Residual Substrate Concentration Research Articles

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture.

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.

Model of dissolved organic carbon distribution for substrate-sufficient continuous culture.

The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism. It appears that the excess substrate could determine metabolic pathways of microorganisms, which further control dissolved organic carbon (DOC) distribution under substrate-sufficient conditions. However, how to quantitatively describe the DOC distribution remains unclear in substrate-sufficient continuous culture. Based on a balanced DOC reaction, a DOC distribution model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. Results showed that a considerable portion of the DOC consumed was directly oxidized to carbon dioxide through energy spilling under substrate-sufficient conditions. The proposed model for the first time quantified the DOC distribution between nongrowth-associated and growth-associated metabolisms of cells. The proposed model was verified with literature data very well.

Modeling of Energy Spilling in Substrate-Sufficient Cultures

A concept of an energy spilling coefficient between anabolism and catabolism was developed. Based on this concept, two models were proposed to describe quantitatively the effect of a substrate on the coefficient for substrate-sufficient continuous and batch cultures. The models have been well verified using literature data obtained from the continuous cultures of Bacillus strains and the writers' own data obtained from both anoxic and aerobic batch cultures of activated sludge microorganisms. It has also been demonstrated that in both types of cultures, with an increasing residual substrate concentration or increasing initial food-to-microorganisms ratio, a decline of observed growth yield (Yobs) was mainly due to energy spilling. The effect of the substrate on the energy spilling coefficient in the substrate-sufficient cultures can be expressed by a Monod type curve. In the continuous cultures, the maximum energy spilling coefficient was found to be 0.7 when the residual methanol concentrations were greater than 10 mmol/L. In the batch cultures, the efficiency of anoxic sludge reached 0.8 as the initial food-to-microorganisms ratio exceeded 10 mg chemical oxygen demand/mg mixed-liquor suspended solids, while that of aerobic sludge was only 60% with the same ratio. These findings reveal that anoxic activated sludge has a higher potential of energy spilling than does aerobic sludge.

Modeling of Energy Spilling in Substrate-Sufficient Cultures

A concept of an energy spilling coefficient between anabolism and catabolism was developed. Based on this concept, two models were proposed to describe quantitatively the effect of a substrate on the coefficient for substrate-sufficient continuous and batch cultures. The models have been well verified using literature data obtained from the continuous cultures of \IBacillus\N strains and the writers’ own data obtained from both anoxic and aerobic batch cultures of activated sludge microorganisms. It has also been demonstrated that in both types of cultures, with an increasing residual substrate concentration or increasing initial food-to-microorganisms ratio, a decline of observed growth yield (\iY\do\db\ds) was mainly due to energy spilling. The effect of the substrate on the energy spilling coefficient in the substrate-sufficient cultures can be expressed by a Monod type curve. In the continuous cultures, the maximum energy spilling coefficient was found to be 0.7 when the residual methanol concentrations were greater than 10 mmol/L. In the batch cultures, the efficiency of anoxic sludge reached 0.8 as the initial food-to-microorganisms ratio exceeded 10 mg chemical oxygen demand/mg mixed-liquor suspended solids, while that of aerobic sludge was only 60% with the same ratio. These findings reveal that anoxic activated sludge has a higher potential of energy spilling than does aerobic sludge.

Kinetics of organic carbon removal by a mixed culture in a membrane bioreactor

The aim of this work was to model the biological activity and anticipate the kinetic behaviour of microorganisms and the overall performance of the process according to a specific model and running parameters. The bacterial inoculum used in these experiments was a mixture of cultures taken from the wastewater treatment plant in Montpellier. The fermentor, used in association with an ultrafiltration separation stage (with a filtration area of 0.2 m 2) had a working volume of 15.8 l. For various working conditions (different solid retention times, different hydraulic retention times and substrate concentrations), the biomass concentration and the residual substrate concentration, expressed in terms of dry weight and chemical oxygen demand, respectively, were measured. The basic idea of modelling was related to the concept of maintenance. The coefficient of maintenance, E, and the theoretical conversion yield, y, were therefore calculated. The values of E and y, measured for total cell recycling experiments and for experiments with various solid retention times, remained similar and were found to equal 0.040 mgCOD mgVSS h −1 and 0.36 mgVSS mgCOD −1, respectively. Determining these two constants and modelling the treatment process made it possible to anticipate the optimal biomass concentration for a defined removal efficiency under different steady-state operating conditions.

Recovery of coal fines from washery and power plant effluents by integrated technique of oil agglomeration and biofilm formation.

The possibility of applying an integrated technique of oil agglomeration and biofilm formation for recovery of coal fines from coal washeries and power plants effluents has been explored. Laboratory experiments with simulated slurries of different Indian coal fines demonstrate that vegetable oils are satisfactory agglomerating agents for recovery of most of the coal fines depending on the nature of coal and type of oil. The agglomeration behaviour of coal fines was assessed in terms of % yield, % organic matter recovery and % ash rejections. Maximum 85% agglomerate recovery was obtained in the agglomeration stage. Residual oil concentrations in some cases were found to exceed the permissible limit. Recovery of residual coal fines and reduction in residual oil concentration in the resultant slurry after oil agglomeration have been attempted using biofilm formation. A laboratory scale treatment reactor was put under complete recirculation to facilitate attached microbial growth on coal particles as carrier under aerobic conditions. The influence of various parameters on attached growth and stable biofilm formation were studied. The growth patterns of attached cell in suspension and consumption pattern of carbon substrate (oil) have been investigated. Steady decline in residual substrate concentration in the slurry with corresponding increase in the growth of attached and free cell mass is observed. The growth process was favoured in pH range of 6.5-7.0. The attached growth was found to be expanded in size in due course of time ultimately leading to the formation of stable biofilm in the treatment reactor which was subjected to the influent total suspended solids loading resulting from oil agglomeration step. Performance of the biofilm reactor in terms of % reduction in total suspended solids and residual oil concentration in the influent slurry was assessed in continuous mode. Complete recovery of coal fines and 60% degradation of oil was observed in the final effluent discharged from the treatment reactor.

Energy uncoupling in microbial growth under substrate-sufficient conditions

It has been observed that the correlation between ATP and biomass formation is very poor, and the observed growth yield is lowered under substrate-sufficient conditions. This indicates that the excess substrate causes uncoupling between anabolism and catabolism, which leads to the dissipation of non-growth energy. However, a quantitative description of such uncoupling remains elusive. Based on a balanced substrate reaction, a growth-yield model in relation to residual substrate concentration for substrate-sufficient continuous cultures was developed. On the basis of this yield model, a coefficient governing the uncoupling of anabolism and catabolism was defined. A model describing the effect of the residual substrate concentration on this uncoupling coefficient was further proposed. These models agree with experimental data very well. It is clearly shown that, under substrate-sufficient conditions, the variation in growth efficiency is mainly due to energy uncoupling rather than to maintenance energy expenditure.

Optimization of Phaffia rhodozyma continuous culture through response surface methodology

Response surface methodology was applied to optimize the growth of the yeast Phaffia rhodozyma in continuous fermentation using peat hydrolysates as substrate. A second-order, complete, factorial design of the experiments was used to develop empirical models providing a quantitative interpretation of the relationships between the two variables studied, dilution rate and pH. Maximum biomass concentration in the fermentor was obtained by employing the following predicted optimum fermentation conditions: a dilution rate of 0.017/h and a pH level of 7.19. A verification experiment, conducted at previously optimized conditions for maximum biomass volumetric productivity (a dilution rate of 0.022/h, and a pH level of 6.90), produced values for biomass concentration, residual substrate concentration, biomass yield, and biomass volumetric productivity that were very close to the predicted values, indicating the reliability of the empirical model. The concentration of the pigment astaxanthin produced by the yeast under the optimized growth conditions was found to be 544 mg astaxanthin/kg dry cell biomass. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 314–320, 1998.

Optimization ofPhaffia rhodozyma continuous culture through response surface methodology

Response surface methodology was applied to optimize the growth of the yeast Phaffia rhodozyma in continuous fermentation using peat hydrolysates as substrate. A second-order, complete, factorial design of the experiments was used to develop empirical models providing a quantitative interpretation of the relationships between the two variables studied, dilution rate and pH. Maximum biomass concentration in the fermentor was obtained by employing the following predicted optimum fermentation conditions: a dilution rate of 0.017/h and a pH level of 7.19. A verification experiment, conducted at previously optimized conditions for maximum biomass volumetric productivity (a dilution rate of 0.022/h, and a pH level of 6.90), produced values for biomass concentration, residual substrate concentration, biomass yield, and biomass volumetric productivity that were very close to the predicted values, indicating the reliability of the empirical model. The concentration of the pigment astaxanthin produced by the yeast under the optimized growth conditions was found to be 544 mg astaxanthin/kg dry cell biomass.

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture.

Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present. The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems. Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.

Model of energy uncoupling for substrate-sufficient culture

The growth yields (Y(obs)) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism, which leads to energy spilling. Although the uncoupling between anabolism and catabolism has already been recognized in the microbiology literature, how to quantitatively describe such uncoupling remains unclear. Based on a balance on substrate reaction, a growth yield model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. On the basis of that yield model, the concept of an uncoupling coefficient between anabolism and catabolism is defined in this work. A model describing the effect of the residual substrate concentration on the uncoupling coefficient of anabolism to catabolism is proposed. This model agrees very well with literature data.

A new enzymatic technique to estimate the efficiency of microbial degradation of pollutants

Dynamics of active sludge microorganism activity in aerotanks under chemostat conditions has been studied. Dependence of microorganism catalase activity has been found to depend on residual substrate concentration in proportion to the biomass of microorganisms. Experimental data and field observations has formed the basis to develop a technique to evaluate in relative units the amount of the substrate consumed by biocenosis of the active sludge in the air tanks of purification facilities.

A Growth Yield Model for Substrate-Sufficient Continuous Culture of Microorganisms

It is generally observed that the growth yields are greater under substrate-limited conditions than under substrate-sufficient conditions in chemostat cultures. However, the Pirt maintenance theory could not correctly explain the effect of excessive substrate on the growth yield. The excess substrate should cause serious uncoupling between anabolism and catabolism leading to energy spilling. Based on a balance on substrate reaction, the total quantity of substrate consumed by microorganisms is taken to be the sums of the quantity of substrate utilization for growth, the quantity of substrate utilization for maintenance and that due to energy spilling. A kinetic model is proposed to describe the dependence of the observed growth yield on residual substrate concentration for substrate-sufficient culture. It is demonstrated that the observed growth yield decreases with increasing the residual substrate concentration. This model describes the literature data satisfactorily.

Transformation of Low Concentrations of 3-Chlorobenzoate by Pseudomonas sp. Strain B13: Kinetics and Residual Concentrations

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.

Steady-state analysis of a fixed bed reactor for urea hydrolysis with microencapsulated urease

The steady-state operation of a fixed bed reactor (FBR) with microencapsulated urease for the regeneration of a dialysate solution was modeled at various enzyme activities to improve the understanding of performance factors. The limiting effects of pH, product inhibition and substrate depletion were also studied individually and in combination under eight case studies for two different microcapsule diameters, 5 and 500 μm. The rapid depletion of substrate at high enzyme activities for a Michaelis-Menten kinetic model lowered the residual substrate concentration ( S r ) to a value below the Michelis-Menten constant K m,0 resulting in dramatic reductions in the intracapsular reaction rate. When the limiting factors were considered (pH, product inhibition and substrate depletion) in the reaction process, reduced urea conversion values were encountered for the level of enzyme activity under consideration. These limiting factors maintained S r at a higher value relative to K m,0 . Hence, the efficiency of the fixed bed reactor decreased from the inlet to the outlet when a Michaelis-Menten kinetic model was considered, and vice versa for other models which accounted for product inhibition and pH-dependent kinetics.

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.

Performance Features for Urea Hydrolysis in A Cstr with Microencapsulated Urease

The factors which influence the steady state performance of a CSTR operation with microencapsulated urease for the regeneration of a dialysated solution have been studied at various enzyme activities. The theoretical model considered the effect of microcapsule diameter, pH-dependent kinetics, and product inhibition and substrate depletion, in relation to urea conversion and the capsule effectiveness factor. The limiting effects of pH, product inhibition and substrate depletion were also studied individually and in combination under eight case studies. The base case which included these three limiting factors in the reaction process, predicted the lowest urea conversion values at the enzyme activities considered. However, the effectiveness factor for each case at a fixed microcapsule diameter depended on the enzyme activity. This behaviour was studied for two different microcapsule diameters, 5 microns and 500 microns. At enzyme activities lower than 1 mM/s, a model considering Michaelis-Menten kinetics alone, predicted the highest effectiveness factors. On the other hand, beyond 1 mM/s enzyme activity, the lowest effectiveness factors were predicted, although higher conversions than that of the base case were achieved. This might be due to the rapid depletion of substrate at high activities when Michaelis-Menten kinetics were considered, leads to residual substrate concentrations (Sr) lower than the Michaelis-Menten constant Km,o; a condition for a dramatic drop in the intraparticle reaction rate. The limiting factors in the base case held Sr at a relatively higher value than Km,o.

Carbohydrate metabolism and physiology of the parasitic protist Trichomonas vaginalis studied in chemostats.

The parasitic protist Trichomonas vaginalis was cultured in chemostats with glucose or maltose as carbon and energy source. The maximum growth rate was about six divisions per day independent of the substrate, and the apparent Km for glucose was 0.375 mM. While growing on maltose, the growth rate depended linearly on the maltose concentration, indicating that in contrast to glucose metabolism a diffusion step is rate-limiting to maltose metabolism. Cultures were examined over a wide range of growth rates under four conditions: utilizing glucose or maltose as carbon and energy source, with the carbon source rate-limiting or present in excess. Cell density, cellular protein and carbohydrate content as well as residual substrate concentration in the culture fluid were measured at each steady state. The protein content was constant at 100 pg protein per cell except when T. vaginalis was cultured under glucose limitation; in the latter case, slow-growing cells had less protein than cells grown at high rates. When growing under glucose limitation T. vaginalis metabolism changed to become more energy efficient at growth rates exceeding about half the maximum rate. The maintenance energy at the low growth rates accounted for approximately half of the total carbon consumption, which is high in comparison to other micro-organisms. At low growth rates the yield on maltose exceeded that on glucose, when expressed in terms of carbon equivalents. The yields on maltose and glucose were equal, but much lower, when the carbon source was not rate-limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Fuzzy control of baker's yeast fed-batch bioprocess: A robustness study

The purpose of the investigation was to determine if, based on the monitoring of the residual substrate concentration, fuzzy control is an effective way of controlling a fermentation process. Various simulations were made using a knowledge model, first to evaluate the performance of the control for the biomass concentration, and then to analyze how it reacted in the presence of disturbances. Results showed high final productivity and biomass concentration. Despite different disturbances, as the feeding glucose concentration changed or the temporary substrate feed-rate increased, the fuzzy controller provided process stability and robustness near the set-point. Moreover, these properties were maintained in the case of noisy measurements.

Kinetics of microbial growth with mixtures of carbon sources

Several investigations have shown that during growth in carbon-limited chemostats the simultaneous utilisation of carbon substrates which usually provoke diauxie under batch conditions, i.e., 'mixed substrate growth,' is probably the rule under ecologically relevant growth conditions. In contrast, the models presently available for the description of the kinetics of microbial growth are all based on the use of single substrates. Systematic studies in chemostat culture have shown that steady-state residual concentrations of individual compounds were consistently lower during mixed substrate growth than during growth with the single substrates. This effect is clearly demonstrated for the case of Escherichia coli growing with mixtures of glucose plus galactose. The data presented indicate that the extent of reduction of steady-state residual substrate concentration is dependent on the proportions of the substrates in the mixture, the nature of substrates mixed and the regulation pattern of enzymes involved in their breakdown. If this behaviour can be shown to be typical for growth under environmental conditions, it may provide an explanation why microbes still grow relatively fast at the low substrate concentrations encountered in nature.