Effect of environmental pH on chain length of lactobacillus bulgaricus.

Culture medium pH was found to affect strongly the chain length of lactobacillus bulgaricus NLS-4 cells. The organism was cultured continuously in glucose-limited complex medium of different pH's with constant agitation at 250 rpm under anaerobic headspace. The cell chains increased their lengths with an increase in pH and yielded clumps of folded filaments at pH above 8.0. Involvement of an autolytic enzyme(s) in the separation of L. bulgaricus cells was confirmed, and the poor synthesis of this enzyme(s) under alkaline culture conditions could explain the pH-related filamentous growth of this organism.     

Enzymatic regulation of glucose catabolism by Lactobacillus plantarum in response to pH shifts in a chemostat

Summary The influence of medium pH on the regulation of glucose catabolism by Lactobacillus plantarum 8014 was examined in anaerobic chemostat cultures. When L. plantarum was grown in a chemostat at pH 5.5, and the pH shifted to pH 7.5, acetate was produced in addition to lactate and acetoin. After the shift, acetate kinase and NAD-dependent lactate dehydrogenase activities increased while the acetoin dehydrogenase and alpha-acetolactate synthase activities decreased. The high acetate kinase activity together with low acetoin dehydrogenase and alpha-acetolactate synthase activities may explain why L. plantarum made more acetate at the expense of acetoin in response to alkaline conditions.Offprint requests to: T.J. Montville     

Effect of dilution rate and carbon availability on Bifidobacterium breve fermentation

Bifidobacterium breve NCFB 2257 was grown in glucose-limited and nitrogen (N)-limited chemostats at dilution rates (D) from 0.04 to 0.60 h^–1, to study the effect of nutrient availability on carbohydrate metabolism. The results showed that D had little effect on fermentation product formation, irrespective of the form of nutrient limitation. However, marked differeces were observed in the distribution of fermentation products, that were attributable to glucose availability. In glucose-limited cultures, formate and acetate were the principal end-products of metabolism. Lactate was never detected under these growth conditions. In contrast, lactate and acetate were mainly formed when glucose was in excess, and formate was not produced. These results are explained by the metabolic fate of pyruvate, which can be dissimilated by either phosphoroclastic cleavage to acetyl phosphate and formate, or alternatively, it may be reduced to lactate. Enzymic studies were made to establish the mechanisms that regulated pyruvate metabolism. The data demonstrated that control was not exercised through regulation of the synthesis and activity of lactate dehydrogenase (LDH), phosphofructokinase or alcohol dehydrogenase. It is possible however, that there was competition for pyruvate by LDH and the phosphoroclastic enzyme, which would determine the levels of lactate and formate produced respectively. These results demonstrate the metabolic flexibility of B. breve, which preferentially uses lactate as an electron sink during N-limited growth, whereas under energy-limitation, carbon flow is directed towards acetyl phosphate to maximise ATP synthesis. Correspondence to: B. A. Degnan     

Mutants of Escherichia coli deficient in the fermentative lactate dehydrogenase.

Mutants of Escherichia coli deficient in the fermentative NAD-linked lactate dehydrogenase (ldh) have been isolated. These mutants showed no growth defects under anaerobic conditions unless present together with a defect in pyruvate formate lyase (pfl). Double mutants (pfl ldh) were unable to grow anaerobically on glucose or other sugars even when supplemented with acetate, whereas pfl mutants can do so. The ldh mutation was found to map at 30.5 min on the E. coli chromosome. The ldh mutant FMJ39 showed no detectable lactate dehydrogenase activity and produced no lactic acid from glucose under anaerobic conditions as estimated by in vivo nuclear magnetic resonance measurements. We also found that in wild-type strains the fermentative lactate dehydrogenase was conjointly induced by anaerobic conditions and an acidic pH. Despite previous findings that phosphate concentrations affect the proportion of lactic acid produced during fermentation, we were unable to find any intrinsic effect of phosphate on lactate dehydrogenase activity, apart from the buffering effect of this ion.     

Effects of pH on murine insulinoma betaTC3 cells

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.     

Effect of carbon source and pH of the growth medium on spore germination inPhycomyces blakesleeanus

Phycomyces blakesleeanus sporangiospores responded differently to activation by physical and chemical stimuli. Spores that were physically (heat shock) activated or chemically (ammonium acetate) activated germinated and grew at pH 4.5 with the hexoses glucose, fructose, galactose, andN-acetylglucosamine, and with glycerol and amino acids. Under these conditions, physically activated spores showed a lower, although significant growth with the hexoses fructose, galactose,N-acetylglucosamine and with glycerol. On the other hand, physically activated spores incubated at alkaline pH (pH 7.3) required glucose to germinate; a requirement not observed with chemically activated spores, which showed significant growth in the other hexoses tested. Both physically and chemically activated spores incubated at pH 7.3 were unable to germinate and grow with amino acids and glycerol. These results suggest that there are different targets for activation of the spores by physical and chemical treatments. The levels of the fermentative enzymes alcohol dehydrogenase and lactate dehydrogenase and of the oxidative enzyme NAD⁺-isocitrate dehydrogenase were higher in cells grown at pH 4.5 in medium containing glucose; however, alcohol dehydrogenase and lactate dehydrogenase appear not to be affected by a change in the pH of the growth medium.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

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

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

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

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

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

Glucose Fermentation by <Emphasis Type="Italic">Propionibacterium microaerophilum</Emphasis>: 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, CO₂, 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 CO₂ 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. Received: 8 April 2002 / Accepted: 8 May 2002     

Factors affecting exocellular polysaccharide production by Lactobacillus delbrueckii subsp. bulgaricus grown in a chemically defined medium

We developed a chemically defined medium (CDM) containing lactose or glucose as the carbon source that supports growth and exopolysaccharide (EPS) production of two strains of Lactobacillus delbrueckii subsp. bulgaricus. The factors found to affect EPS production in this medium were oxygen, pH, temperature, and medium constituents, such as orotic acid and the carbon source. EPS production was greatest during the stationary phase. Composition analysis of EPS isolated at different growth phases and produced under different fermentation conditions (varying carbon source or pH) revealed that the component sugars were the same. The EPS from strain L. delbrueckii subsp. bulgaricus CNRZ 1187 contained galactose and glucose, and that of strain L. delbrueckii subsp. bulgaricus CNRZ 416 contained galactose, glucose, and rhamnose. However, the relative proportions of the individual monosaccharides differed, suggesting that repeating unit structures can vary according to specific medium alterations. Under pH-controlled fermentation conditions, L. delbrueckii subsp. bulgaricus strains produced as much EPS in the CDM as in milk. Furthermore, the relative proportions of individual monosaccharides of EPS produced in pH-controlled CDM or in milk were very similar. The CDM we developed may be a useful model and an alternative to milk in studies of EPS production.     

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.     

Metabolic Control of Anaerobic Glycolysis (Overexpression of Lactate Dehydrogenase in Transgenic Tomato Roots Supports the Davies-Roberts Hypothesis and Points to a Critical Role for Lactate Secretion

Roots of all plants examined so far have the potential for both ethanol and lactate fermentation. A short burst of lactate fermentation usually occurs when plant tissues are transferred from normoxic to anoxic conditions. According to the Davies-Roberts hypothesis, the consequent pH drop both initiates ethanol fermentation and blocks further production of lactate by inhibiting lactate dehydrogenase (LDH). However, the role of LDH in this pH control mechanism is still a matter of debate. To perturb the control system in a defined way, a barley LDH cDNA under the control of the cauliflower mosaic virus 35S promoter was introduced into tomato (Lycopersicon esculentum Mill. cv VFMT) using Agrobacterium rhizogenes. The transgenic root clones expressed up to 50 times the LDH activity of controls. The fermentative metabolism of these clones was compared using roots grown previously in normoxic conditions or roots given a 3-d hypoxic pretreatment. During the transition from normoxia to anoxia, lactate accumulation was no faster and no more extensive in transgenic roots than in controls. Similarly, during prolonged anoxia the flux of 14C from [U-14C] glucose to lactate and ethanol was not modified by the expression of the transgene. However, in both transgenic and control roots, hypoxic pretreatment increased the flux to lactate and promoted lactate export to the medium. These results show that LDH has a very low flux control coefficient for lactate fermentation, consistent with the Davies-Roberts hypothesis. Moreover, they suggest that lactate secretion exerts major control over long-term lactate glycolysis in vivo.     

Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus lactis by Controlled Expression of NADH Oxidase

NADH oxidase-overproducing Lactococcus lactis strains were constructed by cloning the Streptococcus mutans nox-2 gene, which encodes the H₂O-forming NADH oxidase, on the plasmid vector pNZ8020 under the control of the L. lactis nisA promoter. This engineered system allowed a nisin-controlled 150-fold overproduction of NADH oxidase at pH 7.0, resulting in decreased NADH/NAD ratios under aerobic conditions. Deliberate variations on NADH oxidase activity provoked a shift from homolactic to mixed-acid fermentation during aerobic glucose catabolism. The magnitude of this shift was directly dependent on the level of NADH oxidase overproduced. At an initial growth pH of 6.0, smaller amounts of nisin were required to optimize NADH oxidase overproduction, but maximum NADH oxidase activity was twofold lower than that found at pH 7.0. Nonetheless at the highest induction levels, levels of pyruvate flux redistribution were almost identical at both initial pH values. Pyruvate was mostly converted to acetoin or diacetyl via α-acetolactate synthase instead of lactate and was not converted to acetate due to flux limitation through pyruvate dehydrogenase. The activity of the overproduced NADH oxidase could be increased with exogenously added flavin adenine dinucleotide. Under these conditions, lactate production was completely absent. Lactate dehydrogenase remained active under all conditions, indicating that the observed metabolic effects were only due to removal of the reduced cofactor. These results indicate that the observed shift from homolactic to mixed-acid fermentation under aerobic conditions is mainly modulated by the level of NADH oxidation resulting in low NADH/NAD⁺ ratios in the cells.     

End Products of Glucose Fermentation by Brochothrix thermosphacta

Anaerobically, Brochothrix thermosphacta fermented glucose primarily to l-lactate, acetate, formate, and ethanol. The ratio of these end products varied with growth conditions. Both the presence of acetate and formate and a pH below about 6 increased l-lactate production from glucose. Small amounts of butane-2,3-diol were also produced when the pH of the culture was low (相似文献   

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.     

Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum

The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1). However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.