An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y _glucose and Y _O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.     

Growth-rate-dependent bacteriochlorophyll c/d ratio in the antenna of Chlorobium limicola strain UdG6040

The green sulfur bacterium Chlorobium limicola UdG6040 exhibited a significant change in the spectral properties of its antenna when transferred from batch culture to a sulfide-limited chemostat. In steady-state continuous cultures, the in vivo absorption maximum of the culture changed to shorter wavelengths according to the dilution rate. The maximum difference observed was of 15 nm when cells were growing at 0.087 h^–1. HPLC analyses revealed that the observed spectral change was caused by a partial enrichment of the original BChl c-containing antenna with BChl d molecules together with a change in the homolog composition of both pigments. The relative amount of BChl d reached a maximum value of 50% when cells were growing at 0.087 h^–1. The content of BChl d decreased to less than the 22% when the dilution rate was diminished to 0.015 h^–1. An unbalance of pigment synthesis at high dilution rates is suspected to be responsible of the changes observed in the antenna composition. Chlorosomes isolated from Chl. limicola UdG6040 growing at 0.070 h^–1 contain organised pools of BChl c and BChl d in equal amounts. Received: 2 December 1998 / Accepted: 25 February 1999     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Growth of Hansenula polymorpha in a methanol-limited chemostat

Hansenula polymorpha has been grown in a methanol-limited continuous culture at a variety of dilution rates. Cell suspensions of the yeast grown at a dilution rate of 0.16 h^-1 showed a maximal capacity to oxidize excess methanol (QO ₂ ^max ) which was 1.6 times higher than the rate required to sustain the growth rate (Q _O2). When the dilution rate was decreased to 0.03 h^-1, QO ₂ ^max of the cells increased to a value of more than 20 times that of Q _O2. The enzymatic basis for this tremendous overcapacity for the oxidation of excess methanol at low growth rates was found to be the methanol oxidase content of the cells. The level of this enzyme increased from 7% to approximately 20% of the soluble protein when the growth rate was decreased from 0.16 to 0.03 h^-1. These results were explained on the basis of the poor affinity of methanol oxidase for its substrates. Methanol oxidase purified from Hansenula polymorpha showed an apparent K _mfor methanol of 1.3 mM in air saturated reaction mixtures and the apparent K _mof the enzyme for oxygen was 0.4 mM at a methanol concentration of 100 mM.The involvement of an oxygen dependent methanol oxidase in the dissimilation of methanol in Hansenula polymorpha was also reflected in the growth yield of the organism. The maximal yield of the yeast was found to be low (0.38 g cells/g methanol). This was not due to a very high maintenance energy requirement which was estimated to be 17 mg methanol/g cells x h.     

Cell yields of Vibrio succinogenes growing with formate and fumarate as sole carbon and energy sources in chemostat culture

Vibrio succinogenes which gains all the ATP by anaerobic electron transport phosphorylation, was grown in continuous culture on a defined medium with formate and fumarate as sole energy sources. The growth yield at infinite dilution rate (Y ^max) was obtained by extrapolation from the growth yields measured at various dilution rates. With formate as the growth limiting substrate, Y ^max was found as 14 g dry cells/mol formate. Under these conditions growth was limited by the rate of energy supply, because formate is used only as a catabolic substrate (Bronder et al. 1982). The Y _ATP ^max calculated from the ATP requirement for cell synthesis was 18 g dry cells/mol ATP. This gives an ATP/2e ratio of 0.8. The ATP/2e ratio in vitro had been measured as 1 (Kröger and Winkler 1981). It is concluded that growing V. succinogenes gain at least 80% the stoichiometrically possible amount of ATP, when growth is limited by energy supply.     

Rubidium as a probe for function and transport of potassium in the yeast Candida utilis NCYC 321, grown in chemostat culture

Attempts to grow the yeast Candida utilis in continuous culture, using media in which all the potassium had been replaced by other monovalent cations, revealed that neither lithium, sodium, caesium nor ammonium ions could functionally substitute for potassium. However, potassium could be effectively replaced by rubidium which gave (on a molar basis, and under conditions where cation availability limited growth) the same yield of cells as did potassium.Comparison of potassium- and rubidium-limited cultures showed them to be virtually identical in all the measured parameters, with the single exception of the maximum growth rate value which was considerably decreased in the rubidium-containing culture (0.35 h^-1 as compared with 0.55 h^-1).When, with variously-limited chemostat cultures, both potassium and rubidium were supplied in equimolar amounts, these ions were taken up by the cells in a ratio that varied with both the growth rate and the nature of the growth limitation. With glucose-, phosphate- or magnesium-limited cultures, the molar ratio K⁺:Rb⁺ was 1:0.6 (at D=0.1 h^-1) and 1:0.17 (at D=0.5 h^-1). In contrast, ammonia-limited cultures took up increased amounts of rubidium when growing at a low rate such that the ratio was 1:1.2, at D=0.1 h^-1, though still 1:0.17 at the higher growth rate value (D=0.5 h^-1).From a comparison of glucose- and ammonialimited cultures growing first with an equimolar mixture of potassium and rubidium, and then with rubidium alone, it was noted that the yield on oxygen was significantly decreased when potassium was absent.These results are discussed in relation to the transport and possible functions of monovalent cations in micro-organisms. It was concluded that, on the basis of these experiments, some objections could be raised against estimation of potassium transport rates by means of the tracer ⁸⁶Rb.     

Effect of the growth rate on the enzymatic activities of L-lysine-producing cells of Corynebacterium glutamicum

The activity of 6-phosphogluconate dehydrogenase, aspartate kinase and phosphoenolpyruvate carboxylase has been studied at different dilution rates in aerobic continuous culture of Corynebacterium glutamicum. 6-Phosphogluconate dehydrogenase and aspartate kinase reached their maximum values at the lower dilution rates (0.02–0.06 h^–1), when L-lysine was produced. The phosphoenolpyruvate carboxylase activity seemed to be independent of metabolite synthesis. The production of L-lysine was also studied in non-growing cells in batch cultures. In these conditions, statistical analysis revealed significant differences in L-lysine titres when glucose or gluconic acid were used as carbon sources. Higher L-lysine concentration obtained with gluconic acid was found to be associated with a high 6-phosphogluconate dehydrogenase activity.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

n-3 Polyunsaturated fatty acid productivity of the marine microalgaIsochrysis galbana. Growth conditions and phenotypic selection

Two set of isolates were obtained in an isolation/selection programme to select eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) rich strains ofIsochrysis galbana. EPA content was improved up to an average of 5.3% (d.wt) for the second set of isolates. On the other hand, with the aeration rate, pH and irradiance kept at low levels, the growth rate was slow and EPA synthesis was enhanced, but productivity increased when growth rates were maximum. A model relating steady-state dilution rates in chemostat cultures ofI. galbana to internal average irradiance is proposed. The greatest productivities were obtained between 0.0295 h^–1 and 0.0355 h^–1 with 300 mg m^–3 h^–1 for EPA and 130 mg m^–3 h^–1 for DHA.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Two-stage continuous fermentation of Clostridium acetobutylicum: effects of pH and dilution rate

Summary The kinetics of a two-stage continuous fermentation of Clostridium acetobutylicum have been studied. The pH and the dilution rate have been shown to be two essential factors for process optimization. An increase in pH or dilution rate in the first stage decreased solvent production in the second fermentor. To achieve optimal solvent production, the pH had to be maintained at 4.5 in the first stage and between 4.5 and 5.0 in the second stage. Dilution rates of 0.08 h^–1 and 0.04 h^–1,respectively, in the first and second fermentors allowed a high solvent concentration. When the pH was maintained at 4.5 in each stage and when the dilution rates were 0.08 h^–1 and 0.04 h^–1 in the first and second fermentors respectively, 21 g/l solvent concentration was achieved. A conversion yield of 0.36 g solvents/g glucose consumed was obtained with total consumption of glucose. Biomass was only produced in the first stage together with 40% of the solvents, indicating that solvent production had to be induced in the first fermentor. Offprint requests to: J. M. Engasser     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.