Microscopic Studies Of Oil Palm Frond During Processing For Saccharification

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1 ISPUB.COM The Internet Journal of Bioengineering Volume 4 Number 2 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification L Hong, D Ibrahim, I Omar Citation L Hong, D Ibrahim, I Omar. Microscopic Studies Of Oil Palm Frond During Processing For Saccharification. The Internet Journal of Bioengineering Volume 4 Number 2. Abstract In this study, the structural architecture of oil palm frond (OPF) after various pretreatment is evaluated through microscopic studies. Maximal degrees of saccharification of OPF are depended on pretreatment method used. Each pretreatment has its own effect(s) on the fiber of OPF. OPF is a solid waste generated by the palm oil industry that creates a problem for the industry in terms of waste management. This lignocellulosic waste material has many physio-chemical structural and compositional factors that hinder the enzymatic digestion of the cellulose present in the lignocellulosic biomass. A pretreatment is needed to alter or remove these structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase the yield of fermentable sugars. The structural architecture of lignocellulosic fibres after various pretreatment e.g. chemical, biological and biochemical, were evaluated through light microscopic (LM), scanning electron microscopic (SEM) and transmission electron microscopic (TEM) studies. These pretreatments, both separately and intergrated, rendered the OPF biomass more susceptible and accessible to saccharification and increased the production of fermentable sugars. Morphological changes that took place in the lignocellulosic biomass included the removal of inhibitory materials, e.g. triterpenoids, silica, hydrocarbons, etc., production of cracks in the lignocellulosic fibres, and exposure of cellulosis materials by creating pores during pretreatment. The most effective pretreatment was autoclaving followed by enzymatic hydrolysis of the OPF. This combination of treatments resulted in a significant increase in reducing sugar production by creating pores due to the removal of silica and lignin from biomass, as observed in SEM studies. INTRODUCTION Oil palm (Elaeis guineensis) is widely cultivated as a source of oil in West and Central Africa, where it originated, and in Malaysia, Indonesia and Thailand. In Malaysia, oil palm is one of the most important commercial crops. The explosive expansion of oil-palm plantations in these countries has generated enormous amounts of agricultural waste, created problems in replanting, and raised significant environmental concerns. In Malaysia alone during the recent past year, produced about 30 million tonnes of oil-palm biomass, including trunks, fronds, and empty fruit bunches [1]. This estimate was based on a planted area of 2.6 million ha, and a production rate of dry oil-palm biomass of 20,000kg/ha/yr [2], which mean that Malaysia s oil palm industry produced 52 million tonnes of lignocellulosic biomass. This figure is expected to increase substantially when the total planted hectares of oil palm in Malaysia reaches 3.2 million ha. Oil palm frond (OPF) consists primarily of celluloses, hemicelluloses and lignin, and lesser amounts of protein, oil and ash that make up the remaining fraction of the lignocelluloses biomass [3]. The toughness of the native cellulose fibre results because it is embedded in lignin. The hemicelluloses provide the link between lignin and cellulose. This lignin coating, when intact in the plant, reduces the accessibility of the cellulose for digestion by chemical and/or biochemical means for the production of fermentable sugars and liquid fuels [4]. Pretreatment(s) of the lignocellulosic materials before subjecting them to fermentable sugar production via enzymatic hydrolysis can resolve this problem. Various pretreatment processes, i.e. physical, chemical and biochemical [5, 6, 7, 8, 9, 10, 11, 12, 13] are commonly used to remove barriers to cellulose hydrolysis. Thus, it is important to know the morphological, structural and chemical changes that occur during each pretreatment. Therefore the present study was carried-out to investigate the state of morphology of lignocellulosic fibres of OPF through observation with a light microscope (LM), a scanning electron microscope (SEM) and transmission electron microscope (TEM) studies, during the pretreatment of OPF by chemical, physical and biochemical means. 1 of 7

2 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification MATERIALS AND METHODS PRETREATMENT OF OIL PALM FROND (OPF) Dried, powdered form of the OPF (1-2 mm) supplied by the Cedar Food Industry Sdn. Bhd., Sungai Bakap, Penang, Malaysia was used through-out the study. The OPF was pretreated as follows: CHEMICAL DELIGNIFICATION OPF was delignified chemically by alkali treatment. Twenty grams of air-dried OPF was autoclaved with 360 ml of 1 % NaOH, solution at 121 C for 1 hour in a 1000 ml medium bottle. After squeezing the OPF with a nylon cloth to remove excess water, the residual OPF was washed with water and then by air-dried at 45 C. ACID PRETREATMENT Twenty grams of OPF was autoclaved with 360 ml of 1 % of H 2 SO 4 solution at 121 C for 15 minutes in a 1000 ml medium bottle. The treated OPF residual was then washed with water, air-dried at 45 C and used for the saccharification experiment. LIQUID HOT WATER PRETREATMENT Twenty grams of OPF was autoclaved with 360 ml of distilled water at 121 C for 15 minutes in a 1000 ml medium bottle. The treated OPF residual was washed with distilled water, air-dried at 45 C and used for the saccharification experiment SACCHARIFICATION MICROORGANISMS AND CULTURE CONDITIONS The fungus, A. niger USM AI1, was maintained on a potato dextrose (Oxoid, England) agar slant at 37 C until sporulation (5 days). The culture was stored at 4 C until used. Inoculum was prepared by adding 4 ml of sterile distilled water to an agar slants. SUBSTRATE PREPARATION Treated and untreated OPF were dried in oven at 45 C and milled to a desired size by using grinding machine. 5 grams of the dry powder was put into a 250 ml conical flask and autoclaved at 121 C for 15 minutes. SOLID-STATE FERMENTATION (SSF) The sterilized solid substrates, treated and untreated OPF, were inoculated with 1.0 ml of inoculums, and the moisture content adjusted to 120% (v/w) with sterile distilled water. The contents were mixed thoroughly and incubated at room temperature (30±2ºC). Samples as whole flasks in duplicate were withdrawn after 5 days of incubation. FERMENTABLE SUGARS EXTRACTION Crude fermentable sugar from the fermented OPF was extracted by a simple contact method. The fermented OPF was mixed with 100 ml distilled water by shaking for 1 hour at room temperature (30±2ºC) on a rotary shaker at 150 rpm and filtered through filter paper (Whatman No.1). The filtrate and the solids were collected and each used as the crude fermentable sugar for further analysis. ANALYSES The Nelson and Somogyi procedure [14] was used to measure the amount of fermentable sugar. Fungal biomass was estimated by determining the amount of N-acetyl glucosamine released by acid hydrolysis of the chitin, present in the fungal cell wall [15]. MICROSCOPIC STUDIES Samples for microscopic studies were taken from biomass samples resulting from various pretreatments. The small particles remaining after enzymatic saccharification of i.e. OPF treated with hot water also was selected for microscopic observation. LM and SEM studies were carried out with an Olympus BH-2 microscope and a Leica Cambridge S-360 scanning electron microscope, respectively. The TEM study was carried out as described by McDowell & Trump [16], and viewed under a transmission electron microscope (Philips, Jerman). RESULTS OPF had many silica components on its surface (Fig 1). The silica components were arranged in a manner that typifies OPF alone. Similar observations were also made by Hong [17]. The surface of the OPF fibers was smooth if the crude biomass had been subjected directly to enzymatic hydrolysis with A. niger USM AI1 for 5 days. During this process about 32 mg/g dry weight untreated OPF of fermentable sugars was produced. After treatment with liquid hot water, the silica components were gone and the parenchyma cells were exposed (Fig. 2). Many pores formed on the fiber surface as a result of the liquid hot water treatment. We think that the smooth surface of the OPF fiber turned into pores after the liquid hot water because coated matrix - like silica was washed off. Alkali treatment of OPF appears result in complete fusion of the lignocellulosic fibers and the accumulation of cellulosic 2 of 7

3 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification material at irregular intervals along the fused mass (Fig. 3). We observed a partially degraded OPF fiber in which one of the transverse ends had a mass of material that appeared similar to cellulose [4]. Acid-treated OPF contained partially degraded and fused fibers, which could result from chemical reactions between the fibers (Fig. 4). As a result, the biomaterials appeared as a single unified mass. The partially degraded fiber, apparently resulted from an acid reaction the point of the notch, which opened the fiber. As the reaction of the surface materials with the acid, continued the rest of the notch opened and cellulosic material was leaf from the inside of the fibers. If OPF was treated with liquid hot water and then fermented with A. niger USM AI1, spore of the fungus attach to the OPF surface (Fig. 5A). After 3 days of fermentation (Fig. 5B), the fungus was growing on the OPF. Fungal hyphae grow on the surface of the lignocelulosic materials and form a dense hyphal out (Fig. 5C). At this stage, the fungus produces enzymes that degrade the parenchyma cells, which contain cellulosic and hemicellulosic materials, and turns this material into reducing sugars i.e. fermentable sugars. Finally, the fungus enters a death phase, where spores are dropped from the sporangium onto the surface of the OPF and the enzymatic hydrolysis ceases (Fig. 5D). Significant changes in the structure of the fibers have occurred by this stage. Enzymatic hydrolysis of the OPF treated with liquid hot water results from fungal hyphae growing on the OPF and producing an enzyme that breaks down the parenchyma cells (as marked with an arrow) (Fig. 6, 7). Finally, the OPF is degraded and converted to fermentable sugars, primarily monosaccharide. Parenchyma and xylem cells of OPF are arranged regularly (Fig. 6A). The parenchyma cells appear in their native shape with a thin cell wall (Fig. 7A). When the OPF is subjected to enzymatic degradation through solid state fungal fermentation the hyphae of fungus penetrate the substrate, destroying the cell structure and collapsing the cells (Fig. 6B, 7B). Thus hyphal growth contributes to the breakdown of the cell wall of the parenchyma cells. Fermentable sugars are produced after cultivation of the fungus on pre-treated OPF (Table 1). Liquid hot water pretreatment of OPF produced the most fermentable sugar, 127 mg/g substrate, followed by acid-treated OPF (93 mg/g substrate), and alkali treated OPF (44 mg/ g substrate). This 3 of 7 order suggests that a coated hydrocarbon was washed out of the liquid hot water treated OPF. Thus, the available fermentable sugars increased 4.0 fold following enzymatic delignification for five days through solid state fermentation (SSF) with A. niger USM AI1. Figure 1 Table 1: Production of total reducing sugars (fermentable sugars) after enzymatic hydrolysis with USMAI1 Figure 2 Figure 1: SEM micrograph of the control, untreated OPF

4 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification Figure 3 Figure 5 Figure 2: SEM micrograph of the OPF after liquid hot water treatment Figure 4: SEM micrograph of the OPF after acid treatment Figure 4 Figure 3: SEM micrograph of the OPF after alkali treatment 4 of 7 Figure 6 Figure 5: The SEM micrographs of the OPF cultivated with USM AI1 at different cultivation times. A) Day 0; B) Day 3; C) Day 5; D) Day 9

5 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification Figure 7 Figure 6: Micrographs showing light microscopic studies of liquid hot water treated OPF (A) before enzymatic hydrolysis (B) after enzymatic hydrolysis can hydrolyze hemicelluloses to xylose and other sugars and to break down xylose to furfural [10]. Carlos and Ball [20] reported that fungal hyphae can readily colonize plant biomass. The micrographs confirm that enzymatically degradable lignocellulosic substrate produced the observed fermentable sugar. This finding is similar to that of Itoh et al. [21] who reported that white rot fungi were commonly used for the enzymatic hydrolysis of agricultural wastes and to produce fermentable sugars. ACKNOWLEDGEMENTS Figure 8 Figure 7: Micrographs showing TEM studies of liquid hot water treated OPF (A) before enzymatic hydrolysis (B) after enzymatic hydrolysis The work was supported by a Research Grant from the Ministry of Science, Technology and Innovation of Malaysia (MOSTI). The first author thanks the Universiti Sains Malaysia for awarding her a USM fellowship through-out her PhD programme. References DISCUSSION Native cellulosic material is generally associated with secondary metabolites such as hydrocarbons [18]. These secondary metabolites generally are washed out, when the biomass is pre-treated. Different types of pre-treatment caused different morphological changes to the OPF structure. The liquid hot water treatment was expected to solubilize the hemicelluloses, make the cellulose more accessible to enzyme hydrolysis, and not form any inhibitors [13]. Fermentable sugars increased during saccharification if the OPF was subjected to a liquid hot water pretreatment. Currently, alkali-pretreatment with ammonium hydroxide [19] and liquid hot water pretreatment [13] are considered to be a near-term option, among various pretreatment methods. OPF treated with acid yielded the least fermentable sugars following enzymatic saccharification in solid state fermentation. This result suggested that acid pretreatment can extend the notch on cells leading to further loss of lignocellulosic material from the sample as reflected by the low yield of fermentable sugars. Diluted sulfuric acid also 5 of 7 1. Anon: Pulp and Paper International. Annual Report; 1991; 33 (7): Lim KO: Oil palm plantations-a plausible renewable source of energy. RERIC International Energy Journal; 1998; 20: Sjostrom E: Wood chemistry. In Fundamentals and Application. New York: Academic Press Inc.; 1981; pp: Behera BK, Arora M, Sharma DK: Scanning electron microscopic (SEM) studies on structural architecture of lignocellulosic materials of Colotropis procera during its processing for saccharification. Bioresource Technology; 1996; 58: Halliwell G: Cellulosic material and their pretreatment. Proccess Bioconversion Symposium HT Delhi; 1977; pp: Schurz J: How to make native lignocellulosic materials accessible to chemical and microbial attack. Process Bioconversation Symposium HT Delhi; 1977; pp: Lee YY, Lin CM, Johnson T, Chamber RP: Selective hydrolysis of hardwood and hemicelluloses by acids. Biotechnology and Bioengineering Symposium; 1978; 8: Converse AO, Ooshima H, Burns DS: Kinetics of enzymatic hydrolysis of lignocellulosic materials based on the surface area of cellulose accessible to enzyme and enzyme adsorption on lignin and cellulose. Applied Biochemistry and Biotechnology; 1990; 24/25: Ropars M, Marchal R, Pourquie J, Vandecasteele JP: Large-scale enzymatic hydrolysis of agricultural lignocellulosic biomass Part 1: pretreatment procedures. Bioresource Technology; 1992; 42: Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M: Features of promising technologies for pretratment of lignocellulosic biomass. Bioresource Technology; 2005; 96 : Zhang X, Xu C, Wang H: Pretreatment of Bamboo residues with Coriolus versicolor for enzymatic hydrolysis. Journal of Bioscience and Bioengineering; 2007; 104: Cara C, Ruiz E, Oliva JM, Saez F, Castro E: Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification. Bioresource

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7 Microscopic Studies Of Oil Palm Frond During Processing For Saccharification Author Information Lim Sheh Hong School of Biological Sciences, Industrial Biotechnology Research Laboratory (IBRL) Darah Ibrahim School of Biological Sciences, Industrial Biotechnology Research Laboratory (IBRL) Ibrahim Che Omar Faculty of Agroindustry and Natural Resource, Universiti Malaysia Kelantan 7 of 7