ORIGINAL ARTICLE

Production of bioethanol from empty fruit bunches cellulosicbiomass and Avicel PH-101 cellulose

Rose Amira Karim & Azlan Shah Hussain &

Asna Mohd Zain

Received: 25 October 2013 /Revised: 6 February 2014 /Accepted: 6 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Cellulosic ethanol production was carried out usingempty fruit bunches (EFB) via simultaneous saccharificationand fermentation (SSF) method. The EFB was pretreated withalkaline treatment using 0.2 M of sodium hydroxide (NaOH)solution followed by mild acid hydrolysis using 3 % ofsulfuric acid (H2SO4), and enzymatic saccharification usingderived cellulose, Trichoderma reesei prior to fermentationusing Saccharomyces cerevisiae. Acid hydrolysis-pretreatedsamples shows the best substrates to be used in fermentationsince it can produce the highest amount of glucose and highestpercentage of saccharification with 5.3 mg/mL and 48 %,respectively. The EFB hydrolyzate obtained was subjected tofermentation under anaerobic condition. It was found that thehighest ethanol yield was 0.42 mg/mL from acid hydrolyzesample. Optimization of SSF was conducted on filter paperunit (FPU), pH and mass loading effect for bioethanol pro-duction. Highest ethanol productions from cellulose (AvicelPH-101) are 3.1, 3.7, and 4.6 mg/mL using FPU 217, pH 4,and a 5.0-g cellulose loading accordingly.

Keywords Empty fruit bunches . Enzymatic saccharificationand fermentation . Bioethanol . Trichoderma reesei .

Saccharomyces cerevisiae . Avicel PH-101

NomenclatureDNS 3 5-Dinitrosalicyclic acidEFB Empty fruit bunchesFPU Filter paper unit

OD Optical densitySSF Simultaneous saccharification and fermentationRID Refractive index detectorYEPD Yeast-extract-peptone-dextrose

1 Introduction

Since the twentieth century, fossil fuels are the main energysupply to the global industrial and urbanization needs. Fossilfuels originate from deceased organisms deposited from sev-eral million years ago forms ancient treasure to world popu-lation. Fossil fuels already been extracted at optimum capacitywith world reserve will be available for the next 60 to 80 yearsbut in depleting quantity. Combustion of fossil fuels resultedin a net increase of carbon dioxide level in the atmosphere.Environmental effects related to carbon dioxide emission, oneof green house gases include global warming, extreme weath-er, and flooding. Current uses of fossil fuels contributing to83 % energy supply are unsustainable. Energy producer focusto renewable energy sources from biomass for replacing fossilfuels dependent as stated by US. IEA 2011 forecast on naturalgas and renewables continue to be the major sources ofenergy.

Significant agriculture development in Malaysia providesabundant biomass from large-scale agricultural activities suchas palm plantation. In 2011,Malaysia has fivemillion hectaresof oil palm or 14.3 % of total land area with an overall averageof 18.03 tons of fresh fruit bunches (FFB) per hectare pro-duced from the oil palm industry [1]. Palm oil plantation areashas produced more than 66.63 million tons of biomass resi-dues in numerous forms including EFB, mesocarp fiber, shell,palm kernel cakes, trunks, and palm oil mill effluent [2] withEFB representing about 9 % of the total biomass.

R. A. Karim :A. Mohd Zain (*)Chemical Engineering Department, Universiti TeknologiPETRONAS, Bandar Seri Iskandar, 31750 Tronoh, Perak, Malaysiae-mail: asnamz@petronas.com.my

A. S. HussainNPAE, PETRONAS Research S.B., Bandar Baru Bangi,41300 Bangi, Selangor, Malaysia

Biomass Conv. Bioref.DOI 10.1007/s13399-014-0117-7

Biomass can be a very promising alternative source ofrenewable energy as it contains 38–50 % cellulose, 15–35 %hemicellulose, and 10–30 % lignin [3, 4]. Ethanol can beproduced from different lignocellulosic biomass through sev-eral processes, namely, chemical pretreatment in acidic solu-tion and hydrolyze by enzymatic saccharification to produceglucose and fructose before fermentation process to produceethanol [5, 6]. Ethanol production via fermentation is one ofthe complex biochemical processes where yeast or bacteriafeed on simple sugar as substrate for their growth byconverting glucose into ethanol and carbon dioxides as inEq. 1.

C6H12O6→2CH3CH2OH þ 2CO2 ð1Þ

The common microorganism used in fermentation processis Saccharomyces cerevisiae that converts sugar into alcohol[7]. During ethanol fermentation, most of the yeast cells sufferenvironmental stress such as nutrient deficiency, temperature,rate of agitation, and pH condition [8–10]. The current workoptimized bioethanol production fromEFB cellulosic biomassand Avicel PH-101 cellulose via simultaneous saccharifica-tion and fermentation (SSF). Avicel PH-101 was used as acomparison to the EFB biomass, as it has a high cellulosecontent of 99.5 % for ethanol production. Optimization pa-rameters of the Avicel PH-101 ethanol production are FPU,pH, and mass loading.

2 Materials and method

2.1 EFB characterization

Oil palm EFB used as rawmaterial for ethanol production wascollected from local oil palm plantation. The EFB weregrounded using Power Master Machine with fiber length of0.04 in. The EFB was characterized for moisture content bymodified ASTM E1755 by oven drying at 105 °C and weighto the nearest 0.1 mg. The ash content was measured byplacing cellulose in platinum crucible and heated in the fur-nace at 575 °C±25 °C. CHNS analyzer was used to determinetotal carbon (C), nitrogen (N), and C/N ratio. The metalcontent in the EFB was determined using PerkinElmer induc-tively coupled plasma optical emission spectrometry (ICP-OES) optima 8,300. Closed-vessel microwave acid digestionwas performed at 105 °C prior to EFB metal analysis.

2.2 Enzymatic saccharification

2.2.1 Delignification

The substrate was suspended in 0.2MNaOH solution with theratio of 1:10 and kept at room temperature for 18 h for

delignification process. The solution was filtered, and residuewas repeatedly washed with tap water until the pH valuebecomes neutral (pH 7). The residue was then dried at 50 °Cfor acid/enzyme hydrolysis experiments.

2.2.2 Acid hydrolysis

The alkali pretreated substrate was subjected to 3 % sulfuricacid at 130 °C for 30 min. The contents were filtered, andresidue was repeatedly washed with tap water until the pHbecame neutral and the sample was subsequently dried at50 °C.

2.2.3 Enzymatic hydrolysis

100 mL of untreated (control), alkali, and acid hydrolysis-pretreated samples were hydrolyzed with 128 FPU/mL ofderived cellulase, Trichoderma reesei in 0.05-M citrate buffer(pH 4.8) at a substrate concentration of 5 %. The pretreatedsubstrates were soaked in the buffer for 2 h before it wasmixed with the enzyme. Sodium azide 0.005 % was intro-duced to the mixture to prevent any contamination or micro-bial activity. The flasks were incubated at 50 °C on an orbitalshaker at 150 rpm for 60 h. Aliquots of 1 mL were sampledevery 12 h and centrifuged, and the supernatant were analyzedfor reducing sugars in acid hydrolyzate.

2.3 Simultaneous saccharification and fermentation

The selected yeast used in the SSF study was S. cerevisiaeATCC 96581 obtained from the American Type Culture;Manassas United States. The ampoules were stored in liquidnitrogen at −70 °C.

2.3.1 Inoculums preparation

S. cerevisiae was initially grown on yeast-extract-peptone-dextrose (YEPD) medium and was incubated at a temperatureof 30 °C and agitated at 190 rpm for 24 to 36 h (Innova 40). Inthis study, YEPD medium consisted of a 10-g Bacto-peptone,5-g yeast extract, 10-g dextrose, and 500-mL distilled water.After the incubation period, harvested cells were centrifugedat 3,500 rpm for 15 min. The pellet was then rinsed twice withsterilized saline solution before being re-suspended in steril-ized saline solution to yield an optical density (OD) of 0.5 at600 nm (Shimadzu UV-1601PC). The standardizedS. cerevisiae was used for a subsequent study.

2.3.2 Fermentation process

Fermentation by S. cerevisiae was performed under anaerobicconditions of deionized water (pH 3.5) medium. The mediumwas hydrolyzed with 5 mg/mL derived cellulase, T. reesei

Biomass Conv. Bioref.

(Celluclast 1.5, Novozymes A/S Bagsvaerd, Denmark) withacid hydrolysis-pretreated substrate. Deionized water contain-ing the substrates was sterilized at 121 °C for 15 min. Thefermentation was carried out in a 250-mL heavy wall filteringflask since the tube is used to connect from the flask to the gas-washing bottle fritted cylinder containing sterile sodium hy-droxide solution to prevent the presence of oxygen in order tomaintain the anaerobic conditions. The flask was incubatedfor 72 h.

Off-line pH control was done by addition of 2 M sodiumhydroxide solution, keeping the pH around 12. The total liquidvolume was 106.5 mL including 100 mL of deionized water(pH 3.5), 0.5 g of T. reesei, 1.0 g of acid pretreatment sub-strates, and 5 mL of inoculum. Fermentation was conducted at30 °C on a shaker at 100 rpm. Supernatant of 1 mL was takenout, centrifuged, and analyzed by high-performance liquidchromatography (HPLC).

Ethanol was analyzed using a calcium-based ion-exchangecolumn (Hiplex Cal) at 80 °C with 0.6 mL/min eluent of100 % filtered and degassed deionized water. Glucose, fruc-tose, and mannose were analyzed using plumbum-based ion-exchange column (HiplexPb) at 80 °Cwith 0.6 mL/min eluentof 100 % filtered and degassed deionized water using refrac-tive index detector (RID).

3 Results and discussion

3.1 EFB characterization

The EFB which has 6.38 % moisture and 1.39 % ash contentwas demoistured for characterization and bioethanol produc-tion. To ensure EFB was totally dried, weighing was repeateduntil the difference in weight recorded was less than 0.003 g.Metal analysis was carried out to determine the amount ofmetal impurities that may affect conversion of biomass tobioethanol. The composition of EFB is tabulated in Table 1.

The total C and N contents were 44.72 and 1.425 %,respectively, while C/N ratio was at 31.39. Based on the metalcontents in the EFB, potassium was found to give the highestvalue which is 2,451.78 mg/Kg. This is followed by calcium(436.29 mg/Kg), magnesium (251.68 mg/Kg), phosphorus(237.80 mg/Kg), iron (128.44 mg/Kg), and boron(21.89 mg/Kg). Manganese, zinc, and copper were foundbelow 0.1 mg/Kg.

3.2 Enzymatic saccharification

3.2.1 Analysis of enzymatic saccharification by DNS methodusing UV–vis

Fig. 1 shows the results of reducing sugars in acid hydrolyzateby using 3,5-Dinitrosalicyclic acid (DNS) method from

enzymatic hydrolysis of untreated, alkali treated, and acidhydrolysis. The absorbance values of the samples were mea-sured using UV-visible spectrophotometer (Shimadzu UV-1601PC) at 575 nm.

Maximum sugar yield per substrate for each pretreatmentsample were acid hydrolysis with 5.26 mmol/mL, alkali treat-ed with 4.35 mmol/mL, and finally the untreated substratewith 1.73 mmol/mL. Acid hydrolysis pretreatment with 3 %of sulfuric acid had increased the total reducing sugar in acidhydrolyzate over the incubation period.

The percentage of saccharification or hydrolysis can becalculated by dividing the reducing sugars in acid hydrolyzatewith total holocellulose in pretreated samples. As totalholocellulose was not measured, percentage of saccharifica-tion was quantified using Eq. 2 [11].

%Saccharification ¼reducing sugar

mg

mL

� �x0:9x100

initial substratemg

mL

� � ð2Þ

Table 1 EFBcomposition Parameter Value

Carbon, C (%) 44.72

Nitrogen, N (%) 1.42

Phosphorus, P (mg/Kg) 237.80

Potassium, K (mg/Kg) 2451.78

Magnesium, Mg (mg/Kg) 251.68

Calcium, Ca (mg/Kg) 436.29

Boron, B (mg/Kg) 21.89

Iron, Fe (mg/Kg) 128.44

Manganese, Mn (mg/Kg) <0.1

Copper, Cu (mg/Kg) <0.1

Zinc, Zn (mg/Kg) <0.1

C/N ratio <0.1

0.00

1.00

2.00

3.00

4.00

5.00

6.00

12 24 36 48 60 72 84

To

tal r

edu

cin

g s

ug

ar (

mg

/mL

)

Time (hours)

acid hydrolysis

alkali treated

untreated

Fig. 1 Total amount of reducing sugars in acid hydrolyzate analyzedusing the DNS method

Biomass Conv. Bioref.

Based on Fig. 2, the highest percentage of saccharificationwas obtained from acid hydrolysis with 47.37 % followed byalkali treated with 39.11 % and finally untreated substrateswith 15.59 %.

Kinetic rate of reaction of enzyme in acid hydrolysisis higher compared to other substrates. The amount ofreducing sugars produced for acid hydrolysis increaseswith time. At 72 to 84 h, only a small increment wasobserved for every substrate as time proceeds. It showsthat substrate is a limiting reactant. For the improve-ment of enzymatic hydrolysis, it is necessary to opti-mize the critical process parameters such as optimumcellulase loading, temperature, saccharification time, andsubstrate to liquid ratio.

Making cellulose accessible to the enzymes is an im-portant factor to increase the rate of hydrolysis. Therefore,chemical pretreatment, usually alkaline and acid hydroly-sis pretreatment are necessary before enzymatic hydroly-sis. Chemical pretreatment not only removes lignin butalso acts as a swelling agent, which will enhance surfacearea of the substrate accessible for enzymatic action.

3.2.2 Analysis of enzymatic saccharification using HPLCmethod

Determination of oligosaccharides and monosaccharides fromeither acid hydrolysis or enzymatic hydrolysis of cellulosederived from different types of chemical treatment can bedetermined by RID HPLC. The choice of column for separa-tion of these oligosaccharides andmonosaccharides is of equalsignificance as retention time and resolution quality, and thelimit of quantification will be highly dependent on it.

Fig. 3 shows the results of total reducing sugars in acidhydrolyzate from enzymatic hydrolysis of untreated, alkalitreated, and acid hydrolysis. Based on this method, pretreatedsample performance is dissimilar with the results from the

DNS method. It was observed that two common sugars ofglucose and fructose were quantified by HPLC at retentiontime of 7.4 and 5.8 min, accordingly. Enzymatic hydrolysisalso produced reducing sugars which are hexose sugars; glu-cose and fructose. Standard calibration curves of glucose andfructose were used to extrapolate unknown sugars in thesamples.

Fig. 4 shows the percentage of saccharification for eachpretreated sample using the HPLC method. The enzymatichydrolysis by the DNS method exhibited different perfor-mance from the HPLC method. The DNS method couldmeasure any reducing compounds based on its reaction mech-anism and widely used with its simplicity and inexpensivereagents. The DNS method exhibits gradual increment ofreducing sugars over 12 to 24 h. On the contrary, the HPLCmethod indicates two optimum peaks of reducing sugars foreach sample.

HPLC analysis of sample aliquots must be executed im-mediately, or else, the aliquots must be kept in a freezer toretard the enzymatic reaction. Frozen supernatants need to be

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

12 24 36 48 60 72 84

Per

cen

t sa

chh

arif

icat

ion

(%

)

Time (hours)

Acid hydrolysis

Alkali treated

Untreated

Fig. 2 Percentage of saccharification in acid hydrolyzate analyzed usingthe DNS method

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

12 24 36 48 60 84 108

To

tal r

edu

cin

g s

ug

ar (

mg

/mL

)

Time (hours)

acid hydrolysis

alkali treated

untreated

Fig. 3 Total amount of reducing sugars in acid hydrolyzate analyzedusing the HPLC method

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

12 24 36 48 60 84 108

Per

cen

t sa

cch

arif

icat

ion

(%

)

Time (hours)

AcidhydrolysisAlkali treated

Untreated

Fig. 4 Percentage of saccharification of reducing sugars in acid hydro-lyzate analyzed using the HPLC method

Biomass Conv. Bioref.

thawed and agitated before the HPLC analysis. HPLC allowsanalysis of the sample in a shorter time and achieves a higherdegree of resolution. DNS takes longer assay times, and validonly at low levels of hydrolysis.

The comparison of HPLC and DNS methods are presentedto show the real time progress of saccharification percentagein producing total reducing sugars.

3.3 SSF process

Generally, formation of lignocellulosic biomass should under-take several processes consisting of chemical pretreatment;acid and alkali, enzymatic saccharification, and fermentationof sugar to ethanol [12]. Since acid hydrolyze pretreatedsubstrates produced the highest total reducing sugars andpercentage of saccharification during enzymatic saccharifica-tion, the substrates were subsequently being used for fermen-tation process.

The first fermentation phase (0 to 27 h) in Fig. 5 shows thatboth glucose reduction and ethanol production is very slow.The glucose and ethanol yield curves reveal that during thefirst couple of hours, the inoculated yeast cells, S. cerevisiae,went through a process of adjusting themselves to the newenvironment of the fermentation. Little amount of glucose wasconsumed, and ethanol production was barely measurable.The pH value of sodium hydroxide solution remains constantduring this time.

In the second phase from 34 to 52 h, ethanol yield increasedwhile significant amount of glucose was consumed and etha-nol concentration noticeably progressed. The pH value wasfound to decrease by three units caused by the production ofcarbon dioxide. At 96 h, the graph shows maximum ethanolproduction at 0.42 mg/mL. However, insignificant changes inglucose consumption were observed since the amount ofglucose left is 0.69 mg/mL compared to 0.71 mg/mL at 84 h.

It was concluded that the yeast also consumed other sugarssuch as fructose or xylose since at that time, those sugars werealso formed from cellulose saccharification. At 105 h, ethanolproduction depleted to 0.1 mg/mL, albeit higher glucose re-mains available. This might indicate that other fermentablesugars such as fructose, xylose, and mannose were first hy-drolyzed into glucose before converted into ethanol.

Besides ethanol and glucose, there are also xylitol andmannose found at 28.0 and 15.8 min retention time, accord-ingly. These two compounds also keep on increasing overtime.

Major fermentable sugars from hydrolysis of most cellu-losic biomass are D-glucose, D-fructose, and D-xylose exceptthat softwood also contains significant amounts of mannose.However, industrial S. cerevisiae cannot metabolize xylose.Hence, xylose amount accumulated during the process. In acidhydrolysis-pretreated substrates, it contains hemicellulose andalso cellulose. Xylitol can be produced from xylose/

0

1

2

3

4

5

6

7

12 hours 24 hours 36 hours 48 hours 60 hours

Am

ou

nt

of

sug

ars

(mg

/mL

)

FPU 77

FPU 128

FPU 154

FPU 217

Fig. 6 Glucose consumption by S.cerevisiae at different FPU values atpH 4 and 100 rpm

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 50 100 150

Co

nce

ntr

atio

n (m

g/m

L)

Time (hours)

Glucose

Bioethanol

Fig. 5 Kinetics of ethanol fermentation from acid-treated cellulose bySaccharomyces cerevisiae

Table 2 HPLCconditions for Hi-PlexCacolumn

Mobile phase DI—water

Flow rate (mL/min) 0.6

Injection (μL) 20

Column temperature (°C) 80

RID temperature (°C) 35

Column pressure (bar) 35.5

Analysis time (min) 45

Separation, N 26361

0

0.5

1

1.5

2

2.5

3

3.5

12 hours 24 hours 36 hours 48 hours 60 hours

Am

ou

nt

of

eth

ano

l (m

g/m

L)

FPU 77

FPU 128

FPU 154

FPU 217

Fig. 7 Ethanol production from cellulose hydrolyzate at different FPUvalues at pH 4 and 100 rpm

Biomass Conv. Bioref.

hemicellulose hydrolyzate by chemical reduction or microbialfermentation. That indicates for the existence of xylitol afterthe fermentation process. The uses of yeasts, classified asfacultative anaerobes, are capable of performing fermentationin both aerobic and anaerobic conditions.

3.4 Effect of FPU value to the production of bioethanol

The experiment is carried out with different filter paper units(FPU) of derived cellulase, T. reesei. One FPU refers to theamount of enzyme that releases 1 μmol glucose per minuteduring hydrolysis reaction. The FPU values used in the ex-periment were 77, 128, 154, and 217. This experiment aimedto study the effect of FPU value to the production of ethanol.The sample analytes were analyzed using HPLC conditions asin Table 2.

Glucose and fructose were detected by RID HPLC at 14.32and 20.51 min, accordingly. However, ethanol eluent wasdetected at 23.59 min. SSF produced reducing sugars ofhexose; glucose and fructose and ethanol itself. Unknownproducts were interpolated by glucose and fructose calibrationcurve with confidence of RSD<0.01.

Fig. 6 shows the graph of total glucose produced from theseparate SSF process from 24 to 60 h. Generally, as the FPUvalues increased, the amount of glucose significantly reduced

especially at higher FPU. FPU 217 recorded the lowestamount of glucose at 60 h with 1.52 mg/mL. Higher FPUprovides a higher amount of derived cellulase, T. reesei; thus,more glucose is expected since the cellulose is changed intoglucose with the help of cellulase, but as incubation timeprogresses, these glucose already depleted to the minimum.Fig. 7 shows the amount of ethanol production from thefermentation process. Higher FPUs are able to produce moreethanol than a lower FPU with 3.10 mg/mL produced fromFPU 217. Furthermore, ethanol yield gradually increased withincubation time at pH 4. Ethanol production was optimized byFPU 217 at a 60-h incubation with 947.12 % fermentationefficiency. The amount of an FPU value would directly affectthe production of ethanol. Initially, the OD used for eachsample is 1.0, and the value was increased to 1.72 (24 h),2.04 (36 h), 2.47 (48 h), and 2.89 (60 h). Hence, as fermen-tation time progresses, the amount of glucose produced de-creased simultaneously as S. cerevisiae convert the glucoseinto ethanol.

3.5 Effect of pH value to the production of bioethanol

The effect of different initial pH on glucose consumption byS. cerevisiaeATCC 96581 and ethanol yield using Avicel pH-101 are shown in Figs. 8 and 9. From the graph in Fig. 9, thehighest ethanol yield was obtained at pH 4 with a maximum

0

0.5

1

1.5

2

2.5

3

3.5

4

12 hours 24 hours 36 hours 48 hours 60 hours

Am

ou

nt

of

eth

ano

l (m

g/m

L)

Ph 4

Ph 6

Ph 7

Ph 8

Fig. 9 Ethanol production from cellulose hydrolyzate at different initialpH at 30 °C and 100 rpm

0

1

2

3

4

5

6

12 hours 24 hours 36 hours 48 hours 60 hours

Am

ou

nt

of

sug

ars

(mg

/mL

)

Ph 4

Ph 6

Ph 7

Ph 8

Fig. 8 Glucose consumption by S. cerevisiae at different pH at 30 °C and100 rpm

0

2

4

6

8

10

12

14

16

12 hours 24 hours 36 hours 48 hours 60 hoursAm

ou

nt

of

sug

ars

(mg

/mL

)

0.5 g1.0 g2.0 g5.0 g

Fig. 10 Glucose consumption by S. cerevisiae at different mass loadingof cellulose at pH 4 and 100 rpm

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

12 hours 24 hours 36 hours 48 hours 60 hours

Am

ou

nt

of

eth

ano

l (m

g/m

L)

0.5 g

1.0 g

2.0 g

5.0 g

Fig. 11 Ethanol production from cellulose hydrolyzate at different massloading at pH 4 and 100 rpm

Biomass Conv. Bioref.

ethanol concentration of 3.76 mg/mL followed by 3.17 mg/mL at pH 6 at a 60-h incubation. The respective ethanolformation for both pH were 0.06 mg/h (pH 4) and 0.05 mg/h(pH 6). Fermentation of the cellulose hydrolyzate at initial ofpH 7 and pH 8 produced a lower ethanol concentration cor-responding to ethanol formation of 0.04 mg/h for both pH,respectively. It was due to inactive S. cerevisiae activity tofacilitate fermentation process in the alkaline medium. Fig. 8also shows that glucose consumption rate was optimized atinitial pH 4 which correlated to highest glucose utilizationover 60 h of incubation. Instead, Fig. 9 shows higher ethanolconcentration of 3.76 mg/mL in pH 4 sample. The fermenta-tion efficiency was calculated based on the ratio of ethanolyield obtained against theoretical maximum ethanol yield.The highest ethanol fermentation efficiency was obtained atpH 4 with 599 %. High fermentation efficiency was due to thepresence of other simple sugars of mannose and arabinose inthe hydrolyzate; thus, contribute to a higher concentration ofethanol obtained in the process. As fermentation of cellulosehydrolyzate at pH 4 showed the highest ethanol production,this pH value was used in all of the following experiments.

The effect of initial pH has been reported to show a signif-icant influence on fermentation, mainly on yeast growth,fermentation rate, and by-product formation [13–15]. Theresults obtained from this study shows that the most suitableinitial pH value for ethanol production from cellulose was pH4. It was found that increment of pH value was able to reduceethanol production rate and glucose consumption rate. Thiscurrent study is in agreement with other studies reported thatthe growth of yeast and fermentation process perform best innatural or slightly acidic environment [16]. This study indi-cates that lower ethanol concentration is produced at higherpH value. Lower ethanol productivity may be associated to itslower metabolic rate of yeast cell used [17]. Increment in pHvalue will increase the permeability of the cell membraneresulting in a reduction of the rate of sugar fermented enzymeproduction. The lower ethanol yield and sugar conversionobtained at higher pH value were also probably due to theformation of undesired product such as glycerol and organicacid during the fermentation process.

3.6 Effect of mass loading of substrate to the productionof bioethanol

The effects of different mass loading of cellulose on glucoseconsumption by S. cerevisiae and ethanol production fromcellulose hydrolyzate are illustrated in Figs. 10 and 11.Fig. 10 shows the amount of glucose consumed byS. cerevisiae in producing ethanol. From the graph, it wasclearly shown that at higher loading of cellulose, more glucosehas been consumed compared to lower mass loading. Decre-ment of glucose was also higher at higher mass loading ofcellulose over time, from 14.86mg/mL (24 h) to 11.21mg/mL

(60 h) observed with 5.0 g mass loading. Fig. 11 depicted thehighest ethanol concentration which was 4.63 mg/mL obtain-ed with cellulose mass loading of 5.0 g and reduced to3.76 mg/mL at 2.0 g which corresponded to an ethanol for-mation of 0.07 and 0.06 mg/h, respectively.

4 Conclusions

Delignified EFB fibers by acid hydrolysis has the highestreducing sugar in acid hydrolyzate compared to the untreatedor alkali-treated sample analyzed using DNS and HPLCmeth-od. The maximum sugar produced was 5.18 mg/mL. It showsthat acid hydrolysis is the best treatment process to removelignin from EFB. Besides, ethanol can be produced from SSFmethod. The optimum ethanol yield from acid-hydrolyzedEFB sample was 0.42 mg/mL achieved at pH 3.5 and 30 °C.The optimum ethanol yield from Avicel PH-101 cellulose are3.1, 3.7, and 4.6 mg/mL achieved by FPU 217, pH 4, and5.0 g of cellulose mass loading, accordingly. Similar work onother parameters such as temperature, agitation rate, and othertypes of substrate by SSF should be carried out to optimizeethanol production.

Acknowledgement The current research was supported by theUniversiti Teknologi PETRONAS and PETRONAS Research Sdn. Bhd.

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