JOURNAL OF FERMENTATION AND BIOENGINEERING VOI. 72, NO. 1, 61-63 . 1991

Effect of Heavy Metals on Volatiles Production in Alcoholic Fermentation by Saccharomyces cerevisiae

M A R I E - N O E L L E PONS* AND SIMONE C H A N E L

Laboratoire des Sciences du G~nie Chimique, CNRS-ENSIC-INPL, BP541 F-54001 Nancy, Cedex, France

Received 8 January 1991/Accepted 8 April 1991

The effect of inhibition by metals (Cu, Co, Cd and Pb) on the production of volatiles during alcoholic fermentation was investigated by means of a gas membrane sensor connected to a gas chromatograph. Acetal- dehyde kinetics were strongly affected by this inhibition, which increased also the fermentation duration and decreased the ethanol and isoamyl alcohol yields.

In spite of the fact that many heavy metals are essential to the metabol ism of microorganisms as they are involved (a) in a good number of enzymes and other proteins, the same metal elements can be highly toxic and disturb the metabo- lism of the microorganisms (1-3). The presence in agri- cultural substrates of elements such as copper , cadmium, cobal t or lead arises from the nature of the soil the proxim- ity of car traffic (leaded-gasoline) (4), fertilizers and pesti- cides (5) as for example in the t reatment of vines against ~ mildew by copper sulphate. In the product ion of bioetha- nol by fermenta t ion of agricultural substrates by Sac- charomyces cerevisiae, increasing copper concentrat ions in bro th decrease e thanol product ion (6), the yeast being sen- sitive to copper (7) and also to cadmium and cobalt (8). The effect of metal elements on fermentat ion by-products such as ethyl acetate or fusel alcohols is also of interest: (b) they are of course a part of the flavour of alcoholic 180 beverages, par t ic ipate in b ioethanol product ion and are key-parameters for downst ream processing control (such 150 as disti l lation). The purpose of this note is to examine the ~ 120 effects of metal elements on alcoholic fermentat ion by S. cerevisiae with par t icular regard to the kinetics of produc- =o 90 tion of volatiles (acetaldehyde, ethyl acetate, e thanol , n- propyl , isobutyl and isoamyl alcohols) on a natural sub- ~ strate (red-grape juice). They were moni tored via a gas membrane sensor connected to a gas chromatograph (9).

The experiments were run in a 4 l jacketed stainless steel reactor at constant temperature (30°C). Mild agitat ion (magnetic stirrer) was mainta ined throughout the fermenta- tion. A condenser was placed at the gas outlet to prevent (c) the loss of volatiles. Commercia l pasteurized red grape 20 juice (Cidou TM, Haguenau, France) made f rom concen- trate was used. The grapes were harvested in 1988 for ex- 15 periments with copper and in 1989 for the experiments with the other metal elements. Metal elements were added as salts. The inoculum was prepared by suspension of a ~ 10 given amount of a commercial dry S. cerevisiae (Fermipan TM from Soci6t6 des Produi t s du Mais, Clamart , France) in 5 50 ml of fruit j u i c e + 5 0 ml of tap water over a period of 15 min at 30°C. The initial b iomass concentra t ion was 3 g • of dry y e a s t / / f o r experiments with copper and 5.5 g of dry 0 y e a s t / / f o r the other experiments. No steril ization was per- formed. The detect ion of volatiles by gas membrane sensor has been given in details elsewhere (9). The metal elements quantif icat ion was per formed by atomic absorp t ion spec-

* Corresponding author.

61

8O

cc.e ~ , , • °6S° EtOH

4o 6%

0 5 10 15 20 25 30 Time (h)

o 150

,120

E ' 9 0

60

30

• 0 35 4O

" £r~m " .RI.LLmlI,.-- --

• i i i i IIFL~ II.l~ lamOH+ArnOH

2" I'll'LLI I B u O H

60 ,m

30 i o ~do'- - I ~ eth# ~et~t~ ^ J a C ~ • . . . . . . . ,,gK(g(((t¢¢.(i.((g(((t((i.((gi[(g(((• • U I I I l i l l l l l J l l ' l ' [ i l i t t~ t-~''~ I I I I i

0 5 10 15 20 25 30 35 Time (h)

acetaldehyde ¢ CL.e6(ir¢¢l¢6e¢¢ ( | •e - . . f Ce., r ~¢CCCCCCC.CCCC C C C CCC'¢CCc CC CC 0

~ccccCCC~ ~'q,e

¢ ~6¢¢¢m ~ - E t O H

I 4O

150

100

50

I I I I 0 5 10 15 20 25

T i m e (h)

FIG. 1. Kinetics of production of ethanol and other volatiles in fermentation with or without addition of copper. (a and b) Copper was no added in experiment no. 0; (c) copper was added to the initial concentration of 95 mg/l in experiment no. 1.

62 J. FERMENT. B I O E N G . , PONS AND CHANEL

TABLE 1. Effect of copper addition on the production of ethanol and other volatiles

Run Cu juice in Initial Cu Final Cu Ethanol Acetaldehyde max. acetate Ethyl Propanol isobutanol lsoamyl alcohol no. (rag//) (mg//) (mg//) (g//) (mg/l) (mg//) (mg//) (rag//) (rag//)

0 0.41 0.41 0.42 81 131 17 35 54 184 1 0.39 95 75 11 211 1 0 0 0 2 0.22 56 0.55 51 228 12 43 30 82 3 0.36 51 0.56 56 304 12 28 39 57 4 0.38 27 0.6 67 168 16 21 44 81 5 0.36 25 0.6 53 238 12 0 41 106 6 0.36 14 0.41 81 177 17 0 45 142

TABLE 2. Effect of cadmium addition on the production of ethanol and other volatiles

Cd in Initial Final Ethanol Acetaldehyde Ethyl Propanol Isobutanol Isoamyl Run juice Cd Cd max. acetate alcohol no. (rag//) (mg/I) (mg//) (g//) (mg//) (mg//) (mg//) (mg/l) (mg//)

0 0.01 0.01 0.01 80 125 15 25 85 250 1 0.06 1500 771 29 110 7 19 35 20 2 0.08 835 623 28 147 8 24 38 24 3 0.06 294 232 57 190 8 39 48 48 4 0.01 177 112 73 213 10 46 44 63 5 0.01 115 62 68 124 10 38 56 90 6 0.01 62 30 82 120 12 31 69 145 7 0.02 16 12 78 120 14 28 102 258

t r o p h o t o m e t r y ( V a r i a n S p e c t r A A 20) fo r coba l t , c a d m i u m a n d c o p p e r . L e a d was d e t e r m i n e d by p l a s m a e m i s i o n s p e c t r o m e t r y ( B e c k m a n D C P S p e c t r a s p a n V).

T a b l e 1 s u m m a r i z e s the c o p p e r c o n t e n t in p u r e ju ice a n d in b r o t h as well as the p r o d u c t i o n o f vola t i le m e t abo l i t e s . In a n o r m a l r u n w i t h o u t c o p p e r a d d i t i o n ( run 0) the ace ta lde - hyde cu rve versus t ime exh ib i t s a n i n t e r m e d i a r y m a x i m a l va lue , w h i c h c o r r e s p o n d s to t he m a x i m a l va lue o f e t h a n o l p r o d u c t i o n (9) (Fig. l a a n d b). H i g h c o p p e r i nduces a s t r o n g i n h i b i t i o n (Fig. lc) : in r u n 1 a c e t a l d e h y d e is in fac t p r o d u c e d at first bu t its r e c o n s u m p t i o n s t o p p e d . R u n 1 was e n d e d by the o p e r a t o r a f t e r 50 h as the c o n c e n t r a t i o n s o f the vola t i les were n o t c h a n g i n g a n y m o r e . C o p p e r was s t rong ly a b s o r b e d by the cells except in r u n 1 a n d the res id- ual f inal c o p p e r c o n c e n t r a t i o n was the s ame fo r runs 0 a n d 2 t o 5 .

T h e t ime at w h i c h the a c e t a l d e h y d e m a x i m a l c o n c e n t r a - t i on is r e a c h e d a p p e a r s to be a n i n d i c a t i o n o f the i n h i b i t i o n level as s h o w n in Fig. 2. Th i s m a x i m a l va lue is lower for lit- tle i n h i b i t e d runs . T h e t o t a l f e r m e n t a t i o n t ime increases a lso wi th i n h i b i t i o n . C o p p e r level affects the f inal e t h a n o l

6 0 100

501 • • • 80 / 40, ~

30 ~ m

20 o - o

~ .29 10 ~ o ~ O ~ o

0 , , , , 0 0 20 40 60 80 100

Initial Cu content (mg/t)

FIG. 2. Effect of copper content on fermentation time, and etha- nol production. Symbols: i , total time of fermentation; ©, time when the amount of acetaldehyde reached the maximum value; o , the final concentration of ethanol.

c o n c e n t r a t i o n . A m o n g the o t h e r vola t i les , i s o b u t a n o l is n o t s t r ong ly affected by c o p p e r c o n t e n t (Tab le 1). O n the o t h e r h a n d , the i soamy l a l coho l c o n c e n t r a t i o n (wh ich ac- tua l ly inc ludes ac t ive amy l a l coho l ) is g rea t ly dec reased un- der h igh c o p p e r c o n c e n t r a t i o n s : th is c an be r e l a t ed p r o b a - bly to the i n h i b i t i o n o f a c e t y l - C o A syn thes i s , th is com- p o u n d b e i n g i nvo lved derec t ly in i soamyl a l coho l syn thes i s

60 ¸

5 0

40 .

m ~ 2 9

10

O l 0

~(te; • "o ,t¢~-(z'.,.

: ~e ,cc~.,.ctct6t ~c~ ~ E t O H

. eta ehyde

I 1 I 1 I I I I I

5 10 15 29 25 30 35

Time (h)

.2OO

,150

100

50 <

0 4 0 4 5 5 0

50

%4o v

E 2 9 ¸

¢.,r&g~'.=.,.~ J o u u ~ ¢c2*%(~rdOe'o • • ~ 7,

( .£~C0 I~lll L i B" n I lL 'JR. • • 0 Ltf'O • • li I.• " c(b t; • mm m .m ,-m .'

.(o me m.• c m I a m O H + A m O H

~0 0 i i l i l l l ] l l I

o) 10 ' (~o ° ethyl acetate o 0 . . . . . . W~((((t(L((~@L((((((((~(~¢~((((((~(((~ ((~fo

U I l l l l l l l l l lH l l l l [ l l i I I I I I I i I 0 5 10 15 29 25 30 35 40 45 50

Time (h)

FIG. 3. Kinetics of production of ethanol and other volatiles in fermentation with addition of cadmium. Cadmium was added to the initial concentration of 294 mg/I.

VoL. 72, 1991 NOTES 63

TABLE 3. Effect of cobalt addition on the production of ethanol and other volatiles

Co in Initial Final Ethanol Acetaldehyde Ethyl Propanol Isobutanol Isoamyl Run juice Co Co max. acetate alcohol no. (mg//) (mg//) (mg//) (g//) (mg//) (rag//) (mg//) (mg//) (mg//)

0 0 0 0 80 125 15 25 85 250 1 0 19 12 78 126 14 19 89 234 2 0 48 42 77 98 14 22 92 220 3 0 56 40 81 117 14 22 93 244

TABLE 4. Effect of lead addition on the production of ethanol and other volatiles

Pb in Initial Final Ethanol Acetaldehyde Ethyl Propanol Isobutanol lsoamyl Run juice Pb Pb max. acetate alcohol no. (mg//) (mg//) (mg//) (g//) (mg//) (mg//) (mg//) (mg//) (mg//)

0 0 0 0 80 125 15 25 85 250 1 0 125 120 78 191 15 26 86 227 2 0 125 120 80 208 14 29 81 225 3 0 250 120 80 162 14 26 85 203

a n d n o t in i s o b u t a n o l syn thes i s (10). S imi la r o b s e r v a t i o n s were m a d e fo r t he i n h i b i t i o n by

c a d m i u m (Tab le 2). A d d i t i o n o f c a d m i u m increases the fer- m e n t a t i o n t ime a n d the m a x i m a l a c e t a l d e h y d e c o n c e n t r a - t i on whi le r e d u c i n g t he f inal e t h a n o l level. H o w e v e r the t ime at w h i c h the m a x i m a l a c e t a l d e h y d e c o n c e n t r a t i o n is r e a c h e d does n o t inc rease a n d r e m a i n s close to 6 h (Fig. 3). As w i th c o p p e r t h e syn thes i s o f i s oam y l a l c o h o l is s t r ong ly affected by c a d m i u m . U n l i k e c o p p e r the c a d m i u m i n t a k e by t he cells is r e l a t ed to the in i t ia l level o f c a d m i u m (Fig. 4).

Cobalt does not affect the fermentation kinetics al- though Akrida-Demertzi et al. (6) indicate that levels h i g h e r t h a n 15 m g / l s h o u l d h a v e a n i n h i b i t o r y effect ( T a b l e 3). Ne i t he r the f inal e t h a n o l level n o r the f e r m e n t a t i o n t i m e was mod i f i ed . A l t h o u g h on ly t h r ee tes ts were m a d e wi th a d d e d coba l t , t he i n t a k e seems c o n s t a n t w h a t e v e r the in i t ia l c o n c e n t r a t i o n .

F ina l ly lead i n h i b i t i o n was i nves t i ga t ed ( T a b l e 4). No s t r o n g effect was o b s e r v e d a l t h o u g h a s l ight ly h ighe r max- ima l va lue o f a c e t a l d e h y d e a n d s l ight ly smal le r va lues o f i soamy l a l c o h o l were o b t a i n e d , w h i c h , c o n s i d e r i n g the cha rac t e r i s t i c s o b s e r v e d w h e n t he cu l t u r e is i n h i b i t e d by c o p p e r o r c a d m i u m , ind ica t e s the onse t o f i n h i b i t i o n . It s h o u l d be m e n t i o n e d t h a t diff icult ies occu r ed d u r i n g the tes ts w i th lead due to the i n t e r a c t i o n o f the P b 2~ sal t w i th g rape ju ice subs tances . Th i s cou ld have p r e v e n t e d a s t ronger

=o ~a

t -

FIG. 4.

1000.

100.

10.

1-

0,1.

0,01 - : : : : / i 0,01 0,1 1 10 100 lO00 10000

Initial Cd content (mg/t)

Relation between initial and final cadmium content.

inhibition of the fermentation. Inhibition of alcoholic fermentation by metals such as

c o p p e r , lead a n d c a d m i u m is c lear ly seen wi th ace ta lde - hyde kinet ics , w h i c h are easily m o n i t o r e d via a gas m e m - b r a n e sensor c o n n e c t e d to a gas c h r o m a t o g r a p h . T h e me ta l s affect the f e r m e n t a t i o n d u r a t i o n a n d the e t h a n o l a n d i soamy l a l c o h o l yields. These resu l t s were o b t a i n e d u n d e r b i o e t h a n o l f e r m e n t a t i o n c o n d i t i o n s (h igh i n o c u l u m ra te a n d f e r m e n t a t i o n t e m p e r a t u r e ) a n d we are c u r r e n t l y i nves t i ga t i ng t he me ta l s effect u n d e r oeno log ica l f e r m e n t a - t i on c o n d i t i o n s ( low i n o c u l u m ra te a n d f e r m e n t a t i o n t em- pe ra tu re ) .

The authors are thankful to Mr. P. Bourret and Mrs. B. Bourdon- neux from the Laboratoire de Chimie Physique Industrielle (ENSIC) for the use of the plasma emission spectrometer.

REFERENCES

1. Cook, A.H. : The chemistry and biology yeasts, p. 535. Aca- demic Press, New York (1958).

2. Cooney, J. ,I. and Wuertz, S.: Toxic effects of tin compounds on microorganisms. J. Indust. Microbiol., 4, 375-402 (1989).

3. Henderson, G. E., Evans, 1. H., and Bruee, I. J.: Vanadate inhi- bition of mitochondrial respiration and H + ATPase activity in Saccharomyces cerevisiae. Yeast, 5, 73-77 (1989).

4. Lay, H. and Lieh, W.: The contents of zinc, cadmium, lead and copper in the most, wine and by-products of wine-making. Wein Wissenshaft, 43, 2, 107-115 (1988).

5. Solanellas, F. and Bordons, A.: Copper retention by a strain of Bacillus. J. Industrial Microbiol., 3, 205-209 (1988).

6. Akrida-Demertzi, K., Demertzis, P. G., and Koutinas, A. A.: pH and trace elements content in raisin extract industrial-scale alco- holic fermentation. Biotechnol. Bioeng., 31, 666-669 (1988).

7. Ross, I. S.: Effect of glucose on copper uptake and toxicity in Sac- charomyces cerevisiae. Trans. Br. Mycol. Soc., 69, 77-81 (1977).

8. Norris, P. R. and Kelly, D. P.: Accumulation of cadmium and cobalt by Saccharomyces cerevisiae. J. Gen. Microbiol., 99, 317- 324 (1977).

9. Ports, M. N., Piehon, D., and Authier, M.: Monitoring of alco- holic fermentations of fruit juices via a gas membrane sensor. J. Ferment. Bioeng., 68, 282-285 (1989).

10. Webb, A. D. and Ingraham, J. L.: Fusel oil. Adv. Appl. Micro- biol., 5, 317-353 (1963).