Saccharification of Microwave Pretreated Kenaf (Hibiscus Cannabinus) Using Rumen Fluid Isobel Rae S. Impas, Rachiel Ruth C.

Tan, Mark Reinhard P. Young Thesis Adviser: Dr. Florinda T. Bacani

ABSTRACT In this study, rumen fluid was used as the source of enzyme for saccharification of Kenaf bast fiber (KBF). Furthermore, this study introduced microwave pretreatment as its primary pretreatment method. Preliminary experiments were done to obtain the best values of the rumen fluid (RF) and substrate concentrations which were found to be 3% (v/v) and 10 g/L respectively among values of 1%, 3%, and 5% (v/v) for enzyme concentration and 10, 20, and 30 g/L for the substrate concentration. Process parameters for microwave pretreatment were identified to be the irradiation time and fiber concentration. 32 factorial design was used with parameter values of 10, 20, and 30 minutes irradiation time and 20, 30, 40 g/L fiber concentration. The highest sugar yield was found when irradiation time is 10 minutes with fiber concentration of 20 g/L with the value of 0.0318 g total sugar per gram kenaf bast fiber, 3.83% higher than the untreated fiber. Though the obtained total sugar yield is minimal, further research is encouraged to investigate possible solutions to increase total sugar yield. INTRODUCTION To achieve energy security and meet local demand of fuels, biofuels containing bioethanol are now found to be one of the best solutions. The process of biethanol production involves the fermentation of sugars from cellulosic fibers. In this study, Kenaf Bast Fiber was utilized as the primary fiber for saccharification using Carabao rumen fluid as its primary enzyme source. The factors which may affect the saccharification of kenaf bast fiber using rumen fluid, such as its reaction time, rumen fluid concentration, and fiber concentration, was tested to find the best condition for the process. To further improve the saccharification of the fiber, microwave pretreatment is applied to the fiber varying its pretreatment parameters such as concentration and irradiation time to determine the factors that could affect rumen fluid saccharification of kenaf bast fiber in terms of percent yield of sugar. METHODOLOGY Preliminary experiments were made to attain the best rumen fluid concentration by varying the %(v/v) amount of RF with the values of 1%, 3%, and 5%, keeping the substrate concentration constant at 10 g/L where sugar concentration was measured at specific time intervals. Following the same procedure, the best substrate concentration was attained by keeping the substrate concentration constant and varying the values of the substrate with 10 g/L, 20 g/L and 30 g/L. Results show that the values of the best RF concentration is 3% (v/v) and best substrate concentration is 10 g/L, with reaction time of 60 hours. The values obtained in the preliminary experiments were then used as the basis for the actual experiment. In the actual experiment comes the introduction of microwave pretreatment where the irradiation time was varied with 10, 15, 20 minutes and fiber concentration of 20 g/L, 30 g/L and 40 g/L using 32 experimental design. Each run lasted for 60 hours, with 10 g/L microwave pretreated substrate and 3%(v/v) RF. Sugar analysis was done using Dinitrosalicylic Acid (DNS) Method to quantify total sugar yield and High Performance Liquid Chromatography (HPLC) for glucose yield. RESULTS AND DISCUSSION For the preliminary experiment, the highest sugar yield obtained while testing for the best RF concentration was 3.06% (g/g) with 3% RF concentration while best substrate concentration, however, was found to be 10 g/L where in the saccharification curve peaked at a reaction time of 60 hours. The curves obtained from the experiments were found to have irregular behavior since in rumen fluid hydrolysis, different microorganisms are involved, and a multiphase curve can be expected. Jimenez-Flores et al. (2010) mentioned that a multiphase curve can be observed in rumen fluid hydrolysis due to the growth rate of different microorganisms in the fluid. As such, it is possible that the product of the saccharification of the biomass was utilized by the rumen bacteria as their food. Consequently, this variation in the growth rate of the rumen microorganisms could have led to the different rates of production and consumption of the sugar products thus making the curves irregular. In the actual experiment, the highest total sugar yield obtained was 3.1772% (g/g) with microwave pretreated fiber at a fiber concentration of 20 g/L and irradiation time of 10 minutes which shows a 3.83% increase in sugar yield as compared to the saccharified untreated KBF. The highest sugar yield obtained from the pretreated kenaf bast fiber did not show a significant increase as compared to the untreated fiber used in the preliminary experiments. This may be due to the inability of the pretreatment to completely delignify the fiber. Other reason could be that delignification was not enough to increase fiber digestibility. According to Demeyer (1981), delignification can increase the rate of attachment of bacteria to plant cell walls however increased attachment is not automatically followed by digestion. The study also mentioned that although lignin content is related to the rate and extent of plant cell wall carbohydrate digestion, other characteristics associated with type, site, and extent of lignification are involved. CONCLUSION AND RECOMMENDATIONS The application of microwave pretreatment of lignocellulosic biomass was used to increase the yield of hydrolysis to establish the use of rumen fluid for saccahrification process. In studies using rumen fluid hydrolysis; particle size reduction was proven to be effective enough to produce significant amounts of sugars necessary for ethanol production. In this study, the introduction of microwave pretreatment instead of particle size reduction only to kenafbast fiber increased the total sugar yield by 3.84% compared to untreated kenafbast fiber after rumen fluid hydrolysis. The percentage of increase was not too significant; this may be due to the inability of the substrate to be grinded or milled into very fine particles. The microwave pretreatment done in this study may have been able to delignify the fiber; but delignification of the fiber does not guarantee fiber digestibility since although lignin content is related to the rate and extent of plant cell wall carbohydrate digestion, other characteristics associated with type, site and extent of lignification are involved (Demeyer, 1981). This idea leads to another factor that should be considered which is the substrate selection. Kenafbast fiber was found to be theoretically viable source of lignocellulosic biomass for ethanol production. However, kenafbast fiber is not as effective as other commonly used lignocellulosic biomass such as rice straw. In fact, a preliminary runwas conducted with a different substrate, particularly rice straw. The result of the run can be found in Appendix E.6 where the highest total sugar yield was found to be 24.5% higher than that of the highest total sugar yield obtained using kenafbast fiber. This verified the effectiveness of the experimental methodology for saccharification employed throughout the study. Based from the results concluded in this study, it is not further recommended to pursue research. However, other studies which can use kenaf bast fiber may be done to promote its utilization can be done. REFERENCE Demeyer, D.I. (1981). Rumen microbes and digestion of plant cell wall. Agriculture and Environment, 6, 295-337. Jimenez-Flores, R., Fake, G., Carroll, J., Hood, E., & Howard, J. (2010). A novel method for evaluating the release of fermentable sugars from cellulosic biomass. Enzyme and Microbial Technology, 47, 206-2011.

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