Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Colony formations in a halotolerantBrevibacterium sp. JCM 6894 on solid medium with different pH values

Viable cells of a halotolerantBrevibacterium sp. JCM 6894 grown in a liquid medium with pH 7.1 were enumerated as the colony-forming cells on three kinds of agar media with different pH values. Unexpectedly they were lower at neutral pH rather than acidic or alkaline pH. This tendency was invariable regardless of the changes in the concentrations of nutrients in the agar medium as well as in the growth phases of the cells. From the comparison of cell growth between liquid and solid media with different pHs, we notified the importance of the pH changes in liquid medium accompanied with growth. Effects of salts and pH of the liquid medium on protonmotive force (Δp) was estimated from membrane potentials (ΔΨ) and proton gradients (ΔpH) of the strain JCM 6894. In the absence of salts, Δp of the strain JCM 6894 was the largest at neutral pH, which was conflicting with the result of cell viability. The addition of NaCl led to the reduction of Δp at acidic pH, mainly due to the dissipation of ΔΨ, which seems to be consistent with the lower numbers of colony formed at acidic pH in the presence of NaCl.     

Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.     

Effect of pH on the regulatory characteristics of energy metabolism in human erythrocytes

The glucose consumption rate versus ATP content in human red cells (regulatory patterns of glycolysis) and ATP concentration versus glucose uptake rate in red cell suspension (regulatory patterns of total ATPases), when the rate of glucose uptake is constant and lower than the rate of glucose consumption at physiological conditions, were measured at different pH values. The shape of both types of kinetic curves was found to be dependent on the pH of the incubation medium but the same for the red cells taken from different donors. It is supposed that at alkaline pH, glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase reactions become the rate-limiting steps of glycolysis instead of hexokinase and phosphofructokinase under physiological conditions.     

Effect of pH on Bacillus thermoamylovorans Growth and Glucose Fermentation

The effect of pH on the growth and physiology of Bacillus thermoamylovorans, a new moderately thermophilic and non-spore-forming bacterium isolated from palm wine, was studied. Growth occurred from pH 5.4 to 8.5, with optimum growth at 7.0. During the exponential growth phase at optimum pH, glucose was consumed at the maximum rate (qs), 17.87 mmol g(sup-1) h(sup-1), and was mainly fermented into acetate, ethanol, and formate (76.5% of metabolites produced). In acidic or alkaline conditions, glucose specific consumption rates were considerably reduced (qs = 8.06 mmol g(sup-1) h(sup-1) at pH 5.6 and 2.85 mmol g(sup-1) h(sup-1) at pH 8.4), and a switch in glucose metabolism toward lactate production (62.6% of metabolites produced at pH 5.6 and 41.2% of those produced at pH 8.4) was observed. Moreover, optimum cellular yield (Y(infx/ATP)), 14.8 g mol(sup-1), and optimum energy yield (Y(infATP/s)), 2.65 mol mol(sup-1), were observed at neutrality. The results of this study were compared with published data about lactic acid bacteria; this comparison allowed us to complement our previous taxonomic study of B. thermoamylovorans and to identify additional phenotypic differences between B. thermoamylovorans and lactobacilli.     

Toward a Solution for Acid Mine Drainage Treatment: Role of Electron Donors in Sulfate Reduction at Low pH

Abstract

Sediment historically impacted by acid mine drainage was exposed to different initial pH and electron donors to investigate the effect that both conditions had on the performance and fingerprint of the community from naturally acidic sediments. Batch experiments were fed with either acetate, lactate, or glycerol at initial pH of 5, 4, or 3, under sulfate-reducing conditions. The performance results indicated that sulfide production efficiency was above 85% in the treatments fed with lactate and glycerol at pH 5 and 4. However, acetate consumption efficiency was greater than 85% only in the treatments with acetate at pH 5 and lactate at pH 5 and 4. Glycerol fed treatments successfully produced sulfide even at initial pH?=?3. Sulfide production rates were related to the initial pH in treatments fed with lactate and acetate and independent of the pH in the glycerol fed treatments. 16S rRNA gene T-RFLP analysis of the enriched communities indicated that the initial pH could explain the differences of the microbial community fingerprint obtained after 90?days. This study points out the fact that acidic stress is a heavy burden for the development of sulfate-reducing microorganisms, especially for those that use acetate as substrate.     

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.     

The effects of microcarrier culture on recombinant CHO cells under biphasic hypothermic culture conditions

A Chinese hamster ovary (CHO) cell line, producing recombinant secreted human placental alkaline phosphatase (SEAP) was investigated under three different culture conditions (suspension cells, cells attached to Cytodex 3 and Cytopore 1 microcarriers) in a biphasic culture mode using a temperature shift to mild hypothermic conditions (33 °C) in a fed-batch bioreactor. The cell viability in both the suspension and the Cytodex 3 cultures was maintained for significantly longer periods under hypothermic conditions than in the single-temperature cultures, leading to higher integrated viable cell densities. For all culture conditions, the specific productivity of SEAP increased after the temperature reduction; the specific productivities of the microcarrier cultures increased approximately threefold while the specific productivity of the suspension culture increased nearly eightfold. The glucose and glutamine consumption rates and lactate and ammonia production rates were significantly lowered after the temperature reduction, as were the yields of lactate from glucose. However, the yield of ammonia from glutamine increased in response to the temperature shift.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K+, Na+ and ATP on the gastric (H+ + K+)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min-1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min-1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min-1. Additions of 5 mM K+ or Na+, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K+, but not on Na+. Alkaline pH increased the rate of dephosphorylation. K+ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K+ was inhibitory. Below pH 7.0 Na+ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na+ inhibited both activities. The effect of extravesicular pH on transport of H+ was investigated. At pH 6.5 the apparent Km for ATP was 2.7 microM and increased little when K+ was added extravesicularly. At pH 7.5, millimolar concentrations of K+ increased the apparent Km for ATP. Extravesicular K+ and Na+ inhibited the transport of H+. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H+-producing reactions as possible candidates as physiological regulators of (H+ + K+)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO2 and the subsequent formation of H+ and HCO3-. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H+/ATP ratio was about 1 at pH 8.0. When CO2 was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO2. The results indicate a possible co-operation in the production of acid between the H+ + K+-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Rapid transient growth at low pH in the cyanobacterium Synechococcus sp

The thermophilic cyanobacterium Synechococcus sp. strain Y-7c-s grows at its maximum rate at a high pH (pH 8 and above) the does not show sustained growth below pH 6.5. However, rapidly growing, exponential-phase cells from high-pH cultures continued to grow rapidly for several hours after transfer to pH 6.0 or 5.0. This transient growth represented increases in mass and protein, but cells failed to complete division. Viability loss commenced well before the cessation of growth, and cells at pH 5.0 showed no net DNA synthesis. When irradiated by visible light, cells at pH 6.0 and 5.0 maintained and internal pH of 6.9 to 7.1 (determined by 31P nuclear magnetic resonance spectroscopy) and an extremely high ATP/(ATP + ADP) ratio even after growth had ceased. Cells exposed to a low pH did not show an increase in the spontaneous mutation rate, as measured by mutation to streptomycin resistance. However, cells already resistant to streptomycin were more resistant to viability loss at a low pH than the parental type. Cultures that could grow transiently at a low pH had higher rates of viability loss than nongrowing cultures in light or darkness. The retention of a high internal pH by cells exposed to a low pH suggested that a low pH acted initially on the cell membrane, possibly on solute transport.     

Insights into pH‐induced metabolic switch by flux balance analysis

Lactate accumulation in mammalian cell culture is known to impede cellular growth and productivity. The control of lactate formation and consumption in a hybridoma cell line was achieved by pH alteration during the early exponential growth phase. In particular, lactate consumption was induced even at high glucose concentrations at pH 6.8, whereas highly increased production of lactate was obtained at pH 7.8. Consequently, constraint‐based metabolic flux analysis was used to examine pH‐induced metabolic states in the same growth state. We demonstrated that lactate influx at pH 6.8 led cells to maintain high fluxes in the TCA cycle and malate‐aspartate shuttle resulting in a high ATP production rate. In contrast, under increased pH conditions, less ATP was generated and different ATP sources were utilized. Gene expression analysis led to the conclusion that lactate formation at high pH was enabled by gluconeogenic pathways in addition to facilitated glucose uptake. The obtained results provide new insights into the influence of pH on cellular metabolism, and are of importance when considering pH heterogeneities typically present in large scale industrial bioreactors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:347–357, 2015     

Effect of culture pH on the extracellular production of 5-aminolevulinic acid by Rhodobacter sphaeroides from volatile fatty acids

Summary 5-Aminolevulinic acid(ALA) production by Rhodobacter sphaeroides was investigated at various pH with levulinic acid addition using a volatile fatty acids medium prepared from the mandarin orange peel supplemented with glycine. At neutral pH (6.8 and 7.0), extracellular ALA production was up to 16 mM, while low production of ALA(less than 3.5 mM) was observed at acidic pH (lower than 6.5) and less than 3.9 mM of ALA produced at alkaline pH (higher than 7.5). The higher ALA synthase activity observed at neutral pH might enhance the ALA production compared with that observed in acidic and alkaliphilic cultures.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

Water pH and metabolic parameters in silver catfish (Rhamdia quelen)

This study evaluated the effects of an acute change in water pH (from pH 7.5 to 4.0, 5.0, 6.0, 7.5, 8.0 or 9.0) on several biochemical parameters in juveniles of the silver catfish, Rhamdia quelen. Ammonia levels decreased in the liver and increased in the muscle with increasing water pH. In the kidney, lower ammonia levels were observed at neutral pH. An increase in water pH decreased the glucose, glycogen and lactate levels in the liver and kidney (except for glycogen levels in the kidney and lactate levels in the liver, which presented lower levels at neutral pH). In muscle, the glucose and glycogen levels decreased with increasing water pH, whereas lactate levels tended to be lower at neutral pH. Gill and kidney Na⁺/K⁺-ATPase activities tended to increase in alkaline water, and the highest value was observed in fish exposed to pH 9.0. The optimal levels of the analyzed biochemical parameters occurred at neutral pH. In conclusion, exposure to acidic and alkaline pH changes the metabolic parameters of silver catfish as well as gill Na⁺/K⁺-ATPase activity.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Effect of hyperosmolarity on recombinant protein productivity in baculovirus expression system

A lot of strategies were applied to improve recombinant protein productivity in the baculovirus expression system. In this study we propose for foreign protein production fed-batch cultivation method at hyperosmotic environment induced by increased NaCl content. Obtained results suggested relatively high tolerance and adaptation abilities of Tn-5 insect cells exposed to hyperosmotic stress. The cells under hyperosmotic conditions increased the specific rate of glucose consumption and lactate production. The release of additional energy and precursors as a result of increased metabolism by osmotically stressed culture was involved in recombinant protein synthesis. Recombinant nucleoprotein productivity in nutritional feeding cultures exposed to hyperosmolarity was about 72% higher than that obtained in batch culture at physiological osmolarity, but 31% was a result of feeding and the rest 41% was a result of hyperosmolarity and increasing Na(+) concentration.     

Survival of T-lymphocytes under alkalosis and autocrine factor deficiency

Alkalosis associated with elevated pH is characteristic of many clinical pathologies. Respiratory alkalosis is a result of hyperventilation, i.e., reduced partial CO₂ pressure in alveolar air and blood. Yet another type of alkalosis, i.e., metabolic alkalosis, is associated with an absolute or relative increase in the levels of alkaline compounds in the organism. Despite high toxicity of the latter, mechanisms whereby these compounds exert their toxic effects remain obscure. In multicellular organisms, cell survival is controlled by a vast variety of factors, such as autocrine survival factors (AF) specifically targeted at cells that secrete them. Our previous studies (Lutsenko and Diachkova, 2003) demonstrated that AF control cell survival and energy metabolism in T-lymphocytes. In this study, combined effects of AF deficiency and alkalosis (pH 8.3) on cell survival, intracellular content of ATP and mitochondrial transmembrane potential of T-lymphocytes were studied using an IL-2-dependent cell line CTLL-2. It was found that in the absence of AF deficiency, alkalosis had no effect on survival of cultured CTLL-2 cells. The main mechanism of protection of CTLL-2 cells against cytotoxic effects of alkalosis was an enhanced anaerobic glycolysis and consequential increase in the lactate production. In contrast, alkalosis combined with AF deficiency caused a substantial decrease of cell survival, which lowered down to 53% after 6 h and to about 10 % after 20 h of culturing under these conditions. The ATP content dropped down sharply under the AF deficiency even at pH 7.3 but gradually restored to the initial level within the next 2-3 h; cell survival was at a high level under these conditions. Alkalosis combined with the AF deficiency notably worsened the functional state of the cells; ATP content in them remained at a low level over the whole period of the alkaline stress. After a 2-h incubation under alkalosis and AF deficiency, 23% of cells contained depolarized mitochondria; lactate production was notably suppressed. The data obtained suggest that the reduction of the intracellular ATP level in CTLL-2 cells under alkalosis and AF deficiency are due to inhibition of anaerobic glycolysis and mitochondrial dysfunction. Cell death developed predominantly via the necrotic rather than the apoptotic pathway.     

Metabolic arrest and its regulation in anoxic eel hepatocytes

Some vertebrates depress overall metabolism in an abrupt and reversible fashion when challenged with anoxia, ensuring stabilization of cellular [ATP] and long-term survival, but little is known about the eliciting stimuli (e.g., change in O2, adenylates) and downstream effectors responsible for metabolic arrest. Accordingly, eel (Anguilla anguilla) hepatocytes were treated with inhibitors of putative components of the oxygen/metabolite-sensing pathway(s) and exposed to anoxia (Po2=0 mmHg). Anoxia in untreated cells caused a remarkable 85-fold decrease in ATP production rate, but cellular ATP levels stabilized following an initial steep drop. Reoxygenation of cells after 4 h of anoxia caused a fast metabolization of accumulated lactate and reestablishment of preanoxic ATP levels. Unlike physiological anoxia, pharmacological inhibition of the electron transport chain in the presence of oxygen caused extensive cellular ATP depletion, though no loss in viability. In contrast, cellular lactate (i.e., ATP) production rate was affected similarly by either treatment, suggesting that anaerobic glycolysis is regulated by a stimulus other than oxygen tension per se, whereas the continuous matching of ATP consumption and a rapidly ceasing mitochondrial ATP supply require a physiological relevant change in oxygen tension. Protein kinases, notably kinase C (PKC) and A (PKA), have been proposed as key downstream regulators of stress-induced defense mechanisms, but anoxic cell viability, metabolic rate, and [ATP] were not significantly affected by inhibitors of PKC and PKA. Likewise, inhibition of the upstream PKC-activating enzymes phospholipase C (PLC) and phosphatidylinositol 3-kinase (PI 3-K) had no effect on recorded parameters. Anoxic cell survival in complex organisms may, in vivo, also depend on stress hormones released from distant oxygen-sensing cells. Accordingly, adrenaline elevated anaerobic energy production but, apparently, also elevated ATP consumption because cellular ATP levels during oxygen deprivation were slightly lowered by adrenergic stimulation.