Control of Acid Resistance in Escherichia coli

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.     

Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli.

Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pH-inducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.     

Mechanisms of acid resistance in enterohemorrhagic Escherichia coli.

Enterohemorrhagic strains of Escherichia coli must pass through the acidic gastric barrier to cause gastrointestinal disease. Taking into account the apparent low infectious dose of enterohemorrhagic E. coli, 11 O157:H7 strains and 4 commensal strains of E. coli were tested for their abilities to survive extreme acid exposures (pH 3). Three previously characterized acid resistance systems were tested. These included an acid-induced oxidative system, an acid-induced arginine-dependent system, and a glutamate-dependent system. When challenged at pH 2.0, the arginine-dependent system provided more protection in the EHEC strains than in commensal strains. However, the glutamate-dependent system provided better protection than the arginine system and appeared equally effective in all strains. Because E. coli must also endure acid stress imposed by the presence of weak acids in intestinal contents at a pH less acidic than that of the stomach, the ability of specific acid resistance systems to protect against weak acids was examined. The arginine- and glutamate-dependent systems were both effective in protecting E. coli against the bactericidal effects of a variety of weak acids. The acids tested include benzoic acid (20 mM; pH 4.0) and a volatile fatty acid cocktail composed of acetic, propionic, and butyric acids at levels approximating those present in the intestine. The oxidative system was much less effective. Several genetic aspects of E. coli acid resistance were also characterized. The alternate sigma factor RpoS was shown to be required for oxidative acid resistance but was only partially involved with the arginine- and glutamate-dependent acid resistance systems. The arginine decarboxylase system (including adi and its regulators cysB and adiY) was responsible for arginine-dependent acid resistance. The results suggest that several acid resistance systems potentially contribute to the survival of pathogenic E. coli in the different acid stress environments of the stomach (pH 1 to 3) and the intestine (pH 4.5 to 7 with high concentrations of volatile fatty acids). Of particular importance to the food industry was the finding that once induced, the acid resistance systems will remain active for prolonged periods of cold storage at 4 degrees C.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance.

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Arginine deiminase system and bacterial adaptation to acid environments

The arginine deiminase system in a variety of streptococci and in Pseudomonas aeruginosa was found to be unusually acid tolerant in that arginolysis occurred at pH values well below the minima for growth and glycolysis. The acid tolerance of the system allowed bacteria to survive potentially lethal acidification through production of ammonia to raise the environmental pH value.     

Characterization of L-aspartate uptake by Streptomyces hydrogenans.

Multiple transport systems for L-aspartic acid exist in Steptomyces hydrogenans. The intracellular accumulation of L-aspartate against a concentration gradient was immediately inhibited by proton conductors, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone, 2,4-dinitrophenol or nigericin. Transport activity was gradually lost when inhibitors of protein synthesis were added. L-Aspartate transport had two pH optima at 6.5 and 4.5. At pH 6.5, two saturable transport components with different Km and Vmax values could be resolved by kinetic studies. A high-affinity system (system I) preferred the L-isomers of the anionic forms of aspartic and glutamic acid. At the same pH, a second, low-affinity system (system II) operated, which was presumably less specific than system I and also able to accept, at high concentrations, neutral amino acids. At pH 4.5, the Lineweaver-Burk plot revealed only a single catalytic component, with Km and Vmax values similar to those of system II. Again, in contrast to system I, this component showed high affinity for neutral amino acids. The data suggest that L-aspartic acid and L-glutamic acid are transported by this system as neutral zwitterionic molecules.     

Acid tolerance of an acid mine drainage bioremediation system based on biological sulfate reduction

The acid tolerance response of an AMD bioremediation system based on sulfate reduction was investigated. Efficient sulfate reduction was observed with a maximum sulfate reduction rate of 12.3±0.8 mg L(-1) d(-1) and easily available organic carbon was released during high acid treatment with an initial pH of 2.0. The rapid increase in sulfate reduction was observed when the extreme acid treatment with an initial pH of 1.0 was stopped. Column experiment on acid shock showed that efficient sulfate reduction was maintained while precipitation of Cu or Zn still occurred during extreme or high acid shock. More than 98% of Cu and 85% of Zn were removed in the high acid column experiment with influent pH of 2.0. The majority bacteria in the remediation system used for high acid drainage belonged to genera Clostridiaceae, Eubacterium, Pseudobutyrivibrio, and Clostridium. These findings showed high acid tolerance of the straw remediation system.     

Gene expression profiling of the pH response in Shigella flexneri 2a

The pH response of Shigella flexneri 2a 301 was identified by gene expression profiling. Gene expression profiles of cells grown in pH 4.5 or 8.6 were compared with the profiles of cells grown at pH 7.0. Differential expression was observed for 307 genes: 97 were acid up-regulated, 102 were acid down-regulated, 91 were base up-regulated, and 86 were base down-regulated. Twenty-seven genes were found to be both acid and base up-regulated, and 29 genes were both acid and base down-regulated. This study showed that (1) the most pH-dependent genes regulate energy metabolism; (2) the RpoS-dependent acid-resistance system is induced, while the glutamate-dependent acid resistance system is not; (3) high pH up-regulates some virulence genes, while low pH down-regulates them, consistent with Shigella infection of the low gut; and (4) several cross-stress response genes are induced by pH changes. These results also illustrate that many unknown genes are significantly regulated under acid or basic conditions, providing researchers with important information to characterize their function.     

Biochemical Conversion of Uridine Diphosphate Glucose to Uridine Diphosphate 3-Ketoglucose by Washing Cells of Agrobacterium tumefaciens

The relation between the rate of increase in nonprotein nitrogenous compounds (NPN) of rabbit muscle and muscle pH ranging from 5.9 to 7.2 was examined during the post-mortem storage. Muscle of a high ultimate pH was prepared by the injection of ICH₂COOH into the vein. The more the muscle pH kept away from 6.3, the more NPN increased. Therefore, it has been suggested that the post-mortem proteolysis is mainly attributed to the acid proteolytic system comprising cathepsins in muscles at a pH lower than 6.3 and to the neutral proteolytic system in muscles at a pH higher than 6.3.

The ratio of the increment of ninhydrin positive materials to that of Cu-Folin phenol reagent positive materials among NPN was relatively large in muscles at a high pH. This result has suggested that the neutral proteolytic system was more abound in exopeptidase activity than acid proteolytic system.     

Transport of Tricarboxylic Acids in Salmonella typhimurium

Salmonella typhimurium possesses at least three inducible transport systems for the tricarboxylic acids (citric, isocitric, cis-aconitic, and tricarballylic). The first system was induced by citrate, isocitrate, or cis-aconitate, and transported citric acid and isocitric acid. The second system was also induced by the same acids as in the first system and transported cis-aconitic acid. This system required Mg(2+) ions and was stable at pH 8.4 but unstable at pH 7.0. The metal ion was replaced with Sr(2+) or Ca(2+) ions but not with Ba(2+) ions. The third system was induced by tricarballylate and transported citric acid, cis-aconitic acid, and tricarballylic acid.     

Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium

Salmonella typhimurium possesses an adaptive response to acid that increases survival during exposure to extremely low pH values. The acid tolerance response (ATR) includes both log-phase and stationary-phase systems. The log-phase ATR appears to require two components for maximum acid tolerance, namely an inducible pH homeostasis system, and a series of acid-shock proteins. We have discovered one of what appears to be a series of inducible exigency pH homeostasis systems that contribute to acid tolerance in extreme acid environments. The low pH-inducible lysine decarboxylase was shown to contribute significantly to pH homeostasis in environments as low as pH 3.0. Under the conditions tested, both lysine decarboxylase and σ^s-dependent acid-shock proteins were required for acid tolerance but only lysine decarboxylase contributed to pH homeostasis. The cadBA operon encoding lysine decarboxylase and a lysine/cadaverine antiporter were cloned from S. typhimurium and were found to be 79% homologous to the cadBA operon from Escherichia coli . The results suggest that S. typhimurium has a variety of means of fulfilling the pH homeostasis requirement of the ATR in the form of inducible amino acid decarboxylases.     

华南重要人工林及地带性森林凋落物酸碱缓冲能力

植物残体可以修止土壤酸度,但其作用机理还有很大争议,本研究评估森林凋落物作为酸碱缓冲体系的能力,它可能是调节土壤酸度的重要机理之一。凋落物材料取自华南重要的人工林类型及一个地带性顶极森林群落,将材料用不同pH酸溶液提取,并用酸碱进行滴定。结果表明,凋落物本身是一个极强的酸碱缓冲体系,它使浸提液酸度保持不变,并对添加的酸碱起显著的缓冲作用。首次发现两个豆科树种凋落物马占相思（Acacia mangium Willd)与大叶相思(Acacia auriculaiformis A.Cunn)具有很高的pH值(约pH6．0),约高于土壤酸度2个pH单位,每年凋落于地表的枯落物层通过缓冲机理可以提高雨水0．1至0．4pH单位。其它人工林凋落物酸度约为pH4．0,与它们生长的土壤酸度相似。溶液中无机离子组成上的差异不能完全解释凋落物pH格局,但高钠低硝态氮可能是两个豆科树种高pH的一个重要原因。地带性顶极群落12个树种加权酸度为pH3．90,明显低于土壤酸度(pH4．19),因而,凋落物是林下土壤进一步变酸的驱动因素;发现一个种凋落物(山竹子(Garcinia oblongifolia))酸度极低(pH3．19)并且缓冲力极强,该种植物生长的土壤将难于通过施用石灰等措施对酸度进行调节,它影响下的土壤环境将影响其它植物的入侵。观光木(Tsoongiodendron odorum Chun)凋落物酸度碱向最远离土壤酸度,这种凋落物酸度与环境酸度间的不一致可能对植物形成某种长期生态压力,并成为该种致濒危的一个因素。因而,植物残体是一个极强的酸碱缓冲体系,它以化学缓冲机理可以影响环境酸度,但可能只有少数种通过这一机理使土壤致酸或致碱,因为多数植物残体与土壤酸度基本相似。     

华南重要人工林及地带性森林凋落物酸碱缓冲能力

植物残体可以修正土壤酸度,但其作用机理还有很大争议,本研究评估森林凋落物作为酸碱缓冲体系的能力,它可能是调节土壤酸度的重要机理之一.凋落物材料取自华南重要的人工林类型及一个地带性顶极森林群落,将材料用不同pH酸溶液提取,并用酸碱进行滴定.结果表明,凋落物本身是一个极强的酸碱缓冲体系,它使浸提液酸度保持不变,并对添加的酸碱起显著的缓冲作用.首次发现两个豆科树种凋落物马占相思(Acacia mangiumWilld)与大叶相思(Acacia auriculaiformis A.Cunn)具有很高的pH值(约pH6.0),约高于土壤酸度2个pH单位,每年凋落于地表的枯落物层通过缓冲机理可以提高雨水0.1至0.4pH单位.其它人工林凋落物酸度约为pH4.0,与它们生长的土壤酸度相似.溶液中无机离子组成上的差异不能完全解释凋落物pH格局,但高钠低硝态氮可能是两个豆科树种高pH的一个重要原因.地带性顶极群落12个树种加权酸度为pH3.90,明显低于土壤酸度(pH4.19),因而,凋落物是林下土壤进一步变酸的驱动因素;发现一个种凋落物(山竹子(Garcinia oblongifolia))酸度极低(pH 3.19)并且缓冲力极强,该种植物生长的土壤将难于通过施用石灰等措施对酸度进行调节,它影响下的土壤环境将影响其它植物的入侵.观光木(Tsoongiodendron odorum Chun)凋落物酸度碱向最远离土壤酸度,这种凋落物酸度与环境酸度间的不一致可能对植物形成某种长期生态压力,并成为该种致濒危的一个因素.因而,植物残体是一个极强的酸碱缓冲体系,它以化学缓冲机理可以影响环境酸度,但可能只有少数种通过这一机理使土壤致酸或致碱,因为多数植物残体与土壤酸度基本相似.     

Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid Hh, and regulation of pectolytic enzymes

Erwinia chrysanthemi is a phytopathogenic bacterium that causes soft-rot diseases in a broad number of crops. The PhoP-PhoQ system is a key factor in pathogenicity of several bacteria and is involved in the bacterial resistance to different factors, including acid stress. Since E. chrysanthemi is confronted by acid pH during pathogenesis, we have studied the role of this system in the virulence of this bacterium. In this work, we have isolated and characterized the phoP and phoQ mutants of E. chrysanthemi strain 3937. It was found that: (i) they were not altered in their growth at acid pH; (ii) the phoQ mutant showed diminished ability to survive at acid pH; (iii) susceptibility to the antimicrobial peptide thionin was increased; (iv) the virulence of the phoQ mutant was diminished at low and high magnesium concentrations, whereas the virulence of the phoP was diminished only at low magnesium concentrations; (v) in planta Pel activity of both mutant strains was drastically reduced; and (vi) both mutants lagged behind the wild type in their capacity to change the apoplastic pH. These results suggest that the PhoP-PhoQ system plays a role in the virulence of this bacterium in plant tissues, although it does not contribute to bacterial growth at acid pH.     

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.     

Modeling and optimization in anaerobic bioconversion of complex substrates to acetic and butyric acids

Cheese-processing wastewater was biologically treated to produce short-chain organic acids in laboratory scale continuously stirred tank reactors. A constant inoculum system was used to mimimize the experimental error due to the use of inconsistent inoculum. The inoculum system was operated with dilute cheese-processing wastewater with 5000 mg soluble chemical oxygen demand/L at pH 6.5 and 35 degrees C at 0.5 days hydraulic retention time. Response surface methodology was successfully applied to determine the optimum physiological conditions where the maximum rates of acetic and butyric acid production occurred. These were pH 7.01 at 36.2 degrees C and pH 7.26 at 36.2 degrees C, respectively. The lack of overall predictability for butyric acid production meant that the response surface was much more complicated than that of acetic acid; therefore, a small change in pH or temperature could cause large variations in the response of butyric acid production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54:451-460, 1997.