Use of acetaldehyde in the fermentative production of ethanol

Abstract

The instant invention relates to processes and systems for the fermentative production of ethanol. The ethanol is produced by fermenting a fermentable carbohydrate with a yeast, whereby acetaldehyde is externally supplied to the yeast cell for reducing glycerol by-product formation by the yeast cell, for improving the performance of yeast at high ethanol levels and/or for suppression of infections during the fermentation. The acetaldehyde that is externally supplied to the fermentation medium can be produced by catalytic oxidation of ethanol. Advantageously, acetaldehyde production from ethanol is integrated in a system for ethanol production. Thus, in another aspect the invention relates to a system for producing ethanol, e.g. an ethanol plant, which system, in addition to the usual means for fermentative production of ethanol, comprises a means for producing acetaldehyde by catalytic oxidation of ethanol. The invention further relates to a process for disinfecting fermentation equipment such as fermenters and bioreactors, as well as fermentations feedstocks, wherein the equipment and/or feedstocks is disinfected with high concentrations of acetaldehyde, which are then diluted to non-toxic concentrations by addition of the fermentation medium.

Claims

1. A process for producing ethanol comprising: a) fermenting a medium with a yeast cell in a fermenter, whereby the medium contains or is fed with a source of a fermentable carbohydrate and with a source of acetaldehyde, and whereby the yeast cell ferments the fermentable carbohydrate and the acetaldehyde to ethanol; and, b) recovery of the ethanol from the medium, wherein the acetaldehyde is present in or fed into the medium at least during a stage in the process when the growth rate of the yeast cell is at least 0.005 h.sup.1.

2. The process of claim 1, wherein: a) in a first phase of the process before a threshold ethanol concentration in the medium is reached, the rate of the acetaldehyde fed into the medium is controlled to maintain an acetaldehyde concentration of at least 0.0009 kg/m.sup.3 and, preferably no more than 1.0 kg/m.sup.3; and, b) in a second phase of the process after the threshold ethanol concentration in the medium is reached, the rate of the acetaldehyde fed into the medium is controlled to maintain an acetaldehyde concentration of no more than 0.3 kg/m.sup.3, and preferably at least 0.01 kg/m.sup.3, and wherein the threshold ethanol concentration is between 40 and 100 kg/m.sup.3.

3. The process of claim 2, wherein the acetaldehyde concentration in the medium is monitored on-line in an off-gas stream from the fermenter, preferably using a mass spectrometer or a gas chromatograph.

4. The process of claim 3, wherein the acetaldehyde concentration in the medium is monitored on-line in an off-gas stream from the fermenter using a mass spectrometer or a gas chromatograph.

5. The process of claim 1, wherein the acetaldehyde is fed into the medium in a liquid form or in gaseous form.

6. The process of claim 5, wherein the acetaldehyde that is fed into the medium in gaseous form is mixed with at least a part of the off-gas stream from the fermenter that is recycled back into the fermenter.

7. The process of claim 1, wherein the yeast cell is of a genus selected from the group consisting of Saccharomyces, Kazachstania and Naumovia.

8. The process of claim 7, wherein the yeast cell belongs to a species selected from the group consisting of Saccharomyces cerevisiae, S. bayanus, S. bulderi, S. cervazzii, S. cariocanus, S. castellii, S. dairenensis, S. exiguus, S. kluyveri, S. kudriazevii, S. mikatae, S. paradoxus, S. pastorianus, S. turicensis, and S. unisporus.

9. The process of claim 1, wherein the yeast cell ferments under anoxic conditions.

10. The process of claim 7, wherein the yeast cell has one or more modifications selected from the group consisting of: a) a genetic modification that increases resistance to acetaldehyde as compared to a corresponding unmodified parent strain, whereby preferably the cell with increased resistance to acetaldehyde is obtained by one or more of: i) evolutionary engineering; ii) a genetic modification that increases specific NADH-dependent alcohol dehydrogenase activity, whereby preferably the alcohol dehydrogenase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 9; iii) a genetic modification that increases the specific NADH-dependent alcohol dehydrogenase and glutathione-dependent aldehyde dehydrogenase activities, whereby preferably the bifunctional NADH-dependent alcohol dehydrogenase and glutathione-dependent aldehyde dehydrogenase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 10; iv) a genetic modification that increases the intracellular glutathione level in the cell, whereby preferably, the genetic modification comprises at least the overexpression of a gene encoding a -glutamylcysteine synthetase, whereby preferably the -glutamylcysteine synthetase has an amino acid sequence with at least 70% sequence identity to SEQ ID NO: 11; and, v) a genetic modification that increases the intracellular lysine level in the cell, whereby preferably the genetic modification confers resistance to S-2-aminoethyl-L-cysteine, or the genetic modification comprises reducing or eliminating the expression of a gene encoding a amino acid sequence with at least 70% sequence identity to SEQ ID NO: 12; b) a genetic modification that reduces or eliminates endogenous aldehyde dehydrogenase activity, whereby preferably the genetic modification reduces or eliminates expression of endogenous S. cerevisiae ALD6 gene or an orthologue thereof; c) a genetic modification that reduces or eliminates NADH-dependent glycerol synthesis, whereby preferably the genetic modification is a modification that reduces or eliminates the expression of one or more of the S. cerevisiae GPD1, GPD2, HOR2 and RHR2 genes or orthologues thereof; d) a genetic modification that reduces or eliminates transport of glycerol, whereby preferably the genetic modification is a modification that reduces or eliminates the expression of the S. cerevisiae FPS1 gene or an orthologue thereof; and, e) a genetic modification that introduces into the cell at least one of: i) expression of an exogenous xylose isomerase gene, which gene confers to the cell the ability to isomerize xylose into xylulose; and, ii) expression of exogenous genes coding for a L-arabinose isomerase, a L-ribulokinase and a L-ribulose-5-phosphate 4-epimerase, which genes together confer to the cell the ability to convert L-arabinose into D-xylulose 5-phosphate, whereby the cell further preferably comprises genetic modifications that increase the specific activities of one or more of xylulose kinase, ribulose-5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, transketolase and transaldolase; and a genetic modification that reduces or eliminates unspecific aldose reductase activity.

11. The process of claim 1, wherein the process for producing ethanol is preceded by a process for disinfecting the fermenter that is carried out prior to adding a least one of the medium and the yeast cell to the fermenter, wherein the process for disinfecting the fermenter comprises the steps of: i) supplying to the fermenter an amount of acetaldehyde resulting in a concentration of acetaldehyde of at least 1 kg/m.sup.3, and incubating the acetaldehyde in the fermenter for at least 5 minutes, whereby the amount of acetaldehyde supplied is such that upon supply of the medium and the yeast cell to the fermenter, the concentration of acetaldehyde is diluted to no more than 2.0 kg/m.sup.3; ii) supplying medium and optionally yeast cells to the fermenter in an amount to dilute the acetaldehyde to a concentration of no more than 2.0 kg/m.sup.3; whereby, preferably, step i) the acetaldehyde is supplied into the fermenter in gas phase and/or the acetaldehyde is brought into the gas phase and or kept in the gas phase in the fermenter.

12. The process of claim 1, wherein the acetaldehyde is produced by catalytic oxidation of ethanol, preferably using a catalyst comprising one or more of a noble metal, an alloy thereof and oxides thereof, in the presence of oxygen, wherein preferably the noble metal is selected from silver, copper, platinum and gold.

13. The process of claim 12, wherein the acetaldehyde is produced by catalytic oxidation of a part of the ethanol obtained in b) of claim 1, whereby preferably the acetaldehyde is produced at a site in the vicinity of the site where the ethanol is produced.

14. A system for producing ethanol in a process according to claim 1, wherein the system comprises a means for fermentation of a medium to an ethanol-containing beer, a means for distillation for recovery of ethanol from the beer and a means for supplying acetaldehyde to the medium, and wherein, the system comprises a reactor holding a catalyst as defined in claim 8, for producing acetaldehyde by catalytic oxidation of ethanol.

15. The system of claim 14, wherein: a) the system is configured to produce acetaldehyde by catalytic oxidation from a part of the ethanol obtained from the means for distillation, optionally after storage of the ethanol; and, b) optionally, the system is configured to supply the acetaldehyde produced in a) to the medium, optionally after storage of the acetaldehyde.

16. The system of claim 14 or 15, wherein the system comprises a means for monitoring the acetaldehyde concentration and optionally the ethanol concentration, in the fermentation medium and a means for controlling the rate of the acetaldehyde supply into the medium in the fermenter, wherein preferably, the means for controlling the rate of acetaldehyde supply into the medium receives input from the means for monitoring the acetaldehyde concentration, and optionally the ethanol concentration, to maintain an acetaldehyde concentration in the medium in accordance with the process of claim 1, wherein preferably, the means for controlling the rate of acetaldehyde supply into the medium further receives input from the means for monitoring the ethanol concentration in the medium to further control the acetaldehyde concentration in the medium as a function of the ethanol concentration in accordance with the process of claim 2.

17. The system of claim 16, wherein the means for controlling the rate of acetaldehyde supply into the medium receives input from the means for monitoring the acetaldehyde concentration to control an acetaldehyde concentration in the medium in accordance with a process of claim 1, wherein preferably, the means for controlling the rate of acetaldehyde supply into the medium further receives input from the means for monitoring the ethanol concentration in the medium to further control the acetaldehyde concentration in the medium as a function of the ethanol concentration in accordance with the process of claim 2.

Description

DESCRIPTION OF THE FIGURE

[0160] A schematic illustration of an ethanol plant with a facility for catalytic oxidation of part of the ethanol produced in the plant to acetaldehyde and with a facility for supply of the acetaldehyde to the fermenter. In a fermenter (1) of e.g. 2000 m.sup.3, a medium comprising a fermentable carbohydrate and acetaldehyde is fermented with a yeast whereby the yeast cell ferments the fermentable carbohydrate and the acetaldehyde to ethanol. The ethanol-containing beer (9) is transferred to a distillation unit (2) for recovery of the ethanol (10), which is stored in an ethanol storage tank (3). Ethanol form the storage tank can be shipped for sales (11). A part (12) of the ethanol from the storage tank (3) is directed to a saturator (4), of e.g. 0.4 m.sup.3, wherein air (13) is saturated with ethanol. The air saturated with ethanol (14) is directed to a conversion column comprising e.g. a silver catalyst (5) of e.g. 0.8 m.sup.3, wherein the ethanol is oxidized to acetaldehyde (15), which can be held and optionally diluted with buffer in holding tank (6) of e.g. 10 m.sup.3. Heat/energy (16), that is generated by the exothermic catalytic oxidation in (5), can be used elsewhere in the plant, e.g. in the distillation unit (2). Diluted acetaldehyde (17) from the holding tank (6) is supplied to the fermenter (1) by dosage controller (7), which receives input (18) about the concentrations of acetaldehyde and ethanol in the fermentation medium, as detected by a detection unit (8) in the carbon dioxide off-gas stream (19) from the fermenter (1). The detection unit (8) can be a flame ionization detector.

EXAMPLES

Methods and Materials

Strains

[0161] S. cerevisiae CEN.PK2-1C or similar CEN.PK strains are obtainable from EUROSCARF at the University of Frankfurt, Germany, e.g. via email Euroscarf@em.uni-frankfurt.de or at: http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html.

[0162] S. cerevisiae CBS 8066 is obtainable from CBS-KNAW, Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre Centraal Bureau, Utrecht, the Netherlands, at www.cbs.knaw.nl.

[0163] The commercial yeast Thermosacc is obtainable from Lallemand Biofuels & Distilled Spirits, Duluth, Ga. 30097, USA (www.lallemandbds.com).

[0164] The commercial yeast Ethanol Red is obtainable from Phibro Animal Health Corporation, Ethanol Performance Group, Teaneck, N.J. 07666-6712, USA (www.ethanolperformancegroup.com).

[0165] The commercial yeast Fermiol Super HA Thermosacc is obtainable from Enzyme Development, New York.

Metabolite Analyses

[0166] Supernatant obtained by centrifugation of culture samples was analyzed for glucose, ethanol, acetaldehyde, glycerol, acetic acid, succinic acid, pyruvic acid and 2,3-butanediol via HPLC analysis on a Waters Alliance 2690 HPLC (Waters, Milford, USA) containing a Biorad HPX 87H column (Biorad, Hercules, USA). The column was eluted at 60 C. with 0.5 g/l H.sub.2S0.sub.4 at a flow rate of 0.6 ml min.sup.1 . Detection was by means of a Waters 2410 refractive-index detector and a Waters 2487 UV detector. Initial and final glycerol concentrations were further determined using an enzymatic determination kit (Rbiopharm AG, Darmstadt, Germany).

[0167] For dry weight measurements nitrocellulose filters (pore size, 0.45 m; Gelman Sciences, Inc., Ann Arbor, Mich.) were used. Samples were harvested at desired cultivation times. After removal of the medium by filtration, the filters were washed with demineralized water and dried in an R-7400 Microwave Oven (Sharp Inc., Osaka, Japan) for 15 min. This procedure yielded the same dry weight data as drying of filters at 80 C.

Enzyme Activity Analyses

[0168] Cell extracts for activity assays were prepared and analyzed for protein content as described by Postma et al., (1989, Appl. Environ. Microbiol. 55(2):468).

[0169] Acetaldehyde dehydrogenases (NAD.sup.+ and NADP.sup.+) (EC 1.2.1.5 and EC 1.2.1.4, respectively) activity was measured at 30 C. by monitoring the oxidation of NADH or NADPH at 340 nm. The assay mixture contained potassium phosphate buffer (pH 8.0) (100 mM), pyrazole (15 mM), dithiothreitol (0.4 mM), KCl (10 mM), and NAD.sup.+ or NADP.sup.+ (0.4 mM). The reaction was started with 0.1 mM acetaldehyde.

[0170] For glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) activity determination, cell extracts were prepared as described above except that the phosphate buffer was replaced by triethanolamine buffer (10 mM, pH 5). Glycerol-3-phosphate dehydrogenase activities were assayed in cell extracts at 30 C. as described previously (Blomberg and Adler, 1989, J. Bacteriol. 171: 1087-1092.9).

[0171] Glycerol 3-phosphatase was assayed as described previously (Norbeck et al. 1996. J. Biol. Chem. 271:13875-13881). Briefly, cell-free extracts were incubated in 20 mm Tricine-HCl (pH 6.5), 5 mM MgCl.sub.2, and 10 mm dl-glycerol 3-phosphate in a total volume of 1.0 ml. After starting the reaction, samples of 90 l were withdrawn at different time points and the reaction was stopped by adding 10 l of 50% HClO.sub.4. Inorganic phosphate was analyzed according to a previous study (27), and the reaction rate was calculated from the slope of a linear plot of released phosphate versus time. All glassware used was immersed overnight in 1 M HCl and rinsed thoroughly in distilled water, to eliminate phosphate contamination.

Cultivation Procedures

[0172] The mineral salts medium employed was based on standard media (Bruinenberg et al. 1983. Journal of General Microbiology 129:965-971; Verduin et al 1990. Journal of General Microbiology 136:395-403). It contained the following per liter of demineralized water: (NH.sub.4).sub.2SO.sub.4, 5 g; KH.sub.2PO.sub.4, 3 g; MgSO.sub.4.7H.sub.2O, 0.5 g; EDTA, 15 mg; ZnSO.sub.4.7H.sub.20, 4.5 mg; CoCl.sub.2 6H.sub.2O, 0.3 mg; MnCl.sub.2 4H.sub.20, 1 mg; CuSO.sub.4 5H.sub.20, 0.3 mg; CaCl.sub.2. 2H.sub.20, 4.5 mg; FeSO.sub.4 7H.sub.20, 3 mg; Na.sub.2MoO.sub.4. 2H.sub.2O, 0.4 mg; H.sub.3BO.sub.3, 1 mg; KI, 0.1 mg; and 0.025 ml silicone antifoam (BDH). After heat sterilization at 120 C. and cooling, filter sterilized vitamins were added: biotin, 0.05 mg; calcium pantothenate, 1 mg; nicotinic acid, 1 mg; inositol, 25 mg; thiamin.HCl, 1 mg; pyridoxine.HCl, 1 mg; and para-aminobenzoic acid, 0.2 mg. Ergosterol and Tween 80 were dissolved in pure ethanol and steamed at 100 C. for 10 minutes before they were added to the medium to give final concentrations of 10 and 400 mg/l, respectively, and a final concentration of 38 mM ethanol. A glucose solution was heat-sterilized separately at 110 C. for 20 minutes and liquid acetaldehyde was used as such. The sterilized glucose solution was added to the sterile mineral salts medium to give the required final concentration.

[0173] Media for plates (1.5% agar) for S. cerevisiae were the mineral salts medium supplement with 2% glucose and for lactic acid bacteria the de Man, Rogosa and Sharpe (MRS) medium. Cycloheximide (0.1 g/l) was added to the plates in case mixed cultures of yeast and lactic acid bacteria were studied.

[0174] Small-scale batch cultivation of the yeast for testing the effect of acetaldehyde (1.7 mM) was done at 30 C. in capped bottles of 30 ml. Control bottles did not receive addition of acetaldehyde. Carbon dioxide was allowed to escape from the bottles by inserting a needle in the septum of the cap. The bottles were incubated stationary. A sample per bottle was taken at the start of the experiment and after 90 minutes of incubation.

[0175] Small-scale batch cultivation of the yeasts for optimizing acetaldehyde tolerance was done at 30 C. in 100-ml Erlenmeyer flasks. The flasks contained 20 ml mineral salts medium supplied with glucose (12 g/l) and they were incubated on a rotary shaker at 400 r.p.m. Air could enter in the flasks via cotton plugs.

[0176] Semi-anoxic batch cultivation of the yeasts together with lactic acid bacteria was done at 30 C. in 100-ml Erlenmeyer flasks. The flasks contained 50 ml mineral salts medium supplied with glucose (12 g/l) and they were incubated without shaking. Air could enter in the flasks via cotton plugs.

[0177] Anoxic chemostat cultivation of the yeasts was done at 30 C. in a fermenter with a working volume of 1 liter and at a stirring speed of 600 r.p.m. The pH was automatically controlled at pH=5 by titration with 2 M KOH. The condenser at the outlet of the gas stream was connected to a cryostat and cooled at 2 C. The condensate was returned into the fermentation vessel. The tubing on the entire fermenter set-up (including medium- and waste-reservoirs) consisted of material (Norprene tubing) that is very poorly permeable for oxygen. The fermenter and the medium reservoir (with a magnetic stirrer) were sparged with certificated ultra-pure nitrogen prior to the addition of acetaldehyde to the reservoir.

[0178] Batch cultivation for testing the effect of acetaldehyde on the production of glycerol and ethanol was done at 30 C. in a bioreactor with a working volume of 1 liter and a headspace of 0.5 liter. The stirring speed was set at 300 r.p.m. and the pH of the mineral salts medium containing 190 g/l glucose was kept at 5 by adding 2 M KOH. The condenser at the outlet of the gas stream was connected to a cryostat and cooled at 2 C. The condensate was returned into the fermentation vessel. The off-gas was monitored continuously with a Series 200ACE Acetaldehyde Analyzer (GOW-MAC Instrument Co). Sampling was from a loop kept at 35 C. for circulating gas from the headspace. Acetaldehyde was added by a peristaltic pump to the bioreactor intermittently as a solution of 25% acetaldehyde in water. The solution entered in the aqueous phase via a needle at the outlet of the tubing. The addition of the acetaldehyde solution was controlled and set as based on the measurements of the acetaldehyde concentration in the gas phase.

[0179] Batch cultivation for testing the effect of acetaldehyde at higher ethanol concentrations was done at 30 C. in a bioreactor with a working volume of 1 liters and a headspace of 0.5 liter. The stirring speed was set at 300 r.p.m. and the pH of the mineral salts medium containing 280 g/l glucose was kept at 5 by adding 2 M NaOH. The condenser at the outlet of the gas stream was connected to a cryostat and cooled at 2 C. The condensate was returned into the fermentation vessel. The off-gas was monitored continuously for both ethanol and acetaldehyde with a Series 200ACE Acetaldehyde Analyzer (GOW-MAC Instrument Co). Sampling was from a loop kept at 35 C. for circulating gas from the headspace. Acetaldehyde was added to the bioreactor once the ethanol concentration in the aqueous phase had reached 80 g/l as calculated from the concentrations in the gas phase. It was added by a peristaltic pump to the bioreactor intermittently as a solution of 10% acetaldehyde in water. The solution entered in the aqueous phase via a needle at the outlet of the tubing. The addition of the acetaldehyde solution was controlled and set as based on the measurements of the acetaldehyde concentration in the gas phase.

Example 1

Modification of Host Cells By Evolutionary Engineering

[0180] S. cerevisiae CEN.PK2-1C was subjected to evolutionary engineering with the aim of obtaining organisms that had acquired an enhanced tolerance to acetaldehyde. The organisms were cultivated under oxic conditions in a batch-wise mode. The initial pH of the mineral salts medium was set at 6 by titrating with KOH and glucose was added at 12 g/l. In the first round of incubations, acetaldehyde was added to this medium to reach a concentration of 0.2 g/l. After 3 days of incubation, an aliquot of 0.5 ml was taken from the culture and transferred into fresh medium (50 ml), now containing 0.3 g/l acetaldehyde. Subsequently, this procedure was repeated weekly with ever increasing acetaldehyde concentrations in the medium. The strains obtained in this way, had acquired increasing tolerance towards acetaldehyde. Strain S. cerevisiae CEN.PK2-1C now grew in the batch system in the presence of 0.5 g/l acetaldehyde without a noticeable change in growth rate as compared to the parent strain in the absence of acetaldehyde. The newly acquired strains retained their ability to withstand higher acetaldehyde levels also when cultivating them anoxically in mineral salts medium with glucose and for prolonged periods of times. This subculturing was by taking an aliquot of 0.5 ml from a culture that had consumed all glucose and by transferring this inoculum to fresh medium (50 ml). This procedure was repeated ten times before the stability towards acetaldehyde was assessed.

Example 2

Modification of the Host Cells Along with Lactic Acid Bacteria By Evolutionary Engineering

[0181] S. cerevisiae CEN.PK2-1C was subjected to evolutionary engineering analogous to the procedure described above, except that now semi-anoxic conditions were employed. Eight strains of lactic acid bacteria, belonging to Lactobacillus, Pediococcus, Leuconostoc and Weissella, were inoculated as well. The dynamics of the quantitative growth of the 9 organisms thus present as co-culture was followed during the procedure of evolutionary engineering. During each successive transfer step, the suspension after growth was plated on media that allow growth of either S. cerevisiae or of the 8 strains of lactic acid bacteria. In time, with increasing concentrations of acetaldehyde in the subsequent batches, the lactic acid bacteria disappeared from the incubations. Cultivation and subsequent transfers of the co-culture containing the 9 organisms was also done in the absence of acetaldehyde. Under this circumstance, the lactic acid bacteria remained present in the system.

Example 3

Anoxic Chemostat Fermentations Under Glucose Limitation in Either the Absence or Presence of Acetaldehyde

[0182] Strain S. cerevisiae CBS 8066 was cultivated in chemostat culture at a dilution rate of 0.11 h.sup.1. The glucose concentration in the medium reservoir was 24 g/l (137 mM) in each run. Three separate steady states were obtained in either the absence or presence of acetaldehyde. The effect of the aldehyde on the fermentation was tested by adding the compound to the medium reservoir after a steady state situation in its absence (medium and samples 1) had been established. In a first acetaldehyde run, a concentration of 6 mM (0.26 g/l) acetaldehyde was included in the medium reservoir (medium and samples 2). The second acetaldehyde run contained 15 mM (0.65 g/l) acetaldehyde in the reservoir (medium and samples 3). In each of the three steady states (after 3-5 volume changes), the fermentation medium in the fermenter was analyzed for various compounds as summarized in Tables 1 and 2.

TABLE-US-00001 TABLE 1 Chemostat experiment: metabolite concentrations in the feed (medium) and in duplicate (A and B) chemostat samples of the three steady states (1 = no acetaldehyde supplied; 2 = 6 mM acetaldehyde supplied; and 3 = 15 mM acetaldehyde supplied). butane- pyruvic succinic glucose acetaldehyde glycerol ethanol acetic acid diol acid acid (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) Medium 1 137.78 0.00 <1 37.86 n.d. n.d. 2.13 n.d. Medium 2 136.00 6.00 <1 38.27 n.d. n.d. 2.14 n.d. Medium 3 136.27 14.79 <1 38.49 n.d. n.d. 2.12 n.d. Sample 0.42 0.02 15.55 229.51 0.41 n.d. 0.54 0.45 1A Sample 0.44 0.03 15.34 227.99 0.28 n.d. 0.49 0.44 1B Sample 0.48 0.03 9.47 243.13 0.64 n.d. 0.56 0.43 2A Sample 0.46 0.03 9.60 244.74 0.54 n.d. 0.51 0.45 2B Sample 0.44 0.04 4.04 255.35 0.78 n.d. 0.48 0.45 3A Sample 0.48 0.04 4.02 255.25 0.69 n.d. 0.48 0.50 3B n.d. = not detected

TABLE-US-00002 TABLE 2 Effect of acetaldehyde additions on glycerol and ethanol production. glucose acetaldehyde glycerol ethanol consumed consumed produced produced (mM) (mM) (mM) (mM) Steady state 1 138 0 0 0 Steady state 2 136 6 6 15 Steady state 3 136 16 11 27

Example 4

Batch Cultivation for Testing the Effect of Acetaldehyde

[0183] Strain S. cerevisiae CBS 8066 was employed in testing the effect of acetaldehyde on the formation of ethanol and glycerol from glucose at a concentration of 50 g glucose/l. The initial cell concentration was 0.45 g/l dry biomass. Acetaldehyde was supplied to the bottle in a single addition at the start of the incubation. In a separate control run, no acetaldehyde was supplied. The concentrations of acetaldehyde, glycerol and ethanol as determined at the start and at the end of the fermentation, is given in Table 3.

TABLE-US-00003 TABLE 3 Effect of acetaldehyde on the formation of ethanol and of glycerol in a batch fermentation mM mM Glucose at start 280 280 Glucose at end Not determined Not determined Acetaldehyde in 1.76 Acetaldehyde at end 0.12 1.01 Ethanol at end 6.39 6.67 Glycerol at end 1.07 0.58

Example 5

Effect of Acetaldehyde Additions on Batch Fermentations at High Ethanol Concentrations

[0184] The Ethanol Red strain was used in assessing the effect of acetaldehyde at high ethanol concentrations. The mineral salts medium was employed with the addition of yeast extract at 1 g/l. The initial cell concentration was 0.5 g/l dry biomass. Air was supplied to the headspace of the fermenter at 15 ml/min during the first 2 hours of cultivation after which conditions were kept anoxic. Glucose was added initially at 230 g/l. Acetaldehyde was added once the ethanol concentration in the fermentation had reached 80 g/l. The concentration was monitored from the headspace and controlled in the fermenter liquid between 0.06 and 0.1 g/l. In a control experiment, no acetaldehyde was supplied. The time required to reach 98 g/l of ethanol in the liquid as determined from its gas phase concentration was taken as criterion in assessing the effect of acetaldehyde. Without acetaldehyde it took 180 minutes to reach 98 g/l ethanol whereas in the presence of acetaldehyde this level had been reached in 158 minutes. The total amount of acetaldehyde supplied during the interval between 80 and 98 g/l ethanol was 0.7 g.