Enzymic saccharification of some pretreated agricultural wastes

Zentra lhl . iIlikrobiol. 143 ( 19 ~ 8), 55 --62 VEB Gust av E ischer Verlag .Iena [B ot any and Nutrition D epartment, Wom en's College, Ain Sh am s Univ ersity, H eliopolis, Cairo, Egypt]

E nzymic Saccharification of Some Pretreated Agricultural Wast es SAWSAN 1\1. A. EL-GAi\Il\IAL and MONA A. SADEK

w iu, 3 Fig nres Summary Cellu losic wastes, artichoke lea ves an d stalks, sugar-cane bagasse a nd fennel seeds after ex t ra ct ion of essent ial oils were treated wit h various con cent rati ons of peracet ic acid at 100 °0 , 60 °0 a nd room temp era ture severa l tim es, wash ed with water a nd ethanol and air dri ed. The d egree of enzy mat ic solub ilization of each t reated cellulosic wast e was meas ure d wit h A sp ergill us nig er cellulase (E ndo -l ,4-B -Gluc anase; 1,4-(1,3 ; 1,4)-P-D-glucan 4·glucanohydrolase ; EO 3. 2.1.4). Artichoke waste and suga r-ca ne bagasse were solubilized more effect-ively by the en zymethan fenne l wast e. Data are presen ted describi ng t he effect of ti me, enz yme a nd subst ra t e co ncentration on th e rat e of en zymic hydrolysis. I nfrared spect ra of t he t reated a nd u ntrea ted cellulosic materials were recorded.

Zusammenfassung Zell uloseabfiille, wie Art.ischo ckenbla tter und -stengel, Zu ekerohrbagasse und F enchelsam en na eh Extraktion del' esse nt iellen Ole wurden mit verschi ede uen K onzent rat ion en v on P eressigs a ure bei 100 °0 ,60 °0 u ud Raumtemperat.ur und verschi edenen Zeit en behandelt, a nseh liel.lend mit \ Vasser und At.hanol gewa sch en und luft getrocknet. D el' Grad del' en zyma t ischen L osun g del' beh andelt en Zellulosea bfiille wurd e mit del' Aspergillus nig er-Zellulase (E ndo-I, 4-B-Glukanase: 1.4-{1 ,3;1,4 )-t/-D -Gluka n -4-Gluka nohydrolase ; Ee 3. 2. 1.4) gem essen . Art ischocken - un d Zucker. roh rabfa lle lost.en sich dureh das E nzym bessel' a ls die del' Fe nch elsam en. Die E rgebn isse verd eut lich en einc n Einflu13 del' Extraktionszcit , des Enzy ms und rler Substratkonzent ration a uf d ie enzymatische H ydrolyse. Von dem behand clten u nd unbehandelt en Zell ulosema t erial wu rde da s I nfrarot spektrum ermittelt.

Th e lignocellulosic mat er ials represent the largest ren ewable source of potentially fermentable carbohydrates on eart h . The chemically unreact ive nature of cellul ose, parti cula rly its resi stance to h ydr olysis, is a major obstacle to inc reased lignocellulose ut iliza tion as fu el an d feed so urce. T he obs tacles to enzym ic sacc ha rifica tion are ligni n/ he mice llulose matrix, cellulose crystallinity an d su rfa ce a rea (LEE et a!' 1982 ; GHAItPVRAY et a!' 1983}. For t his p ur pose a n umber of mechanical a nd che mical p retreatments (TASSINARI et a!' 1982 ; OOSHIMA et al, 1984 ; MIRON a nd BE N-GHEDALIA ] 981 ; }'ARID et a!' ] 984) were dev ised to increase t he yie lds of hy drolysis suga r fr om cellulose. Some pret reatm ent s combin e both physical and chemical effects (GHOSE et a!' HI83). The two competi ng hydrolysis rout es are acid and enzymatic hydrolysis. E nzy matic hydrolysis is m uch more lik ely to preserve int a ct ot.he r plan t com ponents t ha n acid h ydrolysis, thou gh hy dr olysis rates for enzymes t end to be slowe r an d more ex pe nsive (P EREZ et a l. HJ80 ). P retreatments are req uir ed for effect ive enzyma tic hyd rolysis, bu t are not essent ial fo r ac id hydrolysis (R AO et al. 198:1). It is possible, indeed lik ely , t hat pret reatment s wh ich are effe ctive to one ra w mat erial a re not

56

S. AI. A. EL-G_nnrAL and

}I. ~\.

SADEK

equally effective to other materials. Hence optimization of pretreatments as well as selecting optimum conditions for effecient enzyme hydrolysis for specific raw material is required. However, this study investigates the effect of peracetic acid treatment to some cellulosic wastes saccharification with cellulase from Aspergillus niger with the aim of producing sugars for subsequent use in single cell protein production for animal feed. Various parameters such as substrate and enzyme concentrations were also examined during hydrolysis.

Materials andMethods Enzyme The enzyme preparation was Aspergillus niger cellulase [Endo-l,4-B-glucanase; 1,4-(1,3;1,4)P-D-glucan 4-glucanohydrolase; EC. 3.2.1.4], obtained from Sigma chemical company. The activity was 0.9 unit mg- I solid (one unit is equal to one micromole) produced asD-glucose from cellulose at pH 5.0 at 37°C.

Substrate Cellulosic wastes (artichoke leaves and stalks, fennel seed waste, after extraction of essential oils and sugar-cane bagasse of a length of 0.4-0.6 em) were used as substrates for the enzymatic hydrolysis in all experiments.

Chemical analysis of cellulosic waste Moisture content, ash, ether extract, crude fibre and crude protein in cellulosic waste materials were determined according to the A.O.A.C. (1980). Total sugar was determined after inversion, using cone. HCI at 69. oC for 5 min. The resulted reducing sugars were measured after neutralization by SHAFFER and HARTMANN method, A.O.A.C. (1980).

Peracetic acid treatments Mixtures of acetic anhydride, hydrogen peroxide (30 %) and water in different ratios were prepared. 1 g of the cellulosic material was suspended in 10 ml peracetic acid and then heated at 100 DC for 30 min. The sample was filtered and washed with water until acid-free (pH 6.5-7.0), then suspended in a small volume of ethanol, filtered and air dried. The effect of treatment time and temperature was also investigated.

Enzymic hydrolysis Enzymic hydrolysis was carried out in 50 ml flasks containing 4.5 ml sodium acetate buffer 0.05 M, pH 5.0, 100 mg cellulose substrate and 0.5 ml of the enzyme solution (20 mg crude enzyme ml- I ) . The samples were incubated at 37 DC on a rotatory shaker at 120 rev/min for 24 h. Samples were centrifuged to remove solids and the supernatants used for analysis. Sugar yield upon hydrolysis was calculated as gig dry substrate: (reducing sugar mg ml- I ) I(initial wt. of drysubstrate mg ml l}. Reducing sugars were measured as D-glucose with the help of the SOMOGYI (1952) and NELSON (1944) procedure. r

Resultsand Discussion Chemical analysis of cellulosic wastes Table 1 presents the chemical constituents of sugar-cane bagasse, industrial fennel waste and artichoke waste. It is clear that these wastes contain a high percentage of crude fibres which could be hydrolyzed enzymatically to utilizable sugar. Additionally, a relatively high protein content in artichoke waste and the high total sugars in sugar-cane bagasse may maximize their values, assuming that an applied pretreat-

Enzymic: :-hwc:harific:ation of Some Pretreated Agricultut-a l Wastes

57

Table 1. Chemical constituents of bagasse, fennel and artichoke waste (calculated on the wet weight. basis)

% calculated on

the wet weight basis

Contents

Sugar-cane bagase

Fennel waste

Artichoke waste

Moisture Crude Fibers Total sugars Protein Ether extract Ash

5.12 46.85 15.46 0.79 2.77 18.38

24.40 50.88 2.13 1.31 0.133 21.247

4.50 48.92 11.02 5.40 4.60 6.96

ment has no effect on the other constituents. Cellulose fraction resembles nearly 50 % of most plant material, while in essence throwing away the remaining 50 % of the plant material is unlikely to be economically competitive. Enzymic saccharification after treatment with different concentrations of peracetic acid Table 2 presents the degree of saccharification of the pretreated wastes after enzymic hydrolysis. The term reducing sugar refers to all sugar moieties with a free reducing end-group. It is mainly the content of fJ-glucosidase in the enzyme mixture that influences this value. The reducing sugar yield is important, since yeast can only ferment monomers to ethanol. Table 2. Effect of peracetic acid treatment on enzymic degradation of cellulosic material Reducing sugar yield in 24 h gjg solids Treatment

Sugar-cane bagasse

Fennel waste

Artichoke waste

Untreated peracetic acid

0.135

0.042

0.095

Hydrogen peroxide (30 %) : acetic anhydride: H 20

(vjvjv) 0.8 1.5 3.3 5.0

: 0.8 : 1.6 : 3.3 : 5.0

: 8.4 : 6.8 : 4.6 : 0.0

0.190 0.22 0.330 0.425

0.145 0.140 0.280 0.380

0.315 0.330 0.380 0.470

The high concentration of peracetic acid treatment for 30 min at 100°C resulted in a considerable increase in the enzymatic saccharification of sugar-cane bagasse, artichoke and fennel waste. Maximum sugar yield in 24 h gig solids was 0.47, 0.42, 0.38 for artichoke waste, sugar-cane bagasse and fennel waste, respectively. However, the residual pretreated waste could be used as animal fodder if it is digestable and not toxic. Effect of time and temperature of pretreatments In order to save energy, avoid severe corrosion caused by acid at high pretreatment temperature and, finally, to produce a comparatively low value of sugar, a time to obtain maximum sugar yield at a lower temperature of pretreatment was investiga-

58

S. 1\1. A. EL-G.-DDIAL and

xr.

A. SADEK

Table 3. Enzymic saccharification of cellulosic material pretreated with peracetic acid at 60 DC and room temperature for different time Sugar yield in 24 h gjg dry wt Fennel

Artichoke

Bagasse

60 DC

room temp.

60°C

room temp.

60 DC

room temp.

H 202 : acetic anhydride: H 2O 5: 5 : 0 (vjvjv) 30 min 60 min 120 min Overnight

0.130 0.148 0.280 0.360

0.118 0.190 0.242 0.472

0.0702 0.1520 0.1780 0.385

0.140 0.160 0.165 0.220

0.1525 0.229 0.3815 0.4295

0.1180 0.1270 0.1525 0.3035

H 202 : acetic anhydride: H 2O 3.3: 3.3: 4.6 (vjvjv) 30 min 60 min 120 min Overnight

0.102 0.202 0.290 0.347

0.061 0.122 0.225 0.404

0.066 0.070 0.125 0.4045

0.0765 0.102 0.102 0.229

0.1562 0.1910 0.2545 0.425

0.175 0.127 0.175 0.330

H 20 2 : acetic anhydride: H 2O 1.6: 1.6 : 6.8 (vjvjv) 30 min 60 min 120 min Overnight

7.6 mill. 0.132 0.224

0.108 0.082 0.1272 0.189

0.07 0.202 0.409

0.0700 0.0775 0.132 0.159

0.172 0.220 0.3175 0.372

0.108 0.1980 0.1780 0.2035

H 20 2 : acetic anhydride: H 2O 0.8: 0.8 : 8,4 (vjvjv) 30 min 60 min 120 min Overnight

0.142 0.163 0.2032 0.2032

0.108 0.129 0.164 0.178

0.08 0.102 0.152 0.23

0.032 0.038 0.038 0.0765

0.1525 0.1655 0.2035 0.2025

0.152 0.1510 0.1715 0.2035

ted. The results (Table 3) show that sugar formation increased with extending pretreatment time from 30 min to overnight regardless of incubation temperature. Interestingly, maximum sugar yield from artichoke waste was attained from overnight pretreated waste at room temperature. Sugar-cane bagasse and fennel waste, also pretreated overnight at 60°C, released maximum sugar yield. Effeet of i n c u b a t.i o n time, enzyme and substrate e o n oe n t.r a t i o n on enzymic saccharification of pretreated artichoke, fennel and sugarcane bagasse To find out suitable conditions for hydrolysis, sugar production of all pretreated wastes at enzyme concentrations between 1.2 and 8 FPU/ml was studied for successive periods. Aliquots of 0.2, 0.5, 1.0 and 1.3 ml of enzyme solution containing m~.3 mg enzyme ml- t were incubated with 0.2 g peracetic acid treated material for different incubation periods. At the end of each time period the released reducing sugar was measured according to the standard procedure. Fig. 1 shows the suga,r concentration (gig dry wt of waste) in the hydrolysates after enzymic hydrolysis. The curves show that the bulk of the sugar that is eventually solubilized from all sa-mples is released during the first twenty four hours of hydrolysis. Furthermore, extension of the hydrolysis time from 24 h to 48 h does not increase the degree of saccharifi-

0-0 A -A ._.

59

Enzym« Sa cch arificat.ion of :)"me Pret reat ed Agr i('ll!tllral \rust cs

6.0FP(Jmr' 10FP(Jmr' 1.2FP(Jmf-'

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Fig.!. E ffect of time and enzy me conc entra t ion on sa ech arifica t.ion of pret rea t ed wastes .

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cat ion . An increase in enzyme concent ration led to an in creas e in total conversion . R eaction velocity was also proportional t o enzy me concentrations wit hin t he ran ge (1.2-6U) . We conclude t hat 6 FPUml- J would be all ad equate en zym e concentrati on under our conditions. I ncreasing the am ount of enzy me to 8 FPUml- 1 does not p roduce a higher sugar concent ration. Thu s, high cellul ose concentra ti on may counteract sa echa rification by increasing the rate of transgly cosylat ion reaction. Thi s result agrees well wit h those of VAL LANDER and ERIKSSO N (1\:185), who concluded that t he reason for the high enzyme concentration was only a suffi ciently high p-glucosidase activity til t he enzyme mixture. Maximum sugar yi eld in 24 h (gig solid ) was 0.52, 0.47, 0.45 g for artichoke, sugar-can e bagasse and fennel waste, respectively . The observed increase can be exp lain ed by t he higher enzyme sub st rate ratio. To investigate the effect of different concentrations of the pretreated cellulosic wast es an experiment was carried out using va riou s substrate concent rat ions in the range from 1- 6 %. The released sugar concentration (gig dry wt) inc reased almost. linearly with the increase in subst rate concent rat ion t ill B%, thereafter an appreciab le decrease was noticed in all tes ted treated wastes (Fig. 2). VAL LA NDE R and ERIRSON (1985) noticed significa nt inhibition of t he hydrolysis when t he suga r con centration was 6 % or higher. There a re t wo main prod uct. inhibitors in the cellulose system : cellobiose and glucose. It is usually claimed that cellobiose is th e st ronger inhibitor

60

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1\1. A.

EL-GAlIIMAL

and

xr. A. SAD E K

To/al reducing sugar (gig dry wI. ) 0.60 r---- - - - - - - - - - r - - - - - - - - . - -- - - - - - - - - , Artichoke o

Bagasse

/

o

o

0.45

Fennel

,,0

0.30 o

2

602'"

602

Subs/rate concentration

6

(0/0)

Fig. 2. Effect of subs trate conc ent ra t ion on enzym ic sacchari ficat ion of pr etreated wast e (6 U jm l enzyme).

of the two (GHOSH et al. 1982), a nd results presented by LEE a nd FAN (1983) suggest that th e inhibitory eff ect of cellobiose is mainly exerte d on t he initial hydrolysis rat e. Ba sed on th e results presen t ed in Fig. 1 and 2, 6 FPUml-1 enzy me, 3 % cellulosic wast e, and 24 h hydrolysis time could he chose n as the standa rd condit ions for enzym e sacc ha rificat ion. Th ese studies indicat e t hat pera ceti c acid treatm ent of cellulosic wastes has good p ot ential for use in the saccha rificat ion of cellulosic wast es, arti chok e waste, suga rca ne bagasse and fennel wast e. Th e combination of this treatment with other physical treatment may produce a syn ergistic effect and, consequ ently, maximum sugar production and economy, esp ecially with fennel waste. HA N et al. (1981) reported that maximum sugar yield (0.35 g glucose/g dry fiher) was obtained from NaOH treated ga mma radiated sugar-cane bagasse aft er hydrolysis with cellula se from Aspergillus n iger. A maximum suga r yield of 0.25 gig solid was obtained after enzymic saccharification of p eracetic acid treat ed wh ea t st ra w at 100 °C for 30 min (F AK et al. 1981). Varia t ion in chemi cal compos it ion a nd structure (crystallin e or am orphous) affect the eff iciency of pretreatment and t he hydrolys is yield . _F EUCHAK et al . (1980) have sho wn the presence of a close relationship hetween crys tallinity of cellulosic substances a nd enzy mic solubilization. Th e data from the present st udy and the infrar ed spect rum (Fig. 3) of th e pretreat ed cellulosic wast es demonstrat e that the treatm en t produ ced a decrease in the crys ta llinity of the raw materials. It is d ear (Fig. 3) t hat pera ceti c acid treatmen t of t he cellulosic wast e resulted in broad ab sorption in th e region 1,600-1,700 cm: " an d abso rp tion at 1,730 cm" a nd 1720 cm " ! for pr etreated bagasse, fenn el wast e a nd ar ti chok e, respe ctively . Th ese obse rvat ions indi cate oxidation of the hydroxyl gro up t o aldehydic and/or carboxy lic group , i.e. ket oni c car-

'

,

F ig. 3:

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;t,l I

1800

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1600

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1~00

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I

2000

I

1200

I

1000

800

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500

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'tOO

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1

2

2

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Fig. 3. Infrared spectra of untreated and peracetic acid treated cellulosic materials: 1. untreated bagasse; I treated bagasse; 2. untreated fennel waste; "2 treated fennel waste; 3 unt.rea.t.ed art ichoke : 3 t.reat.ed artichoke waste.

4000

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S. 1\'1. A. EL· GAMMAL and .:\1. A. SADEK, Enzymic Sacchar ification of Some Pretrea t ed •..

bonyl vibrations (STRAU GHA K and WALKER 1976). Additi onally, th e infrared spectrum of arti choke waste showed absorption at wave-number 2800 cm-1 corresponding to (-CH) of t he ald ehydi c group, which emphasizes th e oxidation of the hydroxyl groups. The appearance of th ese new groups may cause decrease in the crys t allinity of th e t reate d compound. H ence, cellulose pretreatments ar e important for the supply of sufficiently inexpensive carbohy dra t es to permit fermentation industri es to compete effecti vely with petrochemical routes. Th e sa me products will not be deriv ed usually from starch or sugar crops because of th eir alt ernate higher valu e uses as fuel or feed. How ever, t he fermentability of the enzy mic hydrolyzates of th ese pr etreated wastes is in progress.

References

A.a A ·C.:

Association of offic ial Agri cultural Chemists. Offic ial m et h od s of analysis of the Association of official Analytical Che m ists. 12th ed, Washington, D. C. 1980. FAN, L. T., GHARPURARY, .M. M', and L EE Y. l\£.: The nature of lign ocellulosics and their pretreat. m en t s for enzymatic hydrolysis. Bi ot ech. Bioeng. Symp. no. II (1081),29. FARID, M. A., SHAKER, H. 1\1., an d EL ·D IWANY, A. 1.: Effect of p eracet ic ac id , sodium hydrox ide an d ph osph or ic acid on cellulosic m ateri al s as a pretreat m ent for en zy m at ic hydrolysi s. E nzyme l\1icrob. Techno!. 5 (1984), 421. F EREHAK, J . D ., HAYERDAL, B., an d PYE , E . K .: Enzymatic h ydroly sis of cellulose. Bi ot ech nol. Bioeng. 22 (1980), 1527. GUARPUHAY, .l\I, M.; LE E, Y. H ., a nd FAN, L. '1'. : Structural m odifica t ion of Iignocellulosics by p retreatmen t to enhance enzymic h y drolysis. Bi otechnol. B ioen g. 25 (1083 ), 157. GHOSE, T. K., P ANNIR-S ELVAlII, P . V. , a nd GHOSH, P . : Catalytic solven t delignification of agricui t ural r esidues: organic ca taly sts . B iotech . B ioeng. 25 (1983), 2577. GUOSH, P ., P AlIUfENT, N . B., an d :MARTIN, ,Y o R. B.: Enz. l\licrob. Techno!. 4, 425. c.f. B iot ech. Bi oeng. 27 (1985), 650. H AN, Y . \V., TnIPA, J., CIEGLER, A. , COURTNEY, J., CURRY, ' V. F ., an d L A::IIBRElIIONT, E. N. : The econom ic mi crobial protein s produ ced from cell ulosic waste. B iot ech . Bi oeng . 23 (1981), 2525. L EE, S. B. , SHIN, H . S. , R Yu, D . D . Y., a nd l\IANDELS, :\1.: B iot ech. Bi oeng . 24, 2137. e. I, B iot ech . B ioeng . 27 (1985). L EE, Y. H., and FAN, L . '1'. : K in et ic studies of enzy ma tic h ydrolysis of insoluble cellulose : (11) Analys is of exten de d hydrolysis ti me . Bio techn . B ioen g. 25 (1983), 939. Mmox, T. a nd B EN-GHEDALIA, D. : E ffect of h y dr olyzin g a nd oxidizing ag ent s on t he com posit-ion and degradion of wh ea t straw. B iotechn . Bio eng. 23 (1981), 2863 . N ELSON, N .: A photometric a da ptat ion of the Somogyi m eth od for t he det ermination of glucose. J. Bi o!. Ch ern. 153 (1044), 375. OOSHIl\IA, H., Aso, K. and HARANO, Y.: A method of sacchar ification of cellulose using enzyme. B iot ech . L etters 6 (5) (1084), 289 . .l:'EHEz, J., WILKE, C. R., an d BLANCU, H . 'V.: Paper presented a t t h e seco n d chemical congress of t he nor t h American con ti n ent . La s Vega s, Nevada 1980. RAO, lVI., SEETA, R., and D ESIiPANDE, V .: Effect of pret r eatment on h ydrolysis of cellulose by Penicillium Iun iculosum cell ulos e an d r ecovery of enzyme. B iotech . Bioeng, 25 (1983),1863. SOMOGYI, l\f. : Notes on sugar det ermina t ion . J . Bi oI. Che rn. 195 (105 2),19. STRAUGHAN, B. P. , and ' YALKER, S. : Spectrosco py (vol. 2). Chap m an a n d H all , London 1976, 211. T ASSINARIA, T . H.,MACY, C. F. , and SPANO, L . A .: Tech nology advances for continuo us com pre ss ion mill ing pretrea tmen t of Iign oeellulosics for enzymic h ydrolysis B iot ech. Bi oen g. 24 (198 2), 1495. VALLANDER, L., and E RIKSOX, K. : Enzymi c sacch ar ificat ion of pretreated wheat st ra w. Biot ech. Bi oeng. 27 (19 85), 650. Author s ' address : S. M. A. E L-GA1If!>IAL, Bot any a nd N utrition D epartmen t, W omen's College , Ain Sh ams U n iv er s ity, H eliop ol is, Cair o, E g yp t .