Abstract. Methodology

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1 ISBN Proceedings of The 9 th Joint Conference on Chemistry Hydrolysis Enzyme Production α-amylase and β-glucosidase from Aspergillus niger with Solid State Fermentation Method on Rice Husk, Bagasse and Corn Cob Substrate Heri Hermansyah a, Rizky Ramadhani, Adinda Putri Wisman, Rita Arbianti Abstract Hydrolysis enzyme such as α-amylase and β-glucosidase can be produced from fungi Aspergillus niger and using solid state fermentation method. This research studies fermentation process from fungi A. niger using variety of substrates such as rice husk, sugarcane bagasse and corn cob. The purpose of this research is to produce hydrolysis enzymes which are α-amylase and β- glucosidase using agro-industry waste. Based on research result, Optimum time for fermentation for each substrate is 6 days or 144 hours. The highest activity unit for α-amylase is U/ml from fermentation using corn cob with 6 days fermentation. for β-glucosidase, activity unit is U/ml from fermentation using corn cob with 6 days fermentation. Liquid crude enzyme dried using spray dryer with skim milk matrix produce dry crude enzyme with enzyme retention 85-98% compared to liquid crude enzyme. Activity unit for dry α-amylase is 73,94 U/ml and for dry β-glucosidase is U/ml. This enzyme is stable for hydrolysis process at temperature C. a Bioprocess Engineering, Engineering Faculty, University of Indonesia, Depok 16424, Indonesia. Corresponding author address: kangheri@yahoo.com Introduction Indonesia is an agricultural country and in every year Indonesia can produce agro industrial waste from agriculture, forestry, farming, and industrial sectors. in 2012, Indonesia produced tons of sugar cane with its waste called sugar cane bagasse reaching tons (BPS, 2013). The biomass residues are generally not processed and used optimally and even tend to cause environmental problems. Agricultural waste is organic waste that can produce methane and if released directly into the atmosphere has an impact greater than carbon dioxide. Biomass derived from agricultural waste contain carbon sources are still quite large. Carbon content in biomass such as rice husk was 48.9% (Wannapeera & Pipatmanormai, 2008), so it can be used as substrate for enzyme production process. Hydrolysis enzyme is enzyme used for breaking glycosidic bonds in polysaccharides into simple sugars. This sugar can be utilized in production of bioethanol. Enzymatic hydrolysis can be carried out at mild conditions (temperature C and ph 5) that does not require large energy (Taherzadeh and Karimi, 2007). Hydrolysis enzyme that we use in hydrolysis reaction is α-amylase and β-glucosidase. These α-amylase and β-glucosidase enzymes are obtained from microorganisms which can produce enzymes, such as bacteria, fungi and yeast. This study is focused on the production process of hydrolysis enzyme, α-amylase and β-glucosidase, using different kinds of biomass substrate and comparing the enzyme activity. Microorganisms producing enzyme that we use is from fungi Aspergillus niger. A. niger, which is capable to produce both α-amylase and β-glucosidase enzymes. in this study, agricultural biomass that we used is sugarcane bagasse, corn cobs and rice husk. Agricultural waste is not only used as a substrate fermentation but can be used as support for A. niger to grow. Enzyme activity was analysed using Miller (1959) method which is using DNS reagent. The result will be compared to determine the most optimal method that we can use for scale up enzyme production. Methodology Materials Research The materials used in the study are PDA (Potato Dextrose Agar), (NH 4) 2SO 4, KH 2PO 4, K 2HPO 4, MgSO 4.7H 2O, lactose, maltose, phosphate buffer, DNS (dinitrosalicylic acid), starch and distilled water. The substrate used were corn cobs, rice husks, and bagasse obtained from local farms in Central Java, Indonesia. The microorganism used was Aspergillus nigerobtained from Indonesian Institute of Sciences (LIPI). Preparation of fermentation substrates 402 P a g e Green Chemistry Section 5: Biochemistry, Heri Hermansyah, et al.

2 Proceedings of The 9 th Joint Conference on Chemistry ISBN Substrates are cut up into 1 cm size and then dried using an oven at 70 C for 24 hours. Bagasse needs to be heated in autoclave at 121 C for 15 minutes to remove lignin compounds which are still contained. Each fermentation needs 30 grams of substrate and are mixed with nutrients such as 0.6 g (NH 4) 2SO 4; 018 g KH 2PO 4; 0.18 g K 2HPO 4; 0,03g MgSO 4.7H 2O, maltose and lactose 1 g and 60 ml H 2O. The substrate is then sterilized for 15 minutes at temperature of 121 C in autoclave. Fermentation Fermentation was done by SSF method using substrate from corn cobs, rice husks and bagasse, respectively 30 g each fermenter. The ratio of nutrients and substrate addition was 1:15 in a 250 ml erlenmeyer. Inoculum solution was transferred in the medium 3% (v/v). Incubation is done for 72, 96, 120 and 144 hours at 30 C and ph conditions 7.0. Enzymes Extraction Enzyme extraction was done by adding 0.1 M phosphate buffer ph 7.0 with a ratio of 1:2 (w/v). The solution was mixed and shaken for 30 min and then filtered using muslin cloth. The extract is then centrifuged at 8000 rpm for 20 minutes. Analysis of Enzyme Activity freeze drying method used casein 0.05% (w/v) as matrix and for spray dryer method skim milk 12% (w/v) was added as matrix. Operation condition of spray drying was inlet temperature at 130 C and outlet temperature C with flow rate of hot gas was 1.45 m 3 /min solution. Results and Discussion Effect of Fermentation Time on Enzyme Production Fermentation is done by SSF method with same amount of substrate and operating condition in room temperature in various fermentation time which are 72, 96, 120 and 144 hours. Based on obtained data the highest enzyme activity of α-amylase is on the sixth day or at 144 hours for every substrate we used. in fermentation using rice husk substrate, enzyme activity of α-amylase at 144 hours is U/ml. for fermentation using corn cob, enzyme activity of α- amylase at 144 hours is U/ml. for fermentation using substrate bagasse, enzyme activity at 144 hours is U/ml. Figure 1 shows the effect of fermentation time on enzyme produced based on enzyme activity. Figure 1 show that enzyme activity increases the longer the fermentation time is, and even until 144 hours we have not seen enzyme activity decreasing. Analysis of enzyme activity were done by calculating the amount of glucose decreased from a starch solution by using the DNS reagent (3,5-dinitrosalicylic acid) (Miller, 1959). One enzyme unit (U) means the amount of enzyme required to produce 1 μmol of glucose per minute. Description: ΔE = Absorbance at 540 nm Vf = final volume including DNS solution (ml) Vs = volume of enzyme used (ml) Δt = Time of hydrolysis Σ = extinction coefficient d = diameter of cuvette (1 cm) Testing is done by adding 0.5 ml of enzyme sample and 0.5 ml starch solution 1%, then incubating it for 5 min at 30 C. The addition of 1 ml of DNS reagent is to stop hydrolysis reaction. The solution is then heated at 90 C for 5 minutes to react DNS reagent and the glucose. Absorbance is measured using spectrophotometric UV-Vis with wavelength 540 nm. Drying Drying was used in this research to keep the stability of enzyme. There is two method of drying that we used, freeze drying performed in BPPT Serpong and spray drying performed in LIPI Cibinong. Enzyme drying by Figure 4. Enzyme activity of amylase in various fermentation time Based on obtained data the highest enzyme activity of β-glucosidase is on the sixth day or at 144 hours for every substrate we used. in fermentation using rice husk substrate, enzyme activity of β-glucosidase at 144 hours is U/ml. for fermentation using corn cob, enzyme activity of β-glucosidase at 144 hours is U/ml. for fermentation using substrate bagasse, enzyme activity at 144 hours is U/ml. Figure 1 shows the effect of fermentation time on enzyme produced based on enzyme activity. Figure 2 show that enzyme activity increases the longer the fermentation time is, and even until 144 hours we have not seen enzyme activity decreasing. Green Chemistry Section 5: Biochemistry,Heri Hermansyah, et al. P a g e 403

3 ISBN Proceedings of The 9 th Joint Conference on Chemistry U/ml. Figure 4 below shows the highest enzyme activity between three kinds of substrate was enzyme produced from corn cob Figure 5. Enzyme activity of glucosidase in various fermentation time Aspergillus niger reached stationary phase between day 4 th and 5 th on medium potato dextrose agar (PDA) (Khan and Yadav, 2011). in this study, A. niger grow in agro-industry biomass, where biomass contains mixture of substances such as lignin, cellulose and hemicellulose that cannot be directly processed by this organism. Effect of Variation of Substrate Fermentation of Enzyme Production Other purpose of this study is to obtain the best substrate for enzyme production. Biomass substrate used for fermentation supply a carbon source for the microorganism producing enzyme which is Aspergillus niger. The selection of the substrate that were used in this study was based on their potential amount in Indonesia. Based on the data obtained fermentation using rice husk produce α-amylase with enzyme activity U/ml. Fermentation using corn cob produce α-amylase with enzyme activity U/ml. While bagasse produce α-amylase with enzyme activity U/ml. Figure 3 below shows the highest enzyme activity between three kinds of substrate was enzyme produced by corn cob. Figure 6. Amylase enzyme activity in various type of fermentation substrate Based on the data obtained fermentation using rice husk produce β-glucosidase with enzyme activity U/ml. Fermentation using corn cob produce β- glucosidase with enzyme activity U/ml. While bagasse produce β-glucosidase with enzyme activity Figure 7.β-Glucosidase enzyme activity in various type of fermentation substrate When viewed from amount of carbon source for each substrate, there does not seem to have much difference between them. On rice husk, amount of carbon source reached 48.9% (Wannapeera & Pipatmanormai, 2008). On bagasse, amount of carbon sources reached 45.5% (Arsène, 2013). On corncob, carbon sources reached 46.8% (Wannapeera & Pipatmanormai, 2008). Other factors which affect enzyme production in solid fermentation system is suitable substrate for microorganisms, substrate pre-treatment process, particle size, moisture content, type and size of inoculum (A. Pandey, 1999). Moist content and inoculum is fixed variable in this study. Moist content for each fermenter was 1:2 (v/v) and amount of inoculum was 3% (v/w). Rice husk substrate is unable to produce enzyme with high activity possibly because rice husk has smaller particle size than the other two types of substrates. Size of rice husk approximately was less than 0.5 cm so the inside of fermenter would be compact and cause lack of aeration which impacted on the growth of Aspergillus niger and affect the outcome of enzyme. Whereas in other types of substrates, corn cobs and bagasse, have a larger particle size so that when fermented in erlenmeyer there would have a bit space for aeration. The resulting enzyme in this study still have low enzyme activity for bothα-amylase and β-glucosidase may be due to some enzyme which were damaged. Enzyme should be stored in temperature 4 C, when enzyme was first obtained as well as when doing extraction process. Centrifugation process should be carried out at 4 C. Effect of Enzyme Drying High temperature causes lack of stability and changes in enzymes structure. Drying is one of methods that 404 P a g e Green Chemistry Section 5: Biochemistry, Heri Hermansyah, et al.

4 Proceedings of The 9 th Joint Conference on Chemistry ISBN can make enzymes able to increase the stability in different temperature, but it may cause decrease in enzyme activity or inactivation of the enzyme (Pilosof & Sanchez, 2006). Commonly used drying methods are spray drying and freeze drying. in enzyme drying process it is requiredto know the characteristics of the enzyme and parameters of drying process so it would not reduce enzyme activity. Another thing to consider in drying enzyme is the retention of enzyme activity. Retention of enzyme activity should be close to 100% after drying so the lifetime of the dry enzyme can be longer (Pilosof & Sanchez, 2006). Retention enzymes are enzymes that are still active after drying process. Retention of the enzyme can be calculated by comparing enzyme activity before and after drying process. occurs at mild operating conditions, low temperature up to 100 C, normal pressure, ph 6-8 (Kolusheva & Marinova, 2007). This study is to determine whether the resulting enzyme for both enzymes α-amylase and β-glucosidase have stability in hydrolysis reaction with various temperature. Hydrolysis temperature variations are at 20 C, 30 C, 50 C and 70 C. In obtained data shows that α-amylase has the highest activity when hydrolysis is done at temperature 30 C and have lowest activity at temperature 20 C and 70 C. Based on these data, α-amylase produced can be optimally used for hydrolysis reaction at temperature C and the enzyme α-amylase has no resistance at low temperature ( 20 C) and high temperature ( 70 C). In drying process by spray drying, inlet and outlet temperatures should be controlled in optimal condition so that they can still have high retention of enzyme activity. Moreover outlet temperature higher than the temperature for stable enzyme results in denaturation of the enzyme (Yoshii, 2008). Freeze drying method produces dry enzyme with approximately 0.03 g/ml of liquid extract. Retention of enzyme activity based on Table 1 ranges from 85% to 98%. Table 2 Retention of dry enzyme activity with freeze drying method Type of Enzyme Retention (%) Amylase Glucosidase Rice Husk 84.8 Corn Cob 87.9 Bagasse 98.1 Rice Husk 86.5 Corn Cob 95.0 Bagasse 89.8 Figure 8. Enzyme amylase stability in hydrolysis temperature variation Enzyme β-glucosidase has similar results as α-amylase. Optimal temperature used for hydrolysis reaction is at temperature C and enzyme β-glucosidase has no resistance at low temperature ( 20 C) and high temperature ( 70 C). Results of enzyme drying by spray drying method produces approximately 0.05 g/ml of liquid extract. Retention of enzyme activity ranges approximately 90 to 96%. Table 3. Retention of enzyme activity by spray drying method Type of Enzyme Retention (%) Amylase Bagasse Glycosidase Bagasse Enzyme Stability at Various Temperatures Hydrolysis reaction is used for various types of industries such as for sugar industry, textile, food and other industries. Enzymatic hydrolysis process usually Figure 9. Enzyme glucosidase stability in hydrolysis temperature variation Enzymes Production Scale Up Enzymes that have been made in lab scale is then tested to produce up 100 grams of dried enzymes. The general method is the same as lab scale production. The operation condition and substrate used are based on the results from previous lab scale production. 1. Fermentation Process Green Chemistry Section 5: Biochemistry,Heri Hermansyah, et al. P a g e 405

5 ISBN Solid state fermentation (SSF) is used in enzyme production because of low cost with result high productivity and easy downstream processes. Results of enzyme crude extract produced by using the SSF method can be used directly (Kumar, Lakshmi, & Sridevi, 2013). Based on the results of this study, most optimal fermentation substrate in enzyme production is corn cob with enzyme activity reached U/ml for enzyme α-amylase and U/ml for β- glucosidase. Substrates that have the highest value activity after corn cobs is bagasse with enzyme activity U/ml for α-amylase and U/ml for β- glucosidase. However, in scale up production process, we used bagasse as substrate because the pretreatment process is easier compared to corn cobs so it will be more efficient. Moreover enzyme activity for both enzymes produced from bagasse and corncob does not have much difference. Based on previous study, dried enzyme produced with average mass is ± 0.05 g/ml. So to produce 100 grams of dry enzyme for each enzyme ml liquid extract enzyme is required for each enzyme to produce this much, we need 600 grams substrate for each fermentation process. Enzymes α-amylase and β- glucosidase produced in scale up use tray fermenter and fermentation was done in 6 days. 2. Enzyme Extraction Process Enzyme extraction process was carried out by adding phosphate buffer with ration 1:2 (v/v). Extraction is done in mixing tank using a marine blade with diameter 10 cm for 30 minutes 2000 rpm. The use of marine blade will give axial and radial flow directly. 3. Enzyme Separation Process Separation process has two stages, the first stage is separation process to separate the enzyme and substrate fermentation done by filtration method and the second stage is separation of Aspergillus niger spores and liquid extract enzyme using centrifuge rpm for 5 minutes. 4. Drying Enzyme Extract Enzymes extract that has been obtained are then dried using spray drying method. Matrix is required for drying process to reduce the damage of enzyme during drying process. The type of matrix used for this drying process is skim milk without sugar 12% (v/v). Conclusions Based on this study it can be concluded that the fermentation time for 6 days or 144 hours still shows increase in enzyme activity for enzymes α-amylase and Proceedings of The 9 th Joint Conference on Chemistry β-glucosidase enzymes by using various types of substrate. Type of fermentation substrate with highest enzyme activity for both α-amylase and β-glucosidase is corn cob. Retention of enzyme activity is about 85-98% after drying process. This enzyme has an optimal temperature for hydrolysis process at temperature C. to produce 100 grams of dry enzyme, 600 gram of fermentation substrate is required. References Arsène, M.-A. (2013). Treatments of non-wood plant fibres used as reinforcement in composite materials. Materials Research vol 16. BPS. (2013). Produksi Padi, Jagung, dan Kedelai. Jakarta: BPS. Khan, J. A., & Yadav, S. K. (2011). Production of Alpha Amylases by Aspergillus Niger Using Cheaper Substrates Employing Solid State Fermentation. International Journal of Plant, Animal and Environmental Sciences, Khan, J. A., & Yadav, S. K. (2011). Production of Alpha Amylases by Aspergillus niger Using Cheaper Substrates Employing Solid State Fermentation.International Journal of Plant, Animal and Environment Science, 5. Kolusheva, T., & Marinova, A. (2007). A STUDY OF THE OPTIMAL CONDITIONS FOR STARCH HYDROLYSIS THROUGH THERMOSTABLE α-amylase. Journal of the University of Chemical Technology and Metallurgy, Kumar, M. S., Lakshmi, C., & Sridevi, V. (2013). Production and optimization of Glucoamylase from wheat bran by Aspergillus oryzae NCIM 1212 under Solid State Fermentation. International Journal of Application or Innovation in Engineering & Management (IJAIEM), Volume 2, Issue 10, 318. Pandey, A. (1999). Solid state fermentation for production of industrial enzymes. Current Science Vol 77, Pandey, A., Ashakumary, L., & Selvakumar, P. (1995). Copra waste-a novel substrate for solid state fermentation. Biores. Technol., 51, Pilosof, A., & Sanchez, V. (2006). Drying of Enzymes. in A. Mujumdar, Handbook of Industrial Drying, Third Edition (pp ). Singapore: CRC Press. Roses, R. P., & Guerra, N. P. (2009). Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material. World J Microbiol Biotechnol, Sa adah, Z. (2010). Produksi Enzim Selulase oleh Aspergillus niger Menggunakan Substrate Jerami 406 P a g e Green Chemistry Section 5: Biochemistry, Heri Hermansyah, et al.

6 Proceedings of The 9 th Joint Conference on Chemistry ISBN dengan Sistem Fermentasi Padat. Semarang: Diponegoro University Semarang. Saida, L., & Oberoi, H. S. (2013). Studies on Cellulase Production by Solid state Fermentation using Sweet Sorghum bagasse. Helix Vol. 1, Sandhu, S., & Maiti, T. K. (2013). Cellulase Production by Bacteria: A Review. British Microbiology Research Journal 3(3), Sharada, R. (2013). Production of Cellulase - A Riview. International Journal of Pharmaceutical, Chemical and Biological Sciences, Singh, P., & Pandey, A. (2009). Solid-State Fermentation Technology for Bioconversion of Biomass and Agricultural Residues. in P. Singh, & A. Pandey, Biotechnology for Agro-Industrial Residues Utilisation (pp ). Northern Ireland: Springer Science. Sloth, J. (2007). Formation of Enzyme Containing Particles by Spray Drying. Denmark: Novozymes Bioprocess Academy. Verardi, A., & De Bari, I. (2012). Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies and Future Perspective. in M. Lima, & A. Natalense, Bioethanol (pp ). Brazil: InTech. Wang, C., & Chen, C. (2010). High Production of β- Glucosidase by Aspergillus niger on Corncob. Appl Biochem Biotechnol. Wannapeera, J., & Pipatmanormai, S. (2008). Product yields and characteristics of rice husk, rice straw and corncob during fast pyrolysis in a drop-tube/fixed-bed reactor. Songklanakarin J. Sci. Technol. 30, Yoshii, h. (2008). Effects of protein on retention of ADH enzyme activity encapsulated in trehalose matrices by spray drying. Journal of Food Engineering 87, Zulfatus, S., & Noviana, I. S. (2010). Produksi Enzim Selulase oleh Aspergillus niger Menggunakan Substrate Jerami dengan Sistem Fermentasi Padat. Semarang: Diponegoro University. Taherzadeh, M. J., & Karimi, K. (2007). Enzyme Based Hydrolysis Processes for Bioethanol from Lignocellulasic Material: A Review. Bioresources 2(4), Green Chemistry Section 5: Biochemistry,Heri Hermansyah, et al. P a g e 407