Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture

Carbon-limited chemostat cultures of Klebsiella aerogenes NCTC 418 consumed more oxygen per unit of cell synthesis when growing on mannitol or glycerol than when growing on glucose; and since the maintenance requirements were similar, this suggested that the extra reducing equivalents present in these compounds were oxidized wastefully. By comparison with carbon-limited cultures, carbon-sufficient cultures that were ammonia-, sulphate- or phosphate-limited generally consumed considerably more oxygen per unit of cell synthesis, particularly at low growth rates. Thus, according to the theory of Pirt, these carbon-sufficient cultures had a greatly increased maintenance energy requirement but nevertheless used the remaining energy with a much increased efficiency compared with carbon-limited cultures. This, we suggest, is a false conclusion which stems from the basic assumption that the maintenance requirement does not change with growth rate. Thus we propose an alternative theory which allows for this possibility, and present evidence to show that it may be applicable to both carbon-limited and carbon-sufficient chemostat cultures. Finally we offer an explanation of the high maintenance rate of oxygen consumption found with carbon-sufficient cultures, and consider this phenomenon in relation to the loose coupling between respiration and growth extant in most microbial cultures.     

The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii

Azotobacter beijerinckii was grown in ammonia-free glucose-mineral salts media in batch culture and in chemostat cultures limited by the supply of glucose, oxygen or molecular nitrogen. In batch culture poly-beta-hydroxybutyrate was formed towards the end of exponential growth and accumulated to about 74% of the cell dry weight. In chemostat cultures little poly-beta-hydroxybutyrate accumulated in organisms that were nitrogen-limited, but when oxygen limited a much increased yield of cells per mol of glucose was observed, and the organisms contained up to 50% of their dry weight of poly-beta-hydroxybutyrate. In carbon-limited cultures (D, the dilution rate,=0.035-0.240h(-1)), the growth yield ranged from 13.1 to 19.8g/mol of glucose and the poly-beta-hydroxybutyrate content did not exceed 3.0% of the dry weight. In oxygen-limited cultures (D=0.049-0.252h(-1)) the growth yield ranged from 48.4 to 70.1g/mol of glucose and the poly-beta-hydroxybutyrate content was between 19.6 and 44.6% of dry weight. In nitrogen-limited cultures (D=0.053-0.255h(-1)) the growth yield ranged from 7.45 to 19.9g/mol of glucose and the poly-beta-hydroxybutyrate content was less than 1.5% of dry weight. The sudden imposition of oxygen limitation on a nitrogen-limited chemostat culture produced a rapid increase in poly-beta-hydroxybutyrate content and cell yield. Determinations on chemostat cultures revealed that during oxygen-limited steady states (D=0.1h(-1)) the oxygen uptake decreased to 100mul h(-1) per mg dry wt. compared with 675 for a glucose-limited culture (D=0.1h(-1)). Nitrogen-limited cultures had CO(2) production values in situ ranging from 660 to 1055mul h(-1) per mg dry wt. at growth rates of 0.053-0.234h(-1) and carbon-limited cultures exhibited a variation of CO(2) production between 185 and 1328mul h(-1) per mg dry wt. at growth rates between 0.035 and 0.240h(-1). These findings are discussed in relation to poly-beta-hydroxybutyrate formation, growth efficiency and growth yield during growth on glucose. We suggest that poly-beta-hydroxybutyrate is produced in response to oxygen limitation and represents not only a store of carbon and energy but also an electron sink into which excess of reducing power can be channelled.     

Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae

Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media. By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc- S. cerevisiae does not exhibit prolonged growth on glucose. Pdc- strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures. The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of pyruvate decarboxylase in the synthesis of cytosolic acetyl-CoA.     

Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.     

Kinetics of growth and leukotoxin production by <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis> in continuous culture

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.     

Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism

An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D = 0.03 h(-1)) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D = 0.01 h(-1)) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.     

Regulation of glucose catabolism in Thiobacillus A2 grown in the chemostat under dual limitation by succinate and glucose

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.     

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.     

Hydrogenase does not confer significant benefits to Azotobacter vinelandii growing diazotrophically under conditions of glucose limitation.

The presumed beneficial effect of hydrogenase on growth of diazotrophic bacteria was reinvestigated with carbon-limited chemostat cultures of the hydrogenase-deficient mutant hoxKG of Azotobacter vinelandii and its parent. The results revealed that hydrogen recycling was too low to benefit the cellular energy metabolism or activities of nitrogenase and respiration.     

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two K _m values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH₄ ⁺ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.Abbreviations ADH alanine dehydrogenase - GS glutamine synthetase - GDH glutamate dehydrogenase - GOGAT glutamine oxoglutarate aminotransferase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring

The production of endo-β-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO₂-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucroso, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h^-1 and a high rate of enzyme production of 362 nkat (mg dry mass h)^-1 were attained under nutrient rich conditions in the CO₂-auxostat. The bacteria had the highest specific growth rate and endo-β-1,4-glucanase enzyme production at 50° C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO₂-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.     

Growth yields,polysaccharide production and energy conservation in chemostat cultures of Rhizobium trifolii

Rhizobium trifolii was grown in a defined medium in chemostat cultures. Extracellular polysaccharide production was found in carbon-sufficient as well as in carbon-limited cultures. Extracellular polysaccharide production in limited cultures, asparagine was always totally depleted from the culture medium. Only when the asparagine supply was not sufficient to meet the nitrogen need of the culture, ammonia assimilation took place. Excess organic nitrogen was excreted as ammonia. Whether ammonia assimilation or ammonia excretion took place was also dependent on the growth rate. Respiration-coupled proton translocation measurements showed the presence of three energy conserving sites in an electron transport chain which is branched. Assuming a H⁺/P ratio of 4, a P/O ratio of 2.33 was found. Growth yield calculations indicated a P/O ratio of approximately 2. Sulphate limitation in the chemostat culture resulted in a decrease in the efficiency of oxidative phosphorylation and in a less stringent coupling between growth and energy yielding processes.The investigations were supported in part by the Foundation for Fundamental Biological Research (BION), which is subsidized by The Netherlands Organisation for the Advancement of Pure Research (ZWO).     

Regulation of alcohol-oxidizing capacity in chemostat cultures of Acetobacter pasteurianus

Acetobacter pasteurianus LMG 1635 was studied for its potential application in the enantioselective oxidation of alcohols. Batch cultivation led to accumulation of acetic acid and loss of viability. These problems did not occur in carbon-limited chemostat cultures (dilution rate = 0.05 h^–1) grown on mineral medium supplemented with ethanol, L-lactate or acetate. Nevertheless, biomass yields were extremely low in comparison to values reported for other bacteria. Cells exhibited high oxidation rates with ethanol and racemic glycidol (2,3-epoxy-1-propanol). Ethanol- and glycidol-dependent oxygen-uptake capacities of ethanol-limited cultures were higher than those of cultures grown on lactate or acetate. On all three carbon sources, A. pasteurianus expressed NAD-dependent and dye-linked ethanol dehydrogenase activity. Glycidol oxidation was strictly dye-linked. In contrast to the NAD-dependent ethanol dehydrogenase, the activity of dye-linked alcohol dehydrogenase depended on the carbon source and was highest in ethanol-grown cells. Cell suspensions from chemostat cultures could be stored at 4°C for over 30 days without significant loss of ethanol- and glycidol-oxidizing activity. It is concluded that ethanol-limited cultivation provides an attractive system for production of A. pasteurianus biomass with a high and stable alcohol-oxidizing activity.     

Unique kinetic properties of phenol-degrading variovorax strains responsible for efficient trichloroethylene degradation in a chemostat enrichment culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities. To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter(-1) day(-1)) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter(-1) day(-1) (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.     

Effect of bioaugmentation and supplementary carbon sources on degradation of polycyclic aromatic hydrocarbons by a soil-derived culture

The degradation of polycyclic aromatic hydrocarbons (PAHs) by an undefined culture obtained from a PAH-polluted soil and the same culture bioaugmented with three PAH-degrading strains was studied in carbon-limited chemostat cultures. The PAHs were degraded efficiently by the soil culture and bioaugmentation did not significantly improve the PAH degrading performance. The presence of PAHs did, however, influence the bacterial composition of the bioaugmented and non-bioaugmented soil cultures, resulting in the increase in cell concentration of sphingomonad strains. the initial enhancement of the degradation of the PAHs by biostimulation gradually disappeared and only the presence of salicylate in the additional carbon sources had a lasting slightly stimulating effect on the degradation of phenanthrene. The results suggest that bioaugmentation and biostimulation have limited potential to enhance PAH bioremediation by culture already proficient in the degradation of such contaminants.     

Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.