Unstructured model for free and immobilized cell culture without pH control of Bifidobacterium animalis subsp. lactis Bb 12—Inhibitory effect of the undissociated organic acids

A model was developed to describe growth and organic acids production of Bifidobacterium animalis growing without pH control in free and immobilized cell culture. The Verlhust model was considered for growth, and to account for the inhibition observed at acidic pH, the Luedeking–Piret production model was modified by introducing an additional term involving the undissociated form of the organic acids, acetic and lactic acids, the main inhibitory species. To describe the relationship between pH and both the dissociated and the undissociated forms of organic acids, the Henderson–Hasselbach equation was considered. The model was found to satisfactory describe experimental growth and production data recorded during free and immobilized cell cultures. The part of each acid produced can be deduced from the calculated production data, since a constant lactic to acetic acid mass ratio was found, 1.29 and 1.66 during free and immobilized cell cultures. Owing to the acidic pH values recorded, 4.43 at lowest, higher amounts of undissociated acetic acid were produced, leading to a higher inhibitory effect of this acid if compared to lactic acid.     

Influence of Medium Buffering Capacity on Inhibition of Saccharomyces cerevisiae Growth by Acetic and Lactic Acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (ΔpH), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.     

Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids

Acetic acid (167 mM) and lactic acid (548 mM) completely inhibited growth of Saccharomyces cerevisiae both in minimal medium and in media which contained supplements, such as yeast extract, corn steep powder, or a mixture of amino acids. However, the yeast grew when the pH of the medium containing acetic acid or lactic acid was adjusted to 4.5, even though the medium still contained the undissociated form of either acid at a concentration of 102 mM. The results indicated that the buffer pair formed when the pH was adjusted to 4.5 stabilized the pH of the medium by sequestering protons and by lessening the negative impact of the pH drop on yeast growth, and it also decreased the difference between the extracellular and intracellular pH values (Delta(pH)), the driving force for the intracellular accumulation of acid. Increasing the undissociated acetic acid concentration at pH 4.5 to 163 mM by raising the concentration of the total acid to 267 mM did not increase inhibition. It is suggested that this may be the direct result of decreased acidification of the cytosol because of the intracellular buffering by the buffer pair formed from the acid already accumulated. At a concentration of 102 mM undissociated acetic acid, the yeast grew to higher cell density at pH 3.0 than at pH 4.5, suggesting that it is the total concentration of acetic acid (104 mM at pH 3.0 and 167 mM at pH 4.5) that determines the extent of growth inhibition, not the concentration of undissociated acid alone.     

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.     

Effects of pH and acetic acid on homoacetic fermentation of lactate by Clostridium formicoaceticum

Clostridium formicoaceticum homofermentatively converts lactate to acetate at 37 degrees C and pH 6.6-9.6. However, this fermentation is strongly inhibited by acetic acid at acidic pH. The specific growth rate of this organism decreased from a maximum at pH 7.6 to zero at pH 6.6. This inhibition effect was found to be attributed to both H(+) and undissociated acetic acid. At pH values below 7.6, the H(+) inhibited the fermentation following non-competitive inhibition kinetics. The acetic acid inhibition was found to be stronger at a lower medium pH. At pH 6.45-6.8, cell growth was found to be primarily limited by a maximum undissociated acetic acid concentration of 0.358 g/L (6mM). This indicates that the undissociated acid, not the dissociated acid, is the major acid inhibitor. At pH 7.6 or higher, this organism could tolerate acetate concentrations of higher than 0.8M, but salt (Na(+)) became a strong inhibitor at concentrations of higher than 0.4M. Acetic acid inhibition also can be represented by noncompetitive inhibition kinetics. A mathematical model for this homoacetic fermentation was also developed. This model can be used to simulate batch fermentation at any pH between 6.9 and 7.6.     

Individual and Combined Effects of pH and Lactic Acid Concentration on Listeria innocua Inactivation: Development of a Predictive Model and Assessment of Experimental Variability

In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH₀) and undissociated lactic acid ([LaH]₀). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH₀ and [LaH]₀ are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.     

Effect of hydrogen sulfide on growth of sulfate reducing bacteria

A culture of sulfate reducing bacteria (SRB) growing on lactate and sulfate was incubated at different pH values in the range of 5.8-7.0. The effect of pH on growth rate was determined in this pH range; the highest growth rate was observed at pH 6.7. Hydrogen sulfide produced from sulfate reduction was found to have a direct and reversible toxicity effect on the SRB. A hydrogen sulfide Concentration of 547 mg/L (16.1 mM) completely inhibited the culture growth. Comparison between acetic acid and hydrogen sulfide inhibition is presented and the concomitant inhibition kinetics are mathematically described. (c) 1992 John Wiley & Sons, Inc.     

Measurement of the Effects of Acetic Acid and Extracellular pH on Intracellular pH of Nonfermenting, Individual Saccharomyces cerevisiae Cells by Fluorescence Microscopy

The effects of acetic acid and extracellular pH (pH_ex) on the intracellular pH (pH_i) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer’s wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pH_ex values was controlled by perfusion with 2 g of total acetic acid per liter at pH_ex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pH_i of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pH_ex 6.5, the pH_i of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pH_ex 3.5, 4.5, and 5.6, the pH_i of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pH_i remained constant. The decreases in pH_i were dependent on the pH_ex; i.e., the decreases in pH_i at pH_ex 5.6 and 6.5 were significantly smaller than the decreases in pH_i at pH_ex 3.5 and 4.5.     

Kinetics of the methanogenic fermentation of acetate

Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, q(ch(4) ) = q(m)S/[K(s) + S + (S/K(i))], where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, K(s), of 4.0 muM for the acclimatized sludge and 104 muM for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 muM (pH 7.50) for the acclimatized sludge and 4.43 muM (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri.     

Regulatory mechanisms of cellular respiration. III. Enzyme distribution in the cell. Its influence on the metabolism of pyruvic acid by bakers' yeast

The rate of the aerobic metabolism of pyruvic acid by bakers' yeast cells is determined mainly by the amount of undissociated acid present. As a consequence, the greatest rate of oxidation was observed at pH 2.8. Oxidation, at a slow rate, started at pH 1.08; at pH 9.4 there was no oxidation at all. The anaerobic metabolism, only a fraction of the aerobic, was observed only in acid solutions. There was none at pH values higher than 3. Pyruvic acid in the presence of oxygen was oxidized directly to acetic acid; in the absence of oxygen it was metabolized mainly by dismutation to lactic and acetic acids, and CO₂. Acetic acid formation was demonstrated on oxidation of pyruvic acid at pH 1.91, and on addition of fluoroacetic acid. Succinic acid formation was shown by addition of malonic acid. These metabolic pathways in a cell so rich in carboxylase may be explained by the arrangement of enzymes within the cell, so that carboxylase is at the center, while pyruvic acid oxidase is located at the periphery. Succinic and citric acids were oxidized only in acid solutions up to pH 4. Malic and α-ketoglutaric acids were not oxidized, undoubtedly because of lack of penetration.     

Distinct Effects of Sorbic Acid and Acetic Acid on the Electrophysiology and Metabolism of Bacillus subtilis

Sorbic acid and acetic acid are among the weak organic acid preservatives most commonly used to improve the microbiological stability of foods. They have similar pK_a values, but sorbic acid is a far more potent preservative. Weak organic acids are most effective at low pH. Under these circumstances, they are assumed to diffuse across the membrane as neutral undissociated acids. We show here that the level of initial intracellular acidification depends on the concentration of undissociated acid and less on the nature of the acid. Recovery of the internal pH depends on the presence of an energy source, but acidification of the cytosol causes a decrease in glucose flux. Furthermore, sorbic acid is a more potent uncoupler of the membrane potential than acetic acid. Together these effects may also slow the rate of ATP synthesis significantly and may thus (partially) explain sorbic acid''s effectiveness.     

Modeling the specific growth rate of Lactobacillus plantarum in cucumber extract

Extensive empirical research has been published on the fermentation of vegetables, but little predictive modeling of the process is available. The objectives of this study were to assess the effects of key variables involved in cucumber fermentation and to develop models for predicting the growth of Lactobacillus plantarum in pure and mixed culture fermentations. The growth medium for the studies was cucumber juice. The effects of various concentrations of lactic, acetic, and hydochloric acids and sodium chloride on growth at 30° C were determined in batch culture. Limiting conditions for growth were pH 3.37 (lower limit), 69 mm undissociated lactic acid, 150 mm undissociated acetic acid, or 11.8% NaCl. Acetic acid was stimulatory to growth at low concentrations (up to 40 mm) but inhibitory at higher concentrations. Lactic acid was more inhibitory than acetic acid, whether total or undissociated concentrations were used as the basis of comparison. A predictive equation for specific growth rate was developed, tested, and shown to predict growth of L. plantarum in batch processes reasonably well.Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U. S. Department of Agriculture or North Carolina Agricultural Research Service, nor does it imply approval to the exclusion of other products that may be suitable Correspondence to: H. P. Fleming     

Growth inhibition of a phytopathogenic fungus, Colletotrichum species by acetic acid

Acetic, oxalic, malic, and citric acids significantly inhibited the growth of Colletotrichum, gloeosporioides, a phytopathogenic fungus, and acetic acid showed the strongest inhibition with no growth at 50 mM. The growth inhibition by these organic acids was closely related with the inhibition of respiration, as tested using three species, C. gloeosporioides, C. coccodes, and C. dematium. Optimum growth of C. gloeosporioides was observed around pH 6.0. The inhibition of growth by acetic acid accelerated along with a decrease in pH from 6.0 to 4.0, suggesting that the inhibition might be more enhanced by undissociated form of acetic acid. Despite of growth inhibition by acetic acid, the fungus was able to grow in a normal medium when acetic acid was eliminated, implying that the growth inhibition may be resulted from an acetic acid-mediated inhibition of respiration than a structural damage of cell. Catalase activity of the fungus increased in response to 0.1% hydrogen peroxide, but addition of this together with 30 mM acetic acid brought about a decrease in the activity. The fungus which showed no grow at 30 mM acetic acid or 0.5% hydrogen peroxide began to grow after the elimination of these. But the fungus added simultaneously by these two compounds did not grow at all despite the elimination of these. Thus, controlling of Colletotrichum might be developed using acetic acid which is generally less dangerous than chemical reagents.     

Individual cells of Saccharomyces cerevisiae and Zygosaccharomyces bailii exhibit different short-term intracellular pH responses to acetic acid

The effects of perfusion with 2.7 and 26 mM undissociated acetic acid in the absence or presence of glucose on short-term intracellular pH (pH(i)) changes in individual Saccharormyces cerevisiae and Zygosaccharomyces bailii cells were studied using fluorescence-ratio-imaging microscopy and a perfusion system. In the S. cerevisiae cells, perfusion with acetic acid induced strong short-term pH(i) responses, which were dependent on the undissociated acetic acid concentration and the presence of glucose in the perfusion solutions. In the Z. bailii cells, perfusion with acetic acid induced only very weak short-term pH(i) responses, which were neither dependent on the undissociated acetic acid concentration nor on the presence of glucose in the perfusion solutions. These results clearly show that Z. bailii is more resistant than S. cerevisiae to short-term pH(i) changes caused by acetic acid.     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

Individual and combined effects of ph and lactic acid concentration on Listeria innocua inactivation: development of a predictive model and assessment of experimental variability

In food technology, organic acids (e.g., lactic acid, acetic acid, and citric acid) are popular preservatives. The purpose of this study was to separate the individual effects of the influencing factors pH and undissociated lactic acid on Listeria innocua inactivation. Therefore, the inactivation process was investigated under controlled, initial conditions of pH (pH0) and undissociated lactic acid ([LaH]0). The resulting inactivation curves consisted of a (sometimes negligible) shoulder period followed by a descent phase. In a few cases, a tailing phase was observed. Depending on the conditions, the descent phase contained one or two log-linear parts or had a convex or concave shape. In addition, the inactivation process was characterized by a certain variability, dependent on the severity of the conditions. Furthermore, in the neighborhood of the growth/no growth interface sometimes contradictory observations occurred. Overall, the individual effects of the influencing factors pH and undissociated lactic acid could clearly be distinguished and were also apparent based on fluorescence microscopy. Appropriate model types were developed and enabled prediction of which conditions of pH0 and [LaH]0 are necessary to obtain a predetermined inactivation (number of decimal reductions) within a predetermined time range.     

Combined effect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast

Summary The internal pH of Saccharomyces cerevisiae IGC 3507 III (a respiratory-deficient mutant) was measured by the distribution of [¹⁴C]propionic acid, when the yeast was fermenting glucose at pH 3.5, 4.5 and 5.5 in the presence of several concentrations of acetic acid and ethanol. Good correlation was obtained between fermentation rates and internal pH. For all external pH values tested, the internal pH was 7.0–7.2 in the absence of inhibitors. The addition of acetic acid and/or ethanol resulted in a decrease of fermentation rate together with a drop in internal pH. Internal pH did not depend on the concentration of total external acetic acid but only on the concentration of the undissociated form of the acid. Ethanol potentiated the effect of acetic acid both with respect to inhibition of fermentation and internal acidification.     

Modeling of Growth of Lactobacillus sanfranciscensis and Candida milleri in Response to Process Parameters of Sourdough Fermentation

We investigated the effect of the ecological factors pH, temperature, ionic strength, and lactate, acetate, and ethanol levels on Candida milleri and two strains of Lactobacillus sanfranciscensis, organisms representative of the microflora of sourdough. A mathematical model describing the single and combined effects of these factors on the growth of these organisms was established in accordance with the following criteria: quality of fit, biological significance of the parameters, and applicability of the in vitro data to in situ processes. The growth rates of L. sanfranciscensis LTH1729 and LTH2581 were virtually identical under all conditions tested. These organisms tolerated >160 mmol of undissociated acetic acid per liter. Growth occurred in the pH range of 3.9 to 6.7 and was completely inhibited by 4% NaCl. C. milleri had a lower optimum temperature for growth (27°C) than the lactobacilli. The growth of the yeast was not affected by pH in the range of 3.5 to 7, and up to 8% NaCl was tolerated. Complete inhibition of growth occurred at 150 mmol of undissociated acetic acid per liter, but acetate at concentrations of up to 250 mmol/liter exerted virtually no effect. The model provides insight into factors contributing to the stability of the sourdough microflora and can facilitate the design of novel sourdough processes.