycle (δ) and the molar fraction of TCA cycle in acetyl-CoA metabolism (γ). The maximum ratio of 0.32 mol glucose per mol glycerol is needed to convert glycerol completely to 1,3-propanediol under anaerobic conditions if glycerol neither enters oxidation pathways nor forms biomass. The ratio can be reduced under microaerobic conditions. The experimental results of batch cultures demonstrate that the biomass concentration and yield of 1,3-propanediol on glycerol could be enhanced by using glucose as a co-substrate. The theoretical analysis reveals the relationship between yield of 1,3-propanediol to glycerol, ratio of glucose to glycerol and respiratory quotient (RQ). These results are helpful for the experimental design and control.     

Carbon metabolism in Bradyrhizobium japonicum bacteroids

Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly.     

Oxygen Response of the Wine Yeast Saccharomyces cerevisiae EC1118 Grown under Carbon-Sufficient,Nitrogen-Limited Enological Conditions

Discrete additions of oxygen play a critical role in alcoholic fermentation. However, few studies have quantitated the fate of dissolved oxygen and its impact on wine yeast cell physiology under enological conditions. We simulated the range of dissolved oxygen concentrations that occur after a pump-over during the winemaking process by sparging nitrogen-limited continuous cultures with oxygen-nitrogen gaseous mixtures. When the dissolved oxygen concentration increased from 1.2 to 2.7 μM, yeast cells changed from a fully fermentative to a mixed respirofermentative metabolism. This transition is characterized by a switch in the operation of the tricarboxylic acid cycle (TCA) and an activation of NADH shuttling from the cytosol to mitochondria. Nevertheless, fermentative ethanol production remained the major cytosolic NADH sink under all oxygen conditions, suggesting that the limitation of mitochondrial NADH reoxidation is the major cause of the Crabtree effect. This is reinforced by the induction of several key respiratory genes by oxygen, despite the high sugar concentration, indicating that oxygen overrides glucose repression. Genes associated with other processes, such as proline uptake, cell wall remodeling, and oxidative stress, were also significantly affected by oxygen. The results of this study indicate that respiration is responsible for a substantial part of the oxygen response in yeast cells during alcoholic fermentation. This information will facilitate the development of temporal oxygen addition strategies to optimize yeast performance in industrial fermentations.     

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen‐limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set‐up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen‐activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox‐sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred‐tank bioreactors. Biotechnol. Bioeng. 2013; 110: 573–585. © 2012 Wiley Periodicals, Inc.     

Metabolism of the thermophilic bacterium Oceanithermus profundus

The metabolism of the novel facultatively anaerobic thermophilic bacterium Oceanithermus profundus was studied during growth on maltose, acetate, pyruvate, and hydrogen. The utilization of carbohydrates was shown to proceed via the glycolytic pathway. Under microaerobic growth conditions, the metabolism of O. profundus grown on maltose depended on the substrate concentration. At an initial maltose concentration of 1.4 mM, O. profundus carried out oxygen respiration, and in the presence of 3.5 mM maltose, facilitated fermentation occurred, with the formation of acetate and ethanol and limited involvement of oxygen. The use of pyruvate and acetate occurs via the TCA cycle. In cells grown on acetate, the activity of glyoxylate pathway enzymes was revealed. Depending on the energy-yielding process providing for growth (oxygen respiration or nitrate reduction), cells contained cytochromes a and c or b, respectively. The results obtained demonstrate the plasticity of the metabolism of O. profundus, which thus appears to be well-adjusted to the rapidly changing conditions in deep-sea hydrothermal vents.     

The tricarboxylic acid cycle, an ancient metabolic network with a novel twist

The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH(2)) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and alpha-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of alpha-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing.     

Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.     

Shewanella oneidensis MR-1 fluxome under various oxygen conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.     

Effect of the global redox sensing/regulation networks on Escherichia coli and metabolic flux distribution based on C-13 labeling experiments

Escherichia coli has several elaborate sensing mechanisms for response to the availability of oxygen and the presence of other electron acceptors. Among them, the one component Fnr protein and the two-component Arc system coordinate the adaptive responses to oxygen availability. To systematically investigate the contribution of Arc- and Fnr-dependent regulation in catabolism, glucose-limited chemostat cultures were conducted on wild-type E. coli, an arcA mutant, an fnr mutant, and an arcAfnr double mutant strains under a well-defined semi-aerobic condition. The metabolic flux distributions of the cultures of these strains were estimated based on C-13 labeling experiments. It was shown that the oxidative pentose phosphate (PP) pathway was functioning at low level under semi-aerobic condition. The fluxes through pyruvate dehydrogenase (PDH) and tricarboxylic acid (TCA) cycle were found to be lower in the arcA mutant and the arcAfnr double mutant strains than that in the wild-type strain, although the expression of the genes involved in these pathways have been proved to be derepressed in the mutant strains ([Shalel-Levanon, S., San, K.Y., Bennett, G.N., 2005a. Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions. Biotechnol. Bioeng. 92, 147-159; Shalel-Levanon, S., San, K.Y., Bennett, G.N., 2005c. Effect of oxygen, and ArcA and FNR regulators on the expression of genes related to the electron transfer chain and the TCA cycle in Escherichia coli. Metab. Eng. 7, 364-374]). The significantly higher lactate production in the arcAfnr double mutant strain was shown to be an indirect effect caused by the reduced pyruvate formate-lyase (PFL) and PDH fluxes as well as the intracellular redox state.     

Metabolic flux variation of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment.

The technique of metabolic flux analysis was implemented to elucidate the flux balancing of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment. The results showed that the majority of the substrate (97.70 +/- 0.49%) was funneled into the glycolytic pathway, while the remainder was subdivided between the pentose phosphate pathway and pathways for polysaccharide synthesis. At the pyruvate node, 87.30 +/- 1.38% of the flux was channeled through the reaction governed by pyruvate decarboxylase. Fluxes through the pyruvate dehydrogenase bypass were maintained at a constant level (82.65 +/- 1.47%) irrespective of the configuration of the fermentation setup. Activity through the TCA "cycle" was replenished by the reaction catalyzed by pyruvate carboxylase and by the transport of cytosolic oxaloacetate across the mitochondrial membrane. The CO(2) evolution rate varied as fermentation progressed; however, the yield coefficient of CO(2) remained at a constant value. Although a constant yield of ethanol (0.42 g of ethanol/g of glucose) was obtained, operations of the TCA cycle were gradually switched from partially reductive to partially oxidative pathways from the first fermenter to the fourth fermenter.     

Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using ¹³C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with ¹³C NMR for metabolic flux analysis to reduce the use of ¹³C-labeled substrates and to obtain more-accurate flux values.     

Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.     

Evolutionary optimization of metabolic pathways. Theoretical reconstruction of the stoichiometry of ATP and NADH producing systems

The structural design of ATP and NADH producing systems, such as glycolysis and the citric acid cycle (TCA), is analysed using optimization principles. It is assumed that these pathways combined with oxidative phosphorylation have reached, during their evolution, a high efficiency with respect to ATP production rates. On the basis of kinetic and thermodynamic principles, conclusions are derived concerning the optimal stoichiometry of such pathways. Extending previous investigations, both the concentrations of adenine nucleotides as well as nicotinamide adenine dinucleotides are considered variable quantities. This implies the consideration of the interaction of an ATP and NADH producing system, an ATP consuming system, a system coupling NADH consumption with ATP production and a system consuming NADH decoupled from ATP production. It is examined in what respect real metabolic pathways can be considered optimal by studying a large number of alternative pathways. The kinetics of the individual reactions are described by linear or bilinear functions of reactant concentrations. In this manner, the steady-state ATP production rate can be calculated for any possible ATP and NADH producing pathway. It is shown that most of the possible pathways result in a very low ATP production rate and that the very efficient pathways share common structural properties. Optimization with respect to the ATP production rate is performed by an evolutionary algorithm. The following results of our analysis are in close correspondence to the real design of glycolysis and the TCA cycle. (1) In all efficient pathways the ATP consuming reactions are located near the beginning. (2) In all efficient pathways NADH producing reactions as well as ATP producing reactions are located near the end. (3) The number of NADH molecules produced by the consumption of one energy-rich molecule (glucose) amounts to four in all efficient pathways. A distance measure and a measure for the internal ordering of reactions are introduced to study differences and similarities in the stoichiometries of metabolic pathways.     

Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under anaerobic and microaerobic conditions

The Arc system is a two-component regulatory system composed of ArcA and ArcB in Escherichia coli. In the present study, the effects of arcA and arcB genes knockout on the TCA cycle activation in E. coli were investigated for the anaerobic and microaerobic conditions. Under anaerobic condition, the TCA cycle was up-regulated along with high lactate production, together with up-regulation of LDH for arcB mutant as compared with the parent strain. Due to down-regulation of aceE, aceF and lpdA genes which code for PDHc and low activity of Pfl in arcB mutant, the glycolysis as well as oxidative pentose phosphate pathway was down-regulated under anaerobic condition. The TCA cycle enzymes were further up-regulated when nitrate was added by modifying the redox state along with lower lactate production for arcB mutant. Different from the case of anaerobic condition, the glycolysis was activated under microaerobic condition, which may be partly due to the increased activity of PDHc encoded by aceE, F and lpdA genes. Under microaerobic condition, the TCA cycle genes together with their corresponding enzymes were up-regulated for arcB mutant as compared with the parent strain. These characteristics were further enhanced in arcA mutant as compared with the case of arcB mutant. The up-regulation of the TCA cycle together with down-regulation of cydB gene expression caused higher redox state in the arcA/B mutants, which in turn repressed the TCA cycle. Then the TCA cycle could be further increased by the addition of nicotinic acid (NA).     

Substrate utilization for lactate and energy production by heat-shocked L929 cells

The hypothesis that heat shock protein (HSP) induction depends on inhibition of respiration was tested by examining the effects of heat shock on tricarboxylic acid (TCA) cycle function. In control L929 cell cultures, glucose and exogenous pyruvate were converted primarily to lactate, and glutamine was extensively oxidized, accounting for more than one-half of the calculated ATP production. During heat shock at 42 degrees C, lactate production from all of the labeled substrates and total unlabeled lactate production increased significantly while oxygen consumption increased slightly. TCA cycle oxidation of pyruvate decreased during this period while that of glutamine increased. Uncoupling of oxidative phosphorylation caused large increases in oxygen consumption at both 37 degrees C and 42 degrees C, indicating that the capacity of the respiratory chain is not exceeded during heat shock. The net effect of these alterations in substrate utilization were decreased ATP generation and increased NADH utilization. Both 14CO2 and lactate production declined during the 24-h period after cultures were returned to 37 degrees C. On the basis of these data, we conclude that while inhibition of respiration plays no apparent role, other metabolic consequences of heat shock related to energy metabolism may be involved in HSP induction.     

Relationship between culture redox potential and culture fluorescence inCorynebacterium glutamicum

Summary Transient experiments were performed during a batch fermentation ofCorynebacterium glutamicum ATCC 14296 in which the flow of air to the reactor was cut off. The fluorescence of the culture was observed to vary linearly with the culture redox potential during the transient. This observation was made below the sensitivity limit of a dissolved oxygen probe. It appears that redox potential in conjunction with fluorescence measurements are of utility in studying extra- and intracellular reduction states, particularly in the microaerobic regime.