Effect Of Alkali Pretreatment and Enzymatic

Effect Of Alkali Pretreatment and Enzymatic Saccharification on Bagasse Reducing Sugar For Bioethanol Production by Kismurtono, M1). and Sutikno2) Abstract-Some efforts have been carried out to find out renewable sources of fuel due to fossil fuel depletion in the past decade. One of the efforts is conversion of bagasse into bioethanol as oil substitution. Bagasse has to be separated from its lignin before saccharification with cellulase enzymes. Objectives of this study were to find out the best concentrations of alkali and enzyme as well as the best incubation time for bagasse saccharification. To achieve the objectives, two treatments - namely concentration and submersion time into NaOH solution, and concentration and incubation time of cellulase - were applied in this study. Bagasse was dried up to constant weight, ground up to 40 meshes, and submersed into 0 – 1.0 M NaOH solution at 121oC for 60 minutes. Bagasse was then analyzed to determine its delignification. Seven and half percent bagasse with highest lignin degradation was then saccharified with 0 – 20 FPU cellulase at 50oC and 100 rpm for 36 hours. After saccharification, bagasse reduced sugar content was measured. The best treatment was submersed bagasse into 1.0 M NaOH solution at 121oC for 15 minutes and then saccharified with 20 FPU cellulase at 50oC, and 100 rmp for 18 hours. The treatment yielded a reduced sugar concentration of 19.4 g/L.

Keywords: bagasse, cellulase, reduced sugar, lignocellulose, NaOH. 1)

2)

. Technical Implementation Unit for Development of Chemical Engineering Processes Gunung Kidul, Yogyakarta, Indonesia. . Lecturer of The Department of Agricultural Product technology, the University of Lampung, Indonesia.

I. INTRODUCTION Since fossil fuels are depleted, alternative sources of fuel have been investigated in past two decades [1]. Bioethanol produced from agricultural products is considered to compensate fuel shortage [2]. Agricultural products such as sugarcane [3], corn [4], or cassava [5] have been successfully utilized as raw materials of the first generation bioethanol production. However, demand for these agricultural products, which are also used as food sources, will not be sufficient to meet the need for fuel bioethanol [6]. To overcome this problem, agricultural solid waste such as bagasse [7], [8], empty palm fruit bunch [9], cotton stalks [10] can be used as raw materials of the second generation bioethanol production. Sugar cane bagasse is one of the main solid agroindustrial wastes in Indonesia. Indonesia produces 39.539.944 ton bagasse yearly [11]. The bagasse consists of 45.8% cellulose, 20.4% hemicelluloses, and 21.6% lignin [12]. According to Badger formula [13], the bagasse can be converted into 7.131.221 kL bioethanol. Before conversion into bioethanol

through fermentation, bagasse has to be pretreated and saccharified. Pretreatment was carried out to remove lignin and hemicelluloses, to reduce cellulose crystallites, and to increase porosity of the materials [14]. Saccharification is performed to hydrolyzed cellulose into reduced simple sugar. Pretreatment using NaOH and saccharification with cellulase at Indonesian bagasse were studied in this research; and the objectives of this study were to find out the best pretreatment and the best saccharification conditions which yielded the highest reduced sugar concentration.

II. MATERIALS AND METHODS Materials Bagasse was obtained from PT Gunung Madu Plantation, Central Lampung, Lampung, Indonesia. Before use, the bagasse was analyzed to determine its cellulose, hemicellulose, and lignin contents. Cellulase (Cellulast 1.5L) was obtained from Balai Besar Teknologi Pati (BBTP) at Sulusuban, Central Lampung, Lampung, Indonesia. All other reagents were of analytical grade. Pretreatment using NaOH Pretreatmen using NaOH was performed according to Sutikno et al [7]. Bagasse was dried up to constant weight and grounded up to 40 meshes. One and half grams of grounded bagasse were put into 100 ml Erlenmeyer flask, added with 30 ml NaOH solution at concentrations of 0, 2.5, 5.0, 7.5, and 1.0 M. After mixing up to homogenize, the solutions were heated using autoclave at 121oC for 0, 15, 30, 45, and 60 minutes. The solution was filtered with filter paper and washed with 300 ml water. The residues (holocelluloses) were dried at 105oC for 24 hours. Bagasse delignification was measured according to Misson et al method [9]. Saccharification using enzyme Saccharification using enzyme was conducted according Samsuri et al [8] with modification. Three gram samples of bagasse holocellulose were poured into a 100 ml Erlenmeyer flask, added with 33,6 mL citrate buffer at a pH of 4,8, added 6,4 ml of enzymes at a concentration of 0, 5, 10, 15, and 20 FPU, and then incubated at a temperature of 50oC in shaker water bath (Polyscience) at a speed of 100 rpm for 0, 6, 12, 18, 24, 30, and 36 hours. After incubation, the filtrate was

taken to determine its reduced sugar content. The reduced sugar was analyzed according to Nelson-Somogy method [15].

The highest degree of delignification (> 96%) occurred when bagasse was submersed into 1.0 M NaOH solution and heated at 121oC for at least 15 minutes. Combination of alkali and high temperature resulted in almost completely

III. RESULTS AND DISCUSSION Materials

Pretreatment using NaOH Bagasse was submersed into 0 – 1.00 M sodium hydroxide and heated at 121oC for 0 – 60 minutes. After cooling and filtering, the residue (holocelullose) was analyzed to determined degrees of bagasse delignification. Bagasse delignification varies from 0 to 98.1% (Fig. 1). When bagasse was heated in water – 0 M NaOH - for 60 minutes, there was no delignification. It agrees to the finding of Mosier et al [20] who stated that liquid hot water itself could not remove lignin from lignocellulosic material. The delignification occurred when it is heated in NaOH solution. McIntosh and Vancov [21] stated that the use of alkali causes the degradation of ester and glycosidic side chains resulting in structural alteration of lignin, cellulose swelling, partial decrystallization of cellulose, and partial salvation of hemicelluloses. In addition to alter lignin structure and increases accessible surface area, alkali solution is able to remove lignin from lignocellulosic material [20]. The higher NaOH concentration and the higher submersion time resulted in the higher degree of delignification (Fig. 1). Alkali can remove lignin from lignocellulose material [20]. The more concentration of alkali will remove higher lignin from the materials due to intensive reactions. The longer reaction between alkali and lignocellulosic material will also result in the higher lignin removal from the materials. Increasing NaOH concentration significantly improved lignin degradation capacity when temperature and residence time were also increased and combined [22].

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Before use, bagasse was analyzed to determine its cellulose, hemicelluloses, and lignin contents. The analysis was performed according to Chesson Methode [18]. Cellulose, hemicelluloses, and lignin contents of the bagasse were 46.3%, 20.7%, and 21.9%, respectively. These results were relatively the same as other researchers’ finding. Sutikno et al [7] reported that bagasse contained 51.12% cellulose, 20.82% hemicelluloses, and 18.84% lignin. Septiani [12} stated that bagasse composted of 45.96% cellulose, 20.37% hemicelluloses, and 21.56% lignin. Canilha et al [17] found that cellulose, hemicelluloses, and lignin contents of bagasse were 45.0%, 25.8%, and 19.%, respectively. Chemical composition of lignocellulosic materials depends on the variety, location, and agricultural practices used to grow the crops [18] as well as on the methods employed for the lignocellulosic analysis [19].

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Fig. 1. Delignification of bagasse after submerging into 0 – 1,0 M NaOH solution at 121oC for 60 minutes. lignin removal. Other researchers performed different treatment combinations and found different degree of delignification, too. Submerging wheat straw into 0.375 M NaOH solution at 20oC for 144 hours yielded 60% delignification [23]. Misson and coworkers [9] submerged 5 g oil palm empty fruit bunch (EFB) into 0.1M NaOH solution and stirred at 90 rpm at 27oC for 48 hours; they found that degree of delignification was 65%. The degree of delignification increased up to 99% when they added with 0.1M H2O2 solution after 24 hours reaction and let the reaction at the same condition (stirred at 90 rpm at 27oC) for other 24 hours. Thus, degree of delignification depends on the treatment combinations given to the lignocellulosic materials. Saccharification using enzyme Holocelluloses resulting from the best treatment (submersion into 1.0 M NaOH solution and heated at 121oC for at least 15 minutes) were saccharified with cellulase at different enzyme concentrations and incubation times. The saccharification was carried out at a temperature of 50oC and at a shaking speed of 100 rpm for 60 hours. After saccharification, the substrate was taken and analyzed for measuring its reduced sugar content. Results of the sugar contents were presented in Fig. 2.

As can be seen at Fig. 2, the higher cellulase concentration yielded the higher reduce sugar concentration, too. Without enzyme (0 FPU), bagasse reduced sugar was very low (