Mathematics and Computers in Science and Industry

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1 Study of enzymatic hydrolysis and liquid glucose production by solid state fermentation from rice hull using Trichoderma Species (Study of rice hull using Trichoderma Species) Arezou Ghadi Department of Chemical Engineering, Islamic Azad University, Ayatollah Amoli Branch, Amol, Iran Azam Sinkakarmi Department of Chemical Engineering, Applied Scientific University Babol, Iran Abstract This study investigates enzymatic hydrolysis of rice hull by Trichoderma Reesei to produce liquid glucose from cellulose under solid state fermentation. Medium fortifying effects on the rate of enzyme activity on (FPA, CMCASE) were studied and the optimum condition of main culture medium and optimization method for production of glucose were investigated. Results showed that in terms of activity of filter paper and activity of specific filter paper, medium containing pure rice husk moistened with liquid basal is optimum and then medium containing pure rice husk moistened with water has the highest level of activity. Therefore if the aim is to achieve the highest glucose concentration and highest concentration of filter paper, basal liquid medium must be used in this fermentation, and if the goal is to achieve the highest degree of carboxymethyl cellulase activity, it is enough to moisten substrate medium containing shell rice with tap water and moniter solid substrate fermentation for 4 days at 30 c. Keywords Rice husk, Trichoderma species, solid substrate fermentation. I. INTRODUCTION In the last few decades, many efforts have been spent in the study of enzymes with cellulolytic activity as potential sources in obtaining energy from an abundant and renewable cellulose especially the cellulase system of the fungus Trichoderma sp. It has been known that cellulases from Trichoderma sp. can be effectively hydrolyze crystalline cellulose []. Lignocellulosic substrates, such as rice husk are the agricultural wastes and by-products of rice hulling plants. The major parts of them are cellulose polymerase, hemi cellulose and lignin which can be used to domesticate animals and birds as well as producing some of other microbial metabolites such as cellulolytic enzymes or ethanol [2-5]. In recent years, studies has been done to enhance the nutritional value of rice husk in some countries. Researches in Nigeria (7) show that the fermentation of rice husk using Trichoderma fungi for 40 days can cause a significant increase in amount of crude protein, energy and mineral content such as sodium and potassium and it can decrease the amount of crude fiber [6]. In Zaid et al., (9) report, the amount of crude protein in the fermented rice husk increased about 97% and the amount of crude fiber has reduced about 45% [7]. In fermentation of rice husk, solid state fermentation (S.S.F) and liquid state fermentation (LSF) or submerged fermentation (SMF) can be used; each of which, due to the type of method, has its own advantages and disadvantages. Solid state fermentation means control of growth of microorganism on wet solid substrate in absence of free water. This method is usually cheaper while equipment used in liquid state fermentation (L.S.F) is more expensive. Solid state fermentation (S.S.F) method does not need big space, so that operations can be done in a pan bioreactor or even in an Erlenmeyer. The greatest advantage of solid state fermentation is that this method does not need soluble substrate rather enough, just it is crucial to moisten the substrate a little, in fact we do not need to free water. The desired product is concentrated so its purification is easier. Another advantage of SSF, due to the high concentration of microorganisms and low humidity of medium, is that it significantly reduces the microbial contamination. Also, the amount of material in SSF is less than SMF and enzymes show less susceptible to catabolic inhibition or stimulation. However, the SSF method is associated with some limitations, including limited use of microorganisms capable of growth in semi-humid environment and information about the increasing scale of production in this way is negligible [8,9]. II. MATERIAL AND METHODS For this experimental research fungal strain Trichoderma reesei (PTCC 542) as lyophilized ampoule was purchased from industrial scientific research organization and transferred to physiologic serum under ISBN:

2 the sterile hood. Strains were cultured in steep medium of Malt extract Agar (MEA), and incubated at 4 C for longterm maintenance. Husk was used in this study as substrate and as carbon source. A. Prepration of substrate For preparation of substrate, husk has been incubated at 4 0 C and then grinded, the resulting rice husk particles size was less than 2 mm. B. Prepration of pre culture After elementary operation, 2.5 gr of rice husk powder was mixed with 7 cc water in the Erlenmeyer (500 ml). After preparation of solid bed, sterilization step 2 C and 5 psi in the autoclave is necessary. After that, the temperature of the contents of the flasks should be brought to room temperature. C. Fermentation of culture Spore suspension prepared from slants containing 3 strains that mentioned above with sterile distilled water, and using sterile pipet at near the flame, impregnation operation from slant to pre culture media took place and then, pre culture flasks were incubated 30 C for 5 days. D. Preparation of main culture After the flasks were placed in the autoclave and cooled at room temperature in the completely sterile conditions, impregnation from preculture to main culture took place. With sterile loop, a part of growth fungi colony on a solid bed was transferred to flask that contained main culture and was mixed and this process was repeated three or four times till 5% growth fungi colony was transferred. Then, after preparing fungi suspension with physiologic serum, amount of 0 micro liter of this suspension was put between lam and Lamel. Using optical microscope, the number of fungal spores in each square was counted and dilution factor that depends on the volume of physiological serum was applied. Number of spores was calculated. When the numbers of spores on a main culture reach to 8 05 wet weight, again cultures were incubated at at 30 C for 5-7 days. After the mentioned time finished, the culture was taken out from incubator. E. Separation of enzymatic solution For separation of raw enzymatic solution, first buffer citrate at ph= 4.8 was prepared, and then, about 5 fold of solid substrate weight, buffer citrate was added to flasks (5 2.5=25 ml). Afterward, the flasks were put in the shaker for 5 mins, until the contents are equal. Then the solution was passed from Watman filter paper No.. to assure of no presence of fungi spores, the passed solution from filter should be centrifuged at 4 0 C and 4000 rpm for 20 min. This prevents turbidity in the experiment. 94By this way, enzymatic solution that contains a lot of liquid sugar (glucose), can be separated. Indeed, during fermentation, oozed cellulose enzyme from Trichoderma fungi, hydrolyze the existed cellulose in the rice husk and convert it to glucose (lump sugar). F. Measurement of glucose and protein concentration in raw enzymatic solution For measurement of glucose concentration in raw enzymatic solution, kit of glucose oxidase (production of Man Company), which is an enzymatic method for lump sugar measurement was used. The produced glucose concentration in crud enzymatic solution (mg/dl), was calculated according to the existed direction in the kit. For the measurement of amount solute protein, Loury method has been used. Bovine Serum Albumin (BSA) solution is as a standard solution and at the end the amount of samples absorption read at 578 wave length and with using BSA standard curve, protein concentration in the unknown sample (mg/ml) was obtained. G. Measurment of enzyme activity This research is on the basis of calculation of filter paper activity and after that calculation of endogluconase activity. With respect to the references in this field, experiments related to activity measurement done and at last, the amount of activity (unit/ml) was calculated. Statistical analysis: To analyze the data and comparison the amount of produced glucose in cultural groups under different incubation times and temperatures, the statistical software SPSS and ANOVA were used and P<0.05 was considered significant H. Optimize the assay of enzyme activity For optimization of enzyme activity during 4 days at 30 c, during which Trichoderma reesei microorganism reaches its maximum growth [0], medium was considerd the effects on various substrates by adding some combination of mineral rich salts. I. Calculation of filter paper Since the sample is liquid enzyme and 0.5 ml of it is diluted with ml citrate buffer after that allowed to react for hour in the oven and then according to the glucose oxidase kit. to measure the glucose level 0 ml or 0.0 μl of it was tested so calculation of filter paper is obtained following equation: (U/ml)FPA = () J. Calculate the specific activity of filter paper Specific activity (SP.A) is the ratio of the concentration of the enzyme on protein concentration and unit is defined as units / mg prot. (SP.A) = (2) ISBN:

3 THE PROTEIN CON CENTRA TION IN THE Mathematics and Computers in Science and Industry K. Calculate the molecular activity of filter paper Molecular activity is defined as units/μmol. To calculate the molecular activity of filter paper, it is sufficient to divide the (u / ml) FP on (μ mol / ml) FP. Methods of medium optimization 6 medium were numbered respectively and operated as follows: -gr nolignin rice crust moist within 7.48 ml basal medium. 2- gr nolignin rice crust moist within 7.48 ml of tap water. 3- gr rice crust (pure culture) 7.48 ml 4-gr purified rice crust moist within 7.48 ml of tap water gr rice crust mixture with 2.2 gr wheat husk and moist within 7.48 ml basal medium gr wheat crust mixture with 2.2 gr wheat husk (ratio of 8 to 2) and moist within 7.48 ml of tap water. After preparing the cultures autoclave them for 20 min in 2 C. After cooling the cultures, from T.reseei pure culture under sterile conditions, fertilization operation is done to mediums and then to autoclave flasks containing medium cultures in optimum time and temperature (30 c temperature for 4 days)[0] then the media is removed from the incubator and 55 ml citrate buffer is given to them and placed on shaker and then the same operation is performed (filtration, centrifugation filter) finally what is achieved is raw enzymatic solution from 6 cultures. First optical absorption of sample obtained by glucose oxidase method in λ=500 nm then with Lowry method concentration of protein in the crude enzyme solution was measured and ultimately FPA and CMCase activity was obtained. Results of produced glucose concentration in the 542 mediums to optimize fungal medium is shown in table. III. 2BRESULT AND DISCUSSION A. 2BMeasuring of enzymatic soluble protein of 542 species by lowry's method for optimization of fungal growth medium Samples and dilutions is prepared according to the Lowry method. Bovine serum albumin standard samples adsorption standard is done in λ=578 nm wavelength so that standard curve with equation OD = Cprot is used. The protein concentration in the solution of 6 sample enzyme is shown in table 2. TABLE I. TABLE II. THE PROTEIN CONCENTRATION IN THE SOLUTION OF 6 SAMPLE ENZYME Medium tested protein concentration mg/ml No lignin in liquid basal medium containing rice crust Medium containing no lignin rice crust with water 0.04 Liquid basal medium containing pure rice crust 0.3 Medium containing pure rice crust with water Shell liquid basal medium containing wheat and rice husk Mixed medium crust of wheat and rice flakes with water 0.4 Rice husk is an organic source and has high percent of protein, after that medium containing pure wheat crust contains high protein concentration. B. 3BCalculation of filter paper activity, specific activity and molecular activities of filter paper In terms of activity of filter paper and specific activity of filter paper, medium containing pure rice husk which soaked in liquid basal is optimum medium and then medium containing pure rice husk that has been moistened with water has the highest level of activity. Activity, specific activity, molecular activity and relative activity of tested media cultures are shown in table3. C. 4BCalculation of activity, specific activity and molecular activity of carboxymethyl cellulase The filter paper activity and specific activity of filter paper medium containing pure rice husk which soaked in liquid basal is optimum medium and then medium containing pure rice husk that has been moistened with water has the highest level of activity. Activity, specific activity, molecular activity and relative activity of CMCase in tested media culture is shown in table4. TABLE І. RESULTS OF PRODUCED GLUCOSE CONCENTRATION IN THE 542 MEDIUMS TO OPTIMIZE FUNGAL MEDIUM Culture No lignin wheat crust Basal medium Glucose concentration in the crude enzyme solution mg/di or mg/00cc 2.35 Absorbance of standard solution (OD) Absorbance of sample (OD) No lignin wheat crust Free basal medium Pure wheat crust Basal medium Pure wheat crust Free basal medium Wheat flakes mixed with rice husk Basal medium Wheat flakes mixed with rice husk (Free basal medium ISBN:

4 TABLE ІІІ. ACTIVITY, SPECIFIC ACTIVITY, MOLECULAR ACTIVITY AND RELATIVE ACTIVITY OF CMCASE IN TESTED MEDIA CULTURE Standard solution OD s = Number of tested culture medium OD sample content CMCase Activity CMCase (u/ml) Activity of filter paper Specific activity SP.A(units/mg propt) Molecular activity (units/µmol) Relative activity TABLE ІV. ACTIVITY, SPECIFIC ACTIVITY, MOLECULAR ACTIVITY AND RELATIVE ACTIVITY OF CMCASE IN TESTED MEDIA CULTURE Standard solution OD s = Number of tested culture medium Activity of filter paper Specific activity of filter paper Molecular activity F.P Relative activity IV. 3BCONCLUSION Enzymatic hydrolysis of rice husk of production of glucose (sugar liquid) has been studied. 542 T.reseei strain has maxium growth if incubated in optimum time and tempreture (30 c in 4 days) on the pure rice husk substrate which didn t lignin formation and moist with liquid basal medium and has the highest enzymatic activity and glucose production. However highest glucose concentration obtained in the crude enzyme solution which substrate contains only pure rice husk that miosted with tap water. If to the rice husk medium addamount of wheat husk (ratio of 8 to 2) and the surface of substrate is wet with tap water glucose produced approximately 2-fold increase compared to before. 5BREFERENCES [] Gaden, E.L., Mandels, M.H., Reese,E.T., Spano,L.A.Eds, 976, Enzymaticconversion of cellulosic maerials: Technology and Applications, Biothechnol. Bioeng. Symp.No.6, Jhon Wiley and sience, Newyork P.39.J. [2] I RR. Singhania, PK. Sukumaran, A. Pilli, et al. Solid state fermentation of lignocellusic substrate for cellulase production by Trichoderma reesei, NRRL460. Indian J Biotech 6, 5, [3] LR. Lyud, PJ. Weimer, WH. Van Zyl, IS. Pretorious. Microbial cellulose utilization: Fundamentals and biotechnology, Microbial Mol Biol Rev 2, 66, [4] Y. Sun, J Cheng. Hydrolysis of lignocellulosic materials for ethanol production: A review, Bioresour technol 2, 83, -. [5] J Zaldivar, J Nielson, L Olsson. Fuel ethanol production from lignocelluloses: A challenge for metabolic engineering and process integration, APPL Microbiol Biotechnol, 56, [6] A Z Aderolu, A Iyaya, AA Onilude. Changes in nutritional value of rice husk during Trichoderma viride degradation. Bulgarian J Agricultural Science, 7, 3, [7] AA Zaid, O Ganiyat. Comparative utilization of biodegraded and undegraded rice husk in Clarias gariepinus diet, African J Biotechnol 9, 8 (7): [8] C Krishna. Solid-state fermentation systems An overview, Critical Rev Biotechnol, 5, 25, -30. ISBN:

5 [9] CN Aguilar, G Gutiérrez-Sánchez, PA Rrado-Barragán, R Rodríguez-Herrera, JL Martínez-Hernandez, JC Contreras-Esquivel. Perspectives of solid state fermentation for production of food enzymes. American J Biochem Biotechnol 8, 4 (4): [0] A.Ghadi, S. Mahjoub, R. Mehravar, Management of Glucose Production Process from Rice Husk by Solid State Fermentation Method, 20 International Conference on Biotechnology and Environment Management IPCBEE vol.8 (20) (20)IACSIT Press, Singapoore ISBN: