Improving the ethanol yield by reducing glycerol formation using cofactor regulation in Saccharomyces cerevisiae

To increase ethanol yield and decrease glycerol production in Saccharomyces cerevisiae, the strategies of direct cofactor-regulation were explored. During anaerobic batch fermentations, the yeast expressing Bacillus cereus gapN gene, encoding non-phosphorylating NADP(+)-dependent glyceraldehyde-3-phosphate dehydrognease, produced 73.8?g ethanol?l(-1), corresponding to 96% of theoretical maximum yield compared to 92% for the wild type. The yeast expressing Escherichia coli frdA gene encoding the NAD(+)-dependent fumarate reductase, exhibited a 22% (relative to the amount of substrate consumed) increase in glycerol yield in medium containing 2?g fumarate?l(-1). The yeast expressing mhpF gene, encoding acetylating NAD(+)-dependent acetaldehyde dehydrogenase, produced 74.5?g ethanol?l(-1), corresponding to 97.4% of theoretical maximum yield while glycerol decreased by 40% when acetic acid was added before inoculation. This strain represents a promising alternative for ethanol production with lignocellulosic hydrolysates where acetate is available at significant amounts.     

Acceleration of high gravity yeast fermentations by acetaldehyde addition

In high gravity Saccharomyces cerevisiae fermentations containing 300 g glucose l^–1, daily addition of acetaldehyde to a total of 93 mM shortened the time required to ferment the first 250 g glucose l^–1 from 790 h to 585 h. Acetaldehyde feeding had no effect on the ethanol yield but increased by 135%, 78% and 77% the final concentrations of 2,3-butanediol, 2-methylpropanol and acetate, while decreasing that of glycerol by 14%. Controlled acetaldehyde feeding has potential as a technique for accelerating high gravity fuel or industrial ethanol fermentations and may be useful in preventing incomplete fermentations.     

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.     

On-line measurement of the substrate concentrations in <Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentations using FT-IR/ATR

The glycerol and methanol concentrations in Pichia pastoris fermentations were measured on-line using Fourier transform infrared spectroscopy and an attenuated total reflection probe. Partial least squares regression was used to obtain calibration models. The models were regressed on synthetic multi-component spectra and semi-synthetic fermentation broth spectra. These were obtained by spectral addition. The accuracy for the on-line measurement of glycerol, given as standard error of prediction (SEP), was determined to 0.68 g/l, and the SEP of methanol was 0.13 g/l. We show how reliable calibration models are obtained relatively effortlessly by replacing extensive sampling from the reactor with simple mathematical manipulations of the model regression spectra.     

Transport and intracellular accumulation of acetaldehyde in saccharomyces cerevisiae

The rate of acetaldehyde efflux from yeast cells and its intracellular concentration were studied in the light of recent suggestions that acetaldehyde inhibition may be an important factor in yeast ethanol fermentations. When the medium surrounding cells containing ethanol and acetaldehyde was suddenly diluted, the rate of efflux of acetaldehyde was slow relative to the rate of ethanol efflux, suggesting that acetaldehyde, unlike ethanol, may accumulate intracellularly. Intracellular acetaldehyde concentrations were measured during high cell density fermentations, using direct injection gas chromatography to avoid the need to concentrate or disrupt the cells. Intracellular acetaldehyde concentrations substantially exceeded the extracellular concentrations throughout fermentation and were generally much higher than the acetaldehyde concentrations normally recorded in the culture broth in ethanol fermentations. The technique used was sensitive to the time taken to cool and freeze the samples. Measured intracellular acetaldehyde concentrations fell rapidly as the time taken to freeze the suspensions was extended beyond 2 s. The results add weight to recent claims that acetaldehyde toxicity is responsible for some of the effects previously ascribed to ethanol in alcohol fermentations, especially Zymomonas fermentations. Further work is required to confirm the importance of acetaldehyde toxicity under other culture conditions. (c) 1993 John Wiley & Sons, Inc.     

Response of wine yeast (Saccharomyces cerevisiae) aldehyde dehydrogenases to acetaldehyde stress during Icewine fermentation

AIMS: We previously reported that the aldehyde dehydrogenase encoded by ALD3 but not ALD6 was responsible, in part, for the increased acetic acid found in Icewines based on the expression profile of these genes during fermentation. We have now completed the expression profile of the remaining yeast aldehyde dehydrogenase genes ALD2, ALD4 and ALD5 during these fermentations to determine their contribution to acetic acid production. The contribution of acetaldehyde stress as a signal to stimulate ALD expression during these fermentations was investigated for all ALD genes. The expression of glycerol-3-phosphate encoded by GPD2 was also followed during these fermentations to determine its role in addition to the role we already identified for GPD1 in the elevated glycerol produced during Icewine fermentation. METHODS AND RESULTS: Icewine juice (38.5 degrees Brix, 398 +/- 5 g l(-1) sugar), diluted Icewine juice (20.8 degrees Brix, 196 +/- 4 g l(-1) sugar) and the diluted juice with sugar levels equal to the original Icewine juice (36.6 degrees Brix, 395 +/- 6 g l(-1) sugar) were fermented in duplicate using the commercial wine yeast K1-V1116. Acetic acid and glycerol production increased 8.4- and 2.7-fold in the Icewine vs the diluted juice fermentation, respectively, accompanied by a fourfold transient increase in acetaldehyde in the Icewine condition during the first week. Both mitochondrial aldehyde dehydrogenases encoded by ALD4 and ALD5 were expressed, with ALD5 expression highest at the start of all fermentations and ALD4 expression increasing during the first week of each condition. ALD2, ALD4, ALD5 and GPD2 showed no differential expression between the three fermentation conditions indicating their lack of involvement in elevating acetic acid and glycerol in Icewine. When yeast fermenting the diluted fermentation was exposed to exogenous acetaldehyde, the transient spike in acetaldehyde increased the expression of ALD3 but this response alone was not sufficient to cause an increase in acetic acid. Expression of the other aldehyde dehydrogenases was unaffected by the acetaldehyde addition. CONCLUSIONS: The aldehyde dehydrogenases encoded by ALD2, ALD4 and ALD5 do not contribute to the elevated acetic acid production during Icewine fermentation. Expression of GPD2 was not upregulated in high sugar fermentations and does not reflect the elevated levels of glycerol found in these wines. Acetaldehyde at a concentration produced during Icewine fermentation stimulates the expression of ALD3, but has no impact on the expression of ALD2, -4, -5 and -6. Upregulation of ALD3 alone in the dilute fermentation is not sufficient to increase acetic acid in wine and requires additional responses found in cells under hyperosmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work confirms that increased acetic acid and glycerol production during Icewine fermentation follows upregulation of ALD3 and GPD1 respectively, but upregulation of ALD3 alone is not sufficient to increase acetic acid production. Additional responses of cells under osmotic stress are required to increase acetic acid in Icewine.     

Inhibition and stimulation of yeast growth by acetaldehyde

Summary Acetaldehyde at above about 0.3 g/l inhibited yeast growth, suggesting that it may contribute to product inhibition in alcohol fermentations when present at high concentrations intracellularly. The toxic effects of acetaldehyde and ethanol were not mutually reinforcing, acetaldehyde appearing to alleviate slightly the effects of ethanol. In support of this, low concentrations of acetaldehyde greatly reduced the lag phase in ethanol-containing medium and increased the specific growth rate.     

Monitoring of ethanol during fermentation of a lignocellulose hydrolysate by on-line microdialysis sampling, column liquid chromatography, and an alcohol biosensor

During a 70-h fermentation of a lignocellulose hydrolysate, the ethanol produced was monitored on-line using a microdialysis probe as an in situ sampling device. The dialysate components were then separated in a column liquid chromatographic system and the ethanol was selectively detected by an amperometric alcohol biosensor. The result was compared with two off-line analysis methods: one chromatographic method with refractive index (RI) detection and one enzymatic method based on spectrophotometric detection. The two methods base on enzymes were shown to give lower values than the chromatographic method based on RI detection, which is discussed n terms of selectivity. The investigated on-line setup was found to be a flexible system for monitoring of fermentations, allowing a sampling frequency of at least 12 h(-1) and with a delay between sampling and detection of less than 5 min. (c) 1994 John Wiley & Sons, Inc.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

The role of acetaldehyde and glycerol in the adaptation to ethanol stress of Saccharomyces cerevisiae and other yeasts

Ethanol inhibition is a commonly encountered stress condition during typical yeast fermentations and often results in reduced fermentation rates and production yields. While past studies have shown that acetaldehyde addition has a significant ameliorating effect on the growth of ethanol-stressed Saccharomyces cerevisiae , this study investigated the potential ameliorating effect of acetaldehyde on a wide range of ethanol-stressed yeasts. Acetaldehyde does not appear to be a universal ameliorating agent for yeasts exposed to ethanol stress. It is also shown that as a result of an ethanol stress, most yeasts rapidly produce glycerol as an alternative means of NAD⁺ regeneration rather than having a specific requirement for glycerol. The results strongly suggest that both ethanol and acetaldehyde exposure have a direct effect on the cellular NAD⁺/NADH ratio, which can manifest itself as modulations in glycerol production.     

Determination of Growth and Glycerol Production Kinetics of a Wine Yeast Strain Saccharomyces cerevisiae Kalecik 1 in Different Substrate Media

Summary Glycerol has been known as an important by-product of wine fermentations improving the sensory quality of wine. This study was carried out with an endogenic wine yeast strain Saccharomyces cerevisiae Kalecik 1. The kinetics of growth and glycerol biosynthesis were analysed at various initial concentrations of glucose, fructose, and sucrose in a batch system. Depending on the determined values of Monod constants, glucose (K_s = 28.09 g/l) was found as the most suitable substrate for the yeast growth. Initial glucose, fructose and sucrose concentrations necessary for maximum specific yeast growth rate were determined as 175 g, 100 l, and 200 g/l, respectively. The yeast produced glycerol at very high concentrations in fructose medium. Fructose was determined as the most suitable substrate for glycerol production while the strain showed low tendency to use it for growth. S. cerevisiae Kalecik 1 could not produce glycerol below 200 g/l initial sucrose concentration. When natural white grape juice was used as fermentation medium, maximum glycerol concentration and dry weight of the yeast were determined as 9.3 g/l and 11.8 g/l, respectively.     

Glycerol production by immobilised cells of Pichia farinosa

Summary Cells ofPichia farinosa were immobilised in calcium alginate and K-Carrageenan and their ability to produce glycerol from glucose under aerobic conditions with acidic as well as alkaline pH was investigated. An average glycerol production rate of 0.07 g/l.h was obtained with immobilised cells (IMC) in shake flasks. Continuous glycerol production in a fluidised bed reactor (FBR) under steady state operation gave a glycerol concentration of 13.5 g/l in the product stream.     

Production of ethanol from molasses at 45 °C using alginate-immobilized Kluyveromyces marxianus imb3

The thermotolerant, ethanol-producing yeast strain, Kluyveromyces marxianus IMB3, has been immobilized in calcium alginate matrices. The ability of the biocatalyst to produce ethanol from cane molasses originating in Guatemala, Honduras, Senegal, Guyana and the Philippines was examined. In each case the molasses was diluted to yield a sugar concentration of 140?g/l and fermentations were carried out in batch-fed mode at 45?°C. During the first 24 hours, the maximum ethanol concentrations obtained ranged from 43–57?g/l with optimum production on the molasses from Honduras. Ethanol production during subsequent re-feeding of the fermentations at 24-hour intervals over a 120-hour period, decreased steadily to concentrations ranging from 20–36?g/l and it was found that ethanol productivity remained highest in fermentations containing the molasses from Guyana. When each set of fermentations was re-fed at 120?h and allowed to continue for 48?h, ethanol production again increased to a maximum with concentrations ranging from 25–52?g/l. It was also found however, that increasing the time between re-feeding at this stage in fermentation had a detrimental effect on the functionality of the biocatalyst.     

Improved selectivity of microbial biosensor using membrane coating. Application to the analysis of ethanol during fermentation

A ferricyanide mediated microbial biosensor for ethanol detection was prepared by surface modification of a glassy carbon electrode. The selectivity of the whole Gluconobacter oxydans cell biosensor for ethanol determination was greatly enhanced by the size exclusion effect of a cellulose acetate (CA) membrane. The use of a CA membrane increased the ethanol to glucose sensitivity ratio by a factor of 58.2 and even the ethanol to glycerol sensitivity ratio by a factor of 7.5 compared with the use of a dialysis membrane. The biosensor provides rapid and sensitive detection of ethanol with a limit of detection of 0.85 microM (S/N=3). The selectivity of the biosensor toward alcohols was better compared to previously published enzyme biosensors based on alcohol oxidase or alcohol dehydrogenases. The biosensor was successfully used in an off-line monitoring of ethanol during batch fermentation by immobilized Saccharomyces cerevisiae cells with an initial glucose concentration of 200 g l(-1).     

Acetaldehyde stimulation of the growth of Saccharomyces cerevisiae in the presence of inhibitors found inlignocellulose-to-ethanol fermentations

The addition of small quantities of acetaldehyde to fermentations containing inhibitory concentrations of furfural, acetate and other compounds typically present in lignocellulosic hydrolyzates significantly reduced the lag phase of yeast growth and stimulated ethanol production. Similar effects were observed when acetaldehyde (0.06 g l⁻¹) was added to fermentations of a birch wood hydrolyzate produced by steam/acid pretreatment. Acetaldehyde addition appears to have potential as a low-cost alternative (or adjunct) to current procedures for medium detoxification in lignocellulose-to-ethanol fermentations, particularly those in which high inhibitor concentrations are generated through recycling of the culture broth. Journal of Industrial Microbiology & Biotechnology (2000) 25, 104–108. Received 18 March 2000/ Accepted in revised form 02 June 2000     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Glycerol fermentation by a new 1,3-propanediol-producing microorganism:Enterobacter agglomerans

 According to their ability to synthesize 1,3-propanediol from glycerol, two species were isolated from the anoxic mud of a distillery waste-water digestor: Clostridium butyricum and Enterobacter agglomerans. The latter, a facultatively anaerobic gram-negative bacterium, is described for the first time as a microorganism producing 1,3-propanediol from glycerol. The products of glycerol conversion by E. agglomerans were identified using nuclear magnetic resonance. A 20-g/l glycerol solution was fermented mainly to 1,3-propanediol (0.51 mol/mol) and acetate (0.18 mol/mol). Ethanol, formate, lactate and succinate were formed as by-products. Gas production was very low; 1,3-propanediol production perfectly balanced the oxido-reduction state of the microorganism. Acetate was the predominant metabolite generating energy for growth. High-glycerol-concentration fermentations (71 g/l and 100 g/l) resulted in an increase of the 1,3-propanediol yield (0.61 mol/mol) at the expense of lactate and ethanol production. Specific rates of glycerol consumption and 1, 3-propanediol and acetate production increased whereas the growth rate decreased. The decrease in ATP yield was linearly correlated with the specific rate of 1,3-propanediol production. Incomplete glycerol consumption (about 40 g/l) was systematically observed when high glycerol concentrations were used. The unbalanced oxido-reduction state, the low carbon recovery and the detection of an unknown compound by HPLC observed in these cases indicate the formation of another metabolite, which is possibly an inhibitory factor. Received: 17 November 1994 / Accepted: 15 December 1994     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy

Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.