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.     

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.     

On-line estimation of sugar concentration for control of fed-batch fermentation of lignocellulosic hydrolyzates by Saccharomyces cerevisiae

A feed control strategy, based on estimated sugar concentrations, was developed with the purpose of avoiding severe inhibition of the yeast Saccharomyces cerevisiae during fermentation of spruce hydrolyzate. The sum of the fermentable hexose sugars, glucose and mannose, was estimated from on-line measurements of carbon dioxide evolution rate and biomass concentration by use of a simple stoichiometric model. The feed rate of the hydrolyzate was controlled to maintain constant sugar concentration during fed-batch fermentation, and the effect of different set-point concentrations was investigated using both untreated and detoxified hydrolyzates. The fed-batch cultivations were evaluated with respect to cellular physiology in terms of the specific ethanol productivities, ethanol yields, and viability of the yeast. The simple stoichiometric model used resulted in a good agreement between estimated sugar concentrations and off-line determinations of sugar concentrations. Furthermore, the control strategy used made it possible to maintain a constant sugar concentration without major oscillations in the feed rate or the sugar concentration. For untreated hydrolyzates the average ethanol productivity could be increased by more than 130% compared to batch fermentation. The average ethanol productivity was increased from 0.12 to 0.28 g/g h. The productivity also increased for detoxified hydrolyzates, where an increase of 16% was found (from 0.50 to 0.58 g/g h).     

Increased heterologous protein production by Saccharomyces cerevisiae growing on ethanol as sole carbon source

Saccharomyces cerevisiae is a widely used host organism for the production of heterologous proteins, often cultivated in glucose-based fed-batch processes. This production system however has many factors limiting the productivity, mainly towards the end of the fermentation. For the optimised production of a Camelid antibody fragment this process was evaluated. In shake flask cultivations, it was found that ethanol has a strong effect on productivity increase and therefore glucose and ethanol fed-batch fermentations were compared. It appeared that specific heterologous protein production was up to five times higher in the ethanol cultivation and could be further optimised. Then the key characteristics of ethanol fed-batch fermentations such as growth rate and specific production were determined under ethanol limitation and accumulation and growth limiting conditions in the final phase of the process. It appeared that an optimal production process should have an ethanol accumulation throughout the feed phase of approximately 1% v/v in the broth and that production remains very efficient even in the last phase of the process. This productivity increase on ethanol versus glucose was also proven for several other Camelid antibody fragments some of which were heavily impaired in secretion on glucose, but very well produced on ethanol. This leads to the suggestion that the ethanol effect on improved heterologous protein production is linked to a stress response and folding and secretion efficiency.     

Long-term repeated fed-batch ethanol fermentation in aerated condition

In this study, we attempted to assess the process stability of long-term fed-batch ethanol fermentation in the absence and presence of aeration (0.33 vvm). To examine the effect of aeration, a long-term repeated fed-batch operation was conducted for 396 h to mimic a long-term industrial bioethanol production process. In this long-term repeated fed-batch ethanol fermentation experiments, withdrawal-fill operation were conducted every 36 h for 10 repeat cycles. The whole operation was stably sustained in a quasi-steady state. The average maximal cell concentration and the average maximal ethanol production during operation were increased by 81.63 and 12.12%, respectively, when aeration was used. In addition, since aeration was carried out, the average ethanol yield slightly decreased by 4.03% and the average specific ethanol production rate decreased by 46.75% during operation. However, the average ethanol productivity increased by 17.53% when aeration was carried out. After 396 h of long-term repeated fed-batch ethanol fermentation, 1,908.9 g of ethanol was cumulatively produced when aeration was used, which was 12.47%, higher than when aeration was not used (1,697.2 g). Meanwhile, glycerol production was greatly decreased during long-term repeated fed-batch ethanol fermentation, in which the glycerol concentration in the culture broth decreased from about 34∼15 g/L. Thus, we can conclude that cell growth was greatly improved by overcoming ethanol inhibition and glycerol production was remarkably decreased when aeration was carried out, although aeration in ethanol fermentation decreased the specific ethanol production rate and ethanol yield.     

Ethanol from whey: Continuous fermentation with cell recycle

The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.     

Control of nitrate concentration in fermentations of Corynebacterium glutamicum

A nitrate control system has been devised for the maintenance of stable nitrate concentrations throughout fed-batch fermentations of Corynebacterium glutamicum. The feedback control system was based on the use of a nitrate-ion-selective electrode to directly monitor the nitrate levels in the fermentor and an automatic controller to activate a nitrate feed pump. The electrode which was used for controlling the nitrate level was stable through-out the fermentation period. The apparent maximum specific growth rate, biomass production, protein production, biomass yields on glucose and nitrate, and amino acid production were all optimal at approximately 50mM nitrate.     

Inline noninvasive Raman monitoring and feedback control of glucose concentration during ethanol fermentation

Raman spectroscopy as a process analytical technology tool was implemented for the monitoring and control of ethanol fermentation carried out with Saccharomyces cerevisiae. The need for the optimization of bioprocesses such as ethanol production, to increase product yield, enhanced the development of control strategies. The control system developed by the authors utilized noninvasive Raman measurements to avoid possible sterilization problems. Real-time data analysis was applied using partial least squares regression (PLS) method. With the aid of spectral pretreatment and multivariate data analysis, the monitoring of glucose and ethanol concentration was successful during yeast fermentation with the prediction error of 4.42 g/L for glucose and 2.40 g/L for ethanol. By Raman spectroscopy-based feedback control, the glucose concentration was maintained at 100 g/L by the automatic feeding of concentrated glucose solution. The control of glucose concentration during fed-batch fermentation resulted in increased ethanol production. Ethanol yield of 86% was achieved compared to the batch fermentation when 75 % yield was obtained. The results show that the use of Raman spectroscopy for the monitoring and control of yeast fermentation is a promising way to enhance process understanding and achieve consistently high production yield.     

Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant

An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step allowed us to obtain high biomass concentrations before continuous fermentation. The continuous anaerobic process successfully incorporated a cell-recycle system to optimize the fermentor productivity. Under conditions permitting a low residual sugar concentration (≤1%), maximum productivity (13.6 g liter⁻¹ h⁻¹) was gained from 15% substrate in the continuous feed at a dilution rate of 0.2 h⁻¹. Complete fermentation of highly concentrated feed solutions (20%) was also demonstrated, but only with greatly diminished fermentor productivity (5.5 g liter⁻¹ h⁻¹).     

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L^-1 glutathione with glutathione content and yield of 4.3% and 25.6 mg_glutathione/g_glucose, respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L^-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.     

A contribution to optimal control of fed-batch biochemical processes

The problem of looking for high efficient modern control strategies in fermentation technology is very urgent, nowdays. Particular attention should be paid to the processes in fed-batch mode. Both, optimal feedforward and feedback control approaches are suggested. A contribution is considered to have been made in the feedback control where continuous and discrete versions are treated as well. The control laws are carried out by a variation calculus problem and a polynomial pole placement synthesis solution, respectively. All the algorithms result in an optimal substrate feed rate profile. On the basis of recursive least squares identification of the model coefficients an adaptive discrete-time control strategy is proposed. Some satisfying simulation results are dealt with.     

On-line monitoring and controlling system for fermentation processes

A personal computer-based on-line monitoring and controlling system was developed for the fermentation of microorganism. The on-line HPLC system for the analysis of glucose and ethanol in the fermentation broth was connected to the fermenter via an auto-sampling equipment, which could perform the pipetting, filtration and dilution of the sample and final injection onto the HPLC through automation based on a programmed procedure. The A/D and D/A interfaces were equipped in order to process the signals from electrodes and from the detector of HPLC, and to direct the feed pumps, the motor of stirrer and gas flow-rate controller. The software that supervised the control of the stirring speed, gas flow-rate, pH value, feed flow-rate of medium, and the on-line measurement of glucose and ethanol concentration was programmed by using Microsoft Visual Basic under Microsoft Windows. The signal for chromatographic peaks from on-line HPLC was well captured and processed using an RC filter and a smoothing algorithm. This monitoring and control system was demonstrated to be effective in the ethanol fermentation of Zymomonas mobilis operated in both batch and fed-batch modes. In addition to substrate and product concentrations determined by on-line HPLC, the biomass concentration in Z. mobilis fermentation could also be on-line estimated by using the pH control and an implemented software sensor. The substrate concentration profile in the fed-back fermentation followed well the set point profile due to the fed-back action of feed flow-rate control.     

Fed-batch production of concentrated fructose syrup and ethanol using Saccharomyces cerevisiae ATCC 36859

A fed-batch process is used for the production of concentrated pure fructose syrup and ethanol from various glucose/fructose mixtures by S. cerevisiae ATCC 36859. Applying this technique, glucose-free fructose syrups with over 250 g/l of this sugar were obtained using High Fructose Corn Syrup and hydrolyzed Jerusalem artichoke juice. By encouraging ethanol evaporation from the reactor and condensing it, a separate ethanol product with a concentration of up to 350 g/l was also produced. The rates of glucose consumption and ethanol production were higher than in classical batch ethanol fermentation processes.     

Monitoring and control of Gluconacetobacter xylinus fed-batch cultures using in situ mid-IR spectroscopy

A partial least-squares calibration model, relating mid-infrared spectral features with fructose, ethanol, acetate, gluconacetan, phosphate and ammonium concentrations has been designed to monitor and control cultivations of Gluconacetobacter xylinus and production of gluconacetan, a food grade exopolysaccharide (EPS). Only synthetic solutions containing a mixture of the major components of culture media have been used to calibrate the spectrometer. A factorial design has been applied to determine the composition and concentration in the calibration matrix. This approach guarantees a complete and intelligent scan of the calibration space using only 55 standards. This calibration model allowed standard errors of validation (SEV) for fructose, ethanol, acetate, gluconacetan, ammonium and phosphate concentrations of 1.16 g/l, 0.36 g/l, 0.22 g/l, 1.54 g/l, 0.24 g/l and 0.18 g/l, respectively. With G. xylinus, ethanol is directly oxidized to acetate, which is subsequently metabolized to form biomass. However, residual ethanol in the culture medium prevents bacterial growth. On-line spectroscopic data were implemented in a closed-loop control strategy for fed-batch fermentation. Acetate concentration was controlled at a constant value by feeding ethanol into the bioreactor. The designed fed-batch process allowed biomass production on ethanol. This was not possible in a batch process due to ethanol inhibition of bacterial growth. In this way, the productivity of gluconacetan was increased from 1.8 x 10(-3) [C-mol/C-mol substrate/h] in the batch process to 2.9 x 10(-3) [C-mol/C-mol substrate/h] in the fed-batch process described in this study.     

A numerical evaluation of a hollow fiber extractive fermentor process for the production of ethanol

The economics of a process for the production of ethanol employing a hollow fiber extractive fermentor have been investigated. A computer simulation of the process incorporating a mathematical model of the fermentor was used to calculate the mass and energy balances. The results of the process simulation were read into a computer spreadsheet programmed with the economic calculations from which a final ethanol product cost was obtained. The process was found to be as competitive as conventional fermentation processes even at the currently high cost--$4/sq ft--of hollow fibers. It was determined that the 1986 price of 46.2 cents/L of ethanol produced by the process would be reduced by 1.8 cents/L for every $1/sq foot drop in the price of hollow fibers. A comparison of this process with conventional fermentation processes indicates that its potential savings lie in its ability to use a concentrated sugar feed, and the fermentor's increased productivity and ability to produce a concentrated ethanol stream which is removed by the extracting solvent.     

Production of ethanol from molasses at 45 °C using Kluyveromyces marxianus IMB3 immobilized in calcium alginate gels and poly(vinyl alcohol) cryogel

The thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus IMB3 has been immobilized in calcium alginate gel and poly(vinyl alcohol) cryogel (PVAC) beads. The immobilized preparations were used as biocatalyst in fed-batch reactor systems for prolonged periods. The substrate utilized in each case consisted of sugar cane molasses diluted to yield a sugar load of 140?g/l. During the first cycle the maximum ethanol concentration produced by the alginate system was 57?g/l, representing 80% of the maximum theoretical yield. In the system employing the PVAC-immobilized biocatalyst, ethanol production increased to a maximum of 52–53?g/l, representing 73% of the maximum theoretical yield. In both cases, maximum ethanol concentration was achieved within a 72-hour period. When each system was operated on a fed-batch basis for a prolonged period of time the average ethanol concentrations produced in the alginate- and the PVAC-immobilized systems were 21 and 45?g/l, respectively. The results suggest that the PVAC-based immobilization system may provide a more practical alternative to alginate for the production of ethanol by K. marxianus IMB3 in continuous or semi-continuous fermentation systems.     

Kinetics of sugars consumption and ethanol inhibition in carob pulp fermentation by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in batch and fed-batch cultures

The waste materials from the carob processing industry are a potential resource for second-generation bioethanol production. These by-products are small carob kibbles with a high content of soluble sugars (45–50%). Batch and fed-batch Saccharomyces cerevisiae fermentations of high density sugar from carob pods were analyzed in terms of the kinetics of sugars consumption and ethanol inhibition. In all the batch runs, 90–95% of the total sugar was consumed and transformed into ethanol with a yield close to the theoretical maximum (0.47–0.50 g/g), and a final ethanol concentration of 100–110 g/l. In fed-batch runs, fresh carob extract was added when glucose had been consumed. This addition and the subsequent decrease of ethanol concentrations by dilution increased the final ethanol production up to 130 g/l. It seems that invertase activity and yeast tolerance to ethanol are the main factors to be controlled in carob fermentations. The efficiency of highly concentrated carob fermentation makes it a very promising process for use in a second-generation ethanol biorefinery.     

Demonstration of the Crabtree effect in Phaffia rhodozyma during continuous and fed-batch cultivation

By monitoring cell yield and fermentation products during fed-batch and continuous growth, Pfaffia rhodozyma was shown to exhibit the Crabtree effect. In fed-batch culture at feed concentrations of 27 and 55 g glucose/l there was good agreement between the observed biomass formation and that predicted by a mass balance model. At 125 g glucose/l in the feed, biomass formation was less than predicted and fermentation products such as ethanol and acetic acid accumulated in the culture medium. In continuous culture with a feed concentration of 10 g glucose/l, the Crabtree effect became apparent at a dilution rate of 0.1 h -1 . Aerobic fermentation did not occur provided the sugar substrate was maintained at a concentration of less than 0.5 g/l. Although the cell yield coefficient was reduced from 0.5 g/g to 0.16 g/g during aerobic fermentation, the carotenoid content of the cells was unaffected.     

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.