Research Journal of Environmental Seienoes 6 (6): 878-886, 2011 ISSN 1819-3412 / DOL: 10,3923frjes.2011.573.586 © 2011 Academie Journals Ino, Biothanol Production from Enzymatically Saccharified Empty Fruit Bunches Hydrolysate using Saccharomyces cerevisiae ‘Mohd Asyraf Kassim, 'Loh Soh Kheang, 'Nasrin Abu Bakar, *Astimar Abdul Aziz and *Rosnah Mat Som ‘Bnerey and Environment Unit, *Agro Product Unit, Engineering and Processing Division, Malaysian Palm Oil Board (MPOB), 6, Porsiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia Corresponding Author: Mohal Asyraf Kassim, Energy and Environment Unit, Engincering and Processing Division, ‘Malaysian Paim Oil Board (MPOB), 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia, ABSTRACT Cellulosie ethanol production was eavried out using Empty Fruit Bunches (EFB) via Separate Hydrolysis and Fermentation (SHF) approseh. In this study, the EFB was initially pretreated with alkaline treatment using 1% (wy) sodium hydroxide (NaOH) followed by mild acid hydrolysis using 0.7% (viv) sulfurie acid (H,80,) and enzymatic saecharification prior to fermentation with Saccharomyces cerevisiae. Pretreatment of EFB via alkaline and acid hydrolysis produced 18.3840.18 g L of xylose and 0.548018 g L” of glucose. A total of 16.45.86 ¢ L and 3.8542.2 g L* of glucose and xyloso respectively was produced during the enzymatic saccharification. The EFB hydrolysate obtained was subjected to fermentation in batch mode and the influence of pH, temperature and rate of agitation on biosthanal production were evaluated. From optimization works of these various fermentation parameters, it was found that the highest ethanol yield (¥,,) was 0.51 g ethanollg glucose achieved from 50 g L“ of acid enzymatically saccharified EFB hydrolysate at pH 4, 80°C and 100xpm was recorded at 72h of incubation. This study showed that EFB generated from palm biomass can be used as a lignocellulosic ethanol production feedstock for the future. Key words: Empty Fruit Bunches (EFB), pre-treatment, hydrelysis, fermentation, ethanol INTRODUCTION Malaysia is one of the largest. producers of palm oil in the region and the product: has contributed the biggest. income to the country far many years, With the rapid growth of palm oil production in Malaysia, the amount of biomass residues generated also has shown a corresponding inerease. As in 2010, the oil palm planted area in the country is 4.8 million heetares. The total average of 18,03 tones Fresh Fruit Bunches (FFB) per hectare palm plantation has been produced from the palm oil industry (Choo, 2011). Based on this figure, palm oil plantation areas has produced a total of more than 66.63 million tonnes of biomass residues such as Empty Fruit Bunches (2FB), mesocarp fiber, shell, palm kernel cakes, fronds, trunks and Palm Gil Mill Effluent (POME) in 2010 (Goh et al., 2008) In a country that has a significant amount of agricultural activities, biomass can be a very promising alternative source of renewable energy. With increased awareness on reducing B78Res, J. Environ. Sci., 5 (6): 578-586, 2011 greenhouse gas emissions, conversion of biomass residues into renewable energy such as ethanol, bio-cil, biogas, syngas and bichydrogen has attracted global attention. Biomass residues are valuable as an immediate and relatively cheap energy feedstock for development of a bio-energy industry (Ibeto et a, 2011). Besides, the utilization of biomass also offers an environmentally friendly way of disposing the unwanted by-products generated from palm oil mills. EFB is one of the major biomass types generated in the oil palm industry. From 85.71 million tonnes of Fresh Fruit Bunches (FFB) produced in 2009, an estimated amount of 6.76 million tonnes of dried EFB was generated from palm oil mills. Generally, EFB is lignocelulosie materials consisting of a mixture of carbohydrate polymers, i.c., colluloso and hemicellulose (Taherzadeh and Karimi, 2007), It is estimated that EFB comprise of 44.2% cellulose, 88.5% hemicellulose and 20.4% lignin, respertively (Limikalsom ef al., 1997; Aziz et al., 2002). Basically, cellulose is @ polymer of ¢-D-1,4-linked anhydrous glucose unit, In contrast, hemicellulose is a random, amorphous copolymer comprising several sugar monomers such as glucose, fructose, xylose and mannose, Hence, EFB with high contents of cellulose and hemicellulose can be potentially converted into ethanol, In most of the ethanol production using various different lignocellulosic biomass, two main processes ie. pretreatment involving alkaline and acid hydrolysis and enzymatic saecharification to produce glucose and xylose followed by fermentation with yeast to produce ethanol (Demirbas, 2005), Ethanol production via fermentation is a complex biochemical process with yeast or bacteria utilizing fermentable sugar as substrate for their growth and converting them to ethanol, carbon dioxide and other metabolic end product, Several factors that can effect ethanol formentation should be considered. During ethanol fermentation, most of the yeast cells used suffer from various stresses, including environmental stress such as glucose concentration, nutrient deficiency, temperature, rate of agitation and pH (Graves et al, 2008; Avisra et ai., 2008; Yah et al., 2010) Temperature in particular has a crucial effect, as it can affect the development of yeast used, yields of ethanol and the types of fermentation by-products (Sener ef al., 2007). ‘Temperature can also affect the sensitivity of the yeast in relation to alcohol concentration, growth rate, rate of fermentation, enzyme and membrane function (Torija et al., 2003), Moreover, pH and rate of agitation also have been reported to be important factors for ethanol production from lignocellulosic hydrolysate (Fleet and Heard, 1993; Liu and Shen, 2008). Thus, the purpose of this study was to explore the potential of EFB as an ethanol feedstock via Separate Hydrolysis and Fermentation (SHF) and to determine the optimum condition of physical parameters, i.e, pH, temperature and agitation rate that can affect the production of ethanol from EEB hydrolysate, MATERIALS AND METHODS EFB preparations: EFB was collected from a palm oil mill located in Padang Jawa, Klang. The bunch was dried at, 105°C and eut into smaller picees. It was then milled, sieved and separated in fractions using a test sieve shaker (Endecotts EFL, 2000). The particle size of EFB used for this, study was 91-106 pm, EFB characterization: Dried EF'B was initially delignified according to ASTM 1104-66 to produce holocellulose, followed by removal of the hemicellulose fraction using ASTM D1103-60, For holocellulose, approximately 4.0 g of the ground EFB were mixed with distilled water and treated with 2.0 mL acetic acid and 6.0 g sodium chlorite at, 70°C for 4h, The mixture was then filtered 574Res, J. Environ. Sci., 5 (6): 578-586, 2011 using filter paper and dried at, 103°C for 24 h. Determination of holocellulose was carried out using dry weight method. A total of 2.0 g of dried holocellulose obtained were dissolved in 50 mL, 17.5% sodium hydroxide solution and then a total of 70 mL were added in the mixture in purpose to separate hemicellulose from the holocellulose and leaving c-cellulose, The insoluble «cellulose was filtered, then washed with 50 mL 8.8% sodium hydroxide and dried at 108°C for 24 h. Determination of e-cellulose was carried out using dry weight. method, Pretreatment: A total of 5 of treated pulverized EFB was initially soaked with 1% (w/v) sodium hydroxide (NaOH) at. 100°C for 2h, The treated EFB was then washed with hot water prior to drying the sample at 103°C for 24h, For the acid hydrolysis proves, a total of 5 g of dried EFB was hydrolysed with 100 ml. 0.7% (viv) H,SO, and autoclaved at, 125°C for 120 min (Hirayama HVE 50). The treated EF'B was then washed with hot water prior to drying at. 108°C for 24 h. For enzymatic saccharification EFB from the acid hydrolysis process was soaked in a 100 mL of acetate buffer solution (pH 4.8) and mixed with cellulase (70 FPU ml) (Novozymes) at a temperature of 48°C and rate of agitation 160 1pm for 48h, The EFB hydrolysate obtained was used for fermentation and determination of optimum fermentation process parameters. Inoculums preparation: Saccharomyces cerevisiae was initially grown on Yeast-peptone-glucose (YPG) and was incubated ata temperature of 85°C and a rate of agitation 150 rpm for 18to 24h (innova 40). In this study, YPG medium consisted of @ L™); 10 g yeast extract, 20 g peptone and 20 g glucose, After the incubation period, the cells were then harvested by centrifugation at 3000 rpm at 4°C for 15 min (Hettich, Universal 82 R), The pellet was then rinsed twico with sterilized saline solution before being re-suspended in sterilized saline solution to yield an Optical Density (OD) of 1.0 at. 600 nm (Hirayama Model U-200), The standardized S. cerevisiae was used for subsequent study. Determination of optimum parameters: Fermentation of EFB hydrolysate from the pretreatment process was carried out by using S, cerevisea ATCC 24860. A total of 150 ml, of EFB hydrolysate from enzymatic saccharification was prepared in a 250 mL conical flask. A total of 10% (wiv) of standardized active S. cerevisiae was added to the hydrolysate prior to incubation into a shaker. To determine the effect of physical parameters on ethanol production, three series of experiments were carried out, For each series of experiments, EFB hydrolysate was harvested every 12to 24h interval. The harvested samples were filtered using a 0.45 yum membrane filter and then the filtered samples were put in 2.5 mL. vials prior to being analyzed. Effect of initial pH: The first series of experiments was to determine the effect of initial pH on ethanol production. The ranges of pH used were pH 4, 6, 7 and 8, Initially, the pH of EFB hydrolysate was adjusted using 1 M hydrochloric acid (HCD or 1 M sodium hydroxide (NaOH) prior to inoculating with standardized S. cerevisiae. ‘The samples were incubated at 80°C with an agitation rate of 100 rpm, Effect of temperature: A second series of experiments was to determine the effect of temperature on ethanol production. Three different temperatures, i.e., 50, 35 and 40°C were used in this study and the EFB hydrolysate was incubated at the optimum pH obtained from the pH optimization study and a rate of agitation of 100 rprn. B76