influence of media composition on xylitol fermentation by

8/6/2019 Influence of Media Composition on Xylitol Fermentation By

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B I O T E C H N O L O G Y L E T T E R SV o l um e 1 7 N o . l l ( N o v .1 9 9 5 ) p . 1 2 2 3 - 1 2 2 8r e c e o v e d 2 0 t h S e p t e m b e r .

I N F LU E N C E O F M E D I A C O M P O S I T IO N O N X Y L I T O L F E R M E N T A T I O N B YCandida gui l iie rmon di i U S I N G R ES P O N SE S U R F AC E M E T H O D O L O G Y*I.C. RO BE RT O 1, S. SA TO 2 I .M. de M A N C I L H A TM,M.E.S . TAQUEDA3

1Dept of Biotechn ology-FA EN QU IL, 12600-000, Lorena, S.P., 2Faculty of PharmaceuticalScien ces and 3Polytechnic School, D ep of Chemical Engineering, U SP-S~o Paulo, S.P.4 DeptO o f Foo d Engineering, UF V, Vi~osa, M.G., Brazil

S U M M A R Y

The bioconversion of xylose to xyli tol by the yeast C a n d i d a g u i l l i e r m o n d i i FT I20037 wa s evaluated under different nutri tional condit ions using rice straw hem icellulosehydrolysate. Statistical designs w ere used to dete rmin e the fermentation m ediu mcomposit ion. Am mo nium sulfate and rice bran have be en identified as required nutrients inthe hydrolysate since there wa s a significant interaction betw een them. In the presenc e o fbo th nutrients, th e xylitol yield factor (Yp/s) and volum etric productivities (Q p) w ere 0.68g/g and 0.54 g/L.h, respectively.

I N T R O D U C T I O N

Rice straw contains a significant portion o f hemicellulose fraction that can be easilyhyd rolyze d to its constitue nt carbohydrates. Th e hydrolysis product contains a mixture o fsugars w ith xylose as the ma jor compo nent (Robe rto et a l . , 1994 ). In rece nt years, severalstudies have been performed aimed at the utilization of the xylose-rich hemicellulosicfraction as a substrate for the production of ethanol (Roberto et a l . , 1991, Lawford andRousseau, 1993), microbial protein (Meyer et a l . , 199 2), acetic acid (Brownell and N akas,1991) and xylitol (Strehaino and Dupuy, 199 0, Felipe et a l . , 1993, Rober to et a l . , 1994,Rober to e t a l . , 1995 ). The lat ter represents a product of w ide application in the food,pharmaceutical and de ntal industry ow ing to its equivalent sweetining pow er to suc rose andits anticariogenic prope rties (Bar, 198 6, Pep per and O linger, 198 8). Many yea st species areable to c onvert xylose to xylitol with go od yield (G ong e t a / . , 1983, Barbosa et a l . , ] 988).The yeast C a n d i d a g u i l l i e r m o n d i i has bee n frequently used to study this bioconve rsion in

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synthetic me dium due to i ts h igh performance (Meyrial e t a l . , 1991 , Nolleau and Preziosi-Belloy, 1993). W e have rece ntly repo rted the feasibil ity of con vert ing xylose to xyli tol by C.g u i l l i e r m o n d i i FTI 2 0037 using rice straw hydrolysate (Robe rto e t a l . 1994) . How ever , nostudies of the effect of nutri t ional condit ions have been invest igated yet . Normally,hemicellulosic hydrolysate.are supplem ented with the same nutrients em ployed for syntheticmedia. T he presen t repor t a ims at def in ing the m edium components for the product ion ofxylitol using th e rice straw hem icellulose hydrolysate.

Factorial designs have bee n largely emplo yed for th e optimization of thefermentat ive process due to the possibi li ty of studing several variables w ith a reduc ednum ber of experiments. T his me thod ology also al low s one to verify the individual factoreffects an d their interactions for the response. In this paper, w e ha ve studied the effects ofnutrient addit ions on rice straw hydrolysate for xyli tol production by the yeast C.g u i l l i e r m o n d i i using response surface m ethodology.

M A T E R I A L A N D M E T H O D SM i c r o or g a n i sm a n d I n o c u l u m P r e p a r a t io n

C a n d i d a g u i l l i e r m o n d i i FTI 20037, selected by Barbosa e t a l . , 1988 was used forthis study. Cultures w er e maintained at 4C o n malt extract ag ar slants. Th e inoculu m w asprepared by grow ing cells in 125-mL E rlenmeye r f lasks containing 50 m L o f medium w iththe fol low ing com posit ion in g/L: xylose, 20; 0 q I - I 4 ) 2 S O 4 , 3 ; CaCI2 x 2 H 2 0 , 0.1 and ricebran, 20. Incub ation wa s at 30C o n a rotary shaker at 200 rpm for 24 hours. Th e culturesw ere inocu lated to obtain an ini tial cell conce ntrat ion of 0.5 g/L.

H e m i c eH u l o s e H y d r o l y s a teRice straw hemicellulose hy drolysate wa s obtained by acid hydrolysis according tothe method descr ibed by Rober to e t a l . , 199 5. The con centrated hydrolysate wa s o ver-titrated with NaOH (pellets) to a pH 10 and adjusted to 5.0 by sulfuric acid. (72%w/w).After each change in pH, the precip ita te wa s rem oved by cen t ri fugat ion at 1000 g for 15min. The chemical com positio n o f rice straw hydrolysate w as in g/L: xylose, 79.3; glucose,22.6; .... t~:._,o.,_,_,nose, 13 .4; furfura l, 0.13 ; hy drox ym ethylfu rfural, 0.5 1 an d ace tic a cid, 1.82.

F e r m e n t a t i o n C o n d i t i o n sFerm entat ion wa s carried out unde r semiaerobic cond it ions using 125-mLErlenmeyer flasks c losed wi th co t ton-w ool p lugs, contain ing 50 mL of m edia prepared wi thhydrolysate supp lem ented by differe nt nutritional com binations. T he ferm entation s w ereperform ed at 30C on a rotary shaker at 200 rpm fo r 72 hours.

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E x p e r i me n ta l D e s i g n s a n d S ta t i s t ic a l A n a l y s i sA 23 factorial design (Box e t a l . , 1978 , Barros e t a l . , 1995)) was used to s tudy the

effec t o f the combina t ion of m edium com ponents on xy li to l p roduc t ion . R ice b ran ,am mo nium sulfate and calcium chloride wer e selected as experimen tal factors . Th e levels ofthese factors were 0 and 20 g/L; 0 and 3 g/L and 0 and 0.1 g/L, respectively. Theexperimental data were analysed by the response surface regress ion procedure (Statis t icasoftware). This meth od is based upo n the use o f the polynomial m odel:Y = bo + X biXi + X bijXiXj + eiwhere Y is the response variable (dependent); b , the regress ion coeffic ients , X, theinde pen den t variables o r experimental fact or levels and e, experimental error.A n a l y t i ca l M e t h o d s

Xylose and xy l itol concen tra tions were de te rmined by H PLC as descr ibed prev iously(Rober to e t a l . 1995).

R E S U L T S A N D D I S C U S S IO NTable 1 shows the dis tribuition of the factor levels in the experiments accord ing to a

23 design and the data for the response under s tudy. The results indicate that C a n d i d ag u i l l i e r m o n d i i was able to a ccum ulate xylitol in a ll the m edia s tudied. Ho we ver, the am ountof xy l i to l fo rmed and xy lose consumption were s t rongly dependent on the mediumcomposition.

When the hydro lysa te was supplemented wi th ammonium su lfa te and r ice b ran(medium 6) the va lues o f xy li to l concen tra t ion and xy lose consumption were 39 .3 g /L and90 % , respectively. These results decrea sed to 30 g/L and 74 % (med ium 3), w hen thesenutrients w ere om itted from hydrolysate. Such results dem onstrate the importance of thepresence of both nutrients in the medium for the bioconvers ion of xylose to xylitol by 6".g u i l l i e r m o n d i i . The effect of nitrogen sources on xylitol prod uction by this yeast have beenwel l documented (Barbosa e t a l . , 1988 ; Silva e t a l . , 1994) . Accord ing to Barbosa e t a l .(1988) small increases in xylitol yields w ere observed by the addition of casam ino acids oryeast extract in the fermentation medium . This observation agrees w ith our results s ince ricebran and yeast ex tract have s imilar com positions (Miller and Churchill 1986).

Th e addition of the calcium chloride to the rice s traw hyd rolysate did not pro m oteany positive effect on the param eters evaluated. Since in synthetic med ium the prese nce ofthis compound is essential for the cell fisiological functions these results suggest thatcalcium ch loride is a lready present in this hydrolysate .

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T a b le 1 - E x p e r ime n ta l F a c to r i a l De s ig n a n d R e s u l ts .M e d i a Xl a X2 X3 XY ~ lb X y lo s ~P r o d u c e d ( g / L ) C o n s u m p t i o n ( % )

t - - 30.21 732 + 35.35 803 + - 30.94 744 + + 37 .77 845 - + 36.72 886 + + 39 .34 907 + + 35.91 868 + + + 40 .05 869 0 0 0 37.70 78

aX1 = Rice bran; X2 = Calcium chloride; X3 = Am mon ium ulfatebAveragevalues o f triplicatesT h e e x p e r ime n ta l r e s u l ts p r e s e n t e d i n T a b le 1 w e r e u s e d t o e s t ima te th e m a in e f f e c t s

o f v a r i a b le s a n d t h e i r i n t e r a c ti o n e f f e c ts . T h e r e g r e s s io n c o e f f i c i e n ts f o r a p o ly n o mia l mo d e lin o r d e r t o p r e d i c t a n d q u a n t i f y t h e l e v e l o f t h e f a c to r s o n t h e x y l i t o l p r o d u c t io n a n d x y lo s ec o n s u m p t io n wa s a l s o e s t ima te d . T h e e f f e c t s f o r a mm o n iu m s u lf a te a n d r i c e b r a ns u p p le me n ta t i o n we r e p o s i t i v e a n d s ig n i f i c a n t ( T a b le 2 ) . T h e mo d e l a l s o i n d i c a t e s t h ee x i s t e n c e o f a n e g a t iv e i n t e ra c t i o n b e tw e e n th e s e f a c to r s . T h e a n t a g o n i s ti c e f f e c t o f t h ein t e r a c ti o n b e twe e n a m mo n iu m s u l f a t e a n d r i c e b r a n i n d i c a t e t h a t s imu l t a n e o u s i n c r e a s e s inth e l e v e ls o f t h e s e f a c to r s d o n o t i n c r e a s e x y l it o l p r o d u c t io n a n d x y lo s e c o n s u m p t io n b ya m o u n t s a s l a r g e a s t h o s e e x p e c t e d b a s e d o n s e p a r a t e in c r e a s e s in t h e s e f a c to r s .

T h e a n a ly s is o f v a r i a n c e p r e s e n t e d i n T a b le 3 d e m o n s t r a t e s t h a t t h e p o ly n o m ia lmo d e l is a d e q u a t e f o r d e s c r ib in g t h e r e l at i o ns h ip s b e twe e n th e r e s p o n s e u n d e r s t u d y a n d t h ee x p e r ime n ta l f a c to r s. T h e r e g r e s s io n m o d e l w a s s ign i fi c an t ( p


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T a b l e 3 - A n a l y s is o f V a r i a n c e f o r t h e R e g r e s s i o n M o d e l s o f X y l i t o l P r o d u c t i o n a n d X y l o s eC o n s u m p t i o n .

S o u r c e D e g r e e o f F r e e d o n S u m o f S q u a re sX y l i t o l X y l o s e

M o d e l ( r e g r e s s io n ) 7 2 7 2 . 5 6 7 9 6 . 9 6R e s i d u a l ( e r r o r ) 1 6 3 6 . 6 2 1 2 6 . 6 7T o t a l 2 3 3 0 9 . 1 8 9 2 3 . 6 2

F - r a t i o 1 7 . 0 1 1 4 . 3 8p - v a l u e 0 . 0 0 0 0 0 . 0 0 0 0

R 2 0 .88 0 .86Tests for curvature using cen ter points showed that squared terms are not im portant for these m odels.

S i n c e t h e c a l c i u m c h l o r i d e c o n c e n t r a t i o n i s n o t i n v o l v e d i n a n y s i gn i f ic a n t t e r m s o ft h e m o d e l s , re s p o n s e s u r fa c e s c a n b e e x p r e s s e d u s i n g o n l y t h e r i c e b ra n a n d a m m o n i u ms u l fa t e va r ia b l es . F i g u r e 1 s h o w s t h e r e s p o n s e s u r f a c e o f x y l it o l p r o d u c t i o n a n d x y l o s ec o n s u m p t i o n a t ta i n e d w i t h t h e r e g r e s s i o n e q u a t i o n : y ~ = 8 2 . 6 2 + 2 .3 8 X ~ + 4 . 7 1 X 3 -1 .8 8 X ~X 3 a n d y 2 = 3 5 . 7 8 + 2 . 3 4 X ~ + 2 . 2 3 X 3 - 0 . 66 X ~ X ~ w e r e y~ a n d y 2 a r e p r e d i c t e d x y l o s ec o n s u m p t i o n a n d x y l i to l p r o d u c t i o n , r e s p e c t i v e ly , a n d X I a n d X 3 a re c o d e d l e v el s f o r r ic eb r a n a n d a m m o n i u m su l fa t e, r e sp e c t i v e ly . T h e c o n t o u r p lo t s s h o w t h e b e h a v i o u r o f t h ex y l it o l c o n c e n t r a t i o n a n d x y l o s e c o n s u m p t i o n b y C.guil l iermondii w h e n a m m o n i u m s u lf at ea n d r i c e b r a n a r e a d d e d i n t h e h y d r o l y s a t e .

81 56: : , O

f " ~ . f , , ~

F i g u r e 1 - I n f l u e n c e o f a m m o n i u m s u l fa t e a n d r ic e b r a n c o n c e n t r a t i o n o n t h e x y l i to lp r o d u c t i o n ( A ) a n d x y l o se c o n s u m p t i o n (B ) b y C . guil l iermondii .

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C O N C L U S I O NThe com pos i t ion o f f e rmen ta t i on med ium us ing r i c e s t raw hyd ro ly sat e a s a subs tr a terequi res r ice bran and am m onium sul fa te supplementa tion . Ho wev er , the opt im um leve ls o fthese nut r ien ts can be de termined through addi t iona l exper iments . F ina l ly , the responsesu rf ace m e thodo logy cou ld be app l ied t o t he op t imiza ti on o f o the r f ac to rs , such a s pH ,aera t ion leve l and inoculum concent ra t ion which may a l so p lay impor tan t ro les in th i s

b ioconvers ion .A C K N O W L E D G M E N T

The authors acknowledge f inanc ia l ass i s tance f rom CNPq/Braz i l and Dr Roy E.Brun s fo r the he lpfu l rev is ion o f th i s paper .

REFERENCES

1. B ar, A. (1986). Alternative Sweeteners. In: Xyli tol, O 'Brein N abors, L. ;Gelardi ,R. , eds , Marce l D ekk er Inc , N ew Yo rk , Ch apter 10 , pp . 185-216.2. Ba rbosa, M.F.S. , Me deiros, M.B., M ancilha, I .M ., Schneider , H. and Lee, H.(1988) , d.Ind. M icrobiol., 3, 241-251.3. Barros Nero, B. , Scarminio, I .S. , Bruns, R,E. (1995). Planejamento eOtimizaqao de Experimentos. Campinas : ed i tora da Unicamp.4 . Box , G.E.P . , Hu nter W .G. and Hu nter , J .S . (1978). Statistics for Experimenters,W iley and Sons , New York .5. Bro wn ell , J .E. and Na kas, J .P. (199 1), lnd. Microbiol. , 7, 1-6.6 . Fd ipe , M .G.A. , Manci lha , I .M . , Vi to lo , M . , Ro ber to , I .C . an d Si lva , S .S . (1993) ,Arquivos d e Biologia e Tec nologia, 36(1 ) , 103-114.7 . Gon g, C.S ., ClaypooI, T .A. M c Cracken , L .D. , M aun, C.M. , Ueng, P .P . an dTsao, G.T. (1983), Biotechnol Bioeng., 25, 85-102.8 . Law ford , H.G. and Rou sseau , J .D. (1993), Biotechnol. L ett., 15(5), 505-510.9. M eyer, P.S. , du Preez, J .C. and Kil ian, S.G. (199 2), Syst . AppZ MicrobioZ,15(1), 161-165.10. M eyrial, V . , Delgenes, J .P. , M olet ta , R. a nd Nav arro, J .M. (1991), Biotechnot.Lett. 13(4), 281-286.11. M il ler , T.L . , Churchil l, B .W (1986). Substrates for large-scale fermentation. In :M anua l o f Indus t ri a l M ic rob io logy and B io t echno logy , A .L . Dem a in and N .A .Solom on, eds ., W ashington: Am er ican Socie ty of Microbio logy , pp . 130-131.12. N olleau , V. , Preziosi-BeU oy, L. , Delgenes, J .P. an d Nav arro, J .M . (1993), Curr.MicrobioL, 27, 191-197.13. Pepper, T. and O linger, P.M. (1988), Food TechnoL, 42(10), 98-106.14. R ob erto, I .C. , Lacis , L., Barb osa, M .F.S. and Mancilha, I .M. (1991), ProcessBiochem., 26, 15-21.15. R obe rto, I .C. , Fel ipe, M.G .A., M ancilha, I .M., V itolo, M., Sato, S. and Si lva,S.S. (1995), Biores Technol., 51, 255-257.16. R ob erto, I .C. , M ancilha, I .M., Souza, C.A., Felipe, M .G.A ., Sato, S. and Castro,H.F. (1994), Biotechnol. L ett., 16(11), 1211-1216.17. Si lva, S.S. , M ancilha, I .M., Qu eiroz, M .A., Fel ipe, M .G.A., R ob erto, I .C andVi to lo , M. (1994) , d. Basic Microbiol. , 34(3), 205-208.18. Strehaino, P. and Dupuy, M.L. (1990), French Patent 2,641,545.

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influence of media composition on xylitol fermentation by

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