Steam explosion was found to be an effective method of improving the enzymatic hydrolysis of cellulose in the oil

palm stem. The pretreatment also caused the degradation of hemicelluloses and Tendered them water-soluble. By

aqueous extraction of the exploded material 23-31 % hemicelulosa with xylose contents up to 83% after acid

hydrolysis coultd be recovered. This paper also discusses some m0rphological changes which occurred in the

pretreatment

Ishihara, M.

Mansor, Halimathon

Sudo, K.

Universiti Putra Malaysia Institutional Repository

Pertanika 13(2),165-170 (1990)Preliminary Studies on the Steam Explosion Pretreatmentof the Oil Palm StemHAUMAHTON MANSORI, K.SUDO' AND M.ISHIHARA'IForest Resem"ch Institute tvlalaysiaKepong, 52109 Kuala Lump"", Malaysia'Wood Chemistry Department.Forest1y and Forest Products Resem'ch lnstituu, P.o. Box 16,Tsukuba, Nonn Kenkyu Danchi-Nai, Ibamki-Ken,305JapanKey words: steam explosion, oil palm stem, enzymatic hydrolysis, hemicellulose, soluble lignin,Klason lignin.ABSTRAKPel£tupan stim telah didapati sebagai suatu kaedah yang berkesan bagi meningkatkan hidrolisis emim ke atasselulosa di dalam batang kelapa sawit. Pengolahan ini juga menyebabkan degmdasi hemiseluwsa danmenjadikann_va lamt di dala.m air. Melalui pengestrakan akueus ke atas bahan yang dil£tupstimkan, 23-31 %hemiselulosa yang mengandungi xilosa sehingga 83 %, bol£h diperol£hi se!epas hidrolisis asif. Perubahan-pentbahan nunfologi yang berlaku di dalam pengolahan ini juga dibincangkan.ASTRACTSUam explosion was found to be an effective method ofimpmving the enzymatic hydrolysis of celluwse in the oilpalm stem. The pretTeatment also caused the degradation ofhemicelluloses and Tendered them wateT-soluble. B)'aqueous extraction of tlu! exploded matl!1;a~ 23-31 % hl!1nicelluwses with ,,-}'wse contents up to 83 % afteT acidhydrolysis coultl be TecoveTed. This papeT also discusses some m01f!hological changes which occurred in thepretreatment.INTRODUCTIONThe economic life of the oil palm (ElaeisguineensisJacq.) is approximately 25 years afterwhich its felling and replanting results in alarge availahility of the oil palm stem (OPS).In order to converL OPS into value-addedmaterials, studies have been conducted on itscharacteristics and properties (Killmann andUrn 1985; Lim and Khoo 1986), pulp andpapermaking (Khoo and Lee 1985) andproduction of reconstituted board (Chew andOng 1985; Rahim et al. 1987). Studies on thecarbohydarate content of OPS (Halimahtonand Abdul Rashih 1990) revealed that thislignocellulosic material provides 50-60%monosaccharides after acid hydrolysis; thus itcould also turn out to be a source of freesugars, especially glucose and xylose.Steam explosion has been known to beone of the most promising pretreatments ofvarious lignocellulosic materials in order to in-crease the rate and extent of cellulose hydro-lysis for production of sugars for fermentationor digestible fiber for use as animal feed(Schults et al. 1983; Playne 1984; Tanahashi etal. 1985; Rolz et al. 1986; Kling et al. 1987)This paper describes the investigation onthe possibility of applying steam explosion asa pretreatment technique for OPS and reportson the effectiveness of the process in terms ofenhancemen t of enzymatic hydrolysis andhemicelluloses solubilisation.HALI\1AI-rro:\' MANSOR, K. SUDa :\:'\D 1\1. ISHIHAR..-\MATERIALS AND METHODSEqllijJlllenlA batch type of stcam explosion was used inthis study. The apparatlls consists of a reactor,a collecting chamber and a steam generator,all of which are made of stainless steel. Thesteam generator can produce pressures of upto 40 kgf/cm' (Nitto Koatsu Corp., Japan).Alaterial<;The oil palm stem (OPS) used in this study'was collected from a plantation near Kepong,Selangor. The stem was divided into threeportions, namely, the bottom (about 10% fromthe base), middle (50% from the base) andtop (approximately 80% from the base). Thewood samples were air-dried for two daysbefore they \\-'ere cut into matchstick-size chips.The chips were then oven-dried (40°C) foranother two days for the pretreatmcnt.Substrate PretreatmentPretreatment by steam explosion wasconducted by heating the OPS (300g) withsuperheated steam. The lignocellulosics wereheated for a predetermined period, after whichthe treated material was exploded and releasedinto the collecting chamber by instantaneousdecompression of the reactor. The pre-treatment time, which corresponded to theresidence time of the; material in the reactor,was varied with the pretreatment steampressure. After the process, the effects of thepretreatment on hemicelluloses solubilisationand enhancement of enzymatic hydrolysis wereevaluated.Enzymatic SaccharificationThe exploded wood (200mg moisture-freebasis) was hydrolysed in a O.lM acetate buffersolution (pH 5.0, 10ml) containing 50mg of acommercial enzpne preparation, "rvleiselase-P"(Meiji Scika Co. Ltd.) derived from Tri-chordenna viride at 40'C for 48 h. Thesaccharification yield was based on the totalpolysaccharides in the treated sample. Theenzymatic hydrolysate was analysed by HPLCfor the determination of glucose and otherreducing sugars.Determination of Water Solubles and Dioxane LigninThe steam-exploded wood (I OOg) wasextracted with distilled water (300ml) at 70'Cfor 2 h. The mixture was filtered and thesolution reduced to a small volume, underreduced pressure, freeze-dried, 'weighed andanalysed by epc and HPLC. About 300m] of90% dioxane solution was added to the water-extracted sample and the mixture was heatedat 7QoC for 4 h. The dioxane extracts wereevaporated under reduced pressure andweighed.Acid Hydrolysis oJ nTafer-soluble ExtractsThi s was conducted according to the methodof Paice et al. (1982). The water extracts (5mg)were mixed with trifluoroacetic acid (2M,1.6ml) and the resulting solution was kept atroom temperature for 48 h. It was then heatedin air at 90°C for 2 h. The acid was removedunder reduced pressure and the hydrolysisproducts dissolved in distilled water andanalysed by HPLC.Detennination of Klason Lignin and HolocelluloseThe determination of Klason Lignin wasconducted according to TAPPI T222 in TAPPITesting Procedures (Anon. 1978). The contentof holocellulosc was determined by the acidchlorite method (Browning 1967).Analysis by HPLCThe relative amount of monosaccharides in theenzymatic hydrolysates and the acid hydro-lysates of the water extracts was determinedusing a liquid chromatograph (Dionex 2000i,USA) with an ion-exchange column (DionexAG-7 and AS--7, USA) and a pulsed am-perometric detector. Distilled water was usedas an eluant with a flow rate of 1.0 rnl/min.To obtain adequate sensitivity of theelectrochemical detection, 0.3N sodiumhydroxide solution was added to the columneluant ,..nth a flow rate of 1.0 ml/min.Analysis by' GPCSolutions of the water extracts (l00 ~l) wereanalysed for the presence of oligosaccharidesusing gel permeation chromatograph (Shi-madzu, LC-3A) with two connected columns166 PERTAN1KA VOL 13 NO.2, ]990STE..-\;"1 EXPI.OSION PRETR.EA.T:\IE~~rOF THE OIL I'A1.;"1 STE;"'1(Shodex !onpak KS-802, Showa Denkok KK.,Japan; length 30mm x diameter 8mm).Distjlled \\,ater was used for the elution, at aflow rate of 0.8 rnl/min. The eluant wasmonitored by refractive index.Examination by Smnning Electron MicroscopyFiber samples selected for study were mountedon brass specimen stubs. The fiber sampleswere then coated with platinum using an IonSputter JEOL JFC-llO. The samples wereexamined on a JEOL. Model JXA-840A scan-ning electron microscope with a I O~kvacceleration voltage.RESULTS AND DISCUSSIONThe analyses of the steam-exploded OPS aregIven in Table 1. Only the middle and thebOltom section of the stem were investigatedand not the top portion since the quantity ofthe latter WdS found to be insufficient for thepretreaunen1.A part of the steam-exploded fiber washydrolysed with a commercial cellulase, yield-ing a hydrolysate comprising mainly glucose(75-100%) and xylose (8-22%) (Table 3). Theresidual polysaccharides of the steam-explodedwood were highly susceptible to enzymatic at-tack compared to the corresponding untreatedwood (Table 2). Based on the percentage ofenzymatic susceptibilhy. the most efficientoperating conditions were 20 kgf/cm:? (4min)for the middle part of Lhe OPS and 25 kgf/em:? (3 min) for the hottom portion. Underthese reaction conditions, the level of enzy-matic digestibility of the steam-exploded fiberwas increased by altering the conditions ofTABl.E lEnzymatic susceptihility and chemical composition of steam exploded oil palm stemSamples Condition Suscep- "Vater K.L. S.L.Pressure Time tibility extract(kgf/cm) (min) (%)' (%) (%) (%)~1iddle 20 4 85.9 22.5 26.4 43.125 3 67.5 30.! 31.1 25.3Bonom 20 4 81.0 30.5 31.9 29.925 3 98.3 27.9 27.7 20.0a Bascd on the residual polysaccharides in the fibre materialK.L. Klason lignin; values hased on the residue.S.L. Soluble lignin-coli tent of extractable lignin with 90% dioxane to the total lignin in steamed wood; values basedon the residue.TABLE 2Enzymatic saccharification and chemical composition of the untreated oil palm stem.SamplesMiddleBottomMoisture Suscep- Hot-water K.L. Holocellulosecontent tibilit)' solubles(%) (%r" (%)'b (%) (%)<4.6 36.8 30.0'" 31.1" 16.33 51.45.1 25.4 19.3 20.0 20.58 68.6a Based on the residual polysaccharides in the fibre materialb Based on the oven-dried materialc Based un extr<\ctivcs-frce woodmeald Based on difference in weight of the sample before and after lhe extractionc The weight of the water extract afler evaponnion under reducer! pressure.PERTANIKA VOL. 13 NO.2. 1990 167HALlMAHTON 11ANSOR, K SUDO AND M. ISHIHARATABLE 3Composition of reducing sugars in the enzymatic hydrolysates of steam-expoded oil palm stemSamplesMiddleBottomCondition Reducing Sugars (ppm)Pressure Time Ara Gal Glc Xyl(kgf/cm') (min)to 10 433.3 76.110 20 707.3 104.920 4 525.0 72.225 3 935.610 to 43.5 11.4 1426.2 220.410 20 632.8 53.720 4 253.0 72.825 3 8.6 696.3 198.8Man26.828.6Ara arabinoseGal galactoseGlc glucoseXyl xyloseMan mannosesteam explosion. The cellulose in the pretrea-ted fibre became more susceptible to enzy-matic attack probably as a result of hemicel-lulose extraction and lignin redistribution. Asubsequent loss in susceptibility, brought bystructural collapse or further lignin redistri-bution and condensation, resulted in a corres-ponding loss in digestibility (Wong et al. J988).The Klason lignin content of the residualcellulosic materials ranged from 26-32%, indi-cating that the lignin was retained mostly inthe residues after the pretreatment. Quite ahigh portion of the lignin could be extractedwith 90% dioxane. However, earlier work hasestablished that the removal of lignin did notincrease the enzymatic digestibili ty of steatn-exploded materials (Shimizu et al. 1983; PuIset al. 1983).Table 4 shows the main sugar componentsin the water extracts after acid hydrolysis withtrifluoroacetic acid. Depending on the part ofthe OPS, 23-32% hemicelluloses with xylosecontents up to 83% could be recovered byaqueous extraction of the steam-explodedmaterials. Glucose seems to be the other majorsugar present in the extracts while galactose,arabinose and fructose Were found in verysDlaIl or trace amounts. In the case of themiddle part of OPS, the amount of xyloseincreased and that of glucose decreased withincreasing steam pressure. However, for theboltom part of OPS both the sugars decreasedwith increasing steam pressure. These resultsdiffer from those of A.mangium andrubberwood in which the amounts of xyloseand glucose in the water extracts decreasedand increased respectively under more drasticreaction conditions (Halimahton et al. 1990).Morphological Characteristics of the Steam-explodedOPSThe morphology of the steam-exploded OPSwas found to be significan tly different fromthat of the steam-exploded Acacia mangium andruhbenvood (Hevea brasiliensis) (Halimahton etal. 1990). For the latter two species, at lowsteam pressure the steam-exploded materialconsisted mainly of fiber bundles; withincreasing steam pressure fully separated fiberscould be observed in addition to fiberfragments. Under more drastic conditions, theproducts appeared to be a dark brown to blackamorp~ousmass, much like mud.However, for OPS, the appearance of thesteam-exploded material was the same at 20kgf/cm' for 4 min and at 25 kgf/cm' for 3min. The material ,vas observed to consist ofundegraded fiber bundles and a pale brownfibrous mass. Electron microscopy showed thatthe fibrous mass consisted of separated fibersand fiber fragments, with some of the fibersentangled into clusters together ,vith scatteringof parenchyma cells (Fig. I) . There were also168 PERTAN1KA VOL. 13 NO.2, 1990STEAM EXPLOSION PRETREATMENT OF THE OIL PALM STEMTABLE 4Sugar composition in the water extr<lcts after steam explosion and acid hydrolysisConditionPressure Time(kgf/cm') (min)Reducing Sugars (%)"SamplesMiddleBouom202520254343WaterExu<lcts(%) Glc22.5 68.530.1 61.830.5 17.427.9 4.4Xyl Gal31.538.282.668.0Ara12.6Fmc13.1a Based on the total solubilised sugarsGlc glucoseXyl xyloseGal galactoseAm arabinoseFruc fruclOseFig 1: Wood fiber of oil palm stem, after steamexplosion at 25 kgf/cm2 fur 3 1nin. Parenchymacells are seen scattered among the fiber fragments.regions containing inlact fibres as shown in Fig.2. An electron micrograph of a vascular bundleis shown in Fig 3. The vascular bundles seemedto be unaffected by the pretreatment.The reason for the resistance displayed hythe vascular bundles in the oil palm stemtowards steam explosion \vas that the steam vvasunable to penetrate the wood chips sufficien tlyduring the process. This arose because thechips used had been thoroughly dried and asa result they could not swell easily when incontact with steam. Hence, it it suggested thatthe use of fresh, green or soaked wood samplesshould be used for the pretreatment in thecase of the oil palm stem.Fig 2: Wood fiber of oil palm stem after steam explosionat 25 kgf/an'l for 10 min. The area "A" shows abundle of inlact fibers.Fig 3: Vascular bundle of steam-exploded oil !Jalm stem,al 25 kgf/cm' for 3 minCONCLUSIOThe results indicate that steam explosion as apretreatment for the oil palm stem appears tohave some promise for practical application.PERTANlKA VOL 13 NO.2, 1990 169HAl.Ji\:lAHTOl\' MANSOR, K. SUDO AND M. ISHIHARAThe process seems to be veIJ' effective inpreparing the substrate for further enzymaticand biological degradation, not on ly becauseof a high degree of delignification but also ofthe enhanced susceptibility of cellulose toenzymatic hydrolysis. This study also lndicatesthat a marked improvement of sugar yield canbe made when OPS is pretreated by steamexplosion.ACKNOWLEDGEMENTSThis work is part of the training programmebetween Forest Research Institute Malaysia andthe Japan International Cooperation AgencyUTCA}. The authors wish to express theirthanks to Dr. T. Fujii for the electronmicroscopic analysis,REFERENCESAr-:O;.lyr.IOUS. 1978. TAPI)1 Testing Procedures, TAPPIAtlanta, USA.BROWNtI\'G, B.L. 1967. A1ethods of Wood Chemistry, Vol.II, New York: lnrerscience.CHE\-V, L.T. and C.L ONG. 1985. Particleboards fromOil Palm Trunk. Malaysian Forester 48(2): 130-136.HALI~lAHTON ?vlANSOR and ABDUL R'\SHIH AHMAD.1990. Carbohydrates in the Oil Palm Stem andtheir Potential Utilisation. In press.HALIMAHTON MANSOR, SUDO, K. and M. ISHIHARO\,1990. Steam Explosion Pretreatment of twoMalaysian Hardwoods. In press.KHOO. K.C AND T.w. LEE. 1985. Sulphate Pulpingof the Oil Palm Trunk. In Proceedings NationalSymposium on Oil Palm B)"jJroducU for Agio-BasedIndustrits, Kuala Lumpur, Porim, 11: 57-66.Kn.LMANN, 'N. and S.c. LIM. 1985. Anatomy andProperties of Oil Palm Stem. [n ProaedingsNational Symposium on Oil Palm By-Products forAgro-Based hldusl1ies. Kuala Lumpur, Porim11,1842KJ.I"c., S.H., C.C Nno, M.A F'ERRARA,j.CR. TORRES,D.B MAGALHAES, and D.D.¥. Ry~. 1987.Enhancement of Enzymatic Hydrolysis of SugarCane Bngasse by Steam Explosion Pretreatment.Biflff'(!molofj)' and Bioengineering, 29: 1035-1039.LIM, S.C. and K.c. KHOO. 1986. Characteristics ofOil Palm Trunk and its Potential Utilizatiun.J\1aloysioll Forester 49(1 ):3-23.PAIC., M.G., L. JURASEK, and M. DESROCHERS. 1982.Simplified Analysis of Wood Sugars. Tappi,7(7),103-106.PL,nNt:, MJ. 1984. Increased Digestibility of Bagasseby Pretreatment with Alkalis and SteamExplosion. Biotechnology and Bioellgineering 26:426433.PULS, j., C. AnA, and H.H. DIETRICHS. 1983.Chemicals and Ruminant Feed from Ligno-celluloses by the Steaming-Extraction Process.Journal 01 Applied Polymer Science: Applied Pol)'merSymposium 37,685-695.R-\HI~I SUDlN, AnDUL RA1AK MOHD ALI and ZAKARl.'\MOHO. A\IIN. 1987. Chemical Components inOil Palm Trunk Influencing Wood-Cementboard Manufacture. Paper presented atthe Asian Science and Technology Congress, 14-17October, Kuala Lumpur.ROLZ, C., M.C ARRIOlA, J. VALLADARES, and S. DECABRARE. 1986. Effects of SOffie Physical andChemical Pretreatments on the Compositionand Enzymatic Hydrolysis and Digestibility ofLemon Grass and Citronella Bagasse.Ag>ieulluml Wastes 18,145-161.SCHULTZ, T.P., CJ. BIERMANN, and G.D. MCGINNIS.1983. Steam Explosion of Mixed HardwoodChips as a Biomass Pretreatment. Ind. Eng.Chem. Prod. Res. Del'. 22:344-348.SHIMIZU, K., K., SUDO, S. NAGASAWA, and M.ISHIHARA. 1983. Enzymatic Hydrolysis of Woods.VB. Enzymatic Susceptibility of AutohydrolysedWoods. Mokmai Gakkaishi 29(6 ),428-437.T.-\.1"IIAHASHI, M., T., HIGUCHI, H. KOBAYASHI. Y.TOGAMUR-\. and M. SHIMADA. 1985. Charac-terization and utrilional Improvement ofSteam-exploded Wood. as Ruminant Feed,Cellulose Chem. and Technol. 19:687-696.WONG, K-KY, K-F. DEVERELL, K.L "'lACKIE, TACL.ARK and L.A. DONALDSON. 1988. The Rela-tionship between Fiber Porosity and CelluloseDigestibility in Slcam-exploded PilluS radiota.Biotedmolog)' alld Bioenginpelillg. 31:447-456.(RPreivfd J7 Februa')'. 1990)IiO PERTANlKA VOL. 13 NO.2, 1990

Preliminary Studies on the Steam Explosion Pretreatment

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Preliminary Studies on the Steam Explosion Pretreatment of the Oil Palm Stem

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