Fuzzy control of ethanol concentration its application to maximum glutathione production in yeast fed-batch culture

A fuzzy logic controller (FLC) for the control of ethanol concentration was developed and utilized to realize the maximum production of glutathione (GSH) in yeast fedbatch culture. A conventional fuzzy controller, which uses the control error and its rate of change in the premise part of the linguistic rules, worked well when the initial error of ethanol concentration was small. However, when the initial error was large, controller overreaction resulted in an overshoot.An improved fuzzy controller was obtained to avoid controller overreaction by diagnostic determination of "glucose emergency states" (i.e., glucose accumulation or deficiency), and then appropriate emergency control action was obtained by the use of weight coefficients and modification of linguistic rules to decrease the overreaction of the controller when the fermentation was in the emergency state. The improved fuzzy controller was able to control a constant ethanol concentration under conditions of large initial error.The improved fuzzy control system was used in the GSH production phase of the optimal operation to indirectly control the specific growth rate mu to its critical value mu(c). In the GSH production phase of the fed-batch culture, the optimal solution was to control mu to mu(c) in order to maintain a maximum specific GSH production rate. The value of mu(c) also coincided with the critical specific growth rate at which no ethanol formation occurs. Therefore, the control of mu to mu(c) could be done indirectly by maintaining a constant ethanol concentration, that is, zero net ethanol formation, through proper manipulation of the glucose feed rate. Maximum production of GSH was realized using the developed FLC; maximum production was a consequence of the substrate feeding strategy and cysteine addition, and the FLC was a simple way to realize the strategy. (c) 1993 John Wiley & Sons, Inc.     

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1⁻¹ on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1⁻¹. The specific antibody production rate had a maximum value at about 25 mmol·1⁻¹ glutamine concentration.     

Evaluation of the maximum specific growth rate of a yeast indicating non-linear growth trends in batch culture

The maximum specific growth rate (μ_max) of an ethanolic D-xylose-fermenting yeast, Pichia stipitis, showing non-linear growth trends in batch culture, was calculated using the rate equation μ₂ = (1/Δt) ln(x ₂/x ₁). The absolute error Δμ, affecting μ₂, was derived using an equation given by Borzani (1994). Based on the assumption of linearity of growth curves between two closest time points, the relation between the two rate formulae, μ₁ = (1/xˉ)dx _t /dt and μ₂ = (1/Δt) ln(x ₂/x ₁) was established. In a particular condition, when μ₁ = μ₂, an equation has been developed, the roots of which are the specific growth rates at different time points. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adaptive control of specific growth rate based on proton production in anaerobic fed-batch culture

Summary This paper develops a practical and useful computer control scheme to control the specific growth rate as accurately as possible by measuring the released protons in anaerobic alcohol fermentation. In the case of current study, most of the released protons are due to the uptake of cationic ammonium ion via the conversion: NH₄ ⁺ NH₃(cell) + H⁺. Correlating the Proton Production (PP) and the Proton Production Rate (PPR) with specific growth rate () proved PP as a better measured variable. Using a simple adaptive control algorithm, was successfully controlled in a Zymomonas mobilis fed-batch culture.     

Maximizing production of glutathione by amino acid modulation and high-cell-density fed-batch culture of Saccharomyces cerevisiae

In this paper the high-cell-density fed-batch culture and optimal amino acid modulation were combined together to enhance glutathione production in Saccharomyces cerevisiae T65. Ethanol concentration in the broth was an important parameter for feedback control in fed-batch culture. Low ethanol concentration was propitious to both the cell growth and glutathione synthesis. The feedback control of a low ethanol concentration was an efficient way to realize high-cell-density culture and the biomass reached 140 g/L after 57 h fermentation. With optimal amino acid addition to elevate the glutathione content continually, the maximum glutathione yield achieved 2190 mg/L.     

Inferential control of the specific growth rate in fed-batch cultivation processes

Inferential control algorithms and systems for set-point control of the specific growth rate in fed-batch cultivation processes are presented. Realization of the proposed control systems does not require mathematical model and a priori knowledge of the culture of microorganisms under control. Feed-back signal in PID control loops of the investigated systems is based on measurements of the O₂ concentration in exhaust gas and the air supply rate, however, measurements of the other technological parameters related to the culture growth dynamics can also be applied.     

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.     

Continuous ethanol production by flocculating yeast in the fluidized bed bioreactor

Abstract: Continuous fermentation by a highly flocculant strain of the yeast Saccharomyces cerevisiae was carried out in a tower fluidized-bed bioreactor. The synthetic and molasses media with a total sugar concentration of 17% (w/v) were used for fermentation. Different dilution rates were tested. Stable cell densities of 50 kg m^-3(dry weight) were maintained for all dilution rates. The ethanol productivity was increasing linearly with dilution rates up to 15—20 kg m^-3 h^-1. Aeration of the culture stabilized flocculating activity and viability of yeast and also permitted long-term operation of the bioreactor.     

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo-l-prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.     

Lack of correlation between glutathione turnover and amino acid absorption by the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae was grown in the presence of 1.0 mM l-methionine and the half-life of degradation of glutathione determined for the strains Σ1278b (444 min) and the amino-acid-uptake deficient mutant 2512c (368 min). There is no significant difference in these values, yet the rate of uptake of l-methionine is 5–7 times lower in the mutant. In neither strain is the turnover of glutathione sufficient to account for amino acid uptake. We conclude that there is no correlation between the γ-glutamyl cycle and amino acid uptake by this east.     

Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration

The effect of glucose concentration on erythritol production by Torula sp. was investigated. The maximum volumetric productivity of erythritol was obtained at an initial glucose concentration of 300 g l⁻¹ in batch culture. The volumetric productivity was maximal at a controlled glucose concentration of 225 g l⁻¹, reducing the lag time of the erythritol production. A fed-batch culture was established with an initial glucose concentration of 300 g l⁻¹ and with a controlled glucose concentration of 225 g l⁻¹ in medium containing phytic acid as a phosphate source. In this fed-batch culture, a final erythritol production of 192 g l⁻¹ was obtained from 400 g l⁻¹ glucose in 88 h. This corresponded to a volumetric productivity of 2.26 g l⁻¹ h⁻¹ and a 48% yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 248–252. Received 26 September 2000/ Accepted in revised form 16 January 2001     

分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响

研究了2．5L罐分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响,确定了分批培养时生产谷胱甘肽合成酶系的最佳pH和最佳温度。研究结果表明：在发酵液的pH为7．2和温度为37℃时,谷胱甘肽合成酶系产量和细胞干重达到最大,分别为690．6U／L和3．77g／L。采用变温控制对菌体的生长和谷胱甘肽合成酶系的合成并没有明显的优点。     

Isothermic variation of the specific growth rate of Saccharomyces cerevisiae in batch culture

The specific growth rate () of a respiration-deficient mutant of Saccharomyces cerevisiae growing under defined experimental conditions in batch culture (mineral medium plus glucose and vitamins at 25°C) varied from experiment to experiment over a wide range (0.10–0.24 h^-1) and showed a normal distribution. Neither the age of the culture, the history of the inoculum, nor experimental error accounted wholy for the variability of . The variation was positively correlated with the specific rate of glucose transfer and negatively with the specific rate of production of non-fermentative CO₂. The yield decreased with implying higher maintenance requirements in batch culture (4.7 mmoles g^-1 h^-1) than in continuous culture (0.8 mmoles g^-1 h^-1). It was concluded that the strain is capable of establishing any one of several steady states of growth under the same experimental conditions, each steady state displaying some buildin inertia with respect to change. The variations of the specific rates of glucose transfer and non-fermentative CO₂ production, and of the yield appeared to be consequences rather than causes of the variation of . The ultimate causes of the variation of remained unidentified.Part of a doctoral thesis submitted by J. Martinez-Peinado to the University of Navarra Spain     

Effects of methanol concentration on expression levels of recombinant protein in fed-batch cultures of Pichia methanolica

The methylotrophic yeast Pichia methanolica can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase (AUG1) promoter. Methanol concentrations during the induction phase directly affect cellular growth and protein yield. Various methanol concentrations controlled by an on-line monitoring and control system were investigated in mixed glucose/methanol fed-batch cultures of P. methanolica expressing the human transferrin N-lobe protein. The PMAD18 P. methanolica strain utilized is a knock-out for the chromosomal AUG1 gene locus, resulting in a slow methanol utilization phenotype. Maximum growth of 100 g of dry cell weight per liter of culture was observed in cultures grown at 1.0% (v/v) methanol concentration. Maximum recombinant gene expression was observed for cultures controlled at 0.7% (v/v) methanol concentration, resulting in maximum volumetric production of 450 mg of transferrin per liter after 72 h of elapsed fermentation time.     

Optimal strategy for controlling the spread of HIV/AIDS disease: a case study of South Africa

HIV/AIDS disease continues to spread alarmingly despite the huge amounts of resources invested in fighting it. There is a need to integrate the series of control measures available to ensure a consistent reduction in the incidence of the disease pending the discovery of its cure. We present a deterministic model for controlling the spread of the disease using change in sexual habits and antiretroviral (ARV) therapy as control measures. We formulate a fixed time optimal control problem subject to the model dynamics with the goal of finding the optimal combination of the two control measures that will minimize the cost of the control efforts as well as the incidence of the disease. We estimate the model state initial conditions and parameter values from the demographic and HIV/AIDS data of South Africa. We use Pontryagin's maximum principle to derive the optimality system and solve the system numerically. Compared with the practice in most resource-limited settings where ARV treatment is given only to patients with full-blown AIDS, our simulation results suggest that starting the treatment as soon as the patients progress to the pre-AIDS stage of the disease coupled with appreciable change in the susceptible individuals’ sexual habits reduces both the incidence and prevalence of the disease faster. In fact, the results predict that the implementation of the proposed strategy would drive new cases of the disease towards eradication in 10 years.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Fractionation of yeast extract by nanofiltration process to assess key compounds involved in CHO cell culture improvement

Yeast extract (YE) is known to greatly enhance mammalian cell culture performances, but its undefined composition decreases process reliability. Accordingly, in the present study, the nature of YE compounds involved in the improvement of recombinant CHO cell growth and IgG production was investigated. First, the benefits of YE were verified, revealing that it increased maximal concentrations of viable cells and IgG up to 73 and 60%, respectively compared to a reference culture. Then, the analyses of YE composition highlighted the presence of molecules such as amino acids, vitamins, salts, nucleobase, and glucose that were contained in reference medium, while others including peptides, trehalose, polysaccharides, and nucleic acids were not. Consequently, YE was fractionated by a nanofiltration process to deeper evaluate its effects on CHO cell cultures. The YE molecules already contained in reference medium were mainly isolated in the permeate fraction together with trehalose and short peptides, while other molecules were concentrated in the retentate. Permeate, which was free of macromolecules, exhibited a similar positive effect than raw YE on maximal concentrations. Additional studies on cell energetic metabolism underlined that dipeptides and tripeptides in permeate were used as an efficient source of nitrogenous substrates. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:875–882, 2015     

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y _m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.Abbreviations CFU colony-forming units - PCA perchloric acid - TCA trichloroacetic acid