IMPROVEMENT OF DIGESTIBILITY AND STRUCTURAL CHANGES OF OIL PALM EMPTY FRUIT BUNCHES BY Pleurotus floridanus AND PHOSPHORIC ACID

PRETREATMENT
Dissertation Summary As requirement for Doctoral degree Program Study of Bioteknology

Proposed by Isroi 08/275457/SMU/00535

to Graduate Schoof of UNIVERSITAS GADJAH MADA YOGYAKARTA 2013

IMPROVEMENT OF DIGESTIBILITY AND STRUCTURAL CHANGES OF OIL PALM EMPTY FRUIT BUNCHES BY Pleurotus floridanus AND PHOSPHORIC ACID PRETREATMENT
Abstract

Oil palm empty fruit bunches (OPEFB) is abundance and not optimally utilized as lignocellulose-based products. OPEFB has low digestibility and difficult to process into its derivatives. This study aims to improve digestibility of OPEFB through biological pretreatment with white-rot fungi. Stages of this study were (1) selection white-rot fungi which selectively degrade lignin, (2) enhancement the digestibility of OPEFB by Mn2+ and Cu2+ addition, (3) improvement digestibility of OPEFB by a combination of biological pretreatment with phosphoric acid pretreatment. All the studies were conducted on laboratory scale. Observations were made on the changes of dry weight, lignin, cellulose, hemicellulose, physical structure by SEM analysis, functional groups by FTIR analysis, and the crystallinity of cellulose. Screening is done on Polyota sp, Pleurotus sp and sp Agraily. Pleurotus sp chosen for further experiments and identified as P. Floridanus LIPIMC966, because it can alter the lignin content from 19.63% to 15.22%, hemicellulose from 14.77% to 12.63%, and increase cellulose from 39.92% to 56.04%. The addition effect of Mn2+ and Cu2+ on the biological pretreatment using P. Floridanus could reduces the dry weight from 27.43% to 32.88%; lignin content up to 43.17% (Mn2+) and 34.08% (Cu2+), hemicellulose content up to 32.82%, while cellulose content remained constant. Combination of biological pretreatment and phosphoric acid were evaluated based on the changes in components of lignocellulose, a structural and morphology. Carbohydrate degradation after biological pretreatment, phosphoric acid, and a combination of biology and phosphoric acid are 7.88%, 35.65%, and 33.77%, respectivelly. Pretreatment changed the hydrogen bonding of the cellulose and linked between lignin and carbohydrates, which is related to the crystallinity of the cellulose. The crystallinity of the cellulose as indicated by lateral order index after pretreatment are 2.77 (without pretreatment), 1.42 (biology), 0.67 (phosphoric acid), and 0.60 (a combination of biology and phosphoric acid), respectively. Phosphoric acid pretreatment damaged the structure and morphology of the OPEFB fibers shown by SEM analysis. Pretreatments have increased digestibility of the OPEFB 4 (biology), 6.3 (phosphoric acid), and 7.4 (biology and phosphoric acid)-fold compared with whitout pretreatment. Keywords: oil palm empty fruit bunches, Pleurotus floridanus, Cu, Mn, structural changes, digestibility

INTRODUCTION Oil palm empty fruit bunches (OPEFB) are abundant and not optimally utilized as lignocellulose-based product. Indonesia is the largest palm oil producer in the world that produces 20.7 million metric tons of OPEFB (FAOSTAT 2012). OPEFB are composed of 39.13% cellulose, 23.40% hemicellulose, and 34.37% lignin (Isroi et al. 2013 ). Having high carbohydrate content makes OPEFB are potential as a feedstock of lignocellulosic derived products. OPEFB has low digestibility and difficult to be processed into its derivatives product. The low lignocellulosic digestibility caused by several factors, such as: the content and composition of lignin, cellulose crystallinity, degree of polymerization, pore volume, acetyl groups bound to the hemicellulose, surface area and particle size of the biomass (Alvira et al. 2010, Anderson and Akin 2008, Rivers and Emert 1988). OPEFB require pretreatment stage to change the structure and to break down the lignin barrier making cellulose more accessible to hydrolytic enzymes. Research to get the right pretreatment method for OPEFB is needs to be done, so that the high potential OPEFB could utilize into lignocellulosic derivative products. Pretreatment of lignocellulose can be done through physical, chemical and biological methods or a combination of these methods (Alvira et al. 2010, Taherzadeh and Karimi 2008). Biological pretreatment utilize the ability of white rot fungi (WRF) or its enzymes to break down lignin and altered the structure of lignocellulose (Hatakka A.I. 1983, Taniguchi et al. 2005). Biological pretreatment has several advantages such as: i) low energy requirement, ii) low capital investment, iii) no chemical requirement, iv) mild environmental conditions, v) specific to substrate, vi) simple process and equipment requirement (Kirk & Chang, 1981; Sun & Cheng, 2002). WRP are grouped into selective and non selective groups. WRP selective is WRP that degrade lignin more than cellulose and hemicellulose, whereas nonselective is WRP that degrade all components of lignocellulose on comparable amount. Biological pretreatment influenced by several factors, such as the addition of cations (Mn2+ dan Cu2+) (Camarero et al. 1996, Palmieri et al. 2000). The addition of cations could increase production and activity of ligninolytic enzyme, lignin degradation and improve digestibility of cellulose. Some isolates WRP successfully isolated by Indonesian Biotechnology Research Institute for Estate Crops ( IBRIEC ) and have the ability to degrade lignin, i.e.: Polyota sp , sp Agraily , and Pleurotus sp . Selectivity of these WRP isolates is unknown. Selection of the appropriate selective WRP isolates for OPEFB is needed to develop biological pretreatment method with the addition of cations (Mn2+ dan Cu2+). Biological pretreatment has some disadvantages compared with the physical/chemical pretreatment, such as: lower of sugar yield (Taherzadeh & Karimi, 2008). Digestibility of lignocellulose could be improved through a combination of biological pretreatment with chemical pretreatment (Itoh et al. 2003, Ma et al. 2010, Taniguchi et al. 2010, Yu et al. 2010). One of the chemicals that can be used for

pretreatment is phosphoric acid. Phosphoric acid can solubilize cellulose and fractionate lignocellulose. Phosphoric acid pretreatment reported efficient in reducing cellulose crystallinity and increase the production of biogas from OPEFB (Nieves et al. 2011). Phosphoric acid pretreatment of lignocellulosic materials has been reported to increase fractionation and digestibility of lignocellulose (Zhang et al. 2007). Combination of biological pretreatment with phosphoric acid pretreatment needs to be tested in order to improve OPEFB digestibility. Combination of biological pretreatment with phosphoric acid pretreatment has been not reported in the literature. Lignocellulosic biomass undergoes physical and chemical changes after pretreatment. These changes include alteration in the content of lignin, cellulose, hemicellulose, reduction of cellulose crystallinity, pore area increase, damage to the surface area and also changes in the functional groups. Analysis of physical and chemical changes in the structure and composition of OPEFB after pretreatment is needed to understand the mechanism of increasing of OPEFB digestibility and designing appropriate pretreatment to produce optimal pretreatment process. General aims of this study is to increase digestibility of OPEFB with a combination of biological pretreatment using WRP and phosphoric acid pretreatment. WRP was selected from three collections of WRP isolates. Cation (Mn2+ dan Cu2+) was added to biological pretreatment in order improve delignification of OPEFB. Alteration in lignin, cellulose, hemicellulose, the degree of crystallinity, changes in the physical structure and functional groups were analyzed to determine the characteristics changes that contribute to increasing the digestibility of OPEFB. MATERIALS AND METHODS 1.1. Microorganisms and Medium 1.1.1. Microorganisms Pleurotus sp, Polyota sp and sp Agraily were obtained from the Indonesian Biotechnology Research Institute for Estate Crops (IBRIEC). All WRPs were grown and maintained using Potato Dextrose Agar media (PDA, Badco) and incubated for approximately one week before being used as inoculum for biological pretreatment. Pleurotus sp identified by LIPIMC (LIPI Microbial Collection) as Pleurotus floridanus LIPIMC996. Yeast Saccharomyces cerevisiae CBS 8066 was obtained from Centraalbureau voor Schimmelcultures (Delft, the Netherlands). Yeast cultures maintained on YPD agar medium containing 20 g/L agar (Scharlau), 10 g/L yeast extract (Scharlau), 20 g/L peptone (Fluka), and 20 g/L D-glucose (Scharlau) as a source carbon and stored at 4 C. 1.1.2. Media Medum for the growth and maintenance of WRP was a medium potato dextrose agar (PDA, DIFCO Laboratories, Detroit, MI). The composition of liquid

medium for biological pretreatment were: (a) medium 1: 7 g/L KH2PO4, 1.5 g/L MgSO4.7H2O, 1.0 g/L CaCl2.H2O; (b) medium 2: 7 g/L KH2PO4, 1.5 g/L MgSO4.7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L CuSO4.5H2O; (c) medium 3: 7 g/L KH2PO4, 1.5 g/L MgSO4. 7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L MnSO4.H2O; (d) medium 4: 7 g/L KH2PO4, 1.5 g/L MgSO4.7H2O, 1.0 g/L CaCl2.H2O, 0.015 g/L CuSO4.5H2O, 0.015 g/L MnSO4.H2O. Pretreatment OPEFB in the control treatment without microbial inoculation media was added 1. Medum 1, 2, and 3 was used in the phase 1 and 2. Medium 4 was used in the Phase 3. 1.1.3. Tandan Kosong Kelapa Sawit (TKKS) OPEFB obtained from palm oil mill Doloksinumbah plantation, PTPN IV, North Sumatra. OPEFB chopped approximately 5 cm and sun dried (moisture content <5%). This study uses two OPEFB samples taken in different times and analyzed by different methods. OPEFB characteristics used in this study were shown in Table 1. Table 1. Characteristics of oil palm empty fruit bunches (OPEFB) Sampel 1*) Sampel 2**) Lignin (%) 23,89 34,37 Cellulose (%) 40,37 39,13 Hemicellulose (%) 20,06 23,04 Hot water soluble (%) 14,47 Ash (%) 1,219 Water content (%) 51.045 *) Analyzed by Chesson-Datta method, used in Phase 1 and 2 studies **) Analyzed with NREL method, used in the phase 3 study OPEFB sample 1 and sample 2 OPEFB taken in different times. 1.2. Phases of Research The study was conducted in three phases as shown in Figure 1. Phase 1 was the selection of WRP for biological pretreatment of OPEFB which has a high selectivity for lignin degradation. Phase 2 was the effect of the addition of cations (Mn2+ and Cu2+) to the biological pretreatment. WRP selected from phase 1 was used on phase 1 and 2. Phase 3 was combination of biological pretreatment with phosphoric acid.

Figure 1. Flow chart and phases of the research 1.3. Pretreatment Methods Biological pretreatment for research on Phase 1 and 2 were conducted on solid state fermentation without aeration and without stirring. Fifty gr of OPEFB was weighted and added to the liquid media OPEFB appropriate treatment to reach 60% moisture content. OPEFB further sterilized using an autoclave at a temperature of 121oC for 30 minutes. Four pieces of culture WRP ( 5mm) were inoculated aseptically. The cultures were incubated at room temperature for 6 weeks. OPEFB was harvested, washed, dried, and milled at the end of the incubation and then used for dry weight and other lignocellulosic components analysis. Biological pretreatment for Phase 3 studies conducted with solid state fermentation without aeration and without stirring. Selected WRP isolated that obtained from phase 1 was used on OPEFB biological pretreatment. Two hundred gr of OPEFB was added 120 mL media then sterilized. Water content in the media OPEFB was approximately 60%. Culture of P. floridanus LIPIMC996 was inoculated aseptically. The cultures were incubated at 31C for 28 days in an incubator cabinet. OPEFBs were harvested at the end of the incubation and frozen at temperatures <0 C to stop the growth of WRP. OPEFB samples were dried with a vacuum dryer (Freezone 7670530, Labconco, Kansas City, MO, USA) at a temperature of -52C for 6 hours and then milled using ball milling (Retsch MM400, Retsch GmbH, Haan, Germany) at a frequency 29.6 s-1 for 4 min. OPEFB dry weight was determined based on the difference of oven dry weight (ODW) at the beginning and at the end of the pretreatments. All experiments were performed in duplo experiment.

1.4. Phosphoric Acid Pretreatment Methods Phosphoric acid pretreatment wes carried out according to the method described in reference (Nieves et al. 2011, Zhang et al. 2007). Samples were stored at temperatures < 0 C before used for hydrolysis or further analysis. 1.5. Enzymatic Hydrolysis Enzymatic hydrolysis OPEFB Sample on Phase 2. Enzymatic hydrolysis OPEFB sample was based on the method of NREL (Renewable Energy Laboratory, USA) with slight modifications (Selig et al. 2008). Hydrolytic enzymes used were a commercial enzyme (Cellulast , 64 FPU / ml and 58pNPGU/ml - glucosidase , Novozyme Co. ) at a dose of 60 FPU enzyme / g cellulase and 64 pNPGU / g Glucosidase. All samples were shaken with a shaker water bath at 50oC for 72 hours and then filtered. Supernatant obtained was then used for glucose analysis. Digestibility ( % ) was calculated by the following equation: (1) where glucose ( g ) was the mass of glucose in the fluid after hydrolysis and cellulose ( g ) was the mass of cellulose in the substrate. Enzymatic hydrolysis samples OPEFB in Phase 3 studies using the same method as above with a few modifications. Hydrolytic enzymes used were commercial enzyme Cellic CTec2 (148 FPU / mL , Co Novozymes , Bagsvaerd , Denmark ) at a dose of enzyme 30 , 60 , and 90 FPU / g cellulose. Digestibility (%) of the initial cellulose was calculated by dividing the glucose produced by the initial cellulose that is used by the following equation: (2) where glucose (g) is the amount of glucose in the fluid after the initial cellulose hydrolysis and ( g ) is the content of cellulose in OPEFB before getting pretreatment. All experiments were carried out duplicate and error bar was presented as the standard deviation. 1.6. Simultaneous Saccharification and Fermentation Simultaneous Saccharification and Fermentation (SSF) carried out by the method of NREL (Dowe and McMillan 2008) using a commercial enzyme Cellic CTec2 ( 148 FPU / mL , Co Novozymes , Bagsvaerd , Denmark ) at a dose of 60 fpu enzyme / g cellulose. Cellulose concentration used was 6% in the 0:05 M citrate buffer pH 4.8. SSF done with volume 100ml to 250ml erlemeyer equipped with a gas trap (bubble trap). SSF was carried out at 31oC in a water bath shaker for 72 hours. Ethanol production was observed every day. 1.7. Analytical Methods Chemical analysis of the OPEFB components (lignin, hemicellulose, and

cellulose) in Phase 1 and 2 studies were conducted using Chesson - Datta methods (Datta 1981). Cellulose, hemicellulose, and lignin content of OPEFB on Phase 3 study was determined by the method of NREL (Sluiter A. et al. 2011). Ash was determined using the furnace overnight at 575 C (Sluiter A. D. et al. 2008). Dry weight was determined as oven dry weight (ODW) after drying the sample at 105 3C for 24 hours in accordance with TAPPI method T264 cm - 97 standard test (TAPPI 2002). Fungal growth during the pretreatment was estimated based on the dry weight of fungal biomass (Kumar et al. 2006). Biomass analysis was conducted to study the Phase 1 and Phase 2. Changes in functional group observed by the adsorption change of the IR spectrum (infra red) by OPEFB samples at a specific wavelength ( Jeihanipour , Karimi et al . 2009). IR spectra measurements was performed using FTIR spectrometer ( Impact 410 , Nicolet Instrument Corp. , Madison , WI ) , 32 scans , resolution 4 cm - 1 in the range of 600-4000 cm - 1 and controlled by softwere Nicolet OMNIC 4.1 ( Nicolet Instrument Corp. , Madison , WI ) and analyzed using eFTIR ( EssentialFTIR , USA ) softwere. Evaluation of changes in the physical structure of the OPEFB sample surface before and after pretreatment was visualized using Scanning Electron Microscopy analysis (SEM) Model JEOL JSM - 820 ( JEOL Ltd. , Akishima , Japan). Monosaccharides (cellobiose, glucose, xylose, mannose, Galactose and Arabinose) were analyzed using HPLC system equipped with an autosampler (WalterTM 717, Milford, USA), UV detector (WalterTM 485, Milford, USA) , and ELS detector (WalterTM 2424, Milford, USA) . Monosugar separated using a Bio Rad Aminex HPX - 87P column (Aminex HPX - 87P, Bio - Rad, USA) , pure water as the mobile phase with a flow rate of 0.6 ml min - 1 under isothermal conditions at 85oC . A Bio - Rad Carbo - P column protector (column guard, Bio - Rad, USA) was used and placed outside the main column at room temperature. Ethanol concentrations were analyzed using HPLC system equipped with an autosampler ( WalterTM 717, Milford , USA), UV detector (WalterTM 48, Milford, USA), and ELS detector (WalterTM 2424, Milford, USA). Monosugar separated using a Bio - Rad Aminex HPX - 87H column ( Aminex HPX - 87H , Bio - Rad, USA ) , 0.025M H2SO4 as the mobile phase with a flow rate of 0.6 ml min - 1 under isothermal conditions at 85oC . A Bio - Rad Carbo - P column protector (column guard, Bio - Rad , USA ) was used and placed outside the main column at room temperature . Ethanol standards were dissolved in 0.025M H2SO4 concentration in some and used as a comparison to calculated ethanol concentration in the sample. The data were analyzed by statistical analysis. Data shown were the average of each test. Value of standard deviation (SD) were calculated and displayed to determine the error of each data. Each data also performed analyzes of variance (analysis of variance, ANOVA) to determine the significance different of treatment effects on control. Correlation analysis of data from several experiments conducted to determine the statistical relationships between data.

RESULTS AND DISCUSSIONS 1.8. White-rot fungi Selection for Biological Pretreatment of Oil Palm Empty Fruit Bunch OPEFBs were changes physically and chemically after biological pretreatment. Pretreated OPEFB becomes brighter and softer than un-pretreated OPEFB. Visual color change is one of the characteristics of lignocellulosic degradation by WRP (Hatakka Annele 2001). Decreased levels of lignin by WRP likely cause discoloration of the lignocellulose (Bajpai 2004, de Jong et al. 1997). Changes in lignin, cellulose, and hemicellulose content are shown in Figure 2. The content of lignin and hemicellulose decreased significantly, whereas cellulose increased significantly after biological pretreatment. Lignin content decreased significantly from 19.63% (initial content) up to 15.32% (Pleurotus sp), 16.63% (Polyota sp) and 18.07% (Agraily sp). Hemicellulose content from the lowest on each treatment were Pleurotus sp (12.63%), Polyota sp (14.26%) and Agraily sp (15.18%). The content of cellulose (%) on each treatment were Pleurotus sp (56.04%), Agraily sp (44.13%) and Polyota sp (42.03%).
70 60 50 Kandungan (%) 40 30 20 10 0 Kontrol Pleurotus sp Polyota sp Agraily sp Lignin Selulosa Hemiselulosa

Figure 2.

The percentage of lignin, hemicellulose, and cellulose content of palm empty fruit bunches (OPEFB): (a) without biologIcal pertreatment (control), (b) Pleurotus sp , (c) Polyota sp , (d) Agraily sp. Biological pretreatment was performed on solid state fermentation without aeration and at room temperature for 4 weeks.

All third isolates of WRP could degrade lignin, but which showed the highest decrease in lignin was Pleurotus sp. The content of hemicellulose (%) showed a slight decrease in each isolate WRP. Hemicellulose degradation occurs in the same relative proportion to the degradation of biomass, so the percentage of hemicellulose content to the total biomass decreased slightly. Changes in cellulose content (%) after pretreatment OPEFB was vary for each isolate WRP. Increasing in the percentage of

cellulose after pretreatment biological biomass also reported in reference (Xu et al. 2010). This increasing occurred due to degradation of other components (lignin and hemicellulose) were higher than the degradation of cellulose, so it would be proportionally increased the cellulose content. Pleurotus sp degraded lignin approximately 22 % of the initial content of lignin and the results were comparable with the results reported in the literature, namely by 25 % after biological pretreatment for 60 days (Taniguchi et al. 2005). Highest decreasing in lignin and hemicellulose content, and the highest increasing in cellulose content by Pleurotus sp suggested that Pleurotus sp was more selective in degrading lignin than the two other isolates. Similar results were also reported by Kerem et al. (1992) that P. ostreatus selectively degrade lignin more than Phanerochaete chrysosporium. Some literature reported that Pleurotus sp isolates produced ligninolytic enzymes (Lac, MnP , and VP) as well as hydrolytic enzymes (Chen et al. 2010, Goudopoulou et al. 2010, Martnez et al. 2005, Tinoco et al. 2011). Pleurotus sp isolates were selected for further biological pretreatment OPEFB phase 2 & 3 and identified as Pleurotus floridanus with collection number LIPIMC 966. 1.9. Effect of Addition of Manganese (Mn2+) and copper (Cu2+) on Biological Pretreatment of Oil Palm Empty Fruit Bunch Using Pleurotus floridanus LIPIMC966 1.9.1. Effect of Biological Pretreatment on Dry Weight and Lignocellulosic Components Dry weight of OPEFB after biological pretreatment for 42 days incubation are shown in Figure 3. It is generally observed that all three treatments results in the reduction of ODW of OPEFB, with the total reductions are 32.88%, 29.08%, and 27.43% for treatment with Mn2+ addition, treatment with Cu2+ addition, and treatment with no nutrient addition, respectively. The ODW decline was a decreasing in the total of lignocellulosic biomass includes reduced lignin content, cellulose, hemicellulose, and other components. WRP degraded solid components into less complex structures, water soluble materials and gaseous products that result in decreasing of lignocellulosic biomass dry weight. Pretreated OPEFB were analyzed for its components, i.e. hot water soluble (HWS) materials, hemicelluloses, cellulose, and lignin. The data are presented in Figure 4. It is shown that all components reduced subjects to biological degradation by P. floridanus in all three different treatments at different rate of reduction. Hot water soluble (HWS) consists of several components, such as carbohydrates, proteins, and inorganic compounds. Activity of P. floridanus in this work has reduced the HWS up to about 50% during 42 days of incubation. Both treatments with Mn2+ addition and Cu2+ addition have accelerated the HWS reduction to some extends (Figure 4 A).

30 Berat Kering ( gr) 25 20 15 10 5 0 0 7 14 21 28 Hari ke35 42 49

Kontrol

Cu

Mn

Figure 3.

Decrease of dry weight (ODW) of oil palm empty fruit buches (OPEFB) during pretreatment using Pleurotus sp with (a) without nutrient addition (control), (b) CuSO4 (Cu2+), and (c) MnSO4 (Mn2+). Biological pretreatment performed by solid state fermentation without aeration and at room temperature

Similar results are also found for degradation of hemicellulose (Figure 4B) and lignin (Figure 4D), in which the components are reduced and the addition of Mn2+ increased the reduction rate. Mn2+ treatment showed a faster decline rate compared to other treatments at day 21 and then remained relatively constant until day 42. Control treatment showed a slower rate of decline, but the decline continued until day 42 and a decrease in the biggest hemicellulose than other treatments. However, as shown in Figure 3C, all three treatments shown slightly reduction on the content of cellulose in the OPEFB (Figure 4 C). Decrease in hemicellulose and lignin content on this phase 2 study confirm the phase 1 experiment showed that isolates of P. floridanus was more degrade hemicellulose and lignin than cellulose. In other words P. floridanus was more selective in lignin degradation, HWS, and hemicellulose than cellulose. The fact that addition of Mn2+ and Cu2+ accelerates the degradation of most components in lignocellulosic materials by fungi has also been observed by other researchers (Janusz et al. 2006, Levin et al. 2007, Tychanowicz et al. 2006). Addition of certain concentration of Mn2+ and Cu2+ can induce and control ligninolytic enzymes production resulted in improvement of lignin degradation. Mn2+ concentration can affect MnP and LiP activities, whereas Cu2+ can affect Lac activities (Isroi et al. 2011). Mn in the growth medium plays an important role in regulating manganese peroxidase (MnP) and lignin peroxidase (LiP) activities. MnP production dominates in the presence of Mn2+, and conversely LiP production dominates in the absence of Mn2+. MnP can diffuse into the lignified cell wall and oxidises non phenolic structure compounds. Laccase oxidises phenolic structures of lignin.

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Figure 4.

Change component content OPEFB , ie : hot water soluble (HWS) (A) , hemicellulose (B) , cellulose (C) , and lignin (D) during pretreatment with Pleurotus floridanus LIPIMC996 without the addition of cations (control), with the addition of CuSO4 (Cu2+) , and the addition of MnSO4 (Mn22+) . Biological pretreatment performed by solid fermentation culture, without aeration , and at room temperature.

1.9.2. Effect of Biological Pretreatment on Physical and Structural Characteristics Inoculation of OPEFB with P. floridanus LIPIMC996 results in changing on physical characteristics of OPEFB, i.e. it turns into lighter color (from dark brown), it is more brittle and easier to grind. The colour change may be used as an indication of lignin reduction or removal. The peak of IR Spectrum at certain wavelength could lower, higher and/or shifted which indicates the alteration of certain functional groups associated with that wavelength. Analysis of FTIR spectra shown in Figure 5 and bands assignment are described in Table 1. Some bands associated with polysaccharides and cellulose were little changed for all pretreatment, namely: 3450-3000, 1456, 1162-1158, 897, and 769 cm - 1. These adsorption bands were consistent with the content of cellulose OPEFB that were not degraded by fungi (Figure 4 C). Peak at 640 cm - 1, 760 cm - 1 and 1,366 cm - 1 are associated with significant changes in cellulose structure after pretreatment. Intensity at a wavelength of 1739-1738 cm - 1 (polysaccharide) significantly decreased after pretreatment. Bonds between lignin and carbohydrates may exist in this peak (Takahashi dan Koshijima 1988). Hemicellulose and lignin degradation by fungi can break the bond between carbohydrates and lignin that can

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contribute to the decrease in adsorption at 1739-1738 cm - 1 peak.

Figure 5.

FTIR spectra of biological pretreated OPEFB with P. floridanus in the control treatment, and Cu 2+ and Mn2+ for: a) 0 days, b) 7 days, c) 14 days d) 21 days, and e) 28 days. Crystallinity of cellulose could be predicted using intensities ratio of certain bands at the IR spectra, that are A1418/A895 known as Lateral Order Index (LOI) (O'Connor, Dupre et al 1958;. Hurtubise dan Krassig 1960). LOI value of biological pretreated OPEFB shown in Figure 9. Crystallinities of cellulose were decreased during pretreatment. Meanwhile, decreasing rate of OPEFB pretreated with Mn2+ and Cu2+ addition shown higher than without cations addition. As indicating by FTIR analysis of cellulose IR band, although there is no significant degradation of cellulose but structure of the cellulose could be changes, such as their crystallinity. Bands at wavelengths of 1,595 and 1,505 cm-1 are associated with significant changes of lignin after pretreatment with Mn2+ and Cu2+. Meanwhile, the intensity at 1.032 cm-1 also decreased after pretreatment with the addition of Mn 2 Absorption of IR spectra at wavenumber 1422-1424 cm-1 indicating presence of the syringyl type lignin (the major type of hardwood lignin) that absorb only at near band 1230 cm-1 (Pandey and Pitman 2003). The observation at these wavenumber showed significant decrease in the lignin content, indicating loss of C-C, C-O, and C=O stretching (G condensed > G estherified). Through FTIR spectra analysis, biological pretreatment

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of OPEFB displayed significant changes in its functional groups in various regions, particularly in G unit and S unit of lignin, suggesting deformation of biomass during biological pretreatment. Table 1. Assignment of FTIR-Absorption Bands (cm-1) to various components of oil palm empty fruit bunches according to literature Source Ref. Wavenumber Assignments -1 (cm )
670 715 858-853 897 C-O out-of-plane bending mode Rocking vibration CH2 in Cellulose I C-H out of plane deformation in position 2,5,6 Anomere C-groups C(1)-H deformation, ring valence vibration C-O valence vibration Aromatic C-H in plane deformation, G>S; plus C-O deformation in primary alcohols; plus C=O stretch (unconj.) C-O-C assimetric valence vibration C-C plus C-O plus C=O strech; G condensed > G etherified C=O stretch, OH i.p. bending G-ring plus C=O strectch O-H blending of alcohol groups C-H deformation vibration CH2 of pyran ring symmetric scissoring ; OH plane deformation vibration Aromatic skeletal vibrations with C-H in plane deformation CH2 scissoring C-H in pyran ring symmetric scissoring; OH plane deformation vibration Cellulose Cellulose G-Lignin Polisakarida (Schwanninger et al. 2004) (Schwanninger et al. 2004) (Fackler et al. 2010) (Fackler et al. 2010, Fengel 1992) (Schwanninger et al. 2004) (Schwanninger et al. 2004)

996-985 1035-1030

Lignin

1162-1125

Polisakarida

1230-1221

Polisakarida

(Fackler et al. 2010, Schwanninger et al. 2004) (Fackler et al. 2010, Fengel 1992) (Faix O. and Bttcher 1992) (Faix O. 1991) (Fackler et al. 2010) (Fengel 1992) (Schwanninger et al. 2004) (Faix Oskar et al. 1991) (Fengel 1992)

1227-1251 1270-1260 1315 1375 1470-1455

G-Lignin Karbohidrat Cellulose

1430-1416

Lignin

1460

Cellulose

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Wavenumber Assignments (cm-1)

1515-1505 1605-1593 Aromatic skeletal vibrations; G>S Aromatic skeletal vibrations plus C=O stretch; S>G; G condensed > G etherified C O stretch in conjugated psubstituted aryl ketones CO stretch unconjugated (xylan) Asymetric CH2 valence vibration CH2, CH2OH in Cellulose from C6 Symmetric CH2 valence vibration Hydrogen bonded O-H valence vibration; O(3)H...O(3) intermolecular in cellulose

Source Lignin Lignin

Ref. (Faix Oskar et al. 1991) (Faix Oskar et al. 1991) (Faix Oskar et al. 1991) (Faix Oskar et al. 1991) (Schwanninger et al. 2004) (Schwanninger et al. 2004) (Schwanninger et al. 2004) (Schwanninger et al. 2004)

1675-1655 1738-1709 2940-2850 2980-2835 2981-2933 3338

Lignin Polisakarida

Cellulose

Cellulose

1.9.3. Effect of Biological Pretreatment on Digestibility OPEFB digestibility calculated by equation 1 was shown in Figure 6. OPEFB digestibility increased with increasing incubation time of biological pretreatment using P. floridanus. As shown in the figure, all samples show no significant difference on its digestibility at 0 day of incubation, i.e. between 17.22 22.00 %. Sample pretreated with no cation addition could reach digestibility of 30.97% following 28 days of incubation. Sample treated with Cu and Mn addition have maximum digestibility of about 60.27% and 55.67%, respectively. The fact that samples pretreated with Cu2+ and Mn2+ addition have higher percentage of digestibility indicates that Cu2+ and Mn2+ addition increased their susceptibility to hydrolysis. The increase of digestibility is significantly observed for hydrolysis period up to 21 days, after that the digestibility only changed slightly.

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70 60 Digestibilitas (%) 50 40 30 20 10 0 0 7

Kontrol

Cu

Mn
42 49

14 21 28 35 Waktu inkubasi (hari)

Figure 6.

Hydrolysis results OPEFB samples that have received biological pretreatment using P. floridanus LIPIMC996 a) without addition of cations (control), b) the addition of CuSO4 (Cu2+), c) the addition of MnSO4 (Mn2+). Hydrolysis was used the enzyme cellulase (60 FPU / g substrate) and -glucosidase (64 pNGU/g substrate), temperature 50 C, for 72 hours. Oil Palm Empty Fruit Bunch Structural Changes after Pretreatment using Pleurotus floridanus and Phosphoric Acid

1.10.

1.10.1. Effect of Pretreatment on Biomass Components Results of analysis of the composition and content OPEFB before and after pretreatment combination with P. floridanus and phosphoric acid are presented Figure 7. The percentage content of components of lignocellulose OPEFB only slightly changed due to fungal pretreatment but significantly changed due to phosphoric acid pretreatment, and pretratment combination with fungal followed by phosphoric acid. Hemicellulose content showed the lowest percentage in the second both pretreatment using phosphoric acid, which is 9:09%. The reduction of total solid was showed significant changes after pretreatment. Biological pretreatment using P. floridanus showed the lowest dry weight (1.31%) and the lowest decrease in total carbohydrate (7.88%) compared with the two other pretreatment.

15

Figure 7.

Profile of components of oil palm empty fruit bunches (OPEFB) after pretreatment. ASL: acid soluble lignin, AIL: acid insoluble lignin.

The content of hemicellulose was the most affected by phosphoric acid pretreatment and pretreatment combinations at 18%. Degradation of total solids after phosphoric acid pretreatment was approximately 55%, whereas for the combination of fungal pretreatment sebesaar 64% phosphoric acid. Loss of total carbohydrate of the two treatments was 35% (fungal pretreatment) and 33% (fungal pretreatment followed by phosphoric acid pretreatment). Based on these data fungal pretreatment is more advantageous in terms of loss of carbohydrate level lower than phosphoric acid pretreatment and fungal pretreatment followed by phosphoric acid pretreatment. Fungal pretreatment application to OPEFB provide a greater amount of carbohydrates and relatively more environmentally friendly than the other two pretreatment. 1.10.2. Pretreatment Effects on Structures of OPEFB The structural changes of OPEFB were analysed based on FTIR spectra of the untreated and pretreated materials. The results are shown in Figure 8. Determination and shifting each band corresponding to the literature listed in Table 2. Fourteen bands were conserved in all of the samples in the range of 6001,800 cm1 and 2,8003,700 cm1. Bands at wavenumbers 2,918, 2,985, and 648 cm1 with high intensity were only found in untreated and fungal pretreated OPEFB. Bands that only appeared in samples pretreated with phosphoric acid and fungal followed by phosphoric acid were 1,224, 998 and 666 cm1.

16

Figure 8.

FTIR spectra of oil palm empty fruit bunches (OPEFB) in the wavelength range from (a) 2800-3800 cm-1 and (b) 600-1800 cm-1. Information line: without pretreatment (red line), fungal pretreatment (green line), phosphoric acid pretreatment (light blue line), fungal pretreatment followed by phosphoric acid (brown line). Assignments of IR band maxima to various components of oil palm empty fruit bunches according to literature.

Tabel 2.
Untreated OPEFB

Fungal pretreatment

Phosphoric acid pretreatment

Fungal followed by phosphoric acid pretreatment

667

Assignments

Source

Ref.

648

666

666

716

C-O out-ofplane bending mode Rocking vibration CH2 in Cellulose I CH2 vibration in Cellulose I C-H out of plane deformation in position 2,5,6 Anomere Cgroups C(1)-H deformation, ring valence vibration C-O valence vibration

Cellulose

Cellulose

770

770

769

769

Cellulose

(Schwanni nger et al. 2004) (Schwanni nger et al. 2004) (Schwanni nger et al. 2004) (Fackler et al. 2010) (Fackler et al. 2010, Fengel 1992) (Schwanni nger et al. 2004)

849

851

850

851

G-Lignin

897

896

895

895

Polisakari da

998

997

17

Untreated OPEFB

Fungal pretreatment

Phosphoric acid pretreatment

Fungal followed by phosphoric acid pretreatment

Assignments

Source

Ref.

1,032

1,033

1,022

1,022

1,159

1,159

1,158

1,158

1,224

1,223

Aromatic C-H in plane deformation, G > S; plus CO deformation in primary alcohols; plus C=O stretch (unconj.) C-O-C assimetric valence vibration C-C plus C-O plus C=O strech; G condensed > G etherified C=O stretch, OH i.p. bending G-ring plus C=O strectch O-H blending of alcohol groups C-H deformation vibration Aromatic skeletal vibrations with C-H in plane deformation CH2 scissoring C-H in pyran ring symmetric scissoring; OH plane

Lignin

(Schwanni nger et al. 2004)

Polisakari da

(Fackler et al. 2010) (Fackler et al. 2010, Fengel 1992) (Faix O. and Bttcher 1992) (Faix O. 1991) (Fackler et al. 2010) (Fengel 1992)

Polisakari da

1,241

1,237

1,243

1,245

1,266 1,321

1,267 1,326

1,267 1,315

1,267 1,315

G-Lignin Karbohidr at Cellulose

1,375

1,371

1,370

1,372

1,418

1,418

1,420

1,419

Lignin

(Faix Oskar et al. 1991)

1,462

1,457

1,455

1,459

Cellulose

(Fengel 1992)

18

Untreated OPEFB

Fungal pretreatment

Phosphoric acid pretreatment

Fungal followed by phosphoric acid pretreatment

Assignments

Source

Ref.

1,511

1,507

1,506

1,506

1,593

1,609

1,608

1,607

1,640

1,646

1,654

1,663

1,735

1,735

1,735

1,735

2,850

2,850

2,850

2,850

2,918

2,918

2,918

2,918

3,338

3,345

3,346

3,351

deformation vibration Aromatic skeletal vibrations; G>S Aromatic skeletal vibrations plus C=O stretch; S>G; G condensed > G etherified C O stretch in conjugated psubstituted aryl ketones CO stretch unconjugated (xylan) Asymetric CH2 valence vibration Symmetric CH2 valence vibration Hydrogen bonded O-H valence vibration; O(3)H...O(3) intermolecular in cellulose

Lignin

(Faix Oskar et al. 1991)

Lignin

(Faix Oskar et al. 1991)

Lignin

(Faix Oskar et al. 1991) (Faix Oskar et al. 1991) (Schwanni nger et al. 2004) (Schwanni nger et al. 2004)

Polisakari da

Cellulose

(Schwanni nger et al. 2004)

A strong and broad absorption was observed at a wavenumber of around 3,300 cm . This wavenumber was assigned to hydrogen bonded (O-H) stretching absorption. O-H stretching region at a wavenumber of 3,0003,600 cm-1 of OPEFB spectra was more identical to the O-H stretching region from cellulose I than cellulose II. The valence vibration of hydrogen-bonding of OH groups of cellulose I is the sum of three different hydrogen-bonds: intramolecular hydrogen bond of 2OHO-6, intramolecular hydrogen bond of 3-OHO-5, intermolecular hydrogen bond of 6-OHO-3 (Schwanninger et al. 2004). Relatively high band in this
-1

19

wavelength interval decreased as a result of a decrease in hydrogen bonding and contains cellulose . Hydrogen bonding bands in the wavelength range of 2800-3800 cm-1 shows the same trend as the degradation of cellulose after pretreatment. The highest cellulose loss was observed in fungal followed by phosphoric acid pretreatment as indicated with the lowest intensity on O-H stretching absorption. A strong intensity band at wavenumbers 2,985 and 2,918 cm-1 was found in untreated and fungal pretreated OPEFB at these two wavenumbers are similar to IR spectra from hardwood and hardwood lignin (Fackler et al. 2010), which suggests that lignin structure in OPEFB is similar to hardwood lignin. Decrease in IR intensity at both wavelengths are on OPEFB who received pretreatment with phosphoric acid and fungal pretreatment combination with phosphoric acid showed a large change in the structure of the CH2 groups. Infrared spectra in the wavelength range of 1,150 and 1,750 cm-1 clearly shows two distinct spectral groups (Figure 8b). The band at a wavenumber of around 1,735 cm-1 was assigned to an unconjugated carbonyl originated from the uronic acid of the xylans in hemicellulose (Fackler et al. 2010). In this peak, there may exist linkages between lignin and carbohydrate (Fengel 1992). IR intensities at this wavenumber diminished after fungal pretreatment. Interestingly, it showed shoulder peaks after phosphoric acid pretreatment and fungal followed by phosphoric acid pretreatment. These peaks at wavenumber 1,735 cm-1 confirmed slight changes in hemicellulose content after fungal pretreatment and a high loss of hemicellulose after phosphoric acid pretreatments and fungal followed by phosphoric acid pretreatment (Figure 8b). Structural changes in lignin and loss of aromatic units were shown by the intensities in the changes in the 1,646, 1,593 and 1,506 cm-1 bands. Fungal pretreatment increased the intensity of the 1,646 cm-1 band and decreased the intensity of the bands at 1,593 and 1,506 cm-1. These changes suggest a split between the benzylic - and -carbon atoms by fungal pretreatment (Fackler et al. 2010). Both phosphoric acid pretreatment and fungal followed by phosphoric acid pretreatment showed similar intensities for the bands at 1,646, 1,607, 1,593, and 1,506 cm-1. These spectra explained the fact shown in Tables 1 and 2 that pretreated OPEFB by phosphoric acid pretreatment and fungal followed by phosphoric acid pretreatment had a similar loss and the percentage of ASL. IR intensity decreased at wave numbers 1,462 and 1,418 cm-1, but increased at wavenumber 1,321 cm-1 after fungal pretreatment. IR intensities of these bands were reduced after phosphoric acid pretreatment and fungal followed by phosphoric acid pretreatment. Different intensities were also found in the bands near wavenumbers 1,267 and 1,236 cm-1. The intensities at these bands did not change after fungal pretreatment, but reduced after phosphoric acid pretreatment. A band at 1,267 cm-1 was assigned to the guaiacyl of lignin. A band at 1,235 cm-1 was attributed to a combination of a deformation of syringyl and cellulose. The decrease in intensity at wavenumber 1,235 cm-1 was greater than that at wavenumber 1,267 cm-1 after

20

phosphoric acid pretreatment. This suggests that syringyl was more solubilized by phosphoric acid than guaiacyl lignin. The band at wavenumber 1,375 cm-1 was assigned to C-H deformations in cellulose and hemicellulose. The intensity of this band was slightly decreased after fungal pretreatment and it showed a slight loss of cellulose and hemicellulose content. A higher decrease in intensity was found after phosphoric acid pretreatment, which could be related to the high loss of hemicellulose content. Decreasing intensities were also found in the band at wavenumber 1,159 cm-1, which was assigned to C-O-O- > C-O-C asymmetric vibration of cellulose and hemicellulose. All the pretreated OPEFB samples showed lower intensities than the untreated OPEFB. Changes in intensity were also found in the band at around wavenumber 1,032 cm-1 that was assigned to the C-O stretch in cellulose and hemicellulose. Intensity of this band was slightly increased after fungal pretreatment. On the other hand, it shifted to 1,021 cm-1 and decreased in intensity after phosphoric acid pretreatment. The shifting and decreasing at this band might be attributed to decreased hemicellulose content after phosphoric acid pretreatment.

Figure 9.

FTIR spectra (a) and second derivative spectra (b) at wavenumber 770 cm1 (CH2 vibration in Cellulose I) and 716 cm1 (CH2 vibration in Cellulose I ). Lines assignment were: untreated (red line), fungal pretreatment (green line), phosphoric acid pretreatment (light blue line), fungal followed phosphoric acid pretreatment (light brown line).

The peak at a wavenumber around 895 cm-1 was assigned to C-H-O stretching of the -(1-4)-glycosidic linkage. Intensities of this peak were increased after fungal pretreatment and phosphoric acid pretreatment, but decreased by fungal followed by phosphoric acid pretreatment (Figure 8b) shows peaks at wavenumbers of around 750 cm-1 and 716 cm-1 that were assigned to rocking vibration CH2 in cellulose I and cellulose I , respectively. Crystalline cellulose I is composed of two allomorphs, Cellulose I (triclinic) and Cellulose I (monoclinic) (O'Sullivan 1997). Peaks at 769 cm1 were clearly observed in all spectra. A clear peak at wavenumber 716 cm -1 was

21

only found in untreated OPEFB spectra which then became a shoulder peak after pretreatments. Second derivative spectra revealed that peaks at a wavenumber around 769 cm-1 for cellulose I was showed constant intensities after pretreatment. However, peaks at a wavenumber of 716 cm-1 for cellulose I were decreased significantly after pretreatment ( Figure 9b ). Different methods have been proposed to characterize and quantify the crystallinity of cellulose using the ratio of the intensities of certain bands at the IR spectra, i.e,: 2,900, 1,429, 1,372, 894 and 670 cm-1. The IR A1418/A895 known as Lateral Order Index (LOI) is the ratio between absorbance at wavenumber 1,418 and 895 cm-1 (Hurtubise and Krassig 1960, O'Connor et al. 1958). LOI value of untreated, fungal pretreated, phosphoric acid pretreated, and fungal followed by phosphoric acid pretreated OPEFB are 2.78, 1.42, 0.67, and 0.60, respectively. Untreated OPEFB has the highest value and the greatest decrease was achieved by phosphoric acid pretreatment. There is no significant difference between the LOI values of phosphoric acid and fungal pretreatment followed by phosphoric acid pretreatment. The LOI showed a linear correlation with the hemicellulose content The correlation of LOI and hemicelluloses was probably due to the fact that the band at 894 cm-1 was assigned to the anomeric carbon group frequency in hemicellulose and cellulose (O'Connor et al. 1958). Results of this analysis also was suggests that the crystallinity of cellulose associated with hemicellulose content. 1.10.3. Effect of pretreatment on OPEFB Morphology Photomicrographs of untreated OPEFB and fungal-pretreated OPEFB are presented in Figure 10. The strand surface of untreated OPEFB has round-shaped spiky silica-bodies. The silica bodies were found in great number and attached relative uniformly around the fibre surface. Fungal pretreated OPEFB shows that some of silica bodies were removed from the strand surface and left empty holes at the bottom of silica-bodies creatures (Figure 10b). The surfaces of fungal pretreated OPEFB are rugged and partially broken faced. Mycelium growth was found in fungal pretreated OPEFB (Figure 10c,d). Mycelium grows outside and penetrates inside the OPEFB strand. Figure 11 presents photomicrographs of untreated, fungal pretreated, phosphoric acid pretreated, and fungal followed by phosphoric acid pretreated OPEFB after being ball-milled. The particle size of pretreated OPEFB varied. Untreated and fungal-pretreated OPEFB showed larger particle size compared to OPEFB pretreated by phosphoric acid and fungal followed by phosphoric acid pretreatment (Figure 11a,b).

22

SB

b
EM

d
M

Figure 10.

Fiber surface of untreated Oil palm empty fruit bunches (OPEFB). (a) untreated OPEFB, (b) fungal pretreated OPEFB, (c) fungal pretreated OPEFB strand covered by fungal mycellium, (d) cross section of fungal pretreated OPEFB. SB = silica body, EH = empty hole, M = mycelium.

Some silica bodies were partially removed in the untreated OPEFB, but the removal was higher in the fungal pretreated OPEFB. It seems that silica bodies were easier to remove by ball mill in the fungal pretreated OPEFB than in the untreated OPEFB. Biological pretreatment was likely to loosen the bond between silica bodies and the surface of OPEFB fibers. Silica bodies were not found on both of the OPEFB preetreated using phosphoric acid. Phosphoric acid and fungal followed by phosphoric acid pretreated samples shows small size and non-uniform particles (Figure 11 c, d). Strands of OPEFB are completely broken after phosphoric acid pretreatment and fungal followed by phosphoric acid pretreatment. The photomicrographs revealed that strands of OPEFB pretreated by phosphoric acid and fungal followed by phosphoric acid pretreatment were weaker and easier to grind than OPEFB strands pretreated by the other methods.

23

Figure 11.

Morphological changes of OPEFB surface before and after pretreatment. All samples were size-reduced using ball milling before hydrolysis and fermentation. (a) untreated OPEFB, (b) fungal pretreated OPEFB, (c) phosphoric acid pretreated OPEFB, (d) fungal followed by phosphoric acid pretreatmented OPEFB.

1.10.4. Cellulose Digestibility Figure 12 shows the digestibility of untreated and pretreated OPEFB after 72 h enzymatic hydrolysis. The digestibility was calculated based on initial cellulose content prior to hydrolysis. Digestibilitas is calculated based on the initial cellulose content OPEFB before pretreatment (equation 2). It is shown that untreated OPEFB had very low digestibility (4.66%), which could be caused by its high lignin and high hemicellulose contents, as well as high crystallinity of cellulose. Digestibilitas OPEFB example that gets pretreatment is as follows: 18.85% (fungal pretreatment), 29.15% (phosphoric acid pretreatment), and 34.64% (pretreatment mushroom-phosphoric acid). Digestibilitas it increased respectively by 400% (pretreatment mushrooms), 630% (phosphoric acid pretreatment), and 740% (pretreatment mushroom-phosphoric acid) times compared with digestiblitas OPEFB who did not receive pretreatment. Moreover, it is comparable to digestilitas digestilitas OPEFB after a pretreatment with ammonia (Ammonia Fiber Expansion, AFEX) pretreatment (58%) (Lau et al. 2010), alkali pretreatment (69.69%) (Piarpuzn et al. 2011), pretreatment superheated steam (66.33%) (Bahrin et al. 2012) and sodium hydroxide-sodium pretreatment hypoclorite (60%) (Hamzah et al. 2011). Digestibilitas OPEFB after biological pretreatment for 28 days with P. floridanus Digestibility higher than that in the Japanese pine-pretreatment with Stereum

24

hirsutum for eight weeks (13.56%) (Lee et al. 2007).

40 35 30 25 20 15 10 5 0 34,64 29,15 18,85 4,66

Digestibilitas selulosa (%)

Kontrol

Pretreatment Pretreatment Pretreatment Jamur asam fosfat jamur-asam fosfat

Figure 12.

Digestibility of cellulose (%) of oil palm empty fruit bunches (OPEFB) in the enzymatic hydrolysis process (based on initial cellulose content after pretreatments). Error bars are standard deviation. Hidrolysis was used commercial enzyme Cellic CTec2, at 50oC, for 72 h.

The digestibility of OPEFB after pretreatment has an inverse correlation with LOI. Digestibility is enhanced as crystallinity of the cellulose is reduced as shown by the lower LOI value. The IR spectra of fungal pretreatment samples indicate that the fungus might attack the linkages between lignin and carbohydrate that exist in hemicellulose. Results of correlation analysis between digestibilitas with LOI values indicate an inverse correlation where digestibilitas OPEFB increases with decreasing value of LOI. Increasing cellulose digestibility was due to several changes in the structure of OPEFB, such as the decreasing of hemicellulose content, breaking down the bonds between lignin and cellulose, decreasing of cellulose crystallinity and increasing of cellulose I. Cellulose I is meta-stable and more reactive than cellulose I (O'Sullivan 1997). This possibility makes OPEFB more reactive and more easily hydrolyzed. OPEFB pretreated by phosphoric acid and fungal followed by phosphoric acid methods showed relatively high lignin proportion up to 44.66%. This finding stresses the fact that lignin seems not the only recalcitrant factor of OPEFB. Available surface area and accessibility to cellulose of OPEFB after pretreatment contribute to improved digestibility of lignocellulosic materials (Rollin et al. 2010). 1.10.5. Bioethanol Production Production of bioethanol (ethanol yield) was shown in Figure 13. Bioethanol production by SSF method of sample OPEFB showed a similar pattern with OPEFB digestibility (Figure 12). Production of bioethanol from the highest were combination fungal pretreatment-phosphoric acid, phosphoric acid pretreatment, fungal

25

pretreatment, and without pretreatment.

90 80 70 60 50 40 30 20 10 0 0
Kontrol Pretreatment Jamur - Asam Fosfat

Yield Etanol (%)

24

48 Jam ke-

72
Pretreatment Jamur Pretreatment Asam Fosfat

96

Figure 13.

Percentage of bioethanol production from the theoretical maximum production (ethanol yield ) of oil palm empty fruit bunches (OPEFB) by the method of SSF (simultaneous saccharification and fermentation).

Yeast will ferment glucose resulted from enzymatic hydrolysis of cellulose to ethanol. Cellulose digestibility cellulose will increase in line with increased production of ethanol by yeast. Increase the ethanol yield of each treatment at the 72h than control treatment was 222 % (fungal pretreatment), 642 % (phosphoric acid pretreatment), and 701 % (fungal pretreatment and phosphoric acid). Ethanol yield has significant positive linear correlation ( r2 = 0.99 ) with OPEFB digestibility which means that the increasing digestibility will be followed by an increasing in ethanol production. Ethanol yield resulting from this study is higher than the yield of ethanol that reported in some literature. Yield of ethanol from biological pretreated OPEFB were 6 g / L higher than the yield of ethanol from the alkali pretreated OPEFB ( 4 g / L ) (Piarpuzn et al. 2011). Increasing the yield of ethanol of the combination pretreated OPEFB increased 7.01 times. This increasing was higher than the increasing of water hyacinth (Eichhornia crassipes) were pretreated with alkali and WRP which the increasing was only 1.34 times (Ma et al. 2010). CONCLUDING REMARKS Third WRP isolates have varying selectivity to degrade lignin, cellulose and hemicellulose. Pleurotus floridanus isolates showed the highest degradation of lignin and lowest cellulose degradation. P. floridanus was more selectively to degrade lignin than other isolates. The addition of Cu2+ and Mn2+ could increase lignin degradation by P. floridanus. Lignin content was degraded up to 46.62 % within 42 days of incubation. Physical, chemical, and structural of OPEFB was changing after pretreated with P. floridanus, phosphoric acid , and a combination of biological and

26

phosphoric acid pretreatment. Some functional groups mainly syringyl and guaiacyl lignin units undergo significant changes. Cellulose crystallinity of OPEFB was decreased . Important structural changes observed by FTIR analysis were reduction of hydrogen bond (OH), unconjugated carbonyl absorption, the absorption peak ( peak ) for cellulose and hemicellulose , and cellulose peaks decrease I. Digestibility OPEFB and ethanol production has increased very significantly on a combination of biological pretreatment and phosphoric acid. Degradation of lignin and hemicellulose, reduction of cellulose crystallinity, decreased cellulose I, and particle size reduction and contribute to the increase in ethanol production digestibilitas. Refernces Alvira P, Toms-Pej E, Ballesteros M, Negro MJ. 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresource Technology 101: 4851-4861. Anderson WF, Akin DE. 2008. Structural and chemical properties of grass lignocelluloses related to conversion for biofuels. J Ind Microbiol Biotechnol 35: 355366. Bahrin EK, Baharuddin AS, Ibrahim MF, Razak MHA, Sulaiman A, Abd-Aziz S, Hassan MA, Shirai Y, Nishida H. 2012. Physicochemical property changed and enzymatic hydrolysis enhancement of oil palm emtpy fruit bunches treated with superheated steam. BioResources 7(2): 1784-1801. Bajpai P. 2004. Biological Bleaching of Chemical Pulps. Critical Reviews in Biotechnology 24 (1): 1-58. Camarero S, Bockle B, Martnez M, Martnez A. 1996. Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius. Applied and Environmental Microbiology 62: 1070-1072. Chen M, Yao S, Zhang H, Liang X. 2010. Purification and Characterization of a Versatile Peroxidase from Edible Mushroom Pleurotus eryngii. Chinese Journal of Chemical Engineering 18: 824-829. Datta R. 1981. Acidogenic Fermentation of Lignocellulose-Acid Yield and Conversion of Components. Biotechnology and Bioengineering XXIII: 2167-2170. de Jong E, Chandra RP, Saddler JN. 1997. Effect of a Fungal Treatment of The Brightness and Strength Properties of A Mechanical Pulp from Douglas-Fir. Bioresource Technology 61: 61-68. Dowe N, McMillan JD. 2008. SSF Experimental Protocols Lignocellulosic Biomass Hydrolysis and Fermentation. National Renewable Energy Laboratory. Report no. NREL/TP-510-42630. Fackler K, Stevanic JS, Ters T, Hinterstoisser B, Schwanninger M, Salmn L. 2010. Localisation and characterisation of incipient brown-rot decay within spruce

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