Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger

Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal. We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A. niger grown in computer-controlled bioreactors. On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled.     

The effect of different dissolved oxygen tensions on growth and enzyme activities of Campylobacter sputorum subspecies bubulus

Campylobacter sputorum subspecies bubulus was grown in batch cultures in which the dissolved oxygen tension (d.o.t) was maintained at various constant levels. At a range of d.o.t. from 0.002 to 0.05 atm, which allowed good growth (mean generation time approximately 1.5 h), L-lactate was preferentially consumed before D-lactate. L-lactate oxidation was accompanied by equimolar acetate production during exponential growth. A value for YL-lactate (g dry weight bacteria per mol L-lactate) of 54 was determined. Net acetate production stopped when C. sputorum started to use D-lactate after consumption of L-lactate. When a culture growing exponentially at the expense of L-lactate was shifted from a d.o.t. of 0.02 atm to a d.o.t. of 0.15 atm, growth was impaired, and L-lactate consumption and corresponding acetate production diminished. This decrease correlated with a loss of lactate dehydrogenase activity after the shift. Campylobacter sputorum appeared to possess cytochromes of the b- and c-type and a carbon monoxide-binding pigment. Evidence is given that the principal site of oxygen damage is lactate dehydrogenase rather than the cytochrome chain.     

Effect of extra aeration on extracellular enzyme activities and ATP concentration of dairy Pseudomonas fluorescens

The effect of forced aeration on extracellular enzyme synthesis during batch growth of a Pseudomonas fluorescens strain of dairy origin on pyruvate mineral salts medium at 7 degrees C was studied. Measurement of oxygen tension, electron micrographs to estimate cell volume, luciferase determination of ATP and plate counts were performed in the course of incubation. Cells from the stationary phase of growth had lower energy status (in terms of intracellular ATP concentration) in the cultures receiving surplus aeration. Those cells produced three times more extracellular proteinase and lipase than control cells. Onset time for production of both enzymes coincided with a sharp fall of intracellular ATP levels.     

Effects of Glucose, pH, and Dissolved-Oxygen Tension on Bacillus cereus Growth and Permeability Factor Production in Batch Culture

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

Cybernetic model of the growth dynamics of Saccharomyces cerevisiae in batch and continuous cultures.

Growth of Saccharomyces cerevisiae on glucose in aerobic batch culture follows the well-documented diauxic pattern of completely fermenting glucose to ethanol during the first exponential growth phase, followed by an intermediate lag phase and a second exponential growth phase consuming ethanol. In continuous cultures over a range of intermediate dilution rates, the yeast bioreactor exhibits sustained oscillations in all the measured concentrations, such as cell mass, glucose, ethanol, and dissolved oxygen, the amounts of intracellular storage carbohydrates, such as glycogen and trehalose, the fraction of budded cells as well as the culture pH. We present here a structured, unsegregated model for the yeast growth dynamics developed from the 'cybernetic' modeling framework, to simulate the dynamic competition between all the available metabolic pathways. This cybernetic model accurately predicts all the key experimentally observed aspects: (i) in batch cultures, duration of the intermediate lag phase, sequential production and consumption of ethanol, and the dynamics of the gaseous exchange rates of oxygen and carbon dioxide; and (ii) in continuous cultures, the spontaneous generation of oscillations as well as the variations in period and amplitude of oscillations when the dilution rate or agitatin rate are changed.     

Effects of glucose, pH, and dissolved-oxygen tension on Bacillus cereus growth and permeability factor production in batch culture.

The production of a Bacillus cereus enterotoxin, measured as rabbit skin permeability factor (PF), in response to differences in glucose availability, pH, and dissolved oxygen tension was studied in a 1-liter batch fermentor system. Glucose had to be present for toxigenesis to occur. In uncontrolled fermentation an increasing inhibition of PF production and growth occurred as pH dropped occurred below 6.5. Optimum pH for toxigenesis was 7.0 to 7.5, and fermentations maintained at this level yielded 10- to 20-fold more PF than comparable uncontrolled fermentations. PF production was appreciably diminished at or below pH 6.0 and at or above pH 8.5. Peak PF titer was associated with a drop in acid output, and the titrant utilization profile could be used as an indication of this point. Productivity was greatest in the early exponential phase of growth and decreased to zero at the transition phase. Differences in dissolved oxygen tension affected both the maximum productivity early in the fermentation and the rate of its decrease as growth progressed. The optimum dissolved oxygen tension for toxigenesis was 0.002 atm, and the most rapid growth occurred at 0.10 atm. Productivity and growth were reduced under anerobic conditions, whereas a hyperoxic environment severely reduced productivity, but not growth. Postexponential-phase loss of toxic activity coincided with a rapid increase in cellular oxygen demand. Neither was inhibited by the presence of glucose. However, PF loss was completely prevented by stringent oxygen limitation. Extracellular proteolytic activity did not appear to be responsible for the loss of toxic activity.     

Monitoring of haploid maize cell suspension culture conditions in bioreactors

Maize (Zea mays L.) haploid cells were cultivated in a 1500 ml aerated and stirred batch bioreactor using modified BM medium. Cell growth was highly affected by pH and dissolved oxygen, and we observed two fairly distinct growth phases. During the first two days after inoculation at pH 5.8, oxygen consumption was high and the cells lowered the pH to a value around 4.3. After this period the pH stabilized at 4.5 and the dissolved oxygen reached a steady level. Decreasing dissolved oxygen concentration leads to lower growth rate and to higher pH. Both events mean stress conditions for the cell culture and probably result in increased genetic variability, and the loss of regeneration capacity. The stress condition during the adaptation phase can be eliminated by decreasing the pH of the medium to 4.7 before inoculation and by keeping dissolved oxygen above 40%. These conditions provide prolonged exponential growth dynamics and the cell suspensions could be the basis of large scale cultures also.Abbreviations 2,4-d 2,4-dichlorophenoxyacetitc acid - NAA naphthalene acetic acid     

GROWTH AND DOMOIC ACID PRODUCTION BY PSEUDO‐NITZSCHIA SERIATA (BACILLARIOPHYCEAE) UNDER PHOSPHATE AND SILICATE LIMITATION1

Pseudo‐nitzschia seriata (Cleve) H. Peragallo isolated from Scottish west coast waters was studied in batch culture with phosphate (P) or silicate (Si) as the yield‐limiting nutrient at 15°C. This species produced the neurotoxin domoic acid (DA) when either nutrient was limiting but produced more when stressed by Si limitation during the stationary phase. Under P‐limiting conditions, exponential growth stopped after P was reduced to a low threshold concentration. Under Si‐limiting conditions, fast exponential growth was followed by a period of slower exponential growth, until Si became exhausted. A stationary phase was observed in the P‐limited but not the Si‐limited cultures, the latter showing a rapid decrease in cell density after the second exponential growth phase. Si‐limited cultures exhibited a further period of active metabolism (as indicated by increases in chl and carbon per cell) late in the experiment, presumably fueled by regenerated Si. DA production was low in exponential phase under both conditions. In P‐limited cultures, most DA was produced during the immediate postexponential phase, with little or no new DA produced during later cell senescence. In contrast, although a substantial amount of DA was produced during the slower exponential phase of the Si‐limited cultures, DA production was even greater near the end of the experiment, coincident with the period of chl synthesis and increase in carbon biomass. Comparison of the magnitude of toxin production in the two nutrient regimes indicated a greater threat of P. seriata‐generated amnesic shellfish poisoning events under Si rather than P nutrient limitation.     

Effects of growth rate and oxygen tension on glucose dehydrogenase activity inAcinetobacter calcoaceticus LMD 79.41

The regulation of the synthesis of the quinoprotein glucose dehydrogenase (EC 1.1.99.17) has been studied inAcinetobacter calcoaceticus LMD 79.41, an organism able to oxidize glucose to gluconic acid, but unable to grow on both compounds. Glucose dehydrogenase was synthesized constitutively in both batch and carbon-limited chemostat cultures on a variety of substrates. In acetate-limited chemostat cultures glucose dehydrogenase levels and the glucose-oxidizing capacity of whole cells were dependent on the growth rate. They strongly increased at low growth rates at which the maintenance requirement of the cells had a pronounced effect on biomass yield. Cultures grown on a mixture of acetate and glucose in carbon and energy-limited chemostat cultures oxidized glucose quantitatively to gluconic acid. However, during oxygen-limited growth on this mixture glucose was not oxidized and only very low levels of glucose dehydrogenase were detected in cell-free extracts. After introduction of excess oxygen, however, cultures or washed cell suspensions almost instantaneously gained the capacity to oxidize glucose at a high rate, by an as yet unknown mechanism.     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Utilization of unhydrolyzed cheese whey for the production of extracellular polysaccharide by Xanthomonas cucurbitae PCSIR B-52

A strain of Xanthomonas cucurbitae PCSIR B-52 produced extracellular polysaccharide using partially deproteinized cheese whey without hydrolysis. A synthetic lactose-salt medium was also utilized to determine the optiomum level of lactose desirable for successfull fermentation. The amount of extracellular polysaccharide was maximised at 7.8 gl⁻¹ in the presence of 40 gl⁻¹ lactose. The bacterium efficiently consumed cheese whey, particularly in the presence of corn steep liquor and penicillin waste mycelium in shaken flasks. The polysaccharide, bacterial cell mass and viscosity gradients were improved as a result of efficient oxygen transfer in a mechanically agitated fermentor. A depletion in dissolved oxygen tension resulted during the exponential growth phase. The fermentation pattern of extracellular polysaccharide was also studied by repeated batch process.     

Production of type 5 capsular polysaccharide by Staphylococcus aureus grown in a semi-synthetic medium

The concentration of the type 5 capsular polysaccharide (CP) antigen of Staphylococcus aureus can be measured directly in cultures or cell suspensions by a two-step inhibition enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies. CP was synthesized during growth on a variety of carbon substrates and its production was not affected by the nature of the carbon source. High levels of yeast extract inhibited CP formation. CP was synthesized in batch culture at the same rate during exponential growth as in the post-exponential phase. Post-exponential CP production contributed at least half the final amount of CP measured. This phenomenon was observed in different culture media, although the specific yield of polysaccharide varied from one medium to another. Post-exponential CP production was observed in the pH range 6-7, but not at pH 8. Post-exponential production was strictly dependent on oxygen availability and did not occur under anaerobic conditions.     

Chemostat production of pediocin SM‐1 by Pediococcus pentosaceus Mees 1934

Production of the bacteriocin pediocin SM‐1 by Pediococcus pentosaceus Mees 1934 was investigated in pH‐controlled batch and chemostat cultures using a complex medium containing glucose, sucrose or fructose. In chemostat cultures operated at 150 rpm, 30°C, 60% dissolved oxygen tension, pH 6.5, and D = 0.148 h^?1, the pediocin titer reached 185 AU/mL representing an increase of 32% compared with batch cultures in which glucose was used as the carbon source. Pediocin biosynthesis was markedly affected by the growth rate of the producer microorganism. For all carbon sources tested, pediocin production appeared to take place only at dilution rates lower than μ_max. However, only glucose supported production at the very low dilution rate of 0.05 h^?1 indicating a direct regulation of pediocin biosynthesis by the carbon source. Glucose supported higher biomass productivity and higher pediocin titers and yields compared with the other sugars used. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1481–1486, 2015     

Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures

Wild-type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4-L stirred-tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic- and organic-nitrogen concentrations were measured, and oxygen consumption and CO(2) production rates were obtained by a gas-balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in +/-5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen-limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking-Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.     

Influence of dissolved oxygen concentration on growth,mitochondrial function and antibody production of hybridoma cells in batch culture

The effects of dissolved oxygen concentration on hybridoma cell growth, metabolism, and antibody production were studied. A mouse hybridoma cell line producing an IgG1 directed at a consensus -interferon was grown in batch cultures in a 5 dm³ stirred bioreactor at dissolved oxygen (DO) concentrations of 5, 30, 90 and 95% or air saturation. High oxygen tension (95% of air saturation) reduced specific growth rate without affecting cell viability. At lower dissolved oxygen levels, specific growth rates were approximately independent of DO, although changes in mitochondrial function and antibody production were observed. Flow cytometry assays of a fluorescent mitochondria-specific marker (Rhodamine 123) show significant single-cell heterogeneities during late exponential growth and greater average fluorescence in cultures grown at 95% DO. The quantity of cell-surface immunoglobulin, measured by an immunofluorescent flow cytometric technique, was the same at high (95%) and low (5%) dissolved oxygen concentrations. Myeloma cells of the type used in constructing the above hybridoma line were much less sensitive to dissolved oxygen level. Specific respiration rate, pyruvate utilization rate, cytochrome oxidase activity, and succinate-cytochrome c oxidoreductase activity were significantly greater (62–116%) for the hybridoma cells than for the myeloma cells in T-flask cultures.     

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.