Growth kinetics of Escherichia coli with galactose and several other sugars in carbon-limited chemostat culture

Kinetic models for microbial growth describe the specific growth rate (mu) as a function of the concentration of the growth-limiting nutrient (s) and a set of parameters. A typical example is the model proposed by Monod, where mu is related to s using substrate affinity (Ks) and the maximum specific growth rate (mu max). The preferred method to determine such parameters is to grow microorganisms in continuous culture and to measure the concentration of the growth-limiting substrate as a function of the dilution rate. However, owing to the lack of analytical methods to quantify sugars in the microgram per litre range, it has not been possible to investigate the growth kinetics of Escherichia coli in chemostat culture. Using an HPLC method able to determine steady-state concentrations of reducing sugars, we previously have shown that the Monod model adequately describes glucose-limited growth of E. coli ML30. This has not been confirmed for any other sugar. Therefore, we carried out a similar study with galactose and found steady-state concentrations between 18 and 840 micrograms.L-1 for dilution rates between 0.2 and 0.8.h-1, respectively. With these data the parameters of several models giving the specific growth rate as a function of the substrate concentration were estimated by nonlinear parameter estimation, and subsequently, the models were evaluated statistically. From all equations tested, the Monod model described the data best. The parameters for galactose utilisation were mu max = 0.75.h-1 and Ks = 67 micrograms.L-1. The results indicated that accurate Ks values can be estimated from a limited set of steady-state data when employing mu max measured during balanced growth in batch culture. This simplified procedure was applied for maltose, ribose, and fructose. For growth of E. coli with these sugars, mu max and Ks were for maltose 0.87.h-1, 100 micrograms.L-1; for ribose 0.57.h-1, 132 micrograms.L-1, and for fructose 0.70.h-1, 125 micrograms.L-1.     

Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria.

Seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria, including Pseudomonas, Alcaligenes, and Bordetella spp., were compared on the basis of growth kinetics. Estimates of maximum growth rate (mu max, k1) and half-saturation growth constant (Ks, k3) were obtained by fitting substrate depletion curves to a four-parameter version of the integrated Monod equation. Estimates of Ks ranged from 2.2 micrograms/ml (10 microM) to 33.8 micrograms/ml (154 microM), and estimates of mu max ranged from 0.20 h-1 (Td = 3.5 h) to 0.32 h-1 (Td = 2.2 h). Estimates of mu max, but not Ks, were affected by changes in initial inoculum density. Maximum growth rates (mu max) were also estimated from turbidity measurements. They ranged from 0.10 h-1 (Td = 6.9 h) to 1.0 h-1 (Td = 0.7 h). There was no correlation between estimates of mu max derived from substrate depletion curves and those derived from turbidity measurements (P = 0.20).     

Kinetic comparison of seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria.

Seven strains of 2,4-dichlorophenoxyacetic acid-degrading bacteria, including Pseudomonas, Alcaligenes, and Bordetella spp., were compared on the basis of growth kinetics. Estimates of maximum growth rate (mu max, k1) and half-saturation growth constant (Ks, k3) were obtained by fitting substrate depletion curves to a four-parameter version of the integrated Monod equation. Estimates of Ks ranged from 2.2 micrograms/ml (10 microM) to 33.8 micrograms/ml (154 microM), and estimates of mu max ranged from 0.20 h-1 (Td = 3.5 h) to 0.32 h-1 (Td = 2.2 h). Estimates of mu max, but not Ks, were affected by changes in initial inoculum density. Maximum growth rates (mu max) were also estimated from turbidity measurements. They ranged from 0.10 h-1 (Td = 6.9 h) to 1.0 h-1 (Td = 0.7 h). There was no correlation between estimates of mu max derived from substrate depletion curves and those derived from turbidity measurements (P = 0.20).     

Nonlinear estimation of Monod growth kinetic parameters from a single substrate depletion curve.

Monod growth kinetic parameters were estimated by fitting sigmoidal substrate depletion data to the integrated Monod equation, using nonlinear least-squares analysis. When the initial substrate concentration was in the mixed-order region, nonlinear estimation of simulated data sets containing known measurement errors provided accurate estimates of the mu max, Ks, and Y values used to create these data. Nonlinear regression analysis of sigmoidal substrate depletion data was also evaluated for H2-limited batch growth of Desulfovibrio sp. strain G11. The integrated Monod equation can be more convenient for the estimation of growth kinetic parameters, particularly for gaseous substrates, but it must be recognized that the estimates of mu max, Ks, and Y obtained may be influenced by the growth rate history of the inoculum.     

Nonlinear estimation of Monod growth kinetic parameters from a single substrate depletion curve

Monod growth kinetic parameters were estimated by fitting sigmoidal substrate depletion data to the integrated Monod equation, using nonlinear least-squares analysis. When the initial substrate concentration was in the mixed-order region, nonlinear estimation of simulated data sets containing known measurement errors provided accurate estimates of the mu max, Ks, and Y values used to create these data. Nonlinear regression analysis of sigmoidal substrate depletion data was also evaluated for H2-limited batch growth of Desulfovibrio sp. strain G11. The integrated Monod equation can be more convenient for the estimation of growth kinetic parameters, particularly for gaseous substrates, but it must be recognized that the estimates of mu max, Ks, and Y obtained may be influenced by the growth rate history of the inoculum.     

Temperature-dependent growth kinetics of Escherichia coli ML 30 in glucose-limited continuous culture.

Detailed comparison of growth kinetics at temperatures below and above the optimal temperature was carried out with Escherichia coli ML 30 (DSM 1329) in continuous culture. The culture was grown with glucose as the sole limiting source of carbon and energy (100 mg liter(-1) in feed medium), and the resulting steady-state concentrations of glucose were measured as a function of the dilution rate at 17.4, 28.4, 37, and 40 degrees C. The experimental data could not be described by the conventional Monod equation over the entire temperature range, but an extended form of the Monod model [mu = mu(max) x (s - s(min))/(Ks + s - s(min))], which predicts a finite substrate concentration at 0 growth rate (s(min)), provided a good fit. The two parameters mu(max) and s(min) were temperature dependent, whereas, surprisingly, fitting the model to the experimental data yielded virtually identical Ks values (approximately 33 microg liter(-1)) at all temperatures. A model that describes steady-state glucose concentrations as a function of temperature at constant growth rates is presented. In similar experiments with mixtures of glucose and galactose (1:1 mixture), the two sugars were utilized simultaneously at all temperatures examined, and their steady-state concentrations were reduced compared with to growth with either glucose or galactose alone. The results of laboratory-scale kinetic experiments are discussed with respect to the concentrations observed in natural environments.     

基因工程菌对阿特拉津的生物转化及其影响因素

研究考察了基因工程菌转化阿特拉津的共代谢碳源、转化动力学和影响因素。结果表明,作为共代谢碳源,葡萄糖优于乙酸盐,碳源浓度对转化影响不大,对工程菌生长影响显著。阿特拉津比转化速率与工程菌初始密度无关,与阿特拉津初始浓度有关,用Monod方程拟合转化动力学,求得方程参数为V_(max)=0．168/h,Ks= 30．49mg/L。降低温度会显著降低阿特拉津比转化速率;偏碱性的条件下,阿特拉津转化率较高,酸性条件严重抑制阿特拉津转化;盐度在一定范围内不影响转化活性;Co~(2 )、Fe~(2 )、Fe~(3 )和Zn~(2 )促进阿特拉津转化,Mn~(2 )、Ni~(2 )和Cu~(2 )抑制阿特拉津转化。菌体细胞对阿特拉津的吸附和转化作用呈正相关关系。     

Long-term competition between sulfate reducing and methanogenic bacteria in UASB reactors treating volatile fatty acids

The competition between acetate utilizing methane-producing bacteria (MB) and sulfate-reducing bacteria (SRB) was studied in mesophilic (30 degrees C) upflow anaerobic sludge bed (UASB) reactors (upward velocity 1 m h-1; pH 8) treating volatile fatty acids and sulfate. The UASB reactors treated a VFA mixture (with an acetate:propionate:butyrate ratio of 5:3:2 on COD basis) or acetate as the sole substrate at different COD:sulfate ratios. The outcome of the competition was evaluated in terms of conversion rates and specific methanogenic and sulfidogenic activities. The COD:sulfate ratio was a key factor in the partitioning of acetate utilization between MB and SRB. In excess of sulfate (COD:sulfate ratio lower than 0.67), SRB became predominant over MB after prolonged reactor operation: 250 and 400 days were required to increase the amount of acetate used by SRB from 50 to 90% in the reactor treating, respectively, the VFA mixture or acetate as the sole substrate. The competition for acetate was further studied by dynamic simulations using a mathematical model based on the Monod kinetic parameters of acetate utilizing SRB and MB. The simulations confirmed the long term nature of the competition between these acetotrophs. A high reactor pH (+/-8), a short solid retention time (<150 days), and the presence of a substantial SRB population in the inoculum may considerably reduce the time required for acetate-utilising SRB to outcompete MB.     

Multiple substrate growth kinetics of Leptothrix discophora SP-6

The growth parameters of Leptothrix discophora SP-6 were quantified on the basis of the steady-state concentrations and utilization rates of pyruvate, dissolved oxygen, and concentration of microorganisms in a chemostat operated at 25 degrees C, pH 7.2, and an agitation rate of 350 rpm. The results showed that the microbial growth was limited by both pyruvate and dissolved oxygen. A combined growth kinetics model using Monod growth kinetics for pyruvate and Tessier growth kinetics for oxygen showed the best correlation with the experimental data when analyzed using an interactive multiple substrate model. The growth kinetics parameters and the respective confidence limits, estimated using the Monte Carlo simulation, were mu(max) = 0.576 +/- 0.021 h(-1), K(sMp) = 38.81 +/- 4.24 mg L(-1), K(sTo) = 0.39 +/- 0.04 mg L(-1), Y(X/p) = 0.150 (mg microorganism mg(-1) pyruvate), Y(X/o) = 1.24 (mg microorganism mg(-1) oxygen), the maintenance factors for pyruvate and oxygen were m(p) = 0.129 (mg pyruvate consumed mg(-1) microorganism h(-1)) and m(o) = 0.076 (mg oxygen consumed mg(-1) microorganism h(-1)), respectively.     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp.

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of p-cresol degradation by an immobilized Pseudomonas sp

A p-cresol (PCR)-degrading Pseudomonas sp. was isolated from creosote-contaminated soil and shown to degrade PCR by conversion to protocatechuate via p-hydroxybenzaldehyde (PBA) and p-hydroxybenzoate (PHB). Cells of the Pseudomonas sp. were immobilized in calcium alginate beads and in polyurethane foam. The relationship between the PCR concentration and the PCR transformation rate was investigated in batch and continuous culture bioreactors. The biodegradation kinetics of PBA and PHB also were investigated. In batch culture reactors, the maximum PCR degradation rate (Vmax) for the alginate-immobilized Pseudomonas sp. cells was 1.5 mg of PCR g of bead-1 h-1 while the saturation constant (Ks) was 0.22 mM. For PHB degradation, the Vmax was 0.62 mg of PHB g of bead-1 h-1 while the Ks was 0.31 mM. For polyurethane-immobilized Pseudomonas sp. cells, the Vmax of PCR degradation was 0.80 mg of PCR g of foam-1 h-1 while the Ks was 0.28 mM. For PHB degradation, the Vmax was 0.21 mg of PHB g of foam-1 h-1 and the Ks was 0.22 mM. In a continuous column alginate bead reactor, the Vmax for PCR transformation was 2.6 mg g of bead-1 h-1 while the Ks was 0.20 mM. The Vmax and Ks for PBA transformation in the presence of PCR were 0.93 mg g of bead-1 h-1 and 0.063 mM, respectively. When PHB alone was added to a reactor, the Vmax was 1.48 mg g of bead-1 h-1 and the Ks was 0.32 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

A kinetic model for surfactant inhibition of pentachlorophenol biodegradation

A kinetic model is used to describe the effect of the nonionic surfactant Tergitol NP-10 (TNP10) on pentachlorophenol (PCP) biodegradation by Sphingomonas chlorophenolica sp. strain RA2. Different initial biomass to initial substrate ratios ranging from 13 to 418 were tested with 23 TNP10 concentrations ranging from 0 to 1500 mg/L. Tests were also conducted at 10 degrees C and 20 degrees C. No PCP biodegradation inhibition was observed at concentrations below the critical micelle concentration (CMC) of 50 mg/L. TNP10 concentrations above 100 to 200 mg/L were increasingly inhibitory to PCP biodegradation rates. This inhibition was best described by the Monod kinetic equation wherein the effect of TNP10 inhibition is reflected in the half-saturation constant (Ks). The value of the Ks increased from between 1.5 and 13.5 mg/L with no surfactant present to 44 to 131 mg/L at 1000 mg/L TNP10. Using a standard competitive inhibition approach, the inhibition constant for TNP10 was approximately 100 mg/L at both 10 degrees C and 20 degrees C.     

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.     

Growth kinetics of Pseudomonas putida G7 on naphthalene and occurrence of naphthalene toxicity during nutrient deprivation

The objectives of this work were (1) to demonstrate how the chemostat approach could be modified to allow determination of kinetic parameters for a sparingly soluble, volatile substrate such as naphthalene and (2) to examine the influence of the interactions of various nutrients on possible growth-inhibitory effects of naphthalene. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. Naphthalene was found to be toxic to P. putida G7 in the absence of a nitrogen source or oxygen. The death rate of cells grown on minimal medium plus naphthalene and then exposed to naphthalene under anoxic conditions was higher than that observed under oxic conditions in the absence of a nitrogen source. The presence of necessary nutrients for the biodegradation of PAH compounds is indicated to be important for the survival of microorganisms that are capable of PAH degradation. The amounts of ammonia and oxygen necessary for naphthalene biodegradation and for suppression of naphthalene toxicity were calculated from growth yield coefficients. A chemostat culture of P. putida G7 using naphthalene as a carbon and energy source was accomplished by using a feed augmented with a methanol solution of naphthalene so as to provide sufficient growth to allow accurate evaluation of kinetic parameters. When naphthalene was the growth-limiting substrate, the degradation of naphthalene followed Monod kinetics. Maximum specific growth rate (micrometer) and Monod constant (Ks) were 0.627 +/- 0.007 h-1 and 0.234 +/- 0.0185 mg/L, respectively. The evaluation of biodegradation parameters will allow a mathematical model to be applied to predict the long-term behavior of PAH compounds in soil when combined with PAH transport parameters.     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

Kinetics of microbial growth on pentachlorophenol

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

Kinetics of microbial growth on pentachlorophenol.

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization by an enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monod equation was found to describe the relationship between the specific growth rate and substrate concentration. Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients are low, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60 micrograms/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (KI = 1,375 micrograms/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can be used to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.     

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.     

Growth efficiency and the specific rate of the yeast Hansenula polymorpha during continuous cultivation on methanol

The growth of Hansenula polymorpha DL-1 in the chemostat (under methanol limitation) and turbidostat was measured. Cultivation with different specific rates of growth mu made it possible to determine the maximum yield of biomass Ys(max)=0.425 and the level of expendables required to maintain Ms=0.023 hr-1. The following parameters describing mu as a function of the concentration of methanol S in the fermenter were found: muo=0.154 hr-1 (maximum growth rate), Ks=1.31 mg/l, Ki=5.35 g/l. The paper emphasizes a very low value of the saturation constant Ks derived from the above experiments and reviews the literature data on the kinetic characteristics of various methanol-grown yeast.     

Kinetics of biotransformation of 1,1,1-trichloroethane by Clostridium sp. strain TCAIIB.

Batch experiments were conducted to examine the effects of high concentrations of 1,1,1-trichloroethane (TCA) on the biotransformation of TCA by Clostridium sp. strain TCAIIB. The biotic dehalogenation of TCA to 1,1-dichloroethane by nongrowing cells was measured at 35 degrees C, and the data were used to obtain the kinetic parameters of the Monod relationship half-velocity coefficient Ks (31 microM) and the coefficient of maximum rate of TCA biotransformation (kTCA; 0.28 mumol per mg per day). The yield of biomass decreased with an increase in the TCA concentration, although TCA concentrations up to 750 microM did not completely inhibit bacterial growth. Also, kTCA was higher in the presence of high concentrations of TCA. A mathematical model based on a modified Monod equation was used to describe the biotransformation of TCA. The abiotic transformation of TCA to 1,1-dichloroethene was measured at 35 degrees C, and the first-order formation rate coefficient for 1,1-dichloroethene (ke) was determined to be 0.86 per year.