APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1994, p. 1519-15240099-2240/94/$04.00+0Copyright C 1994, American Society for Microbiology

Effects of Particulate Materials and Osmoprotectants on

Very-High-Gravity Ethanolic Fermentationby Saccharomyces cerevisiae

K. C. THOMAS,* S. H. HYNES, AND W. M. INGLEDEWDepartment ofApplied Microbiology and Food Science, University of Saskatchewan, Saskatoon,

Saskatchewan, Canada S7N OWO

Received 9 December 1993/Accepted 2 March 1994

The effects of osmoprotectants (such as glycine betaine and proline) and particulate materials on thefermentation of very high concentrations of glucose by the brewing strain Saccharomyces cerevisiae (uvarum)NCYC 1324 were studied. The yeast growing at 20°C consumed only 15 g of the sugar per 100 ml from a minimalmedium which initially contained 35% (wt/vol) glucose. Supplementing the medium with a mixture of glycinebetaine, glycine, and proline increased the amount of sugar fermented to 30.5 g/100 ml. With suchsupplementation, the viability of the yeast cells was maintained above 805% throughout the fermentation, whileit dropped to less than 12% in the unsupplemented controls. Among single additives, glycine was more effectivethan proline or glycine betaine. On incubating the cultures for 10 days, the viability decreased to only 55% withglycine, while it dropped to 36 and 27%, respectively, with glycine betaine and proline. It is suggested thatglycine and proline, known to be poor nitrogen sources for growth, may serve directly or indirectly as

osmoprotectants. Nutrients such as tryptone, yeast extract, and a mixture of purine and pyrimidine basesincreased the sugar uptake and ethanol production but did not allow the population to maintain the high levelof cell viability. While only 43% of the sugar was fermented in unsupplemented medium, the presence ofparticulate materials such as wheat bran, wheat mash insolubles, alumina, and soy flour increased sugar

utilization to 68, 75, 81, and 82%, respectively.

Research in recent years has shown that the application ofvery-high-gravity (VHG) fermentation technology for indus-trial scale production of fuel alcohol is a distinct possibility(19). The term "gravity" (actually specific gravity) is commonlyused in the fermentation industry to indicate the dissolvedsolids content of the fermentation medium. The progress offermentation is usually monitored by measuring the specificgravity of the medium. VHG technology for fuel alcoholproduction is defined as "the preparation and fermentation tocompletion of mashes containing 27 or more grams of dis-solved solids per 100 g mash" (38). Saccharomyces cerevisiae,the yeast commonly used for ethanolic fermentation, is notvery tolerant to diminished water activity (13), yet at 20°C itcan completely ferment wheat mashes containing 38 to 39%(wt/vol) dissolved solids, yielding over 23% (vol/vol) ethanol(38). The amount of sugar this yeast can ferment in a definedmedium, however, is considerably less, even though all re-

quired nutrients are provided in adequate amounts. It has beensuggested that yeasts grown in synthetic media produce a

substance which inhibits their growth (17). However, theidentity of this substance has not been established. It is alsopossible that an unknown nonnutritional factor or factorsavailable in complex media such as grain mashes, brewer'swort, or grape juice play a role in stimulating yeast growth andfermentation in VHG media. The effect of such factors maynot be as apparent in low- or normal-gravity media since all ofthe sugars from these media are consumed by the yeast

* Corresponding author. Mailing address: Department of AppliedMicrobiology and Food Science, University of Saskatchewan, Saska-toon, Saskatchewan, Canada S7N OWO. Phone: (306) 966-8869. Fax:(306) 966-8898. Electronic mail address: THOMASK@SASK.US-ASK.CA.

irrespective of the presence or absence of the growth-promot-ing particulate materials.

It has been reported that the addition of insolubles such as

grape solids, bentonite, or diatomaceous earth to clarifiedgrape juice caused the fermentation to finish more rapidly andto a greater degree of completion (16). Alcoholic fermentationof grape juice has also been shown to be stimulated bymicrocrystalline cellulose and yeast cell wall preparations (27).Ethanol productivity from molasses was increased by theaddition of -y-alumina (18) or soy flour (43). Various particu-late materials such as trub, diatomaceous earth, activatedcarbon, pea flour, and Chromosorb W have been reported tostimulate yeast growth and increase the rate of sugar utilizationfrom brewer's worts (34).An important consideration in VHG fermentation is that the

yeast is subjected to considerable osmotic stress which reducesthe growth and increases the loss of cell viability (8, 28, 40).Successful VHG fermentation is therefore dependent on theyeast's ability to withstand increased osmotic stress and totolerate high concentrations of ethanol. The mechanism ofosmotolerance in S. cerevisiae has not been studied in detail. Itis known, however, that this nonosmotolerant yeast (1) synthe-sizes glycerol in response to increased salt concentrations inthe medium, whereas one or more polyols are synthesized as

compatible solutes by osmotolerant yeasts (7).Certain components of complex media appear to alleviate

the effects of osmotic stress on yeast growth and viability. Forexample, by increasing the concentration of peptone and yeastextract in the medium, a yeast strain's tolerance to osmoticstress and elevated temperature can be improved (36). In yeastextract, the active ingredient that plays a part in the osmoreg-ulation appears to be betaine (12). Dulaney et al. (12) isolatedand purified glycine betaine from yeast extract and showed thatit contained about 110 ,ug of betaine per g of yeast extract.

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1520 THOMAS ET AL.

They demonstrated that the amount of betaine present in yeastextract was sufficient to relieve inhibition of bacterial growth indefined medium of high osmolarity, although it had no effecton the performance of some osmotolerant yeasts (12).

Several low-molecular-weight organic compounds are

known to function in the osmoregulation of bacteria, fungi, andplants. These compounds include polyols for yeasts (1, 7, 32,42); glutamate (26), glutamine (2), N-acetylglutamylglutamine(11), and N-ox-carbamoylglutamine amide (30) for bacteria;and betaine (10-12, 24, 30) and proline (2, 10, 24) for plantsand bacteria.

In this report, we show that the addition of certain insolublematerials to a modified Wickerham's medium increased therate of sugar fermentation and the maximum amount of sugar

fermented by a brewing strain of yeast. In addition, we showthat amendment of glycine betaine, proline, glycine, or a

combination of these three compounds resulted in maintainingthe viability of cells at high levels throughout the course offermentation.

MATERIALS AND METHODS

An industrial strain of lager yeast, S. cerevisiae (uvarum)NCYC 1324, was grown at 20°C with shaking in 250-ml sidearmflasks containing 100 ml of modified Wickerham's medium(44). In some cases, where particulate materials were notadded, the experiments were conducted in duplicate with 15 mlof medium contained in test tubes and incubated with shaking(30 oscillations per min) in a temperature gradient incubator(Scientific Industries, Inc., Mineola, N.Y.) set to run isother-mally at 20°C. Various analyses were done in duplicate, and theresults were analyzed statistically. The medium contained 1.94M (35% [wt/vol]) glucose, 10 mM (NH4)2SO4, 50 mMKH2PO4, 50 mM MgSO4, and other minerals and vitamins atthree and five times the normal concentrations, respectively.The initial pH of the medium was adjusted to 5.2. Theinoculum was prepared by growing the yeast to mid-exponen-tial phase at 20°C in 100 ml of the medium described above.The cells were harvested by centrifugation and resuspended in10 ml of fresh medium. The experimental flasks were theninoculated to give 107 cells per ml. Growth was monitored bymeasuring absorbance with a Klett-Summerson colorimeterequipped with a red filter or by counting the cells. Sugarutilization was monitored by measuring, at 20°C, the decreasein specific gravity of the supernatant liquid (10,300 x g for 15min) with a digital density meter (DMA-45; Anton Paar KG,Graz, Austria). Total numbers of cells were determined micro-scopically, and viabilities were estimated by the methylene bluetechnique (39). Where necessary, the initial dissolved solidsconcentrations were adjusted to the same osmolarity by addingthe nonmetabolizable sugar alcohol sorbitol to the medium.Glycerol and ethanol levels were measured by analyzing thesupernatant portion of the medium by high-performance liquidchromatography as described previously (38).

All routinely used chemicals and microbiological mediumingredients were purchased from local suppliers. Particulatematerials used in this study were either purchased or preparedin the laboratory. Wheat bran (Quaker) and defatted soy flour(Vita, Winnipeg, Manitoba, Canada) were purchased fromlocal stores. Alumina (80/200 mesh) and sea sand (primarilyquartz) were obtained from Fisher Scientific Co. Mash solidswere prepared by freeze-drying washed insoluble materialsobtained during the preparation of wheat mash as describedpreviously (39). Ash was obtained by incinerating yeast extract.

TABLE 1. Effect of various particulate materials on glucoseutilization, cell growth, and cell viability of S. cerevisiae (uvarum)

NCYC 1324a

Amt of dissolved MaximumSupplementb solids (g/100 ml) no. (10") % ViabilitySupplement (g/100 ~~~of cells ± ±SE'

Initial Final SE

None (control) 36.4 20.2 194 ± 1 4.9 ± 1.3Wheat bran 37.2 11.9 266 ± 8 14.2 ± 2.4Mash solids 36.4 9.1 189 ± 18 69.7 ± 4.1Alumina 36.1 6.8 218 ± 9 56.6 ± 0.5Sea sand 34.8 16.1 178 ± 6 21.4 ± 2.1Soy flour 36.9 6.6 231 ± 6 74.1 ± 2.7

a Yeast was grown at 20°C in modified Wickerham's medium containing 35 to36% (wt/vol) glucose.

b All supplements were added to give a concentration of 2% (wt/vol).c Viability was determined by the methylene blue technique at the end of 9

days of fermentation.

RESULTS

Particulate materials stimulate yeast growth and fermenta-tion. Although the total amount of glucose used by the yeastwith a particular additive in the medium remained about thesame, the times taken to reach the end of fermentation variedslightly between experiments, and therefore the results fromdifferent experiments could not be averaged. The resultsreported here are averages from single experiments. Variousparticulate materials added to the defined VHG mediumstimulated fermentation, as indicated by the greater disappear-ance of dissolved solids (Table 1). While only 44.5% of thetotal sugar was fermented in 9 days in the unsupplementedcontrol, addition of 2% soy flour or 2% alumina increased theamount of sugar consumed to 82%. Other additives hadintermediate effects on fermentation. Even sea sand had amoderate stimulatory effect. Part of the stimulatory effect, itappears, is not mediated through the provision of nutrients.With all additives except sea sand, cell viability was maintainedat high levels for longer periods of time (Table 1).

Soluble nutrients provide or act as osmoprotectants for theyeast. Nutrients such as tryptone, yeast extract, and a mixtureof purine and pyrimidine bases also stimulated yeast growthand fermentation (Table 2). The normal-gravity minimal me-dium (<12% sugar) contained enough nutrients to allow theproduction of greater amounts of biomass than that observedin a medium which contained 35% sugar (data not shown).This suggested that the stimulatory effect of the supplementsdescribed above was probably mediated, not through provisionof nutrients, but by providing something which protected theyeast from the deleterious osmotic effect. Ergosterol andTween 80, known to stimulate yeast growth under oxygen-deficient conditions, were without effect. In these studies, wedid not exclude oxygen from fermentors since the presence ofoxygen did not adversely affect the ethanol yield. This clearlysuggested that, under VHG fermentation conditions, synthesisof sterols and unsaturated fatty acids by the yeast continuedunimpaired. Ash obtained from yeast extract had no effect onfermentation-probably because the medium already con-tained an excess supply of all required minerals.

Proline, glycine betaine, and glycine promote yeast growthand improve cell viability in VHG medium. The effects ofproline and glycine betaine, two proven osmoprotectants inplants and bacteria (24), on growth and metabolism of yeast inVHG medium were studied. Glycine, a precursor of glycinebetaine (41), was also included in this study. Glycine andproline are not used as sources of nitrogen by Saccharomyces

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TABLE 2. Effect of various soluble supplements on glucoseutilization, cell growth, and cell viability of S. cerevisiae (uvarum)

NCYC 1324"Amt of dis-solved solids Maximum growth % Viability

Supplement' (g/100 ml) (Klett units) ± SE ± SEC

Initial Final

None (control) 35.1 20.2 340 ± 7 11.6 ± 2.1Tryptone 35.2 0.6 483 ± 6 10.8 ± 1.0Yeast extract 35.1 1.0 488 ± 9 31.2 ± 0.9Adenine-uracil-cytosine 35.1 5.9 477 ± 2 55.3 ± 0.9Ash 35.1 19.0 390 ± 4 4.0 ± 0.3Ergosterol-Tween 80 35.4 20.9 360 ± 7 6.8 ± 0.1

a Yeast was grown at 20°C in modified Wickerham's medium containing 34 to35% (wt/vol) glucose.

b Tryptone and yeast extract were added at 1% (wt/vol). The adenine-uracil-cytosine supplement contained a 10 mM concentration of each component. Theamount of ash added was equivalent to 1% (wt/vol) yeast extract. The ergosterol-Tween 80 (a source of oleic acid) supplement contained 0.1 and 3% (wt/vol)ergosterol and Tween 80, respectively.

c Viability was determined by the methylene blue technique at the end of 9days of fermentation.

spp. under fermentation conditions (33), although with lowconcentrations of glucose (high concentrations of glucoserepress aerobic metabolism in yeasts) and under aerobicconditions, proline may serve as a nitrogen source for yeastgrowth. Addition of these three compounds singly or togetherstimulated yeast growth (Fig. 1A) and fermentation (Fig. 1B).Glycine was more effective than proline and glycine betaine. Inthe control, only 43% of the sugar was fermented by the yeast,but when all three compounds were present, 87% of the sugarwas consumed. Sugar consumption was about 65% when glycinewas the only additive. Ethanol production was proportional tothe amount of glucose removed from the medium by the yeast.With the control, the ethanol yield was 8.8% ± 0.3% (vol/vol),while in the presence of all three additives, 18.0% ± 0.6%(vo/vol) ethanol was realized. With glycine as the only additive,an ethanol yield of 13.1% ± 0.7% (vol/vol) was obtained.A mixture of glycine betaine, glycine, and proline (30 mM

each) increased the viability of the yeast and maintained it athigh levels throughout the course of fermentation (Fig. 2). Theosmolality of the medium was maintained at the same level byadding sorbitol, a sugar alcohol not used by the yeast. Eachsupplement, when added alone, was less effective in maintain-ing the cell viability than a mixture of all three. Glycine was thebest single additive.

Osmoprotection by glycine is concentration dependent. Thestimulatory effect of glycine on yeast growth increased withincreasing concentration up to 40 mM (Table 3). Furtherincrease in the concentration did not improve growth orfermentation. The amount of sugar consumed (from a mediumwhich initially contained 35 g of glucose per 100 ml) in 13 daysat 20°C was 49% in unsupplemented controls, while it in-creased to 92% with glycine supplementation at concentrationsof 40 mM. The viability of yeast cells also increased withincreasing glycine concentration and was maintained at 80% orhigher at a concentration of 40 mM or higher (Table 3).

DISCUSSION

The rate and extent of utilization of sugar by S. cerevisiae(uvarum) NCYC 1324 from a defined VHG medium wereinfluenced by the two factors studied here. First, the presenceof insoluble materials such as wheat bran, wheat mash in-

0C 400

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S 200

2 100a,

0

E00C,

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o

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0

cn

la00n._

0 100 200 300Time (hours)

0 100 200

Time (hours)300

FIG. 1. Effects of glycine betaine (0), glycine (C1), and proline (a)on the growth (A) of S. cerevisiae (uvarum) NCYC 1324 in minimalmedia containing 35% (wt/vol) glucose and their effects on sugarutilization (B). Each compound was added at a concentration of 30mM. Osmolality in all cases was adjusted to the same level by addingsorbitol. 0, control; A, betaine plus glycine plus proline.

solubles, soya flour, alumina, or sea sand in the mediumpromoted cell growth and accelerated fermentation. Second,soluble additives such as tryptone, yeast extract, and a mixtureof purine and pyrimidine bases stimulated sugar utilization andethanol production.

It is clear that the insoluble materials did not stimulatefermentation through provision of nutrients, since alumina andeven sea sand (quartz) increased the fermentation perfor-mance of the yeast. Several workers (3, 16, 18, 27, 34) haveshown that ethanolic fermentations of various kinds are stim-ulated by the additions of particulate materials. The mecha-nism of such stimulation is not, however, clearly understood.One suggestion is that particulate materials may serve asnucleation sites for the formation of CO2 bubbles, which onreaching a certain size escape into the atmosphere, therebyminimizing the inhibitory effect of this metabolic product onyeast growth (3, 22, 34). Supersaturation of medium withcarbon dioxide has been shown to affect the uptake of certainamino acids and utilization of carbohydrates by yeasts, al-though these effects are to some degree strain dependent (22).Another beneficial effect of particulate materials is thought tobe through the "enlargement of the inner surface of thefermenting medium" (27).

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001%%

0A>l-mi-O

m

0 100 200 300

Time(hours)FIG. 2. Effect of glycine betaine (0), glycine (O), and proline (U)

on viability of S. cerevisiae (uvarum) NCYC 1324 during VHGfermentation in minimal medium. Each of the supplements mentionedabove was added at a concentration of 30 mM. 0, control; A, betaineplus glycine plus proline.

Inhibition of yeast growth by metabolic CO2 may become aserious problem under VHG fermentation conditions. First,the VHG medium because of its high carbohydrate content isconsiderably more viscous than normal-gravity media. Forexample, a 35% glucose solution at 20°C is about 3.4 timesmore viscous than a 5% glucose solution (q = 1.14 and 3.94 cPfor S and 35% glucose solutions, respectively). Because of theirhigh protein and, in some cases, high P-glucan contents,viscosities of VHG grain mashes are considerably higher thanthose of simple sugar media with equivalent sugar contents.Carbon dioxide may not escape from a viscous medium asreadily as from a normal-gravity fermentation broth. Second,for maximum productivity, VHG fermentation is carried out attemperatures close to 20°C rather than at 30 to 35°C asnormally employed in fuel alcohol production. Low fermenta-tion temperature increases the solubility of CO2 and theviscosity of the medium. Both increased viscosity and lowfermentation temperature may cause retention of greateramounts of CO2 in the medium, and this may enhanceinhibition of yeast growth.

It is known that addition of peptone and yeast extract canimprove the stress and temperature tolerance of yeast (36),

TABLE 3. Effect of various concentrations of glycine on glucoseutilization, cell growth, and viability of S. cerevisiae (uvarum)

NCYC 1324a

Concn of Maximum amt of Maximum growthglycine (mM) glucose consumed (Klett units) ± SE N Viability ± SEC

(gIl00 ml)'

0 17.3 365 ± 5 13 ± 2.35 20.0 415 ± 4 28 ± 2.510 23.7 435 ± 7 40 ± 1.120 25.9 465 ± 7 53 ± 2.340 32.4 475 ± 5 78 ± 1.280 32.1 475±7 85±0.3

Yeast was grown at 20°C in modified Wickerham's medium which initiallycontained 35% (wt/vol) glucose.

b Glucose consumption was determined at the end of 13 days of fermentation.c Viability was determined by the methylene blue technique at the end of 10

days of fermentation.

although the mechanism of this protection is not understood.The improvement in fermentation observed with yeast extractdid not result from an increased supply of minerals, sinceaddition of ash obtained from yeast extract or addition ofincreased concentrations of minerals or trace elements had noeffect on the fermentation. Yeast extract (and possibly othercomplex ingredients such as tryptone and peptone) may play adual role: it may supply growth factors to nutritionally inade-quate media, and it may stimulate microbial growth that couldoccur in its absence (35). It is apparent that it is the second rolethat was important in the stimulation of fermentation observedin the present study. This role may be through the supply ofpreformed molecules (amino acids, nucleic acids, vitamins,etc.), or it may be through the provision of compounds thatprotect the yeast cells. The role of soy flour is difficult to assess,since it contains both soluble and insoluble components andboth may have contributed to the stimulation.

While tryptone, yeast extract, and nucleic acids stimulatedyeast growth and fermentation, they did not prevent loss of cellviability (Table 2). This suggested that the mechanisms ofstimulation of fermentation under VHG conditions need notbe coupled to the protection of yeast cells from osmotic stress.Much work in the past has been concerned with the physio-logical response of the yeast to osmotic stress, and very little isknown about how yeast cells can be protected by the exoge-nous addition of osmoprotectants or by stimulating the synthe-sis of osmoprotectants. Increased synthesis of glycerol has beencited as a means of osmoregulation in S. cerevisiae (5, 13, 21,32). In the present study, glycerol production varied between 1and 1.2% (wt/vol), irrespective of the nature or amount ofvarious additives (data not shown). It is not clear whether thesynthesis of glycerol is a response to osmotic stress or whetherit is a means of protecting the yeast cells. In general, compat-ible solutes are synthesized to counteract the effects of osmoticstress and to maintain cell turgidity (6). Since most of theglycerol produced by S. cerevisiae under stress is excreted intothe medium (6, 23), it is likely that glycerol production is not ameans of protecting the yeast but is a response to eliminate theexcessive reducing power generated in glycolysis. Reduction ofdihydroxyacetone to glycerol provides a convenient means ofdisposing of reducing equivalents (31). Since anabolic activitiesare decreased in S. cerevisiae under osmotic stress (29), reduc-ing equivalents generated are not used to a great extent forgrowth but are channelled for the reduction of dihydroxyac-etone to glycerol. There is some evidence to suggest a stimu-lation of synthesis of glycolytic enzymes in S. cerevisiae underosmotic stress (13, 36a) while there is a reduction in theactivities of enzymes of the pentose phosphate pathway (36a).A number of factors affect the osmotolerance of S. cerevisiae.

An important factor appears to be the physiological state ofthe cells (15). Generally, nongrowing cells are more stresstolerant than their actively growing counterparts (14), and ithas been suggested that respiratory metabolism confers greaterstress tolerance to yeast cells (25). The mechanism of thisincreased stress tolerance is unclear. Exogenously added tre-halose has been shown to offer cryoprotection to yeast (4) andto increase the dehydration resistance of stationary-phase cells(14). In the present study, exogenously added trehalose did notaffect growth, cell viability, or fermentation (data not shown).We are in agreement with the suggestion that trehalose is notan osmoregulator in growing yeasts (29), although it is synthe-sized in response to nutrient limitation, desiccation, heatshock, and other forms of stress (9, 14, 25).The results presented here show that unrecognized pro-

tectants may be involved in the osmoregulation of S. cerevisiae.In a previous report (37), we suggested that proline, produced

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OSMOPROTECTANTS AND ETHANOLIC FERMENTATION 1523

through the catabolism of arginine, may confer osmoprotec-tion to S. cerevisiae during fermentation of VHG wheatmashes. The results in this report show that exogenously addedproline stimulated yeast growth and increased cell viability.About the same degree of response was obtained when glycinebetaine was added. Both these compounds have been exten-sively studied as osmoprotectants in bacteria. Very little infor-mation is available, however, regarding their osmoprotectiveeffect on S. cerevisiae, although proline has been shown to havesome cryoprotective effect (4).

It was surprising to see that exogenously added glycinebetaine, a compound actively synthesized by yeast (12) (it is acomponent of yeast extract), had only a moderate effect inprotecting the yeast from osmotic stress. This compound is aneffective osmoprotectant in several bacteria (10), but no no-ticeable effect was observed when it was added to growingosmotolerant yeasts (12). It is possible that the reducedeffectiveness of exogenously added glycine betaine in S. cerevi-siae may be related to the difficulty of transporting thispositively charged molecule into yeast cells. Glycine, on theother hand, was the most effective single additive in combatingthe osmotic stress in S. cerevisiae. Glycine has been used as anutrient supplement during wine fermentation (20), althoughits effect on osmoregulation was not recognized. It is interestingthat where exogenously added glycine betaine served as aneffective osmoprotectant, glycine was without effect, as, forexample, in the case of Pseudomonas aeruginosa (11). Sinceglycine and proline are not used by S. cerevisiae as sources ofnitrogen (33), it is likely that these amino acids may have otherbiological roles. We suggest that one of these roles is to servedirectly or indirectly as osmoprotectants. As reported in the caseof some bacteria (41), glycine in yeast may serve as the precursorof glycine betaine, which may be the actual intracellular osmo-protectant. This aspect is currently under investigation.

ACKNOWLEDGMENTS

We gratefully acknowledge research support from the NaturalSciences and Engineering Council of Canada and the Western GrainsResearch Foundation.

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