An extracellular sensor and an extracellular induction component are required for alkali induction of alkyl hydroperoxide tolerance in Escherichia coli

Escherichia coli K12 transferred from pH 7.0 to pH 9.0 gains alkylhydroperoxide (AHP) tolerance. The aim here was to establish whether extracellular components (ECs) are needed for such induction. Therefore, the effects of removing ECs during incubation at pH 9.0 were tested and the abilities of culture filtrates to induce tolerance were examined. First, AHP tolerance did not appear, at pH 9.0, if cultures were subjected to continuous filtration or dialysis, against the same medium, suggesting that an EC might be needed. Second, neutralized filtrates from pH 9.0-grown cultures induced tolerance at pH 7.0, and these filtrates were inactivated by dialysis, filtration or heating but not by protease. Thus, pH 9.0 filtrates have a small non-protein extracellular induction component (EIC), which acts as an alarmone, 'warning' cells of stress and preparing them to resist it. Filtrates from pH 7.0-grown cultures did not induce AHP tolerance at pH 7.0 but if incubated at pH 9.0 without organisms, gained such ability. It is proposed that pH 7.0 filtrates have an EIC precursor (termed an extracellular sensing component, ESC), which senses alkaline pH, and is converted by it to the EIC. The ESC in pH 6.0 filtrates was distinct from that in pH 7.0 filtrates; there may be several oligomeric (or conformational) forms of this ESC. As the EIC is small, it can diffuse away from the alkalinized region and induce tolerance in unstressed organisms.     

An extracellular stress-sensing protein is activated by heat and u.v. irradiation as well as by mild acidity, the activation producing an acid tolerance-inducing protein

During growth of Escherichia coli in broth at pH 5·0, an extracellular protein termed an extracellular induction component (EIC) appears in the medium, this component being essential for acid tolerance induction. The present study establishes that the EIC arises from an extracellular precursor which is formed during growth at pH 7·0, and that conversion of the precursor to the EIC occurs at pH 5·0 (and other mildly acidic pH values) in the absence of organisms. On the basis that this precursor is produced by non-stressed cells as well as by stressed ones, and that it is converted to the EIC (which in turn induces the tolerance response) by the stress, the precursor can be considered to be a stress sensor, the first extracellular stimulus sensor to be reported. The EIC formed at pH 5·0 was inactivated at pH 9·0. This inactivation probably involved conversion back to the precursor as EIC was reformed if the alkali-inactivated component was incubated at pH 5·0. Both mild heat treatments (exposure to 40–55 °C) and u.v. irradiation also activated the precursor; the active induction component formed by the mild heat treatments was reversibly inactivated at pH 9·0 and so it seems likely that the component formed by heat treatment is similar or identical to the EIC produced at acidic pH. In contrast, the EIC produced by u.v. irradiation was not inactivated at pH 9·0, suggesting that it is different in some way to the EICs produced from the precursor by acidity or by heat treatment. It is likely that many responses affecting stress tolerance involve the functioning of such extracellular sensors, as similar components were shown to be involved in the acid tolerance responses induced at pH 7·0 by glucose, l -aspartate and l -glutamate. Extracellular stimulus sensors may also be needed for other inducible responses.     

Properties of an L-glutamate-induced acid tolerance response which involves the functioning of extracellular induction components

Escherichia coli became more acid tolerant following incubation for 60 min in a medium containing L-glutamate at pH 7.0, 7.5 or 8.5. Several agents, including cAMP, NaCl, sucrose, SDS and DOC, prevented tolerance appearing if present with L-glutamate. Lesions in cysB, hns, fur, himA and relA, which frequently affect pH responses, failed to prevent L-glutamate-induced acid tolerance but a lesion in L-glutamate decarboxylase abolished the response. Induction of acid tolerance by L-glutamate was associated with the accumulation in the growth medium of a protein (or proteins) which was able to convert pH 7.0-grown cultures to acid tolerance, and the original L-glutamate-induced tolerance response was dependent on this component(s). Acid tolerance was also induced by L-aspartate at pH 7.0 and induction of such tolerance was dependent on an extracellular protein (or proteins). The L-glutamate and L-aspartate acid tolerance induction processes are further examples of a number of stress tolerance responses which differ from most inductions in that extracellular components, including extracellular sensors, are required.     

Adaptive acid tolerance response of Streptococcus sobrinus

Streptococcus mutans and Streptococcus sobrinus are the bacteria most commonly associated with human dental caries. A major virulence attribute of these and other cariogenic bacteria is acid tolerance. The acid tolerance mechanisms of S. mutans have begun to be investigated in detail, including the adaptive acid tolerance response (ATR), but this is not the case for S. sobrinus. An analysis of the ATR of two S. sobrinus strains was conducted with cells grown to steady state in continuous chemostat cultures. Compared with cells grown at neutral pH, S. sobrinus cells grown at pH 5.0 showed an increased resistance to acid killing and were able to drive down the pH through glycolysis to lower values. Unlike what is found for S. mutans, the enhanced acid tolerance and glycolytic capacities of acid-adapted S. sobrinus were not due to increased F-ATPase activities. Interestingly though, S. sobrinus cells grown at pH 5.0 had twofold more glucose phosphoenolpyruvate:sugar phosphotransferase system (PTS) activity than cells grown at pH 7.0. In contrast, glucose PTS activity was actually higher in S. mutans grown at pH 7.0 than in cells grown at pH 5.0. Silver staining of two-dimensional gels of whole-cell lysates of S. sobrinus 6715 revealed that at least 9 proteins were up-regulated and 22 proteins were down-regulated in pH 5.0-grown cells compared with cells grown at pH 7.0. Our results demonstrate that S. sobrinus is capable of mounting an ATR but that there are critical differences between the mechanisms of acid adaptation used by S. sobrinus and S. mutans.     

Fatty acid specificity of immobilized kid goat pre-gastric esterase: Effects of pH

Summary Kid goat pre-gastric esterase immobilized in a hollow fiber reactor was used to hydrolyse butteroil at buffer pH values of 5.0, 6.0 and 7.0. Overall hydrolysis proceeded fastest at pH 6.0, but changes in volatile fatty acid ratios with pH suggest that the same enzyme:substrate system can produce different flavor profiles, e. g., at pH 6.0 the relative rate of production of undesirable soaplike flavors is minimized.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer.

Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium. Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0. Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0. DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0. Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Cytoplasmic pH response to acid stress in individual cells of Escherichia coli and Bacillus subtilis observed by fluorescence ratio imaging microscopy

The ability of Escherichia coli and Bacillus subtilis to regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. The E. coli cytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In an E. coli biofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells. B. subtilis cells were acid shifted from pH 7.5 to pH 6.0. In B. subtilis, unlike the case with E. coli, cytoplasmic pH showed no "overshoot" but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individual B. subtilis cells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.     

Effect of pH and ionic strength on the catalytic and allosteric properties of native and chemically modified ox liver mitochondrial glutamate dehydrogenase.

Inorganic phosphate, a strong activator of glutamate dehydrogenase at pH 8.0–9.0, is an inhibitor at pH 6.0–7.6. The extent of inhibition increases with the decrease of pH. The same effect is shown by other electrolytes, including Tris-hydroxymethyl-aminomethane and NaCl.The combined effect of pH and ionic strength also alters the allosteric characteristics of the enzyme. Lowering the pH minimizes the activation by high concentrations of NAD; phosphate partially restores this activation. The allosteric activation by ADP disappears at pH around neutrality; in the pH range 6.0–7.0, ADP becomes a strong inhibitor, the inhibition being enhanced by the addition of ionic compounds. Similarly, the extent of allosteric inhibition by guanosine 5′-triphosphate (pyro) (GTP), which is maximal at pH 9.0, decreases at lower pH values and a slight activation is observed in the presence of electrolytes at pH 6.0.Glutamate dehydrogenase, selectively desensitized by dinitrophenylation in the presence of ADP, can be activated by ADP at pH 9.0, but is no longer inhibited by the same effector at pH 6.0, high salt concentration. The densensitized enzyme is not inhibited by GTP at pH 9.0, but is activated by this effector at pH 6.0 in the presence of ionic compounds. Conversely, GTP-protected dinitrophenylated glutamate dehydrogenase is desensitized only to the effect of the activating modifier, ADP at pH 9.0, GTP at pH 6.0, high salt concentration. These findings suggest that the conformation of each allosteric site of glutamate dehydrogenase is changed by pH and ionic strength so that it keeps its specificity for the ligand which brings about a given effect, activation or inhibition, independently from its chemical structure.     

The in vitro interaction of naphthyridinomycin with deoxyribonucleic acids

The binding of naphthyridinomycin (NAP) to deoxyribonucleic acid was investigated using radioisotope labeled antibiotic. Dithiothreitol (DTT) enhances complex formation in a concentration dependent fashion but was found to be slightly inhibitory at concentrations above 10 mM. [C3H3]-NAP-DNA complexes, formed in the presence or absence of reducing reagents, were stable to Sephadex G-25 chromatography and precipitation with ethanol, indicating a strong bond formed between the drug and DNA. Time course studies showed that the difference between the binding of activated and non-activated antibiotic was a DTT-dependent burst. This was followed by a second phase of binding which was similar in both the activated and non-activated antibiotics. The activation of the antibiotic by DTT was a reversible reaction at pH 7.9. The activated form at pH 5.0 was extremely stable and did not revert to the unactivated form even after an 8-h incubation period. Antibiotic-DNA complex formation was pH independent between pH 5.0 and 7.0 for activated NAP. The non-activated antibiotic bound to DNA much better at pH 5.0 than at physiological pH values. Release of antibiotic from complexes (as followed by long term dialysis) formed in the presence of DTT and at pH 5.0 was biphasic, suggesting that the drug can bind to DNA in more than one way. A constant rate of antibiotic release was observed at pH 7.9 with or without DTT. At pH 2.0 and pH 12.0, greater than 95% of the antibiotic is released from the complexes. Most of the acid released antibiotic is NAP while most of the base released antibiotic had decomposed to a more polar compound. NAP binds well to calf thymus DNA, poly(dG) . poly(dC), and T4 DNA but shows significantly less affinity for poly(dA) . poly(dT), poly(dA . dT) . poly(dA . dT), poly(dG), poly(dC), poly(dI) . poly(dC) or poly(dG . dC) . poly(dG . dC). This specificity of NAP for DNA is similar to that observed for the pyrrolo(1,4)benzodiazepine antibiotics and saframycin A and S; all of which bind to double stranded DNA through their carbinolamine or masked carbinolamine functionalities. Two mechanisms which can explain the need for activation of NAP are also proposed.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Novel acid sensitivity induced in Escherichia coli at alkaline pH

Transfer of pH 7.0-grown Escherichia coli to pH 9.0 led to rapid acid sensitivity induction (ASI), the response being fully accomplished within 15 min at 37°C in broth. Only a slight increase in acid sensitivity occurred at pH 8.2 but the response was substantial at pH 8.4 and complete at pH 9.0 with no further sensitization at pH 9.5–10.5. ASI was not prevented by lesions in rpoH, katF, ompR, relA, spoT, fur, phoU, phoM (CreC), phoB/R, unc(atp), phoP or cadA and was unaffected by nalidixic acid, L-leucine or iron starvation or excess. Full acid sensitivity was maintained for at least 2 h after a shift from pH 9.0 back to pH 7.0. ASI did not depend to a major extent on PhoE derepression and increased acid sensitivity of alkali-induced strain C75a ( phoE⁺ ) probably did not involve use of a new outer membrane proton pore.     

Factors influencing biohydrogenation and conjugated linoleic acid production by mixed rumen fungi

The objective of this study was to evaluate the effect of soluble carbohydrates (glucose, cellobiose), pH (6.0, 6.5, 7.0), and rumen microbial growth factors (VFA, vitamins) on biohydrogenation of linoleic acid (LA) by mixed rumen fungi. Addition of glucose or cellobiose to culture media slowed the rate of biohydrogenation;only 35-40% of LA was converted to conjugated linoleic acid (CLA) or vaccenic acid (VA) within 24 h of incubation, whereas in the control treatment, 100% of LA was converted within 24 h. Addition of VFA or vitamins did not affect biohydrogenation activity or CLA production. Culturing rumen fungi at pH 6.0 slowed biohydrogenation compared with pH 6.5 or 7.0. CLA production was reduced by pH 6.0 compared with control (pH 6.5), but was higher with pH 7.0. Biohydrogenation of LA to VA was complete within 72 h at pH 6.0, 24 h at pH 6.5, and 48 h at pH 7.0. It is concluded that optimum conditions for biohydrogenation of LA and for CLA production by rumen fungi were provided without addition of soluble carbohydrates, VFA or vitamins to the culture medium; optimum pH was 6.5 for biohydrogenation and 7.0 for CLA production.     

Humic acid and iron uptake by plants

Summary The behaviour of ferric EDTA and ferric citrate in nutrient solution and their interaction with humic acid was investigated at various hydrogen ion concentrations using the technique of membrane ultrafiltration to separate small iron species from high molecular weight products of hydrolysis and to estimate the binding of iron by humic acid. Ferric EDTA was found to be of small molecular size at all pH values between 5.0 and 7.0 whilst ferric citrate solutions contained an increasing proportion of high molecular weight material as pH was increased from 5.0 to 7.0. Some iron present in solutions of both ferric EDTA and ferric citrate was bound by humic acid at all pH values from 5.0 to 7.0. Studies were also made of the uptake of iron by wheat roots from nutrient solutions containing either ferric EDTA or ferric citrate and of the effect of humic acid on uptake. More iron was absorbed from ferric EDTA than from ferric citrate at all pH values. Increasing pH between 5.0 and 7.0 resulted in a progressive decrease in the uptake of iron in both cases. The presence of humic acid depressed iron absorption from both solutions at all pH values.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Survival of a Salmonella typhimurium poultry isolate in the presence of propionic acid under aerobic and anaerobic conditions

Propionic acid is commonly found as a fermentation product in the gastrointestinal tracts of food animals and has also been used to limit the microbial contaminants in animal feeds. Because propionic acid is known to have antibacterial activity, the propionic acid encountered by foodborne pathogens during their life cycles may play an important role in inhibiting the survival of the pathogens. The survival patterns of Salmonella typhimurium poultry isolate were determined both in aerobic and anaerobic tryptic soy broth (TSB; pH 5.0 or 7.0) containing various concentrations of propionic acid (0-200 mM). The levels of recovered cells were consistently greater at pH 7.0 compared to those at pH 5.0. For the first 4 days, the levels were significantly decreased by incubation under anaerobic conditions as compared to aerobic condition at pH 7.0 (P<0.05). However, there were fluctuations of cell populations with different patterns depending on both concentrations and growth conditions. To characterize the nature of the capability which allowed the cell multiplication following decreases in cell population during incubation at pH 7.0, the cells isolated from the outgrowth cultures were tested for survival in aerobic or anaerobic TSB (pH 5.0 or pH 7.0) containing propionic acid (50 mM). The outgrowth isolates did not show significant differences in the level of recovered cells in the presence of propionic acid when compared to the wild type strain (P>0.05), suggesting that the cells in the outgrowth cultures did not harbour mutation(s) conferring increased resistance to propionic acid. In addition, the level of recovered cells of isogenic rpoS mutant strain of S. typhimurium was not significantly different from that of the wild type strain in the same assay conditions (P<0.05). The results of this study show that the bactericidal activity of propionic acid on S. typhimurium can be affected by environmental conditions such as acidic pH levels and anaerobiosis in food materials and gastrointestinal tracts. However, S. typhimurium is also able to multiply in the presence of sublethal concentrations of propionic acid at neutral pH during prolonged incubation under both aerobic and anaerobic conditions.     

Exposure of Escherichia colito acid habituation conditions sensitizes it to alkaline stress

R.J. ROWBURY AND N.H. HUSSAIN. 1996. Escherichia coli transferred from pH 7.0 to pH 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response. Alkali sensitization was reduced at pH 5.0 and virtually abolished at pH 6.5. The response was triggered by cytoplasmic rather than external or periplasmic acidification and de novo protein synthesis was needed. Alkali sensitivity failed to appear at pH 5.5 plus DNA gyrase inhibitors and was markedly reduced by himA, himD, hns, ompC and nhaA lesions. A tonB deletion mutant showed alkali sensitivity at pH 7.0. Alkali sensitivity induction was not subject to catabolite repression nor was it appreciably affected by a relA lesion. Acid-induced cells were more sensitive to alkali damage to both DNA and β-galactosidase and to alkali inhibition of β-galactosidase induction. Alkali sensitization induced at pH 5.5 may involve NhaB loss.     

Effect of lysozyme on mycobacteria

The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.     

Mechanism of acid tolerance in a rhizobium strain isolated from Pueraria lobata (Willd.) Ohwi

The Rhizobium sp. strain PR389 was isolated from the root nodules of Pueraria lobata (Willd.) Ohwi, which grows in acidic (pH 4.6) yellow soil of the Jinyun Mountains of Beibei, Chongqing, China. While rhizobia generally have a pH range of 6.5-7.5 for optimum growth, strain PR389 grew in a liquid yeast extract - mannitol agar medium at pH 4.6, as well as in a pH 4.1 soil suspension, suggesting acid tolerance in this specific strain of rhizobium . However, at pH 4.6, the lag phase before vigorous growth was 40 h compared with 4 h under neutral conditions (pH 7.0). For PR389, the generation time after the lag phase remained the same at different pH levels despite the different durations of the lag phase. Except in the pH 4.4 treatment, the pH of the culturing media increased from 4.6, 4.8, 5.0, and 5.5 to neutral and slightly alkaline after 70 h of culture. Chloramphenicol was added to determine if protein production was involved in the increasing pH process. Chloramphenicol significantly inhibited PR389 growth under acid stress but had little effect under neutral conditions. Proton flux measured during a short acid shock (pH 3.8) revealed that this strain has an intrinsic ability to prevent H(+) from entering cells when compared with acid-sensitive rhizobia. We propose that the mechanism for acid tolerance in PR389 involves both intracellular and extracellular processes. When the extracellular pH is lower than pH 4.4, the cell membrane blocks hydrogen from entering the cell. When the pH exceeds 4.4, the rhizobium strain has the ability to raise the extracellular pH, thereby, potentially decreasing the toxicity of aluminum in acid soil.     

Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH?<?6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5–7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.     

Extracellular acidification leads to reactive oxygene species formation in rat brain synaptosomes

Formation of reactive oxygen species in rat brain synaptosomes was studied using DCFDA fluorescent dye at lowered extracellular pH. It has been shown that decrease in pH value from 7.4 to 7.0 and up to 6.0 leads to increase of fluorescence that is indicative of oxidative stress. The effect is observed regardless of whether Ca ions are present in incubation medium or no. Acidification of the incubation medium induces quenching of fluorescence of previously oxidized form of the dye in experiments without synaptosomes This evidences that increase of dye fluorescence is really associated with reactive oxygen species accumulation. Thus, it has been demonstrated that pH declined up to 7.0 in the incubation medium is sufficient to induce the formation of reactive oxygen species in synaptosomes.