Ethanol Production during Batch Fermentation with Saccharomyces cerevisiae: Changes in Glycolytic Enzymes and Internal ph

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1987, p Vol. 53, No /87/ $2./ Copyright , Americn Society for Microbiology Ethnol Production during Btch Fermenttion with Scchromyces cerevisie: Chnges in Glycolytic Enzymes nd Internl ph K. M. DOMBEK AND L.. INGRAM* Deprtment of Microbiology nd Cell Science, University of Florid, Ginesville, Florid Received 12 Jnury 1987/Accepted 26 Mrch 1987 During btch fermenttion, the rte of ethnol production per milligrm of cell protein is mximl for brief period erly in this process nd declines progressively s ethnol ccumultes in the surrounding broth. Our studies demonstrte tht the removl of this ccumulted ethnol does not immeditely restore fermenttive ctivity, nd they provide evidence tht the decline in metbolic rte is due to physiologicl chnges (including possible ethnol dmge) rther thn to the presence of ethnol. Severl potentil cuses for the decline in fermenttive ctivity hve been investigted. Vibility remined t or bove 9%, internl ph remined ner neutrlity, nd the specific ctivities of the glycolytic nd lcohologenic enzymes (mesured in vitro) remined high throughout btch fermenttion. None of these fctors ppers to be cuslly relted to the fll in fermenttive ctivity during btch fermenttion. Scchromyces cerevisie is used extensively in btch fermenttions to convert sugrs to ethnol for the production of beverges nd biofuels. Despite the obvious importnce of this process, the physiologicl constrints which limit the rte of glycolysis nd ethnol production re not fully understood (7, 15, 23). Identifiction of these constrints represents n importnt step towrd the development of improved orgnisms nd process conditions for more rpid ethnol production. Such improvements could result in n increse in the ethnol production cpcity of existing fermenttion plnts nd reduction in the cost of future fcilities. S. cerevisie is cpble of very rpid rtes of glycolysis nd ethnol production under optiml conditions, producing over 5 mmol of ethnol per h per g of cell protein (11, 12). However, this high rte is mintined for only brief period during btch fermenttion nd declines progressively s ethnol ccumultes in the surrounding broth (7, 15, 23). Erlier studies hve identified requirement for lipids (2, 8, 26) or moleculr oxygen for lipid biosynthesis (1, 4, 5) in mny fermenttion broths s being essentil for the mintennce of high fermenttive ctivity. Mgnesium is n essentil cofctor for mny of the glycolytic enzymes nd hs lso been identified s limiting nutrient in fermenttion broth contining peptone nd yest extrct (11, 12). Supplying these nutritionl needs reduces but does not eliminte the decline in fermenttive ctivity during btch fermenttion. The bsis for the decline in fermenttion rte is not fully understood. Since the ddition of ethnol to cells in btch cultures nd in chemostts cuses dose-dependent inhibition of ethnol production (7, 11, 12), most investigtions hve focused on ethnol s n inhibitor (7, 15, 22). Ethnol is known to lter membrne permebility nd disrupt membrne function in vriety of biologicl systems (7, 15). In yests, ethnol cuses n increse in hydrogen ion flux cross the plsm membrne of cells suspended in wter (6). This incresed hydrogen ion flux hs been proposed s being responsible for the ethnol-induced decline in trnsport rtes observed under similr conditions (2, 17-19). * Corresponding uthor Evidence hs been ccumulting which indictes tht the presence of ethnol my not be the only fctor responsible for the decline in fermenttive ctivity. The replcement of fermenttive broth contining ethnol with fresh medium lcking ethnol did not immeditely restore fermenttive ctivity (11). In comprehensive study, Millr et l. (22) demonstrted tht concentrtions of ethnol below 12% (vol/vol) do not denture glycolytic enzymes or cuse pprecible inhibition of ctivity in vitro under substrtesturting conditions. Since ethnol does not ccumulte within yest cells but rpidly diffuses cross the cell membrne (1, 13), direct inhibition of glycolytic enzymes by intrcellulr ethnol is unlikely during fermenttions which produce 12% (vol/vol) ethnol or less. In this pper, we hve exmined three physiologicl fctors (glycolytic nd lcohologenic enzymes, internl ph, nd vibility) s possible cuses for the decline in fermenttive ctivity during btch fermenttion. MATERIALS AND METHODS Orgnism nd growth condition. S. cerevisie KD2 (petite strin) ws used in this study nd ws grown in complex medium supplemented with.5 mm mgnesium sulfte s previously described (1, 12). Btch fermenttions (initil opticl density t 55 nm of.35;.1 mg of cell protein per ml) with 2% glucose were crried out in 3-ml Spinner bottles t 3 C with 1% inoculum from 12-h culture. Anlyses of fermenttion broth. Cell protein ws determined by the method of Lowry et l. (2). Glucose, ethnol, nd cell mss were mesured s described (12). Respirometry mesurements. The rte of glycolysis nd ethnol production ws estimted by mesuring crbon dioxide evolution with Gilson differentil respirometer (Gilson, Middleton, Wis.). This rte is tken s equivlent to glycolytic flux, ssuming the production of 2 mol of ethnol nd crbon dioxide per mol of glucose consumed. Results re expressed s micromoles of crbon dioxide evolved per hour per milligrm of cell protein (12). Enzyme nlyses. Activities of glycolytic nd lcohologenic enzymes were determined in 2-ml smples removed t vrious times during btch fermenttion. Cells were hr- Downloded from on October 5, 218 by guest

2 VOL. 53, 1987 L- 1. o ^- _ 2 5. > e. 1. z z.5 OWo C1) U z c.1 I I -I.5 CD WI I t I n A m m I.5 r- l -Ii z I.1 FIG. 1. Alcohol production by strin KD2 during btch fermenttion with 2% glucose nd.5 mm mgnesium sulfte. vested by centrifugtion t 1, x g for 3 s t 4 C nd wshed in n equl volume of 5 mm potssium phosphte buffer (ph 7.4). All subsequent steps were crried out t 4 C. The pellet ws suspended in the sme buffer contining 2 mm mercptoethnol nd 2 mm EDTA nd disrupted with.1-mm glss beds using Mini-Bed Beter (Biospec Products, Brtlesville, Okl.; five periods of disruption, 1 min ech, with cooling on ice between tretments). Cell debris ws removed by centrifugtion t 1, x g for 5 min, nd the superntnt ws ssyed immeditely for enzymtic ctivities. Only two enzymes t time were ssyed in ech btch fermenttion experiment to void potentil problems which could result from storge of cells or extrcts. Pyruvte decrboxylse nd ll glycolytic enzymes were ssyed spectrophotometriclly by the methods of Mitr nd Lobo (21) s modified by Clifton et l. (9). All enzymes were ssyed under substrte-sturting conditions except triose phosphte isomerse, which ws ssyed with 1 mm substrte. The mounts of coupling enzymes were djusted s needed to ensure liner rection rte. Alcohol dehydrogense ws ssyed by mesuring the oxidtion of ethnol s described by Mitr nd Lobo (21) but using buffer t ph 8.7 contining 75 mm sodium pyrophosphte, 75 mm semicrbzide hydrochloride, nd 21 mm glycine (3). Determintion of internl ph nd membrne energiztion. The mesurements of internl ph nd At were performed using 7-['4C]benzoic cid nd [3H-phenyl]tetrphenyl phosphonium bromide, respectively. Protocols were similr to those described by Crtwright et l. (6) except tht cells were incubted in their ntive growth medium rther thn distilled wter nd.4-,um-pore-size polycrbonte filters were used insted of mixed cellulose ester filters. Cell volumes were determined s previously described (1). These rnged from 2.23,il/mg of cell protein for cells removed from the 12-h stge of btch fermenttion to.86 [li/mg of cell protein for cells removed from the 48-h stge. As control for dventitious binding of rdioctive compounds, cells were permebilized with combintion of ethnol, toluene, nd Triton X-1 s described by Slmon (25), wshed with 5 mm phosphte buffer, resuspended in ntive broth, nd processed. This tretment resulted in % ETHANOL PRODUCTION BY S. CEREVISIAE 1287 complete collpse of ApH nd loss of membrne potentil. Clcultions were performed s described by Rottenberg (24). Mterils. Yest extrct, peptone, nd gr were obtined from Difco Lbortories, Detroit, Mich. Glucose, coupling enzymes, coenzymes, nd substrtes were purchsed from Sigm Chemicl Co., St. Louis, Mo. Inorgnic slts were obtined from Fisher Scientific Co., Orlndo, Fl. Absolute ethnol ws supplied by AAPER Alcohol nd Chemicl Co., Shelbyville, Ky. Rdioctive compounds were purchsed from New Englnd Nucler Corp., Boston, Mss. RESULTS Reversibility of the decline in fermenttive ctivity. Figure 1 shows representtive btch fermenttion with 2% glucose beginning with low inoculum. Growth s mesured by cell protein ws exponentil for the first 12 h nd becme sttionry between 18 nd 24 h. During these growth periods, reltively low concentrtions of ethnol hd ccumulted (<5% [vol/vol]), well below the minimum inhibitory concentrtion of dded ethnol for growth (8% [vol/vol]). Ethnol production proceeded exponentilly for the initil 12 h (1% [vol/vol] ccumulted ethnol). Cells were removed t vrious times during btch fermenttion, nd the rte of ethnol production per milligrm of cell protein ws determined (Fig. 2). Cells were most ctive t the erliest times mesured, 12 h, nd declined by 5% fter 24 h (6.5% [vol/vol] ccumulted ethnol). Approximtely 4% of the fermenttive ctivity remined fter the ccumultion of 1% (vol/vol) ethnol (3 g of remining glucose per liter). The brupt, finl decline in ctivity reflects the ner-complete exhustion of glucose, the substrte. Removl of ethnol from cells by wshing nd suspending in fresh mcdium resulted in only modest increse in fermenttive ctivity in ll but the highest level of ccumulted ethnol. The pprent increse in ctivity in the cells which hd ccumulted 12.1% (vol/vol) ethnol ws primrily due to the restortion of fermentble substrte glucose. Loss of vibility ws exmined s possible cuse for the filure of wshing to restore full fermenttive ctivity (dt not shown). Cell number prlleled the increse in cell protein (Fig. 1) nd incresed exponentilly for the initil 12 h, reching mximum of 3 x 18 cells per ml fter 18 h. Cell number remined constnt for the remining period of fer- 6 Io. m 4 ~> W2 A FIG. 2. Chnges in fermenttive ctivity of cells during btch fermenttion. Fermenttive ctivity is expressed s micromoles of crbon dioxide evolved per hour per milligrm of cell protein. Symbols:, ctivity mesured in ntive broth;, ctivity mesured fter cells were suspended in fresh medium contining 2% glucose. Downloded from on October 5, 218 by guest

3 1288 DOMBEK AND INGRAM APPL. ENVIRON. MICROBIOL. Sc 8 -c c W B I.- h.. CP2 z Ul rl I 11 E s T FIG. 3. Effects of ethnol exposure on the fermenttive ctivities of 12- nd 24-h cells. Fermenttive ctivity is expressed s micromoles of evolved crbon dioxide per hour per milligrm of cell protein. Cells were hrvested fter 12 h (A) or 24 h (B) nd suspended in fresh medium contining vriotis concentrtions of ethnol, nd their fermenttive ctivity ws mesured fter 1 min t 3C. A prllel set of smples ws exposed to ethnol for 1 min, hrvested by centrifugtion, wshed once, nd suspended in fresh medium lcking ethnol. Brs denote representtive stndrd devition for n verge of three determintions. Symbols:, cells in the presence of dded ethnol;, cells exposed to ethnol nd suspended in fresh medium. menttion, with 9% vibility fter 48 h s mesured by the exclusion of methylene blue dye (12). In control experiment, we investigted the inhibition of ethnol production by dded ethnol nd its reversibility fter wshing (Fig. 3). Cells were hrvested nd suspended in fresh medium contining vrious concentrtions of ethnol. Cells from the 12-h period (Fig. 3A) were more ctive nd more sensitive to inhibition by dded ethnol thn cells from the 24-h period (Fig. 3B). Ethnol cused progressive, dose-dependent inhibition of fermenttion in both, with 12-h cells being more sensitive. Inhibition by ethnol ws immedite nd ppered complete within the first 1 min. No further decline in ctivity ws observed during subsequent 2 h of incubtion (3 C) with 1% dded ethnol (dt not shown). The concentrtions of ethnol required to inhibit 5% of fermenttive ctivity were 6.5 nd 9% (vol/vol), respectively, for 12- nd 24-h cells. The inhibition of fermenttion cused by exposure to TABLE 1. Specific ctivities of glycolytic enzymes t the pek of fermenttive ctivity (12 h) nd fter 5% decline (24 h) Enzyme * * ~~~~~s Sp ct (plmol/min per mg of protein) (SD)" 12-h cells 24-h cells Glycolytic flux (hexose)b 1..5 Hexokinse.84 (.5) 1.1 (.1) Phosphoglucose isomerse 4.2 (.6) 3.3 (.1) Phosphofructokinse.64 (.3).53 (.5) Fructose diphosphte 1.4 (.1) 1.2 (.1) ldolse Glycolytic flux (triose)b Triose phosphte isomerse 11 (2) 97 (2) Glycerldehyde-3-phosphte 16 (1) 18 (1) dehydrogense Phosphoglycerte kinse 11 (1) 12 (1) Phosphoglycerte mutse 6.2 (.5) 9. (.8) Enolse 3. (.2) 3.2 (.4) Pyruvte kinse 1 (3) 8.4 (1.4) Pyruvte decrboxylse 1.1 (.2).92 (.4) Alcohol dehydrogense 4.8 (.4) 3.8 (.4) Cells were removed from btch fermenttions fter 12 nd 24 h. Stndrd devitions re bsed upon determintions from three seprte btch fermenttions. b Glycolytic flux for hexose nd triose intermedites ws estimted from mesurements of fermenttion rte. concentrtions of ethnol bove 5% (vol/vol) for 1 min ws only prtilly reversed by resuspension in fresh medium lcking ethnol (Fig. 3), indicting tht exposure to ethnol dmged the cells in some wy. Agin, 12-h cells ppered more sensitive to ethnol dmge thn 24-h cells. Longer incubtion periods with 1% ethnol before wshing resulted in further loss of ctivity, with only 4% of the originl ctivity remining fter 2 h. Chnges in the levels of glycolytic nd lcohologenic enzymes during btch fermenttion. The specific ctivities of the enzymes involved in lcohol production (under substrte sturting conditions) re listed in Tble 1 for cells hrvested fter 12 h (the most ctive stge of fermenttion) nd 24 h (5% mximl ctivity). For comprison, the rtes of glycolytic flux for hexose nd triose intermedites hve been included (bsed on rtes of crbon dioxide evolution). The ctivities of ll but three of these re clerly in excess of tht required to support the mesured rtes of glycolytic flux. The exceptions were hexokinse t 12 h nd phosphofructokinse nd pyruvte decrboxylse t both 12 nd 24 h. However, the true in vivo ctivities must be sufficient to support the mesured rtes of crbon dioxide evolution except for smll contribution from nbolic processes. It is of interest to compre the reltive ctivities of ech enzyme t these two times. After 24 h, glycolytic flux hd declined by pproximtely hlf while the specific ctivities of six enzymes remined unchnged, four hd declined by pproximtely 2%, nd two hd incresed. Figure 4A illustrtes the chnges in the specific ctivities of these enzymes throughout btch fermenttion, reltive to tht of 12-h cells (1%). An nlogous plot of fermenttion rte is included for comprison. None of the specific ctivities declined drmticlly during btch fermenttion. At times beyond 12 h, with the exceptions noted bove, ll enzymes were in excess of the mesured fermenttion rtes. Phosphofructokinse declined to the gretest extent with 2% drop in ctivity fter 48 h. The specific ctivities of three enzymes incresed by more thn 2%: phosphoglucomutse (1% increse), hexokinse (5% increse; not shown), nd enolse (5% increse; not shown). All other enzymes exhibited similr increse of up to 2%. Figure 4B illustrtes the chnges in the mounts of these enzymes present per milliliter of broth reltive to tht t 12 h. Anlogous plots of soluble cell protein nd fermenttive ctivity per milliliter re included for comprison. The pek Downloded from on October 5, 218 by guest

4 VOL. 53, 1987 ETHANOL PRODUCTION BY S. CEREVISIAE O lo CM s 12 A U iu 2U 3U 4 ou A 8 m. t 1~s u.i 24 i 2 < FIG. 4. Chnges in the levels of glycolytic nd lcohologenic enzymes during btch fermenttion with 2% glucose. Cells were removeo from vrious stges of fermenttion nd disrupted, nd the ctivities of individul enzymes were determined under substrte-sturting conditions. Vlues re expressed reltive to 12-h cells, the time t which the highest ctivity per milligrm of cell protein ws observed. Brs denote representtive stndrd devition for n verge of three determintions. (A) Chnges in the specific ctivities of representtive enzymes. (B) Chnges in the ctivities per milliliter of culture of representtive enzymes. For comprison, nlogous plots of the chnges in fermenttion rte (A nd B) nd the chnges in the mount of soluble cell protein (B only) hve been included. Symbols: A, phosphoglucomutse; r, glycerldehyde-3-phosphte dehydrogense; *, triose phosphte isomerse;, phosphofructokinse; *, glycolysis; *, soluble cell protein. of fermenttive ctivity on volumetric bsis occurred fter 18 h. Although the rte of fermenttion declined beyond 18 h, the ctivities of ll of the glycolytic enzymes continued to increse until 3 h, the pek of soluble proteins. These increses in ctivities roughly prlleled the increses in soluble proteins. The ctivities of phosphoglucomutse, enolse (not shown), hexokinse (not shown), nd glycerldehyde-3-phosphte dehydrogense incresed more rpidly thn soluble cell protein, consistent with the observed increses in specific ctivities of these enzymes during fermenttion. With the exception of phosphoglucomutse, which declined more rpidly, the rtes of decline of the glycolytic enzyme ctivities per milliliter prlleled tht of the bulk soluble cell proteins, indicting neither preferentil retention nor degrdtion of these centrl ctbolic ctivities. Chnges in internl ph nd membrne energiztion during btch fermenttion. Although ethnol is the principl, reduced fermenttion product from the metbolism of glucose by S. cerevisie, orgnic cids re lso produced which lower the externl ph of the fermenttion broth to ph 3.5 (Fig. SA). Since the ph optim for glycolytic enzymes re ner neutrlity or bove (9), the filure of S. cerevisie to mintin lrge ApH during the ccumultion of ethnol could explin the rpid decline in the fermenttive ctivities of cells despite the bundnce of glycolytic nd lcohologenic enzymes. However, this does not pper to be the cse. The ApH of yest cells increses coincident with the decrese in the externl ph, mintining reltively constnt internl ph of between 6.7 nd 7. throughout btch fermenttion (Fig. SA). Similrly, At lso incresed during btch fermenttion, resulting in n overll increse in proton motive force (Fig. SB). These results were somewht surprising since previous workers hve shown tht ethnol increses the permebility of yest suspended in wter to hydrogen ions (6). To further exmine this point, we determined the effect of dded ethnol on the internl ph of cells from vrious stges of fermenttion (Fig. 6). Ethnol concentrtions of 15% (vol/vol) or bove were required to cuse mesurble decline in internl ph. The ddition of 2% (vol/vol) ethnol to 12- nd 24-h cells cused complete collpse of ApH. The ApH of cells from 36- nd 48-h cultures ws considerbly more resistnt to 2% (vol/vol) dded ethnol, consistent with n dpttion of older cells. DISCUSSION The fermenttion of glucose to ethnol represents series of coordinted enzymtic rections. This process is internlly blncing nd thermodynmiclly fvorble provided tht cellulr enzymes consume the net ATP generted from substrte-level phosphoryltion. The requirements for this process include glucose, functionl enzymes, coenzymes (NAD+, thimine pyrophosphte, ADP, ATP), cofctors (Mg2+, Zn2+), pproprite internl ph, functionl membrne to mintin the concentrtion of rectnts nd enzymes, nd glucose uptke system. Indeed, fermenttion cn proceed well in concentrted preprtions of disrupted cells (14, 27). Downloded from on October 5, 218 by guest 7.1I &C i A P%~~~~~~~ "~~~~~ k B Al~~~~~~~~m -15 : K -1 O 3< 3.. s m s TME (h) 2. 2.C FIG. 5. Chnges in internl ph nd membrne energiztion during btch fermenttion on 2% glucose. Brs denote representtive stndrd devition for n verge of three determintions. (A) Internl ph (), externl ph (-), nd ApH (A). (B) Membrne energiztion. Symbols:, proton motive force; *, ApH; A, APV. D m

5 129 DOMBEK AND INGRAM _J 1 z W O 4 I-- Z 2-1t_ h 36 h 48 h 24 h FIG. 6. Effects of dded ethnol on ApH. Cells were removed from vrious stges of btch fermenttion (indicted on grph), hrvested, nd suspended in fresh medium contining vrious concentrtions of ethnol t 3 C for 1 min, nd ApH ws determined. Why then does the rte of glycolysis in vible yest cells decline during btch fermenttion? Two nutritionl fctors hve been identified previously which reduced but did not eliminte the ethnol-ssocited decline in ctivity (7, 11, 12). Our results with dded nd ccumulted ethnol indicte tht physiologicl chnges such s ethnol dmge, rther thn n immeditely reversible effect of ethnol, pper responsible. Added ethnol inhibited fermenttion, but wshing did not restore full ctivity. Similrly, the replcement of ethnol-contining broth from the middle to lter stges of fermenttion with fresh medium did not immeditely restore fermenttive ctivity. The exposure of cells to ethnol in some wy dmged their bility to produce ethnol. The extent of this dmge ppers relted to both ethnol concentrtion nd the durtion of exposure. We hve exmined cell vibility, internl ph, nd individul enzymes involved in glycolysis nd lcohologenesis s sites for chnges (including ethnol dmge) which could be responsible for the loss of ethnol productivity during btch fermenttion. No pprecible loss of cell vibility ws observed during 48-h btch fermenttions. The ctivities of glycolytic nd lcohologenic enzymes mesured in vitro remined high nd did not pper limiting, consistent with erlier reports of the persistence of hexokinse nd lcohol dehydrogense ctivity (16). The specific ctivities of mny of these continued to increse even fter increses in totl cell protein hd ended, suggesting tht they my be preferentilly synthesized. Only modest loss of totl ctivity (per milliliter) ws observed during the ltter stges of fermenttion, consistent with low rte of turnover of these enzymes. The internl ph of the cell ws mintined ner neutrlity despite cidifiction of the broth nd the ccumultion of over 12% ethnol. This ltter observtion ws contrry to expecttion bsed upon erlier studies with cells suspended in wter (6). These erlier studies hd demonstrted tht ethnol enhnced the lekge of protons (6), with n cidifiction of the cytoplsm below the optiml ph for glycolytic nd lcohologenic enzymes. Although such enhnced lekge my lso occur in fermenttion broth, the mintennce of high internl ph in broth contining ethnol indictes tht such lekge must be offset by the ction of hydrogen ion pumps such s ATPse. Cells from the lter stges of fermenttion were more resistnt to inhibition by ethnol nd to the disruptive effects of ethnol on membrne integrity (s mesured by proton lekge). During btch fermenttion, cells my be undergo- 2 APPL. ENVIRON. MICROBIOL. ing progressive dpttions to ccumulted ethnol. Chnges in the lipid composition of yest cell membrnes hve been observed in response to ccumulted ethnol nd hve been proposed s n importnt fctor involved in such dpttion (2, 7, 15). The results of our investigtions do not identify the cuse for the decline in fermenttive ctivity during btch fermenttion but rther nrrow the rnge of remining fctors. Although the ctivities of glycolytic nd lcohologenic enzymes ssyed in vitro under substrte-sturting conditions remined high during btch fermenttion, the in vivo ctivities of these enzymes within the cell cnnot be ccurtely predicted. The ctivities of some of these re subject to modultion by llosteric effectors in ddition to constrints imposed by the vilbility of individul substrtes, cofctors, nd coenzymes. Further studies re now under wy to explore the levels of these low-moleculr-weight intrcellulr constituents. ACKNOWLEDGMENTS This reserch hs been supported in prt by the Florid Agriculturl Experiment Sttion (publiction no. 7838), by grnts from the Deprtment of Energy, Office of Bsic Energy Sciences (FG5-86ER3574) nd the Ntionl Science Foundtion (DMB ), nd by the Deprtment of Agriculture, Alcohol Fuels Progrm (86-CRCR ). LITERATURE CITED 1. Andreson, A. A., nd T. J. B. Stier Anerobic nutrition of Scchromyces cerevisie. II. Unsturted ftty cid requirement for growth in defined medium. J. Cell. Comp. Physiol. 43: Bevn, M. J., C. Chrpentier, nd A. H. Rose Production nd tolernce of ethnol in reltion to phospholipid fttycyl composition in Scchromyces cerevisie NCYC 431. J. Gen. Microbiol. 128: Bernt, E., nd I. Gutmn Ethnol determintion with lcohol dehydrogense, p In H. U. Bergermeyer (ed.), Methods of enzymtic nlysis, vol. 3. Acdemic Press, Inc., New York. 4. Buttke, T. M., S. D. Jones, nd K. Bloch Effect of sterol side chins on growth nd membrne ftty cid composition of Scchromyces cerevisie. J. Bcteriol. 144: Buttke, T. M., nd A. L. Pyle Effects of unsturted ftty cid deprivtion on neutrl lipid synthesis in Scchromyces cerevisie. J. Bcteriol. 152: Crtwright, C. P., J.-R. Juroszek, M. J. Bevn, F. M. S. Ruby, S. M. F. De Moris, nd A. H. Rose Ethnol dissiptes the proton-motive force cross the plsm membrne of Scchromyces cerevisie. J. Gen. Microbiol. 132: Csey, G. P., nd W. M. Ingledew Ethnol tolernce in yests. Crit. Rev. Microbiol. 13: Csey, G. P., C. A. Mgnus, nd W. M. Ingledew High-grvity brewing: effects of nutrition on yest composition, fermenttive bility, nd lcohol production. Appi. Environ. Microbiol. 48: Clifton, D., S. B. Weinstock, nd D. G. Frenkel Glycolysis mutnts in Scchromyces cerevisie. Genetics 88: Dombek, K. M., nd L.. Ingrm Determintion of intrcellulr concentrtion of ethnol in Scchromyes cerevisie during fermenttion. Appl. Environ. Microbiol. 51: Dombek, K. M., nd L.. Ingrm Nutrient limittion s bsis for the pprent toxicity of low levels of ethnol during fermenttion. J. Ind. Microbiol. 1: Dombek, K. M., nd L.. Ingrm Mgnesium limittion nd its role in the pprent toxicity of ethnol during yest fermenttion. Appl. Environ. Microbiol. 52: Guijrro, J. M., nd R. Lguns Scchromyces cerevi- Downloded from on October 5, 218 by guest

6 VOL. 53, 1987 sie does not ccumulte ethnol ginst concentrtion grdient. J. Bcteriol. 16: Hrden, A Alcoholic fermenttion. Longmns, Green nd Co., New York. 15. Ingrm, L. O., nd T. M. Buttke Effects of ethnol on micro-orgnisms. Adv. Microb. Physiol. 25: Lrue, F., S. Lfon-Lfourcde, nd P. Ribereu-Gyon Reltionship between the inhibition of lcoholic fermenttion by Scchromyces cerevisie nd the ctivities of hexokinse nd lcohol dehydrogense. Biotechnol. Lett. 6: Leo, C., nd N. vn Uden Effects of ethnol nd other lknols on the glucose trnsport system of Scchromyces cerevisie. Biotechnol. Bioeng. 24: Leo, C., nd N. vn Uden Effects of ethnol nd other lknols on the generl mino cid permese of Scchromyces cerevisie. Biotechnol. Bioeng. 26: Leio, C., nd N. vn Uden Effects of ethnol nd other lknols on pssive proton influx in the yest Scchromyces cerevisie. Biochim. Biophys. Act 774: Lowry,. H., N. J. Rosebrough, A. L. Frr, nd R. J. Rndll Protein mesurement with the Folin phenol regent. J. ETHANOL PRODUCTION BY S. CEREVISIAE 1291 Biol. Chem. 193: Mitr, P. K., nd Z. Lobo A kinetic study of glycolytic enzyme synthesis in yest. J. Biol. Chem. 246: Millr, D. G., K. Griffiths-Smith, E. Algr, nd R. K. Scopes Activity nd stbility of glycolytic enzymes in the presence of ethnol. Biotechnol. Lett. 9: Moulin, G., H. Boze, nd P. Glzy Inhibition of lcoholic fermenttion. Biotechnol. Bioeng. 25: Rottenberg, H The mesurement of membrne potentil nd ApH in cells, orgnelles nd vesicles. Methods Enzymol. 55: Slmon, M Appliction of the technique of cellulr permebiliztion to the study of the enzymtic ctivities of Scchromyces cerevisie in continuous lcoholic fermenttion. Biotechnol. Lett. 6: Thoms, D. S., J. A. Hossk, nd A. H. Rose Plsm membrne composition nd ethnol tolernce in Scchromyces cerevisie. Arch. Microbiol. 117: Welch, P., nd R. K. Scopes Studies on cell-free metbolism: ethnol production by yest glycolytic system reconstituted from purified enzymes. J. Biotechnol. 2: Downloded from on October 5, 218 by guest