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Effect of Temperature and Fermentation Time of Crude Cellulase Production by Trichoderma Reesei on Straw Substrate
Available online at www.sciencedirect.com
ScienceDirect Energy Procedia 65 (2015) 368 – 371
Conference and Exhibition Indonesia - New, Renewable Energy and Energy Conservation (The 3rd Indo-EBTKE ConEx 2014)
Effect of Temperature and Fermentation Time of Crude Cellulase Production by Trichoderma reesei on Straw Substrate Vita T Rosyidaa*, A Wheni Indrianingsiha , R Maryanaa, Satriyo K Wahonoa a
Technical Implementation Unit for Development of Chemical Engineering Processes- Indonesian Institute of Sciences, Jl. Jogja-Wonosari Km. 32 Gading, Playen, Gunungkidul, Yogyakarta 55861, Indonesia
Abstract Research was conducted to determine the effect of temperature and fermentation time of crude cellulase production by Trichoderma reesei on straw substrate. The research was arranged in a randomized complete factorial design consisting of two factors. The first factor is the temperature of fermentation consists of two levels, 27 oC and 37 oC. The second factor is the time fermentation consists of three levels, namely 5 d, 7 d, and 9 d. The results showed that the temperature and fermentation period significantly affect the observed parameters on crude cellulase production by Trichoderma reesei. The optimal treatment combination to produce crude cellulase with maximum activity is at 27 oC and fermentation time of 9 d with an average value of cellulase activity (filter paperase) and soluble protein respectively 0.041 193 7 unitgmL̢1 and 0.021 613 mggmL̢1.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 V.T. Rosyida, A.W. Indrianingsih, R. Maryana, S.K. Wahono. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014. Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 Keywords: Crude cellulase; fermentation; straw; Trichoderma reesei
* Corresponding author. Tel.: +62 085 228 583 165; fax: +62 274 391 168 E-mail address: [email protected]; [email protected]
1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 doi:10.1016/j.egypro.2015.01.065
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
Nomenclature d L one oil barrel min wg g v-1
day liter = 159 L (litre) minute weight per volume
1. Introduction Today the scarcity of fuel oil sourced from fossil is increasing. This is because fossil fuel is a resource that is not renewable, also coupled with the high demand for the fossil fuel of which is the energy source for many manufacturers in public life. The high use of fuel derived from fossils in Indonesia is still very high, causing Indonesia still relies heavily on fossil fuels. According to Pertamina’s data, national fuel consumption during 2000 to 2011 was about 312 to 364 m oil barrel equivalent and increased every year. Most of the fuel consumption is used for the transportation sector. This is exacerbated by an increase in the number of vehicles from year to year. The fossil fuels produce more pollution, so it is not environmentally friendly [1]. Increase of the energy needs must be balanced with long-term sustainable supply of renewable energy [2]. Also, the increasing levels of environmental pollution that comes from burning fossil fuels cause renewable fuel that environmentally friendly are urgently needed. One of the renewable energy is energy that derived from biomass. Biomass energy sources derived from plants, agricultural and forestry waste [3] or from farm waste and domestic waste [4]. One kind of the utilization of biomass energy is the conversion of biomass [5]. The biomass conversion results could be biogas, bioethanol or charcoal, etc. There is also biofuel resulted from biomass conversion results [6]. Bioethanol also be a new potential options for use in industrial waste and agricultural waste and biomass energy are successfully converted [7,8]. Bioethanol itself is a result of the conversion of renewable biomass, in addition to the production that involves a lot of agricultural waste and industrial waste [9]. Production of bioethanol and other biomass-based energy also utilize plant cell wall as a biomass source [5]. The human ability to convert [10] is very dependent on the complexity of the existing system of one enzyme cellulase enzyme class [3]. Cellulase enzyme production took a total cost of 40 % from the bioethanol synthesis process [11]. To reduce the cost of production, one of the solutions is to use an enzyme [9]. The crude enzyme can be obtained from microorganisme [3] one of which is a type of filamentous fungi of the genus Trichoderma [12,3]. Fungi of the genus Trichoderma are known to degrade plant cell walls with cellulase enzymes [13]. Cellulase enzyme itself is an inducible-type enzyme to be produced by microorganisms during the process of development of the cellulosic material in the living microorganisms, including Trichoderma to hydrolyze lignocellulosic [13,11] Based on the description above, cellulase has an important role in the bioconversion of cellulose to glucose as material for ethanol production, so it requires optimization for the production of raw cellulases from fungus Trichoderma reeesei. Treatment was conducted by fermentation process for 5 d, 7 d, and 9 d, at temperature 27 °C and 37 °C which is expected to generate an optimal raw cellulase enzyme that can be used to convert the lignocelluloses materials to glucose. This study was conducted to determine the optimum combination of fermentation time and temperature for producing raw cellulase enzymes with high activity of fungus Trichoderma reesei. 2. Material and method 2.1. Strains, culture media and chemicals Microbial fungi strain which was used is Trichoderma reesei FNCC 6012. Strain was obtained from Microbiology Laboratorium of PAU, Food and Nutrition Division, Gadjah Mada University. Medium for maintenance and rejuvenation of culture medium was Potato Dextrose Agar (PDA). The substrate is dried straw. Chemicals used were: (NH4) 2SO4 (Merck), KH2PO4 (Merck), Urea, CaCl2, MgSO4.7H2O, FeSO4.7H2O, MnSO4, ZnSO4.7H2O, Peptone (Merck), Bovin Serum Albumin (BSA) (Merck), H2SO4 (Merck), NaH2PO4 (Merck), MgCl2 (Merck), Dinitrosalicylic Acid (DNS), citrate buffer, HCl and distilled water. Substrate in the form of straw was weighed and dried under sunlight till the water content around 10 %. The straw was cut into small pieces with a size of 1 cm. 2.2. The design of experiments The research experimental design was arranged in a completely randomized factorial design consisting of two factors. The first factor was the temperature of fermentation storage which was 27 oC and 37 oC. The second factor was the length of fermentation consists of three levels, namely 5 d, 7 d, and 9 d. Data obtained from each treatment were analyzed by analysis of variance, if the treatment significantly affected the observed parameters then followed by Duncan's test.
369
370
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
2.3. Preparation of Trichoderma reseei culture The culture was prepared by inoculating fungi that had been rejuvenated (from the stock culture) into the agar plate medium (PDA) which has been prepared in advance. Pure culture of Trichoderma reesei spores was grown in the agar plate media (1 loop). The pure cultures were incubated at 25 oC to 27 oC for 7 d. 2.4. Production and testing of cellulase enzyme activity Trichoderma reesei pure culture was grown on agar plate media and then incubated for 7 d. Sterile distilled water (10 mL) was added to the Trichoderma reesei cultures in agar plate, then it was shaked so that the spores released into the liquid phase. Two grams of dry straw substrate was included in the 50 mL erlenmeyer. The mineral nutrient solution contained of 1.4 g . L̽ 1 (NH4)2.SO4; 2 g . L̢1 KH2PO; 0.3 g . L̢1 urea; 0.3 g . L̢1 CaCl2; 0.6 g . L̢1 MgSO4.7H2O; 0.009 g . L̢1 FeSO4.7H2O; ̢ ̢ ̢ 1.4 g . L 1 MnSO4; 0.001 4 g . L 1 ZnSO4.7H2O; 0.75 g . L 1 Pepton. Medium that already contain nutrient and mineral solution was added to 50 mL of distilled water, and then the pH was arranged to pH 4. It was covered with cotton, sterilized at 121 oC for 15 min in an autoclave. Spore suspension of Trichoderma reesei were added to the fermentation medium at concentration of 10 % (w.v̢1) then fermented [14]. Harvesting was conducted at the end of fermentation at 5 d, 7 d and 9 d. The results of fermentation in erlenmeyer was stirred and shaken and filtered with a filter paper. The filtrate was centrifuged at room temperature and extracted supernatant (rough cellulose) were ready to be analyzed. In this study the observed parameters included: evaluation of cellulase activity (filter paperase) [15] and soluble protein analysis by Lowry method [16]. 3. Results and discussion 3.1. Cellulase activity (filter paperase) Testing of filter paperase activity can reflect the general cellulase activity because the substrate used for testing was Whatman filter paper no. 1 (crystal fibers properties) that involves the activity of C1 which acts as an activator of crystalline cellulose into reactive cellulose [17]. Based on the analysis of variance, it showed that the interaction of treatment and the concentration of bagasse fermentation time was highly significant (p < 0.01) on cellulase activity. The average value of cellulase activity can be seen in Table 1. Table 1. The average value of cellulase activity (filter paperase) (units . mL-1 filtrate) Fermentation time Temperature of incubation (OC) (d) 27 5 0.035 6 b 7 0.041 4 a 9 0.041 1 a Note: the same letters on the average value showed no significant difference (p > 0.05)
37 0.036 9 b 0.042 1 a 0.032 7 c
3.2. Soluble protein Based on the analysis of variance, it showed that the interaction of substrate concentration and fermentation time was significantly affect (p < 0.05) to the soluble protein content. The average value of soluble protein can be seen in Table 2. Table 2. The average value of soluble protein content (mg protein/filtrate) (mg . mL-1 filtrate) Fermentation time Temperature of incubation (OC) (d) 27 5 0.009 67 d 7 0.011 55 d 9 0.021 61 a Note: the same letters on the average value showed no significant difference (P > 0.05)
37 0.011 82 d 0.015 33 c 0.018 33 b
Tables 1 and Table 2 showed that the value of cellulase activity and the average value of the highest soluble protein was 0.041 1 unitg mL-1 and 0.021 61 mggmL-1, which was obtained with 9 d of fermenation at temperature of 27 oC. This is due to the fact that the longer the fermentation time, the higher substrate hydrolysis, thus the protein levels also increased so that cellulase production was also increasing. The lowest cellulase activity was 0.032 7 unitg mL-1 which was obtained from 9 d of fermentation at temperature of 37 oC. The lowest average value of soluble protein was 0.009 67 mggmL-1 with 5 d of fermentation at temperature 27 oC. 4. Conclusion The results showed that room temperature storage and fermentation time significantly affect the observed parameters on cellulase production by raw Trichoderma reesei. The best combination treatment to produce raw cellulase enzyme with optimal
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
371
activity was at 27 oC of storage temperature and 9 d of fermentation time with an average value of cellulase activity (filter paperase) and soluble protein were 0.041 1 unitsg mL-1 and 0.021 61 mggmL̽1, respectively.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
Sugiyono A. Pemanfaatan biofuel dalam penyediaan energi nasional jangka panjang [Utilization of biofuels in the long-term national energy supply]. Prosiding Seminar Teknologi untuk Negeri. 2005:78-86 [Bahasa Indonesia] Balat M, Balat H. Recent trends in global production and utilization of bio-ethanol fuel. Applied Energy Journal 68. 2009: 2273-2282 Wackett LP. Biomass to fules via microbial transformation. Current opinion in Chemical Biology Journal 12. 2008: 187-193 Abbasi T, Abbasi SA. Biomass energy and the environmental impacts associated with its production and utilization. Renewable and Sustainable Energy Reviews Journal 14. 2010: 919-937 Paola DC, Isabella DB, Patrizia R. 2011. Latest frontiers in the biotechnologies for etanol production fromlignocellulosic biomass, In: Aurelio M, Bernades S editors. Biofuel Production-Recent Develpoments and Prospects. Intech. Croatia. 2010: 163-165 Fortman JL, Chhabra S, Mukhopadhyay A, et al. Biofuel alternatives to ethanol: pumping the microbial wall. Trends in Biotechnology 26(7). 2008 : 375381 Aimaretti NR, Ybalo CV, Rojas ML, Plou FJ, Yori JC. Production of bioethanol from carrot discards. Biosource Technology Journal 123. 2012: 727-732 Leathers TD. Enzymatic saccharification of defatted corn germ. Biotechnology Letters Journal 26. 2004: 203-207 Siwarasak P, Pajantagate P, Prasertlertrat K. Use of Trichoderma reesei RT-P1 crude enzyme powder for ethanol fermentation of sweet sorghum fresh stalks. Biosource Technology Journal 107. 2012: 200-204 Singhania RR, Pate AM, Sukumaran RK, Larrochee C, Pandey A. Role and significance of beta-glucosidases in the hydrolysis ofcellulose for bioethanol production. Biosoure Technology Journal 127. 2013: 500-507 Gutierrez-Correa M, Portal L, Moreno P, Tengerdy RP. Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane baggase. Biosource Technology Journal 68. 1999: 173-178 Seiboth B, Ivanova C, Seidl-Seiboth V. Trichoderma reesei: A fungal enzyme producer for cellulosic biofuels, In: Aurelio M, Bernades S editors. Biofuel Production-Recent Develpoments and Prospects. Intech. Croatia. 2010: 309-315 Ilmen M, Saloheimo A, Onnela M, Penttila ME. Regulation of celluse gene expression in the filamentous fungus Trichoderma reeei. Applied and Environmental Microbiology Journal 63(4). 2007: 1298-1306 Hardjo S, Indrasti NS, Bantacut T. Biokonversi Pemanfaatan Limbah Industri Pertanian [Bioconvertion of agricultural industrial waste utilization]. PAU Pangan dan Gizi. IPB Bogor. 1989 [Bahasa Indonesia] Mandels M, Andreotii, Roche C. Measurement of saccharifying cellulase. Biotechnology Bioengineering Symposium 6. 1976: 21-31 Lowry OH, Roser AF, Randall R.Protein measurement with folin phenol reagent. Biology Chemistry Journal 242.1951: 265-275 Darwis AA, Sukara E. Isolasi, Purifikasi dan Karakteristik Enzim [Isolation, purification and characteristics of enzymes]. PAU Bioteknologi IPB Bogor. 1990 [Bahasa Indonesia]
ScienceDirect Energy Procedia 65 (2015) 368 – 371
Conference and Exhibition Indonesia - New, Renewable Energy and Energy Conservation (The 3rd Indo-EBTKE ConEx 2014)
Effect of Temperature and Fermentation Time of Crude Cellulase Production by Trichoderma reesei on Straw Substrate Vita T Rosyidaa*, A Wheni Indrianingsiha , R Maryanaa, Satriyo K Wahonoa a
Technical Implementation Unit for Development of Chemical Engineering Processes- Indonesian Institute of Sciences, Jl. Jogja-Wonosari Km. 32 Gading, Playen, Gunungkidul, Yogyakarta 55861, Indonesia
Abstract Research was conducted to determine the effect of temperature and fermentation time of crude cellulase production by Trichoderma reesei on straw substrate. The research was arranged in a randomized complete factorial design consisting of two factors. The first factor is the temperature of fermentation consists of two levels, 27 oC and 37 oC. The second factor is the time fermentation consists of three levels, namely 5 d, 7 d, and 9 d. The results showed that the temperature and fermentation period significantly affect the observed parameters on crude cellulase production by Trichoderma reesei. The optimal treatment combination to produce crude cellulase with maximum activity is at 27 oC and fermentation time of 9 d with an average value of cellulase activity (filter paperase) and soluble protein respectively 0.041 193 7 unitgmL̢1 and 0.021 613 mggmL̢1.
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license © 2015 V.T. Rosyida, A.W. Indrianingsih, R. Maryana, S.K. Wahono. Published by Elsevier Ltd. (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014. Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 Keywords: Crude cellulase; fermentation; straw; Trichoderma reesei
* Corresponding author. Tel.: +62 085 228 583 165; fax: +62 274 391 168 E-mail address: [email protected]; [email protected]
1876-6102 © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Peer-review under responsibility of the Scientific Committee of EBTKE ConEx 2014 doi:10.1016/j.egypro.2015.01.065
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
Nomenclature d L one oil barrel min wg g v-1
day liter = 159 L (litre) minute weight per volume
1. Introduction Today the scarcity of fuel oil sourced from fossil is increasing. This is because fossil fuel is a resource that is not renewable, also coupled with the high demand for the fossil fuel of which is the energy source for many manufacturers in public life. The high use of fuel derived from fossils in Indonesia is still very high, causing Indonesia still relies heavily on fossil fuels. According to Pertamina’s data, national fuel consumption during 2000 to 2011 was about 312 to 364 m oil barrel equivalent and increased every year. Most of the fuel consumption is used for the transportation sector. This is exacerbated by an increase in the number of vehicles from year to year. The fossil fuels produce more pollution, so it is not environmentally friendly [1]. Increase of the energy needs must be balanced with long-term sustainable supply of renewable energy [2]. Also, the increasing levels of environmental pollution that comes from burning fossil fuels cause renewable fuel that environmentally friendly are urgently needed. One of the renewable energy is energy that derived from biomass. Biomass energy sources derived from plants, agricultural and forestry waste [3] or from farm waste and domestic waste [4]. One kind of the utilization of biomass energy is the conversion of biomass [5]. The biomass conversion results could be biogas, bioethanol or charcoal, etc. There is also biofuel resulted from biomass conversion results [6]. Bioethanol also be a new potential options for use in industrial waste and agricultural waste and biomass energy are successfully converted [7,8]. Bioethanol itself is a result of the conversion of renewable biomass, in addition to the production that involves a lot of agricultural waste and industrial waste [9]. Production of bioethanol and other biomass-based energy also utilize plant cell wall as a biomass source [5]. The human ability to convert [10] is very dependent on the complexity of the existing system of one enzyme cellulase enzyme class [3]. Cellulase enzyme production took a total cost of 40 % from the bioethanol synthesis process [11]. To reduce the cost of production, one of the solutions is to use an enzyme [9]. The crude enzyme can be obtained from microorganisme [3] one of which is a type of filamentous fungi of the genus Trichoderma [12,3]. Fungi of the genus Trichoderma are known to degrade plant cell walls with cellulase enzymes [13]. Cellulase enzyme itself is an inducible-type enzyme to be produced by microorganisms during the process of development of the cellulosic material in the living microorganisms, including Trichoderma to hydrolyze lignocellulosic [13,11] Based on the description above, cellulase has an important role in the bioconversion of cellulose to glucose as material for ethanol production, so it requires optimization for the production of raw cellulases from fungus Trichoderma reeesei. Treatment was conducted by fermentation process for 5 d, 7 d, and 9 d, at temperature 27 °C and 37 °C which is expected to generate an optimal raw cellulase enzyme that can be used to convert the lignocelluloses materials to glucose. This study was conducted to determine the optimum combination of fermentation time and temperature for producing raw cellulase enzymes with high activity of fungus Trichoderma reesei. 2. Material and method 2.1. Strains, culture media and chemicals Microbial fungi strain which was used is Trichoderma reesei FNCC 6012. Strain was obtained from Microbiology Laboratorium of PAU, Food and Nutrition Division, Gadjah Mada University. Medium for maintenance and rejuvenation of culture medium was Potato Dextrose Agar (PDA). The substrate is dried straw. Chemicals used were: (NH4) 2SO4 (Merck), KH2PO4 (Merck), Urea, CaCl2, MgSO4.7H2O, FeSO4.7H2O, MnSO4, ZnSO4.7H2O, Peptone (Merck), Bovin Serum Albumin (BSA) (Merck), H2SO4 (Merck), NaH2PO4 (Merck), MgCl2 (Merck), Dinitrosalicylic Acid (DNS), citrate buffer, HCl and distilled water. Substrate in the form of straw was weighed and dried under sunlight till the water content around 10 %. The straw was cut into small pieces with a size of 1 cm. 2.2. The design of experiments The research experimental design was arranged in a completely randomized factorial design consisting of two factors. The first factor was the temperature of fermentation storage which was 27 oC and 37 oC. The second factor was the length of fermentation consists of three levels, namely 5 d, 7 d, and 9 d. Data obtained from each treatment were analyzed by analysis of variance, if the treatment significantly affected the observed parameters then followed by Duncan's test.
369
370
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
2.3. Preparation of Trichoderma reseei culture The culture was prepared by inoculating fungi that had been rejuvenated (from the stock culture) into the agar plate medium (PDA) which has been prepared in advance. Pure culture of Trichoderma reesei spores was grown in the agar plate media (1 loop). The pure cultures were incubated at 25 oC to 27 oC for 7 d. 2.4. Production and testing of cellulase enzyme activity Trichoderma reesei pure culture was grown on agar plate media and then incubated for 7 d. Sterile distilled water (10 mL) was added to the Trichoderma reesei cultures in agar plate, then it was shaked so that the spores released into the liquid phase. Two grams of dry straw substrate was included in the 50 mL erlenmeyer. The mineral nutrient solution contained of 1.4 g . L̽ 1 (NH4)2.SO4; 2 g . L̢1 KH2PO; 0.3 g . L̢1 urea; 0.3 g . L̢1 CaCl2; 0.6 g . L̢1 MgSO4.7H2O; 0.009 g . L̢1 FeSO4.7H2O; ̢ ̢ ̢ 1.4 g . L 1 MnSO4; 0.001 4 g . L 1 ZnSO4.7H2O; 0.75 g . L 1 Pepton. Medium that already contain nutrient and mineral solution was added to 50 mL of distilled water, and then the pH was arranged to pH 4. It was covered with cotton, sterilized at 121 oC for 15 min in an autoclave. Spore suspension of Trichoderma reesei were added to the fermentation medium at concentration of 10 % (w.v̢1) then fermented [14]. Harvesting was conducted at the end of fermentation at 5 d, 7 d and 9 d. The results of fermentation in erlenmeyer was stirred and shaken and filtered with a filter paper. The filtrate was centrifuged at room temperature and extracted supernatant (rough cellulose) were ready to be analyzed. In this study the observed parameters included: evaluation of cellulase activity (filter paperase) [15] and soluble protein analysis by Lowry method [16]. 3. Results and discussion 3.1. Cellulase activity (filter paperase) Testing of filter paperase activity can reflect the general cellulase activity because the substrate used for testing was Whatman filter paper no. 1 (crystal fibers properties) that involves the activity of C1 which acts as an activator of crystalline cellulose into reactive cellulose [17]. Based on the analysis of variance, it showed that the interaction of treatment and the concentration of bagasse fermentation time was highly significant (p < 0.01) on cellulase activity. The average value of cellulase activity can be seen in Table 1. Table 1. The average value of cellulase activity (filter paperase) (units . mL-1 filtrate) Fermentation time Temperature of incubation (OC) (d) 27 5 0.035 6 b 7 0.041 4 a 9 0.041 1 a Note: the same letters on the average value showed no significant difference (p > 0.05)
37 0.036 9 b 0.042 1 a 0.032 7 c
3.2. Soluble protein Based on the analysis of variance, it showed that the interaction of substrate concentration and fermentation time was significantly affect (p < 0.05) to the soluble protein content. The average value of soluble protein can be seen in Table 2. Table 2. The average value of soluble protein content (mg protein/filtrate) (mg . mL-1 filtrate) Fermentation time Temperature of incubation (OC) (d) 27 5 0.009 67 d 7 0.011 55 d 9 0.021 61 a Note: the same letters on the average value showed no significant difference (P > 0.05)
37 0.011 82 d 0.015 33 c 0.018 33 b
Tables 1 and Table 2 showed that the value of cellulase activity and the average value of the highest soluble protein was 0.041 1 unitg mL-1 and 0.021 61 mggmL-1, which was obtained with 9 d of fermenation at temperature of 27 oC. This is due to the fact that the longer the fermentation time, the higher substrate hydrolysis, thus the protein levels also increased so that cellulase production was also increasing. The lowest cellulase activity was 0.032 7 unitg mL-1 which was obtained from 9 d of fermentation at temperature of 37 oC. The lowest average value of soluble protein was 0.009 67 mggmL-1 with 5 d of fermentation at temperature 27 oC. 4. Conclusion The results showed that room temperature storage and fermentation time significantly affect the observed parameters on cellulase production by raw Trichoderma reesei. The best combination treatment to produce raw cellulase enzyme with optimal
Vita T. Rosyida et al. / Energy Procedia 65 (2015) 368 – 371
371
activity was at 27 oC of storage temperature and 9 d of fermentation time with an average value of cellulase activity (filter paperase) and soluble protein were 0.041 1 unitsg mL-1 and 0.021 61 mggmL̽1, respectively.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
Sugiyono A. Pemanfaatan biofuel dalam penyediaan energi nasional jangka panjang [Utilization of biofuels in the long-term national energy supply]. Prosiding Seminar Teknologi untuk Negeri. 2005:78-86 [Bahasa Indonesia] Balat M, Balat H. Recent trends in global production and utilization of bio-ethanol fuel. Applied Energy Journal 68. 2009: 2273-2282 Wackett LP. Biomass to fules via microbial transformation. Current opinion in Chemical Biology Journal 12. 2008: 187-193 Abbasi T, Abbasi SA. Biomass energy and the environmental impacts associated with its production and utilization. Renewable and Sustainable Energy Reviews Journal 14. 2010: 919-937 Paola DC, Isabella DB, Patrizia R. 2011. Latest frontiers in the biotechnologies for etanol production fromlignocellulosic biomass, In: Aurelio M, Bernades S editors. Biofuel Production-Recent Develpoments and Prospects. Intech. Croatia. 2010: 163-165 Fortman JL, Chhabra S, Mukhopadhyay A, et al. Biofuel alternatives to ethanol: pumping the microbial wall. Trends in Biotechnology 26(7). 2008 : 375381 Aimaretti NR, Ybalo CV, Rojas ML, Plou FJ, Yori JC. Production of bioethanol from carrot discards. Biosource Technology Journal 123. 2012: 727-732 Leathers TD. Enzymatic saccharification of defatted corn germ. Biotechnology Letters Journal 26. 2004: 203-207 Siwarasak P, Pajantagate P, Prasertlertrat K. Use of Trichoderma reesei RT-P1 crude enzyme powder for ethanol fermentation of sweet sorghum fresh stalks. Biosource Technology Journal 107. 2012: 200-204 Singhania RR, Pate AM, Sukumaran RK, Larrochee C, Pandey A. Role and significance of beta-glucosidases in the hydrolysis ofcellulose for bioethanol production. Biosoure Technology Journal 127. 2013: 500-507 Gutierrez-Correa M, Portal L, Moreno P, Tengerdy RP. Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane baggase. Biosource Technology Journal 68. 1999: 173-178 Seiboth B, Ivanova C, Seidl-Seiboth V. Trichoderma reesei: A fungal enzyme producer for cellulosic biofuels, In: Aurelio M, Bernades S editors. Biofuel Production-Recent Develpoments and Prospects. Intech. Croatia. 2010: 309-315 Ilmen M, Saloheimo A, Onnela M, Penttila ME. Regulation of celluse gene expression in the filamentous fungus Trichoderma reeei. Applied and Environmental Microbiology Journal 63(4). 2007: 1298-1306 Hardjo S, Indrasti NS, Bantacut T. Biokonversi Pemanfaatan Limbah Industri Pertanian [Bioconvertion of agricultural industrial waste utilization]. PAU Pangan dan Gizi. IPB Bogor. 1989 [Bahasa Indonesia] Mandels M, Andreotii, Roche C. Measurement of saccharifying cellulase. Biotechnology Bioengineering Symposium 6. 1976: 21-31 Lowry OH, Roser AF, Randall R.Protein measurement with folin phenol reagent. Biology Chemistry Journal 242.1951: 265-275 Darwis AA, Sukara E. Isolasi, Purifikasi dan Karakteristik Enzim [Isolation, purification and characteristics of enzymes]. PAU Bioteknologi IPB Bogor. 1990 [Bahasa Indonesia]