HYDROLYSIS OF SAGO (METROXYLON SAGO ROTTB.) PITH POWDER BY SULFURIC ACID AND ENZYME AND FERMENTATION OF ITS HYDROLYZATE BY PICHIA STIPITIS CBS 5773, SACCHAROMYCES CEREVISIAE D1/P3GI, AND ZYMOMONAS MOBILIS FNCC 0056 INTO BIOETHANOL.

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Rat_uSAFITRI1, Ba_mban_gMARWOTO2, Fenn_yFIRSTIANT_Y1, _Jett_yNURHA_JATI1 1_{Dept of Biology, FMIPA, University of Padjadjaran, Bandung, Indonesia}

P_{hone :+6}222₇₇₉₆₄₁2_{, Email: ratusafitrie@yahoo.com} 2

BPP_T S_erpong-_Indonesia

C_orrespondingauthoremail: ratusafitrie@yahoo.com A_bstract

Thepurposeofthisstudywastodeterminehydolysisefficiencyofsagopithpowderandfermentationefficiencyby P.

Stipitis CBS 5773, S.cerevisiaeD_{1/P3GI, andZ}.mobilis FNCC 0056..Thisresearchwasexperimentallyandconsistof

hydrolisisprocesssagopithpowderandfermentationofitshydrolyzate. Sagopithpowderhascontent 77,5% starch,

4,63% cellulose, 4,86% hemicelluose, lignin 3,07%, dan water 10,12%. In gelatination process in temperatureat

120˚C, theyieldof sugarconcentration 0,62% anddextroseequivalent (DE) 0,89% obtainedfrom thesizeof 100

mesh. Sago gelatine hydolized bysulfuric acid acid, Į-amylase, hemicellulase, cellulase, dan amyloglukosidase

producesugarconcentration 53,28% andD_{E 68,52%}. Atfermentationprocessin sugarconcentration 5% withcontent

of glucose 4.17 %, fermentationby Pichiastipitis, Saccharomycescerevisiae, andZymomonasmobilisprovideethanol

fermentationefficiency 23,5%, 37,65%, dan 32,13% respectively. At fermentationprocesswith sugarconcentration

10% with contentof glucose 8,28 %, fermentationby Pichiastipitis, Saccharomycescerevisiae, and Zymomonas

mobilisyielding ethanolfermentationefficiency 9,30%, 50,85% and 20,65% respectively.

K_ey w_ords:Hydrolysis,SagoPithPowder,fermentation, Pichiastipitis CBS 5773, SaccharomycescerevisiaeD1/P3GI,

Zymomonasmobilis FNCC 0056

INTRODUCTION

Indonesia currently has a high dependence on fossil fuels and has at least three serious problems that the depletion of petroleum reserves, the instability of fuel prices due to high demand and rising greenhouse gas (CO2) from burning fossil fuels so that Indonesia needs alternative and renewable energy sources. On the other side of Indonesia as a tropical country that has many resources, especially carbohydrates and cellulose from agricultural waste. Especially for Eastern Indonesia has vast forest sago has great potential for renewable energy sources and energy independence.

Bio-ethanol is ethanol produced by fermentation by various microorganisms (yeasts and bacteria) [1]. Currently ethanol is used as 'fuel additive' (fuel mixture), it can even replace non-renewable fuel conventionally.

One of substrate which is potential and environmentally friendly to produce ethanol is

a sago plant. Although S_{ago need long age} harvest is about 6 years but can produce starch in large quantities. S_{ago is Indonesia plant} native, can grow on various soil types, such as: dry soil, clay, swamp or soil flooded. S_ago palm acreage in Indonesia is very wide which is about 1.128 _{million ha [}2_]. S_{eedling density of} about 1480 trees / ha when the crop yield and 125-140 trees / year [3]. (Each of the sago tree containing an average of 2₀₀-400 kg sago starch, so can obtain 30-_{60 tonnes of sago}

starch / ha [4]. (Bintoro, 2_{003). Trunk of sago} pith without peel has weight an average 850 kg

with starch content 2_{9 percent, water content 50} percent and fibers ranged 2_{1% [5]. Overall} utilization of sago for ethanol production is perceived to be more effective because of the stem consists of the sago pith which is containing starch and fiber. S_{ago starch} content about 8_{4.7 to} 8_{5.9% with the} percentage of amylose 27 % and amylopectin 73%[2_{], while the sago fiber composed of} cellulose and hemicellulose, when hydrolyzed Scientific Papers, Animal Science, Series D, vol. LV

completely into simple sugars, especially glucose[2_].

In this study uses S_{ago (}S_ago M_etroxylon

Rottb.) obtained from K_{ibin, Banten.} S_{ago is} processed (self-preparative) sago flour. Based on proximate test, the pith of sago contain of starch about 77.50%,and fiber 12_{:56%, water} 1_0.12_{%. Total amount of sugar contained in the} pith flour equal to the amount of carbohydrate. Total sugars of pith flour in size 50 mesh is equal to 60.47% (w / w), it meansthat every 1 _g of the pith flour contained 0.604 g of carbohydrates. While the total sugar contained in the pith flour 100 mesh size is equal to 77.76% (w / w) means that in every 1 _{g of the} pith of flour contained 0.778 _{g of} carbohydrates. W_{ith high carbohydrate content}

means sago is very potential as a major source of sugar for ethanol fermentation.

S_{everal groups of microorganisms have the} ability to produce ethanol from carbohydrates through the fermentation process. Some microorganisms such as Pichia stipitis can ferment xylosa . S_{accharomyces cerevisiae, and} Z. mobilis has the ability to ferment hexose (C6) [6]. While Z.mobilis is a type of bacteria that can produce ethanol from hexose sugars and fermentation capabilities faster than S_. cerevisiae [7].

On lignocellulose biomass is necessary to pre

-treatment such as desizing of the pith flour and temperature of gelatinization. Optimum temperature of the pre-_{treatment can increase}

the acid and enzyme -_{hydrolysis which is}

expected to produce sugars from cellulose then fermented into ethanol. Concentration of ethanol produced is influenced by the type and concentration of sugar. Because microorganisms have different preferences and tolerances to concentration of sugar.

Pre_-treat_ment. P_re-_{treatment of lignoselulose}

needs to be done prior to enzymatic or acid hydrolysis. Pretreatment aims to solve, dissolve, hydrolyze and separate components of cellulose, hemicellulose, and lignin [8_{]. (}S_aha, 2_003). V_{arious methods for biomass} pretreatment methods such lignoselulosik autohydrolisis,treatment with organic solvent, thermo mechanical; milling, refining, cutting, extortion, acid treatment; acid solution (H2S_{O4, HCl), concentrated acids (H}2S_O4,

HCl),treatment with alkali; Sodium hydroxide, ammonia, hydrogen and peroxide alkaline [8_]. The use of acid for the initial treatment materials can lower the cost for enzyme. Gelatini_zation and _Sacc_harification. _The conversion process of starch, cellulose, and hemicellulose into sugars through the stages of gelatinization and hydrolysis. S_{tages of} gelatinization facilitate the action of the enzyme hydrolyzes the substrate. G_{elatinization} is influenced by temperature, substrate type and concentration of the substrate itself. The smaller of gelatin concentration the higher of reducing sugar content. Because acid is difficult to diffuse to the high starch concentrations.

Hydrolysis is the decomposition of polysaccharides into monosakarida. Hydrolysis carried out by crushing the raw material of sago mixed with water, acids or enzymes such as termamyl, at pH 6.5 and heated at a temperature of 90oC. Further saccharification process using dextrozim for 24 hours at pH 4.5 and temperature 60 ° C[9] (Bujang, Ishizakhi, and G_oh P_ing Y_au, 2_006).

Hydrolysis can be performed using acids, enzymes, or mixtures of acids and enzymes. The acid used can be either concentrated acid or dilute acid. Acid will break down the starch molecules randomly and sugars produced mostly reducing sugars. At the optimum conditions for starch molecules will produce 88_{% glucose. However, in practice, using the} acid hydrolysis of starch to glucose would only result in the value of DE (Dextrose Equivalent) 55%. If the value of DE (Dextrose Equivalent) above 55%, it will produce furfural compounds and acid hydroximethyl levulinat which can inhibit the fermentation process.

Hydrolysis of polysaccharides into monosaccharides using enzyme carbohydrase, namely: D-amylase, hemiselulase, cellulase, and amiloglukosidase. The amount of enzyme amiloglukosidase at full dose (0.56 P l / g) were used to hydrolyze starch (100%), whereas the amount of cellulase enzymes on the full dose (0.8_{3 P l / g) were used to hydrolyze pure} cellulose Hydrolysis of starch and cellulose will produce glucose and additional products in the form of sugar that is hydroximethyl furfural derivatives. Hemicellulose hydrolysis would produce two types of sugar, pentose and

hexose. This research will study the influence of particle size reduction in pith flour of sago, gelatinization temperature, hydrolysis effectiveness of combination of acid and enzyme in hydrolysis and fermentation capabilities of P_ichia stipitis Sacchromyces c_erevisiae and Zymomonas mobilis in hydrolyzate results of sulfuric acid hydrolysis 6

M followed by the enzyme in hydrolysis. MAT_ERIALAND_METHOD

Pre_-treat_ment and H_ydrol_ysis. _{The research} was carried out experimentally in the laboratory and conducted in three stages: (1₎ P_re

-treatment: consists of optimization of particle size reduction (50 mesh and 100 mesh) of pith flour of sago, optimization of temperature in gelatinization which are 90qC, 1_00qC, 11_0qC, dan 12_{0qC C for} 2_{0 min in} 1 _{atm pressure (}2_). Acid and enzymatic hydrolysis in G_{elatin pith} flour. In the hydrolyzate of the pre treatment that produces the highest reducing sugar content was added 6 M _{sulfuric acid,}

hereinafter incubated for 1 _{hour at} 12₀o_{C in this} hydrolizate (I) was . measured the concentration of reducing sugar and Dextrose equivalent (DE). Once hydrolyzed by 6 M

sulfuric acid hidrolisate (I) is cooled to 2₅0_C and adjusted to pH 6.0, then Į-amylase enzyme is added as much as 0.17 ml / g (volume enzyme/ g substrate), incubated at 1₀₄0C _{for 60} minutes with a pressure of 1 _{atm (HII). The} hydrolyzate II measured the concentration of reducing sugar and dextrose equivalent (DE).

Hydrolyzate II is heated at a temperature of 1210_{C with a pressure of} 1 _{atm for} 1_{0 minutes.} Then added with hemiselulase as much as 1_/3 dose. Hydrolysed further incubated at 55qC with agitation of 150 rpm for 270 minutes (hydrolyzate III) hydrolyzate III had cooled 2₅0_{C. Then the pHwas adjusted to 4.}8_{. After} that cellulase enzymes are added as much as 0.55 ml / g and amyloglucosidase 0.37 ul g (volume enzyme / g substrate). S_ubsequently incubated at 600C for 48 _{h with agitation} 1₃₀ rpm. At this stage, concentration of reducing sugar and DE is measured.(Gerhartz,1990). Fermentation by Pichia stipitis CBS _5773, S_{accharomyces cerevisiae D}1_/P₃G_{I, and}

Zymomonas mobilis FNCC0056 a single

culture to ferment sugars hydrolizate pith of sago starch hydrolyzate.

The reducing sugar content measurement method done by DNS_. S_{ago as much as} 1_{5 g} gelatin dissolved in distilled water until the volume is 2_{5 ml. The next sample is introduced} into a centrifuge tube and centrifuged at 3500 rpm for 2_{0 minutes. Clear sample solution of} 1 ml pipetted into a test tube inserted. Then into the test tube was added 3 ml of DNS _(3.5 Dinitrosalicylic acyd). After that, samples were homogenized with a vortex and heated in a boiling water bath for 5 minutes. After five minutes of heated, then cooled in an ice bath. After the absorbance was measured using a spectrophotometer at a wavelength of 550 nm. Calculation of Dextrose Equivalent (DE) using the formulaDextrose Equivalent (DE) = reducing sugar concentration of the sample (%) x 1_{00%/ Total sugar concentration (%).}

Pre_paration of _Substrate _Fer_mentation and fer_mentation. _{The hydrolyzate which is having} highest DE (Dextrose Equivalent) and reducing sugars content. then adjusted to 5% and 1_0% ofreducing sugar content.. Each hydrolyzate is added with fermentation medium containing (per liter): yeast extract 4g, K_H2PO4 2_{g, (NH4)} 2SO4 3g, MgSO4.7H2O1g, and pepton 3, 6 g (S_{anchez et al.,} 2₀₀2_{). pH was adjusted to pH} 7. For fermentation by P_{. stipitis CB}S _{5773 and} S. Cerevisiae D1/P3GI pH medium was adjust to pH 5, and medium for Z. mobilis FNCC 0056 pH adjusted to ph 7. Fermentation medium were sterilized. For fermentation every cultures (Pichia stipitis CBS 5773 S. Cerevisiae D1_/P3G_{I and Z. mobilis FNCC 0056) as much} as 1_{0% put into a fermentation substrate,} which is containing hydrolyzate with concentration sugar are 5% and 10%. Then cultures in medium shake-incubated at 30o C for 72 _{h with agitation} 1_{50 rpm. During} incubation, samples were taken at 0, 6, 12_, 18_, 24, 30, 36, 48, 60, and 72 hours for measurement of ethanol and reducing sugar content.

RESULT_SANDD_ISC_USSION_S

O_pti_mi_zation of desi_zin_g of _parti_kel of _pit_h flo_ur and te_mperat_ure inc_ubation in gelatini_zed P_{ith flour used in this study had a}

hemicellulose 4.86%, lignin 3.07% and water 1_0.12_%. Prior to the hydrolysis step, the substrate made gelatinization. G_{elatinization is} a mechanism of entry of water into the starch granules so as to facilitate the action of the enzyme hydrolyzes the substrate. G_{elatinization} is influenced by temperature, substrate type and concentration of the substrate itself [1_0]. Treatment with temperature 90˚C, 100˚C, 11_{0˚C, and} 12_{0˚C of the pith flour of in 50 and} 1_{00 mesh resulted a reducing sugar} concentrations were as follows.

Table1_{. Duncan'smultiplerangetestanalysisof}

Concentrationofreducingsugarinsizeofpithflour50 meshand1_00mesh.

Tempof Gelatinization

(˚C)

Cons. OfRed. sugar(%)in50 meshflour

Cons. Of

Red.sugar(%)in

100mesh

90 0,28a 0,25a

100 0,35b 0,27a

110 0,42c 0,40b

120 0,53d 0,62c

From the results of Duncan's multiple range test analysis can be seen that the increase in gelatinization temperature, the greater the concentration of sugar produced. The results of starch gelatinization of sago pith 50 mesh at 120˚C is a reducing sugar concentration of 0.53%. While on 100 mesh at 120˚C obtained 0.62_{%. Concentration of sugar produced from} the pith of sago in 1_{00 mesh size larger than 50} mesh. 1_{00 mesh size, mean grain size of pith} starch particles is 175ȝ. While the 50 mesh

size, mean grain size of pith starch particles is 350ȝ.. The smaller a particle, the greater the

absorption area [11_].

Table2_{. Duncan'smultiplerangetestanalysisof} Dextrose equivalent (DE)at varioustemp. on50mesh

and1_00mesh

Tempof G_el.

(˚C)

Dextrose equivalent (DE)

(%) insize50

mesh

Dextrose equivalent (DE)

(%) insize1₀₀

mesh

90 0,51_{a 0,3}1_a

1_{00 0,57b 0,3}2_b

11_{0 0,69c 0,5}2_c

12_{0 0,}8_{5d 0,}8_9d G_{rain flour} 1_{00 mesh has a smaller particle size} will absorb more heat and water, therefore the more that comes out of the starch amylose so that the concentration of sugar produced more

than 50 mesh size. De_xtrose equivalent (DE) indicates the number of starch polymers that have been cut into glucose molecules are much simpler. Dextrose equivalent can be obtained from the ratio of the concentration of sugar samples with a total sugar concentration. From the analysis of Duncan's multiple range test t is known that increasing the temperature of gelatinization, the greater the dextrose equivalent (DE) is generated. From starch gelatinization of sago pith flour 50 mesh obtained the highest DE 0.8_{5% at} 12_{0˚C and}

DE8_{9% at} 1_{00 mesh. This is due to the high} temperature will facilitate the breaking of carbon-hydrogen bond, so that more polymer is cut starch into glucose[12_]. G_{elatin then is} hydrolyzed. chemically and enzymatically, Chemical hydrolysis using sulfuric acid 6M _and

the pH was adjusted to 2.

Tabel3: Conc. ofred. sugarand (DE) ofsubstratis hydrolyzedbysulfuricacid6M_andamylase,

hemicellulase,cellulaseandamyloglucosidase TreatmentofHydrolysis Cons. Of

red. sugar. DE(%)

Sulfuricacid6M 22,6 28,63

Į-_{amylase 33,0}2 42,47

Hemicellulase 33,94 43,65

Cellulase and

Amyloglucosidase 53,28 68,52

DE = dextroseequivalent

Concentration of reducing sugar produced in the pith flour of sago is hydrolyzed by sulfuric acid is measured by DNS method. Hydrolysis using sulfuric acid 6M produce concentration of reducing sugars 22_.2_{6% and DE}2 8_{.63% .} On the acid hydrolysis, acid breaks the bond glycosidic pith flour of sago at random but consecutive to the smallest molecule called glucose. However, the results of a reducing sugar and DE obtained indicates that not all pith flour of sago can be hydrolyzed by acid. This is in accordance with the statement [13] that the conversion of starch using acid will only generate a ma_ximum of DE 55%.

Hydrolysis by enzymes

Hydrolysates I generated at acid hydrolysis using sulfuric acid, and then hydrolyzed by the enzyme Į-amylase with doses 0,17 ul/g, which lasted for 60 minutes at a temperature of 1_04ºC and pH 6.0, hydrolysis using Į-amylase enzyme, obtained hydrolyzate II ith reducing

sugars obtained amounted to 33.02%, and DE 42_{.47%. Increased concentrations of reducing} sugars due to the breakdown amylose and amylopectin in starch by Į-amylase which does not occur at optimum in the acid hydrolysis. Į -amylase is an endo-enzyme that can break the bond of alpha-₍1_{,4) glycosidic randomly. Break} down of amylose by Į-amylase into maltose and maltotriosa that occur at random. Then form of glucose and maltose as the final result.

W_{hile working of} Į-amylase on the amylopectin would produce glucose, maltose, and various types of oligosaccharide [15].

Hydrolysates II produced by Į-amylase then hydrolyzed by hemicellulase. Hydrolysis by hemicellulase for 2₇0_{minutes at a temperature} of 55ºC and pH 6.0. Concentrations of enzyme were added at 1/3 of the recommended enzyme concentration is equal to 0.001 _{g / g substrate.} Hydrolysis by hemicellulase obtained hydrolyzate III.

The main purpose hemicellulase, fibers which is containing hemicellulase will produce simple sugar such as hexose and pentose. The use of hemicellulase is able to raise of a reducing sugar concentration and DE, reducing sugar from 33.02_{% to 33.94% and DE from} 42_{.47% to 43.65%.. The use of hemicellulase} not only able to contribute of increase in reducing sugar concentration of the hydrolysis of hemicellulose but also helps the action of the enzyme cellulase..

H_ydrol_ysis _by cell_ulase en_zymes and A_mylo_gl_ucosidase_{. The addition of cellulase} done because in the previous stage, the hydrolysis of cellulose was not optimal. While the addition amiloglukosidase made to hydrolyze dextrins obtained from the previous stage so as to produce glucose. Provision of both types of enzyme was carried out simultaneously as cellulase and amiloglukosidase have synergistic activity.Hydrolysis by cellulase enzymes and amiloglukosidase (saccharification) was held at a temperature of 60 º C and pH 4.8_{. DX} dextrozyme enzyme dosage was 0.37 ml / g (dose of 2_{/3), while the number of Celluclast} 1.5 L is inserted is 0.55 ml / g (dose of 2/3). S_{accharification carried out for 4}8 _hours.

Hydrolysis using dextrozyme DX and Celluclast 1_{.5 L produces a reducing sugar}

concentration [% (w / w)] 53.28% and DE 68_.52_{% after 4}8 _{hours of incubation.}

Cellulase enzymes play a role at the beginning of the process is made starch granules more open so that amyloglucosidase a chance to hydrolyze starch granules inside which is have higher of sensitivity towards amiloglukosidase activity[1_{5]. Reducing sugar concentration} increased, this is due to cellulase enzymes break down cellulose into selobiosa. S_elobiosa is then split into glucose. In addition, DX dextrozyme breaking glycosidic bond Į-1, 4 and Į-1, 6 produce monomers of glucose. Bond of Į-1 ,4-glycosidic found in maltose and maltotriosa which is the result of a process of amylose and amylopectin liquefaction while bonds Į-1 ,6-glycosidic which is the dextrins present in the process liquefaction of amylopectin.

At this stage of the hydrolysis of the resulting concentration of DE for 68_.52_{%. This is} consistent with the statement Langlois and Dale (1_{940) in Tjokroadikoesoemo [}1_{6] that the} hydrolysis process using a combination of acid and enzymes can increase the value of DE. Initially carried out by acid hydrolysis process to DE 55%, then uses the enzyme hydrolysis followed by amilolitik to DE 61-_{65%. In this}

study, DE values greater than 65% can be caused by the addition of the cellulolytic enzyme hydrolysis process, the cellulase and hemicellulase so that the amount of glucose produced more.Hydrolyzates of sugar which is the result of hydrolysis, then set the concentration to 10% and 5%. HPLC analysis of the results obtained that 1_{0% of sugar} hydrolizate consists of glucose 8_.28_{% and} maltotetraosa 1_.72_%. S_{ugar concentration of} 5% consisted of glucose 4.17% and maltotetraosa 0.8_3%. S_{ugar is then fermented} by P.stipitis, S.cerevisiae, and Z.mobilis a single culture.

Efficienc_y fer_mentation of _Pichia stipitis, Zymomonas mobilis and Sacchromyces cerevisiae in fer_mentation of red_ucin_gs_ugars of _hydrol_yzate of _pit_h of sa_go starc_h. Concentration of sugar and ethanol at the end of the process of ethanol fermentation of hydrolyzate sugar by Pichia stipitis.

concentration of 5%, containing glucose 4,17% glucose and maltotetraosa 0.8_3%. While the concentration of sugar hydrolyzate medium 1_{0% containing glucose} 8_.28_{% glucose and} 1.76% maltotetrosa.

Fig1_{. Reducingsugarandethanolconcentrationchanges}

duringfermentationhydrolyzateofpithofsagostarchby P.stipitis

During the fermentation process takes place, the concentration of sugar hydrolyzate 5% containing glucose 4,1_{7% is used up by} P.stipitis at the 60 hours. While the initial sugar hydrolyzate concentration 1_{0%, after 7}2 _hours as much as 8.28% glucose used by P.stipitis and leaving glucose 4.73%, this means only 3.55% used by P.stipitis. This suggests that P.st_ipit_is less tolerant to sugar concentration 10%. Sugar is used as a nutrient and for ethanol fermentation. On medium hydrolyzate sugar 5% containing glucose 4,1_{7% produces} ethanol only 0.98_{% which began to form after} 36 hours,meaning that up to 36 hours glucose used for nutrition. From the fermentation is well known efficiency of glucose fermentation by P_{ichia stipitis only} 2_{3.5%, while according} to theory, 1_{00% sugar when fermented will} produce 50% ethanol [12].

Concentration of ethanol in the fermentation by P.st_ipit_is sugar in a medium of sugar hydrolyzate 1_{0% containing glucose} 8_.28_%, produces ethanol only as much as 0.77% for 72 hours and ethanol is formed at 48_{, it means up} to 36 hours, glucose used as a nutrient. Concentration of ethanol produced in the hydrolyzate 10% less than the ethanol produced in the sugar hydrolyzate 5%. thus the efficiency of fermentation by P.stipitis on glucose medium containing 8_.28_{% only 9.30%.}

Efficiency of fermentation by Pichia stipitis is very low, it is presumed P.stipitisless able to ferment hexose sugars. The best ability to ferment pentose sugars. M_{ore glucose is used to}

increase the cell biomass. Also P.stipitis have a low tolerance to high sugar concentration. This can be seen in 1_{0% glucose medium, P}.stipitis lag phase longer than the medium of 5%. Concentration of red_ucin_gs_ugarand et_hanol at t_he end of t_he _process of et_hanol fer_mentation of _hydrol_yzate s_ugar _by

Saccharomycescerevisiae

Fig2. Reducingsugarandethanolconcentrationchanges duringfermentationhydrolyzateofpithofsagostarchby

S. cerevisiae

Hydrolyzate sugar concentration both at 5% and 1_{0%, during the fermentation process,} glucose is used up by the S.cerevisiae for 36 and 48 _{hours. This shows} S_{.cerevisiae has the} ability to ferment hexose sugars, particularly glucose very well. S_{.cerevisiae has invertase} enzyme that acts to convert glucose into ethanol[1_{7] .} S_{ugar medium consisting of} glucose 4.1_{7%, fermentation by} S_{.cerevisiae for} 72 _{hours produced} 1_{.57% ethanol and ethanol} have started to form after 18 hours. Efficiency of fermentation in sugar hydrolyzate 5% by S_{.cerevisiae as much as 37.6%.} W_{hile the}

concentration of ethanol produced by S_{.cerevisiae on fermentation of sugar} hydrolyzate 10% in medium containing glucose 8_.28_{%, producing ethanol as much as 4.}21_{% for} 72 _{hours. Fermentation efficiency of 49.}2_% reaching almost 50%, this corresponds to the theory that 100% of sugar when fermented will produce 50% ethanol [12_].

Concentration of red_ucin_gs_ugarand et_hanol at t_he end of t_he _process of et_hanol fer_mentation of _hydrol_yzate s_ugar _by

Zymomonasmobilis.

During the fermentation process, hydrolyzate sugar concentration 5% containing glucose -2 0 2 4 6 8 10

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time (hour)

F in a l c o n s . O f s u g a r (% )

Cons. In 5 % Of Sugar hydrolizate Cons. In 10 % Of Sugar hydrolizate

-0.2 0 0.2 0.4 0.6 0.8 1 1.2

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time (hour)

C o n s o f E th a n o l (% )

Cons. Of etanol in initial cons of sugar 5% Cons. Of etanol in initial cons of sugar 10%

-2 0 2 4 6 8 10

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time(Hour)

F in a l c o n s . O f s u g a r (% )

Cons. In5% OfSugarhydrolizate Cons. In10% OfSugarhydrolizate

-1 0 1 2 3 4 5

06121824303642485460667278 Time(Hour) C o n s o f e th a n o l (% )

Cons. Of etanol in initial cons of sugar 5 % Cons. Of etanol in initial cons of sugar 10 %

4.17% is used up by Z.mobilis after 36 hours and the ethanol yield of 1_.34%.

Fig3. Reducingsugarandethanolconcentrationchanges duringfermentationhydrolyzateofpithofsagostarchby

Z.mobilis.

Glucose is used for cell growth and ethanol fermentation substrate. W_{hereas in sugar}

hydrolysates 1_{0% containing glucose} 8_, 28_% still remaining glucose. .37%. This is due to bacterial cells die after 36 hours, while ethanol produced as much as 1_{.34% for 7}2 _{hours, the} ethanol begins to form after 6 hours. Based on amount of ethanol that is formed is known that the sugar hydrolyzate 5% fermentation efficiency Z.mobilis reachs 32_.1_{3% while on} the sugar hydrolyzate 1_{0%, ethanol} fermentation efficiency reach 2_0.65%. According to[18]. Z.mobilis has a good ability to ferment glucose, the results are even greater than the S_{.cerevisiae due to the formation of} ethanol by the Entner Doudoroff. In this study the fermentation of sugars hydrolyzate b

Z.mobilis obtained a little amount of ethanol, because the medium pH dropped to 3:37 is so low that bacterial cell death occurred.

CONCL_USION_S

1_{.Desizing of pith of sago starch particles in} the size of 100 mesh, and gelatinization temperature at 12_{0 ˚ C, can increase the} reducing sugar and dextrose equivalent (DE).

2.Hydrolysis by acids and enzymes can be applied in synergy so as to increase the activity on the hydrolysis of lignocellulosic materials.

3.Efficiency of fermentation in the sugar hydrolyzate sugar concentration of 5% containing of glucose 4.1_{7% Pichi}a_stipitis,

Sa_{cchromyces cerevisi}a_{e and Zymomo}na_s

mobilis, respectively are 2_{3.5%, 37.65% and}

32.13%. While in the hydrolyzate sugar concentration1_{0%of sugar with glucose} content 8_.28_{%, fermentation efficiency of} Pichia _stipitis,Sa_{cchromycescerevisi}a_{e and}

Zymomonas mobilis respectively are 9.3%,

50.8_{5% and} 2_0.65%. AC_KNO_WL_ED_GEMENT_S

This research work was carried out with the support of DP2M - _Directorate G_{eneral of}

Higher Education, Ministry of National of education through Hibah K_ompetitif Penelitian –P_{rioritas Nasional Batch}

I.no.303/SP2_H/PP_/DP2M_/V_I/2_009. REFERENC_ES

[1_]Najafpour,G_{.Ddan J.}K_. Lim. 2002. Evaluation and

Isola_tion _{of Eth}an_{olProducerStr}ain _{SMP-6. Regional}

S_{ymposiumonChemicalEngineering}2₀₀2_.

[2_{] Budianto,J.} 2_{003. Tek}n_ologi Sa_{gu B}a_gi Agribisnis

dan _Keta_hananP_{angan. Sem}ina_rNa_siona_lSa_guUn_tuk

Keta_hanan_Pan_gan_{. hal. 5}-1_5.

[3] Doelle, H. W_. 1₉₉8_. S_ocio-_{economic M}icrobia_l ProcessStra_{tegiesfor A Sust}aina_bleDevelopmen_tUsin_g En_viron_men_ta_{lly Cle}an _Techn_{ologies: S}a_gopa_{lm A} Ren_ewa_{ble Resource.} Livestock Research for Rural Development,V_ol. 1_0,No. 1_,January.

[4] Bintoro,H. M_{. H.} 2_{003. Pote}n_siPeman_faa_tan_Sa_gu Untuk Industri dan Pangan. Seminar Nasional: Sagu

UntukKetahananPangan. hal. 16-19.

[5]Yasin, A.Z. 2003. Pengelolaan Agrobisnis Sagu di

Riau. Prosiding Seminar Nasional Sagu: Sagu untuk

KetahananPangan,Manado,6Oktober2003.

[6] Haagensen,F. D. 2005. Enzymes For Biomass And

Forestry. http://www.novozymes.com. Diakses pada tanggal20Desember2008jam11.00WIB.

[7] Davis,L.,P. Rogers,J. Pearce danP. Peiris. 2006. Evaluation of Zymomonas-Based Ethanol Production From a Hydrolysed Waste Starch Stream. Elsevier BiomassandBioenergy. 30:8₀₉-81_4.

[8_] S_{aha Balal.C.} 2_{003. Hemicellulose Bioco}n_version_.

ReviewPaper. J. Ind. Microbiol. Biotechnol30:2₇₉-2₉1_. S_{ocietyfor Industrial}Microbiology.

[9] Bujang,K_{, Ishizaki A,}G_{oh, Y.} 2_{006. High Speed} Fermen_ta_tion_{of Eth}an_{ol For Fuel FromS}a_goUtilisin_g The Ishiza_{ki Process.} M_{eeting and interview with} M_OS_{TI (}M_{inistry of} S_{cience, Technology and}

Innovation).

[1_{0] Fengel,Dietrichdan}G_. W_agener. 1_{995. K}a_{yu Kimi}a Ultra_{struktur Re}a_ksi-rea_{ksi. Yogayakarta:}G_adjahmada UniversityP_ress.

[11_{] Handayani,B H.} 2_{006. Hidrolisis P}a_{ti D}a_gu (Metroxylon _sa_{gu Rottb}.) Secara Enzima_{tis d}an _Asa_m Serta _Fermen_ta_{i Hidrolis}a_tn_ya _Men_ja_{di Et}an_{ol Oleh} Strain_{S C FNCC 3012 d}an _Isola_{t B}a_{kteri As}a_{l Empulur}

-1 0 1 2 3 4 5 6 7 8 9

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time (Hour)

F

in

a

l

C

o

n

s

.o

f

S

u

g

a

r

Finalcons.Ofsugarwithinitialcons.Ofsugar5% Finalcons.Ofsugarwithinitialcons.Ofsugar10%

-0.2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

06 12 18 24 30 36 42 48 54 60 66 72 78 Time(Hour)

C

o

n

s

o

f

E

th

a

n

o

l

Consofethanolwith InitialConsofSugar5% Consofethanolwith InitialConsofSugar10%

Sagu.Skripsi. Jurusan Bilogi FakultasMatematikadan

Ilmu Pengetahuan Alam.

[12] Hidayat,N., M.C.Padaga dan S. Suhartini. 2006.

Mikrobiologi Industri.Yogyakarta

[13] Judoamidjojo, M., A.A.Darwis, danE.G.Sa’id.

1992. Teknologi Fermentasi.Jakarta: PenerbitRajawali.

[14] Winarno, F. G. 1983. Enzim Pangan. Penerbit Gramedia Pustaka Utama.Jakarta

[15] Haska, N. 1995. Alcohol Production From Sago

Starch Granules by Simultaneous Hydrolyzation and

Fermentation Using A Raw Starch Digesting Enzyme

From Aspergillus sp. No. 47 and Saccharomyces cerevisiae No. 32. In the Fifth International Sago

Symposium: ISHS ActaHolticulturae 389.

[16] Tjokroadikoesoemo, P.S.1986.HFS dan Industri

Ubi KayuLainnya.Jakarta: PenerbitGramedia

[17] Sardjoko.1991. Bioteknologi Latar Belakang dan

Beberapa Penerapannya.Jakarta : Gramedia

[18] Gunasekaran P. dan K.C Raj. 2002. Ethanol

Fermentation Technology Zymomonas mobilis.

Department of Microbial Technology, School of Biological Sciences MaduraiKamaraj University. India.

http://tejas.serc.iisc.ernet.in/currsci/jul 10/article14.htm.

hexose. This research willstudy the influence of particlesizereductionin pithflourof sago,

gelatinization temperature, hydrolysis

effectiveness of combination of acid and

enzyme in hydrolysis and fermentation

capabilities of P_ichia stipitis Sacchromyces c_erevisiae and Zymomonas mobilis in hydrolyzateresultsofsulfuricacidhydrolysis6

Mfollowedbytheenzymeinhydrolysis. MAT_ERIAL AND _METHOD

Pre_-treat_ment and H_ydrol_ysis. The research wascarriedoutexperimentallyinthelaboratory and conducted in three stages: (1₎ P_re

-treatment: consists of optimization of particle sizereduction(50meshand100mesh)of pith flour of sago, optimization of temperature in gelatinization which are 90qC, 1_00qC, 11_0qC, dan12_0qCCfor2_0minin1_atmpressure(2_). Acid and enzymatichydrolysis in G_{elatin pith} flour. In the hydrolyzate of the pre treatment

that produces the highest reducing sugar

content was added 6 M _{sulfuric acid,}

hereinafterincubatedfor1_hourat12₀o_Cinthis

hydrolizate (I) was . measured the

concentration of reducing sugar and Dextrose

equivalent (DE). Once hydrolyzed by 6 M

sulfuric acid hidrolisate (I) is cooled to 2₅0_C andadjustedtopH6.0, thenĮ-amylaseenzyme

is added as much as 0.17 ml / g (volume

enzyme/gsubstrate),incubatedat1₀₄0C_for60 minutes with a pressure of 1 _{atm (HII). The} hydrolyzate II measured the concentration of reducing sugarand dextrose equivalent(DE). Hydrolyzate II is heated at a temperature of 1210_{Cwithapressureof}1_atmfor1_0minutes. Thenaddedwith hemiselulaseasmuchas1_/3 dose. Hydrolysed further incubated at 55qC with agitation of 150 rpm for 270 minutes (hydrolyzate III) hydrolyzate III had cooled 2₅0_{C. Then the pHwas adjustedto 4.}8_{. After} that cellulase enzymes are added as much as 0.55 ml / g and amyloglucosidase 0.37 ul g (volume enzyme / g substrate). S_ubsequently incubated at600Cfor 48 _{h withagitation} 1₃₀ rpm. At this stage, concentration of reducing sugarandDEismeasured.(Gerhartz,1990). Fermentation by Pichia stipitis CBS _5773, S_{accharomyces cerevisiae D}1_/P₃G_{I, and} Zymomonas mobilis FNCC0056 a single

culture to ferment sugars hydrolizate pith of sagostarchhydrolyzate.

The reducing sugar content measurement

method done byDNS_. S_{ago as much as} 1_{5 g} gelatin dissolved in distilled water until the volumeis2_{5ml. Thenextsampleisintroduced} intoa centrifugetube and centrifuged at3500 rpmfor2_{0minutes. Clearsamplesolutionof}1 ml pipettedintoatest tubeinserted. Theninto the test tube was added 3 ml of DNS _(3.5 Dinitrosalicylicacyd). Afterthat,sampleswere

homogenized with a vortex and heated in a

boiling water bath for 5 minutes. After five minutes of heated,then cooled in anice bath.

After the absorbance was measured using a

spectrophotometeratawavelength of550nm. CalculationofDextroseEquivalent(DE)using

the formulaDextrose Equivalent (DE) =

reducingsugarconcentrationofthesample(%) x1_{00%/Totalsugarconcentration(%).}

Pre_paration of _Substrate _Fer_mentation and fer_mentation. Thehydrolyzatewhichishaving highestDE(DextroseEquivalent)andreducing sugars content. then adjusted to 5% and 1_0% ofreducing sugarcontent.. Each hydrolyzateis added with fermentation medium containing (perliter):yeastextract4g,K_H2PO42g,(NH4)

2SO4 3g, MgSO4.7H2O1g, and pepton 3, 6 g

(S_anchezetal.,2₀₀2_{). pHwasadjusted topH} 7. ForfermentationbyP_{. stipitisCB}S_5773and S. CerevisiaeD1/P3GIpHmediumwasadjust to pH 5, and medium for Z. mobilis FNCC

0056 pH adjusted to ph 7. Fermentation

mediumweresterilized. Forfermentationevery cultures(PichiastipitisCBS5773S. Cerevisiae D1_/P3G_{Iand Z. mobilisFNCC0056)asmuch} as 1_{0% put into a fermentation substrate,}

which is containing hydrolyzate with

concentration sugar are 5% and 10%. Then cultures in medium shake-incubated at 30o C for 72 _{h with agitation} 1_{50 rpm. During} incubation,samplesweretakenat0,6,12_,18_,

24, 30, 36, 48, 60, and 72 hours for

measurement of ethanol and reducing sugar

content.

RESULT_S AND D_ISC_USSION_S

O_pti_mi_zation of desi_zin_g of _parti_kelof _pit_h flo_ur and te_mperat_ure inc_ubation in

gelatini_zed P_{ith flourusedin this studyhada}

hemicellulose 4.86%, lignin 3.07% and water 1_0.12_%. Prior to the hydrolysis step, the substrate madegelatinization. G_{elatinizationis} a mechanism ofentry of water into thestarch granules so as to facilitate the action of the enzymehydrolyzesthesubstrate. G_{elatinization} isinfluencedbytemperature,substratetypeand concentration of the substrate itself [1_0]. Treatment with temperature 90˚C, 100˚C, 11_0˚C,and12_{0˚Cofthepithflourofin50and}

1_{00 mesh resulted a reducing sugar}

concentrationswereasfollows.

Table 1_{. Duncan's multiple range test analysis of}

Concentration of reducing sugar in size of pith flour 50 mesh and 1_{00 mesh.}

Tempof

Gelatinization

(˚C)

Cons. OfRed.

sugar(%)in50

meshflour

Cons. Of

Red.sugar(%)in

100mesh

90 0,28a 0,25a

100 0,35b 0,27a

110 0,42c 0,40b

120 0,53d 0,62c

Fromtheresultsof Duncan'smultiplerangetest

analysis can be seen that the increase in

gelatinization temperature, the greater the concentrationofsugarproduced. Theresultsof starch gelatinization of sago pith 50 mesh at 120˚C is a reducing sugar concentration of 0.53%. Whileon 100meshat 120˚C obtained 0.62_{%. Concentrationof sugarproduced from} thepithofsagoin1_{00meshsizelargerthan50} mesh. 1_{00 meshsize, mean grain size of pith} starch particles is 175ȝ. While the 50 mesh

size, mean grainsizeofpithstarchparticlesis 350ȝ.. The smaller a particle, the greater the

absorptionarea [11_].

Table 2_{. Duncan's multiple range test analysis of}

Dextrose equivalent (DE)at various temp. on 50 mesh and 1_{00 mesh}

Tempof

G_el.

(˚C)

Dextrose equivalent(DE)

(%)insize50

mesh

Dextrose equivalent(DE)

(%)insize1₀₀

mesh

90 0,51_{a 0,3}1_a

1_{00 0,57b 0,3}2_b

11_{0 0,69c 0,5}2_c

12_{0 0,}8_{5d 0,}8_9d

G_rainflour1_{00meshhasasmallerparticlesize} willabsorb moreheat andwater,thereforethe more that comes outof the starch amyloseso that theconcentration of sugarproduced more

than 50 mesh size. De_xtrose equivalent (DE) indicates the number of starch polymers that havebeencutintoglucosemoleculesaremuch simpler. Dextrose equivalent can be obtained from the ratio of the concentration of sugar sampleswithatotalsugarconcentration. From the analysis of Duncan's multiplerange test tisknownthat increasingthetemperature of gelatinization, the greater the dextrose equivalent (DE) is generated. From starch

gelatinization of sago pith flour 50 mesh

obtained the highest DE 0.8_{5% at} 12_{0˚C and} DE8_{9% at} 1_{00mesh. This isdueto the high} temperature will facilitate the breaking of carbon-hydrogenbond,sothatmorepolymeris cut starch into glucose[12_]. G_{elatin then is} hydrolyzed. chemically and enzymatically, Chemicalhydrolysisusingsulfuricacid6M_and

thepHwasadjustedto2.

Tabel 3: Conc. of red. sugar and (DE) of substrat is hydrolyzed by sulfuric acid 6 M _{and amylase,}

hemicellulase, cellulase and amyloglucosidase

TreatmentofHydrolysis Cons. Of

red. sugar. DE (%)

Sulfuricacid6M 22,6 28,63

Į-_{amylase 33,0}2 42,47

Hemicellulase 33,94 43,65

Cellulase and

Amyloglucosidase 53,28 68,52

DE = dextroseequivalent

Concentration of reducing sugar produced in thepithflourofsagoishydrolyzed bysulfuric acidismeasuredby DNS method. Hydrolysis using sulfuricacid 6M produceconcentration of reducing sugars 22_.2_{6% and DE}2 8_{.63% .} On the acid hydrolysis, acid breaks the bond glycosidic pith flour of sago at random but consecutive to the smallest molecule called glucose. However, the results of a reducing sugar and DE obtained indicates that not all pith flour of sago can behydrolyzed by acid. This is in accordance with the statement [13] that the conversion of starch using acid will onlygenerateama_ximumof DE55%. Hydrolysisbyenzymes

Hydrolysates I generated at acid hydrolysis usingsulfuricacid,andthenhydrolyzedbythe enzymeĮ-amylasewithdoses0,17ul/g,which lastedfor60minutesatatemperatureof1_04ºC

and pH 6.0, hydrolysis using Į-amylase

sugars obtained amounted to 33.02%, and DE 42_{.47%. Increased concentrations of reducing}

sugars due to the breakdown amylose and

amylopectininstarchbyĮ-amylasewhichdoes notoccuratoptimumintheacidhydrolysis. Į -amylaseis anendo-enzyme thatcan break the bondofalpha-₍1_{,4)glycosidicrandomly. Break} down of amylose by Į-amylase into maltose and maltotriosa that occur at random. Then formofglucoseandmaltoseasthefinalresult.

W_{hile working of} Į-amylase on the

amylopectin would produceglucose, maltose, and various types of oligosaccharide [15]. Hydrolysates II produced by Į-amylase then hydrolyzed by hemicellulase. Hydrolysis by hemicellulase for2₇0_{minutesatatemperature} of55ºCandpH6.0. Concentrationsofenzyme wereaddedat1/3oftherecommendedenzyme concentration isequalto0.001_{g /gsubstrate.} Hydrolysis by hemicellulase obtained hydrolyzateIII.

The main purpose hemicellulase, fibers which is containing hemicellulase will produce simple sugarsuchas hexoseandpentose. The

use of hemicellulase is able to raise of a

reducingsugarconcentrationandDE,reducing sugar from 33.02_{% to 33.94% and DE from} 42_{.47% to 43.65%.. The use of hemicellulase}

not only able to contribute of increase in

reducing sugarconcentration of thehydrolysis ofhemicellulosebutalsohelpstheactionofthe enzymecellulase..

H_ydrol_ysis _by cell_ulase en_zymes and A_mylo_gl_ucosidase_{. The addition of cellulase}

done because in the previous stage, the

hydrolysisofcellulosewasnotoptimal. While

the addition amiloglukosidase made to

hydrolyze dextrins obtained from the previous stage so as to produce glucose. Provision of

both types of enzyme was carried out

simultaneously as cellulase and

amiloglukosidase have synergistic

activity.Hydrolysis by cellulase enzymes and amiloglukosidase(saccharification)washeldat a temperature of 60 º C and pH 4.8_{. DX}

dextrozyme enzyme dosage was 0.37 ml / g

(dose of 2_{/3), while the number of Celluclast} 1.5 L is inserted is 0.55 ml / g (dose of 2/3). S_{accharification carried out for 4}8 _hours. Hydrolysis using dextrozyme DX and Celluclast 1_{.5 L produces a reducing sugar}

concentration [% (w / w)] 53.28% and DE 68_.52_{% after4}8_{hoursofincubation.}

Cellulaseenzymesplaya roleatthebeginning of the process is made starch granules more

open so that amyloglucosidase a chance to

hydrolyzestarchgranules insidewhichishave higher of sensitivity towards amiloglukosidase activity[1_{5]. Reducing sugar concentration} increased, this is due to cellulase enzymes break down celluloseintoselobiosa. S_elobiosa is then split into glucose. In addition, DX dextrozyme breaking glycosidic bond Į-1, 4 andĮ-1,6producemonomersofglucose. Bond of Į-1 ,4-glycosidic found in maltose and maltotriosawhich istheresult of a process of

amylose and amylopectin liquefaction while

bonds Į-1 ,6-glycosidic which is the dextrins

present in the process liquefaction of

amylopectin.

Atthis stageofthe hydrolysisofthe resulting concentration of DE for 68_.52_{%. This is} consistentwiththestatementLangloisandDale (1_{940) in Tjokroadikoesoemo [}1_{6] that the} hydrolysisprocessusingacombinationofacid and enzymes can increase the value of DE. Initially carriedoutbyacid hydrolysisprocess to DE 55%, then uses the enzyme hydrolysis followed by amilolitik to DE 61-_{65%. In this}

study, DE values greater than 65% can be

caused by the addition of the cellulolytic

enzyme hydrolysis process, the cellulase and hemicellulase so that the amount of glucose producedmore.Hydrolyzatesofsugarwhichis

the result of hydrolysis, then set the

concentration to 10% and 5%. HPLC analysis of the results obtained that 1_{0% of sugar} hydrolizate consists of glucose 8_.28_{% and} maltotetraosa 1_.72_%. S_{ugar concentration of}

5% consisted of glucose 4.17% and

maltotetraosa 0.8_3%. S_{ugar is then fermented} by P.stipitis, S.cerevisiae, and Z.mobilis a singleculture.

Efficienc_y fer_mentation of _Pichia stipitis,

Zymomonas mobilis and Sacchromyces

cerevisiae in fer_mentation of red_ucin_g s_ugars of _hydrol_yzate of _pit_h of sa_go starc_h. Concentration of sugarand ethanol atthe end of the process of ethanol fermentation of hydrolyzate sugar by Pichia stipitis. Hydrolyzate fermentation medium sugar

concentrationof5%,containingglucose4,17% glucose and maltotetraosa 0.8_3%. While the

concentration of sugar hydrolyzate medium

1_{0% containing glucose} 8_.28_{% glucose and} 1.76% maltotetrosa.

Fig 1_{. Reducing sugar and ethanol concentration changes}

during fermentation hydrolyzate of pith of sago starch by P.stipitis

During the fermentation process takes place,

the concentration of sugar hydrolyzate 5%

containing glucose 4,1_{7% is used up by}

P.stipitisatthe60hours. Whiletheinitialsugar hydrolyzateconcentration1_{0%, after7}2_hours as much as 8.28% glucose used by P.stipitis and leaving glucose 4.73%, this means only 3.55% used by P.stipitis. This suggests that

P.st_ipit_is less tolerant to sugar concentration 10%. Sugar is used as a nutrient and for ethanol fermentation. On medium hydrolyzate sugar 5% containing glucose 4,1_{7% produces} ethanol only0.98_{% whichbegantoform after} 36hours,meaningthatup to36hoursglucose used for nutrition. From the fermentation is well knownefficiencyof glucosefermentation

byP_{ichia stipitisonly} 2_{3.5%, while according} to theory, 1_{00% sugar when fermented will}

produce50% ethanol [12].

Concentrationofethanolinthefermentationby

P.st_ipit_is sugar in a medium of sugar hydrolyzate 1_{0% containing glucose} 8_.28_%,

producesethanolonlyasmuchas0.77% for72 hours andethanolisformedat48_{,itmeans up}

to 36 hours, glucose used as a nutrient.

Concentration of ethanol produced in the

hydrolyzate10% lessthantheethanolproduced inthesugarhydrolyzate5%. thustheefficiency of fermentation by P.stipitis on glucose mediumcontaining8_.28_{% only9.30%.} Efficiencyof fermentation by Pichia stipitisis very low,it is presumed P.stipitisless able to ferment hexose sugars. The best ability to fermentpentosesugars. M_{oreglucoseisusedto}

increasethecellbiomass. AlsoP.stipitishavea lowtolerancetohighsugarconcentration. This canbeseenin1_{0% glucosemedium,P}.stipitis lagphaselongerthanthemediumof5%. Concentration of red_ucin_g s_ugar and et_hanol at t_he end of t_he _process of et_hanol fer_mentation of _hydrol_yzate s_ugar _by

Saccharomycescerevisiae

Fig 2. Reducing sugar and ethanol concentration changes during fermentation hydrolyzate of pith of sago starch by

S. cerevisiae

Hydrolyzate sugar concentration both at 5% and 1_{0%, during the fermentation process,} glucose is used up by the S.cerevisiae for 36 and 48 _{hours. Thisshows} S_{.cerevisiae has the} ability to ferment hexose sugars, particularly glucose very well. S_{.cerevisiae has invertase}

enzyme that acts to convert glucose into

ethanol[1_{7] .} S_{ugar medium consisting of} glucose4.1_{7%,fermentationby}S_{.cerevisiaefor} 72 _{hours produced} 1_{.57% ethanol and ethanol} havestartedtoform after18 hours. Efficiency of fermentation in sugar hydrolyzate 5% by S_{.cerevisiae as much as 37.6%.} W_{hile the}

concentration of ethanol produced by

S_{.cerevisiae on fermentation of sugar}

hydrolyzate10% inmediumcontainingglucose 8_.28_{%,producingethanolasmuchas4.}21_{% for} 72 _{hours. Fermentation efficiency of 49.}2_% reaching almost 50%, this corresponds to the theorythat100% ofsugarwhenfermentedwill

produce50% ethanol [12_].

Concentration of red_ucin_g s_ugar and et_hanol at t_he end of t_he _process of et_hanol fer_mentation of _hydrol_yzate s_ugar _by

Zymomonasmobilis.

During the fermentation process, hydrolyzate sugar concentration 5% containing glucose -2 0 2 4 6 8 10

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time (hour)

F in a l c o n s . O f s u g a r (% )

Cons. In5% OfSugarhydrolizate Cons. In10% OfSugarhydrolizate

-0.2 0 0.2 0.4 0.6 0.8 1 1.2

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time (hour)

C o n s o f E th a n o l (% )

Cons. Ofetanolininitialconsofsugar5% Cons. Ofetanolininitialconsofsugar10%

-2 0 2 4 6 8 10

0 6 12 18 24 30 36 42 48 54 60 66 72 78 Time(Hour)

F in a l c o n s . O f s u g a r (% )

Cons. In5% OfSugarhydrolizate Cons. In10% OfSugarhydrolizate

-1 0 1 2 3 4 5

06121824303642485460667278 Time(Hour)

C o n s o f e th a n o l (% )

Cons. Ofetanolininitialconsofsugar5% Cons. Ofetanolininitialconsofsugar10%

4.17% is usedup byZ.mobilisafter 36 hours andtheethanolyieldof1_.34%.

Fig 3. Reducing sugar and ethanol concentration changes during fermentation hydrolyzate of pith of sago starch by

Z.mobilis.

Glucose is used for cell growth and ethanol fermentation substrate. W_{hereas in sugar}

hydrolysates 1_{0% containing glucose} 8_, 28_% still remaining glucose. .37%. This is due to bacterialcellsdieafter36hours,whileethanol producedas much as 1_{.34% for 7}2 _{hours, the} ethanolbeginsto formafter6hours. Basedon amountofethanolthatisformedisknownthat the sugar hydrolyzate 5% fermentation efficiency Z.mobilis reachs 32_.1_{3% while on}

the sugar hydrolyzate 1_{0%, ethanol}

fermentation efficiency reach 2_0.65%. Accordingto[18]. Z.mobilishas agoodability to fermentglucose,theresultsareevengreater than the S_{.cerevisiae due to the formation of} ethanolbytheEntnerDoudoroff. Inthisstudy the fermentation of sugars hydrolyzate b Z.mobilis obtained a little amount of ethanol, becausethemediumpHdropped to3:37 isso lowthatbacterialcelldeathoccurred.

CONCL_USION_S

1_{.Desizing of pith of sago starch particles in} the size of 100 mesh, and gelatinization temperature at 12_{0 ˚ C, can increase the} reducing sugar and dextrose equivalent (DE).

2.Hydrolysis by acids and enzymes can be applied in synergy so as to increase the activityon the hydrolysisof lignocellulosic

materials.

3.Efficiency of fermentation in the sugar hydrolyzate sugar concentration of 5%

containing ofglucose 4.1_{7% Pichi}a_stipitis,

Sa_{cchromyces cerevisi}a_{e and Zymomo}na_s mobilis,respectivelyare2_{3.5%,37.65% and}

32.13%. While in the hydrolyzate sugar

concentration1_{0%of sugar with glucose}

content 8_.28_{%, fermentation efficiency of}

Pichia _stipitis,Sa_{cchromycescerevisi}a_{e and} Zymomonas mobilisrespectively are 9.3%, 50.8_{5% and}2_0.65%.

AC_KNO_WL_ED_GEMENT_S

This research work was carried out with the support of DP2M - _Directorate G_{eneral of} Higher Education, Ministry of National of educationthroughHibahK_ompetitifPenelitian

–P_{rioritas Nasional Batch}

I.no.303/SP2_H/PP_/DP2M_/V_I/2_009.

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[11_{] Handayani,B H.} 2_{006. Hidrolisis P}a_{ti D}a_gu (Metroxylon _sa_{gu Rottb}.) Secara Enzima_{tis d}an _Asa_m Serta _Fermen_ta_{i Hidrolis}a_tn_ya _Men_ja_{di Et}an_{ol Oleh} Strain_{S C FNCC 3012 d}an _Isola_{t B}a_{kteri As}a_{l Empulur}

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