Specific Growth Rate Constant Research Articles

Mixed-feed exponential feeding for fed-batch culture of recombinant methylotrophic yeast

A simple, accurate model capable of predicting cell growth and methanol utilization during the mixed substrate fed-batch fermentation of Mut S recombinant Pichia pastoris was developed and was used to design an exponential feeding strategy for mixed substrate fed-batch fermentation at a constant specific growth rate. Mixed substrate feeding has been shown to boost productivity in recombinant fed-batch culture of P. pastoris, while fixed growth rate exponential feeding during fed-batch culture is a useful tool in process optimization and control.

Effect of feed rate on growth rate and antibody production in the fed-batch culture of murine hybridoma cells.

Batch and fed-batch cultures of a murine hybridomacell line (AFP-27) were performed in a stirred tankreactor to estimate the effect of feed rate on growthrate, macromolecular metabolism and antibodyproduction. Macromolecular composition was foundto change dynamically during batch culture ofhybridoma cells possibly due to active production ofDNA, RNA and protein during the exponential phase.Antibody synthesis is expected to compete with theproduction of cellular proteins from the amino acidpool. Therefore, it is necessary to examine therelationship between cell growth in terms of cellularmacromolecules and antibody production. In this study,we searched for an optimum feeding strategy bychanging the target specific growth rate in fed-batchculture to give higher antibody productivity whileexamining the macromolecular composition. Concentratedglucose (60 mM) and glutamine (20 mM) in DR medium(1:1 mixture of DMEM and RPMI) with additional aminoacids were fed continuously to the culture and thefeed rate was updated after every sampling to ensureexponential feeding (or approximately constantspecific growth rate). Specific antibody productionrate was found to be significantly increased in thefed-batch cultures at the near-zero specific growthrate in which the productions of cellular DNA, RNA,protein and polysaccharide were strictly limited byslow feeding of glucose, glutamine and other nutrients. Possible implications of these results are discussed.

Phenol degradation byRalstonia eutropha: Colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.

Phenol degradation by Ralstonia eutropha: colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations.

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.

Phenol degradation by Ralstonia eutropha: Colorimetric determination of 2‐hydroxymuconate semialdehyde accumulation to control feed strategy in fed‐batch fermentations

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a μmax of 0.41 h−1. Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h−1, close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m3/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 407–415, 1999.

Egg production rates of eight calanoid copepod species during summer 1997 off Newport, Oregon, USA

Measurements of hydrography, water transparency, chlorophyll (Chl)<it>a</it> and egg production rates (EPRs) by females of<it>Calanus marshallae</it> Frost, <it>Calanus pacificus</it> Brodsky, <it>Eucalanus californicus</it> Johnson, <it>Epilabidocera longipedata</it> Sato,<it>Pseudocalanus mimus</it> Frost, <it>Centropages abdominalis</it> Sato, <it>Acartia longiremis</it> Lilljerborg and <it>Paracalanus parvus</it> (Claus) were estimated at weekly intervals between 17 July and 2 September 1997. Production of eggs was determined in 24 h incubations to examine the effects of environmental variability on EPR, to detect the possibility of food limitation of EPR, and to evaluate the hypothesis that growth rates of females are size dependent. During the study, an anomalous downwelling event occurred, possibly in response to the 1997 El Ni&ntilde;o, which allowed us to determine how El Ni&ntilde;o events affect EPRs of coastal copepods. The larger copepods <it>Calanus marshallae, Calanus pacificus</it> and <it>Centropages abdominalis</it> showed the highest egg production and specific growth rates during the period of active upwelling (18 July-13 August, water temperatures 8-13<IMG SRC="/math/deg.gif">C, Chl <it>a</it> concentration 4.7-16.2 <IMG SRC="/math/mu.gif"> l^-1 and water transparency 3-5 m). After 27 August, the 1997-98 El Ni&ntilde;o arrived off Oregon, creating a downwelling situation. Upwelling winds ceased, the thermocline intensified, temperature and transparency increased (to &gt;18&deg;C and 16 m), and Chl<it>a</it> declined to &lt;2 &mgr;g l^-1. Densities of the common coastal species declined greatly as well. <it>Paracalanus parvus</it> became the dominant species, and <it>Eucalanus californicus, Epilabidocera longipedata</it> and <it>Corycaeus anglicus</it> became common in our samples. EPRs for the larger boreal copepods (<it>Calanus</it> and <it>Centropages</it>) declined greatly during El Ni&ntilde;o;; the smaller copepods,<it>Pseudocalanus mimus, A.longiremis</it> and<it>Paracalanus parvus</it>, showed low but relatively constant egg production and specific growth rates during both upwelling and downwelling events. Over the entire study period, only three species produced eggs at its maximum rate (24-28 eggs female^-1 day^-1),<it>Pseudocalanus mimus</it> which averaged 4 eggs day^-1. All other species had EPRs that were two or five times below their maximum EPR. Thus, EPRs were not related to body size, contrary to our expectations. Hatching success was variable among species. Values as low as 20-40% were found for all species at least once during the study period, suggesting that occasionally a substantial portion of egg production may not be viable.

活性汚泥から分離した有殻葉状仮足類Ａｒｃｅｌｌａ ｖｕｌｇａｒｉｓの増殖に及ぼす環境因子の影響

Testacealobosia, Arcella vulgaris is well known as the typical protozoa found in the activated sludge and biofilm when nitrification is in progress. The effects of good source, water temperature, pH and phosphate buffer concentration on specific growth rate of A. vulgaris isolated from activated sludge were examined. A. vulgaris was successfully cultured when Chlorella powder suspension media and sludge bacteria were used as food sources. The growth parameters wwere determined using Chlorella powder suspension media. The specific maximum growth rate and half-saturation constant were 1.05d-1 and 1.4TOCmg·l-1, respectively. The growth of A. vulgaris was observed from 10°C to 36°C with optimal growth at 32°C. A. vulgaris was able to grow when the concentration of phosphate buffer was higher than 2/75M and pH value ranged from 4.2 to 9.0. The results show that food concentration and water temperature are the important parameters for the growth of A. vulgaris.

Effect of surface materials on initial biofilm development

The effect of the support characteristics on the substrate degradation kinetics and on the adherence and growth of biofilms is a subject of considerable importance in wastewater treatment. Many works deal with the kinetics, but less work has been reported concerning the surface differences. In this paper, the results of an experimental programme studying the biofilm growth in rotating biological reactors (RBC) with different solid material in each biodisk, are presented. Results from two phases have been obtained; the first phase of colonization, of about 1–2 days, was followed by a growth phase with a specific growth rate constant between 1·1–1·3 d −1. The biofilm dry matter (DM) ranged between 7 mg DM/cm 2 for the glass particle and diatomite earth support materials, and 9 mg DM/cm 2 for the sand disk and 10 mg DM/cm 2 for the activated carbon disks. All these results indicated that carbon was the best of the tested support materials for biofilm formation. No dependence was observed between position of the biodisk in the RBC and the organic layer formation or the initial biofilm development.

Enhancement ofCandida rugosa lipase production by using different control fed-batch operational strategies

Simulation studies have predicted that maximum lipase activity is reached with fed-batch operation strategies. In this work, two different fed-batch operational strategies have been studied: constant substrate feeding rate and specific growth rate control. A constant substrate feeding rate strategy showed that maximum aqueous lipolytic activity (55 U/mL) was reached at low substrate feeding rates, whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. In the second fed-batch strategy studied, a feedback control strategy has been developed based on the estimation of state variables (X and mu) from the measurement of indirect variables such as CER by means of mass spectrometry techniques. An on-off controller was then used to maintain the specific growth rate at the desired value by adjusting the substrate feeding rate. A constant specific growth rate strategy gave higher final levels of aqueous lipolytic activity (117 U/mL) at low specific growth rates. At higher specific growth rates the enzyme remained accumulated inside the cell, as was observed with a constant feeding fed-batch strategy. With a constant specific growth rate strategy, lipase production by Candida rugosa was enhanced 10-fold compared to a batch operation. Purification studies have demonstrated that lipolytic and esterasic specific activity ratios of Candida rugosa isoenzymes can be modified by using different operational conditions. These studies have also showed that the isoenzymes obtained in a controlled growth rate strategy are around three- to four-fold more active than those obtained in a constant feeding rate strategy.

Growth kinetics ofPseudomonas 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.

Improvement of heterologous protein productivity using recombinantYarrowia lipolytica and cyclic fed-batch process strategy

A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h).

Low Temperature Effects on Anaerobic Microbial Kinetic Parameters

Anaerobic sequencing batch reactors (ASBRs) were operated in the temperature range of 5 to 25°C and 24, 16, 12, 8, 6 hour hydraulic retention times (HRTs) over a period of two years to evaluate the effects of different temperatures on the microbial kinetic parameters K, Ks, Kd, Yg and μm. The values of K, Kd and μm were found to decrease, and the half saturation constant, Ks was found to increase with a decrease in temperature in accordance with the Arrhenius equation. Temperature coefficient, θ values for substrate utilization rate, maximum specific growth rate, decay rate and half saturation constant were 1.083, 1.062, 1.076 and 1.05 respectively. Through the determination of kinetic parameters, a design engineer may be able to predict the performance of ASBR treatment systems over a wide range of low temperatures.

On-Line Monitoring and Optimal Adaptive Control of the Fed-Batch Baker's Yeast Fermentation

In this paper an experimental validation of on-line monitoring and optimal adaptive control strategies for the baker’s yeast fermentation is presented. The online monitoring strategy consists of an on-line biomass measurement (based on a capacitance measure), which is exploited to estimate the specific growth rate by using an observer based estimator. The model based control algorithm is an adaptive linearizing control law, which keeps the estimated specific growth rate constant during the fermentation. Special attention is devoted to the tuning of the estimator and the controller.

Factors affecting the fermentative production of a lysozyme-binding antibody fragment inEscherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery.

Analysis of microbial I growth data using a spreadsheet

This paper describes the use of computer spreadsheets for the simple mathematical determination of microbial growth parameters. By carrying out laboratory growth experiments using non-pathogenic microorganisms, the student can enter results into a spreadsheet and, using the functions of the spreadsheet, calculate growth parameters such as specific growth rate constant, doubling time, and yield coefficients. The spreadsheet can readily be used for different sets of data from different microorganisms and/or different growth conditions.

A kinetic model of growth and product formation of the anaerobic microorganism Thermoanaerobacter thermohydrosulfuricus

A model is developed to describe the growth, product formation and enzyme synthesis of the microorganism Thermoanaerobacter thermohydrosulfuricus. It differentiates between extracellular enzymatic starch degradation and the microorganism's constant specific growth rate, combined with the ingestion of maltose and maltotriose (the products of starch cleavage). The formation of growth dependent products like ethanol, acetate and the synthesis of the cell-associated enzyme, amylopullulanase, are formulated with constant yield coefficients. In contrast, both the non-growth dependent product lactate, and extracellular enzyme activity are linked to an intracellular substrate concentration. Most of the parameters used in the model are evaluated from batch culture or literature. The calculated results are compared with experimental data. Keywords: Continuous culture; Thermoanaerobacter thermohydrosulfuricus; Starch; Model

Filamentous growth in activated sludge.

Bulking and foaming in activated sludge have been associated with filamentous overgrowth. Filamentous Nocardia amarae and nonfilamentous Pseudomonas auruginosa were cultured using fatty acids (C2-C24) as the sole carbon. N. amarae could utilize all acids tested for growth, whereas P. auruginosa hardly grew on acids with 12 or more carbons. Maximum specific growth rate and saturation constant of N. amarae on C24, at 0.048 h-1 and 1.520 g COD/L, respectively, were much lower than that of P. auruginosa, showing that N. amarae had a relatively stronger affinity for long-chain fatty acids. N. amarae was competitive in activated sludge processes that receive sewage containing a high proportion of long-chain fatty acids, oils, and fats.

Characterization and application of an on-line flow injection analysis/wall-jet electrode system for glucose monitoring during fermentation

An on-line system for glucose monitoring during fermentation was developed. The system employed flow-injection analysis coupled with a wall-jet electrode. Glucose was converted enzymatically in a tubular reactor packed with controlled pore glass beads containing immobilized glucose oxidase. The hydrogen peroxide produced was detected amperometrically. A filtration/sampling module operated by a peristaltic pump enabled sample withdrawal from the fermenter. The system was interfaced to a programmable language controller which was in turn linked to a microcomputer. The dual injection valve of the flow system was operated in a novel configuration to achieve sample dilution, by dispersion, and standardization simultaneously. The developed system was characterized for dilution factor, stability and linearity of response. After conditioning of the reactor, sample peak heights were found to have a precision of 1.07% (R.S.D., 25 determinations) when the glucose concentration was 1.32 g l −1. Under the conditions studied, linear response to glucose concentration from 0.062 to 4.0 g l −1 was obtained. Sample throughput was about 7 per hour. Interferences from the fermentation media were negligible after filtration through the sampling module and dilution. The on-line system was applied to glucose monitoring during E. coli fermentation. Good results were obtained as shown by the high cell density (OD 600nm = 168 in 9 h fermentation), constant specific growth rate ( μ = 0.66) and lower acetate accumulation (less than 2 g l −1 when OD 600nm was over 50).

Comparative study of anaerobic digestion of olive mill wastewater (OMW) and OMW previously fermented with Aspergillus terreus

A comparative kinetic study was carried out on the anaerobic digestion of olive mill wastewater (OMW) and OMW that was previously fermented with Aspergillus terreus. Two completely-mixed bioreactors were used for the study. The results obtained were evaluated using the Chen-Hashimoto methane production model to obtain values for the maximum specific growth rate (μmax) and kinetic constant (K) of the process for each case studied. The kinetic parameters were found to be influenced by the pretreatment carried out, and were 4.5 and 3.8 times higher for pretreated OMW than that obtained in the anaerobic digestion of untreated OMW. This was significant at 95 % confidence level. This behaviour is believed to be due to the lower levels of phenolic compounds and biotoxicity present in the pretreated OMW, as compared to untreated OMW, resulting in an improved process performance and stability. Finally, the experimental values of methane production were reproduced with deviations equal to or less than 10% in every case.

Comparison of temperature- and isopropyl-β-d-thiogalacto-pyranoside-induced synthesis of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli

Two different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (bFGF) using Escherichia coli TGl as host organism. The bFGF structural gene was cloned into two vectors differing only with respect to the promoter, which was either the bacteriophage λ PRPL- or the E. coli lac-promoter. The resulting expression systems were studied in high-cell density cultures. Cells were grown in a fed-batch procedure with a predetermined exponential feeding rate based on mass balances and kinetic equations to ensure constant specific growth rates. Prior to induction, cells were grown at 30°C. Product formation was induced by either a temperature shift from 30 to 42°C or by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). Under comparable culture conditions—induction of bFGF expression at 41 to 45 g l−1 dry cell weight and the addition of feed medium with identical rates—bFGF accumulated to 4.9 and 1.1 g l−1 using the temperature- and IPTG-inducible expression systems, respectively. The final biomass concentrations obtained using temperature- and IPTG-inducible expression systems were 61 and 135 g l−1 dry cell weight, whereas the specific product concentrations were 80 and 8.1 mg bFGF g−1 dry cell weight, respectively. When a temperature shift was used for product induction, 30% of bFGF was recovered as inclusion bodies in the insoluble cell fraction. IPTG-dependent induction yielded exclusively soluble bFGF.