Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose.

The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained. Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures. When the malolactic bacterium L. oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed. While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production. The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation. However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated. The stimulatory effects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase. While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.     

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.     

苹果酸-乳酸发酵能量产生机理

苹果酸乳酸发酵(MLF)是现代葡萄酒酿造工艺中重要的生物降酸手段。MLF是L苹果酸在乳酸菌的苹果酸乳酸酶催化下转变成L乳酸的酶促反应过程,该过程没有底物水平的磷酸化,但在MLF过程中苹果酸确实能够刺激细菌的比生长速率,增加细菌生物量。进一步的研究表明,代谢能的产生并非由于苹果酸的脱羧反应,而主要源于跨膜的L苹果酸摄入和(或)L乳酸的流出,从而产生跨膜质子电动势(pmf)(一小部分能量可能由L苹果酸的代谢中间产物丙酮酸产生)。按照化学渗透理论,pmf驱动F0F1ATPase合成ATP用于细菌生长。本文还对苹果酸的运输机制进行了综述。     

Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures

The energetics of Saccharomyces cerevisiae were studied in anaerobic glucose-limited chemostat cultures via an analysis of biomass and metabolite production. The observed YATP was dependent on the composition of the biomass, the production of acetate, the extracellular pH, and the provision of an adequate amount of fatty acid in the medium. Under optimal growth conditions, the YATP was approximately 16 g biomass (mol ATP formed)-1. This is much higher than previously reported for batch cultures. Addition of acetic acid or propionic acid lowered the YATP. A linear correlation was found between the energy required to compensate for import of protons and the amount of acid added. This energy requirement may be regarded as a maintenance energy, since it was independent of the dilution rate at a given acid concentration.     

Respiration-linked proton translocation in the obligate methylotroph Methylophilus methylotrophus.

The stoicheiometries of respiration-linked proton translocation in Methylophilus methylotrophus were determined by using both the oxygen-pulse and initial-rate methods. The latter has also been used to determine leads to charge/O quotients (measured as yield K+/O quotients) in order to ascertain whether the leads to H+/O quotients might be underestimated by H+/anion symport. The results suggest that 6H+/O are translocated during NADH oxidation, and that 2H+/O are translocated during the oxidation of methanol to formaldehyde. There was no evidence for underestimation of the leads to H+/O quotients due to H+/anion symport, except by the movement of formic acid during formate oxidation. By comparing these results with the known growth efficiencies of this organism, an leads to H+/ATP quotient of close to 2 g-ions of H+/mol of ATP can be calculated. It is proposed that the respiratory chain in Methylophilus methylotrophus is arranged such that there are three sites of energy conservation for NADH oxidation, each translocating 2H+ and each linked to the synthesis of one molecule of ATP. Only the third site of energy conservation is involved in methanol oxidation.     

Energy transduction in the mitochondrionlike bacterium Paracoccus denitrificans during carbon- or sulphate-limited aerobic growth in continuous culture.

Paracoccus denitrificans was grown in carbon-limited aerobic continuous culture (critical dilution rate (Dc) = 0.48 h-1). The molar growth yield for carbon (succinate or malate) was constant at about 60 over a broad dilution range (growth rate) from 0.10 to 0.48 h-1. Measurements of the stoichiometry of proton translocation associated with the oxidation of endogenous substrates yielded a ratio of protons ejected from the cell per atom of oxygen consumed(leads to H+:O) of 8.55 which decreased to 5.85 in the presence of piericidin A (PA), a specific inhibitor of NADH dehydrogenase (EC 1.6.99.3). With starved cells, the observed leads to H+:O associated with the oxidation of added succinate in the presence of PA was 5.61. These observed leads to H:O's represent an underestimation since no correction was made for proton backflow during the short interval of respiratory activity. Aerobic growth of Pc. denitrificans in the chemostat becomes sulphate limited at entering concentrations of sulphate less than 300 is microM. Neither the maximum specific growth rate (measured at Dc) nor the observed molar growth yield for succinate decreased under sulphate limitation. The NADH oxidase in electron transport particles prepared from sulphate-limited cells was completely inhibited by PA. The stoichiometry of proton translocation associated with malate oxidation was similarly unaffected by sulphate limitation. It is concluded that (a) the respiratory chain of aerobic, heterotrophically grown Pc. denitrificans possesses three sites of energy conservation, including site III, (b) the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling site is 3, and (c) that sulphate limitation does not lead to a loss of proton translocation associated with the cytochrome-independent region of the respiratory chain.     

Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH2 reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3 to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H2, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H2 at final pH 4.0, acetate + ethanol + modest H2 at final pH 6.8, and acetate + lactate + trivial H2 at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H2 yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd(red)) formation is required for proton reduction to H2. Also consistent with this hypothesis is that high H2 production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H2 formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at approximately 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had approximately 3.5 mol ATP/mol glucose) over a wide range of H2 production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.     

Role of malolactic fermentation in lactic acid bacteria

Although decarboxylation of malate to lactate by malolactic enzyme does not liberate biologically available energy (e.g., ATP, NADH), the growth rate of many malolactic bacteria is greatly enhanced by malolactic fermentation. The deacidification of the medium due to malate dissipation cannot fully account for this situation. The chemiosmotic theory postulates that another form of energy could generated by translocation of protons through the membrane coupled to end-product efflux. Konings et al. showed that this theory is indeed applicable to lactate efflux in Streptococcus cremoris at pH 7.0. A similar mechanism could account for the observed increased activity in malolactic bacteria. The study in wild type and mutant strains of Streptococcus lactis unable to carry out malolactic fermentation led us to the following conclusions: (1) under glucose non-limiting conditions, malolactic fermentation helps to maintain pH of the medium at a certain level; (2) during glucose limited growth, malolactic fermentation could be coupled with an energetic process independent from that mentioned above.     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

Proton motive force, energy recycling by end product excretion, and metabolic uncoupling during anaerobic growth of Pseudomonas mendocina.

Batch cultures of Pseudomonas mendocina, grown in rich medium with glucose excess, showed metabolic differences dependent upon whether the growth conditions were aerobic or anaerobic, with or without added electron acceptor. Under anaerobic conditions in the absence of nitrate, P. mendocina reached the stationary phase of growth after 2 or 3 days, followed by a stationary phase of 4 to 5 days. Under these conditions, a mixed-type fermentative metabolism (formic, lactic, and acetic acids) appeared. A fivefold-higher specific rate of glucose consumption and eightfold-higher production of organic acids, compared with aerobic cultures, were shown by this microorganism growing anaerobically in the absence of exogenous electron acceptors. The gradients of organic acid produced by P. mendocina under these conditions reached a maximum (lactate, 180 mV; formate, 150 mV; acetate, 215 mV) between days 2 and 3 of culture. The proton motive force (delta p) decreased during growth from -254 to -71 mV. The intracellular pH remained alkaline during the culture, reaching a steady-state value of 7.9. The gradients of organic acids apparently contributed to the generation of a delta p, which, according to the Energy Recycling Model (P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, FEMS Microbiol. Lett. 5:357-364, 1979), would produce an average energy gain of 1 or 1.5 mol of ATP equivalents per mol of glucose consumed with H+/ATP stoichiometry of 3 or 2, respectively. Low YATP and Yglucose values were observed, suggesting that an uncoupled metabolism exists; i.e., ATP produced by catabolic processes is not directly used for biomass synthesis. This metabolic uncoupling could be induced at least in part by organic acids and the ATP wastage could be induced by a membrane-bound ATPase involved in intracellular pH regulation.     

Energy conservation by succinate decarboxylation in Veillonella parvula.

Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4-3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA:pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.     

Physiology of yeasts in relation to biomass yields

The stoichiometric limit to the biomass yield (maximal assimilation of the carbon source) is determined by the amount of CO2 lost in anabolism and the amount of carbon source required for generation of NADPH. This stoichiometric limit may be reached when yeasts utilize formate as an additional energy source. Factors affecting the biomass yield on single substrates are discussed under the following headings: Energy requirement for biomass formation (YATP). YATP depends strongly on the nature of the carbon source. Cell composition. The macroscopic composition of the biomass, and in particular the protein content, has a considerable effect on the ATP requirement for biomass formation. Hence, determination of for instance the protein content of biomass is relevant in studies on bioenergetics. Transport of the carbon source. Active (i.e. energy-requiring) transport, which occurs for a number of sugars and polyols, may contribute significantly to the calculated theoretical ATP requirement for biomass formation. P/O-ratio. The efficiency of mitochondrial energy generation has a strong effect on the cell yield. The P/O-ratio is determined to a major extent by the number of proton-translocating sites in the mitochondrial respiratory chain. Maintenance and environmental factors. Factors such as osmotic stress, heavy metals, oxygen and carbon dioxide pressures, temperature and pH affect the yield of yeasts. Various mechanisms may be involved, often affecting the maintenance energy requirement. Metabolites such as ethanol and weak acids. Ethanol increases the permeability of the plasma membrane, whereas weak acids can act as proton conductors. Energy content of the growth substrate. It has often been attempted in the literature to predict the biomass yield by correlating the energy content of the carbon source (represented by the degree of reduction) to the biomass yield or the percentage assimilation of the carbon source. An analysis of biomass yields of Candida utilis on a large number of carbon sources indicates that the biomass yield is mainly determined by the biochemical pathways leading to biomass formation, rather than by the energy content of the substrate.     

A theoretical evaluation of growth yields of yeasts

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: --S. cerevisiae had a lower protein content than C. utilis; --uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass.mol ATP-1. In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.     

Mechanism of glutamate uptake in Zymomonas mobilis.

The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Energetics of syntrophic propionate oxidation in defined batch and chemostat cocultures

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

Stoichiometry of proton translocation coupled to substrate oxidation in plant mitochondria

The proton translocation coupled to the electron flux from succinate, exogenous NADH, and NAD⁺-linked substrates (malate and isocitrate) to cytochrome c and to oxygen was studied in purified potato (Solanum tuberosum) mitochondria using oxygen and ferricyanide pulse techniques. In the presence of valinomycin plus K⁺ (used as a charge compensating cation), optimum values of H⁺/2 e⁻ were obtained when low amounts of electron acceptors (oxygen or ferricyanide) were added to the mitochondria (1-2 nanogram [2 e⁻] equivalents per milligram protein). The stoichiometry of proton translocation to electron flux was unaffected in the presence of N-ethylmaleimide, an inhibitor of the Pi/H⁺ symport. With succinate as substrate, H⁺/2 e⁻ ratios were 4.0 ± 0.2 and 3.7 ± 0.3 with oxygen and ferricyanide as electron acceptors, respectively. With exogenous NADH, H⁺/2e⁻ ratios were 4.1 ± 0.9 and 3.4 ± 0.2, respectively. The proton translocation coupled to the oxidation of NAD⁺-linked substrates (malate, isocitrate) was dependent upon the presence of adenylates (ADP, AMP, or ATP). For malate (+ glutamate) oxidation the observed H⁺/2 e⁻ ratios were increased from 3.6 ± 2.2 to 6.5 ± 0.5 in the presence of 20 micromolar ADP.     

The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. An analysis of ADP-induced oxygen jumps by linear nonequilibrium thermodynamics

Uncertainty exists as to the proton stoichiometries of mitochondrial oxidative phosphorylation and consequently as to the ATP stoichiometries. In rat liver mitochondria, ADP/O ratios were determined from the total and extra oxygen consumed during ADP-stimulated respiration under conditions of quantitative conversion of ADP to ATP. For succinate, glutamate plus malate, 3-hydroxybutyrate, and 2-oxoglutarate, respectively, ADP/total O was 1.71, 2.71, 2.61, and 3.45. ADP/extra O was 2.03, 3.04, 3.23, and 4.15. The results were interpreted in terms of linear nonequilibrium thermodynamics. It was shown that ADP/extra O = Z/q where Z is the phenomenological stoichiometry and q is the degree of coupling. q was determined from the dependence of respiratory rate on delta Gp, the phosphorylation potential, and was about 0.98 for all substrates. The results were consistent with ideal ATP/O stoichiometries of 2 for succinate, 3 for glutamate plus malate, 3 or 3 1/4 for 3-hydroxybutyrate, and 4 for 2-oxoglutarate. Taking into account the oxidation-reduction free-energy changes measured across Sites 1 + 2 at static head (J.J. Lemasters, R. Grunwald, and R.K. Emaus J. Biol. Chem. 259, 3058-3063), an ideal ATP/O stoichiometry of 3 1/4 for 3-hydroxybutyrate is proposed. The lower ATP/O for glutamate plus malate is then accounted for by proton translocation linked to glutamate/aspartate exchange. The data suggest a new 13-proton scheme of chemiosmotic coupling in which proton stoichiometries are 3 for the F1Fo-ATPase, 1 for the exchange of ATP for ADP and Pi, and 5, 4, and 4 for Sites 1, 2, and 3.     

The role of sodium ions in methanogenesis. Formaldehyde oxidation to CO2 and 2H2 in methanogenic bacteria is coupled with primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings. Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors. Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores. Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide. With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.     

Determination of the efficiency of oxidative phosphorylation in continuous cultures of Aerobacter aerogenes.

For anaerobic glucose-limited chemostat cultures of Aerobacter aerogenes a values of 14.0 g/mole was found for Ymax/ATP and a value of 6.8 mmoles ATP/g dry weight/hr for the maintenance coefficient. Both values are much lower than those previously determined for tryptophan-limited anaerobic chemostat cultures. It is concluded that generally the largest part of the maintenance energy is not used for true maintenance processes. For aerobic glucose-limited chemostat cultures two phases could be differentiated. Acetate production started at mu values higher than 0.53. The slopes of the curves relating the specific rates of glucose- and oxygen consumption with mu became higher and lower respectively above the mu value of 0.53. Using the YATP values obtained in the anaerobic experiment a P/O ratio of about 1.3 could be calculated for glucose- and tryptophan-limited chemostat cultures. In sulfate-limited chemostat cultures acetate was produced at all growth rates. At high growth rates also pyruvate and alpha-ketoglutarate were produced. With the YATP values obtained in the anaerobic experiment a P/O ratio of about 0.4 was calculated for sulfate-limited chemostat cultures.