Stuck and Sluggish Fermentations

Transcription

1 Stuck and Sluggish Fermentations LINDA F. BISSON* Premature arrest of fermentation constitutes one of the most challenging problems in wine production. The causes of stuck and sluggish fermentations are numerous, troublesome to diagnose, and difficult to rectify. It has become well-established that fermentation rate decreases due to a targeted loss of hexose transport capacity. Factors which have been correlated with incomplete fermentations also regulate transporter expression, turnover and function. Several causes of slow and incomplete fermentations, including ethanol toxicity, have been well-characterized under enological conditions as described herein. Other potential factors that may impact cell growth, viability, and fermentative activity have not been sufficiently evaluated. The role of these factors in problematic enological fermentations is discussed. KEY WORDS: fermentation, stuck fermentation, ethanol toxicity, hexose transport capacity Slow and incomplete alcoholic fermentations are a chronic problem for the wine industry. Incomplete or "stuck" enological fermentations are defined as those leaving a higher than desired residual sugar content in the wine at the end of the alcoholic fermentation. A residual sugar level of less than 0.4% (4 g/l, glucose + fructose) is often considered by winemakers to indicate a complete or "dry" fermentation, but final sugar concentrations are typically below 0.2% at the end of yeast fermentation. A slow or sluggish fermentation is one that requires a longer than average time to reach a low residual sugar content. A routine fermentation will usually go dry within 7 to 10 days (depending upon temperature), while a sluggish fermentation will take considerably longer, perhaps even months, to complete. Sluggish consumption of sugar by the yeast is commonly an indicator of adverse environmental or physiological conditions, and such fermentations may eventually become stuck at a high residual sugar concentration. Stuck fermentations are a serious problem because wines with a high post-fermentation sugar content are very susceptible to microbial spoilage and cannot be bottled until it is known that they are microbially stable. Slow and incomplete fermentations may require special antioxidation operations because of the loss of the protective carbon dioxide blanket of an active fermentation. In addition, such wines tie up fermentor space and may be stylistically unacceptable. Fermentation rate is a function of both the total viable biomass and rate of sugar utilization of the individual cells [91]. Grape juice factors which limit growth or lead to cell death will reduce viable biomass and cause a decrease in sugar utilization that can result in a stuck fermentation. A sluggish fermentation may occur if the rate of fermentation per cell decreases even *Professor and Maynard A. Amerine Endowed Chair, Department of Viticulture and Enology, University of California, Davis, CA [ <lfbisson@ucdavis.edu>; Fax: (530) ]. Acknowledgements: I would like to thank Ralph Kunkee, Rich Morenzoni, Roger Boulton, Nancy Irelan, Christian Butzke, David Block, David Mills, and Laura Lange for critical reading of the manuscript and for sharing unpublished observations. I would also like to thank the reviewers of this manuscript, Drs. Gafner and Sponholz, for their very helpful comments. Finally, I would like to thank the reviewers of an AVF grant on this topic who read an earlier version of this review. Their critical suggestions greatly improved the final version. Manuscript submitted for publication 9 July 1998; revised 18 December Copyright 1999 by the American Society for Enology and Viticulture. All rights reserved. though viable biomass remains high. Numerous factors have been identified that impact cell viability and yield [2,16,73]. Conditions which affect fermentation rate of individual cells have also been identified [16,73] and partially overlap with those factors impacting viability and biomass yield. The circumstances yielding stuck and sluggish fermentations can frequently be alleviated thus allowing the fermentation to go to dryness. However, early and accurate diagnosis of the cause of fermentation arrest are critical in order to eliminate correctly the specific stress affecting the yeast in a timely fashion [16,73]. Once cells lose viability or permanently adapt to the adverse conditions of the environment by reducing rate of sugar consumption, it is very difficult to restore the rate of fermentation. Currently, a slow or incomplete fermentation is not recognizable until the rate of sugar consumption has been observed to decrease. By the time the rate has dramatically slowed, it is often too late to modify the adverse conditions or prevent the stress-induced fermentation arrest of the yeast. Continued incubation of the cells under conditions of stress generally leads to loss of viability and death of the culture. We have noted that re-initiation of fermentations that contain a large population of nonviable cells is particularly challenging [16]. A major impediment to the rapid identification, correct diagnosis, and treatment of a stuck or sluggish fermentation is a lack of basic information on the biology of yeast during grape juice fermentation. A significant amount of information is known about the metabolism, physiology, cell biology, and adaptation to stress in Saccharomyces cerevisiae under laboratory conditions, but this organism has been less thoroughly characterized in the natural environment of grape juice. Unfortunately, laboratory conditions do not mimic the natural environments of yeast. Therefore, the application of this wealth of information is somewhat limited. There has been a long series of important studies on control of fermentations in grape juice [see 5 and references therein], but these studies, while helpful in many aspects, were by necessity empirical in nature and made without benefit of our current knowledge of the intermediary metabolism and molecular genetics of 107

2 BISSON yeast. However, with the completion of the genome sequencing project for Saccharomyces and the development of new technologies for the analysis of whole genome and protein expression [38,124,129,136], it is now possible to thoroughly investigate yeast biology under enological conditions. Metabolic Basis of Stuck and Sluggish Fermentations The metabolic basis of stuck and sluggish fermentations has been fairly well established. The decrease in rate of sugar consumption is correlated with a decrease in sugar uptake capacity [16,74,85,125,128] while the rest of the glycolytic pathway remains intact and fully active. Analysis of an isogenic set of industrial strains of Saccharomyces differing only in ploidy, demonstrated that plasma membrane transport capacity, and not level of internal enzymes, dictates the overall CO 2 production rate [126]. Given the potential toxicity of intracellular free glucose which would occur if a downstream metabolic step of glycolysis were decreased instead [12,16,144], the targeted loss of transport capacity represents an important survival mechanism for the cells. Glucose and fructose consumption are reduced in response to various environmental or cellular conditions of stress. Nutrient limitation (macronutrient and micronutrient), low ph, lack of oxygen, lack of adequate agitation, temperature extremes, presence of toxic substances, presence of other microorganisms, imbalance of cations, and poor strain tolerances (particularly to ethanol or acetaldehyde), which have all been associated with stuck and sluggish fermentations [for review see 2,16,47,73], impact glucose and fructose transporter expression and activity [12]. Yeast balance the rate of entry of sugar into the cell with the rate of catabolism [10]. The overall rate of flux through glycolysis is equivalent to the rate of flux through the transport step [69,85,97,126,128]. A few years ago this was thought to be a consequence of transport being inherently slow as compared to phosphorylation and subsequent metabolism. However, it is now generally accepted that transport processes are not simply slow in a biochemical sense but are exquisitely regulated so as to match, but not exceed, the metabolic needs of the cell [12,14,74,144]. The process of sugar uptake in Saccharomyces has been extensively investigated in the last decade [for review see 12,14,16,67,74], and has been examined under enological conditions [69,85,86,87,125,126, 128,131,132]. Saccharomyces possesses a multigene family of transporters, called the HXT genes [12,14,67,68]. The "HXT" designation stands for hexose transporter [68]. There are 18 members of the HXT family (HXT1 through HXT17 and GAL2), which display a high identity in coding sequence (65% - 99% identical), share common functional motifs and predicted secondary structure [12,14,67,74]. These proteins share the same structural features (12 membrane spanning domains) and conserved residues of members of the Major Facilitator Superfamily [14,74,94]. This family includes transporters from animals and plants in addition to fungi and bacteria, reflecting a high degree of conservation of the structure and function of proteins that translocate sugars across lipid bilayers. Members of this family have been unequivocally shown to translocate glucose across membranes with catabolically relevant kinetics [12,14,114,115]. In addition, the members of the HXT family display differences in regulation at both the transcriptional and posttranslational levels [12,14,64,68,76,102,103,114,115,155,156]. HXT1 and HXT3 encode low affinity transporters (K m mm) while HXT6, HXT7, and GAL2 are high affinity glucose transporters (K = 1-2 mm) [114]. HXT2 and HXT4 have been shown to possess a moderate low affinity (K = 10 mm) [114]. The HXT2 transporter, Hxt2p, apparently can exist in either a low and high affinity state, suggesting that environmental conditions can modify the kinetics of this protein [114]. A kinetic analysis of the remainder of the members of the HXT family has not been reported. Deletion of the HXT2 gene has been shown to result in a sluggish fermentation confirming the critical role of transport in glucose utilization [69]. Related genes, SNF3 and RGT2, have been shown to encode regulatory proteins involved in the detection of glucose and are not predicted to play a direct role in transport for catabolism [12,14,28,77,100,149]. Snf3p functions as a low glucose sensor while the related protein, Rgt2p is a sensor of high glucose concentrations [28,77,100,149]. Both of these proteins are members of the Major Facilitator Superfamily [12,14,74] and regulate expression of members of the HXT family [28,77,100,149]. Other transporter-like proteins have been identified [94], with as yet unknown functions. One of the key factors regulating HXT gene expression is glucose itself [12,102,155,156]. Transporter genes are regulated by both glucose induction and glucose repression at the transcriptional level. Genes regulated by glucose induction are not expressed in the absence of glucose in the medium whereas glucose repressed genes are not expressed at high glucose concentrations, becoming derepressed upon glucose exhaustion. Some genes, like HXT1, are induced by high levels of glucose, while others, HXT2 and HXT4, are repressed by high glucose concentrations. Thus, HXT1 will not be expressed if external glucose levels are too low, while the moderate affinity transporters, HXT2 and HXT4 will not be expressed on high concentrations of glucose. The HXT2 gene represents a special case as this gene is both glucose repressed and glucose induced [102,156]. This pattern of regulation allows for HXT2 expression only under conditions of low glucose. Glucose repression of these genes is mediated by the general glucose repression pathway [156]. Glucose induction involves the removal of a negative transcriptional factor, called Rgtlp, which is mediated by SNF3 under low glucose and RGT2 under high glucose conditions [100,101]. Other factors such as permissive growth conditions, also regulate HXT gene expression [76,155, 156]. We have observed that the level of nitrogen in the medium impacts HXT1 expression and can override

3 STUCK and SLUGGISH FERMENTATION m 109 glucose control [N. M. Fong and L. F. Bisson, unpublished observations]. Further, HXT2 is expressed during enological fermentations under conditions of a normally repressing sugar concentration [P. Vagnoli and L. F. Bisson, unpublished observations]. Other yeasts that have been evaluated have far fewer transporter genes, frequently no more than three [14]. Why, then, are there so many transporters in Saccharomyces? Several explanations have been proposed for the existence of a multigene family of transporters in Saccharomyces. The transport of glucose and fructose in this yeast occurs by facilitated diffusion, meaning that no expenditure of energy is involved, the process is not concentrative, and accumulated free sugar can exit via the same transporter molecules [26,27,49,54,134]. Facilitated diffusion systems are optimally operational only over a narrow substrate concentration range. This is a consequence of two phenomena: the inherent kinetics of the transport process and substrate inhibition [14,86,87,93]. Translocation of a sugar molecule into the cell requires first binding and then recognition of the sugar by the transporter. As a result the transporter must possess a mechanism for substrate identification. Transporters that operate at low substrate concentrations (the high affinity transporters) are thought to have an open structure with more than one substrate recognition or binding site. These proteins are capable of recognizing their substrate from more than one position, meaning that the sugar molecule can associate with the transporter regardless of the orientation of its approach. For example, the molecule could be recognized whether it approached from the carbon 6 or 1 position via a specific binding event to different regions of the transporter. Once the sugar substrate is bound and recognized as correct there is a conformational change, and the transporter adopts an inward or cytoplasmic facing position with subsequent release of the sugar into the cell. This controlled movement of the protein requires a certain amount of plasma membrane fluidity. The membrane lipid composition must allow the conformational change of the transporter protein, but also restrict it so that the empty carrier can readily undergo a second conformational change to again face the outside of the cell. Thus, proper membrane fluidity is critical to maintenance of fermentation rate. It has been assumed that hexose transporter activity is immune to the disruptive effects of ethanol because transport activity is maintained in the presence of ethanol, but this assumption bears further scientific scrutiny. Some members of the HXT family may be designed to work in an ethanol environment, while others might not. At high substrate concentrations, transporters with multiple substrate recognition sites are subject to substrate inhibition. Substrate inhibition occurs when multiple substrate molecules attempt to simultaneously bind to the transporter [93]. In effect this jams the transporter which prevents the requisite conformational change and therefore no sugar is translocated into the cell. Under these conditions, the overall rate of sugar uptake decreases [86,87,93]. At high sugar concentration transporters with a relatively closed structure are required. These transporters are thought to have a single substrate binding site and to be less susceptible to substrate inhibition. Because the transporter is less accessible to the substrate, these transporters display a high K m (the low affinity transporters). As a consequence they are not effective as substrate concentration drops below their K m value. Thus, one reason for the large hexose transporter family is the need to shift from situations of very high substrate concentration to low, while maintaining flux rates into the cell. The strategy of coupling HXT gene expression to external glucose concentrations assures that transporters with a proper affinity and sensitivity to substrate inhibition will be expressed at the appropriate time during fermentation. A second reason for the existence of multiple protein species catalyzing the same catabolic step concerns the speed of adaptation to changing conditions that such an arrangement allows. We have found that multiple transporters are expressed simultaneously by the cells, and transport should be viewed as the summation of these independent activities, which we have termed the "consortium hypothesis" [12]. If it becomes necessary to partially reduce the uptake capacity, this is more easily achieved by eliminating one transporter protein species in its entirety rather than attempting to partially down regulate a single species. For example, if three transporters are all simultaneously expressed and contribute equally to uptake and the cell needs to reduce uptake capacity by a third, it is easier to eliminate one of the species entirely than to fractionally reduce the activity of each protein. In Saccharomyces transport is the main site of control of the rate of sugar fermentation [10]. When environmental conditions become adverse, fermentation rate is decreased due to the specific degradation of the HXT transporters [16,18,75,85,125,128]. We have shown that this degradation occurs in the vacuole and that the HXT proteins are internalized by a unique endocytotic mechanism as has been observed for the galactose and maltose transporters [116; J. S. Mazer and L. F. Bisson, unpublished observations]. Blocking proteolysis of the transporter results in cell death. Thus, continued glucose catabolism under adverse conditions is itself toxic to the cells. While this explains the physiological response of transporter degradation, it means that it will not be feasible to prevent stuck fermentations by simply blocking transporter turnover. Thus, it is instead necessary to identify and correct the exact cause of the adverse condition resulting in transporter loss. It is generally concluded from competition studies that fructose and glucose share the same transporters, meaning that fructose competitively inhibits glucose uptake [26,27,54,134]. The K for fructose is generally m 2.5 to 5 fold greater than the K for glucose, but the V m max of transport of fructose is generally higher than that for

4 BISSON glucose [24,26,27,54,134]. The lower affinity for fructose is thought to be due to the fact that glucose and other sugars are transported in the pyranose rather than the furanose form [27,54,74]. Roughly 30% of the fructose present in solution is in the furanose form, meaning that the actual transport-competent concentration of fructose is below the total concentration [27]. From the difference in kinetics, it is predicted that glucose will be consumed at a faster rate changing the environmental ratio of glucose to fructose from 1:1. As an adaptation to this alteration in relative substrate concentrations, the hexokinase isozyme expressed switches from hexokinase II, which displays an equal reaction rate for both sugars, to hexokinase I which displays a faster reaction rate against fructose, (glucose to fructose ratio of 1:3) [reviewed in 51]. This switch allows the cell to compensate for the change in external glucose to fructose ratio and maintain glycolytic flux. It has recently been suggested that low-level expression of Hexokinase I is associated with strains prone to sluggish fermentations [132]. The substrate specificity of all of the members of the HXT transporter family has not been evaluated in detail. It is certainly possible that a similar change to transporters with a higher affinity towards fructose would have to occur to match the change in hexokinase species in order to increase the rate of flux of fructose into the cell. Some of the HXTs may in fact be "fructophilic" in order to maintain metabolic rates. The central role of transporter activity in the regulation of fermentation rate is thus well established. Loss of transporter activity has been definitively associated with stuck and sluggish enological fermentations in several laboratories [16,18,69,85,125,128]. This loss of activity appears to represent an important survival mechanism preventing the uptake of potentially toxic sugar levels. More detailed information is needed on the factors regulating expression of each of the 18 HXT genes under enological conditions to more clearly define the role of these genes in maintenance of fermentation rate. Factors Affecting Fermentation Rate Several factors have been shown to impact fermentation rate and lead to sluggish or stuck fermentations: nutrient limitation, ethanol toxicity, organic and fatty acid toxicity, presence of killer factors or other microbially-produced toxins, cation imbalance, temperature extremes, pesticide, and fungicide residues, microbial competition and poor enological practices (excessive SO 2 use; excessive must clarification; lack of agitation; lack of attention to temperature) [2,16,47,73]. These factors can interact synergistically, and in combination may be far more inhibitory than any factor individually. For example, the minimum and maximum temperatures of growth are altered by the presence of ethanol, organic acids and fatty acids [79,121,123]. The minimum temperature supporting growth of Saccharomyces is quite ethanol sensitive, and may be elevated to as high as 27 C, depending upon conditions [123]. Tolerance to both ethanol and temperature is very strain dependent. In addition, cells grown at high temperature are susceptible to inhibition by oxygen exposure under non-growing conditions [33]. Thus, correct diagnosis of the cause of a stuck fermentation necessitates consideration of possible synergistic effects in addition to a single individual cause. Nutrient limitation: The best characterized of the conditions leading to stuck and sluggish fermentations is nutrient limitation [2,16,47,53,56,58,73]. Nutritional requirements can be classified as those needed for proliferation and those required for maintenance of metabolically active non-proliferative or stationary phase. These two categories are not mutually exclusive as the same nutrients may be required for both phases. Stationary phase nutrition has not been as well studied as growth nutrition. Nutrients required for periods of non-growth include an energy source, ample amino acids for synthesis of actively degraded proteins, and compounds required to minimize the inhibitory effects of ethanol. The latter class of compounds have been termed "survival factors" [72,73], and include long chain saturated and unsaturated fatty acids and ergosterol. It is important to note that in addition to disruptive effects of plasma membrane activity, ethanol can also disrupt non-membrane cytoplasmic functions through the dielectric disorder of the aqueous phase of proteins [59,60,61]. Several compounds have been suggested to be important in protecting internal processes from ethanol: trehalose, proline and glycine [82,83,147]. Trehalose is made from glucose and proline is plentiful in grape juice, so these two compounds would be predicted to be in excess during grape juice fermentation. In contrast, glycine levels are quite low. Thomas et al. [147] reported a synergistic effect of proline and glycine in fermentation of high gravity media. In light of these findings, the impact of limiting glycine on grape must fermentation, particularly of musts with a high sugar content as would occur with late harvest or Botrytis infection, warrants investigation. The two macronutrients most frequently implicated as causes of stuck fermentations when present in insufficient quantities are nitrogen and phosphate [2,16,47,53,73]. Lack of micronutrient vitamins and minerals have also been shown to limit fermentation rate, as has a deficiency of oxygen. Depending upon the nature and severity of the limitation, starvation for a required nutrient can reduce the rate of cell growth, limit maximal biomass production, decrease viability and affect the fermentation rate of individual cells. In contrast to many other proteins, the sugar transporters maintain high rates of turnover in stationary phase cells [14,15,85,125,128]. Thus, a supply of nitrogen must be available to allow continued resynthesis of these proteins. Since a high ethanol concentration inhibits the translocation of amino acids and other nitrogen sources [146], the nitrogen must be available early in fermentation and stored in the vacuole for later use

5 STUCK and SLUGGISH FERMENTATION- 111 [16]. It has recently been shown that supplementation of stationary phase fermentations with specific amino acids prolongs maximal fermentative activity [82]. Previous work suggests that ammonium does not have this effect [73]. Amino acid additions may enhance the ability to synthesize rapidly degraded proteins such as the glucose transporters. Some amino acids were far more effective than others and than a mixture of amino acids [82]. The effectiveness of the amino acids appeared unrelated to their utility as nitrogen sources supporting growth. One of the most effective amino acids was glycine [82], a very poor nitrogen source for Saccharomyces. This is consistent, however, with reports of the stimulatory value of glycine supplementation in very high gravity fermentations [147]. The nitrogen requirement for maintenance of stationary phase fermentation was shown to differ dramatically by strain, much more so than nitrogen requirements during growth [81]. It has also recently been shown that high ammonium content of juice may inhibit efficient utilization and biomass formation of wine strains under enological conditions [127]. Thus, nitrogen-containing compounds must be in balance for optimal utilization and growth. Numerous studies have underscored the importance of proper nitrogen nutrition for completion of fermentation under enological conditions [1,4,11,53,58,90,91, 119,120,130]. Phosphate limitation has also been shown to impact cell growth and biomass yield as well as directly affecting fermentation rate [16,51,73]. Ample phosphate must be present to maintain cellular pools of Pi, ADP and ATP to drive glycolysis. Imbalances in the AMP/ATP or ADP/ATP ratios have been suggested to play a key role in regulation of glycolytic flux [51]. Hexose transport might also be responsive to this ratio either directly or via the inhibition of glycolysis. Mutational loss of one of the irreversible steps of glycolysis, hexose phosphorylation, phosphofructokinase or pyruvate kinase, results in greatly diminished glucose uptake [12]. Deficiencies and imbalances in minerals and cations can result in reduced fermentation rates [13,39,153]. In fact, treatment of grape juice with metal chelating resins has been proposed as a means to prevent microbial spoilage and fermentation [44]. Minerals serve as cofactors for glycolytic and other enzymatic reactions and limitations of minerals such as zinc and magnesium directly affect sugar catabolism. Calcium limitation increases ethanol sensitivity [92]. High manganese depresses uptake of magnesium and vice versa [13], so a disparity in these ions will lead to a deficiency situation. We have also recently shown that an imbalance of ph and potassium ions can lead to a stuck fermentation in model juice-like media, and such irabalances may be present in grapes from vines that display poor potassium uptake from soil [70]. Saccharomyces is capable of making all essential vitamins except biotin, but research has shown that the presence of other vitamins is highly stimulatory to growth and fermentation [47,73,90,99]. It has recently been demonstrated that Kloeckera apiculata is quite efficient at stripping thiamine from grape juice in a matter of hours, thus leading to a deficient situation for Saccharomyces [9]. The presence of acetic acid has been reported to reduce the ability of Saccharomyces to transport and retain thiamine [57]. Thus, one likely cause of stuck fermentations arising from uninoculated juices is the depletion of thiamine by wild yeasts with the production of acids that inhibit transport of the residual vitamin by Saccharomyces. Sulfur dioxide reacts directly with thiamine, reducing the level of this vitamin. Excessive use of SO 2 may also lead to a deftciency situation, especially if it follows cold settling. This is another example of the synergistic interaction of factors that can lead to poor fermentation performance. Depletion of thiamine may also pose a problem for inoculated fermentations as well [73], especially under conditions favoring the growth of Kloeckera. It is important to note that the nutritional requirements of yeast during grape juice fermentation may be influenced by inhibitory substances present in the medium. Plants produce several inhibitory compounds, the phytoalexins, in response to fungal invasion [78,138]. One class of these compounds is derived from the amino acid phenylalanine [78,138]. It is not surprising, therefore, that one of the groups of mycotoxins produced by the molds during plant infection are inhibitors of phenylalanine biosynthesis and mobilization [66]. Saccharomyces has been reported to be insensitive to these mycotoxins [80], but that analysis was conducted on rich media where amino acids were plentiful. It is possible that Saccharomyces may be impacted by these mycotoxins if phenylalanine levels in the must are low. Indeed, a correlation between fermentation arrest and phenylalanine deficiency has been observed [16; R. B. Boulton, personal communication], which was not able to be replicated in defined media studies. It is possible that the phenylalanine requirement arose because of the presence of mold toxins in the juice. Alternatively, phenylalanine limitation may impact the cells ability to communicate at high density (called quorum sensing [see 50]). We have observed that cells of Saccharomyces at high densities produce relatively large amounts of phenethyl alcohol, which is derived from phenylalanine. It is possible that this compound functions in quorum communication and the inability to produce it may lead to senescence of the culture. Very high levels of phenethyl alcohol (1.5 g/l) have been shown to inhibit a variety of cellular functions in Saccharomyces [8,17]. While the inhibitory concentrations are well above those found in wine (trace to 50 mg/l) [5], it is possible that lower concentrations play a role in the correct entry into the non-proliferative state (see discussion below). Ethanol toxicity and the role of survival factors: Another major cause of stuck fermentations is ethanol toxicity [23,31,59,145,146]. Ethanol appears to impact plasma membrane fluidity in a complex fashion. Ethanol decreases polarity in aqueous surroundings

6 BISSON but increases it in hydrophobic environments [25]. The effect of ethanol on plasma membrane functions, and whether a decrease or increase in fluidity is observed, depends upon the conditions of the assay and the membrane component evaluated [3,23,25,59]. The cell responds to ethanol by producing a membrane rich in unsaturated fatty acids and ergosterol [3,25,35, 59,84,145,148,150]. Unsaturated fatty acid content increases approximately two-fold, while ergosterol levels increase 18-fold [25]. The content of saturated long chain fatty acids also increases [25]. In addition to an increase in unsaturated fatty acids and sterols, the protein content is reduced ethanol-tolerant membranes. The changes in plasma membrane composition have been proposed to alter the plasma membrane fluidity to compensate for the disruptive effects of ethanol on fluidity. However, this conclusion has recently been questioned [59,60,61]. Ethanol is a polar molecule and is not found in high concentrations in nonpolar environments as would occur inside of the lipid bilayer. Instead, it has been suggested that ethanol impacts membrane function by polar, dielectric, and hydrogen bond interactions with the polar head groups of the phospholipids and integral membrane proteins rather than changing plasma membrane fluidity [59,60,61]. The changes in fatty acid desaturation, decrease in protein content and increase in ergosterol level are proposed to occur to counter the disruptive effects of ethanol on phospholipid head groups and proteins, rather than to alter plasma membrane fluidity [59,60,61]. Ethanol sensitivity is a variable property among strains of Saccharomyces. It is also impacted by the availability of sterols and long-chain saturated and unsaturated fatty acids in the medium as these compounds cannot be readily synthesized by the yeast under anaerobic conditions [6,25,30,32,72,73,148,150]. Both fatty acid desaturase and squalene oxidase require molecular oxygen as electron acceptor. Grape must contains unsaturated fatty acids and plant sterols which can be utilized by yeast. Previous work demonstrated that the major grape sterol, oleanolic acid [109,110], could not replace the ergosterol requirement [73,148]. This finding is not surprising since oleanolic acid would not be predicted to supplant the yeast sterol based upon analysis of the structural requirements for the compound [95,96,108]. We evaluated the effect of other plant sterols found in grape, stigmasterol, B-sitosterol, dihydrobrassica sterol, and campesterol. These plant sterols could only replace the so-called "bulk" or structural sterol requirement [95,96,108], but could not replace the regulatory role of ergosterol in control of protein and membrane function. They were not as effective as the yeast ergosterol in formation of an ethanol tolerant membrane and resulted in a more sluggish fermentation, but the fermentation did go to dryness [N. Peay and L. F. Bisson, unpublished observations]. If the grape juice is deficient in fatty acids, these components can be harvested from internal membranes such as those of the mitochondria [reviewed in 16], but this may result in loss of mitochondrial activity and ability to respire [16]. Recent work suggests that the "survival factors" may be critical in maintenance of fermentation rates in spite of the fact that glucose transporters have been reported to be relatively ethanol insensitive [K. Morrisey and L. F. Bisson, unpublished observations]. Further analysis of the factors involved in ethanol sensitivity and tolerance is clearly needed. Ethanol is believed to be toxic to cells because it increases the passive proton flux into the cell, placing stress on the cellular capacity to maintain ph homeostasis [22]. This leakage is thought to occur via the disruptive effects on ethanol on protein structure rather than through general affects on plasma membrane fluidity [59,60,61]. This finding is based upon the observation that passive flux of undissociated acids is not similarly affected by ethanol and membranes of high fluidity are not ethanol tolerant [59,60,61]. It has recently been suggested that the increase in passive proton flux may not play a major role in ethanol toxicity [23,118], since it appears that in ethanol challenge assays (ethanol added to a culture) cell death occurs prior to the appearance of an altered cytoplasmic ph. The impact of ethanol on the plasma membrane and disruption of protein function and accompanying leakage of cytoplasmic components may be more immediately toxic than the ph change of the cytoplasm [7,62,84,122,146]. Also, ethanol affects translocation of other ionic species, Ca 2 and Mg 2, which may be important in toxicity [39,92,153]. Cultures naturally accumulating ethanol rather than being challenged by it are able to adapt the protein composition of the membrane which may be as important of a factor in ethanol tolerance as alteration of the lipid composition [59]. Ethanol will also affect the function and stability of cytoplasmic enzymes [59,60], which may also play a role in toxicity. Ethanol may directly impact transporter function by affecting substrate recognition, binding or the dynamics of the conformational change that is required for substrate translocation into the cell. When considering the toxic effects of ethanol, it is also important to remember that acetaldehyde, the immediate precursor to ethanol in catabolism, is also toxic [139,140], and may be in large part responsible for "ethanol sensitivity" [59,60]. Since alcohol dehydrogenase is a freely reversible enzyme, acetaldehyde will accumulate in parallel to ethanol and is toxic at much lower concentrations [59,60,139,140]. Indeed, strain differences in ethanol tolerance has been associated with cellular levels of acetaldehyde; those strains with the lowest cytoplasmic acetaldehyde level being the most tolerant [59,60]. Low ph: Saccharomyces is tolerant to low ph fermentations and can readily grow in the juice ph range of 2.8 to 4.2 [16,47,52,73]. Below ph 2.8, both growth and fermentation are inhibited [16]. The ethanol, organic and fatty acid tolerances of many strains are reduced at very low ph values [105], and we have seen that the potassium concentration is a key factor in ph tolerance [70]. Saccharomyces excretes protons during fermentation and may reduce the ph of the medium by

7 STUCK and SLUGGISH FERMENTATION m 113 as much as 0.3 units [16]. As described below, ph will have a dramatic effect on the types of bacterial species present and their persistence, which may significantly impact fermentation progression. Extremes of temperature: Exposure to temperature extremes can also inhibit fermentation rates [45,135,142]. The plasma membrane is the main target of the inhibitory effects of high or low temperature [reviewed in 142,154]. Temperature affects membrane fluidity and, therefore, transporter performance. Lower temperatures reduce fluidity and restrict transporter conformational change while high temperatures increase fluidity and result in too great of a dissociation of transporter structure during the conformational change. Since ethanol and temperature target the same cellular function, it is not surprising that their effects are synergistic. A dramatic drop in temperature, as would occur at the end of vigorous fermentation with continued cooling of the tank, should be avoided when concerned with the prevention of stuck fermentations. Abrupt changes in temperature also affect cytoplasmic enzyme activity and organelle structure and function [142,154]. Heat shock leads to the induction of several stress related proteins, which may also be present upon entry into stationary phase [29]. The ability to respond to sudden temperature changes is dependent upon the ability to synthesize the heat shock proteins. If the temperature shock occurs under conditions of nutrient limitation of the yeast, the cells might not be able to compensate for the change in temperature. Zymostatic and zymocidal toxins: Several toxic substances have also been shown to lead to fermentation arrest [2,16,47,73]. Yeast can produce zymocidal substances known collectively as killer factors [reviewed in 157]. Non-Saccharomyces yeasts such as Hansenula and Kluyveromyces produce killer factors which are active against Saccharomyces [89]. Saccharomyces strains can also produce glycoprotein killer factors that are toxic to susceptible strains of Saccharomyces [21,111]. The effect of killer toxins is dependent upon medium composition, and the relative ratios of the sensitive to toxin-producing strains [88]. Conditions may impact killer factor production, activity or sensitivity of the susceptible strains. Bacteria may also produce substances toxic towards Saccharomyces. The bacterial toxin syringomycin produced by the soil bacterium Pseudomonas impacts plasma membrane K, H +, and Ca 2 fluxes in eucaryotic cells, and has been shown to inhibit yeast [143]. Bacillus and Streptomyces, also common soil organisms, have been shown to produce metabolites which limit yeast growth under enological conditions [63]. Both Streptomyces and Bacillus have been found as winery contaminants [16,47,48]. Streptomyces can grow quite well in filter matrices that have not been properly sanitized. However, such a situation can be readily detected due to the distinctive aroma produced by this organism. Molds present on the fruit at harvest may produce mycotoxins to which Saccharomyces is susceptible. It has been suggested that botrytized fruit contains toxic substances [73,117], but the nature of the substance has not been determined in spite of significant research efforts. Saccharomyces is in general resistant to many mycotoxins [80], which is one reason this organism is used in the production of fuel ethanol from mold-infested grains [65]. Saccharomyces was not found to be sensitive to mycotoxins during wort fermentation either [133] so it is unclear if mycotoxins in general would pose a problem under enological conditions. We have conducted vinifications of fruit heavily infested with mold post-harvest and have not noticed a correlation with slow or incomplete fermentations. However, as mentioned above, sensitivity to mycotoxins may be dependent upon the nutritional composition of the medium. A more likely problem caused by mold infestation of fruit is the production of compounds toxic to fungi by the plant when challenged with fungal infection. Plants produce numerous compounds (the phytoalexins) and enzymes (the pathogenesis-related proteins) in response to infection which are designed to eliminate the pathogen [78,138]. This same response might not occur in post-harvest infection. Toxic phenolic compounds, amino acid analogs, and enzymes capable of degradation of fungal cell walls (chitinases and glucanases) can all be produced in response to infection. The phytoalexins are broadly toxic and may even reduce viability of the plant cells producing them [138]. It is highly likely that some of these factors will also impact yeast growth and fermentation since the yeast are members of the same taxonomic family as the filamentous fungi and have a similar cell wall architecture. Organic and medium-chain fatty acids are also inhibitory to Saccharomyces [20,40,42,45,71,73, 112,113,151,152]. These compounds may be produced by bacteria and non-saccharomyces yeast, but they can also be formed by Saccharomyces [42,113]. Under normal fermentation conditions, the concentrations found are not inhibitory, but if mixed culture fermentations (Saccharomyces and non-saccharomyces yeast and bacteria) are conducted, the risk for the appearance of this type of inhibition is greater. As mentioned above, these acids are more toxic at high ethanol and extremes of temperature [152], and may impact vitamin absorption and retention [57] which may affect fermentation by Saccharomyces. Fungicides and pesticides used in the vineyard may negatively affect yeast viability if present at high enough residual concentrations at the time of harvest [16,73]. These compounds may cause an extended lag phase or might not lead to an immediate problem and sluggish fermentations not manifest until later in the fermentation. It has been suggested that high concentrations of heavy metal ions or use of components containing sodium salts may be inhibitory to fermentation and growth. We have not found these substances to have an impact at the levels normally found in juices and musts. However, they are indeed toxic at higher levels. Zymostatic toxins are those that inhibit cell growth

8 BlSSON and metabolic activity but are not immediately lethal. If such compounds are present, the fermentation can usually be re-initiated by blending with other juices or wines, assuming such a practice does not lead to further toxin production. Sometimes it is necessary to first remove the initial biomass prior to attempts to reinitiate fermentation or to treat the stuck wine with yeast ghosts [73]. However, unless it is clear that a toxin is involved and the nature of the toxin is known, this practice may simply result in a larger volume of stuck wine. Microbial incompatibility: Enological fermentations containing initial high populations of non-saccharomyces yeast and bacteria are at high risk for the development of stuck and sluggish fermentations [16,40,47,48,73]. This is due in part to the competition for nutrients and production of toxic substances as described above. However, other factors such as ~ high total cell density may also be important in the reduction of fermentation rate. We and others have found that mixed culture fermentations (Saccharomyces and bacteria) require higher than normal vitamin supplementation, and that the arrested fermentation may not be re-startable until the existing biomass is removed via racking or centrifugation [T. Rynders and L. F. Bisson, unpublished observations]. It is also important to note that there are incompatible pairings of wine yeast and malolactic (ML) bacteria. Some Saccharomyces strains are very susceptible to inhibition by the ML bacteria, while other strains are not. Yeasts inhibited by one bacterium are not necessarily inhibited by all strains of ML bacteria. To avoid this type of fermentation problem, the compatibility properties of the ML bacteria and yeast strains that the winemaker desires to use should be evaluated. It has recently been shown that a novel bacterium, Lactobacillus kunkeei, referred to as the "ferocious Lactobacillus", frequently causes stuck fermentations regardless of the yeast strain(s) present [41,43]. Other factors impact the appearance and persistence of the non-saccharomyces flora. Many bacterial species are unable to grow at the lower ph range found in wine, below 3.4, but will grow above this ph [16,73]. Wild yeasts such as Kloeckera apiculata (perfect form: Hanseniaspora uvarum), are as ph tolerant as Saccharomyces, but are more tolerant of low temperatures [47,52]. We and others [47] have found that holding of must or juice at a low temperature (cold maceration/ extraction for red musts or cold settling for white juices) allows the build up of Kloeckera populations. Fermentation at low temperature gives these organisms a competitive advantage over Saccharomyces. Kloeckera is far more ethanol tolerant at low temperature than at high temperatures. As mentioned above, these practices can lead to vitamin depletion of the juice or must [9]. Enological practices: Several enological practices can also be the cause of fermentation arrest [2,16,73]. Excessive clarification of musts reduces fermentation rate. This appears to be due to multiple factors: the loss of nutrients found in particulate matter such as unsaturated fatty acids and sterols [34,35,46, 55,73], the reduction of vitamins and mineral content via the removal of microbes that have sequestered these components [9], and a decrease in the natural agitation ability of the must [16]. It has also been suggested that must solids serve as nucleating sites for the release of CO 2, and may provide a solid surface upon which the yeast can form a biofilm. Excessive use of sulfur dioxide can also lead to poor fermentation performance, depending upon the ph of the medium and bound and free concentrations of the compound [16,141]. Winery practices such as the addition ofnutrients and aeration may positively impact fermentation performance and reduce the incidence of stuck and sluggish fermentations [2,16,73]. Finally, the type of temperature control employed may be a major contributing factor to fermentation problems. Too large or too small of a temperature differential between the coolant and the desired temperature of the must can result in a sluggish fermentation and even in death of the yeast [16]. The temperature of the must may not be uniform and may be either too low near the sides of the tanks or alternately too high in the center, depending upon heat transfer capability of the system employed [16]. "Warm" fermentations above 30 C should be avoided, as this could result in the rapid generation of an inhibitory temperature depending upon the fermentation rate of the strain and the heat transfer capacity of the system [discussed in 16]. Too much cooling at the later stages of fermentation can also cause fermentation arrest. Depending upon the concentration of ethanol and other components such as fatty and organic acids, the minimum temperature sustaining fermentation will be elevated and the maximum decreased. Late in fermentation, the temperature should be held around 22 C to 25 C (70 F - 78 F), depending upon the yeast strain. Yeast perform better with gradual temperature changes as that allows the cells to adapt properly to the new condition. Temperature swings in excess of 5 C (9 F - 10 F) should be avoided. Other factors: Several additional factors that may impact fermentation rate have been less well characterized. It has been suggested that a high concentration of fructose relative to glucose is inhibitory to yeast [131,132]. Fructose is not as reactive of a sugar as glucose, so the basis of this inhibition is unclear. The high residual concentrations of fructose may be a symptom rather than a cause of stuck and sluggish fermentations. Schfitz and Gafner [132] have suggested that poor utilization of fructose and a tendency to stick during fermentation are associated with a deficiency in level of hexokinase I activity. However, it has been shown that addition of fructose, and the accompanying change in the glucose to fructose ratio (GFR) can inhibit an ongoing fermentation [131] while addition of glucose to alter the GFR to less than 0.1 can stimulate fermentation activity [131]. It is not clear if this is due to substrate inhibition of the transport process or not. Initial high sugar concentrations can lead to a sluggish initiation of fermentation depending upon the affinity

9 STUCK and SLUGGISH FERMENTATION- 115 of the transporters expressed in the inoculum. Thehigh viscosity of some grape musts may inhibit fermentation. Musts from late harvest grapes with a high pectin content can display a very sluggish initiation of fermentation [L. F. Bisson, unpublished observations]. It has recently been suggested that high viscosity media may prevent loss of CO 2, elevating the carbon dioxide pressure of the medium to an inhibitory level [106,147]. High viscosity solutions generally decrease mixing and diffusion rates, which may result in slow growth and diminished fermentation rates. An area that has been largely ignored is the impact of grape must enzymes on the progression of fermentation. Polyphenol oxidase levels vary dramatically with the varietal and the season [16]. This enzyme competes directly with Saccharomyces for available dissolved oxygen [16]. It has been suggested that the real reason that SO 2 is able to stimulate fermentation by Saccharomyces lies in the inhibition of the competing polyphenol oxidase, making oxygen more readily available for Saccharomyces [16] and not in the inhibition of wild (non- Saccharomyces) yeast and bacteria. The ability to accumulate the disaccharide trehalose has also been shown to be important during fermentation [83]. Viability has been correlated with cellular trehalose levels, perhaps protecting yeast against both osmotic stress and ethanol toxicity. Inability to accumulate trehalose as a factor in sluggish and stuck fermentations has not been evaluated. Proline and glycine have also been demonstrated to have an osmoprotective effect [147], and deficiencies in these amino acids may likewise impact fermentation rates and cell viability. It has occasionally been noted by winemakers that certain vineyard locations yield fruit highly susceptible to sluggish fermentations, even when nutrient additions are made to the must. This effect may reflect a particularly high disease pressure of those vines and the resultant phytoalexin content of the juice, or alternately may suggest that these grapes support high wild flora populations. We have recently shown that phenolic compounds found in grape can stimulate or inhibit fermentation rates, depending upon the concentration and the specific compound [R. Hood and L. F. Bisson, unpublished observations]. The biological effects of grape phenolics on yeast have not been well-studied. Cantarelli reported both inhibitory and stimulatory effects on growth and fermentation rates [19]. Sluggish fermentations may arise due to a deficiency of stimulatory phenolics or to an excess of inhibitory ones. Differences in phenolic composition may be one reason that fruit from certain vineyard sites seems prone to fermentation problems. Finally, an area meriting further studies concerns the analysis of cell-cell communication during fermentation. It has been demonstrated that several compounds produced by yeast can signal developmental changes in other yeast in the vicinity. Ammonia pulses produced by neighboring colonies have been demon- strated to limit colony growth towards each other [104]. Carbon dioxide accumulation in a dense population induces sporulation of surrounding cells so that sexual reproduction might occur [98]. Higher alcohols (fusel oils) have been shown to bring about pseudohyphae formation in Saccharomyces at higher concentrations than typically found in wine [36]. Similar types of signaling may be occurring during fermentation, regulating growth and fermentative activity of the population. It has recently been reported in Schizosaccharomyces pombe that the multidrug resistance protein encoded by the fnxl gene plays a role in entry of cells into stationary or non-proliferative phase in response to nitrogen limitation [37]. Cells lacking fnxl fail to respond properly to nitrogen limitation, leading to loss of viability [37]. It was suggested that the fnxl protein releases a signaling molecule that is involved in cell-tocell communication of the deficiency, and that cells do not enter a quiescent phase without receiving both an internal and external nitrogen starvation signal [37]. Failure to properly enter the quiescent state leads to cell death. This observation has important implications, especially for reinitiation of stuck and sluggish fermentations. Saccharomyces possess a gene with significant homology to fnxl [37], and may produce a similar starvation factor affecting neighboring cells. If such a factor exists, it may explain the difficulty in restarting stuck fermentations, as the newly introduced yeast would also respond to these compounds. Reinitiation of nutrient-limited fermentations may require the removal or dilution of these quiescence factors. Settled (but not flocculant), populations of Saccharomyces are not as fermentatively active as dispersed populations and inactive populations quickly settle. Cell-to-cell communication in high density settled populations may differ from interactions in dispersed cultures. Saccharomyces has been shown to undergo apoptosis or programmed cell death typical of higher eucaryotes [137]. The senescence factors appear to be unrelated to those found in mammalian cells and conditions leading to early senescence have not been adequately described [137]. Apoptosis is a phenomenon associated with communal cells. Induction of death in a segment of the population would provide nutrients for the remaining viable cells increasing their chance of survival. Further analysis of yeast apoptosis may provide important information useful in the re-initiation of stuck fermentations. We have noticed that re-starting stuck fermentations may require removal of the existing biomass. Fermentations at full cell density that arrest may be difficult to restart due to the fact that the maximal tolerable culture density has been exceeded. It is highly likely that Saccharomyces will possess some type of cell density or "quorum" sensing ability as has been described in other microorganisms [reviewed in 50]. Conclusions Many factors have been shown to result in slow or incomplete enological fermentations. However, other

10 BISSON causes of fermentation arrest that have not been evaluated merit investigation. While the precise cause of a stuck and sluggish fermentation might not be currently discernible, the type of fermentation arrest can be quite useful at limiting the number of possibilities. Careful attention to numbers of viable Saccharomyces cells and judicious use of cooling and intelligent temperature maintenance in addition to prudent nutrient supplementation should greatly reduce the incidence of slow and incomplete fermentations. Three general areas merit further research: non-proliferative phase nutrition, the impact of plant phenolic compounds and phytoalexins on growth and fermentation rates, and determination of the factors involved in quorum and senescence communication among yeast populations. 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