World Journal of Microbiology and Biotechnology Volume 6 Issue 3 1990 [Doi 10.1007_bf01201297] L. Ban-Koffi; Y. W. Han -- Alcohol Production From Pineapple Waste

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W o r l d J o u r n a l o f M i c r o b i o l o g y a n d B i o te c h n o l o g y 6 , 281-284

Alcohol product ion f rom pineapplewa s te

L . B a n - K o f f i a n d Y . W . H a n

S a c c h a r o m y c e s e r e v i s l a e a n d Z y m o m o n a sm o b i l i s w e r e g r o w n o n p i n e a p p l e w a s t e a n dt h e i r a l c o h o l p r o d u c t i o n c h a r a c t e r i s t i c sc o m p a r e d . T h e p i n e a p p l e w a s t e c o n s i s t e do l 1 9 % c e l l u lo s e , 2 2 % h e m l - c e l l u l o s e , 5 %I ig n i n a n d 5 3 % c e l l s o l u b l e m a t t e r s b u tc o n c e n t r a t i o n o f s o l u b l e s u g a r s , w h i c h i n -c l u d e d 5 . 2 % s u c r o s e , 3 . 1 % g l u c o s e a n d3 . 4 % f r u c t o s e , w a s r e l a t i v e l y l o w a n d p r e -t r e a t m e n t o t th e s u b s t r a te w a s n e e d e d . P r e -t r e a t m e n t o f p i n e a p p l e w a s t e w i l h c e l l u l a s ea n d h e m i - c e l l u l e s e a n d t h e n fe r m a n t a t i o nw i t h S . c e r e v l s l a e o r Z . m o b l l l s p r o d u c e da b o u t 8 % e t h a n o l fr o m p i n e a p p l e w a s t e I n4 8 h .F o r F r e n c h s u m m a r y , s e e n e x t p a g e.L . Ban- Ko f f i i s w i t h t h e I v o r i a n C e n t e r o fT e c h n o l o g i c a l R e s e a r c h , M i n i s t r y o f S c i e n -t i f i c R e s e a r c h , A b i d j a n , I v o r y C o a s t , a n dY . W . H a n i s w i t h t h e U S D A , S o u t h e r n R e -g i o n a l R e s e a r c h C e n t e r , N e w O r l e a n s , L A ,7 0 1 7 9 , U S A . Y . W . H a n i s t h e C o r r e s p o n d i n gA u t h o r .

9 1 9 9 0 R a p i d C o m m u n i c a t i o n s o f O x fo rd L td .

I n t r o d u c t i o nB e c a us e o f c u r re n t i n t e re s t in t he e c onom ic c onve rs io n o f r e ne w a b le r e s ou rc e sin to a l c oho l , r e s idue s o f a num be r o f c rops w e re e va lua te d a s s ubs t ra t e s fo r a l c oho lp r o d u c t i o n ( H a n & C h o 19 73 ; B u ' L o c k 19 79 ; R o g e r s et al . 1979; Weisz &Ma rs ha l l 1980 ; H e r t z ma rk et aL J o n e s et aL 1981). P ine a pp le w a s te w a s one . A b ou t100,000 tons o f p ine a pp le a re p rodu c e d a nnua l ly in the Ivo ry C oa s t a nd a bou t 40to 80% i s d i s c a rde d a s w a s te , be ing c om pos e d o f pe e l, c o re s a nd pom a c e( M u t t a m a r a & N i r m a l a 1 9 8 2 ; T e w a r i et aL 1987) . The s e ma te r i a l s , ha v ing h ighb i o c h e m i c a l o x y g e n d e m a n d ( B O D ) a n d c h e m i c a l o x y g e n d e m a n d ( C O D ) v a l u e s ,c a us e a se r ious po l lu t ion p ro b le m i f no t d i s pos e d o f p rop e r ly (B urba nk &K u ma ga i 1965 ). P ine a pp le w a s te , c ons i s t ing m a in ly o f c e l lu lo s e a nd s t a rc h , w a ss ugge s t e d a s a s ubs t ra t e fo r p roduc t ion o f va lua b le f e rme n ta t ion a nd non -f e r m e n t a t i o n p r o d u c t s ( T e w a r i et aL 1987) . In the pa s t , p ine a pp le w a s te f romc a nne r i e s ha s be e n u t i l i z e d a s t he s ubs t ra t e fo r b rome l in , v ine ga r , w ine , food / fe e dye a s t a nd o rg a n ic a c id s (D e v & Ing le 1982). Th i s pa pe r e va lua te s t he po te n t i a lo f p ine a pp le w a s te a s a s ubs t ra t e fo r e tha no l f e rme n ta t ion .M a t e r i a l s a n d M e t h o d sSubs t ra tePine a pp le s w e re ob ta ine d f rom loc a l ma rke t s i n N e w O r le a ns , U SA . Fo r a na ly t i c a lpu rpos e s , t he p ine a pp le w a s d iv ide d in to th re e pa r t s : c row n ( the top pa r t ) , pu lp( the inne r pa r t ) a nd s k in . Fo r f e rme n ta t ion , t he e n t i r e p ine a pp le w a s g round w i tha W a r ing b l e nd e r a nd s te r i li z e d a t 121~ fo r 20 min .P r e - t r e a t m e n lO n e g e a c h o f c e llu l as e (5.1 U /m g s o l id , S igm a C he mic a l C o . ) a nd he mi -c e l lu l a s e(0.053 U / mg s o l id , S igma C he mic a l C o . ) w e re a dde d to 1 kg o f g roun d p ine a p p lea nd the mix tu re w a s ke p t a t roo m t e mp e ra tu re (25 to 30~ fo r 24 h .Microorganisms and Fermenta tionSaccharomyces cerevisiae ( N R R L Y - 2 0 3 4 ) , g r o w n o n p o t a t o d e x t r o s e b r o t h , a n dZymomonas mobil is (N R R L B -14022) , g row n on a ba s a l me d ium, w e re u s e d a s

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L. Ban-Ko/ fl and Y.W . HanO n a f a i l c r o i t r e S a c c h a r o m y c e s c e r e v is i a ee t Z y m o m o n a s m o b i li s s u r d e s d b c h e t sd ' a n a n a s , e t o n a c o m p a r ( ~ l e s c a r a c t d r -i s t i q u e s d e l e u r p r o d u c t i o n d ' a l c o o l . L ed (~ c he t d ' a n a n a s c o n s i s t a i t e n 1 9 % d ec e l l u lo s e , 2 2 % d ' h 6 m i c e l l u l o s e , 5 % d el i g n i n e e t 5 3 % d e m a t i ( ~ r e s c e l l u l a i r e ss o l u b l e s . M a i s l a c o n c e n t r a t i o n e n s u c r e ss o l u b l e s qu i c o m p r e n a i t 5 . 2 % d e s u c r o s e ,3 . 1 % d e g l u c o s e e t 3 . 4 % d e f r u ct o s e , 6 t a ilr e l a t i v e m e n t f a i b l e . L e p r ( ~ t r a i t e m e n t d us u b s t r a t s ' a v 6 r a i t d o n c n 6 c e s s a i r e . L ep r ( ~ tr a l te m e n t c e s d ( ~ c he ts d ' a n a n a s a v e c l ac e l l u l a s e e t I ' h e m i c e l l u l a s e , s u i v i d e l af e r m e n t a t i o n p a r S . c e r e v i s i a e o u Z . m o b i l iso n t p r o d u i t e n v i r o n 8 % d ' e t h a n o l / ~ p a r t i r d er b s i d u s d ' a n a n a s e n 4 8 h .

inocula. The basal medium consisted of sucrose, 150 g; peptone, 2 g; yeast extract,1.5 g; K2HPO4, 2 g; (NHa)2SO 4. 2 g; MgSO4.H20 , 0.1 g per litre of water.About 1 kg of gro und pineapple waste in 2 1 Fernback flasks was inoculatedwith 100 ml of actively growing culture and kept at room temperature (25 to30~ for up to 72 h. The high-solid fermentation mash contained about 25%(w/v) dry matter and the low-solid fermentation mash contained about 10% (w/v)dry matter. The low-solid fermentation mash was prepared by pressing the groundpineapple waste through three layers of cheesecloth and collecting the liquid.Ana [ytical ProceduresCellulose, hemi-cellulose, lignin, cell soluble matter (CSM), ash and in vitro rumendigestibility were determined by the method of Goering & Van Soest (1970).Crude protein was determined as total N, determined by the micro-Kjeldahlmethod, x 6.25, according to Perrin (1953). Sugars and alcohol were determinedby high-performance liquid chroma tography (HPLC) (Waters sugar analyser,Waters Assoc. Inc. Milford, MA, USA) with a refractive index detector andAmines HPX-87 column (Bio-Rad Corp., Richmond, CA, USA). Water was usedas the mobile phase. Total fermentable sugar was the sum of sucrose, glucose andfructose. Cell growth was determined by plate counts.R e s u l t s a n d D i s c u s s i o nTable 1 shows the chemical and nutritional composition of the various parts ofpineapple. The crown contained less cell soluble matter and more cellulosecompared with other parts. The compositio n of the pulp was similar to that ofthe skin. The amounts of fermentable sugars in pineapple varied depending onthe maturity o f the fruit. A fresh pineapple contained 5.2% sucrose, 3.1% glucoseand 3.4% fructose, which amounts to 11.7% (w/w) of total fermentable sugar.The prote in content was about 4% for all parts of the fruits. The pu lp was mostdigestible (72%); the crown was the least digestible (60%). Digestibility wasinversely proportion al to the cellulose conten t of the sample. The pH of the freshpineapple juice was about 3.4.

Relatively low levels of fermentable sugars (11.7% total sugar) and h igh amountsof fibre necessitated a pre-treatment o f pineapple waste to increase the sugarcontent prior to fermentation. Initial sugar concentrations of more than 20% havebeen suggested as being the necessary minimum for economic alcohol fermentationof grains (Anon. 1980). Cellulase and hemi-cellulase were used to saccharify thefibrous matter, which increased the total sugar content to 14.4% in the substrate,a 23% increase from the initial level (Table 2). Combinations with other pre-

T a b l e 1 . C h e m i c a l a n d n u t r it i o n a l r o f p i n e a p p l e w a s t e ( % d r y m a t t e r ) .*C o m p o n e n t

W h o l e S k i n C r o w n P u l pC e l l u l o s e 19.4 14.0 29.6 14.3H e m i - c e l l u l o s e 22.4 20.2 23.2 22.1Lignin 4.7 1.5 4.5 2.3A s h 0.7 0.6 0.4 0.2CSM 53.4 64.8 42.5 61.4Total sugar 11.7 -- -- --P r o t e i n 4.4 4.1 4.2 4.6D i g e s t i b i l i t y l - 6 3 . 2 6 4 . 3 6 0 .1 7 2 .1* A v e r a g e o f t ri p l ic a t e s a m p l e s .t In v i t ro r u m e n d i g e s t i b i l i t y .

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T a b l e 2 . S u g a r r e l e a s e f r o m p i n e a p p l ew a s t e b y e n z y m e t r e a tm e n t s * .S u g a r U n t r e a t ed E n z y m e t r e a te d *( % ) ( % )S u c r o s e 5.2 1.1G l u c o s e 3.1 6.6F ru ct o se 3.4 6.7T o t a l s u g a r 11.7 14.4* A mi xt u re o f ce l l u l ase (5100 uni ts) a n dh e m i - c e l l u l a s e (53 units) w a s a d d e d t o 1 k go f g r o u n d p i n e a p p l e , a n d t h e t r e a t e d m a -t e r ia l w a s k e p t t o r o o m t e m p e r a t u r e f o r 24 h.

A lco holfrom pineapple wastet r e a tme n t s , s u c h a s h e a t , a c id a n d a lk a l i , m ig h t r e l e a s e mo re s u g a r . T e w a r i et al.(1 9 8 7 ) r e p o r t e d th a t h e a t a n d a c id s a c c h a r i f i e d p in e a p p le w a s te u p to 8 0 %; w h e re a so n ly 5 7 % s a c c h a r i f i c a t io n w a s a c h ie v e d b y c e l lu l a s e t r e a tme n t , T h e e n z y met r e a t m e n t a l s o h y d r o l y s e d s u c r o s e a n d i n c r e a s e d t h e g l u c o s e a n d f r u c t o s e l e v e l s .

F i g u r e 1 d e p i c t s t h e c h a r ac t e r is t i cs o f p i n e a p p l e f e r m e n t e d b y S. cerevisiae a n dZ. mobilis. A l mo s t a ll o f t h e f e rm e n ta b le s u g a r in i t i al ly p re s e n t ( a b o u t 1 4 . 7 % o ft h e f e r m e n t a t i o n m a s h ) w a s c o n v e r t e d t o a l c o h o l a n d c e l l m a s s . S. cerevisiaep r o d u c e d a b o u t 8 % a l c o h o l i n 2 4 h , w h i l e Z. mobilis p r o d u c e d 7 . 6 % a l c o h o l i n4 8 h : t h e s e a l c o h o l c o n c e n t r a t i o n s s t a y e d t h e s a m e t h r o u g h o u t t h e 7 2 h f e r m e n t a -t i o n p e r i o d . T h e f e r m e n t a t i o n c h a r a ct e r is t i cs o f th e b o t h o r g a n i s m s w e r e s i m i la r .I n b o t h c a se s, a b o u t 9 8 % o f th e t o t a l f e r m e n t a b l e s u g a r w a s c o n s u m e d , a n d t h ea l c o h o l y i e l d w a s a l i t t l e m o r e t h a n t h e t h e o r e t i c a l m a x i m u m , p r o b a b l y b e c a u s eo f th e p re s e n c e o f o th e r f e r me n ta b le s u g a r s , b e s id e s s u c ro s e , g lu c o s e a n d f ru c to s e ,i n t h e f e r m e n t a t i o n m a s h . T h e p H o f t h e f e r m e n t a t i o n m a s h s t a y e d t h e s a m et h r o u g h o u t f e r m e n t a ti o n f o r b o t h o r g a n is m s . A d d i t i o n o f e xt ra n i t r o g e n o rp h o s p h o r u s w a s n o t n e e de d f o r g r o w t h o f th e o r g a n is m s . T h e a l c o h o l p r o d u c t i o nra t e , e x p re s s e d a s v o lu me t r i c p ro d u c t i o n e f f i c i e n c y (V P E ) , w a s h ig h e r (4 . 7 - fo ldf o r S. cerevisiae a n d 2 . 7 - fo ld fo r Z. mobilis) f o r h i g h - s o l i d f e r m e n t a t i o n t h a n f o rl o w - s o l i d f e r m e n t a t i o n ( T a b l e 3 ) . S i n c e t h e V P E c o m p a r e s a l c o h o l p r o d u c t i o n p e ru n i t t i m e w i t h t h e u n i t f e r m e n t e r v o l u m e , i t d e t er m i n e s t h e s iz e o f f e r m e n t e rn e e d e d a n d h a s a l a r g e i m p a c t o n f e r m e n t a t i o n c o s t .

S o l id s r e ma in in g a f t e r f e rme n ta t io n c o u ld b e u s e d a s a n ima l f e e d b e c a u s efe rme n ta t io n in c re a s e s th e i r n u t r i t i o n a l v a lu e (p ro te in , a min o a c id s , v i t a min s ,e t c. ) T o t a l u t i li z a t io n o f t h e f e r m e n t a t i o n p r o d u c t i n c re a se s t h e e c o n o m i c f e a s i bi l it yo f a l c o h o l p r o d u c t i o n f r o m p i n e a p p l e w a s t e .C o n c l u s i o nP i n e a p p l e w a s t e h a d a r e l a t iv e l y l o w c o n t e n t o f s u g a r s f o r a l c o h o l f e r m e n t a t i o n .T h e r e f o r e , p r e - t r e a t m e n t t o i n c r e a s e t h e s u g a r l e v e l w a s n e e d e d . F e r m e n t a t i o n

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L . B a n -K o f f a n d Y . IV . H a nTable 3. Compar ison of alcohol yield by h igh- and low-sol id fermentat ion.Organism Total sug ar Alcohol VPE*

( % , w / v ) ( % , w/ v ) (g / I / h )S. cerevisiae

High-so l id1" 14.7 8.1 3.37L o w - s o l i d ~ 1 0 .2 3 . 5 0 . 7 3

Z. mobilisHigh -sol id ' l " 14.7 7.6 1.66Low - s o l i d~ t 10 .2 3 .0 0 .62

* V o l u m e t r i c p r o d u c t i o n efficiency.1 H i g h - s o li d f e r m e n t a t i o n m a s h c o n t a i n e d a b o u t 2 5 % ( w / v ) d ry m a t t e r.:1: L o w - s o l i d f e r m e n t a t i o n m a s h c o n t a i n e d a b o u t 1 0 % ( w / v ) d r y m a t t e r .w i t h a h i g h - s u b s t r a t e c o n c e n t r a t i o n a l s o i n c r e a s ed t h e a l c o h o l y i e ld . O n c e a ne f fi c ie n t p r e - t r e a t m e n t m e t h o d i s d e v e l o p e d p i n e a p p l e w a s t e c o u l d b e u s e d f o r t h ee c o n o m i c p r o d u c t i o n o f a l c o h o l. U s i n g r e s i du e s a s a n i m a l f e e d f u r t h e r i n c re a s e st h e e c o n o m i c f e a s i b i l i t y .

AcknowledgementT h i s i n v e st i g at i o n w a s s u p p o r t e d b y th e U S D A , O I C D , C o c h r a n M i d d l e I n c o m eC o u n t r y P r o g r a m a n d U S D A , S o u t h e rn R e g i o n a l R e s e ar c h C e nt er .

ReferencesANON, 1980 Fuel from Farms: A Guide to Small-scale Ethanol Production, S o l a r E n e r g yResearch Ins t i tu te Repor t No . SERI /SP-451-519 UC-61 . Golden , CO: SERI .BU'LOCK, J .D . 197 9 Indus t r i a l a lcoho l : In Microbial Technology: Cu rrent State, FutureProspects. Symposium of the Society o r Genera l Microbiology, eds . Bu l l , A .T . , E l lwood , D .C .& Ra t ledge , C . Vol . 29 , pp . 309-325 . Cam br idge : Cam br idge Univers i ty P ress .BURBANK, N.C. & KAMAGAI, J .S . 1965 S tudy o f p ineapp le cannery was te . Proceedingsof the 20th-lndustrial W aste Conference, Engin eer ing E x tens ion Ser ies 118 , Vol . 20 , pp .

365-397 . Purdue Univers i ty .D E V , D .K . & I N G LE , U .M . 1 9 8 2 U t i l iz a t io n o f p i n e a p p l e b y - p r o d u c t s an d w a s t e s - - r e -v iew. Indian Food Packer 36, 15-22.GOERING, H .K . & VAN SOEST, P .J . 1970 Forage Fiber Analysis. A g r i c u l t u r e H a n d b o o kNo. 379 , p . 20 . Uni ted S ta tes Dep ar tme nt o f Agr icu l tu re , Wash ing ton , DC : A gr icu l tu ra lResearch Service.HAN, Y.W. & CHO, Y.K. 1983 Ef fec t o f gamm a-ray i r rad ia t ion on a lcoho l p rodu c t ionf r o m c o r n . Biotechnology and Bioengineering25, 2631-2640.HERTZMARK, D., RAY, D . & RICHARDSON, S. 1981 Economic Imp act of Ethanol Fuelfrom Crops, S o l a r E n e r g y R e s e a r c h I n s t i t u t e R e p o r t N o . S E R I / T R - 7 3 4 - 1 3 2 0 . G o l d e n ,C O : S E R I .JONES, R.P., PAMMENT,N . & GREENFIELD,R.F. 1981 Alcoho l f e rmenta t ion by yeast sthe e f fec t o f env i ronmenta l and o the r va r iab les . Process Biochemistry 16, 42-49.MUTTAMARA,S. & NIRMALA,D .J . 1 9 8 2 M a n a g e m e n t o f i n d u s t r ia l w a s t e w a te r i n d e v e l o p -ing na t ions . Proceedings of International Symp osium (Alexandr ia , Egyp t , M arch 1981) . edsS tuckey , D . & Ham za , A . pp . 445-452 . Oxfo rd : Pe rgam on Press .PERRIN, C .G. 1953 Rap id m odi f ied p roced ure fo r the de te rm ina t ion o f Kje ld ah l n i t rogen .Analytical Chemistry 25, 968--971.R O GE R S, P .L . , L E E , K . J . & T R I B E , D .E . 1 9 7 9 K i n et i c s o f a l c o h o l p r o d u c t i o n b yZymomonas mobil# a t h igh sugar concen t ra t ions . Biotechnology Letter s 1, 165-170.TEWARI , H .K . , MARWAHA, S .S ., RUPAL, K . & KENNEDY, J .F . 1987 Bio-u t i l i za t ionof p ineapp le was te fo r e thano l genera t ion . In W oo d and C ellulosics: Industria l utilizationbiotechnology, structu re and p roperti es, eds. Kenn edy , J .F . , Ph i l l ips , G .O . & Wi l l i ams , P .A .pp . 251-259 . Ch iches te r : E l l is Ho rwo od .WEISZ, P.B. & MARSHALL, J . 198 0 Fuel fro m Biomass. A Critical Analy sis of Technologyand Economics. N e w Y o r k : M a r c el D e k k e r .

(Received 22 January 1990 ; revised and accepted 26 Ma rch 1990 )

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