Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Studies on the relationship between specific growth rate and concentration of nitrogen source for heterogeneous microbial populations of sewage origin

Batch experiments were run using heterogeneous populations to determine whether a hyperbolic equation of the type suggested by Monod could be used to depict the relation between specific growth rate, μ, and NH₃-N concentration when ammonia N was the growth-limiting nutrient. The heterogeneous populations employed were developed from sewage seed grown on glucose at various levels of nitrogen and various dilution rates in completely mixed continuous flow reactors. It was found that the hyperbolic function could be used. Values of μ_m in the range of 0.4–0.7 hr^?1 were observed, and values of K_s, in general, ranged from 1.5 to 4.0 mg/l. Variation in the values of these growth “constants” did not follow any discernible pattern related to past growth history (i.e., COD:N ratio or dilution rate at which the cells were previously grown).     

Physiology of myeloma cells grown in glucose-limited chemostat cultures

A recombinant myeloma NS1-derived clone was grown in chemostat cultures in Dulbecco's MEM/Ham's F12 (1∶1) medium containing various concentrations of glucose, at a dilution rate of 0.028 h⁻¹. Serum-supplemented cultures were virtually glucose-limited at a large range of glucose feed concentrations (0.7–5 mM). True glucose-limited cultures, however, were only established at low glucose supply levels to 1.3 mM at a maximum. In cultures obtained at higher glucose concentrations methionine was shown to be the growth-limiting compound. The pattern derived for serum-free chemostat cultures was similar, except that growth yields on glucose were much lower. Glucose was shown to be the growth-limiting substrate in cultures fed with media containing less than 4.5 mM glucose. Upon supplying glucose at higher concentrations such cultures presumably run into methionine and/or tryptophan limitation.     

Effect of high substrate concentration and cell feedback on kinetic behavior of heterogeneous populations in completely mixed systems

Growth kinetics of heterogeneous populations of sewage origin were studied in completely mixed reactors of the once-through type at a high concentration of incoming substrate, 3000 mg/l glucose, and in systems employing cell feedback or sludge recycle at an incoming substrate concentration of 1000 mg/1 glucose. The recycle flow rate employed was 25% of the incoming feed flow, and the concentration of cells in the recycle was maintained as closely as possible at 150% of the cell concentration in the reactor. Studies were made at various dilution rates. Throughout these studies, batch experiments using cells grown at the various dilution rates were made to determine k_s and μ_m values. As in previous studios using heterogeneous populations, the relationship between specific growth rates μ and substrate concentration S was represented better by the Monod equation than by any other which was tested. The growth “constants” μ_m, k_s, and Y were found to fall in the same general range as those determined in previous studies in once-through systems operated at 1000 mg/l glucose. It was observed that cell recycle, even at the relatively low concentration factor employed in these studies, greatly enhanced the flocculating and settling characteristics of the cells.     

药用蕈菌紫芝液体培养的生长特征分析

使用专门设计的ALR ff蕈菌液体培养反应器 ,研究了有关紫芝液体培养过程中菌丝生长的特征。结果表明 ,液体培养时紫芝菌丝生长对温度有较宽的适应范围 2 5℃～ 3 5℃ ,但生长比速率相差很大。通气量由 0 93vvm提高到 1 6 4vvm时 ,菌丝生长比速率明显地增加 ,最大值为 0 0 4 4 4 (h- 1) ;当通气量进一步提高到 2 5 0vvm时 ,生长比速率反而下降。与低糖浓度比较 ,较高糖浓度 (2 80g 1 0 0mL)可以使生长迟滞期缩短 ;培养后期菌丝缠绕成浓密颗粒 ,较高糖浓度可以促进生长。在整个培养过程中 ,糖浓度较低时葡萄糖转化成菌体的生长效率明显高于较高葡萄糖浓度的效率。碳源限制生长的连续培养研究结果 ,进一步反映了菌丝不同生长活性使菌丝物具有不同形态结构。菌体量、限制生长底物、菌体生成率的三者关系与细菌有明显的差异。并且 ,在 0 0 1 0～ 0 2 2 0 (h- 1)稀释率范围内菌丝生长符合Contois方程     

Hydrogen production by immobilized cells in the nozzle loop bioreactor

Summary The continuous production of hydrogen in a Nozzle Loop Bioreactor was investigated using immobilized Rhodospirillum rubum KS-301 with glucose as the growth-limiting substrate. The maximum hydrogen production rate in the experimental range was 91mL/h at dilution rate 0.4h^-1, initial glucose concentration 5.4g/L, and circulation rate 70h^-1 .     

Dynamics and control of continuous microbial propagators to subject substrate inhibition

Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X /YD )[dμ/dS]s }, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.     

Actual and potential rates of substrate oxidation and product formation in continuous cultures ofChromatium vinosum

Kinetics of electron-donor oxidation, storage-polymer formation and growth were studied in continuous cultures ofChromatium under conditions of balanced growth as well as during transient states.Under steady-state conditions, glycogen was accumulated at all dilution rates. This observation is consistent with previously postulated ideas about an ineffective glycogen-synthesis regulation.Upon perturbing the steady states, brought about by injection of extra sulfide into steady-state cultures, the following phenomena were observed immediately, irrespective of the dilution rate: the specific rate of sulfide oxidation increased to the value found in batch cultures, the sulfur-oxidation rate was decreased, the specific glycogen-synthesis rate increased, the increment being higher the lower the dilution rate, but an increase in the specific growth rate, if any, was below the limit of detection. The inverse relationship between the specific rates of glycogen synthesis and growth after removing the substrate limitation is to be explained by a shortage of intermediates, rather than by a growth-rate dependent intrinsic glycogen-synthesis limitation, because upon complete inhibition of growth a further increase in the rate of glycogen synthesis was observed. Essayed in this way, identical glycogen-synthesis rates were found at all dilution rates.Competitive advantages of such an apparently not adapted metabolism in environments with diurnal fluctuations in substrate concentrations are discussed.Non-Standard Abbreviations N_c cell nitrogen - TS total sugar - PHB poly--hydroxybutyrate - D dilution rate - S_R reservoir concentration of the growth-limiting substrate - CAP chloramphenicol     

Factors influencing the susceptibility of Candida albicans to the polyenoic antibiotics nystatin and amphotericin B.

Factors influencing the interaction between Candida albicans and the polyenoic antibiotics nystatin and amphotericin B have been investigated using a K+-specific electrode to measure polyene-mediated efflux of cellular K+. In batch cultures, sensitivity was a function of culture age. Using continuous (chemostat) cultures, the influence of growth-limiting substrate, specific growth rate, growth temperature and growth pH were examined. Carbon-limited cultures showed the highest sensitivity of those substrates tested, and susceptibility increased with growth rate. Within the range 22 to 42 degrees C, growth at lower temperatures resulted in increased sensitivity, whilst a similar trend was observed when the growth pH of cultures was reduced. Further, under the conditions tested, there were considerable variations in free intracellular K+ concentrations.     

Fermentation shifts and metabolic reactivity during anaerobic carbon-limited growth of Klebsiella aerogenes NCTC 418 on fructose,gluconate, mannitol and pyruvate

Klebsiella aerogenes NCTC 418 was grown in chemostat culture under carbon limitation, with fructose, mannitol, gluconate or pyruvate as the growth-limiting substrates, respectively. It was found that under these conditions the carbon sources were fermented with maximal stoichiometry of ATP generation. The Y _ATP values (g cells per mol ATP generated net) were similar for mannitol- and fructose-limited cultures, but gluconate-limited cultures expressed a value that was 20% lower. From these data it was concluded that gluconate-limited cells invest 0.5 ATP in the uptake of 1 gluconate.If the carbon limitation was instantaneously relieved by addition of a saturating amount of the growth-limiting substrate it was found that the response depended on the nature of the carbon substrate, and in particular on the ability to shift the fermentation pattern towards new products. In fructose- and gluconate-limited cultures the specific uptake rate of the carbon source increased substantially, without a concomitant increase in growth rate, and D-lactate appeared as a new fermentation product, in the case of gluconate accompanied by pyruvate. In contrast, with mannitol- and pyruvate-limited cultures the uptake rate of the carbon source and the fermentation pattern did not change. These results are discussed in connection with the functioning of the methylglyoxal by-pass and its role in sustaining metabolic reactivity.     

Growth of Bacteroides fragilis in Continuous Culture and in Batch Cultures at Controlled pH

Bacteroides fragilis NCTC 9343 has been grown in continuous cultures with glucose as growth-limiting factor. At pH 7.0 and at a dilution rate of 0.07 per h, glucose limited growth in concentrations up to 0.6%. Maximal cell yield and productivity were obtained with 0.87% glucose in the inflowing medium. A pH of 7.0 was optimal for growth. With 0.6% glucose in the fresh medium and at pH 7.0, cell yield and productivity were highest at a dilution rate of 0.07 per h and 0.11 per h, respectively. At dilution rates higher than 0.07 per h, glucose was no longer growth limiting, and at dilution rates above 0.11 per h, another compound seemed to have replaced glucose also as energy source. When grown in batch cultures at pH 7.0, the best yields of B. fragilis was achieved with 0.6% glucose in the fresh medium. The highest specific growth rate (mum) determined from viable counts was 0.45, corresponding to a mean generation time of 92 min.     

Control Mechanisms Operative in a Natural Microbial Population Selected for Its Ability to Degrade L-Lysine. III. Effects of Carbohydrates in Continuous-Flow Systems Under Shock Load Conditions

Two naturally selected microbial populations were maintained under continuousflow conditions with glucose or magnesium growth-limiting. The reactors were subjected to shock loads by changing the influent substrate from L-lysine to a mixture of L-lysine and glucose, L-lysine and fructose, or L-lysine and ribose. During the subsequent transient state, the following parameters were examined: lysine chemical oxygen demand (COD), carbohydrate COD, total COD, biological solids concentration, cell protein, enzymatic capability (lysine-degrading enzymes), and the rate of lysine removal. The carbohydrate was then removed from the influent and the same parameters were examined until a new steady state was established. In all cases, glucose and fructose caused a significant repression of the synthesis of lysine-degrading enzymes, resulting in a decrease in the enzymatic capability of the cells. In the carbon-limited reactor, the faster the flow rate, the greater was the repression, whereas, in the magnesium-limited reactor, the slower the flow rate, the greater was the repression. The introduction of ribose into the reactors caused an initial increase in lysine enzymatic capability followed by a slight repression when ribose degradation started.     

Breakdown ofd-glucose by mixed cultures ofEscherichia coli,Desulfovibrio vulgaris,andChromatium vinosum

Experiments with mixed cultures ofEscherichia coli, Desulfovibrio vulgaris, andChromatium vinosum were conducted using a synthetic medium with glucose as the substrate. The bacterial number and the changes in the chemical factors were determined during the development of the mixed culture. (i) Fermentation of glucose byE. coli produces organic acids (formic, acetic, lactic, and succinic) and alcohol. The growth-yield constant K (cell dry weight per weight solid substrate) does not exceed 0.05. (ii) In the mixed culture ofE. coli andD. vulgaris, the reduction of sulfate is accompanied by a total consumption of formate, lactate, and alcohol and an increase in the sulfide and acetate content. (iii) When the three physiologically different species are allowed to grow simultaneously, there is no accumulation of catabolites in the medium and the growth yield constant increases to 0.46. Maximum phototrophic production requires the presence of bothE. coli andD. vulgaris. A low substrate concentration and the simultaneous growth of the three organisms are other factors that contribute towards a high output. The biochemical parameters of the medium are influenced to a large extent by the glucose level. The results suggest that the behavior of the strains is different in pure and mixed cultures.     

Experimental studies on a kinetic model for design and operation of activated sludge processes

Previous experimentation in our laboratory has shown that the classical theory developed for continuous growth of pure cultures in completely mixed aerobic systems in which the recycle cell concentration factor, c (where c = X_R/X), is a selectable system constant, did not provide a suitable model for the heterogeneous (natural) populations of the activated sludge process. Another model was derived in which the recycle cell concentration, X_R was employed as a system constant instead of c, and computational analysis was performed. Laboratory pilot plant experimentation was undertaken in order to determine whether a “steady state” in aerator biological solids concentration, X?, and substrate concentration, S?, could be approached under this mode of operation. Studies were performed at various organic feed concentrations holding dilution rate, D, at 0.125 hr^?1, hydraulic recycle ratio, α, at 0.25, and X^R at 10,000 mg/liter. Also, values of maximum specific growth rate, μ_max, and saturation constant, K_s were determined. It was found that the model approached the steady state condition with heterogeneous populations more closely than did the classical model, and the high degree of treatment efficiency predicted by the model was demonstrated experimentally.     

Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol

During the production of biodiesel, a significant amount of glycerol is generated which currently has little commercial value. A study on the growth and lipid production of Chlorella protothecoides using glycerol as the carbon source was performed to demonstrate the utility of recycling crude glycerol created during biodiesel production. Glycerol was examined as both the sole carbon source and in combination with glucose. Algae cultures grown on only glycerol in shake flasks showed a specific growth rate and final lipid yield of 0.1/h and 0.31 g lipid/g substrate, respectively. The values were similar to those observed on pure glucose, 0.096/h and 0.24 g lipid/g substrate. When the media contained a mixture of glycerol and glucose, simultaneous uptake of the two substrates was observed. Due to the difference in rates of lipid storage, lipid production was 0.077 g lipid/(l h) during growth on glycerol, while growth on glucose had a productivity of 0.096 g lipid/(l h). During growth on the 9:1 mixture of both glucose and glycerol, lipid productivity was 0.098 g lipid/(l h). In order to simulate the use of waste glycerol from biodiesel production the experiments were repeated and similar growth rates, yields, and lipid productivities were achieved. Therefore, we have demonstrated the promise for simultaneous high growth rates and lipid yields of C. protothecoides heterotrophically grown on mixtures of glycerol.     

Substrate inhibition kinetics of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fed-batch cultures operated at constant glucose and maltose concentration levels

Fed-batch culture is the mode of operation of choice in industrial baker’s yeast fermentation. The particular mode of culture, operated at stable glucose and maltose concentration levels, was employed in this work in order to estimate important kinetic parameters in a process mostly described in the literature as batch or continuous culture. This way, the effects of a continuously falling sugar level during a batch process were avoided and therefore the effects of various (stable) sugar levels on growth kinetics were evaluated. Comparing the kinetics of growth and the inhibition by the substrate in cultures grown on glucose, which is the preferential sugar source for Saccharomyces cerevisiae, and maltose, the most common sugar source in industrial media for baker’s yeast production, a milder inhibition effect by the substrate in maltose-grown cells was observed, as well as a higher yield coefficient. The observed sugar inhibition effect in glucostat cultures was taken into account in modeling substrate inhibition kinetics. The inhibition coefficient K _i increased with increasing sugar concentration levels, but it appeared to be unaffected by the type of substrate and almost equal for both substrates at elevated concentration levels.     

Effects of sudden temperature shifts on pure cultures of four strains of freshwater bacteria

Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified asCytophaga johnsonae (C-21),Cytophaga sp. (M-17),Pseudomonas fluorescens (KD), andEnterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. TheC. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereasCytophaga sp. (M-17) shift-up cultures showed a lower andP. fluorescens (KD) a higher growth rate. The mesophilicE. cloacae (BS-2), likeC. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion byP. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase.     

Relationship between substrate concentration,growth rate,and respiration rate of Escherichia coli in continuous culture

Summary Using a continuous flow technique the relationship between growth rate and substrate concentration was investigated with glucose as the limiting factor of a culture of Escherichia coli. Graphical and numerical analysis of the experimental data demonstrated that the application of the Michaelis-Menten equation produced erroneous results, whereas, the constants obtained from the Teissier equation were in agreement with the experimental data. On this basis, new equations defining the steady state cell and substrate concentration in continuous flow cultures were developed and tested against experimental data.Comparison of the specific growth rates, substrate uptake rates and oxygen consumption rates demonstrated that all were directly proportional to each other and could be related to each other by mathematical equations. Specifically it was shown that as the growth rate increased from 0.06 to k _m =0.76 the substrate uptake rate increased from 134 to 1420 mg glucose per gram cell weight per hour and the oxygen consumption rate increased from 48.6 to 505 mg O₂ per gram cell weight per hour. Independent of the growth rate 37% of the carbohydrate consumed were oxidized. The yield factor varied from 0.44 at low growth rates to 0.54 at high growth rates. Analysis of the growth rate-substrate uptake rate relationship indicated that a minimum substrate uptake rate of 55 mg glucose per gram cell weight per hour existed below which cell reproduction would cease. This was supported by the fact that steady state conditions could not be maintained in the culture at D values below 0.02 when the substrate supply rate decreased below 45 mg glucose per gram cell weight per hour.Material contained in this paper was submitted as a thesis in partial fulfillment of the requirements for the Ph. D. degree of Dr. R. S. Lipe.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.