The glycerol and ethanol production kinetics in low-temperature wine fermentation using Saccharomyces cerevisiae yeast strains

Increasing glycerol production in low-temperature wine fermentation is of concern for winemakers to improve the quality of wines. The objective of this study was to investigate the effect of 10 different Saccharomyces cerevisiae on the kinetics of production of glycerol, ethanol and the activities of glycerol-3-phosphate dehydrogenase (GPD) and alcohol dehydrogenase (ADH) in low-temperature fermentation. Ethanol production was influenced by temperature, and it was slightly higher at 13 °C than at 25 °C. Glycerol yields were significantly affected by both temperature and strains. More glycerol was produced at 25 °C than at 13 °C because the activity of GPD was higher at 25 °C than at 13 °C. Glycerol production of the different yeast strains was up to 3.19 and 3.18 g L⁻¹ at 25 and 13 °C, respectively. Therefore, isolating the yeast strains with high glycerol production and adaptation to low-temperature fermentation is still the best method in winemaking.     

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

Glycerol is a major fermentation product of Saccharomyces cerevisiae that contributes to the sensory character of wine. Diverting sugar to glycerol overproduction and away from ethanol production by overexpressing the glycerol 3-phosphate dehydrogenase gene,GPD2, caused S. cerevisiae to produce more than twice as much acetic acid as the wild-type strain (S288C background) in anaerobic cell culture. Deletion of the aldehyde dehydrogenase gene, ALD6, in wild-type and GPD2 overexpressing strains (GPD2-OP) decreased acetic acid production by three- and four-fold, respectively. In conjunction with reduced acetic acid production, the GPD2-OP ald6Delta strain produced more glycerol and less ethanol than the wild-type. The growth rate and fermentation rate were similar for the modified and wild-type strains, although the fermentation rate for the GPD2 ald6Delta strain was slightly less than that of the other strains from 24h onwards. Analysis of the metabolome of the mutants revealed that genetic modification affected the production of some secondary metabolites of fermentation, including acids, esters, aldehydes and higher alcohols, many of which are flavour-active in wine. Modification of GPD2 and ALD6 expression represents an effective strategy to increase the glycerol and decrease the ethanol concentration during fermentation, and alters the chemical composition of the medium such that, potentially, novel flavour diversity is possible. The implications for the use of these modifications in commercial wine production require further investigation in wine yeast strains.     

Glycerol formation during wine fermentation is mainly linked to Gpd1p and is only partially controlled by the HOG pathway

Glycerol 3-phosphate dehydrogenase, a key enzyme in the production of glycerol, is encoded by GPD1 and GPD2. The isoforms encoded by these genes have different functions, in osmoregulation and redox balance, respectively. We investigated the roles of GPD1, GPD2 and HOG1-the kinase involved in the response to osmotic stress-in glycerol production during wine fermentation. We found that the deletion of GPD2 in a wine yeast-derived strain did not affect growth or fermentation performance and reduced glycerol production by only 20%. In contrast, a gpd1delta mutant displayed a prolonged lag phase, and produced 40% less glycerol than the wild-type strain. The deletion of HOG1 resulted in a slight decrease in growth rate and a 20% decrease in glycerol production, indicating that the HOG pathway operates under wine fermentation conditions. However, the hog1delta mutant was not as severely affected as the gpd1delta mutant during the first few hours of fermentation, and continued to express GPD1 strongly. The hog1delta mutant was able to increase glycerol production in response to high sugar concentration (15-28% glucose), to almost the same extent as the wild-type, whereas this response was totally abolished in the gpd1delta mutant. These data show that Gpd1p plays a major role in glycerol formation, particularly during the first few hours of exposure to high sugar concentration, and that GPD2 is only of little significance in anaerobic fermentation by wine yeast. The results also demonstrate that the HOG pathway exerts only limited control over GPD1 expression and glycerol production during wine fermentation.     

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4·7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6·5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8·1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.     

Fermentation properties of a wine yeast over-expressing the Saccharomyces cerevisiae glycerol 3-phosphate dehydrogenase gene (GPD2)

Wine yeasts efficiently convert sugar into ethanol. The possibility of diverting some of the sugar into compounds other than ethanol by using molecular genetic methods was tested. Over-expression of the yeast glycerol 3-phosphate dehydrogenase gene ( GPD2 ) in a laboratory strain of Saccharomyces cerevisiae led to an approximate two-fold increase in the extracellular glycerol concentration. In the medium fermented with the modified strain, acetic acid concentration also increased approximately two-fold when respiration was blocked. A strain deleted for the GPD2 gene had the opposite phenotype, producing lower amounts of glycerol and acetic acid, with the latter compound only reduced during non-respiratory growth. A commercial wine yeast over-expressing GPD2 produced 16.5 g/L glycerol in a wine fermentation, compared to 7.9 g/L obtained with the parent strain. As seen for the laboratory strain, acetic acid concentrations were also increased when using the genetically modified wine yeast. A panel of wine judges confirmed the increase in volatile acidity of these wines. The altered glycerol biosynthetic pathway sequestered carbon from glycolysis and reduced the production of ethanol by 6 g/L.     

The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae

Maintenance of a cytoplasmic redox balance is a necessity for sustained cellular metabolism. Glycerol formation is the only way by which Saccharomyces cerevisiae can maintain this balance under anaerobic conditions. Aerobically, on the other hand, several different redox adjustment mechanisms exist, one of these being the glycerol 3-phosphate (G3P) shuttle. We have studied the importance of this shuttle under aerobic conditions by comparing growth properties and glycerol formation of a wild-type strain with that of gut2Δ mutants, lacking the FAD-dependent glycerol 3-phosphate dehydrogenase, assuming that the consequent blocking of G3P oxidation is forcing the cells to produce glycerol from G3P. To impose different demands on the redox adjustment capability we used various carbon sources having different degrees of reduction. The results showed that the shuttle was used extensively with reduced substrate such as ethanol, whereas the more oxidized substrates lactate and pyruvate, did not provoke any activity of the shuttle. However, the absence of a functional G3P shuttle did not affect the growth rate or growth yield of the cells, not even during growth on ethanol. Presumably, there must be alternative systems for maintaining a cytoplasmic redox balance, e.g. the so-called external NADH dehydrogenase, located on the outer side of the inner mitochondrial membrane. By comparing the performance of the external NADH dehydrogenase and the G3P shuttle in isolated mitochondria, it was found that the former resulted in high respiratory rates but a comparably low P/O ratio of 1·2, whereas the shuttle gave low rates but a high P/O ratio of 1·7. Our results also demonstrated that of the two isoforms of NAD-dependent glycerol 3-phosphate dehydrogenase, only the enzyme encoded by GPD1 appeared important for the shuttle, since the enhanced glycerol production that occurs in a gut2Δ strain proved dependent on GPD1 but not on GPD2. © 1998 John Wiley & Sons, Ltd.     

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh ^d) and diacetyl reductase (dar ^d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh ^d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.     

酿酒酵母关键节点基因缺损对法尼烯合成的影响

以法尼烯为评价效应物,研究了缺损乙醇合成途径、甘油合成途径、胞质乙酰辅酶A转运途径和法尼基焦磷酸消耗支路关键基因对酿酒酵母WHE4菌株合成法尼烯的影响。通过CRISPR-cas9基因编辑技术,获得8株关键基因缺损菌株。结果表明,与WHE4菌株相比,缺损乙醇脱氢酶基因ADH3-6对乙醇和法尼烯产量没有影响;单独缺损甘油三磷酸脱氢酶基因GPD1和GPD2使甘油积累量分别降低了15%和34%,缺损半乳糖激酶基因GAL1、GAL7、GAL10下调了甲羟戊酸途径所有基因转录水平,它们的缺损均不能提高菌株的法尼烯产量;缺损香叶基香叶基焦磷酸合酶基因BTS1和二酰基甘油二磷酸磷酸酶基因DPP1,法尼烯产量提高了29%,在5 L发酵罐补料分批发酵,菌株WHE4-33(WHE4 Δbts1,Δdpp1)的法尼烯产量达到1 578.91 mg/L。该研究对甲羟戊酸途径上游和下游关键节点基因进行了缺损影响法尼烯合成研究,为构建酿酒酵母萜类化合物高效平台提供了参考价值。     

葡萄酒中甘油的生成及其影响因素

甘油具有甜味和粘稠性,是干、半干葡萄酒的主要组成分,对酒的质量具有重要影响。甘油是葡萄酒酵母的正常发酵副产物,主要在葡萄汁发酵初期产生。葡萄酒酵母启动甘油生产原因可能是发酵最初缺少醇脱氢酶,造成氧化还原当量不平衡,细胞生产甘油、消耗NADH以平衡胞内氧化还原电势;葡萄汁中初始糖浓度高造成高渗透压,胞内产甘油作为溶质以抵消高渗透压。不同的酵母之间产甘油能力差别很大。提高甘油量是改进干、半干葡萄酒质量的重要措施之一。筛选高产甘油的葡萄酒酵母、混合菌种发酵、调整葡萄汁成分、改变发酵条件、施加热刺激等均能在不同程度上增加甘油的产量。     

CRISPR/Cas9介导的酿酒酵母ADH2基因中断及反义RNA干扰GPD1的表达

CRISPR/Cas9是一个简单、高效的用于靶向目的基因和无标记的基因组工程的工具。本文通过构建酿酒酵母沉默组件PGK-SGPD1-CYC1,使甘油-3-磷酸脱氢酶I(Glycerol-3-phosphate dehydrogenase,GPD1)基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。应用CRISPR/Cas9基因编辑技术,在中断乙醇脱氢酶Ⅱ(alcohol dehydrogenase Ⅱ,ADH2)基因的同时,定点敲入GPD1基因的反义干扰组件,从而特定地干扰GPD1的表达。采用高效的酵母化学转化法将反应组件敲入酿酒酵母Y1H中,CRISPR/Cas9介导的同源重组效率达43.48%,由此获得了ADH2基因中断和GPD1反义干扰的酿酒酵母突变株。发酵实验结果表明,酿酒酵母突变菌株SG1-1与出发菌株Y1H相比,乙醇产率提高了9.07%,甘油产率下降了12.05%,乙酸产率下降了12.30%,结果表明通过中断ADH2基因及插入GPD1反义干扰组件,既能够中断ADH2基因的功能,减少乙醇转化为乙醛,同时也能在一定程度上干扰GPD1基因的表达,提高乙醇产率。     

Glucose metabolism,enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum

The physiology of Hanseniaspora uvarum K₅ was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h^?1, glucose was completely metabolized in biomass and CO₂. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO₂ were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.     

Overexpression of the genes of glycerol catabolism and glycerol facilitator improves glycerol conversion to ethanol in the methylotrophic thermotolerant yeast Ogataea polymorpha

Production of fuel ethanol is one of the possible ways to utilize crude glycerol, substantial amounts of which are produced by biodiesel industry. Earlier, we have described construction of the recombinant strains of methylotrophic thermotolerant yeast Ogataea polymorpha with simultaneous overexpression of the genes PDC1 and ADH1, which produced increased amounts of ethanol from glycerol. In this work, we have further improved these strains by overexpression of genes involved either in oxidative (through dihydroxyacetone) or phosphorylative (through glycerol-3-phosphate) pathway of glycerol catabolism, as well as heterologous gene coding for glycerol transporter FPS1 from Komagataella phaffii (formerly, Pichia pastoris). Obtained recombinant strains produced up to 10.7 g/L of ethanol (with ethanol productivity 30 mg/g of biomass/hr and yield 132 mg/g of consumed glycerol) from pure glycerol and up to 3.55 g/L of ethanol (with ethanol productivity 11.6 mg/g of biomass/hr and yield 72.3 mg/g of consumed glycerol) from crude glycerol as a carbon source, which is approximately 15 times more relative to that of the O. polymorpha wild-type strain and 2.2 more relative to the earlier constructed strain.     

Transient responses of Candida utilis to oxygen limitation: Regulation of the Kluyver effect for maltose

The facultatively fermentative yeast Candida utilis exhibits the Kluyver effect for maltose: this disaccharide is respired and assimilated but, in contrast to glucose, it cannot be fermented. To study the mechanism of the Kluyver effect, metabolic responses of C. utilis to a transition from aerobic, sugar-limited growth to oxygen-limited conditions were studied in chemostat cultures. Unexpectedly, the initial response of maltose-grown cultures to oxygen limitation was very similar to that of glucose-grown cultures. In both cases, alcoholic fermentation occurred after a lag phase of 1 h, during which glycerol, pyruvate and D-lactate were the main fermentation products. After ca. 10 h the behaviour of the maltose- and glucose-grown cultures diverged: ethanol disappeared from the maltose-grown cultures, whereas fermentation continued in steady-state, oxygen-limited cultures grown on glucose. The disappearance of alcoholic fermentation in oxygen-limited chemostat cultures growing on maltose was not due to a repression of the synthesis of pyruvate decarboxylase and alcohol dehydrogenase. The results demonstrate that the Kluyver effect for maltose in C. utilis does not reflect an intrinsic inability of this yeast to ferment maltose, but is caused by a regulatory phenomenon that affects a key enzyme in maltose metabolism, probably the maltose carrier. The observed kinetics indicate that this regulation occurs at the level of enzyme synthesis rather than via modification of existing enzyme activity.     

影响果酒酵母甘油产量主要因素的研究

发酵果酒时,酵母生成甘油的量受多种因素的影响。试验选取6种果酒酵母,研究了酵母菌株(Y1、Y2、Y3、Y4、Y5、Y6)、发酵温度(15℃和20℃)及添加铵态氮源(100 mg/L(NH4)2HPO4与不同剂量的SO2(100mg/L和150 mg/L)对苹果酒中甘油产量的影响。同时研究了这6种酵母发酵不同果汁(苹果汁和梨汁)的甘油产量的不同。试验结果表明:(1)不同酵母发酵苹果酒的甘油含量具有极显著差异(P>0.01),6种酵母于15℃下发酵苹果酒的甘油含量为(4.42±0.02)-(6.18±0.07)g/L;(2)6种酵母于20℃发酵的苹果酒的甘油含量均高于15℃,但Y2甘油产量差异不显著(P2>0.05);(3)添加100 mg/L的(NH4)2HPO4,Y1、Y2、Y3和Y4发酵苹果酒的甘油含量较对照显著增加(P<0.05),Y5与对照相比显著降低(P5<0.05),Y6与对照相比降低不显著(P6=0.312>0.05);(4)与100 mg/L的SO2添加量相比,添加150 mg/L的SO2,Y2和Y4发酵的苹果酒的甘油含量显著增加(P<0.05),Y1、Y3和Y6增加不显著(P>0.05),而Y5显著降低(P5<0.05);(5)梨汁不添加氮源,添加100 mg/L的SO2后接种6种酵母于15℃下发酵梨酒的甘油含量均显著高于同条件下发酵的苹果酒甘油含量(P<0.01),其中以Y2发酵的苹果酒甘油含量最高,Y4最低,而以Y6发酵的梨酒甘油含量最高,Y4最低。     

Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol. Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+). Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+). Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.     

Dissimilation of organic acids by dairy lactic acid bacteria

One hundred and eleven different strains of lactic acid bacteria, belonging to the genera Leuconostoc, Streptococcus and Lactobacillus, were examined for their ability to degrade 10 organic acids by detecting CO₂ production, using the conventional Durham tube method. All the strains did not break down succinate, glutarate, 2-oxoglutarate, or mucate. Malate, fumarate, citrate, gluconate, tartrate and pyruvate were variably attacked. A malo-lactic fermentation was brought about by two thirds of S. cremoris and S. lactis strains as well as L. casei but not by L. buchneri or L. brevis. One third of the lactic streptococci examined, i.e. S. cremoris AM₂, ML₈ and SK₁₁ were capable of diacetyl production. They required glucose for cleavage of citrate, whereas Leuconostoc citrovorum and S.faecalis did not. S. cremoris differed from S. lactis in not producing CO₂ from gluconate. From all the lactic acid bacteria examined, only L. plantarum dissimilated tartrate. Production of acetoin and diacetyl is a more reliable evidence for assessing the degradation of pyruvate, compared to detection of evolved CO₂. Facultatively heterofermentative lactobacilli and Leuconostoc citrovorum produced acetoin and diacetyl from pyruvate, whereas the obligately heterofermentative lactobacilli did not, a character that would be of taxonomic value. Glucose fermented by both the facultatively and obligately heterofermentative lactobacilli did not prove to be a metabolic source of acetoin and diacetyl. The facultative heterofermenters degraded pyruvate in the presence of glucose with lactate as the major product together with a mean of acetate of 4.1%, ethanol of 7.9%, acetoin of 1.7% and diacetyl of 2.6% yield on a molar basis after 60 days at 30°C. L. brevis produced acetate and lactate. The role of S. cremoris and the facultatively and obligately heterofermentative lactobacilli in flavour development during the ripening of many cheese varieties has been confirmed.     

不同发酵条件对酿酒酵母甘油产量的影响

为提高酿酒酵母的甘油产量,分别考察不同初始葡萄糖和果糖质量浓度、pH值、发酵温度及SO2添加量对酿酒酵母D254甘油产量的影响。对酿酒酵母D254不同发酵初始条件进行单因素试验,其他因素固定条件下,葡萄糖质量浓度180g/L时酵母菌体生长平稳、生长量最高;果糖质量浓度108g/L时酵母甘油产量最高;pH值为3.5更适宜酵母菌体生长和合成甘油;在发酵温度和SO2添加量的单因素试验中也分别得出适宜发酵温度为28℃和适宜SO2添加量为 20mg/L。通过单因素试验,筛选出最利于酿酒酵母D254生长和产甘油的各因素的最佳质量浓度,进行Plackett-Burman发酵条件组合试验,得到发酵条件最佳组合为：初始葡萄糖质量浓度216g/L、果糖质量浓度144g/L、发酵温度32℃、pH 3.0、SO2添加量40mg/L,此条件下,酿酒酵母D254获得最高甘油产量达655.64μmol/L。     

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose-limited chemostat cultures (D=0·05–0·40 h⁻¹) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose-grown shake-flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake-flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady-state chemostat cultures (D=0·10 h⁻¹) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose-limited chemostat cultures (D=0·10 h⁻¹) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady-state growth at μ=0·10 h⁻¹ to growth at μ_max. It is concluded that in K. lactis, as in other Crabtree-negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation. © 1998 John Wiley & Sons, Ltd.