Continuous production of ethanol using immobilized growing yeast cells

Summary Immobilized growing yeast cells were prepared in kappa-carra-geenan gel. Gel beads containing a small number of cells were incubated in a complete medium. The cells grew very well in the gel and the number of living cells per ml of gel increased to over 10 times that of free cells per ml of culture medium. After growing in the gel, the cells formed a dense layer of cells near the gel surface and produced large amounts of ethanol. The conditions for continuous production of ethanol using immobilized growing yeast cells were investigated. The supply of appropriate nutrients for growth was essential for the continuous production. The living cells in the gel were maintained at the high level of 10⁹ per ml of gel and continuous production of ethanol using the complete medium containing 10% glucose was carried out with a retention time of 1 h. In this operation, a stable steady state was maintained for longer than 3 months. The ethanol concentration was 50 mg/ml and the conversion of glucose utilized to ethanol produced was almost 100% of the theoretical yield.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

A parametric study on ethanol production from xylose byPichia stipitis

Characteristics of ethanol production by a xylose-fermenting yeast,Pichia stipitis Y-7124, were studied. The sugar consumption rate and specific growth rate were higher in the glucose-containing medium than in the xylose-containing medium. Specific activities of xylose reductase and xylitol dehydrogenase were higher in the medium with xylose than glucose, suggesting their induction by xylose. Maximum specific growth rate and ethanol yield were achieved at 30 g xylose/L concentration without formation of by-products such as xylitol and acetic acid whereas a maximum ethanol concentration was obtained at 130 g/L xylose. Adding a respiratory inhibitor, rotenone, increased a maximum ethanol concentration by 10% compared with the control experiment. In order to evaluate the pattern of ethanol inhibition on specific growth rate, a kinetic model based on Luong’s equations was applied. The relationship between ethanol concentration and specific growth rate was hyperbolic for glucose and parabolic for xylose. A maximum ethanol concentration at which cells did not grow was 33.6 g/L for glucose and 44.7 g/L for xylose.     

Production of ethanol and biomass from various carbohydrates byKluyveromyces fragilis

Summary Growth ofKluyveromyces fragilis NRC 2475 and the production of ethanol by the yeast were studied in the media containing one of the following sugars: glucose, lactose, galactose, or a glucose-galactose (50% 50%) mixture as a carbon source.The largest biomass yield and the lowest yield of ethanol were obtained in the medium containing glucose. The medium containing galactose gave the lowest yield of biomass and the largest yield of ethanol. When lactose was used for the growth and production of ethanol the obtained results for both biomass and ethanol were between those obtained with glucose and galactose.The ethanol productivities, expressed in terms of ethanol produced either per unit of cells, or per unit of cells and time, were the highest in the system with galactose and the lowest in that with glucose.     

Immobilization and characterization of Saccharomyces cerevisiae in crosslinked,prepolymerized polyacrylamide-hydrazide

Summary Baker's yeast (Saccharomyces cerevisiae) was immobilized in gels made of prepolymerized, linear, water soluble polyacrylamide, partially substituted with acylhydrazide groups. Gelation was effected by the addition of controlled amounts of dialdehydes (e.g. glyoxal). The immobilized yeasts retained full glycolytic activity. Moreover, the entrapped cells were able to grow inside the chemically corsslinked gel during continuous alcohol production. Glyoxal was found to be the most favourable crosslinking agent for this system. the system employed allowed for the free exchange of substrate and products. The gel surrounding the entrapped cells had no effect on temperature stability profile. On the other hand, substantial enhancement in survival of cells in presence of high ethanol concentrations was recorded for the entrapped yeast. The capability of the immobilized yeast to carry out continuous conversion of glucose to ethanol was demonstrated.     

Combined use of three methods for high concentration ethanol production by Saccharomyces cerevisiae

Summary Ethanol concentration and fermentation productivity using Saccharomyces cerevisiae were substantially increased in shake flask cultures with a normal inoculum by combining 3 methods: (a) by making nutrient additions to the standard medium for ethanol production, (b) by immobilizing the cells in alginate beads and (c) by using a glucose step-feeding batch process. Ethanol concentration by free yeast was improved from 5.9% (w/w) to 9.6% (w/w) when a further 0.8% yeast extract and 1% animal peptone were added to the standard 30% (w/v) glucose nutrient medium. This was further increased to 12.8% (w/w) by using alginate immobilized yeast. The ethanol concentration was increased again, to 15.0% (w/w) by using the glucose step-feeding batch process.     

Kinetics of the selective fermentation of glucose from glucose/fructose mixtures using Saccharomyces cerevisiae ATCC 36859

The kinetics of batch fermentation during the growth of S. cerevisiae ATCC 36859 was studied in various glucose/fructose mixtures. It was found that the growth is inhibited equally by glucose and fructose even though fructose is not consumed to any large extent by the yeast under the conditions tested here. The inhibition of growth by the substrate and ethanol is represented by linear equations. These equations were combined with the MONOD expression in order to formulate equations for the biomass growth, glucose and fructose consumption and ethanol production. Parameter estimates were obtained by fitting these equations to batch fermentation data and so developing models which indicate that the growth is completely inhibited when 62 g/l ethanol is produced by the yeast, while glucose consumption and ethanol production continue up to an ethanol concentration of 152 g/l. Products containing a high concentration of fructose are best produced by using a high initial biomass concentration.     

The use of plant cell vesicles for immobilization of yeast cells producing ethanol

The preparation of immobilized living yeast cells adsorbed into or onto delipided specimens of the dwarf duckweed Wolffia arrhiza (Fam. Lemnaceae) is reported. These yeast cell-plant cell conjugates were used for the repeated batch production of ethanol from glucose (143 to 246 g/l) or saccharose (150 g/l). Up to 25 fermentation cycles at 30°C were performed. The cycle time for complete substrate conversion to ethanol was reduced 10-fold by a 5-fold increase of the yeast cell Wolffia conjugate concentration (ε = 0.08 to ε =0.4) ε = volume of cell conjugate/totnl reaction volume. The corresponding ethanol production was 11.5 to 13.5 vol% and 9 vol% respectively. The reported results on the discontinuous ethanol fermentation with Wolffia-immobilized yeast cells open the field for their application in continuous ethanol production processes.     

Effect of carbon sources on the growth and ethanol production of native yeast Pichia kudriavzevii ITV-S42 isolated from sweet sorghum juice

The importance of non-Saccharomyces yeast species in fermentation processes is widely acknowledged. Within this group, Pichia kudriavzevii ITV-S42 yeast strain shows particularly desirable characteristics for ethanol production. Despite this fact, a thorough study of the metabolic and kinetic characteristics of this strain is currently unavailable. The aim of this work is to study the nutritional requirements of Pichia kudriavzevii ITV-S42 strain and the effect of different carbon sources on the growth and ethanol production. Results showed that glucose and fructose were both assimilated and fermented, achieving biomass and ethanol yields of 0.37 and 0.32 gg⁻¹, respectively. Glycerol was assimilated but not fermented; achieving a biomass yield of 0.88 gg⁻¹. Xylose and sucrose were not metabolized by the yeast strain. Finally, the use of a culture medium enriched with salts and yeast extract favored glucose consumption both for growth and ethanol production, improving ethanol tolerance reported for this genre (35 g L⁻¹) to 90 g L⁻¹ maximum ethanol concentration (over 100%). Furthermore Pichia kudriavzevii ITV-S42 maintained its fermentative capacity up to 200 g L⁻¹ initial glucose, demonstrating that this yeast is osmotolerant.

     

Effects of Cultural Conditions on Mononucleotide Phosphorylating Activity of Yeast Cells

It was found that the AMP phosphorylating activity of Candida sp. N–25–2 (a hydrocarbon assimilating yeast) was affected extremely by the liquid volume of cultural medium and the concentration of inorganic salts in medium. The yeast cells having no fermentative activity showed a strong activity of AMP phosphorylation when they were cultured under relative anaerobic conditions. It was observed that the glucose consumption of yeast cells was promoted by the addition of Mg²⁺ ion and AMP into the reaction system, and that the AMP phosphorylation was promoted in the presence of F-1,6-DP or phosphaenolpyruvate.

The cells of Candida sp. N–25–2 grown on glucose medium had a remarkable fermentative activity, while the cells grown on acetate or ethanol medium had a weak activity. On the other hand, it was found that the cells grown at strong aeration on glucose medium were able to produce remarkably the phosphorylated substances from mononucleotides, when F-1,6-DP was added as a phosphate donor. Similar phenomenon was observed in case of the cells grown on the carbon sources such as acetate, ethanol and hydrocarbon.     

Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral Kissiris at 45°C

Thermotolerant Kluyveromyces marxianus var. marxianus IMB3 yeast strain was immobilized on Kissiris (mineral glass foam derived from lava) in column packed reactors, and used for ethanol production from glucose or molasses under continuous culture conditions at temperatures between 40 and 50°C. Both ethanol yield and fermentation efficiency were highest at 45°C and a dilution rate (D) of 0.15/h. Increasing sugar concentration led to an increase in ethanol yield of up to 68.6 and 55.9 g/l on approx. 200g glucose or molasses, respectively. Optimum fermentation efficiency (experimental yields over theoretical maximum yields) however was at about 15% sugar for both glucose and molasses. Slight aeration (25 ml of air/min) through the medium addition line was found advantageous due to its mixing effect and probable maintenance of activity.     

Ethanol-inducible gene expression using gld1 + promoter in the fission yeast Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, the gld1 ⁺ gene encoding glycerol dehydrogenase is repressed by glucose and induced by ethanol and 1-propanol. The promoter region of gld1 ⁺ was cloned into a multicopy vector designated as pEG1 for evaluation as an ethanol-inducible expression vector using EGFP as a model heterologous protein. Expression of EGFP was repressed in the presence of high glucose and induced in the presence of ethanol, low-glucose, and 1-propanol in the absence of glucose. Addition of ethanol to cells harboring pEG1–EGFP was found to be the most effective means for inducing EGFP production. Protein yields were found to increase in proportion to ethanol concentration. As a further test of effectiveness, secreted recombinant human growth hormone was produced using the pEG1 expression vector in medium containing glycerol and ethanol. The pEG1 gene expression system is an effective tool for the production of heterologous proteins under glucose-limiting conditions, including medium containing glycerol as a carbon source.     

Inulin enrichment by fermentation in a flocculating yeast reactor

Summary Simultaneous production of ethanol and fructose enriched syrups was obtained from Jerusalem artichoke extract using a Saccharomyces diastaticus flocculating yeast in a continuous gas-lift reactor with internal biomass recycle. This allowed the production of 42 g/L of ethanol and 70 g/L of inulin containing up to 92% fructose (fructose/glucose ratio of 11). These results can be compared to the batch and chemostat fermentations which gave a higher ethanol concentration but a lower fructose enrichment. Mass transfert limitations can explain both the productivity decrease and the selectivity improvement in the gas-lift reactor.     

Continuous production ofL-isoleucine using immobilized growing Serratia marcescens cells

An immobilized growing cell system was applied to the continuous L -isoleucine production by Serratia marcescens. In the new immobilized-cell systems using the carrageenan gel method. S. marcescens cells in the gel required nutrients and oxygen for growth, and the numbers of living cells per milliliter of gel increased to the levels of that of free cells in the liquid medium. This immobilized growing cell system exhibited high and stable activity for isoleucine production under steady-state conditions. Continuous isoleucine production was carried out by feeding the nutrient medium under aeration into a fluidized bed reactor containing the immobilized cells. In the continuous operation, an efficient production was maintained by automatically controlling the pH of the reaction mixture at 7.5. The productivity of isoleucine increased using multibed reactors. In a two-bed reactor system, the effluent L -isoleucine concentration reached 4.5 mg/ml at a retention time of 10 hr, and a steady state was maintained for longer than 30 days.     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

Ethanol from Zymomonas,a bacterium

Thirteen isolates ofZymomonas were analyzed for their ability to tolerate increasing concentrations of glucose and ethanol. In medium containing 5.0% (v/v) ethanol, four isolates grew well in 15.0% (w/v) glucose. Six cultures tolerated at least 6,0% ethanol. Of all the isolates, 7 preferred glucose and 4 preferred sucrose as a sugar substrate. In a nutrient medium containing mineral salts and high concentrations of pantothenate and biotin ethanol production for 2 isolates was approximately 7.0%. Continuous stirring and growth factors were responsible for this increased ethanol production.