Cinética con Zymmomonas Mobilis

8/13/2019 Cintica con Zymmomonas Mobilis

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Appl Microbiol Biotechnol (1990) 33:31-35 ppliedM icrobiologyBiotechnology Springer-Veriag 1990

Sugar-cane molasses fermentation by Zymomonas mobi l isMon i c a B . D oe | l e an d Hor s t W . D oc i l eDep artm ent of Microbiology, University of Queensland, St. Lucia-Brisbane, Que enslan d 4067, AustraliaReceived 10 Oct ober 1989/Accepted 8 Decemb er 1989

Summary. T wo d i f f e r e n t q u a l i t y t y p e s o f s u g a r - c a n em o l a s s e s c o n t a i n i n g a t o ta l s u g a r c o n t e n t o f 4 8 % -5 0 %(w/ v ) a n d 3 5 % -4 2 % (w/ v ) we re i n v e s t i g a t e d fo r Zymomonas b i o e t h a n o l p r o d u c t i o n . M o l a s s es c o n c e n t r a t i o n so f up to 250 g /1 (1 :3 d i lu t ion ) w ere su ccess fu l ly fe r -m e n t e d w i t h i n 2 4 h d e s p i t e a h i g h e r s a l t c o n c e n t r a t i o ni n t h e l o we r g r a d e m o l a s s e s . H i g h e r m o l a s s e s c o n c e n -t r a t i o n s (3 00 g / l ) l e d t o f ru c t o s e a c c u m u l a t i o n . T h e a d -d i t i o n o f s u c ro s e t o a f i n al s u g a r c o n c e n t r a t i o n o f 1 5 %(w/ v ) l e d t o 1 0 % (v / v ) e t h a n o l w i t h c o n v e r s i o n e f f i c i e n -c ies up to 96%. Sorb i to l l eve l s were neg l ig ib le , bu t in -c r e a s e d u p t o t e n fo l d u p o n a d d i t i o n o f i nv e r t a s e .

IntroductionM o l a s s e s o f a n y t y p e is u n d o u b t e d l y t h e c h e a p e s t f e e d -s t o c k fo r e t h a n o l f e r m e n t a t i o n (M a i o re l l a e t al . 1 98 1 ;Ko s a r i c e t a l. 1 9 8 1) , a n d i s a b y -p ro d u c t o r w a s t e p ro d -u c t o f t h e s u g a r - c a n e i n d u s t ry (P a t u r a u 1 98 2) . T h e t o t a ls u g a r c o n t e n t o f th e v a r i o u s t y p e s o f m o l a s s e s v a r i e sb e t w e e n 4 0 % - 5 0 % ( w / v ) w i t h a n a s h c o n t e n t b e t w e e n6 % -1 5 % . M o l a s s e s h a s b e e n u s e d fo r d e c a d e s t o p ro -d u c e ru m (M u rp h y 1 9 8 4 ) o r e t h a n o l (P o t g e i t e r 1 9 8 1 ) .H i g h e r d e m a n d s f o r e t h a n o l , h o w e v e r , i n c r e a s e d t h es e a rc h fo r b e t t e r f e rm e n t a t i o n c o n d i t i o n s .R e a l i z i n g t h a t i o n i c s a l t s a r e r e q u i r e d b u t a l s o fu n c -t i o n a s i n h i b i t o r s d e p e n d i n g o n th e i r c o n c e n t r a t i o n s( J o n e s a n d Gre e n f i e l d 1 9 8 4 ) , p r e - t r e a t m e n t (Ka p o o rand Mis ra 1980; Rhe e e t a l . 1984; Sa iga l and Viswa na-t h a n 1 9 8 2 ) , f e d -b a t c h (Hs i e a n d S u 1 9 8 0 ; Ko s h i m i z u e ta l . 1984 ; Borzan i 1987) , con t inuous fe rmen ta t ions(M u rd i y a t m o a n d T e d j o wa l i j o n o 1 9 8 7 ; R o g e r s 1 9 8 5 ;Gro t e a n d R o g e r s 1 98 5) , u s e o f h i g h e r y e a s t c o n c e n t r a -t ions (Quei roz e t a l . 1983) , ce l l recyc le (Sedha e t a l .1984 ; Wang e t a l . 1984) , yeas t se lec t ion (Sharma e t a l .1 9 8 0 ; Ha ra l d s o n a n d B j o r l i n g 1 9 8 1 ; C o n v e r t i e t a l .1985) and mixed yeas t (Shvets e t a l . 1985 ; Jones e t a l .Offprint requests to: H. W. Doelle

1 9 8 2 ; Ha ra l d s e n a n d R o s e n 1 9 8 2 ; L a y o k u n 1 9 8 4 ) o ry e a s t a n d Zymomonas (Cruz and Borzan i 1980) cu l tu reswe re i n v e s t i g a t e d t o g e t h e r w i t h t h e a d d i t i o n o f i n ve r -tase (Park and Sa to 1982 ; Vi to lo e t a l . 1985) and thee f f e ct o f f o a m f o r m a t i o n a n d s u p p r e s s i o n ( S c h u g er l1 98 5) t o f i n d wa y s o f i m p ro v i n g t h e f e rm e n t a t i o n .

A t t e m p t s w i t h Z. mobilis a s t h e f e rm e n t i n g m i c ro o r -g a n i s m l e d t o i n c o m p l e t e s u c ro s e h y d ro l y s i s (Gu n a s e -k a ra n e t a l. 1 9 86 ; M u rp h y 1 9 8 8 a -c ) , l o w c o n v e r s i o n e f -f i c i e n c i e s d e s p i t e a d a p t a t i o n a n d n u t r i e n t s u p p l e m e n t a -t i o n (v a n Vu u r e n a n d M e y e r 1 9 82 ), a n d r e q u i r e d e x p e n -s ive desa l t ing p re- t rea tmen t (Rhee e t a l . 1984) . Th is pa-p e r d e s c r i b e s t h e f i r st f u l ly s u c c e s s ful m o l a s s e s f e rm e n -t a t i o n b y t h e Z. mobilis s t r a i n r e p o r t e d t o b e s u c c e s s fu li n s u g a r -c a n e s y r u p b i o e t h a n o l p r o d u c t i o n ( D o e l le a n dDoelle 1989).

Materials and methodsMicroorganism. The organism used throughout these investiga-tions was Z. mobilis UQM 2716, a laboratory strain developedfrom Z. mobilis UQM 2007 (NCIB 11199) and now deposited asATCC 39676 (US patent no. 4797360).Maintenance. Zymomonas mobilis was maintained both in a liquidculture me dium and on ag ar plates stored at 4 C. The liquid cul-ture contained 100 g/1 sucrose, 10 g/1 glucose, and 2 g/1 eac h o fMgSOa-7H20, KH 2PO 4, (NH4)2SO4, casein hydrolysate, andyeast extract. The culture was ke pt at 35 C a nd tra nsferr ed at reg-ular time intervals during the experiments. The agar plates con-tained 40 g/1 glucose as carbon source plus the same maintenancemedium as the liquid culture with t7 g/1 Bacto agar. The cultureswere transferred to another plate every 3-4 weeks.Bioprocess. Initial experi~aents were carried out using 500-mlflasks with the initial pH man ually adjusted to 6.5 with 4.0 MNa OH af ter inoculation: incubation of the standing cultures wasat 35 C. B atch cultivations were carried out in 3-1 Che map (M/in-nedorf, Switzerland) bioreactors with automatic pH a nd tempe ra-ture control. Without the addition of air or nitrogen, a stirrerspeed of 90-100 rpm maintained homogeneous nutrient distribu-tion, and the bioprocess temperature was maintained at 35C.After inoculation the pH was adjusted to 6.5 with no further con-


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32

100~ < 0 .~ 0~

8o~ \o\60 '~O ~E3 ~-' --- ' -- - ~

~/t I~ ~4-0 ~~ D ' D &O20 $ ~ XO.a A--~ , O 0 I

o HOURSFig. 1. Fermentation patte~ of ymomonas mobilisATCC 39676on 250 g/1 B-molasses: O, sucrose; ~, glucose; A , fructose; ~,sorbitol; ~, ethanol

10080

g/L 60 - 'o -~ . 40 \0~ ~ A5 ~ [ ]~ - [ ] ' ~ - ' - ' -~ E ][ ]~ : r . ,~ ~& /A~ Ozv, ~ A . ~ X x~ l

0 4 8 12 I 6 20 24 28 ~2 Z6HOURS

Fig. 2. Fer mentation pattern of Z mobilis ATCC 39676 on 250 g/1C-molasses: (3, sucrose; O, glucose; A, fructose; A, sorbitol; [],ethanol

trol. The feedstock molasses used was of two di fferent types, clas-sified in Australia as B- and C-molasses. Whereas B-molassescontains on average a total sugar concentration of 48%-50% (w/v), which is equivalent to a value of 67%-70% (w/w), C-molassespossesses a much reduced sugar concentration of approximately35%-42% (w/v), which is equivalent to 55%-60% (w/w). The C-molasses could be regarded as closest to blackstrap molasses .The molasses was diluted to the appropriate sugar concentrationand used w ithout heat tre atment, sterilization or nutrient addition.The same procedure was adopted upon additional sucrose addi-tion to bring the total sucrose content to 15% (w/v). Inoculationwas carried out using an 8-h-old Z mobilis culture grown on theabove maintenance medium.Analytical methods Sucrose, glucose, fructose, sorbitol and etha-nol were analysed according to Doelle and Greenfield (1985) us-ing HPLC and gas chromatography. Ethanol was additionallyanalysed by an ebulliometer, and initial sucrose was estimated bythe invertase method (Lyness and Doelle 1981).Conversion efficiency was calculated as follows:Sucrose (g/l ) 0.538 = A[gluco se+fru ctose (g/l)] = B

Ethanol ( fi nal )- ethanol (initial) = CConversion efficiency (%)= C 100/A + B

ResultsA series of different-type mo lasses concentrations waschosen be tween 100-300 g/ l , varying in total sugar con-centrations between 40 g/1 ( = 100 g/ l C-molasses) to144 g/1 ( = 300 g/ l B-molasses). The highest sugar con-centration of C -molasses used was 123 g/1 (= 30 0 g/1C-molasses). The concentrations were ch osen on the un-derstanding that present molasses fermentations withyeast are conducted with t :4 and 1:5 di lutions of theoriginal substrate.

Figures 1 and 2 exhibit typical fermentation graphsobtained with 250 g/ l (w/ v) B- and C-molasses. Thehighest ethanol production rate with B-molasses was0.59 /h or 4.66 g/1 per hour, which dropp ed to 0.32 /h or 2.5 g/1 per hour, whereas with C -molasses the

Table 1. Fer mentation patterns of Zymomonas mobilis ATCC 39676 using various conc entrations of C- molasses (C-mol), C-molasses plusinvertase, C-molasses plus sucrose (= 150 g/l total sugar) and C-molasses plus sucrose in the pres ence of invertaseSerial no. C-Mol Sucrose Sucrose Glucose Fructose Final Final Ferm Conversionadde d added sorb EtOH time efficiency(g/l) (g/l) Initial Final Initial Final Initial Final (g/l) (g/l) (h) (%)(g/l) (g/l) (g/l) (g/l) (g/l) (g/l)CM10 00 100.00 - - 42.09 2.09 4.04 1.10 4.04 0.00 1.65 26.77 15.50 99.90CM10 20 200.00 - - 78.99 3.05 8.08 1.00 8.08 0.15 1.64 49.77 24.00 98.00CM1040 250.00 - - 93.10 3.61 10.10 1.38 10.10 5.93 1.99 53.I7 24.00 88.00CM1060 300.00 - - 110.77 0.17 12.12 2.94 12.12 26.49 4.25 48.98 40.00 68.09CM1010 100.00 - - 45.55 1.64 4.04 0.00 4.04 0.05 3.46 28.63 15.50 99.00CM1030 a 200.00 - - 78.99 3.23 8.08 0.00 8.08 0.19 5.66 49.51 24.00 97.50CM1050 a 250.00 ~ 93.10 3.53 10.10 0.00 10.10 11.96 6.12 52.54 24.00 92.45CM1070 a 300.00 - - 110.77 0.13 12.12 1.16 12.12 40.81 8.08 51.82 40.00 73.38CM600 100.00 103.80 143.64 3.45 - - 4.14 - - 0.00 15.58 68.73 24.00 84.56CM610 200.00 66.00 142.50 0.00 - - 5.72 - - 0.57 10.29 74.26 24.00 92.68CM620 250.00 47.50 t31.48 0.00 - - 9.97 - - 8.54 10.68 73.47 40.00 98.99CM630 300.00 29.00 157.70 0.00 - - 11.62 - - 29.75 12.15 63.99 40.00 7L42CM650 100.00 103.80 165.68 0.00 - - 0.00 - - 0.1 t 21.18 79.00 24.00 85.37CM6 60 a 200.00 66.00 164.16 0.00 - - 3.17 - - 7.87 18.84 80.42 40.00 87.84CM670 250.00 47.50 147.44 0.00 - - 5.66 - - 28.46 17.89 67.15 40.00 81.08CM680 ~ 300.00 29.00 158.84 0.00 - - 9.07 - - 50.31 18.86 54.51 40.00 60.54

0.125 g/1 invertase addedSorb, sorbitol; EtOH, et hanol; ferm = fermentation


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33Table 2. Fermentation patterns of Z. mobilis ATCC 39676 using various concentrations of B-molasses (B-tool) in the presence or absenceof sucrose and invertaseSerial no. B-Mol Sucrose Sucrose Glucose Fructose Final Final Ferm Conversionadded added sorb EtOH time efficiency(g/ t) (g/l ) Initial Final Initial Final Initial Final (g/l ) (g/l) (h) (%)(g/l) (g/l) (g/l) (g/l) (g/l) (g/l)CM 123 0 100.00 -- 46.77 t .22 0.25 1.48 2.98 0.00 0.00 35 .5 5 2 2 .0 0 88.89CM1 500 200.00 - - 93 .54 0 .00 0 .50 0 .80 5 .96 0 .25 4 .78 56 .8 8 16 .0 0 88 .96CM 15 10 250 .00 -- 116.93 0.00 0.63 2.65 7.45 0.90 4.9 8 6 7 .5 5 2 3 .0 0 86.7 8CM 152 0 300.0 0 -- 140.31 0.67 0.75 3.52 8.94 5.24 6.29 7 4 .6 6 2 3 .0 0 81.41CM 165 0 2 5 0 .0 0 30.00 146.88 0.35 3.41 3.96 10.89 t .93 7.79 7 9 .0 0 2 0 .0 0 84.11CM1660a 25 0 .0 0 30.0 0 142.63 1.32 5.44 0.73 12.48 6.21 16.39 8 2 .5 6 2 0 .0 0 90.3 4a 0.125 g/1 invert ase adde d

h i g h e s t p r o d u c t i o n r a t e o c c u r r e d b e t w e e n 6 a n d 1 0 hw i t h 0 . 5 % / h o r 3 . 95 g / l p e r h o u r , w h i c h d r o p p e d t h e re -a f te r to 0 .24%/h or 1 .93 g /1 pe r hour .T h e r e su l t s i n T a b le 1 f o r C - m o la s se s e x h ib i t a v e r yf a m i l i ar t r e n d , w h e r e b y t h e c o n v e r s i o n e f f i c i e n c y d r o p s

f r o m a lm o s t 1 0 0 % to 6 8 . 1 % w i th i n c r e a s in g m o la s se sc o n c e n t r a t i o n . T h e sh a r p e s t d e c l i n e o c c u r r e d b e tw e e n2 5 0 g / l a n d 3 0 0 g /1 m o la s se s w i th t h e f o r m e r g iv in gh ig h e r e th a n o l y i e lds . T h e m a in f e a tu r e o f t h i s f e r m e n -t a t i o n w a s t h e e x t r e m e ly l o w so r b i t o l l ev e l s, w h ic hs l i g h tly i n c r e a se d t o 0 .4 % w i th 3 0 0 g / l m o la s se s . A f u r -t h e r im p o r t a n t o b se r v a t i o n i s r e l a t e d t o t h e a c tu a l f e r -m e n ta t i o n t im e . W h e r e a s 1 0 0 - 2 5 0 g /1 m o la s se s f e r -m e n te d o u t w i th in 2 4 h , w i th 3 0 0 g / l m o la s se s 4 0 hw e r e r e q u i r e d t o o b t a in t h e l o w c o n v e r s io n e f f i c i e n c yof 68.1%.T h e s l o w s u c ro s e h y d r o l y s i s w a s e n h a n c e d b y t h ea d d i t i o n o f 0 . 12 5 g /1 i n v e r t a se . A l th o u g h a s l i g h t i n -

c r e a se i n c o n v e r s io n e f f i c i e n c y a n d e th a n o l y i e ld o c -c u r r e d ( T a b le 1 ), t h e t r e n d s t a y e d t h e s a m e a n d f e r m e n -t a t i o n t i m e d i d n o t i m p r o v e . T h e a d d i t i o n o f i n ve r t a sed id , h o w e v e r , d o u b le t h e so r b i t o l f o r m a t io n .E a c h m o l a s s e s c o n c e n t r a t i o n w a s t h e n s u p p l e -m e n te d w i th su c r o se t o a f i n a l c o n c e n t r a t i o n o f 1 5 0 g /1to t a l su c r o se i n o r d e r t o t e s t th e p o s s ib i l i t y o f a 1 0%( v /v ) e th a n o l y i e ld [ = 7 9 - 8 0 g /1 ( w /v )] . T h i s m e a n t t h eadd i t ion of 103 .8 , 66 .0 , 47 .5 a nd 29 .0 g /1 suc ros e , r e -sp e c t i v e ly . T h e r e su l t s i n T a b le 1 i n d i c a t e t h a t t h e e th a -

glL

1, T \\o

1 A ~ / ~ ~~o~ ~ m ~ ~0 4 8 t2 I6 20 24 28

OURSFig . 3 . Fe rm en ta t i on pa t t e rn o f Z. mobilis AT CC 39676 on 250 g/1B-molasses plus 30 g/ l sucrose: O , sucrose; O , g lucose; A , f ruc-tose; i , sorbi to l ; [3 , e thano l

n o l y i e l d c o u l d b e r e a c h e d w i t h i n v e r ta s e a d d i t i o n t o1 0 0 a n d 2 0 0 g / l m o la s se s , b u t n o t w i th h ig h e r c o n c e n -t r a t i o n s . I n c o m p a r i n g f e r m e n t a t i o n w i t h a n d w i t h o u tsu c r o se a d d i t i o n , t h e c o n v e r s io n e f f i c i e n c i e s o f t h el o w e r m o l a s s e s c o n c e n t r a t i o n s d r o p p e d , w h e r e a s t h eh ig h e r m o la s se s c o n c e n t r a t i o n s i n c r e a se d s ig n i f i c a n t ly ,i n p a r t i c u l a r t h e 2 5 0 g /1 C - m o la s se s . T h e d r o p i n c o n -v e r s io n e f f i c i e n c y w a s u n d o u b te d ly d u e t o t h e i n -c r e a se d so r b i t o l l e v el s , w h ic h v a r i e d b e tw e e n 1 % ( w i th -o u t i n v e r t a se ) a n d 2 % ( w i th i n v e r t a se a d d i t i o n ) .A l t h o u g h t h e s e e x p e r im e n t s s h o w e d t h a t 1 0. 2% ( v /v ) e th a n o l c a n b e o b t a i n e d w i th in 2 4 - 4 0 h u s in g 1 0 0 -2 0 0 g / l ( w /v ) C - m o la s se s p lu s su c r o se w i th a n a p p r o x i -m a te ly 9 6 % c o n v e r s i o n e f f i c i e n c y , t h e y a l so r e v e a l e dth a t i t i s f r u c to se u p t a k e t h a t c a u se s p r o b l e m s in m o -l a s se s f e r m e n ta t i o n . 'W i th su c r o se c o m p le t e ly h y d r o -ly se d w i th a l l m o la s se s c o n c e n t r a t i o n s , t h e r e s id u a lf r u c to se l e v e ls a r e t h e r e a so n f o r r e t a r d a t i o n o f t h e e f fi -c i e n c y i n h ig h e r m o la s se s c o n c e n t r a t i o n s .

B - Mo la s se s i s a h ig h e r q u a l i t y m o la s se s w i th a t o t a lsu c r o se c o n t e n t o f a p p r o x im a te ly 5 0 % ( w /v ) . T h e f e r -m e n ta t i o n p a t t e r n o f 2 5 0 g /1 B - m o la s se s (F ig . 1 ) sh o w sth e s a m e t r e n d a s f o r C - m o la s se s a lo n e . T h e f e r m e n ta -t ion re su l t s a re g iven ~n Tab le 2 . In us ing th e sam e mo -l a s se s c o n c e n t r a t i o n s ( 1 0 0 - 3 0 0 g / l ) , t h e c o n v e r s io n e f fi -c i e n c i e s a r e v e r y m u c h c lo se r a n d w i th in 1 0% c o m p a r e dto 3 0 % in t h e c a se o f C - m o la s se s . I n c o n t r a s t t o t h e C -m o la s se s p a t t e r n , f e r m e n ta t i o n w a s c o m p le t e w i th in

~/L

i o~1 2 0 j l10Or\80 ~0, ~--~_~ ~ - ~ ~[Z]-[~-~-~] I

I Z x20- [~ ' ~ /= ~ ~~ / ~ ~ - ~ b ~ _ ~ _ . ~o ~ ~ , . ~ - ~ - ~ ~ ~ , _ ~0 4 8 12 16 20 24 28HOURS_

Fig. 4 . Ferme nta t ion pat tern o f Z. mobilis A TC C 39676 on 250 g / lB-molasses plus 30 g/ l sucrose and 0 .125 g/1 inver tase : O , su-crose; O, glucose; A, fructose; i, sorbitol; El, ethanol


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342 4 h w i t h c o n v e r s i o n e f f i c i e n c i e s r a n g i n g b e t we e n 8 0a n d 9 0 d e p e n d i n g u p o n th e m o l a s s e s c o n c e n t r a ti o n .F i g u re s 3 a n d 4 d e m o n s t r a t e t h e B -m o l a s s e s f e r -m e n t a t i o n p a t t e rn u s i n g B -m o l a s s e s p l u s s u c ro s e t o at o t a l s u g a r c o n t e n t o f 1 50 g / 1 w i t h a n d w i t h o u t i n v e r -t a s e a d d i ti o n . T h e h i g h e s t e t h a n o l p ro d u c t i o n r a t e swe re v e ry s i m i l a r w i th 0 . 9 4 / h [ = 7 .4 g / 1 p e r h o u r (w /v )] a n d 0 . 9 7 / h [= 7 .6 g / 1 p e r h o u r (w / v ) ] r e s p e c t i v e l y .A t h i gh e r B - m o l a s s e s c o n c e n t r a t i o n s p r o b l e m s w e r ea l s o o b s e rv e d i n r e g a rd t o f ru c t o s e u p t a k e w i t h s o rb i t o ll e v e ls s li g h t ly h i g h e r , a l t h o u g h t o a l e s s p ro n o u n c e d e x -t e n t t h a n w i t h C -m o l a s s e s .

c o n v e r s i o n e f f i c i e n c y a n d t h u s f i n a l e t h a n o l y i e l d s si g -n i f ican t ly (Bor rego e t a l . 1988), bu t ma y be ab le to re -d u c e r e s i d e n c e t i m e (Am i n e t a l . 1 9 8 7 ; Am i n a n dDo e l l e 1 9 8 9 ) , a l t h o u g h t h e s e s y s t e m s h a v e n o t b e e nt r i e d o n f e e d s t o c k s s u c h a s m o l a s s e s . T h e l o we r c e l lb i o m a s s r e q u i r e m e n t o f Z. mob i l i s i n c o m p a r i s o n t oyeas t (Wang e t a l , 1984) cer ta in ly saves the indus t ry re -c y c l i n g e q u i p m e n t c a p i t a l c o s t s . W e we re u n a b l e t oc o n f i rm e a r l i e r r e p o r t s o f e x t e n s iv e f i l a m e n t o u s g ro wt h(Fein e t a l . 1984) on molasses and nor d id the exper i -m e n t s s h o w e x t e n s i v e l e v a n o r f ru c t o -o l i g o m e r fo rm a -t ion (Torres and Bara t t i 1987 ; Vi ikar i 1988) .

i scussion

T h e p r e s e n t e d r e s ul t s d e m o n s t r a t e t h a t Z. mob i l i s s t ra inUQ M 2 7 16 (AT C C 3 9 67 6 ) is n o t o n l y s u i t e d fo r c o rn(Doel le e t a l . 1989) , mi lo (Mi l l i ch ip and Doel le 1989) ,p o t a t o (R i c h a rd s a n d Do e l l e 1 9 8 9 ) a n d s u g a r - c a n e s y -ru p (D o e l l e a n d Do e l l e 1 98 9) , b u t a l s o fo r d i f f e r e n tt y p e s o f m o l a s s e s f e rm e n t a t i o n t o i n d u s t r i a l e t h a n o l .W h e r e a s s u g a r - c a n e s y r u p r e q u i r e d t h e a d d i t i o n o f n u -t r i e nt s (Do e l l e a n d Do e l l e 1 98 9) , B - a n d C -m o l a s s e sh a d n o s u c h a d d i t i o n a l r e q u i r e m e n t , i n c o n t r a s t t o e a r -l i e r r e p o r t s ( v a n Vu u re n a n d M e y e r 1 9 8 2 ) .

A t a l l m o l a s s e s c o n c e n t r a t i o n s u s e d , s u c ro s e h y d ro -l y s i s wa s a l wa y s c o m p l e t e , i n c o n t r a s t t o e a r l i e r r e p o r t sw i t h d i f f e r e n t Z. mob i l i s s t r a i n s (M u rp h y 1 9 8 8 a -c ) . I twa s , h o we v e r , d i s c o v e re d t h a t o n e o f t h e r e a s o n s fo rm o l a s s e s i n h i b i ti o n o f f e r m e n t a t i o n a t c o n c e n t r a t i o n sh ighe r th an 250 g /1 (equal to a 1 : 3 d i lu t ion ) m ay b e in -h i b i ti o n o f s o m e m o l a s s e s c o m p o n e n t o n f r u c t o se u p -t a k e i n t o t h e c e l l o r f ru c t o s e c o n v e r s i o n . S u c h a n i n h i -b i t io n c o u l d b e d u e t o a n e f f e c t o n m e m b r a n e t r a n s p o r to r e i th e r o f t h e t w o e n z y m e s , f r u c t o k i n a s e o r p h o s p h o -g l u c o i s o m e ra s e , s i n c e g l u c o s e c o n v e r s i o n a n d s u c ro s eh y d ro l y s i s we re n o t a f f e c t e d . T h e o b s e rv a t i o n s m a d e b yR h e e e t al . (1 9 84 ) c o u l d t h e re fo re b e d u e t o a n e f f e c t o fi o n s o n f ru c t o s e u t i l i z a t io n . F u r t h e r wo rk i n t h is d i r e c -t ion i s in p rogress .T h e r e s u l t s a l s o c o n f i rm e d e a r l i e r r e p o r t s t h a t b yu s i n g t h e o p t i m a l t e m p e r a t u r e ( L y n e s s a n d D o e l l e1 9 8 0 ) a n d p H (L y n e s s a n d Do e l l e 1 9 8 1 ) s u c ro s e h y d ro -l ys is is i n d e p e n d e n t o f m o n o m e r i c s u g a r u p t a k e a n dt h a t t h e a c t i v i t y o f l e v a n s u c ra s e (L y n e s s a n d Do e l l e1 9 8 3 ) i s d i r e c t l y r e l a t e d t o s u c ro s e c o n c e n t r a t i o n (L y -h e s s a n d Do e l l e 1 9 8 1 ) , p ro v i d e d t h a t g l u c o s e d o e s n o ta c c u m u l a t e (B l a c k b e a rd a n d Do e l l e 1 9 8 3 ) . G l u c o s e a c -c u m u l a t i o n n o t o n l y i n h i b it s s u c ro s e h y d ro y l s i s (B l a c k -b e a rd a n d Do e l l e 1 9 8 3 ) , b u t a l s o l e a d s t o s o rb i t o l f o r -mat ion (Vi ikar i 1988) .I n c o m p a r i n g m o l a s s e s f e r m e n t a t i o n u s i n g S a c c h a -r o my c e s s p e c i e s w i t h Z. mob i l i s t h e l a t t e r a p p e a r s t oh a v e a n a d v a n t a g e i n c o n v e r s io n e f f i c i e n cy ( R o s e1 9 7 6 ) , b u t i s v a s t l y s u p e r i o r i n r e g a rd t o f e rm e n t a t i o nt ime (Pa t i l and Pa t i l 1989) . Z y r n o mo n a s mo b i l i s d o e sn o t r e q u i r e a n y n u t r i e n t a d d i t i o n , i n c o n t r a s t t o y e a s t(Woln iewizc e t a l . 1988 ; Pa t i l e t a l . 1986 , 1989) . Higherc e l l b i o m a s s o f Z. mob i l i s o b t a i n e d t h r o u g h u s e o f f l oc -c u l a n t o r i m m o b i l i z e d s t r a i n s a p p e a r s n o t t o i n c re a s e

ReferencesAmin G, Doelle HW (1989) Vertical rotating immobilized rea ctorof the bacterium Zymomonas mobilis for stable long term con-tinuous ethanol production. Biotechnol Techniques 3:95-100Amin G, D oelle HW, Greenfield PF (1987) Ethanol productionfrom sucrose by immobilized Zymomonas mobilis cells in po-lyurethane foam. Biotechnol Lett 9:225-228Blackbeard JR Doelle HW (1983) The effect of glucose on thesucrose hydrolysing activity of Zymomonas mobilis. Eur JAppl Microbiol Biotechnol 17:261-263Borrego F, Obon JM, Canoves M, Manjon A, Iborr a JL (1988) PHinfluence on ethanol production and retained biomass in apassively immobilized Zymom onas m obilis system. BiotecbnolLett 10:437-442Borzani W (1987) Kinetics of ethanol production during the reac-tor feeding phase in constant fedbatch fermentation of mo-lasses. Biotechnol Bioeng 29:844-849Converti A, Perego P, Loeli A, Parisi F, Borglei M del (1985) Akinetic study of Saccharomyces strains: performance at high

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