Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Metabolism and growth yields in Bacteroides ruminicola strain b14.

Metabolism of D-glucose by Bacteroides ruminicola subsp. brevis, strain B14, has been examined. Growth yield studies gave molar growth yields, corrected for storage polysaccharide, of approximately 66 g (dry weight)/mol of glucose fermented. The storage polysaccharide amounted to about 14% of the total dry weight, or 55% of the total cellular carbohydrate, at full growth. After correcting glucose utilization for incorporation into cellular carbohydrate, measurement of product formation showed that 1.1 succinate, 0.8 acetate, and 0.35 formate are produced and 0.5 CO2 net is taken up during the fermentation of 1 glucose under the conditions used. The implication of these results with respect to adenosine 5'-triphosphate (ATP) molar growth yield calculations is discussed. If substrate-level phosphorylation reactions alone are responsible for ATP generation, then the ATP molar growth yield must be about 23 g (dry weight)/mol of ATP. Alternatively, if anaerobic electron transfer-linked phosphorylation also occurs, the ATP molar growth yield will be lower.     

Efficiency of oxidative phosphorylation in continous cultures of Candida parapsilosis.

By using a gas balance technique, the effective oxidative phosphorylation ratio in glucose-limited continuous cultures of Candida parapsilosis was calculated to be 2.4 +/- 0.2. When this value was used, the molar growth yield with respect to adenosine 5'-triphosphate was found to be 10 g of cells produced per mol of ATP.     

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.     

Induction of citrate lyase in Enterobacter cloacae grown under aerated conditions and its effect on citrate metabolism.

Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained alpha-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.     

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Adenylate energy charge in Acholeplasma laidlawii.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.     

Molar Growth Yields as Evidence for Oxidative Phosphorylation in Streptococcus faecalis Strain 10C1

During the aerobic growth of Streptococcus faecalis strain 10C1, with limiting levels of glucose as the substrate, a molar growth yield (Y) of 58.2 g (dry weight) per mole of glucose was obtained. Under these conditions of growth, glucose was dissimilated primarily to acetate and CO(2). The incorporation of (14)C-glucose into cell material was no greater under aerobic conditions than during anaerobic growth. Assuming an adenosine triphosphate coefficient of 10.5, the aerobic Y cannot be explained solely on the basis of substrate phosphorylation and would appear to substantiate previous enzymatic evidence for oxidative phosphorylation in this cytochromeless species. With mannitol as the substrate, an aerobic Y of 64.6 was obtained. Extracts of mannitol-grown cells contained a nicotinamide adenine dinucleotide (NAD)-linked mannitol-1-phosphate (M-1-P) dehydrogenase. The difference in aerobic Y values with mannitol and glucose as substrates would indicate that the in vivo P/O ratio from the oxidation of reduced NAD generated by the oxidation of M-1-P approximates 0.6. The Y values with pyruvate and glycerol as substrates under aerobic conditions were 15.5 and 24.7, respectively.     

Evidence for cytochrome involvement in fumarate reduction and adenosine 5'-triphosphate synthesis by Bacteroides fragilis grown in the presence of hemin.

Growth of Bacteroides fragilis subsp. fragilis on glucose was very much stimulated by the addition of hemin (2 mg/liter) to the medium. The generation time decreased from 8 to 2 h, and the molar growth yield increased from YM = 17.9 to YM = 47 g (dry weight) of cells per mol of glucose. In the absence of hemin, glucose was fermented to fumarate, lactate, and acetate. The cells did not contain detectable amounts of cytochromes or fumarate reductase. In the presence of hemin, the major products of fermentation were succinate, propionate, and acetate. A b-type cytochrome, possibly a c-type cytochrome, and a very active fumarate reductase were present in the cells. It is concluded from these results that hemin is required by B. fragilis to synthesize a functional fumarate reductase and that the hemin-dependent, enormous increase of the growth yield may be due to adenosine 5'-triphosphate production during reduction of fumarate to succinate.     

Effect of aeration and sodium on the metabolism of citrate by Klebsiella aerogenes.

Anaerobic growth of Klebsiella aerogenes NCDO 711 (NCTC 418) on citrate was dependent on the presence of Na+ in the medium, and fermentation of citrate was mediated via the fermentation pathway enzymes, citrate lyase and a Na+-dependent oxalacetate decarboxylase. This confirms the previous findings on strain NCTC 418. Growth under aerobic conditions was independent of Na+. The mean generation time for cells grown aerobically on either Na+ or K+ citrate medium was about 60 min, with a molar growth yield of about 40 g (dry weight) of cells per mol of citrate utilized. Citrate was apparently metabolized aerobically in both the Na+ and K+ citrate cells via the citric acid cycle, since cell extracts contained alpha-ketoglutarate dehydrogenase but not the citrate fermentation enzymes. The presence of theother enzymes of the citric acid cycle in K. aerogenes was shown in earlier studies. Under aerated conditions (no detectable oxygen tension in the culture), growth was faster on the Na+ citrate medium (mean generation time, 85 min) than on the K+ citrate medium (mean generation time, 120 min). Both cultures grew slower than under aerobic conditions, presumably because of oxygen limitation. Despite the faster growth rate, the molar growth yield of the aerated Na+ citrate culture was one-half that observed for the aerated K+ citrate culture. Citrate was metabolized via the citric acid cycle in cells grown in the K+ citrate medium under aerated conditions since alpha-ketoglutarate dehydrogenase, but not the fermentation enzymes, was detected in extracts prepared from these cells. Metabolism of citrate in the Na+ citrate medium under aerated conditions occurred via both the fermentation pathway (approximately 75 percent) and the citric acid cycle (about 25 percent), as evidenced by (i) the presence of the fermentation enzymes and alpha-ketoglutarate dehydrogenase in extracts of cells grown under these conditions, (ii) a molar growth yield which was intermediate between that obtained for anaerobic and aerated K+ citrate cultures, and (iii) the excretion of acetate, which also occurred in anaerobic cultures but not in aerated K+ citrate or aerobic cultures.     

大肠杆菌突变株QQS101发酵生产琥珀酸初步研究

琥珀酸是一种具有重要应用价值的生物基平台化合物。对大肠杆菌focA-pflB ldhA突变株QQS101在严格厌氧条件下生长和葡萄糖代谢能力进行了考察,比较分析了葡萄糖与大肠杆菌混合酸发酵产物的单位碳的还原程度,认为非严格厌氧条件有利于QQS101发酵葡萄糖积累琥珀酸,进一步对有氧生长碳源进行了对比试验的结果表明,以木糖支持有氧生长,QQS101摇瓶发酵39 h消耗葡萄糖37.6 g/L,琥珀酸的产量达到31.01 g/L,摩尔产率为1.258 mol Succinate/mol Glucose。发酵过程中,丙氨酸的添加能够提高琥珀酸的摩尔产率。     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli.

It is known that in Escherichia coli two dehydrogenases of the flavoprotein kind can participate in the transfer of hydrogens from sn-glycerol 3-phosphate (G3P) to nitrate and that possession of either enzyme is sufficient to permit anaerobic growth on glycerol as carbon source and nitrate as hydrogen acceptor. Results from this study show that under such a growth condition a protein with light-absorption characteristics of cytochrome b1 is induced. If G3P, nitrate, and adenosine diphosphate are added anaerobically to a particulate fraction prepared from these cells, four reactions can be detected: (i) the reduction of the cytochrome b1-like protein, (ii) the formation of dihydroxyacetone phosphate (DHAP), (iii) the formation of nitrite, and (iv) the generation of adenosine 5'-triphosphate (ATP). The anaerobic G3P dehydrogenase system can yield an ATP-DHAP (or ATP-nitrite) molar ratio of about 0.2, whereas the aerobic G3P dehydrogenase system can yield a corresponding ratio of about 0.3. The hydrogen transfer activity is sensitive to respiratory inhibitors such as cyanide, Rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide.     

Maintenance requirements for bacteria growing on C1-compounds

The maintenance coefficient, ms (mmol substrate/g cell dry wt hr), of two distinct groups of C1-utilizing bacteria has been determined by growing the organisms in an aerobic continuous culture limited by different C1 growth substrates. For growth on methanol, ms = 2.5 +/- 0.3 for Pseudomonas C; 3.9 +/- 0.7 for Ps. methylotropha (these bacteria utilize methanol via the ribulose monophosphate pathway of formaldehyde fixation); 1.5 +/- 0.2 for Pseudomonas 1, and 2.3 +/- 0.4 for Pseudomonas 135 (the latter bacteria utilize C1-compounds via the serine pathway). For growth on formaldehyde, ms = 1.5 +/- 0.3 for Pseudomonas 1 and 2.7 +/- 0.7 for Pseudomonas 135, whereas on formate the values for ms are 1.0 +/- 0.2 and 4.4 +/- 1.3; respectively. Although the maintenance coefficients did not differ systematically between the two groups of bacteria, the maintenance requirements per generation of the serine pathway bacteria were considerably higher (8.7 vs. 3.9) owing to their slower growth rate. The maximum molar yield values, YMmax (g cell dry wt/mol substrate utilized), corrected for the maintenance energy of bacteria which utilize C1-compounds via the ribulose monophosphate pathway averaged 19.1 when grown on methanol, while the values for bacteria which use the serine pathway averaged 13.5. On formaldehyde an average value of 11.5 is obtained and on formate the average value was 7.4 in the serine pathway bacteria.     

Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

Glucose degradation, molar growth yields, and evidence for oxidative phosphorylation in Streptococcus agalactiae

In a complex medium with the energy source as the limiting nutrient factor and under anaerobic growth conditions, Streptococcus agalactiae fermented 75% of the glucose to lactic acid and the remainder to acetic and formic acids and ethanol. By using the adenosine triphosphate (ATP) yield constant of 10.5, the molar growth yield suggested 2 moles of ATP per mole of glucose from substrate level phosphorylation. Under similar growth conditions, pyruvate was fermented 25% to lactic acid, and the remainder was fermented to acetic and formic acids. The molar growth yield suggested 0.75 mole of ATP per mole of pyruvate from substrate level phosphorylation. Under aerobic growth conditions about 1 mole of oxygen was consumed per mole of glucose; about one-third of the glucose was converted to lactic acid and the remainder to acetic acid, acetoin, and carbon dioxide. Molar growth yields indicated 5 moles of ATP per mole of glucose. Estimates based on products of glucose degradation suggested that about one-half of the ATP was derived from substrate level phosphorylation and one-half from oxidative phosphorylation. Addition of 0.5 m 2,4-dinitrophenol reduced the growth yield to that occurring in the absence of oxygen. Aerobic pyruvate degradation resulted in 30% of the substrate becoming reduced to lactic acid and the remainder being converted to acetic acid and carbon dioxide, with small amounts of formic acid and acetoin. The molar growth yields and products found suggested that 0.70 mole of ATP per mole of pyruvate resulted from substrate level phosphorylation and 0.4 mole per mole of pyruvate resulted from oxidative phosphorylation.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Ethanol production with Saccharomyces cerevisiae at potassium-limited aerobic growth conditions

Summary The specific ethanol productivity withSaccharomyces cerevisiae grown aerobically in a chemostat at a growth rate of 0.17 hr^–1 was found to increase from zero to 13 mmol/g cell dry matter·h when the potassium content in the substrate used was decreased to 0.05 mol/kg glucose. 78% of the glucose metabolized were converted to ethanol under these aerobic growth conditions.