Enzyme and Microbial Technology 31 (2002) 640647

Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors
M. Mete Altnta a , Kutlu . lgen a, , Betl Krdar a , Z. Ilsen nsan a , Stephen G. Oliver b s
b a Department of Chemical Engineering, Bo azii University, 80815 Bebek-Istanbul, Turkey g School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

Received 20 October 2001; received in revised form 23 May 2002; accepted 7 June 2002

Abstract The production of ethanol from starch has been investigated in a genetically modied Saccharomyces cerevisiae strain, YPB-G, which secretes a bifunctional fusion protein that contains both the Bacillus subtilis -amylase and the Aspergillus awamori glucoamylase activities. The effects of a number of environmental factors on starch degradation, ethanol production, and plasmid stability have been assessed in batch culture. These include initial glucose supply, colony selection methodology prior to inoculation, and medium formulation. Cultures containing 40 g/l starch were observed to degrade starch effectively and produce higher amounts of ethanol in shorter periods. The provision of glucose in the growth medium during the early phases of fermentation resulted in faster growth and higher ethanol productivities. YE-Salts medium was found to support plasmid-containing cells throughout the whole fermentation; only 15% of the recombinant cells had lost the plasmid content by the end of the fermentation of 120 h. Fed-batch cultures produced high yields of ethanol on starch (0.46 g ethanol/g substrate) through the longer production period. 2002 Elsevier Science Inc. All rights reserved.
Keywords: Recombinant; Saccharomyces cerevisiae; Plasmid stability; Glucoamylase; Ethanol; Fed-batch fermentation

1. Introduction Ethanol has been promoted as a solution for a variety of complex problems related to energy and the environment. Compared to fossil fuels, ethanol has the advantages of being renewable, providing cleaner burning and producing no green-house gases. Methods reported for the fermentative production of ethanol from starch include (i) simultaneous saccharication and fermentation with a mixed culture of amylolytic and an ethanol-producing microorganisms [15], (ii) use of amylolytic enzymes from bacteria and fungi for the saccharication of starch, together with the fermenting yeast [68], and (iii) addition of glucoamylase to the fermentation broth, which is a common practice in industry [9]. The use of mixed cultures or the external addition of amylolytic enzymes together with the fermenting microorganism renders it difcult to establish the optimum culture conditions for the production of ethanol. In systems with mixed microorganisms, the ethanol yield decreases because much of the starch is consumed by the growth of the
Corresponding author. Tel.: +90-212-358-1540; fax: +90-212-287-2460. E-mail address: ulgenk@boun.edu.tr (K.. lgen).

amylolytic organism. The use of enzymes, on the other hand, increases the costs of the process and it may be difcult to optimize their use in fed-batch fermentations. An alternative to the above-mentioned strategies, is to use a genetically engineered microorganism that can directly ferment starch into ethanol and hence lead to an increase in the productivity of ethanol. Inlow et al. [10] evaluated the performance of glucoamylase producing recombinant yeast strains for the direct fermentation of soluble starch into ethanol. They produced 44.8 g/l ethanol from 100 g/l starch. Nakamura et al. [11] obtained an ethanol concentration of 24.9 g/l from 100 g/l starch in batch cultures and 28.2 g/l ethanol from 94 g/l starch in fed-batch cultures of recombinant Saccharomyces cerevisiae SR93. In the present study, the recombinant yeast strain, S. cerevisiae YPB-G [12], having both -amylase and glucoamylase as a bifunctional fusion protein, was used for the direct fermentation of starch into ethanol. Preliminary batch experiments were performed to characterize the system. The effect of different substrate feeding strategies on ethanol yields and productivities were studied by fed-batch fermentations, with the aim of increasing the ethanol production period as well as improving the overall product yield compared to simple batch cultures. In order to prevent high plasmid loss during

0141-0229/02/$ see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 6 7 - 9

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prolonged fermentation, the investigation was extended to include inoculum size, and medium improvement. Fermentation characteristics for long-term operation were further improved by selecting the cells retaining their plasmids during prolonged fed-batch fermentation. 2. Materials and methods

0.002% (w/v) histidine. YE-Salts medium was described by Banerjee et al. [13] and contained 0.1% (w/v) yeast extract, 0.1% (w/v) KH2 PO4 , 0.2% (w/v) (NH4 )2 SO4 , 0.05% (w/v) MgSO4 7H2 O and 0.002% (w/v) histidine. Varying amounts of starch were added to both media. Glucose (when supplied) was kept at an initial concentration of 4 g/l in both YPS and YE-Salts media. 2.4. Fermentor setup and operation

2.1. Organism Recombinant S. cerevisiae strain, YPB-G (MATa, his311, his315, leu23, leu2112, can1, mal), which produces the Bacillus subtilis -amylase and Aspergillus awamori glucoamylase (BsAAase/GAase) as a fusion protein was used in this study; it was constructed by de Moraes et al. [12]. The strain was maintained as a frozen suspension in 50% (v/v) glycerol at 80 C. 2.2. Colony selection Samples were streaked onto selective YNBG-S agar plates from a frozen culture. A selection procedure was applied in transferring the cells from agar plates to YNB-G minimal medium so that colonies which showed an amylolytic white halo in the I2 -stained agar plate assay were chosen for the preparation of precultures. In order to increase plasmid stability for long-term operations, colonies were isolated from a prolonged fed-batch culture on starch, and those that retained their recombinant plasmid were selected as the inoculum for further investigations. For this purpose, a fermentation sample was taken from the culture FB1 during the stationary phase of growth at 216 h. Cells were harvested by centrifugation and resuspended in YNBG before plating dilutions on non-selective YPG agar to give single colonies. These were then replica-plated onto YNB-GS agar plates, plasmid-containing colonies (i.e. those showing an amylolytic white halo in the I2 -stained agar plate) were selected. These colonies are then referred to as stressed colonies to distinguish them from regular inocula. Each of these stressed colonies was grown in 10 ml YNB-G medium for 24 h at 30 C. Cultures were then kept as frozen stocks (for not more than 3 months) and used as seeds for further experiments. 2.3. Growth media Precultures were grown in a minimal medium, YNB-G, containing 0.3% (w/v) Yeast Nitrogen Base without amino acids, 2% (w/v) glucose, 0.5% (w/v) (NH4 )2 SO4 and 0.002% (w/v) histidine. The precultures were incubated in orbital shakers at 30 C and 180 rpm. Preculture growth continued until late exponential phase and the inoculum size was controlled so that each fermentor culture received the same initial cell mass. The growth medium, YPS, contained 1% (w/v) yeast extract, 2% (w/v) peptone and A 2.5 l (nominal volume) bioreactor (New Brunswick BioFlo 3000) was used which allowed control of many variables including pH, temperature, impeller speed, dissolved oxygen (DO), and substrate feed rate. The fermentor vessel was autoclaved at 121 C for 25 min, and growth medium was added after sterilization. The glucose stock solution (60 g/l) was autoclaved separately. A 150 ml of shake-ask culture in the late exponential phase was used to inoculate 1.5 l of culture medium. The temperature was kept at 30 C and the pH of the medium was adjusted to 5.60 by addition of either 1 M HCl or 1 M NaOH. Fermentations were carried out without air supply, stirring at 400 rpm; DO was not controlled. Foam control was achieved by addition of Silicone Antifoaming agent (0.3%, v/v). In all fed-batch experiments, the fermentation was rst carried out in batch mode until the starch concentration had dropped to a preselected value. The substrate, an aqueous solution of starch, was then added in discrete pulses. 2.5. Analytical techniques Cell dry weight, the concentrations of glucose, reducing sugars, starch in the medium, intracellular plasmid DNA concentration and the proportion of plasmid-bearing cells were all determined as described by Altnta et al. [14]. s 3. Results and discussion 3.1. Batch fermentations The direct fermentation of starch into ethanol by a recombinant S. cerevisiae strain, YPB-G, which displays -amylase and glucoamylase activities was investigated in a New Brunswick BioFlo 3000 fermenter under controlled conditions (Table 1). Experiments PB1, PB2 and PB3 were inoculated with the stressed cells, P standing for experiments performed to investigate plasmid stability. Cultures PB2 and PB3 were inoculated with 30 ml of preculture (2% inoculum) while 150 ml (10% inoculum) was used for all other cultures. PB3 culture was the only one containing YE-Salts. 3.1.1. Substrate utilization The efciency of enzymatic starch fermentation is limited by the glucoamylase activity [10]. Since the gene encoding

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Table 1 Batch experiments and fed-batch experiments with pulse feeding Experiments B1 B2 B3 B4 PB1 PB2 PB3 PFB1 PFB2 Initial starch (g/l) 30 40 40 50 40 40 40 40 40 Initial glucose (g/l) 4 4 4 4 4 4 4 Total starch (g) 45 60 60 75 60 60 60 120 100 Biomass (g/l) 3.44 3.06 3.22 3.84 3.96 3.90 1.90 5.26 2.48 Maximum ethanol (g/l) 9.5 14.2 13.9 14.9 19.2 19.2 17.1 29.7 29.1 Starch consumption rate (g/h) 0.37 0.52 0.47 0.37 0.57 0.53 0.58 1.15 1.23 YX/S [(g biomass) (g substrate)1 ] 0.115 0.077 0.073 0.071 0.090 0.089 0.043 0.081 0.042 YP/S [(g ethanol) (g substrate)1 ] 0.315 0.355 0.326 0.275 0.437 0.436 0.388 0.459 0.494 qP [(g ethanol/1) (l/h)] 0.080 0.118 0.183 0.127 0.165 0.163 0.187 0.204 0.233 Plasmid stability (% decrease) 42.0 28.6 23.6 24.9 62.9 57.4 15.3 70.9 25.7

the fusion protein on the pPB-G plasmid is under the control of PGK promoter and this constitutive promoter is reported to be active in media containing glucose [15], 0.4% (w/v) glucose was added to batch cultures in order to reduce the batch-to-batch variations in glucoamylase activity. Fig. 1(a)(d) shows the time proles for starch degradation and glucose utilization for batch experiments initiated with (B3 and B4) and without (B1 and B2) a glucose supplement. Starch was totally consumed in experiments B1 and B2 with no glucose supplement while more than 5% of the initial starch remained unconsumed at the end of the fermentation in cultures B3 (3.69 g/l starch after 136 h) and B4 (3.34 g/l starch after 195 h) which were commenced with

initial glucose supply. Since the amount of residual starch in these cultures is approximately equivalent to the initial glucose supplement, this suggests that the growth limiting factor may not be the carbon source in these batch cultures. Starch degradation rates (based on the time interval between the start of the fermentation and the point where there was no further signicant drop in the residual starch concentration) were unaffected by the glucose supplement, but were opitmal in cultures containing 40 g/l initial starch, i.e. 0.50 0.03 g starch/h for B2 and B3 compared to 0.37 g starch/h for B1 and B4. The maximum accumulated glucose concentrations for these two cultures were similar (0.24 g/l). The maximum

Fig. 1. Starch and glucose consumption curves for batch experiments: (a) B1; (b) B2; (c) B3; (d) B4.

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Fig. 2. Biomass and ethanol production curves for batch experiments: (a) B1; (b) B2; (c) B3; (d) B4.

accumulated glucose concentrations increased with increasing initial starch concentration in both unsupplemented and glucose-supplemented cultures (i.e. from 0.18 to 0.24 g/l in B1 and B2; from 0.24 to 0.38 g/l in B3 and B4, respectively). The 4 g/l glucose supplied initially was totally consumed within the rst 20 h of fermentation (Fig. 1(c) and (d)). The nal residual glucose concentrations in these fermentations varied between 0.01 (B1) and 0.10 g/l (B4). 3.1.2. Biomass and ethanol production The variation of biomass and ethanol production with time for the batch cultures are shown in Fig. 2(a)(d). In each experiment, biomass and ethanol curves followed a similar trend and reached stationary phase at about the same time of fermentation. Cultures B2 and B3 which degraded starch more efciently when compared to cultures B1 and B4 also had higher ethanol yields on substrate (0.355 and 0.316 g ethanol/g substrate for B2 and B3, respectively) (Table 1). Culture B4 had the lowest yield of ethanol (0.275 g ethanol/g substrate) as well as a low starch degradation rate (0.37 g starch/h). Again, this demonstrates that an initial starch concentration of 40 g/l in YPS medium seems to be an optimum level for starting batch fermentations. The specic growth rates of the cells in cultures B3 and B4 (0.040 0.003/h), calculated after the consumption

of the initial glucose supplement, are higher than those in cultures B1 and B2 (0.028 0.001/h). A comparison of the biomass and ethanol production curves also indicates that both biomass and ethanol production curves for B3 level off earlier than those for B2. This may be due to the early availability of glucose to the recombinant cells in cultures B3 and B4 allowing the PGK1 promoter to switch on glucoamylase production at an earlier phase of fermentation [15]. This is also supported by the higher ethanol productivities calculated for cultures B3 and B4 (0.183 and 0.127 g ethanol/l/h, respectively) (Table 1). 3.1.3. Plasmid stability The recombinant S. cerevisiae YPB-G preculture was grown in a selective medium (YNB-G) without amino acids in order to maintain the plasmid and, therefore, the amylolytic potential of the culture. (The plasmid carries a LEU2 gene that complements the leucine auxotrophic (leu2) mutation of the host.) The growth and production (fermentation) medium, YPS, used in most experiments reported in Table 1, contains 1% yeast extract and so does not select for the maintenance of the plasmid; the nal frequency of plasmid loss in such cultures varied from 24 to 63%. Since an initial starch concentration of 40 g/l starch was optimal in terms of ethanol production and productivity, it was used in experiments with stressed cells that retained their

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Fig. 3. Starch consumption (a), biomass (b), and ethanol production (c) curves for the batch experiments containing 40 g/l initial starch and commenced with 10% (PB1) and 2% (PB2) inoculum.

plasmid following prolonged growth in fed-batch culture (Section 2.2). Since they contain the same amount of initial starch and glucose, experiments PB1 and B3 are comparable. Culture PB1 degrades starch more effectively (with a starch degradation rate of 0.57 g/h) than culture B3; the maximum attainable biomass concentration is higher by 23% and ethanol concentration is higher by 38% in culture PB1 (Table 1). The recombinant cells also grew faster in PB1 (with a specic growth rate of 0.05/h). However, despite these improvements in fermentation performance, the PB1 culture (inoculated with the stressed cells) in fact showed a higher frequency of plasmid loss than found in the B3 culture, where an inoculum of non-stressed cells was employed. Fig. 3(a)(c) compares the starch consumption, biomass and ethanol production curves for the high- and lowinoculum cultures, PB1 and PB2, respectively. Similar rates of starch degradation and ethanol production were calculated for PB1 and PB2, i.e. 0.57 and 0.53 g starch/h corresponding to 0.165 and 0.163 g ethanol/l/h, respectively. Moreover, the difference in inoculum size had no signicant effect on plasmid stability. The starch consumption, biomass and ethanol production curves for cultures PB2 and PB3 are presented in

Fig. 4(a)(c), respectively. Culture PB3 containing YE-Salts effectively degrades starch with a starch degradation rate of 0.58 g starch/h. The fermentation time for culture PB3 is shorter than that for PB2, and the maximum biomass and ethanol concentrations obtained (1.90 g cell/l and 17.1 g ethanol/l, respectively) are less than those in PB2. Although the yields of biomass (0.043 g biomass/g substrate) and ethanol (0.388 g ethanol/g substrate) in culture PB3 are lower, the ethanol productivity is relatively high (0.187 g ethanol/l/h). The recombinant cells in culture PB3 were observed to maintain a higher percentage of their plasmids at the end of the experiments, i.e. only 15.3% of the cells lost their plasmid after 120 h of fermentation (Table 1). This increase in plasmid stability with decreasing concentrations of yeast extract (in YE-Salts medium) may be attributed to an increased selection pressure due to a decreased concentration of leucine in the medium [16]. 3.2. Fed-batch fermentation Manipulation of yeast physiology coupled with nutrient feeding strategies may signicantly improve productivity in fermentations using recombinant strains [17]. Fed-batch experiments were therefore designed to increase the ethanol

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Fig. 4. Starch consumption (a), biomass (b), and ethanol production (c) curves for the batch experiments containing 40 g/l initial starch in YPS (PB2) and YE-Salts (PB3) medium.

Fig. 5. Starch and glucose consumption curves for fed-batch experiments with 40 g/l initial starch concentrations: (a) PFB1; (b) PFB2; biomass and ethanol production curves for fed-batch experiments with 40 g/l initial starch concentrations. (c) PFB1; (d) PFB2.

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production period as well as to degrade a higher amount of starch efciently in prolonged fermentations. Cultures were initially supplied with 40 g/l starch and 4 g/l glucose in either YPS or YE-Salts medium, since these concentrations were found to support both biomass and ethanol production in the batch experiments (Table 1). In experiment PFB1, three pulse feedings of 20 g starch were made at equal intervals of 12 h to a culture grown in YPS. In experiment PFB2, YE-Salts were used and there were two feedings of 20 g starch at 70 and 88 h of growth, respectively. The systems behaved as a batch reactor in between pulses since the volume remained constant during this period. The initial glucose supplement (4 g/l) was totally consumed in about 20 h in all fed-batch experiments. Table 1 and Fig. 5 summarize the results obtained in fed-batch experiments. The rst feeding in PFB1 was made at a time when 80% of the initial amount of starch had been consumed and resulted in an increase in the starch degradation rate. The starch degradation rate in PFB1 continued to increase following the second feeding (from 1.04 to 1.15 g starch/h). The starch degradation rate was observed to drop in the nal stage of the experiment. In PFB2 (which used the low-leucine YE-Salts medium), although the starch consumption rate was lower (0.62 g/h) between the start of the experiment and the rst feeding, a much higher rate (1.23 g/h) was reached after the rst feeding than was the case for PFB1. Fig. 5(a) and (b) gives the variation of starch and glucose concentrations with time for the cultures PFB1 and PFB2, respectively. The amount of accumulated glucose was observed to increase after the feedings. The residual glucose concentrations in these fermentations varied between 0.55 to 1.30 g/l. Time proles for biomass and ethanol production in culture PFB1 and PFB2 are given in Fig. 5(c) and (d), respectively. The metabolic pathway from starch to ethanol was supported effectively by culture PFB1 yielding the highest amount of ethanol (29.7 g/l). The maximum attainable biomass concentration (2.48 g/l) and the yield of biomass on substrate (0.042 g biomass/g substrate) remained low in culture PFB2 as in culture PB3 that also contained YE-Salts in the growth and fermentation medium. On the other hand, the ethanol yield of 0.494 g ethanol/g substrate in culture PFB2 was the highest among all batch and fed-batch cultures (Table 1). Culture PFB2 also had the highest ethanol productivity (0.233 g ethanol/l/h) similar to culture PB3, which had the highest ethanol productivity (0.187 g ethanol/l/h) among all the batch cultures. Only 26% of the cells grown from the stressed inoculum had lost their plasmid by the end of the experiment.

were investigated for the single-step bioconversion of starch into ethanol. The starch degradation rate and the yield of ethanol on starch were higher in the presence of 40 g/l starch in YPS media than when either higher or lower initial starch concentrations were employed. The use of an initial glucose supplement resulted in faster growth and high ethanol productivities, since the gene encoding the fusion protein on the pPB-G plasmid is under the control of the PGK promoter, which is active in glucose-containing media [15]. Although the non-selective YPS media leads to higher production of biomass (3.90 g cells/l versus 1.90 g cells/l) and ethanol (19.2 g ethanol/l versus 17.1 g ethanol/l) in batch cultures, the YE-Salts medium promotes plasmid maintenance throughout the entire fermentation, with 85% of the recombinant cells retaining the plasmid at the end of 120 h. Fed-batch cultures produced high yields of ethanol on starch through longer production periods. Rather high biomass (5.26 g cells/l) and ethanol (29.7 g ethanol/l) concentrations together with high ethanol productivity (0.204 g ethanol/l/h) were obtained in the fed-batch culture PFB1 with stressed cells. The use of YE-Salts medium with the same culture of cells from a stressed inoculum results in a drastic improvement of plasmid stability. In the fed-batch culture PFB2, 74% of the cells retained their plasmids, and the highest ethanol yield and productivity of 0.494 g ethanol/g substrate and 0.233 g ethanol/l/h were achieved. These results indicate that the strain selection technique utilized in this study was successful in obtaining plasmid stabilities sufcient for long-term applications. As the function of all the genes in S. cerevisiae is not known, the next step should be the identication of the entire metabolic network to better understand the biosynthesis reactions in the metabolism. The application of metabolic ux analysis for S. cerevisiae YPB-G is presently in progress.

Acknowledgments The nancial support for this research was provided by the Bo azii University Research Fund through projects g 01A502, 01S105 and by TBITAK through project MISAG 170. References
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4. Conclusions Cell growth, ethanol production, and plasmid maintenance for a recombinant S. cerevisiae strain, YPB-G, possessing both the -amylase and glucoamylase activities

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