Glucose-limited Chemostat Cultures Research Articles

Nitrogen-limited behaviour of micro-organisms growing in the presence of large concentrations of ammonium ions.

Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5-5) in a glucose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH4+) and NH3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values.

Nitrogen-limited behaviour of micro-organisms growing in the presence of large concentrations of ammonium ions

Cells of Klebsiella pneumoniae NCTC 418 grown at low culture pH values (4.5–5) in a glucose-limited chemostat culture contained elevated levels of glutamate synthase (EC 2.6.1.53). This can be taken as an indication that these cells show the physiology of nitrogen-limited cells, in spite of the fact that high concentrations (about 80 mM) of ammonium ions were present in the culture extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3) through the cell membrane, leading to very low cytoplasmic ammonium (NH4+) and NH3 levels in cells that possess an almost neutral cytoplasmic pH value, but are growing at low culture pH values.

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic and energetic aspects of the growth of Bacillus stearothermophilus in glucose-limited and glucose-sufficient chemostat culture

With a glucose-limited chemostat culture of Bacillus stearothermophilus, increasing the incubation temperature progressively from 45°C to 63°C led to a progressive marked increase in the maintenance rates of glucose and oxygen consumption. Hence, at a fixed low dilution rate the yield values with respect to glucose and oxygen decreased substantially with increased temperature. However, the apparent Y glucose max and $$Y_{{\text{O}}_2 }^{\max } $$ values did not decrease but actually increased with temperature, being highest at 63°C (i.e., close to the maximum growth temperature). With glucose-sufficient cultures growing at a fixed low dilution rate (0.2 h−1) and at their optimum temperature (55°C), glucose and oxygen consumption rates invariably were higher than that of a corresponding glucose-limited culture. Cation (K+ or Mg2+)-limited cultures expressed the highest metabolic rates and with the K+ limited culture this rate was found to be very markedly temperature dependent. As the temperature was increased from 45°C to 63°C the rate of glucose consumption increased 1.8-fold, and that of oxygen consumption by 3.7-fold. The culture pH value also exerted a noticeable effect on the metabolic rate of a glucose-limited culture, particularly at the extremes of pH tolerance (5.5 and 8.5, respectively). A K+-limited culture was less affected with respect to metabolic rate by the culture pH value though the steady state bacterial concentration, and thus the cellular K+ content, changed substantially. These results are discussed in relation to previous findings of the behaviour of this organism in batch culture, and to the behaviour of other thermophilic Bacillus species in chemostat culture.

Stability and expression of a plasmid‐containing killer toxin cDNA in batch and chemostat cultures of saccharomyces cerevisiae

A chimeric plasmid (pYT760-ADH1) containing the yeast killer toxin-immunity cDNA was transformed into a leucine-histidine mutant (AH22) and into four industrial toxin-sensitive yeasts. The chimeric plasmid was very stable and expressed toxin production (89.5 +/- 4.8% killer cells) in two of the transformed yeasts that contained the 2mu plasmid, but was lost within 10 generations from two other transformed pickle yeasts that did not contain the 2mu plasmid. It suggested that plasmid stability was dependent on the presence of the 2mu plasmid which is naturally present in some yeasts. The plasmid was extremely stable (100% killer cells) and expressed more toxin in the mutant strain AH22. The effects of dilution rate, D(h(-1)) on plasmid stability and toxin expression were studied in transformed AH22 (AH22/T3) and Montrachet 522 (522/T1) wine yeast grown in glucose-limited chemostat cultures. The results show that killer toxin production by AH22/T3 cells increased as a function of D(h(-1)) and that plasmid stability reached 100% at D >/= 0.09 +/- 0.01 h(-1). However, with Montrachet 522/T1 transformed cells, 100% plasmid stability was seen at D >/= 0.18 +/- 0.02. h(-1). We also challenged the AH22/T3 in chemostat culture (D = 0.25 h(-1)) with an equal number of untransformed cells (AH22). Transformed cells dominated the population (100%) within 8-10 h of growth, a time equivalent to two mean residence time.

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad Hw, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5-22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.

Effects of oxygen on the growth and metabolism ofActinomyces viscosus

Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h−1) with exposure to variable amounts of oxygen. The Yglucose increased from 62.5 g · mol−1 under anaerobic conditions to 149 g · mol−1 under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [14C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.

Effect of oxygen tension on stability and expression of a killer toxin chimeric plasmid in a chemostat culture of Saccharomyces cerevisiae

The effects of oxygen tension and dilution rate on expression and stability of killer toxin chimeric plasmid (pADH-10A) in transformants of wine yeast (Montrachet 522) were investigated using a glucose-limited chemostat culture. Killer toxin activity increased as a function of dilution rate when transformants were grown in N2 or air, but not in 100% O2. Killer transformants grown in the presence of N2 had 2–4 fold and 10–15 fold higher toxin expression than that in the presence of air and 100% O2, respectively. Plasmid stability increased as a function of dilution rate, regardless of the oxygen tension used. However, transformants grown in air gave the highest plasmid stability.

Biosynthesis of superoxide dismutase and catalase in chemostat culture of Saccharomyces cerevisiae

The effects of oxygen (100%), paraquat (0.5 mM), and copper (0.1 mM) on the growth and the biosynthesis of the antioxidant enzymes, superoxide dismutase (SOD) and catalase, were studied in Saccharomyces cerevisiae grown in glucose-limited chemostat cultures. The effect of dilution rates (D, h−1) on cell mass, glucose consumption, ethanol production, oxygen uptake, and specific activities of SOD and catalase were also investigated at each steady state. SOD was optimally produced at D-values between 0.22 and 0.26 h−1 in the presence of oxygen or paraquat, and at D-values greater than 0.17 h−1 when copper was used. On the other hand, catalase activity decreased with increasing D-values. However, the presence of copper or 100% oxygen repressed catalase activity at low D-values (D 0.22 h−1). We also studied the effect of oxygen concentration on the biosynthesis of SOD and catalase at D=0.1 h−1. The data clearly show that synthesis of SOD and catalase, though correlated with changes in oxygen tension, are independent of one another.

Effects of ethanol and acetate on glucose-limited chemostat cultures of Schizosaccharomyces pombe, a fission yeast

Glucose-limited chemostat cultures of Schizosaccharomyces pombe were studied to assess metabolic changes induced by the presence of ethanol and acetate. Both ethanol and acetate were utilized by the chemostat cultures. Ethanol increased the maintenance rate of glucose assimilation. Acetate reduced the maintenance rate of glucose consumption and cell counts without significantly affecting the glucose requirements for growth and cell yield. Ethanol accumulation in the chemostat cultures was facilitated by high dilution rates and noninhibitory concentrations of acetate.

Establishment of the steady state in glucose-limited chemostat cultures of Klebsiella pneumoniae.

To investigate the relationship between growth rate and concentration of the nutrient that limits growth, 'Klebsiella aerogenes' NCTC 418 (K. pneumoniae) was grown in a glucose-limited chemostat. The actual time required to establish a steady-state glucose concentration exceeded that expected theoretically. Apparently, there is a long-term adaptation of the cells to nutrient limitation. As yet, it is not clear whether this has a phenotypic or genetic origin. In the final steady state, the dependence of the growth rate on glucose concentration could be mathematically described equally well by a hyperbolic and by a logarithmic function.

Methanogenic digestion of glucose plus yeast extract by a defined bacterial consortium: Carbon balances and growth yields in chemostat culture

A methanogenic consortium of bacteria, isolated from anaerobic sewage sludge by growth on glucose and yeast extract and mineral salts, consisted of two strict anaerobes, one of which (the GD strain) degraded glucose and the other was a methanogen. In addition the consortium contained a small population of facultative anaerobes (4 types) which constituted <1% of the total biomass. In glucose-limited chemostat cultures of the consortium, the maximum methane output rate occured with a dilution rate ( D) of 0.1 h −1. With D = 0.10 h −1 the consortium fermented both the glucose and yeast extract giving the following C balance (% C of glucose and yeast extract in the products): acetate, 34.2; biomass, 25.4; CO 2, 13.8; CH 4, 6.5; ethanol, 7.9; butyrate, 7.3; propionate, 3.2 (C recovery, 98.3%; H 2 production, 0.04 mol/Cmol substrate. The GD strain in uncontaminated culture fermented glucose only and gave the following C balance in a glucose-limited steady state chemostat culture with D=0.12 h −1 (% glucose C in the products): acetate, 35.1; ethanol, 23.1; CO 2, 20.6; biomass, 12.3; butyrate, 4.4; propionate, 1.7 (C recovery, 97.2%); H 2 production, 0.293 mol/C mol glucose. The maximum growth yields ( Y G) from the C sources were 0.139 and 0.292 (C-mol biomass/C-mol substrate) for the GD organism and the consortium respectively. The maintenance energies were remarkably small compared with that typical of aerobic bacteria. This prompts the suggestion that the main function of maintenance energy substrate in aerobes is not to provide ATP but rather reducing equivalents to protect cells against O 2 damage. It is concluded that, in the technology of methanogenic conversion of wastes, besides the acidogenic and methanogenic stages, a third stage, for digestion of the biomass formed is required, otherwise the biomass can account for 25% of the substrate C supplied.

Patterns of extension growth of the fission yeast, Schizosaccharomyces pombe

The growth of sausage-shaped cells of the fission yeast, Schizosaccharomyces pombe (strain NCYC 132), was followed in the second or third cycle by time-lapse photomicrography. Experimental cells were harvested from glucose-limited (0.2% glucose EMM3) chemostat culture (dilution rate, 0.125/h) and were plated onto a slide with EMM3 agar (2% glucose). By observing their extension patterns, we found some rules of extension growth. Thus, (1) all sibs with walls newly formed in the previous cycle, whose progenitor cells grew at the old end (followed Mitchison's rule), grow at the old end (also follow Mitchison's rule). (2) Sibs with old walls whose progenitor cell followed Mitchison's rule behave in one of three ways: (i) growth at the old end (follow Mitchison's rule); (ii) growth at the new end (violate Mitchison's rule); or (iii) growth at both ends (bipolar). (3) Both sibs whose progenitor grew at both ends (bipolar) always grow at the old end (follow Mitchison's rule).

Enhanced stability of a 2?-based recombinant plasmid in diploid yeast

The stability of a 2μ-based recombinant plasmid, pJDB219, has been compared in glucose-limited chemostat cultures of two haploid strains ofSaccharomycescerevisiae and a diploid derived from them. The stability of the recombinant plasmid differed in the two haploid hosts but was greatest in the diploid. Enhanced stability in the diploid is probably a function of both the increased copy number and reduced selective burden of the plasmid.

Pantothenate limitation does not induce uncoupled growth of Zymomonas in chemostat culture

Growth coefficients ofZymomonas mobilis were compared in glucose-limited chemostat culture using a complex medium and a defined minimal growth medium at non inhibitory concentrations of ethanol. Under carbon and energy limited conditions in the complex medium containing yeast extract, the max. molar growth yield (YGmax) and maintenance energy coefficient (me) were 10.8 g cell/mol glucose and 8.3 mmol glu/g cell/hr, respectively. Glucose-limited growth in the minimal medium with NH4Cl as nitrogen source promoted slight energetic uncoupling, as reflected in the decrease in the maximum growth yield. The growth yield with respect to calcium pantothenate was calculated to be 1.4×104 g cell/g Ca-pantothenate. However, pantothenate-limited growth did not result in a decrease in growth yield nor an increase in the specific rate of glucose catabolism. Steady-state growth measurements failed to confirm the previously held view of Belaichet al. (1972) that pantothenate deficiency induces energetic uncoupling inZymomonas.

A Radiorespirometric Study on the Contribution of the Hexose Monophosphate Pathway to Glucose Metabolism in Candida utilis CBS 621 Grown in Glucose-limited Chemostat Cultures

Summary: A radiorespirometric study of glucose metabolism in Candida utilis CBS 621 was done using glucose-limited chemostat cultures growing at a dilution rate of 0.1 h−1 with ammonium or nitrate as the nitrogen source. From a steady-state analysis of 14CO2 yields from [1-14C]- and [6-14C]glucose supplied in the medium feed it appeared that during growth with nitrate the flow of glucose through the hexose monophosphate (HMP) pathway was much higher than during growth with ammonium as the nitrogen source. The same phenomenon was apparent from an analysis of the rate of 14CO2 production after administration of small amounts of labelled glucose to samples withdrawn from steady-state cultures. Additionally, these experiments revealed not only that the initial fraction of glucose 6-phosphate routed into the HMP pathway increases, but also that recycling of hexose phosphates via this pathway increases when nitrate is used as the nitrogen source. From a quantitative analysis of the results it is concluded that the contribution of the HMP pathway to glucose metabolism is close to the theoretical minimum required to cover the NADPH requirement for biosynthesis.

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.

Methanol metabolism in Paracoccus denitrificans I. Isolation and characterization of mutants with defects in methanol utilization

Bacillus licheniformis is a facultative aerobic bacterium used in the fermentation industry for the production of enzymes. For a complete description of the production process knowledge of growth parameters, electron-chain composition and efficiency of energy conservation is obligatory. B. licheniformis S1684 is able to produce an alkaline serine protease exocellularly. In glucoselimited chemostat cultures the specific rate of protease production was maximal at a specific growth rate value (Ix) of 0.22. At higher growth rates protease production was repressed (catabolite repression). Dependent on Ix 10-20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products (Tp) was 4.1, showing that products are not more reduced than biomass. Maximum molar growth yields were high and indicated a high efficiency of growth. The values of yg~auX and y ~ x were 83.8 and 53.3, respectively. When y~ax was corrected for the amount of glucose used for product formation a value of 100.3 could be calculated. These high maximum YATP. molar growth yields most probably are caused by a high max In anaerobic batch experiments a YATP of 14.6 could be determined. The efficiency of energy conservation most probably is low, which is indicated by the measured H +/O ratios of about 4 in cells taken at different Ix-values during glucose limitation, indicating that at the most one or two traditional sites of oxidative phosphorylation are active. The strain used appeared to be instable in protease production and cell morphology. Non-protease-producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells are relaxed (tel-) and non-producing cells stringent (rel § In non-protease-producing chemostat cultures y~ax was significantly higher because no glucose was needed for protease production.

Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

Candida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066 were grown in glucose-limited chemostat cultures with formate as an additional energy source. In both yeasts formate was oxidized via a cytoplasmic NAD+-linked formate dehydrogenase. Other formate-oxidizing enzymes could not be detected.

A continuous culture study of the bioenergetic aspects of growth and production of exocellular protease in Bacillus licheniformis

Bacillus licheniformis S 1684 is able to produce an alkaline serine protease exocellularly. In glucose-limited chemostat cultures the specific rate of protease production was maximal at a μ-value of 0.22. Above this growth rate protease production was repressed. Dependent on μ 10–20% of the glucose input was used for exocellular product formation. The degree of reduction of exocellular products $$\bar \gamma _p$$ was 4.1. Maximum molar growth yields were high and indicate a high efficiency of growth. The values of Y glu max and YO 2 max were 83.8 and 53.3, respectively. When Y glu max was corrected for the amount of glucose used for product formation a value of 100.3 was obtained. These high maximum molar growth yields are most probably caused by a high Y ATP max . Anaerobic batch experiments showed a Y ATP of 14.6. Sometimes the used strain was instable in cell morphology and protease production. Non-protease producing cells most probably develop from producing cells by mutation in the rel-gene. Producing cells most probably are relaxed (rel -) and non-producing cells stringent (rel +).