Elementary mode analysis to study the preculturing effect on the metabolic state of Lactobacillus rhamnosus during growth on mixed substrates

Quantification of metabolism through elementary modes offers insights into the working of a metabolic network. We have determined the fluxes of elementary modes through linear optimization using the stoichiometry of the elementary modes as a constraint. We apply this methodology to obtain insights into the effect of preculturing on growth of Lactobacillus rhamnosus on medium containing mixed substrates. L. rhamnosus, a microaerophilic organism, produces flavor compounds such as diacetyl and acetoin during growth on glucose and citrate. The uptake of citrate has been shown to be sensitive to preculturing states of the cells. Elementary modes demonstrated that citrate was utilized by the organism as a sole carbon source. Further, both glucose and citrate was catabolized by this organism through aerobic and anaerobic routes. The flux analysis indicated that only 21 elementary modes were operational during growth of L. rhamnosus on glucose and citrate. Glucose specifically accounted for 6 elementary modes, while the remaining 15 involved citrate as substrate. The modes associated with glucose were mainly operational when cells were precultured on glucose. It was observed that all the 21 modes contributed to the fluxes when the cells were precultured on citrate. The NADH recycling through lactate formation and oxygen uptake were dependent on the preculturing state. The analysis also demonstrated that preculturing on citrate yielded better productivity of diacetyl and acetoin.     

Effect of carbon dioxide on growth of Pseudomonas putida ATCC 11172 on asparagine, citrate, glucose, and lactate in batch and continuous culture

The growth of Pseudomonas putida ATCC 11172 on L-asparagine, citrate, D-glucose, and L-lactate was followed in air and in 40% CO2 + air, using batch and carbon-limited continuous cultures. Batch cultures in air utilized a mixture of the carbon sources simultaneously. However, a change to 40% CO2 favoured the utilization of glucose. The maximum specific growth rate (mumax) in air was about 0.3 h-1 on glucose and 0.6 h-1 on the other carbon sources. In CO2, the mumax for glucose was reduced by 16% compared with almost 60-70% for the others. An order of preference for the different carbon sources in continuous cultures was determined by comparing the dilution rates at which the different carbon sources started to appear in the effluent. Glucose was the first compound to appear as the dilution rate increased (lowest preference when grown in air). In 40% CO2, the mumax for glucose was slightly higher than the others and the recorded preference for glucose in continuous culture was equal to that for citrate but was somewhat lower than that of lactate and asparagine. D-Gluconate and glucono-delta-lactone were produced as a step in the utilization of glucose. The D-gluconate production was enhanced by CO2.     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

Fermentation characteristics of Fusariumoxysporum grown on acetate

In this study, the growth characteristics of Fusariumoxysporum were evaluated in minimal medium using acetate or different mixtures of acetate and glucose as carbon source. The minimum inhibitory concentration (MIC) of acetic acid that F.oxysporum cells could tolerate was 0.8%w/v while glucose was consumed preferentially to acetate. The activity of isocitrate lyase was high when cells were grown on acetate and acetate plus glucose indicating an activation of the glyoxylate cycle. Investigation of the metabolic fingerprinting and footprinting revealed higher levels of intracellular and extracellular TCA cycle intermediates when F.oxysporum cells were grown on mixtures of acetate and glucose compared to growth on only glucose. Our data support the hypothesis that a higher flux through TCA cycle during acetate consumption could significantly increase the pool of NADH, resulting in the activation of succinate-propionate pathway which consumes reducing power (NADH) via conversion of succinate to propionyl-CoA and produce propionate.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Internal supply of coenzyme to an amperometric glucose biosensor based on a chemically modified electrode

A biosensor for glucose using glucose dehydrogenase immobilized on a chemically modified graphite electrode was supplied with coenzyme, nicotinamide adenine dinucleotide (NAD+), through pores in the material. A graphite rod was hollowed out, leaving 0.3 mm at the end contacting the solution, filled with 10 mM NAD+ and pressurized. The response factor was 40% of that obtained when 2 mM NAD+ was mixed with the sample solution in a flow system. The coenzyme consumption was 11 microliters h-1 representing a 500-fold saving compared to supply through the bulk solution. The biosensor had a linear calibration curve from the detection limit, 1 microM, to 2 mM glucose and a repeatability of 0.3%. The graphite electrode was modified by adsorption of a bis-(benzophenoxazinyl)-terephthaloyl derivative in order to be able to oxidize NADH at 0 mV versus Ag/AgCl, 0.1 M KCl.     

Mechanism of citrate metabolism in Lactococcus lactis: resistance against lactate toxicity at low pH

Measurement of the flux through the citrate fermentation pathway in resting cells of Lactococcus lactis CRL264 grown in a pH-controlled fermentor at different pH values showed that the pathway was constitutively expressed, but its activity was significantly enhanced at low pH. The flux through the citrate-degrading pathway correlated with the magnitude of the membrane potential and pH gradient that were generated when citrate was added to the cells. The citrate degradation rate and proton motive force were significantly higher when glucose was metabolized at the same time, a phenomenon that could be mimicked by the addition of lactate, the end product of glucose metabolism. The results clearly demonstrate that citrate metabolism in L. lactis is a secondary proton motive force-generating pathway. Although the proton motive force generated by citrate in cells grown at low pH was of the same magnitude as that generated by glucose fermentation, citrate metabolism did not affect the growth rate of L. lactis in rich media. However, inhibition of growth by lactate was relieved when citrate also was present in the growth medium. Citrate did not relieve the inhibition by other weak acids, suggesting a specific role of the citrate transporter CitP in the relief of inhibition. The mechanism of citrate metabolism presented here provides an explanation for the resistance to lactate toxicity. It is suggested that the citrate metabolic pathway is induced under the acidic conditions of the late exponential growth phase to make the cells (more) resistant to the inhibitory effects of the fermentation product, lactate, that accumulates under these conditions.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

Benzoate degradation via the ortho pathway in Alcaligenes eutrophus is perturbed by succinate.

During batch growth of Alcaligenes eutrophus on benzoate-plus-succinate mixtures, substrates were simultaneously metabolized, leading to a higher specific growth rate (mu = 0.56 h-1) than when a single substrate was used (mu = 0.51 h-1 for benzoate alone and 0.44 h-1 for succinate alone), without adversely affecting the growth yield (0.57 Cmol/Cmol). Flux distribution analysis revealed that succinate dehydrogenase most probably controls the rate of total succinate consumption (the maximum flux being 9.7 mmol.g-1.h-1). It is postulated that the relative consumption rate of each substrate is in part related to modified levels of gene expression but to a large extent is dependent upon the presence of succinate, end product of the beta-ketoadipate pathway. Indeed, the in vitro beta-ketoadipate-succinyl coenzyme A transferase activity was seen to be inhibited by succinate, a coproduct of the reaction.     

Physiological,biochemical, and cytological characteristics of a halotolerant and alkalitolerant methanotroph grown on methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h-1) was twice as high as on methane (0.1 h-1). The maximum growth rate increased with increasing medium salinity and was lower at neutral than at alkaline pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

Citrate efflux in glucose-limited and glucose-sufficient chemostat culture of Penicillium simplicissium

Citrate excretion by Penicillium simplicissimum was investigated in a chemostat. Carbon-limited grown P. simplicissimum did not excrete no citrate. Citrate was excreted, however, when growth was nitrogen-limited. Further effects of nitrogen-limitation were a slightly increased rate of glucose and oxygen consumption. This behaviour is typical for a so-called `overflow metabolism', i.e. the uncoupling of anabolism from catabolism under conditions of carbon excess. Still more citrate was excreted by nitrogen-limited P. simplicissimum when (i) the extracellular osmolarity was increased from 0.2 to 1.5 osm kg^–1 or (ii) when the pH was increased from 4 to 7; or (iii) when the extracellular potassium concentration was lowered from 6 to 0.5 mM. These results were interpreted in terms of a higher energy-consumption under these conditions.     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Effect of amino acids on the heat production and growth efficiency of Streptococcus bovis: balance of anabolic and catabolic rates.

Streptococcus bovis JB1 grew nearly twice as fast (0.9 versus 1.6 h-1) and had a 40% greater growth yield (18 versus 12.5 mg of protein per mmol of glucose) when an ammonia-based medium was supplemented with amino acids, but the glucose consumption rate (88 mumol mg of protein-1 h-1) and specific rate of heat production (2.1 mW/mg of protein) were unaffected. Amino acid availability had little effect on the catabolic rate, but the specific heat decreased 40% (8.8 to 5.2 J/mg of protein). These growth rate-dependent changes in metabolic efficiency were fivefold greater than the maintenance energy. Chloramphenicol (100 mg/l), an inhibitor of protein synthesis, caused a gradual decrease in anabolic (growth) rate, but there was little change in the rate of glucose consumption and the specific heat increased. When growth was inhibited by iodoacetate, the catabolic and anabolic rates both declined and there was not increase in specific heat. On the basis of these results, the benefit of amino acid supplementation was largely explained by the balance of anabolic and catabolic rates. When amino acids were available, the anabolic and catabolic rates were more closely matched and less energy was spilled as heat.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

Effects of Energy Substrates on Nitrate Reduction and Nitrate Reductase Activity in a Ruminal Bacterium, Selenomonas ruminantium

The factors affecting the rate of nitrate reduction and the nitrate reductase content in Selenomonas ruminantium were examined. The rate of nitrate reduction per cell mass was higher when S. ruminantium was grown on lactate than when grown on glucose, and the rate was further enhanced when grown on succinate. The nitrate reduction rate was parallel to the nitrate reductase content in cells, suggesting that the amount of nitrate reductase limits the rate of nitrate reduction. The amount of nitrate reductase was inversely related to growth rate. The growth rate was related to the level of intracellular ATP, which was inversely related to the levels of ADP and AMP. The ratio of NADH to NAD⁺was related to the rate of nitrate reduction and to the amount of nitrate reductase. From these results, it is conceivable that the synthesis of nitrate reductase is regulated in response to the sufficiency of energy and electron supply. Intracellular concentrations of adenine nucleotides and pyridine nucleotides may be the regulating factors. The amount of nitrate reductase was increased by the presence of nitrate, suggesting that the synthesis of nitrate reductase is enhanced by nitrate. In addition, nitrate reduction altered the fermentation pattern as a result of electron consumption.