1981 ethanol inhibition

8/7/2019 1981 ethanol inhibition

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Alcoholic Fermentation: On the Inhibitory Effect ofEthanolM. NOVAK, Institute of Microbiology, Czechoslovak Academy ofSciences, Videnska 1083, 14220 Praha, Czechoslovakia,P. STREHAIANO, M . M O R E N O , Ecole Nationale SupkrieuzeAgronomique, Avenue de Muret, 31000 Toulouse Cedex, France, andG. GOMA,* Institut National des Sciences Appliqukes, Laboratoire deGinie Biochimique, Avenue de Rangueil, 31077 Toulouse Cedex,France

SummaryThe inhibitory effect of ethanol is studied during alcoholic fermentation in strict anaero-biosis (initial dissolved oxygen stripped by gasing pure nitrogen). It is demonstrated thatthe ethano l produ ced during the batch fermentation is more inhibitory than the added ethan ol(in the range of 0 to 72.6 gh te r) . By analogy w ith noncompetitive enzy me kinetic inhibition,the inhibition constant for added ethanol is 105.2g/liter, and 3.8 g/liter for produced etha nol,which exhibits the sam e inhibition effects in all expe rimen ts where e thanol was added . Theme asurem ent of the intracellular alcohol concen tration can explain the dual inhibitory effectsof ethanol.

INTRODUCTIONTh e specific growth rate is usually expressed as a function of concen-tration of the limiting substrate S

P = f(S) (1)

(2 )In the presence of an inhibitor, i.e., generally a su bstan ce that dec rease sthe value of the specific growth r ate , eq . ( 1 ) should be exte nde d to includethe concentration P, i .e. ,

e.g ., by t he well-known Monod expression:p = P.,S/Ws + S)

P = g ( S , P ) (3)Th e analytical sh ap e of the function g ( S , P) depends on the character ofthe inhibitor. In the cas e of ethanol it is known that, with respect to the

* To whom all correspondence should be addressed.Biotechnology and Bioengineering, Vol. XXIII, Pp. 201-21 1 (1981)0 1981 Joh n Wiley & Sons, Inc. 0006-3592/81/0023-0201$01,10


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202 NOVAK ET A L .specific growth rate, it behaves analogously to a noncompetitive inhib-i t ~ r ; ' * * * ~ . ' ~hence in eq. (2) the specific growth rate affects only thevalue of km,but not K , . Thus we may write

(4 )where pmiis the value of the maximal specific growth rate in the presenceof an inhibitor.

Three types of dependence of pmion the ethanol concentration P weredescribed in the literature: a) Linear relation'.3,4

( 5 )(Recently, Ghose and Ty a g P found a linear kinetic pattern for growthand product formation. The substrate was cellulose hydrolysate.)

p = k m i S / ( K , + S)

pmi = k,,, - K,."'Pb) An exponential relation7

pmi = P , ~* exp(-Ki'2'~)c) And a hyperbolic r e l a t i ~ n ~ . ~

pmi = pm - Ki(3)/(Ki'3'f P) (7)Equation (7), which is analogous to the relation describing noncom-

petitive inhibition in enzymatic systems, appears to be most suitable be-cause of the noncompetitive-like inhibition by ethanol; for small valuesof P , eq. (7) transforms into eq. ( 5 ) , as can be ascertained by a Taylorseries expansion. l o

By inserting eq. (7 ) into eq. (4) we obtain a relation describing bothsubstrate limitation and inhibition by ethanol:

(8 )where we used the more common symbol K p instead of K > 3 ) .For highconcentrations of limiting substrate, eq. (8) is reduced to the form:

(9)Again reverting to the analogy with enzyme kinetics, eqs. (7) and (8)are seen to be valid for a constant concentration of inhibitor (usually

introduced from the outside). However, during a batch fermentation theconcentration of the inhibitory product-ethanol-changes owing to theproduction of ethanol. In this case, the value of pmi is not constant, butthe validity of eq. (8) does not change. From this viewpoint there shouldbe no difference between the culture-produced ethanol and the ethanolintroduced from the outside. In fact, it was found that the added ethanolexhibits a smaller inhibitory effect than the produced ethanol."*'2 Oneof the principal reasons is that during rapid fermentation the rate of alcoholdesorption from the cell to the medium is greater than its production rate,and so intracellular accumulation occurs. This phenomenon is most im-portant at higher temperatures and this observation suggests a limitation

p = pm . K , / ( K , + P) . S / ( K , + S)

CI. = kmKp/(Kp+ P)


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ALCOHOLIC FERMENTATION 203by e than ol of diffusion through the cell walls.I3 Th is work c ontr ibute s tothe solution of this problem.

MATERIALS AND METHODST h e microorganism strain used in the e xperim ents was SaccharomycesT h e medium consisted of 5 g K H 2 P0 4 ;2 g (NH4),S04; 0.4 g M g S 0 47 H 2 0 ; 1 g yeast extra ct; 20 g gluco se; distilled wa ter to 1 l iter. T he p Hwas adjusted after sterilization to 3 .7 with H 3 P 0 4 .Fermentation: To study the influence of ethanol addition, 3 liter fer-mentors with a working volume of 1.5 l i ters were used. For other ex-periments 30 liter ferme ntors w ith a working volume of 20 liters were use d.The temperature during the fermentation was maintained at 30C, andagitation at 150 rpm. In order to e nsure anaerobic condi t ions, the mediumwas bubbled with pure nitrogen for 30 min after sterilization and inocu-lation.T he biomass concentration was determined by absorb ance a t 615 nm .1 UOD = 0.98 g dry weight (DW) using the dry matter method.The amount of ethanol was determined in a Perkin-Elmer F 11 gas

chrom atograph with n-butanol a s an internal stand ard. ..T h e intracellular ethano l content wa s measured from 500 ml centrifugedfermentat ion broth. Th e e thanol content of both su pernatant , Psup,andsediment , P s e d (volume: Used), are measured by a gas chromatographicm etho d. Assum ing that 1 g dried cells has a volume of 5 ml (measuredby the C oulter coun ter method ) and considering the dry weight, X , g/literof cells in the fermentation medium, it is possible to calculate the intra-cellular ethanol concentration, P i , by the relation resulting from a massbalance made at t he level of the cells-containing sediment:

cerevisiae UG5 f rom the ENSA T co llection desc ribed by S t r e h a i a n ~ . ' ~

P s e d * Vsed = P i V c e l l s + p s u p ( V s e d - c c e l ls )so

the accuracy of the Pi determ ination is be tter than 10%.RESULTS AND DISCUSSION

Exp eriments w ith different am ounts of ethan ol added at the beginningof fermentation were first performed. The course of the experimentalgrowth cu rve s is depicted in Figure 1. The values of kmi(presented forindividual initial ethanol concentrations in Table I) were obtained from


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204 NOVAK ET AL.

A (U.0D)1

t (h)10 20 30 40

Fig. 1. Growth curves for experiments with added ethanol (optical density( 0 ) 1 , P ,= 0.39 glliter; (9 ) 2, Pa = 36.3 g/liter; (0)3, P, = 57.6 g/liter; ( A ) 4, P,liter, SO = 20 g/liter.

vs. t ime).= 7 2 . 6 g /

- - - - --,t b )

w10 20 30 40Fig. 2. Total ethanol concentration for experiments in Fig. 1 (Pvs . tim e). Sym bols arethe same as in Fig. 1 .


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ALCOHOLIC FERMENTATION 205TABLE IInfluence of Ethanol Addition (P,)on theMaximal Growth R ate (p,') of Yeast

0.3936.357.672.6

0.3240.2540.2010.199

the values of In X for the exponential growth phase by means of the least-squares method.

The total ethanol concentrations in this set of experiments, i.e., theconcentrations of added and produced ethanol, are given in Figure 2. A smentioned before, ethanol added to the culture (P,) is known to exhibita markedly lower inhibitory effect than that produced by the culture ( P p ) .The same holds for the described experiments.

On transcribing eq. (8) into a reciprocal form, we obtainl/p = up, * ( K , + P ) / K p . (S+ K J S

P = Pa + Pp(10)

In our case the concentration of ethanol, P, equalsIn the exponential phase S+K, and similarly P ,+ P p (as evident from Fig.2). By substituting into eq. (10) and rearrangement we thus obtain

( 1 1 )Equation ( 1 1 ) allows a graphical determination of p, and K p (Fig. 3).Using the values given in Table I , we obtain p, = 0.328 hr-' and Kp= 105.2g/liter.The dependence of the specific growth rate on the concentration ofproduced ethanol, Pp , is of a different character. Equation (10) can berearranged in this case to the following form:

(12)where the concentration P , is included in the first constant term on theright-hand side of eq. (12). The dependencies for different P, (as a pa-rameter) are depicted in Figure 4. In the S + K , region the curves arelinear, while in the curving part, the effects of ethanol inhibition togetherwith glucose, limitation prevail. The values of p, and Kp can also beobtained from the linear part of the curves in Figure 4 according to eq.(12). The values of the inhibitory constants are summarized in Table 11;for the sake of differentiation they are denoted as KA. The differences inthe values of K , (105.2 g/liter) and KA are really significant.Because of the neglected effect of produced ethanol concentration

1/p = l/pmi= l/p, + P , / p m i K p

= [(Kp + Pa)/kmKp + Pp/pmKpl * (S + KsYS


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206 N O V A K ET A L .

-5 0 Pa ( I. 1 - 7 1Fig. 3. Variation of the reciprocal maximal growth rate ( l i pm i )vs. the concentration of

ethanol added , P,,fo r experiments described in Figs. I and 2 .

Fig. 4. P p variation of the reciprocal growth rate (Up) with the amount of ethanol pro-duced ( P p )during experiments described in Figs. 1 and 2. (*) 1 , P, = 0.39 g/liter; ( 0 ) 2 , P,= 36.3 g/liter; ( A ) 3 , P, = 57.6 g/liter.


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AL COHOL IC FE RME NT AT ION 201T A B L E 11Influence of Added Ethanol on theInhibitory Co nstant Related to theProduced Ethanol

P'I K ;(g/litel) (g/liter)0.39

36.357.63.163.874.003.88

(although small) on the determined K p value [eq. ( l l ) , Fig. 31, we per-form ed ano ther set of experim ents with the initial glucose concentrationincreased to a level at which the p roduced ethanol concentration corre-spo nd s to the co ncentration of ethanol added in the preceding experim entswithin a n orde r of magnitude. T he results of these experim ents are sh ow nin Figure 5 . T he reciprocal values of the specific growth rate we re plottedagainst the produced ethanol concentration P p in the same manner asdescribed above (Fig. 6). Th e comm on linear part of the curves yieldedthe following values: pm = 0.313 h r - ' a n d Kb = 3.04 g/liter. T he valueof KA corresponds well to the values of Table 11; also the value of pmcorr esp ond s well t o the value obtained in the previous experim ents, thusindirectly proving the assumptions of a noncompetitive-like mode of in-

1-1)

Fig. 5 . Cou rse of biomass and ethano l production of experim ents with higher So. (a) 1 ,Growth curve for SO = 80 g/liter; (3)2 , ethano l production for SO = 80 g/liter; ( A ) 3 , growthcurve for SO = 125 g/liter; (m) 4, ethanol production for So = 125 g/liter.


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208 NOVAK ET A L .

Fig. 6 . Variation of the reciprocal growth rate (Ilk)with the amount of ethanol produced( P p )during experiments described in Fig. 5 .

hibition. The ethanol produced in these experiments evidently exhibitsthe sam e inhibit ing effect a s in th e experim ents w here ethano l was a dded .This effect is approximately 20-25 times stronger than that of the addedethanol itself.Ex plan ation s of this phenomenon result from the comp arison of ethanolconc entration both inside and o utside the cells during the cou rse of batchfermentation. Th is evolution is shown in Figure 7, which gives th e featu resfo r a cultivation with a n initial sub strate concentration of 100 &liter (in-termediate to the 80 and 125 g/liter described previo usly). D uring th e firstpart of growth, when cell multiplication is rapid, the intracellular ethanolcon tent is very high, 127 &liter, a nd d ecre ases slightly to 21 g/liter a t theend of the fermentation. At the same time, the ethanol concentrationreaches 45 g/liter. Obviously, the only ethanol concentration in the fer-me ntation broth-which is classically considered-cannot alone de scr ib ethe product inhibition an d this fact might ca us e som e of the discrepanciesin th e literature: in som e stud ies ethanol was a dd ed , while in oth ers it w asnot.A simple kinetic relationship between the yeast growth rate and theintracellular ethanol content does not exist because when this parameterdecrea ses, i t neither stabilizes nor increase s the growth rate. T he irrever-


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ALCOHOLIC FERMENTATION

rL,d125i

100

75

M

25

209

--DIX

1,25+1

0,75

015

0 , Z I

Fig. 7. Fermentation parameters when SO= 100 g/liter. (e) Growth curve X ; (A ) 2, glucoseconsumption S; ( 3 ) 3, ethanol concentration in fermentation broth, P;(A) 4, intracellularethanol con centrat ion, P,..

s ib le inhib i tory e f fec t observed previ~us ly~seems to be l inked to themaximum intracellular ethanol concentration, which can d enature a partof the en zym e, mainly h exokinase s involved in glycolysis, a s observedby Nago daw ithana and Steinkraus and N ava rro and Durand.From another viewpoint, cell propagation is not observed when theamo unt of added alcohol is greater than 96 gliter, which is a value lowerthan tha t observed inside the cells (127 g/liter). This observation cannotbe explained.

CONCLUSIONSWhen an inhibit ion p henom enon occurs by produ ct synthesis it is nec-

essary to carefully determine the inhibiting mechanism and the model.Considering only the product concentration is not sufficient. Our expe-rienc e, from stud ying bo th cell gro w th inhibition by o rganic acids (thefirst st ep of metha nog ene sis) and by ethan ol indicates that the two inhi-bit ion mechanisms (or den atura tion) are not similar. During organic acidprodu ction, we do not ob serv e intracellular accumulation of products an dth e classical relationships o f biological kinetics ar e reliable. O n th e con-trary, for ethanol fermentation, modeling must be improved. The impli-


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210 NOVAK ET AL.cation of the dual influence of added and p roduced ethan ol is importantto the level of technology of ethanol production. Previously, Nagoda-withana and Steinkrauss ' ' reported that ethanol added to the medium wasmuch less lethal than the same o r smaller quantit ies of ethanol producedby the cell. They postulated that ethanol accumulating in the cells wascontributing to th e higher death rate. It is on e of the few w orks dealingwith th e different toxic effects of pro duced o r added ethano l. In ou r case,the results suggest that the permeability of ethanol from outside the cellto the inside is very low (cf. difference on inhibitory effec t).Th e intracellular effect of ethan ol inhibition o ccu rs at the start of fer-men tation and during the first steps of fermen tation, while a t the end offerm entatio n this phen om eno n is less important a nd negligible. T here fore ,as the intracellular ethanol concentration is very high at the start of theferme ntation, the sensitivity to the e thanol extracellular concen tration islow.The alcoholic fermentation bottleneck appears to be both at the levelof cell concentration in the fermentor and the limitation for ethanol dif-fusion through the cell wall and th e cell mem brane.

PsedS

Nomenclatureinhibition c onstant of eq s. ( 3 4 7 )product inhibition constant of eqs . (8) and (9)inhibition con stant of eq s. (8) and (9) related to the co ncen tration of theonly produced productMonod constantprodu ct conce ntration in the fermentation brothintracellular product concentrationconcentration of added productconcentration of produced productethanol concentration in supernatant (e.g., intracellular ethanol contentcalculations)ethanol concentration in sedimentsubstrate concentration

P yeast specific growth ratePm maximal specific growth ratePml maximal specific growth rate in the presence of inhibitor

Th e autho rs thank the Delegation G enera te de la Re cherch e Scientifique et Tech nique fortheir financial support.References

1 . C . N . Hinshelwood, The Chemical Kinet ics of the Bacterial Cell (Oxford U. P . , Lon-2. I. Holsberg, R. K . Finn, and K . H . Steinkraus , Bio technol . B ioeng . , 9, 413 (1967).3. D . 0. Zines and P. L. Rogers, Biotechnol. Bioeng . , 12, 561 (1970).4. D. 0. Zines and P. L. Rogers, Bio technol. B ioeng . , 13, 293 (1971).5. T. K. Ghose and D. Tyagi, Biotechnol . Bioeng. , 22, 1387 (1979).6 . T. K. Ghose and D. Tyagi, Bio technol . B ioeng . , 22, 1401 (1979).

don, 1946).


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ALC OHOLIC F ER MENTATION 21 17. S . Aiba, M. Sho da, and M . Nagatani, Biotechnol. Bioeng . , 10, 845 (1968).8. N . B . Egamberdiev and N . D. Ierusalimskij, in Conrinuous Cultivation ofMicroor-ganisms, I . Malek, et al . , Ed s. (Academia, Praha, 1969),pp. 517-527.9. S . Aiba and M. Shoda, J . Ferment. Technol. , 47, 790 (1969).

10. J . A. Roels and N. W. F. Kos sen, in Progress in Industrial Microbiology, H . J. Bull,11. T. W. Nagodawithana, J . T. Whitt, and A . J . Cutaia, J . A m . Soc. B r ew . C h e m . , 35,12. T. W. N agodawithana and K. H . Steinkraus, J . A p p l . Environ. Microbiol. , 31, 15813. J . M . Navarro and G . Durand, A n n . Microbiol. B , 129, 215 (1978).14. P. Streha iano, The se, Dr.Ing. , Toulouse, 1973.IS. J . M. N avar ro and G. D urand, und er publication.

Ed. (Elsevier , A msterdan, 1978),pp. 95-203.179 (1977).(1976).

Accepted for Publication April 22, 1980

1981 ethanol inhibition

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