Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l⁻¹. At a glucose concentration of 100 g l⁻¹, SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD⁺/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Effects of ethanol concentration and stripping temperature on continuous fermentation rate

The operation of a pilot plant consisting of a 14-l fermentor, 10-cm packed column and condenser for continuous fermentation and stripping of ethanol was stable for more than 100 days. The feed consisted of a non-sterile solution of 560 g/l glucose with 100 g/l corn steep water. Fouling of the packing in the column with attached growth of yeast cells was controlled by in situ washing at intervals of 3–6 days. A computer simulation of the pilot plant was developed and used to analyze the data. The productivity of the continuous fermentor varied from 14 g ethanol to 17 g ethanol l⁻¹ h⁻¹. The yield was equal to the maximum theoretically possible: 0.51 g ethanol/g glucose consumed. Results are fit to linear models for the effects of ethanol concentration on specific growth rate and cell yield, and for the effect of stripping temperature on specific growth rate. Received: 16 October 1996 / Received revision: 3 January 1997 / Accepted: 24 January 1997     

A simultaneous saccharification and fermentation model for dynamic growth environments

Many mathematical models by researchers have been formulated for Saccharomyces cerevisiae which is the common yeast strain used in modern distilleries. A cybernetic model that can account for varying concentrations of glucose, ethanol and organic acids on yeast cell growth dynamics does not exist. A cybernetic model, consisting of 4 reactions and 11 metabolites simulating yeast metabolism, was developed. The effects of variables such as temperature, pH, organic acids, initial inoculum levels and initial glucose concentration were incorporated into the model. Further, substrate and product inhibitions were included. The model simulations over a range of variables agreed with hypothesized trends and to observations from other researchers. Simulations converged to expected results and exhibited continuity in predictions for all ranges of variables simulated. The cybernetic model did not exhibit instability under any conditions simulated.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts

High-gravity brewing, which can decrease production costs by increasing brewery yields, has become an attractive alternative to traditional brewing methods. However, as higher sugar concentration is required, the yeast is exposed to various stresses during fermentation. We evaluated the influence of high-gravity brewing on the fermentation performance of the brewer’s yeast under model brewing conditions. The lager brewer’s strain Weihenstephan 34/70 strain was characterized at three different gravities by adding either glucose or maltose syrups to the basic wort. We observed that increased gravity resulted in a lower specific growth rate, a longer lag phase before initiation of ethanol production, incomplete sugar utilization, and an increase in the concentrations of ethyl acetate and isoamyl acetate in the final beer. Increasing the gravity by adding maltose syrup as opposed to glucose syrup resulted in more balanced fermentation performance in terms of higher cell numbers, respectively, higher wort fermentability and a more favorable flavor profile of the final beer. Our study underlines the effects of the various stress factors on brewer’s yeast metabolism and the influence of the type of sugar syrups on the fermentation performance and the flavor profile of the final beer.     

An innovative consecutive batch fermentation process for very high gravity ethanol fermentation with self-flocculating yeast

An innovative consecutive batch fermentation process was developed for very high gravity (VHG) ethanol fermentation with the self-flocculating yeast under high biomass concentration conditions. On the one hand, the high biomass concentration significantly shortened the time required to complete the VHG fermentation and the duration of yeast cells suffering from strong ethanol inhibition, preventing them from losing viability and making them suitable for being repeatedly used in the process. On the other hand, the separation of yeast cells from the fermentation broth by sedimentation instead of centrifugation, making the process economically more competitive. The VHG medium composed of 255 g L⁻¹ glucose and 6.75 g L⁻¹ each of yeast extract and peptone was fed into the fermentation system for nine consecutive batch fermentations, which were completed within 8–14 h with an average ethanol concentration of 15% (v/v) and ethanol yield of 0.464, 90.8% of its theoretical value of 0.511. The average ethanol productivity that was calculated with the inclusion of the downstream time for the yeast flocs to settle from the fermentation broth and the supernatant to be removed from the fermentation system was 8.2 g L⁻¹ h⁻¹, much higher than those previously reported for VHG ethanol fermentation and regular ethanol fermentation with ethanol concentration around 12% (v/v) as well.     

Kinetics of ethanol fermentations in membrane cell recycle fermentors

Ethanol fermentation by yeast was carried out in a cell filtration recycle system with a hollow-fiber membrane filter. Maximum biomass concentrations up to 210 g dry wt/L were obtained, but in normal operation concentrations they were between 100 and 150 g/L. The ethanol productivity using 14% glucose feed was 85 g/L h, with an ethanol concentration of 65 g/L and an ethanol yield of over 90%. The ethanol productivity and yeast growth rate decreased as the cell concentration increased beyond a certain level. The cell mass in the reactor was maintained by a proper manipulation of diluticn rate and bleed ratio depending on the growth rate.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Repeated use of yeast biomass in ethanol fermentation of juniper berry sugars

Summary The object of this study was to establish the possibility of using the yeast biomass separated from the fermentation broth at the end of ethanol fermentation of juniper berry sugars as an inoculum in successive batch fermentation processes. A part of the fermentation broth (10% v/v) and a suspension of yeast biomass (separated from the same broth) into the water extract of juniper berries (2 g of wet yeast biomass per liter of water extract) were used as inocula. It was shown that the suspension of yeast biomass could be used as inoculum in successive batch processes without negative effects on the kinetics and ethanol yield, but with positive effects on the capacity and economy of the bioprocess. The addition of ammonium salts at optimum levels did not affect the final ethanol concentrations (4.3–4.4% v/v), but enhanced the specific rate of ethanol production, thus reducing the process duration by about five times.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Intracellular ethanol accumulation in yeast cells during aerobic fermentation: a Raman spectroscopic exploration

Aims: To investigate the intracellular ethanol accumulation in yeast cells by using laser tweezers Raman spectroscopy (LTRS). Methods and Results: Ethanol accumulation in individual yeast cells during aerobic fermentation triggered by excess glucose was studied using LTRS. Its amount was obtained by comparing intracellular and extracellular ethanol concentrations during initial process of ethanol production. We found that (i) yeasts start to produce ethanol within 3 min after triggering aerobic fermentation, (ii) average ratio of intracellular to extracellular ethanol is 1·54 ± 0·17 during the initial 3 h after addition of 10% (w/v) excess glucose and (iii) the accumulated intracellular ethanol is released when aerobic fermentation is stimulated with decreasing glucose concentration. Conclusions: Intracellular ethanol accumulation occurs in initial stage of a rapid aerobic fermentation and high glucose concentration may attribute to this accumulation process. Significance and Impact of the Study: This work demonstrates LTRS is a real‐time, reagent‐free, in situ technique and a powerful tool to study kinetic process of ethanol fermentation. This work also provides further information on the intracellular ethanol accumulation in yeast cells.     

The role of nisin in fuel ethanol production with Saccharomyces cerevisiae

Aims: To investigate the effects of nisin on lactobacilli contamination of yeast during ethanol fermentation and to determine the appropriate concentration required to control the growth of selected lactobacilli in a YP/glucose media fermentation model. Methods and Results: The lowest concentration of nisin tested (5 IU ml^?1) effectively controlled the contamination of YP/glucose media with 10⁶ CFU ml^?1 lactobacilli. Lactic acid yield decreased from 5·0 to 2·0 g l^?1 and potential ethanol yield losses owing to the growth and metabolism of Lactobacillus plantarum and Lactobacillus brevis were reduced by 11 and 7·8%, respectively. Approximately, equal concentrations of lactic acid were produced by Lact. plantarum and Lact. brevis in the presence of 5 and 2 IU ml^?1 nisin, respectively, thus demonstrating the relatively higher nisin sensitivity of Lact. brevis for the strains in this study. No differences were observed in the final ethanol concentrations produced by yeast in the absence of bacteria at any of the nisin concentrations tested. Conclusions: Metabolism of contaminating bacteria was reduced in the presence of 5 IU ml^?1 nisin, resulting in reduced lactic acid production and increased ethanol production by the yeast. Significance and Impact of the Study: Bacteriocins represent an alternative to the use of antibiotics for the control of bacterial contamination in fuel ethanol plants and may be important in preventing the emergence of antibiotic‐resistant contaminating strains.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

The effect of temperature,pH, and initial glucose concentration on the kinetics of ethanol production by Zymomonas mobilis in batch fermentation

Summary Zymomonas mobilis, strain ATCC 10988, was used to evaluate the effects of pH (5.0 to 8.0), temperature (30°C to 40°C), and initial glucose concentration (75 g/l to 150 g/l) on the kinetics of ethanol production from glucose using batch fermentation. Specific ethanol production rate was maximum and nearly constant over a pH range of 6.0 to 7.5. End-of-batch ethanol yield and specific growth rate were insensitive to pH in the range of 5.0 to 7.5. End-of-batch ethanol yield was maximum and nearly constant between 30°C and 37°C but decreased by 24% between 37°C and 40°C. All other kinetic parameters are greatest at 34°C. End-of-batch ethanol yield is maximum at an initial glucose concentration of 100 g/l. Specific growth rate reaches a maximum at 75 g/l, but specific ethanol production rate decreases throughout the range. The optimum initial glucose concentration of 100 g/l gives the highest ethanol yield at a specific ethanol production rate less than 10% below the maximum observed.     

Temperature-Dependent Kinetic Model for Nitrogen-Limited Wine Fermentations

A physical and mathematical model for wine fermentation kinetics was adapted to include the influence of temperature, perhaps the most critical factor influencing fermentation kinetics. The model was based on flask-scale white wine fermentations at different temperatures (11 to 35°C) and different initial concentrations of sugar (265 to 300 g/liter) and nitrogen (70 to 350 mg N/liter). The results show that fermentation temperature and inadequate levels of nitrogen will cause stuck or sluggish fermentations. Model parameters representing cell growth rate, sugar utilization rate, and the inactivation rate of cells in the presence of ethanol are highly temperature dependent. All other variables (yield coefficient of cell mass to utilized nitrogen, yield coefficient of ethanol to utilized sugar, Monod constant for nitrogen-limited growth, and Michaelis-Menten-type constant for sugar transport) were determined to vary insignificantly with temperature. The resulting mathematical model accurately predicts the observed wine fermentation kinetics with respect to different temperatures and different initial conditions, including data from fermentations not used for model development. This is the first wine fermentation model that accurately predicts a transition from sluggish to normal to stuck fermentations as temperature increases from 11 to 35°C. Furthermore, this comprehensive model provides insight into combined effects of time, temperature, and ethanol concentration on yeast (Saccharomyces cerevisiae) activity and physiology.     

Effect of <Emphasis Type="Italic">m</Emphasis>-carbonyl cyanide 3-chlorophenylhydrazone on inorganic polyphosphates synthesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under different growth conditions

The effects of different concentrations of the protonophore uncoupler m-carbonyl cyanide 3-hchlorophenylhydrazone (CCCP) on the synthesis of inorganic polyphosphates (polyP) during the first 0.5 h of hypercompensation in the yeast Saccharomyces cerevisiae VKM Y-1173 growing on media with 2% glucose under low (hypoxia) or high aeration or with 1% (vol/vol) ethanol under high aeration were studied. It was shown that the yeast growth on ethanol was completely inhibited by 5 μM CCCP, while growth on glucose was inhibited by 25 μM CCCP, independently of aeration of the medium. The maximum rate of H2 absorption was shown at 2, 5, and 25 μM CCCP for the cells grown on ethanol, on glucose under high aeration, and on glucose under hypoxia, respectively. Against the decrease of total ATP level and total polyP, CCCP had a nonuniform effect on the synthesis of individual polyP fractions. CCCP maximally inhibited synthesis of the most actively formed fractions: polyPI during growth on glucose under hypoxia, polyPIII during growth on glucose under aeration, and polyPIII and polyPV during growth on ethanol. CCCP had no substantial effect on the synthesis of polyPII and polyPIV fractions, the formation of which seems to be less related to the electrochemical potential gradient of H⁺ ions.