effect ofsodium chloride onbakers' yeast growing in...

Vol. 43, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1982, p. 757-7630099-2240/82/040757-07$02.00/0

Effect of Sodium Chloride on Bakers' Yeast Growing inGelatin

CHIA-JENN WEI,1 ROBERT D. TANNER,1 AND GEORGE W. MALANEY2*Departments of Chemical Engineering' and Civil and Environmental Engineering,2 School of Engineering,

Vanderbilt University, Nashville, Tennessee 37235

Received 6 August/Accepted 21 November 1981

In recent years, industrial fermentation researchers have shifted their attentionfrom liquid to solid and semisolid culture conditions. We converted liquid culturesto the semisolid mode by adding high levels of gelatin. Previous studies on liquidcultures have revealed the inhibitory activity of mineral salts, such as NaCl, onthe fermentation of sugars by yeasts. We made a kinetic study of the effects of 1 to5% (wt/vol) NaCl on the alcoholic fermentations of glucose by Saccharomycescerevisiae in a growth medium containing 16% gelatin. Our results showed thatthe effect of high salt content on semisolid culture is essentially the same as theeffect on liquid culture; i.e., as the salt content increased, the following occurred:(i) the growth of yeasts decreased, (ii) the lag period of the yeast biomass curvelengthened, (iii) the sugar intake was lowered, (iv) the yield of ethanol wasreduced, and (v) the production of glycerol was increased. We observed a newrelationship correlating the area of kinetic hysteresis with ethanol production rate,acetaldehyde concentration, and the initial NaCl concentration.

The fermentation of sugars, especially glu-cose, by Saccharomyces cerevisiae in liquidculture has been one of the most intensivelystudied phenomena in the history of scientificinquiry. Recently, interest in this area has shift-ed to solid and semisolid fermentations for twoprimary reasons: certain products such as fer-mented foods like miso and soy sauce are be-coming more popular worldwide, and stirringand the separation of sugars from natural rawmaterials are often not required with solid mate-rials; therefore, much energy can be saved byusing the natural substrates directly.We studied kinetic changes at a transition

point between the culture in liquid form and insolid form by fabricating a gelatin medium as asemisolid model. A 16% (wt/vol) gelatin concen-tration was selected as the maximum gelatinlevel because preliminary studies had shownthat a higher concentration (i) made the separa-tion of yeast cells from the gel medium (formeasurement of biomass) very difficult, (ii)made pH measurements in the fermenting mashunreliable, and (iii) caused concern about possi-ble aeration of the anaerobic culture after theremoval of sampling plugs since the holes madein the gelled medium by the sampling did notclose completely, allowing diffusion of ambientair into the openings.

Historical review. The high concentrations ofelectrolytes in fermented foods and in raw mate-rials such as molasses-enriched sugar canestalks has motivated studies of the inhibitory

effects of high levels of inorganic salts on sugarfermentations of industrial importance. This in-hibitory action was first reported by Tajima etal. (12) and Umemoto et al. (18). Tajima et al.(12) have suggested that salt tolerance be addedto the list of desirable characteristics of yeaststrains used for alcoholic fermentation of molas-ses. For more convenient experimental method-ology, subsequent researchers have replaced theheterogeneous mixture of inorganic salts foundin molasses with pure sodium chloride.To date, however, few kinetic studies have

been reported on the effects of sodium chlorideon entrapped yeasts growing in semisolid media(16), mainly because it is difficult to track cellsattached to solids. Gelatin medium provides aneasy way to measure the mass of cells by simplymelting the gelatin at 40 to 45°C. The still viablecells are then easily separated from the gel, andthe cell-free liquid is handled as a conventionalliquid medium.Summary of previously reported salt effects.

Elevated levels of inorganic electrolytes in anotherwise satisfactory liquid growth mediumhave been found to influence several parametersof yeast activity. (i) Cell growth and multiplica-tion: (a) the number of viable yeast cells per unitvolume of liquid growth medium decreases assalt content increases, (b) the biomass of theculture (i.e., the total weight of yeast cells perunit volume of liquid growth medium) decreasesas salt content increases, and (c) the length ofthe lag phase (i.e., the incubation period be-

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758 WEI, TANNER, AND MALANEY

tween inoculation of the culture and detectableinitiation of cell growth) lengthens as salt con-centration increases. (ii) Utilization of the pri-mary carbon and energy source is reduced. (iii)Change in concentration of metabolic products:(a) there is a decrease in the production ofethanol as salt content increases and (b) there isan increase in the concentration of other fermen-tation products (such as glycerol, acetaldehyde,etc.) as salt content increases.

MATERIALS AND METHODSFermentors. The fermentations were carried out in

1-liter Pyrex glass jars with approximately 600 ml ofworking volume. Closure was with aluminum foil. Nostirring, aeration, or pH-control devices were usedduring the runs.Growth medium. Maxon and Johnson Synthetic

Medium C (6) was the basal fermentation medium.Sodium chloride was added as American ChemicalSociety-grade NaCl (catalog no. S-271, Fisher Scien-tific Co.). This liquid culture medium was converted tothe semisolid mode by the addition of 16% (wt/vol)gelatin (BBL Microbiology Systems). The final fer-mentation medium contained 10% glucose.

Organism. The fermenting organism was S. cerevi-siae (bakers' yeast) purchased as Fleischmann's dryyeast in foil packets. Each yeast package was usedonly on the day it was opened.The inoculum for 600 ml of growth medium was

prepared by the suspension of 1.2 g of dry yeast in 20ml of sterile Maxon-Johnson Synthetic Medium C atroom temperature.

Fermentation start-up. Maxon-Johnson medium (600ml) with selected salt content (0.0 to 5.0% [wt/vol]NaCI) was poured into a fermentor covered withaluminum foil and steam sterilized at 15 pounds persquare inch gauge for 30 min.The unit was cooled to 60 to 65°C and gelatin was

added. With the temperature held in this range, thecontents of the fermentor were mixed with a magneticstirrer until the gelatin dissolved. At this point thegrowth medium was adjusted to pH 5.0 with sterile 1.0N HCI or 1.0 N NH40H solution.The fermentor and contents were cooled to 40°C and

the 20-ml suspension of yeast cells was added. Theloaded fermentor was then placed in a 25°C constanttemperature bath, and the mixture was stirred at 500rpm to keep the cells dispersed while gelation tookplace. When gel formation was complete, stirring wasstopped and fermentation was continued at 25°C.pH monitoring. The pH of the fermenting mash was

measured by using a miniature combination electrode(Sargent-Welch Scientific Co.) positioned about 5 cmbelow the surface of the growth medium and 2 cm fromthe fermentor wall. The probe was moved occasionallyso that the tip would not be immersed in a trapped gas

pocket, which would lead to faulty readings.Sampling technique. After selected incubation peri-

ods, 20-ml samples of the semisolid fermenting mix-ture were removed by means of a sterile stainless steelspatula. Each sample was transferred to a 50-mi flaskwhich was closed with a rubber septum to minimizethe loss of volatile components. The sample was

liquefied at 40 to 45°C. After being mixed thoroughly,

a 1.0- or 2.0-ml portion of the melted sample wasremoved and diluted with warm distilled water invarious proportions, depending upon the concentra-tion of the component being measured and the sensi-tivity of the analytical method being employed.Biomass of yeast cells. The concentration of yeast

cells in the fermenting mash was measured by theturbidimetric (absorbancy) method. A 2.0-ml portion(1.0-ml portions in later stages of fermentation) of theliquefied sample described above was diluted 10-foldwith 40 to 45°C distilled water in a test tube. Dilutionwas crucial to the separation of yeast cells from the gelmatrix in the subsequent centrifugation at 1,800 rpmfor 10 min. The supernatant cell-free culture mediumwas stored for determination of glucose, ethanol,acetaldehyde, glycerol, and L-lysine.The cell pellet was washed with 10 ml of distilled

water at 40 to 45°C, succeeded by a second centrifuga-tion. The supernatant was discarded. Finally, thewashed cells were suspended in 10 ml of distilledwater. The optical density of the washed cell suspen-sion at 610 nm was measured in a Bausch & LombSpectronic 20 spectrophotometer that had a red lightfilter.A standard curve of optical density versus yeast dry

weight (grams per liter) that covered the appropriaterange of concentrations was made with a series of sixsuspensions prepared from the dry, packaged yeastcells.

Glucose determination. After appropriate dilution ofthe cell-free fermentation mash, glucose was measuredby the Somogyi-Nelson method (4a) with optical den-sity measurements at 425 nm in the Spectronic 20. Thegelatin in the fermentation medium interfered with theassay. Correction for this interference was made byrunning the appropriate blank. Results were expressedas grams of glucose per liter of fermenting medium.Ethanol determination. Ethanol in the diluted cell-

free fermentation mash was estimated by the alcoholdehydrogenase method of Kaplan and Ciotti (5) withreadings made in the Spectronic 20 at 340 nm. Resultswere expressed as grams of glucose per liter offermen-tation growth medium.Acetaldehyde determination. Assay of acetaldehyde

was made on undiluted cell-free fermentation mash byusing aldehyde dehydrogenase (catalog no. 171-832)purchased from Boehringer Mannheim Biochemicals.Readings ofNADH were made in the Spectronic 20 at340 nm. Results were reported as grams of acetalde-hyde per liter of fermentation mash.

Glycerol determination. Glycerol content of the di-luted cell-free fermentation mash was measured by theglycerol kinase method (1) with the Boehringer Mann-heim Biochemicals glycerol UV test kit. Readingswere made in the Spectronic 20 at 340 nm. Resultswere recorded as grams of glycerol per liter offermen-tation mash.

Lysine determination. The intracellular free L-lysinecontent of the yeast cells was measured by microbio-logical assay after extraction by boiling.

Lysine was extracted from an appropriate weight ofyeast cells by suspending the cells in 5 ml of distilledwater in a test tube and holding the suspension in aboiling water bath for 20 min. The cell debris wasremoved by centrifugation.The microbiological assay used was that described

in the Difco manual (4), with Pediococcus cerevisiae

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EFFECT OF NaCl ON BAKERS' YEAST IN GELATIN 759

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FERMENTATION PERIOD, hr

FIG. 1. Effect of NaCl levels on yeast biomass insemisolid growth cultures of S. cerevisiae in ca. 10%glucose. All points represent data points.

(NRRL B-1116) as the test organism. The assay was

made on the extract at 37°C with incubation for 18 h.The final cell concentrations of the test organism were

measured in optical density units in the Spectronic 20at 660 nm. The lysine results were reported as specificfree lysine, i.e., % (wt/wt) L-lysine per unit cell massof yeast.

RESULTS AND DISCUSSIONNumber of yeast cells per unit volume of liquid

growth medium. Combs et al. (3) have studiedthe effects of changing NaCl levels on the multi-plication of Candida albicans, as measured byconventional plate counts with Sabouraud agar.NaCl in 1.0% concentration has essentially no

effect on the 96-h viable cell count. This is notsurprising since 1.0% NaCl solution is essential-ly isotonic physiological saline solution. On theother hand, 3.5% NaCl reduces the 96-h countby 70 to 80%, and 5.0% NaCl by about 90%.

Biomass of yeast cells per unit volume of liquidgrowth medium. In the study of several yeastsisolated from marine environments, Ross andMorris (8) have found that as the concentrationof NaCl in the culture medium increases, theproduction of yeast biomass decreases. Nork-rans (7) has reported that 4.0% NaCl reduces thegrowth of S. cerevisiae by 10 to 15% comparedwith that of the control (0.0% NaCl), whereas8.0% NaCl cuts the biomass to about 90% of thecontrol level. These researchers measured bio-mass by the absorbancy (turbidimetric) method.Combs et al. (3) have investigated changes in

biomass by monitoring dry cell weight per literafter a 48-h incubation period. They have report-ed that biomass falls from 5.61 g/liter at 0.0%NaCl, to 3.40 g/liter at 1.0% NaCl, and to 1.90 g/liter at 5.0% NaCl.

Umemoto et al. (18) have reported that thehigh concentrations of molasses electrolytes re-duce yeast cell growth. Furthermore, they haveshowed that individual pure inorganic salts havea similar effect.Tanner et al. (16) have found that 40 g of NaCl

per liter reduces cell production in a semisolid(40 g of gelatin per liter) growth medium. Figure1 shows results of our studies on the effects ofNaCl on S. cerevisiae growth in gelatin. Thesekinetic trajectories show that inhibition of yeastcell growth occurs in semisolid ferme'ntation asit does in liquid fermentation. At virtually everytime point, the trend is clear; i.e., NaCl quantita-tively reduces the cell content up to about 5%NaCl, where growth becomes negligible.Length of lag phase. Ross and Morris (8) and

Norkrans (7) have plotted absorbancy (as ameasure of cell growth) versus incubation time.The early portions of their curves suggest thatincreasing NaCl content proportionately length-ens the lag period in the yeast growth curve inliquid culture. The curves in Fig. 1 show thatsimilar inhibition occurs in semisolid cultures.

Utilization of primary carbon and energysource. During an investigation of the liquidalcoholic fermentation of Okinawa molasses,Umemoto et al. (18) have observed a so-called"sugar defect"; i.e., sugar in the molasses mashis not converted to alcohol in the expected yieldsbased on glucose equivalents. The higher theconcentration of electrolytes in the mash, thelarger the "sugar defect," i.e., the poorer the"fermentation efficiency." A similar inhibitoryaction has been noted in liquid fermentationsutilizing six different yeasts.

Spencer (9) has reported that glucose con-sumption by the yeast Saccharomyces rouxii isreduced by the presence of 3.1 M (18% wt/vol)NaCl in the liquid growth medium. Brown (2),studying S. cerevisiae, has found that 1.73 M(10% wt/vol) NaCl in liquid culture increasessugar utilization. Tanner et al. (14) have foundthat a concentration of NaCl as low as 0.3 M (ca.1.5% wt/vol) initiates a reduction in the rate ofglucose uptake for liquid cultures of S. cerevi-siae.

It is interesting that when Umemoto et al. (18)added 10-3 M sodium azide to the growth medi-um, the growth of a yeast identified as alcoholicyeast Hakken no. 1 was strongly inhibited,whereas sugar uptake was only slightly reducedfrom that of the control, suggesting that thesugar is used less in cell synthesis than for thesynthesis of noncellular products, such as glyc-erol.

In semisolid culture, an increase in the NaCllevel in the growth environment above 2.0%markedly reduced the rate of glucose uptake byS. cerevisiae, although eventually (by 69 h) the

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FIG. 2. Effect of NaCi levels on glucose uptake insemisolid growth cultures of S. cerevisiae in ca. 10%glucose.

glucose was entirely used (Fig. 2).Decrease in the concentration of ethanol pro-

duced. Tajima and Yoshizumi (10) have reportedthat as the concentration of NaCi in the growthmedium increases from 0.0 M to 1.0 M, theamount of ethanol produced falls from 5.95 to5.05 ml per 100 ml of liquid growth medium.

Results obtained in the present semisolidstudy (Fig. 3) support the results of Tajima andYoshuzumi (10) for salt levels above 2% up to 30h. After 30 h, the differences tend to be negligi-ble. Although initial ethanol production rates areslowed proportionately by increasing salt levels,it appears that ultimate ethanol content is essen-tially the same, regardless of salt concentration.

Increase in the concentration of other fermenta-tion products. In 1967, Umemoto et al. (18)reported that not only do high concentrations ofelectrolytes in liquid culture cause the "sugardefect," but also high concentrations of electro-

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FIG. 4. Effect of NaCl levels on glycerol produc-tion in semisolid growth cultures of S. cerevisiae in ca.10% glucose.

lytes in several yeasts promote high accumula-tions of polyhydric organic compounds, such asglycerol, 2,3-butanediol, arabitol, and erythritol.Tajima and Yoshizumi (10) have found thatSaccharomyces formosensis Nakazawa in 0.25to 1.00 M NaCl converted much of the sugarsubstrate into glycerol, 2,3-butanediol, manni-tol, erythritol, organic acids, vicinal diketone,acetaldehyde, and CO2.Our gelatin study results corroborate previous

reports on the effect of salt in stimulating theproduction of glycerol during the fermentationof glucose by S. cerevisiae in liquid culture (Fig.4). However, it should be noted that the control(0.0% NaCl) curves, after reaching a plateau,subsequently show a gradual drop in glycerolbeginning at about 24 h, at which time theglucose substrate has been exhausted (Fig. 2),suggesting the shift to glycerol as substrate forthe yeast cells. This suggestion is reinforced bythe diauxie curves (Fig. 1) for low salt levels.The same sequence of events is seen in the 1.0,2.0, and 3.0% NaCl curves, with the reduction inglycerol beginning after 30 to 35 h. The concen-tration of glycerol continued to build up in the4.0 and 5.0% NaCl cultures in which (Fig. 2)glucose was not exhausted during the length ofthe run.Brown (2) has discussed evidence for consid-

ering the polyols synthesized by S. cerevisiae asfulfilling several physiological functions in theyeast, including the role of food reserves.The acetaldehyde curves (Fig. 5) corroborate

the previously observed (10, 20) increasingbuildup of acetaldehyde in liquid cultures at highNaCl levels, but only if measurements are madebetween 10 and 17 h or after 40 h. All of thecurves, including the control curve, reach amaximum and then fall off at variable rates.Tempest et al. (17) have reported that the

presence of 4% (ca. 0.67 M) NaCl S. cerevisiae-


FIG. 3. Effect of NaCl levels on ethanol productionin semisolid growth cultures of S. cerevisiae in ca. 10%glucose.



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FIG. 5. Effect of NaCi levels on acetaldehyde pro-duction in semisolid growth cultures of S. cerevisiae inca. 10% glucose.

glucose continuous liquid cultures produce noobservable change in intracellular amino acidpool size or composition. Tanner et al. (15) havereported that 0.15 to 0.6 M NaCl in batch liquidconditions promotes an increase in free intracel-lular lysine. The results (Fig. 6) for semisolidfermentation do not confirm any increase in theproduction of intracellular lysine per cell overthat in the control, but rather, recalling thebiomass data in Fig. 1, total intracellular lysineconcentration decreases with increasing NaCllevels. However, in their studies, Tanner et al.(15) used aerated cultures, whereas this semisol-id study used unaerated cultures. As in the lowNaCl salt cases of glycerol production, anylysine produced, even in the control, appears tobe used later in the fermentation, probably forcell synthesis.The rise in pH of the cultures containing high

levels of NaCl is shown in Fig. 7. The increasedacidity at lower salt concentrations corresponds

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FIG. 7. Effect of NaCl levels on the pH of semisol-id growth cultures of S. cerevisiae in ca. 10% glucose.

to higher cell levels (Fig. 1). What is particularlyinteresting, however, is the fact that a micro pHprobe can be used for on-line pH monitoring ofcells growing in a gel system, as well as in aliquid culture.Mechanisms proposed to explain the inhibitory

effects of NaCI on yeast fermentations. Umemotoet al. (19) have suggested a partial explanation ofthe inhibitory effects of electrotytes in terms ofthe inhibition of yeast (pyruvate) carboxylase,the enzyme that catalyzes the decarboxylationof pyruvate to acetaldehyde. Obviously, if acet-aldehyde production is slowed down, the pro-duction of ethanol is reduced proportionately.They concluded that in the absence of acetalde-hyde, if the fermentation was to continue, ahydrogen acceptor other than acetaldehydemust become available to oxidize the NADH

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FIG. 8. Typical kinetic hysteresis curve relatingthe synthesized acetaldehyde concentration to theethanol production rate at 3% NaCl. Arrows on thecurve indicate time progression.

'0 10 20 30 40 50 60 70FERMENTATION PERIOD, hr

FIG. 6. Effect of NaCl levels on intracellular lysineproduction in semisolid growth cultures of S. cerevi-siae in ca. 10%o glucose.

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ACKNOWLEDGMENTSThis study developed from the United States-Taiwan Coop-

erative Science Program, which included the United States-Republic of China Seminar on Fermentation Engineering heldat the University of Pennsylvania on 30 May to 1 June 1978and the Fermentation Engineering Research visit to Taiwanbetween 6 and 21 June 1979 under the sponsorship of theDivision of International Programs of the National ScienceFoundation (Project no. FCV-147).

S. Y. Huang and C. H. Lin collaborated in this effort. H. H.Wang contributed significantly to the definition of the studydescribed in this report.

I I A I, aLITERATURE CITED

0 1 2 3 4 5 6 1. Boehringer Mannheim Biochemicals. 1979. Methods ofenzymatic food analysis. Boehringer Mannheim Biochem-

SODIUM CHLORIDE CONCENTRATION,% (w/v) icals, Indianapolis, Ind.2. Brown, A. D. 1978. Compatible solutes and extreme water

r.9.Correlation between the area of the kinetic stress in eukaryotic microorganisms, p. 181-242. In A. H.

esis curve (relating acetaldehyde concentration Rose and J. G. Morris (ed.), Advances in microbial physi-anol production rate) and the initial NaCl con- ology, vol. 17. Academic Press, Inc., New York.Ltion. Hysteresis curve for the 3% NaCl case is 3. Combs, T. J., J. J. Guarneri, and M. A. Pisano. 1968. Thein Fig. 8. effect of sodium chloride on the lipid content and fatty

acid content composition of Candida albicans. Mycologia

60:1232-1239.d during the metabolism of glucose in the 4. Difco Laboratories. 1953. Difco manual of dehydrated

len-Meyerhof pathway. It was proposed culture media and reagents, p. 229. Difco Laboratories,he substitute hydrogen acceptor was phos- Detroit, Mich.Yceraldhyde,hnce thesubsequnt pro-

4a.Hodge, J. E., and B. T. Hofreiter. 1962. Determination ofLyceraldehyde, hence the subsequent pro- reducing and carbohydrates. Methods Carbohydr.

on of excess glycerol. Chem. 1:386-388.

:work of Tajima and Yoshizumi (11) shows 5. Kaplan, N. O., and M. M. Ciotti. 1957. Enzymatic deter-r(24- to 48-h) inhibition of acetaldehyde mination of ethanol, p. 253-255. In S. P. Colowick and.rly (24- to 48-h) inhibition of acetaldehyde N. 0. Kaplan (ed.), Methods in enzymology, vol. 3.iction by 1.0 M (6%) NaCl. Figure 5 also Academic Press, Inc., New York.

s rapid production of acetaldehyde in the 6. Maxon, W. D., and M. J. Johnson. 1953. Aeration studiesstages of glucose fermentation in the pres- on propagation of baker's yeast. Ind. Eng. Chem.of 3.0, 4.0, and 5.0% NaCl. There is 45:2554-2560.itionwithventulrecvery t the1.0 ad 7.Norkrans, B. 1966. Studies on marine-occurring yeasts:ition with eventual recovery at the 1.0 and growth related to pH, NaCl concentration and tempera-NaCl levels. ture. Arch. Mikrobiol. 54:374-392.ima and Yoshizumi (11) have suggested 8. Ross, S. S., and E. 0. Morris. 1962. Effect of sodiumthanol production is reduced and acetalde- chloride on the growth of certain yeasts of marine origin.,thanolproducionisreducedand actalde-

J. Sci. Food Agric. 13:467-475.accumulation is increased under highly 9. Spencer, J. F. T. 1968. p. 1-42. In D. J. D. Hockenhull

growth conditions because the salt inhib- (ed.), Progress in industrial microbiology, vol. 7. J. and A.cohol dehydrogenase (the enzyme which Churchill Ltd., London.es acetaldehyde to ethanol). This would 10. Tajina, K., and H. Yoshizumi. 1972. Effects of theacetldehydetoethaol). Ths would

inorganic salt concentration on yeast metabolism in alco-in the lengthening of the lag phase in the holic fermentation. III. Metabolic pathway of abnormalurves in Fig. 3. This is consistent with the fermentation by yeast in the salted medium. J. Ferment.

early accumulation of acetaldehyde in the Technol. 50:764-769.for the 5% NaCl cultures shown in Fig 5 11. Tajima, K., and H. Yoshizumi. 1975. Mechanisms offor the*%NaC*cultures shown in Fig. 5. abnormal fermentation of distiller's yeast in salted medialuld also account for the fact that the (such molasses media) from the point of NAD(P) redox

ol production rate (which is proportional to balances. J. Ferment. Technol. 53:841-853.

lcohol dehydrogenase activity) is signifi- 12. Tajima, K., H. Yoshizumi, and Y. Terashima. 1966. Saltless in the kinetics for late times com- and sugar tolerances of yeast on alcoholic fermentation. I.

with early times in the hysteresis curve slThe inhibition of fermentation by the highly concentratedsalts in molasses. J. Ferment. Technol. 44:77-84.

8). The direction of the kinetic hysteresis 13. Tanner, R. D. 1978. Kinetic hysteresis in enzyme and

has been shown to be useful as an indica- fermentation systems, p. 73-89. In D. Perlman (ed.),a decay in enzyme activity (13). Annual reports on fermentation processes, vol. 2. Aca-adecayinenzymactivity (13).demic Press, Inc., New York.

relationship between the early and late 14. Tanner, R. D., L. D. Richmond, C.-J. Wei, and J. Wood-

mnzyme activities becomes even more pro- ward. 1981. The effect of sodium chloride on the intracel-

:ed when the area of hysteresis curve (an- lular free lysine levels of growing baker's yeast. J. Chem.measure of the difference between the( Tech. Biotech. 31:290-294.measurelo trajectorifferenes)is b een a 15- Tanner, R. D., N. T. Souki, and R. M. Russell. 1977. Aand lower trajectories) is graphed as a fermentation process for producing both ethanol and

ion of the salt level in Fig. 9. This new lysine-enriched yeast. Biotechnol. Bioeng. 19:27-42.

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16. Tanner, R. D., C.-J. Wei, and J. Woodward. 1981. Thedevelopment of a semi-solid fermentation system for theproduction of lysine-enriched yeast and ethanol, p. 323-328. In M. Moo-Young, C. W. Robinson, and C. Verzina(ed.), Advances in biotechnology, vol. 1. Pergamon Press,Inc., Oxford.

17. Tempest, D. W., J. L. Meers, and C. M. Brown. 1970.Influence of environment on the content and compositionof microbial free amino acid pools. J. Gen. Microbiol.64:171-185.

18. Umemoto, S., Y. Irie, and T. Imai. 1967. The effect ofelectrolytes concentrations on alcoholic fermentation of

molasses. I. Glycerol accumulation in the medium causedby high concentrations of electrolytes. J. Ferment. Tech-nol. 45:117-124.

19. Umemoto, S., Y. Irie, and T. Imal. 1967. The effect ofelectrolytes concentrations on alcoholic fermentation ofmolasses. II. Inhibitory effect of high concentrations ofelectrolytes on yeast carboxylase. J. Ferment. Technol.45:241-245.

20. Wei, C.-J., R. D. Tanner, and J. Woodward. 1981. Eluci-dating the transition between submerged culture and solidstate baker's yeast fermentations. Third Symposium onBiotechnology in Energy Production, Gatlinburg, Tenn.

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