Restricted oxygen supply and excretion of metabolites

Summary The effect of oxygen supply on the formation of metabolites by Escherichia coli K12, Enterobacter aerogenes and Brevibacterium lactofermentum was studied. The relative respiration rate (i.e. the ratio, in percent, of the respiration rate during the production phase to that at the end of exponential growth phase) was taken as a measure of cell response to oxygen supply to which the production of metabolites was also correlated. Several metabolites were excreted at distinct relative respiration rates: ethanol, hydrogen gas, acetate, butanoate, capronate, malate, succinate, acetoin, 2,3-butanediol, 2-methylpropanoate, 3-methylbutanoate, formate, cis-aconitate and 2-oxoglutarate. At relative respiration rates above 80% the excretion of metabolites ceased and the substrate was completely oxidized.The effects of oxygen and of NADH, respectively, on synthesis and activity of several enzymes involved in the formation of these metabolites are discussed. The efficiency of aeration in a fermentation process can be controlled by means of analysis of excreted metabolites.     

Excretion of metabolites by hydrogen bacteria I. Autotrophic and heterotrophic fermentations

Summary The excretion of metabolites by 48 wild-type and mutant strains belonging to various species and genera of aerobic hydrogen-oxidizing bacteria was studied. The cells were grown autotrophically and heterotrophically, and samples were analyzed by gas chromatrographic techniques. The following metabolites were identified and quantitatively determined: acetate, ethanol, malate, citrate, lactate, succinate, 2-propanol, 2-methylpropanoate, 3-methylbutanoate, cis-aconitate, acetone, 2-oxoglutarate, isocitrate, butanoate, and methanol. The excretion of the metabolites started when ammonia and oxygen became limiting. The concentrations reached a maximum, whereupon the excreted products were reconsumed.The total concentration of the metabolites identified reached 5 g/l. Maximum concentrations were measured when mutants of Alcaligenes eutrophus lacking the ability to accumulate poly-3-hydroxybutanoate were grown on fructose, gluconate, or lactate in the fermenter under conditions of ammonia limitation and when the carbon source was present in excess.     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

Fermentation enzymes in strains of Paracoccus denitrificans

Various strains of Paracoccus denitrificans grown under conditions of unrestricted oxygen supply contained low but measurable activities of fermentation enzymes such as ethanol dehydrogenase and 2,3-butanediol dehydrogenase. However, when the bacteria were subsequently incubated for up to 22 h under restricted aeration conditions permitting respiration rates of only 10 or 6% of the maximum value to occur, the above enzymes increased in specific activities by 5- or 10-fold to 0.14 mol/min·mg protein. Lactate dehydrogenase was not detected. Six strains tested reacted almost alike.Cells grown anaerobically on fructose in the presence of limiting concentrations of KNO₃ contained specific activities of up to 0.41 (in case of ethanol dehydrogenase) and 0.56 (butanediol dehydrogenase) mol/min·mg protein. Lactate dehydrogenase was only formed at low activity (0.012 mol/min·mg protein) after a long period of incubation.Cells of P. denitrificans strain Stanier 381 grown anaerobically in the chemostat on fructose+KNO₃ with either fructose or nitrate as the limiting factor differed with respect to the specific enzyme activities, too. Ethanol dehydrogenase was high under conditions of nitrate limitation and low under fructose limitation. 2,3-Butanediol dehydrogenase, but not lactate dehydrogenase, was formed in moderate activities.     

Production of optically active 2,3-butanediol by Bacillus polymyxa

Bacillus polymyxa produces (R, R)-2,3-butanediol from a variety of carbohydrates. Other metabolites are also produced including acetoin, acetate, lactate, and ethanol. The excretion of each metabolite was found to depend on the relative availability of oxygen to the culture. When the relative oxygen uptake rate was high, enhanced yields of acetate and acetoin were noted. At an intermediate oxygen availability, the butanediol yield was maximal. When the availability of oxygen was more restricted, higher yields of lactate and ethanol occurred. The cells appeared to regulate themselves such that energy generation is optimal subject to the constraint that the cells do not produce more reducing equivalents than can be oxidized by the electron transport system. The dependence of each product yield on the relative oxygen availability was determined, and this knowledge was used to carry out a fed-batch fermentation that attained a final butanediol concentration of over 40 g/L in 50 h.     

Oxygen-dependent switch-over from respiratory to fermentative metabolism in the strictly aerobic Alcaligenes eutrophus

Summary The metabolism of Alcaligenes eutrophus is both qualitatively and quantitatively affected by the availability of oxygen as is documented by the in vivo excretion of several distinct metabolites. Intermediates of the tricarboxylic acid cycle are produced because the dehydrogenases catalyzing the subsequent steps of metabolism become inhibited in a sequential order by increasing NADH levels which are caused by lack of oxygen. Simultaneously, other enzymes which cannot be detected when the cell's oxygen demand is satisfied, i.e., formate dehydrogenase, fumarate reductase, butanediol, lactate, and ethanol dehydrogenases are induced in a sequential order enabling the cells to produce the corresponding metabolites. The molecular mechanism by which dehydrogenases involved in the fermentative metabolism are derepressed under lack of oxygen is discussed.     

Effect of specific oxygen uptake rate on Enterobacter aerogenes energetics: carbon and reduction degree balances in batch cultivations

The effect of oxygen availability on the metabolism of Enterobacter aerogenes NCIMB 10102 was studied through batch fermentations of glucose performed increasing the specific oxygen uptake rate up to 72.7 mmol(O2) C-mol(DW) (-1) x h(-1). The final concentrations of fermentation products of this biosystem (2,3-butanediol, hydrogen, acetoin, formate, acetate, carbon dioxide, ethanol, lactate, succinate, and biomass) were utilized to check the use of simple carbon mass and reduction degree balances for the study of microbial energetics even in batch cultivations.     

The physiology of Lactococcus lactis subsp. lactis biovar. diacetylactis immobilized in hollow-fibre bioreactors: glucose,lactose and citrate metabolism at high cell densities

Lactococcus lactis subsp. lactis biovar. diacetylactis was selected to study the physiological influences of immobilization and growth to high cell densities. Cells were cultivated on glucose or lactose medium in the presence and absence of citrate. With excess glucose the cells produced mainly lactate as the fermentation product (homofermentative) providing that not all of the substrate was consumed. The population so cultivated was exposed to extreme gradients of pH and lactate concentrations. When the glucose concentration was reduced the population showed a mixed product profile with half of the glucose being fermented to lactate, the remainder to formate, acetate, ethanol and 2,3-butanediol. Inclusion of citrate in the medium shifted the population to homofermentation, with respect to the amount of glucose or lactose consumed. The citrate was metabolized via the pyruvate-formate lyase and -acetolactate synthase routes. The pH of the medium was shown to strongly influence the product profile from citrate, presumably by affecting the activity of the key enzymes of pyruvate metabolism. The lactococci immobilized at high cell densities show product profiles typical of carbohydrate limitation at low dilution rates. Correspondence to: M. R. Smith     

Excretion of metabolites by hydrogen bacteria II. Influences of aeration,pH, temperature,and age of cells

Summary The effects of the aeration rate, the pH value, the temperature of the culture medium and of the age of cells on the excretion of metabolites by mutant strains of Alcaligenes eutrophus were studied. With lactate or gluconate as substrates, ethanol, 3-hydroxybutanoate, succinate, cis-aconitate, 2-oxo-3-methylbutanoate and 2-oxoglutarate were excreted, each at a distinct low aeration rate. Maximum concentrations of metabolites were found at pH 7.0 at 30°C when ammonia was growth limiting and the carbon substrate was present in excess. Excretion occurred only by viable intact cells.     

H2 production of Rhodospirillum rubrum during adaptation to anaerobic dark conditions

Rhodospirillum rubrum is able to produce H₂ during fermentation anaerobically in the dark in two ways, namely through formate hydrogen lyase and through the nitrogenase. After chemotrophic preculture aerobically in the dark formate hydrogen lyase was synthesized after a lag phase, whilst after phototrophic preculture a slight activity was present from the beginning of the anaerobic dark culture. During fermentation metabolism its activity increased noticeably. Hydrogen production through the nitrogenase occurred if the nitrogenase had been activated during phototrophic preculture. It ceased during fermentation metabolism after about 3 1/2 h anaerobic dark culture. The CO insensitive H₂ production by the nitrogenase could be partially inhibited by N₂. Potential activity of this system, however, remained and could be increased under conditions of nitrogenase induction. It seems therefore possible that synthesis of nitrogenase under N-deficiency can occur during fermentation metabolism in the same way as the formation of the photosynthetic apparatus in order to prepare for subsequent phototrophic metabolism.Abbreviations CAP chloramphenicol - DSM Deutsche Sammlung von Mikroorganismen, Göttingen - FHL formate hydrogen lyase - O.D optical density - PFL pyruvate formate lyase     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Optimization of 2,3-butanediol production by Klebsiella oxytoca through oxygen transfer rate control

Production of 2,3-butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy-coupled growth, during which cell growth and oxygen supply are coupled; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3-butanediol productivity occurs during the energy-coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy-coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasing the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3-butanediol productivity of 18%. (c) 1993 John Wiley & Sons, Inc.     

Effects of substrate limitation on product distribution and H2/CO2 ratio inKlebsiella pneumoniae during anaerobic fermentation of glycerol

Product formation during anaerobic degradation of glycerol byKlebsiella pneumoniae DSM 2026, under glycerol limitation and glycerol excess in continugius cultures, has been investigated. Major and minor products and by-products as well as gaseous products were measured. The results indicated a positive correlation between specific glycerol uptake and most product formation rates under glycerol limitation. The production of 1,3-propanediol, lactate, formate, acetate, succinate and the by-products of anaerobic glycerol degradation byK. pneumoniae, acetoin and 2,3-butanediol, was favoured by glycerol excess, while hydrogen generation and ethanol formation were best under glycerol limitation. It was also found that under glycerol limitation the rate of hydrogen evolution was generally higher than the CO₂ production rate while under excess glycerol the reverse was true. Hence, on the basis of the ratio of the specific rates of evolution of H₂ and CO₂ (q _{H 2}/q _{CO 2}), it is possible to infer the existence of glycerol limitation. On the basis of the carbon and available electron balances, which are independent of metabolic pathways, the data are consistent. The NADH₂ balance, which took into consideration the pathways of product formation, was also tested to check the validity of the assumed pathways and to check critically the consistency of the data. Good balances were also obtained.[     

A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.     

Effect of external pH on ethanol oxidation byCandida utilis

The external pH affects both ethanol and oxygen uptake rates by nongrowing cells ofCandida utilis suspended either in distilled water or in phthalate buffer. The buffering properties of organic acids control the maximum rates of exogenous respiration and ethanol uptake. The substrate limitation of ethanol uptake rate and endogenous respiration rate increase proportionally with increasing hydrogen ion concentration in the medium.     

Effect of deletion of 2,3-butanediol dehydrogenase gene (bdhA) on acetoin production of Bacillus subtilis

The present work aims to block 2,3-butanediol synthesis in acetoin fermentation of Bacillus subtilis. First, we constructed a recombinant strain BS168D by deleting the 2,3-butanediol dehydrogenase gene bdhA of the B. subtilis168, and there was almost no 2,3-butanediol production in 20?g/L of glucose media. The acetoin yield of BS168D reached 6.61?g/L, which was about 1.5 times higher than that of the control B. subtilis168 (4.47?g/L). Then, when the glucose concentration was increased to 100?g/L, the acetoin yield reached 24.6?g/L, but 2.4?g/L of 2,3-butanediol was detected at the end of fermentation. The analysis of 2,3-butanediol chiral structure indicated that the main 2,3-butanediol production of BS168D was meso-2,3-butanediol, and the bdhA gene was only responsible for (2R,3R)-2,3-butanediol synthesis. Therefore, we speculated that there may exit another pathway relating to the meso-2,3-butanediol synthesis in the B. subtilis. In addition, the results of low oxygen condition fermentation showed that deletion of bdhA gene successfully blocked the reversible transformation between acetoin and 2,3-butanediol and eliminated the effect of dissolved oxygen on the transformation.     

A model of acetic acid and 2,3-butanediol inhibition of the growth and metabolism ofKlebsiella oxytoca

Summary The main product of fermentation byKlebsiella oxytoca is 2,3-butanediol. This organism also produces acetic acid, ethanol, and acetoin. In this report, product inhibition due to 2,3-butanediol and acetic acid is considered. Although the acetate ion has little effect on growth, acetic acid is a strong inhibitor. Acetic acid inhibits growth more strongly than it inhibits respiration. The neutral product 2,3-butanediol is not a strong inhibitor; its effect on growth is no more than is expected by the decrease in water activity it causes. The effect of 2,3-butanediol on respiration can also be explained by a decreased water activity. It appears that it is possible to accumulate as much as 130 g/L butanediol while as little as 0.45 g/L acetic acid completely inhibits growth.     

Two-carbon assimilative capacity and the induction of isocitrate lyase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was grown on 10% glucose medium and subsequently transferred to fresh medium containing 2- and 3-carbon substrates. Under these conditions, the yeast rapidly acquired an oxidative capacity, as evidenced by oxygen uptake rates and 14CO2 evolution rates during respiration on ethanol or (14C)acetate. The assimilative capacity for 2-carbon substrates developed more slowly and followed the induction of isocitrate lyase. Washed yeast transferred to the basic medium containing no added carbon substrate possessed only low levels of isocitrate lyase after a 6-h adaptation. After 6 h, isocitrate lyase was present at high levels in cells transferred to a range of ethanol concentrations but was present in only low amounts in cells transferred to acetate. The role of ethanol as an inducer of isocitrate lyase is discussed.     

氧供应量对Klebsiella pneumoniae利用混合糖生物合成2,3-丁二醇过程的影响分析

以筛选的肺炎克雷伯氏茵（Klebsiella pneumoniaeUV-86）为对象,考察供氧条件分别对茵体生长、葡萄糖和木糖双底物利用和产物合成的影响。研究发现生物量随氧供应量增加而增加。不同供氧条件对茵体消耗葡萄糖过程的影响较小,而代谢木糖的能力随氧供应量的增大而增强。微氧条件下2,3-丁二醇的生物合成能力最强,2,3-丁二醇产量在1．5wm下达到最高为30．1g／L,是好氧时的2．5倍,最大体积产率为0．485g／（L·h）。不同条件下两底物产物分布有所区别,木糖代谢中乙酸生产增强。因此根据不同阶段代谢特点选择适合的供氧策略可以提高过程产量和产率。