Hydrolysis Of Empty Fruit Bunch Research Articles

Vapor-Liquid Equilibrium (VLE) Curve for Furfural Purification from Oil Palm Empty Fruit Bunch Hydrolysate Solution with the UNIQUAC Model

Furfural is the main Oil Palm Empty Fruit Bunch (OPEFB) hydrolysis product. It can achieve high purity from hydrolysate distillation. Its equipment design can be done by utilizing vapor-liquid equilibrium (VLE) data to achieve high furfural purity. The purpose of this research is to develop a Vapor-Liquid Equilibrium (VLE) curve for furfural purification from Oil Palm Empty Bunch hydrolysate solution with the Universal Quasi Chemical (UNIQUAC) model. Distillation data is obtained from literature and examined on the Universal Quasi Chemical (UNIQUAC) model. Calculations for average deviation and R2 are also made to show the accuracy of the model quantitatively. The modeled VLE curve indicates that the UNIQUAC model and the literature distillation pattern are not ideal, and the UNIQUAC curve pattern closely follows the literature pattern. The average deviation and R2 values for this UNIQUAC model are 0.0954 and 0.297, respectively. These values are still acceptable and show that UNIQUAC can be used as useful model distillation data of furfural and water as the representatives of an OPEFB hydrolysate solution.

Enhanced ethanol production from lignocellulosic hydrolysate using Meyerozyma caribbica biofilm immobilized on modified epoxy foam

In comparison to planktonic cells, microbial biofilm immobilization is known to enhance ethanol production. To maintain this capability during long-term fermentation, the detachment of the biofilm must be prevented. In this study, the enhancement of the biofilm immobilization of the yeast Meyerozyma caribbica YLP01GX on bio-based epoxy foam (EF) was performed through plasma surface modification to improve the production of ethanol from oil palm empty fruit bunch hydrolysate. This newly synthesized EF serves as a suitable carrier due to its superior properties and cost-effectiveness. The modification could induce yeast cell attachment more rapidly with more extracellular polymeric substance (EPS) being produced in the presence of the yeast extract. Therefore, the immobilization period can be shortened. In comparison to untreated EF-immobilized cells (NEF-IC), the modified EF-immobilized cells (MEF-IC) showed an improved binding affinity for proteins in the EPS, which resulted in a higher adhesion force between the carrier and the yeast cell surface. The stable biofilm attachment on the modified EF can prevent biofilm detachment and improve the tolerance of the yeast to inhibitors in the hydrolysate, leading to enhanced ethanol production compared to NEF-IC. Additionally, the MEF-IC was reused several times with similar fermentation activity being observed. This approach offers a more efficient and economical fermentation process.

Preparation of Oil Palm Empty Fruit Bunch Hydrolysate

Malaysia is the second largest palm oil producer and exporter globally. When crude palm oil is produced in both plantations and oil processing mills, a large amount of oil palm empty fruit bunch (OPEFB) is simultaneously produced as a waste product. Here, we describe the preparation of hydrolysate from OPEFB. After OPEFB was hydrothermally treated at 180–200 °C, the resultant liquid phase was subjected to high-performance liquid chromatography analysis, while the solid phase was used for acidic and enzymatic hydrolysis. Hemicellulose yield from the acid-treated solid phase decreased from 153 mg/g-OPEFB to 27.5 mg/g-OPEFB by increasing the hydrothermal treatment temperature from 180 to 200 °C. Glucose yield from the enzyme-treated solid phase obtained after hydrothermal treatment at 200 °C was the highest (234 ± 1.90 mg/g-OPEFB, 61.7% production efficiency). In contrast, xylose, mannose, galactose, and arabinose yields in the hydrolysate prepared from the solid phase hydrothermally treated at 200 °C were the lowest. Thus, we concluded that the optimum temperature for hydrothermal pretreatment was 200 °C, which was caused by the low hemicellulose yield. Based on these results, we have established an effective method for preparing OPEFB hydrolysates with high glucose content.

Catalytic Performance of Sulfonated Carbon Catalysts for Hydrolysis of Palm Oil Empty Fruit Bunch

Utilizing lignocellulosic biomass into valuable products, such as chemicals and fuels, has attracted global interest. One of lignocellulosic biomass, palm oil empty fruit bunch (EFB), has major content of cellulose (30-40%), which is highly potential to be a raw material for fermentable sugar production. In this research, a series of sulfonated carbon catalysts with various concentrations of sulfuric acid (H2SO4, 10-30 v/v%) solutions have been successfully prepared and applied for a single stage of heterogeneous acid-catalyzed hydrolysis over microcrystalline cellulose and EFB under moderate temperature condition and ambient pressure. The catalysts’ physical and chemical properties were characterized by using a Thermogravimetric Analyzer (TGA), X-ray diffractometer, surface area analyzer, and Fourier-transform infrared spectrophotometer. The characterization results showed that sulfonated carbon had relatively similar physical properties with the parent of active carbon. The hydrolysis activity of sulfonated carbon catalysts gave various Total Reducing Sugar (TRS). The effects of sulfate loading amount in catalyst samples and various ionic liquids were investigated. The hydrolysis of pure microcrystalline cellulose powder (Avicel) using 30%-sulfonated carbon (30-SC) catalyst in 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) ionic liquid at 150°C yielded the highest TRS of 16.11%. Subsequently, the catalyst of 30-SC was also tested for hydrolysis of EFB and produced the highest TRS of 40.76% in [BMIM]Cl ionic liquid at 150°C for 4 h. The obtained results highlight the potential of sulfonated carbon catalysts for hydrolysis of EFB into fermentable sugar as an intermediate product for ethanol production.

High Enzymatic Recovery and Purification of Xylooligosaccharides from Empty Fruit Bunch via Nanofiltration

Xylooligosaccharides (XOS) are attracting an ever-increasing amount of interest for use as food prebiotics. In this study, we used efficient membrane separation technology to convert lignocellulosic materials into a renewable source of XOS. This study revealed a dual function of nanofiltration membranes by first achieving a high yield of xylobiose (a main component of XOS) from alkali-pretreated empty fruit bunch (EFB) hydrolysate, and then by achieving a high degree of separation for xylose as a monosaccharide product. Alkali pretreatment could increase the xylan content retention of raw EFB from 23.4% to 26.9%, which eventually contributed to higher yields of both xylobiose and xylose. Nanofiltration increased the total amount of XYN10Ks_480 endoxylanase produced from recombinant Streptomyces lividans 1326 without altering its specific activity. Concentrated XYN10Ks_480 endoxylanase was applied to the recovery of both xylobiose and xylose from alkali-pretreated EFB hydrolysate. Xylobiose and xylose yields reached 41.1% and 17.3%, respectively, and when unconcentrated XYN10Ks_480 endoxylanase was applied, those yields reached 35.1% and 8.3%, respectively. The last step in nanofiltration was to separate xylobiose over xylose, and 41.3 g.L−1 xylobiose (90.1% purity over xylose) was achieved. This nanofiltration method should shorten the processes used to obtain XOS as a high-value end product from lignocellulosic biomass.

Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass

New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.

Lipid production by the yeast Lipomyces starkeyi grown on sugars and oil palm empty fruit bunch hydrolysate

Lipid accumulation by the yeast Lipomyces starkeyi grown on various carbon (C) sources (glucose, xylose, hydrolysate of oil palm empty fruit bunch (OPEFB)) is reported. In media with glucose as the main C-source, a glucose concentration of 60–80 g L−1 was optimal for producing microbial biomass and lipids. Glucose at 60 g L−1 afforded a final dry biomass concentration of 21.2 g L−1 with a 35.8% lipid content. The biomass concentration was reduced to 18.7 g L−1 if the glucose was 80 g L−1 and the lipids were reduced to 11.8%. With pure xylose (80 g L−1), the final biomass concentration was 14.0 g L−1 and the lipid content of the biomass was 42.1%. A 4:1 by mass mixture of glucose and xylose was used to simulate a typical hydrolysate of OPEFB. With this mixture (glucose = 64 g L−1, xylose = 16 g L−1), the maximum biomass concentration was 17.9 g L−1 and the lipids in the biomass exceeded 37.9%. A hydrolysate of OPEFB was prepared by enzymatic hydrolysis of the alkali-pretreated OPEFB. With this hydrolysate as the main C-source, the final yeast biomass concentration was 14.9 g L−1, but the lipid content in the biomass was only 6%. The lipid content of the yeast biomass could be raised to >40% by using a hydrolysate diluted with distilled water. Apparently, dilution alleviated the effects of inhibitory compounds that formed during hydrolysis of lignocellulosic biomass with the commercial enzymes. The lipid yield on C-sources was 0.162 g g−1 in a medium with a C:N mole ratio of 71.7:1.

Sequential chemical and enzymatic pretreatment of palm empty fruit bunches for Candida pelliculosa bioethanol production.

Second-generation bioethanol production process was developed using pretreated empty fruit bunches (EFB). Consecutive acid/alkali EFB pretreatment was performed, first with HCl and then with NaOH with final washing steps for phenolic compounds elimination. Scanning electron microscopy images showed that EFB chemical treatments indeed attacked the cellulose fibers and removed the silica from surface pores. The optimization of enzymatic hydrolysis of EFB's cellulosic fraction was performed with 0.5%-4% v/v of Cellic® CTec2/Novozymes, different EFB concentrations (5%-15%, w/v), and hydrolysis time (6-72H). Optimization essays were carried out in Erlenmeyer flasks and also in a 1L stirred tank reactor. After enzymatic hydrolysis, a hydrolysate with 66g/L of glucose was achieved with 2.2% (v/v) Cellic® CTec2, 15% (m/v) acid/alkaline pretreated EFB after 39H of hydrolysis. A gain of 11.2% was then obtained in the 1L stirred tank promoted by the agitation (72.2g/L glucose). The hydrolysate was employed in bioethanol production by a new isolate Candida pelliculosa CCT 7734 in a separate hydrolysis and fermentation process reaching 16.6 and 23.0g/L of bioethanol through batch and fed-batch operation, respectively. An integrated biorefinery process was developed for EFB processing chain.

Continuous production of biohydrogen from oil palm empty fruit bunch hydrolysate in tubular photobioreactors

Production of hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides was compared in continuously operated tubular photobioreactors illuminated by natural outdoor sunlight (0.15–66 klux; diurnal cycle) and constant indoor artificial light (10 klux; tungsten lamps). In both cases the operating temperature was 35 °C and the organic carbon source was an acid hydrolysate of oil palm empty fruit bunch (EFB), an agroindustrial waste. In the outdoor photobioreactor, under the best production conditions, the daytime feeding rate of the mixed carbon substrate was 48 mL h−1 and the average pseudo-steady state hydrogen production rate was 36 mL H2 L−1 medium h−1. The cumulative hydrogen production was 430 mL H2 L−1 medium. For the indoor photobioreactor fed at the same rate as the outdoor system, the steady state average hydrogen production rate was 43 mL H2 L−1 h−1 and the cumulative hydrogen production was 517 mL H2 L−1 medium. Reducing the feed rate to less than 48 mL h−1, enhanced the biomass concentration, but reduced hydrogen production in both bioreactors. The sunlight-based cumulative hydrogen production was only about 17% less compared to the artificially lit system, but required only 22% of the electrical energy.

Chemical and structural changes of pretreated empty fruit bunch (EFB) in ionic liquid-cellulase compatible system for fermentability to bioethanol.

The pretreatment of empty fruit bunch (EFB) was conducted using an integrated system of IL and cellulases (IL-E), with simultaneous fermentation in one vessel. The cellulase mixture (PKC-Cel) was derived from Trichoderma reesei by solid-state fermentation. Choline acetate [Cho]OAc was utilized for the pretreatment due to its biocompatibility and biodegradability. The treated EFB and its hydrolysate were characterized by the Fourier transform infrared spectroscopy, scanning electron microscopy, and chemical analysis. The results showed that there were significant structural changes in EFB after the treatment in IL-E system. The sugar yield after enzymatic hydrolysis by the PKC-Cel was increased from 0.058g/g of EFB in the crude sample (untreated) to 0.283 and 0.62 ± 06g/g in IL-E system after 24 and 48h of treatment, respectively. The EFB hydrolysate showed the eligibility for ethanol production without any supplements where ethanol yield was 0.275g ethanol/g EFB in thepresence of the IL, while lower yield obtainedwithout IL-pretreatment. Moreover, it was demonstrated that furfural and phenolic compounds were not at the level of suppressing the fermentation process.

FERMENTATION OF BIOETHANOL FROM THE BIOMASS HYDROLYZATE OF OIL PALM EMPTY FRUIT BUNCH USING SELECTED YEAST ISOLATES

Objective: The purpose of this research was to investigate the potential of yeast-producing bioethanol from few type of grapes and durians.Methods: The isolation of potential yeast was carried out from five different types of fruits, namely, red grape, black grape, durian Medan, durianBangkok, and durian Monthong. Optimum fermentation condition was obtained by comparing shaking and non-shaking, detoxifying the hydrolysate,varying temperature, and concentration of N source in the culture medium.Results: The results of an analysis using gas chromatography showed that the ideal conditions for the fermentation of bioethanol were at a temperaturebelow room temperature (+220°C), without shaking, using the hydrolysate, without detoxification, and with a 1% concentration of ammonium acetateas a source of N. Among the isolates, isolate DM1 obtained from durian Medan showed a strong potential to produce bioethanol but at lower levelsthan Saccharomyces cerevisiae. The concentration of ethanol produced was 0.241%.Conclusion: A yeast isolate with a strong potential to produce bioethanol was discovered as the DM1, which isolated from Medanese durian. Theisolate produced 0.241% bioethanol. However, fermentation optimization efforts using an oil palm empty fruit bunch hydrolysate substrate did notyield higher concentrations.

Effective depolymerization of concentrated acid hydrolysis lignin using a carbon-supported ruthenium catalyst in ethanol/formic acid media

Lignin isolated by two-step concentrated acid hydrolysis of empty fruit bunch (EFB) was effectively depolymerized into a high-quality bio-oil using formic acid (FA) as an in-situ hydrogen source and Ru/C as a catalyst in supercritical ethanol. A bio-oil yield of 66.3wt% with an average molecular weight of 822g/mol and an aromatic monomer content of 6.1wt% was achieved at 350°C and a FA-to-lignin mass ratio of 3 after a reaction time of 60min. The combination of Ru/C and FA also resulted in a significant reduction in the oxygen content of the bio-oil by ∼60% and a corresponding increase in the higher heating value (HHV) to 32.7MJ/kg due to the enhanced hydrodeoxygenation activity. An examination of the FA decomposition characteristics revealed that Ru/C provides a greater increase in the rate of hydrogen production from FA, explaining the efficient depolymerization of lignin in a combined system.

Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01

The effect of various biomass derived inhibitors (i.e. furfural, hydroxymethylfurfural (HMF), vanillin, 4-hydroxy benzaldehyde (4-HB) and acetate) was investigated for fatty acid accumulation in Rhodococcus sp. YHY 01. Rhodococcus sp. YHY01 was able to utilize acetate, vanillin, and 4-HB for biomass production and fatty acid accumulation. The IC50 value for furfural (3.1mM), HMF (3.2mM), vanillin (2.0mM), 4-HB (2.7mM) and acetate (3.7mM) was calculated. HMF and vanillin affect fatty acid composition and increase saturated fatty acid content. Rhodococcus sp. YHY 01 cultured with empty fruit bunch hydrolysate (EFBH) as the main carbon source resulted in enhanced biomass (20%) and fatty acid productivity (37%), in compression to glucose as a carbon source. Overall, this study showed the beneficial effects of inhibitory molecules on growth and fatty acid production, and support the idea of biomass hydrolysate utilization for biodiesel production by avoiding complex efforts to remove inhibitory compounds.

Improved microbial oil production from oil palm empty fruit bunch by Mucor plumbeus

This study investigated the effect of cultivation parameters on microbial oil production from hydrolysate of oil palm empty fruit bunch (EFB) using fungus Mucor plumbeus. The parameters selected for evaluation were sugar concentration (30–100g/L), yeast extract concentration (0–13.3%, g yeast extract/g sugar), pH (5–7) and spore concentration (4.3–6.3, log spore number/mL medium). Response surface methodology was used to optimise the cultivation conditions which were based on the oil concentration and oil yield. Sugar concentration was the most influential parameter that affected oil concentration. However, the cultivation at high sugar concentration (∼100g/L) also resulted in ethanol accumulation. The optimum condition for oil yield was found at 30g/L sugar, 0g/L yeast extract and pH 5.0. Cultivation in 1L bioreactor under the optimum conditions resulted in ∼1.8-fold increase in oil yield compared to the shake-flask cultivation. Microbial oil produced from EFB hydrolysate has the potential to be used as the feedstock for biodiesel production from non-food feedstock, with cheaper cost of biodiesel production in comparison to glucose-derived microbial oil.

Microbial Production of Xylitol from Oil Palm Empty Fruit Bunch Hydrolysate: Effects of Inoculum and pH

Considering its high content of hemicellulose, oil palm empty fruit bunch (EFB) lignocellulosic biomass waste from palm oil processing has the potential to be utilized as the raw material for the production of xylitol, a low calorie, low GI, and anti cariogenic alternative sugar with similar sweetness to sucrose. This research explored the possibility of converting EFB to xylitol via green microbial fermentation, in particular the effects of inoculum and initial pH on the fermentation performance. It was observed that the cell concentration in the inoculum and the initial pH affect cell growth and xylitol production. pH 5 was observed to give the best fermentation performance. Further, the fermentation tended to yield more xylitol at higher initial cell concentration. It was also observed that no growth or fermentation inhibitory compounds were found in the EFB hydrolysate obtained from enzymatic hydrolysis of EFB. Thus it can be used directly as substrate for xylitol fermentation.

Implementation of Definite Screening Design in Optimization of In Situ Hydrolysis of EFB in Cholinium Acetate and Locally Produced Cellulase Combined System

Ionic liquids (ILs) have been found to be highly promising for lignocellulosic biomass pretreatment, due to their excellent abilities to dissolve biopolymers. However, they have found to inactivate enzymes. In previous studies, Cholinium-based ILs showed great compatibility with cellulases and thus were introduced for a combined-system. This study aims to find the optimum condition in order to achieve the maximum pretreatment-hydrolysis of empty fruit bunches (EFB). Definitive screening design (DSD) was employed with seven factors that are thought to impact the process including pretreatment temperature, pretreatment time, hydrolysis time, enzyme loading, particle size, biomass loading, and IL/buffer ratio. DSD offered several solutions for optimization in which they were experimentally tested. The maximum sugar concentration (77 g/L) was obtained at 90 °C, 70–116 min of treatment, 36–42 h of hydrolysis, 40–62 Unit/g cellulase loading, 34–35 %, w/v) biomass loading, 220–450 μ particle size, and 10 % (v/v) IL to buffer ratio, respectively. The subsequent optimization by response surface methodology (RSM) revealed the temperature for treatment can drop to 75 °C while fixing the pretreatment time at 100 min. Around 75 unit/g of cellulase and >22.0 % (w/v) of the biomass could be loaded to achieve a minimum of 70.0 ± 7.83 g/L of sugar, equivalent to 0.38 ± 0.08 g glucose/g and 0.48 ± 0.05 g total reducing sugar/g dry EFB. The locally produced cellulase (PKC-Cel) from Trichoderma reesei exhibited promising results in the single-step process and can be used as an efficient approach to be optimized for fermentation to bioethanol production.

Evaluation of several ionic liquids for in situ hydrolysis of empty fruit bunches by locally-produced cellulase.

Lignocellulosic biomasses, exhibit resistance to enzymatic hydrolysis due to the presence of lignin and hemicellulose. Ionic liquids proved their applicability in lignin degradation, however, ionic liquid removal has to be performed to proceed to hydrolysis. Therefore, this study reports an in situ hydrolysis of empty fruit bunches (EFB) that combined an ionic liquid (IL) pretreatment and enzymatic hydrolysis. For enzyme production, palm kernel cake (PKC) was used as the primary media for microbial cellulase (PKC-Cel) from Trichoderma reesei (RUTC30). The obtained enzyme exhibited a promising stability in several ionic liquids. Among few, in choline acetate [Cho]OAc, PKC-Cel retained 63.16 % of the initial activity after 6 h and lost only 10 % of its activity in 10 % IL/buffer mixture. Upon the confirmation of the PKC-Cel stability, EFB was subjected to IL-pretreatment followed by hydrolysis in a single step without further removal of the IL. The findings revealed that choline acetate [Cho]OAc and choline butyrate [Cho]Bu were among the best ILs used in the study since 0.332 ± 0.05 g glucose/g and 0.565 ± 0.08 g total reducing sugar/g EFB were obtained after 24 h of enzymatic hydrolysis. Compared to the untreated EFB, the amount of reducing sugar obtained after enzymatic hydrolysis increased by three-fold in the case of [Cho]OAc and [Cho]Bu, two-fold with [EMIM]OAc and phosphate-based ILs whereas the lowest concentration was obtained in [TBPH]OAc. Pretreatment of EFB with [Cho]OAc and [Cho]Bu showed significant differences in the morphology of EFB samples when observed with SEM. Analysis of the lignin, hemicellulose and hemicellulose showed that the total lignin content from the raw EFB was reduced from 37.8 ± 0.6 to 25.81 ± 0.35 % (w/w) upon employment of [Cho]OAc in the compatible system. The PKC-Cel from T. reesei (RUTC30) exhibited promising characteristics that need to be investigated further towards a single-step process for bioethanol production.

Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source

In this study, a biosugar obtained from empty fruit bunch (EFB) of oil palm by hot water treatment and subsequent enzymatic saccharification was used for undecylprodigiosin production, using Streptomyces coelicolor. Furfural is a major inhibitor present in EFB hydrolysate (EFBH), having a minimum inhibitory concentration (MIC) of 1.9mM, and it reduces utilization of glucose (27%), xylose (59%), inhibits mycelium formation, and affects antibiotic production. Interestingly, furfural was found to be a good activator of undecylprodigiosin production in S. coelicolor, which enhanced undecylprodigiosin production by up to 52%. Optimization by mixture analysis resulted in a synthetic medium containing glucose:furfural:ACN:DMSO (1%, 2mM, 0.2% and 0.3%, respectively). Finally, S. coelicolor was cultured in a fermenter in minimal medium with EFBH as a carbon source and addition of the components described above. This yielded 4.2μg/mgdcw undecylprodigiosin, which was 3.2-fold higher compared to that in un-optimized medium.

Screening conditions for acid pretreatment and enzymatic hydrolysis of empty fruit bunches

Empty fruit bunches were received from Teck Guan, Malaysia and were pretreated and enzymatically hydrolyzed to determine the possible sugar recovery from the biomass. Several different conditions were explored in a screening study. Temperature ranged from 100°C to 150°C, time ranged from 30 to 90min, and acid loading ranged from 0 to 1.3% weight acid/weight liquid. The material was then enzymatically hydrolyzed at three different enzyme loadings 1.67%, 3.33%, and 6.66% (g enzyme/g glucan×100) and total sugar recovery was calculated for both pretreatment and enzymatic hydrolysis. Best pretreatment conditions yielded 81.4% recovery of hydrolyzed xylan. Best glucan conversions in enzymatic hydrolysis were 74.8%. These conversions and recoveries make empty fruit bunches a good potential feedstock for cellulosic ethanol.

Enzyme feeding strategies for better fed-batch enzymatic hydrolysis of empty fruit bunch

Lignin inhibitory becomes a major obstacle for enzymatic hydrolysis of empty fruit bunch conducted in high solid loading. Since current technology required high enzyme loading, surfactant application could not effectively used since it is only efficient in low enzyme loading. In addition, it will increase final operation cost. Hence, another method namely “proportional enzyme feeding” was investigated in this paper. In this method, enzyme was added to reactor proportionally to substrate addition, different from conventional method (“whole enzyme feeding”) where whole enzyme was added prior to hydrolysis process started. Proportional enzyme feeding could increase enzymatic digestibility and glucose concentration up to 26% and 12% respectively, compared to whole enzyme feeding for hydrolysis duration more than 40h. If enzymatic hydrolysis was run less than 40h (25% solid loading), whole enzyme feeding is preferable.