Optimization of microwave assisted alkali pretreatment and enzymatic hydrolysis of Banana pseudostem for bioethanol production

Transcription

1 2011 2nd International Conference on Environmental Science and Technology IPCBEE vol.6 (2011) (2011) IACSIT Press, Singapore Optimization of microwave assisted alkali pretreatment and enzymatic hydrolysis of Banana pseudostem for bioethanol production S.Chittibabu, K.Rajendran, M.Santhanmuthu Periyar Maniammai University Thanjavur , Tamilnadu, India M.K.Saseetharan Government College of Technology Coimbatore , Tamilnadu, India Abstract The aim of this research work is to optimize the microwave assisted alkali pretreatment and enzymatic hydrolysis of banana pseudostem (BPS) for the production of bioethanol. Pretreatment of BPS was performed at different alkali concentration, liquid-solid ratio, temperature and microwave exposure time. Enzymatic hydrolysis of pretreated BPS was done at constant cellulase enzyme loading and yield of reducing sugars (YRS) with respect to time was observed. It was found that when BPS was pretreated by 10 % NaOH with 4:1 liquid to solid ratio at 90 C for 8 min, the yield of reducing sugars reached 84% by enzymatic hydrolysis of 110 h with cellulase enzyme loading of 30 FPU/g of solid. Compared with convection mode of heating of alkali pretreatment, microwave assisted alkali pretreatment and enzymatic hydrolysis was more effective for BPS. Keywords-banana pseudostem; pretreatment; microwave; cellulase; enzymatic hydrolysis; bioethanol I. INTRODUCTION Bioethanol is the most dominant biofuel, considered as a good alternative for liquid transportation fuels with powerful economic, environmental and strategic attributes [1]. Although the energy equivalent of ethanol is 68% lower than that of petroleum fuel, the combustion of ethanol is clean (because it contains oxygen). Worldwide production capacity of ethanol in 2005 and 2006 were about 45 and 49 billion liters per year respectively and the total projected demand in 2015 is over 115 billion liters [2]. At present, sugar and starch based raw materials are used for the production of bioethanol. But increasing human population and fuel demand makes these raw materials insufficient for bioethanol production. Alternatively, bioethanol can be produced from lignocellulosic materials such as agricultural residues, wood and dedicated energy crops which constitute the most abundant global source of biomass [3]. Lignocellulosic biomass comprises about 50% of world biomass and its annual production was estimated as MT [4]. In these, 60% of lignocellulosic biomass came from agriculture and 40 % from the forest residues [5]. Therefore, bioconversion of lignocellulosic biomass to fermentable sugars, followed by fermentation to ethanol, has been considered as an attractive route for the production of low cost bioethanol [6]. Conversion of lignocelluloses to bioethanol consists of three major unit processes: pretreatment of raw materials, enzymatic hydrolysis of pretreated raw materials into fermentable sugars and fermentation of sugars into ethanol. Out of these, pretreatment process utilizes as much as 30% of the total ethanol production cost [7]. Therefore, increasing the pretreatment efficiency can significantly reduce the production cost of ethanol. Pretreatment breaks the lignin structure and disrupts the crystalline structure of cellulose thus enhancing enzyme accessibility to the cellulose during hydrolysis stage [8]. Current pretreatment research is focused on identifying, evaluating, developing and demonstrating promising approaches that primarily support the subsequent enzymatic hydrolysis of the treated biomass with lower enzyme dosages and shorter bioconversion time [9]. In this study, Banana pseudostem (BPS) was used as raw material for the production of bioethanol. BPS is abundantly available agriculture residues in subtropical and tropical regions. India is the largest producer of banana, contributing to 27% of world s banana production [10]. After harvesting, t/ha of banana pseudostem is generated in the field. In India, presently this biomass is dumped on roadside or burnt or left in situ causing detrimental impact on environment. High concentration of holocellulose (72%) with low lignin content (10%) [5] and its easy availability makes BPS as potential lignocellulosic biomass which could be used for the production of bioethanol. The most commonly used chemical pretreatment technique: alkali pretreatment [11] was applied for the pretreatment of BPS in combination with microwave heating. Dilute NaOH treatment of lignocellulosic materials increases the internal surface area, decreases crystalline nature and separates the structural linkages between lignin and carbohydrates [12]. To enhance the alkali pretreatment process, microwave heating was used, because of its capacity to induce explosion among the particles and improves the disruption of recalcitrant structures [13]. The aim of the present study was to optimize the microwave assisted alkali pretreatment process and find out the effects of alkali concentration, liquid-solid ratio (LSR), temperature and time of pretreatment on yield of reducing sugars (YRS) in the enzymatic hydrolysis process of BPS. Experiments were also performed using both microwave heat pretreated and convectional heat pretreated BPS to compare the YRS, rate of enzymatic hydrolysis, cellulase enzyme loading and residual cellulase activity. V2-67

2 II. MATERIALS AND METHODS A. Materials BPS was cut into small pieces, thoroughly washed with water and dried. It was ground and screened to obtain the particles of average size 420 μm. The composition of BPS on dry basis is holocellulose 72.71%, klason lignin 8.88%, acid soluble lignin 1.9%, ash content 8.2%, extracts % and pectin -0.27% [5]. All chemicals used in these experiments were analytically pure grade and obtained from local chemical manufacturers in India. Trichoderma reesei cellulase was used and the activity was determined as FPU/mL.. B. Pretreatment The pretreatment of BPS was carried out in 250 ml Erlenmeyer flask fitted with an insertion hole to fix thermometer. Five grams of screened BPS was taken in the flask and certain volumes of NaOH solution were added. The NaOH loading (based on initial dry material) was varied in the range of 4 12% and LSR was varied between 3:1 and 6:1. IFB domestic microwave oven was used to heat the BPS suspended in alkali solution. The microwave oven was calibrated with water for microwave power, exposure time and temperature. The pretreatment was conducted in the temperature range of 60 to 90 C with 5 to 9 min microwave exposure time. The treated BPS was filtered and washed with water until the ph of the water is neutral and dried at 105 C for 6 h. The oven dried BPS were stored for further usage. For comparing the effect of microwave in the alkali pretreatment process, BPS was also pretreated by using convectional heating method. The pretreatment conditions used were: alkali concentration 10%, LSR - 3:1, temperature 90 C and time of pretreatment 1.5 h [14]. Oil bath heater was used to maintain the temperature during this pretreatment of BPS. C. Enzymatic Hydrolysis The samples were digested by cellulase loading of 15 FPU.g -1 solid at 50 C and ph 4.8 (adjusted using 0.1 M sodium acetate buffer) in a shaker incubator at 150 rpm for 110 h. The initial solid concentration was 2% (w/v). Samples were taken periodically and analyzed for reducing sugars concentration. The enzymatic digestibility was denoted by the yield of reducing sugars (YRS) in % which is defined in (1) [14]. D. Analytical Methods The reducing sugars produced by enzymatic hydrolysis was determined by DNS (3,5- dinitro salicylic acid) method [15]. The cellulase activity was determined by the procedure given in [16] and expressed in FPU/ml. One FPU was defined as the amount of enzyme capable of producing 1 μmol of reducing sugars in 1 min. The reported results are the mean of three experiments. III. RESULTS AND DISCUSSION A. Effect of NaOH loading Concentration of NaOH had significant influence on YRS% as shown in the fig.1. It was found that when NaOH loading was increased from 4% to 10%, then YRS after 90 h of enzymatic hydrolysis was increased from 30% to 65%. Hence, NaOH loading was fixed at 10% for further experiments. Figure 1. Effect of NaOH loading in enzymatic hydrolysis (other conditions: 3:1 LSR, 80 C and 8 min microwave irradiation time) B. Effect of Liquid Solid ratio Higher LSR led to lower NaOH loading in the liquid phase. This resulted in low YRS at high LSR as shown in the fig.2. Maximum YRS was obtained in the LSR of 4:1. YRS % = (W Rs ) / (W Is ) (1) where W Rs weight of reducing sugars produced by enzymatic hydrolysis; W Is weight of initial solids. Experiments were also performed under optimum pretreatment conditions for both microwave heat pretreated and convectional heat pretreated with different enzyme loadings (5 30 FPU/g of solid). After 110 h of enzymatic hydrolysis, samples of liquid phase were collected from each enzyme loading and residual cellulase activity was found out. V2-68

3 Figure 2. Effect of liquid-solid ratio on enzymatic hydrolysis. (other conditions: 10% NaOH, 80 C and 8 min microwave irradiation time) The low YRS were due to insufficient NaOH to act on the cellulose molecules. But at LSR 3:1, the high loading NaOH resulted in low YRS and this probably due to the formation inhibitory compounds which affected the enzyme activity. The homogeneity of the system was not maintained when LSR below 3:1. C. Effect of temperature The effect of pretreatment temperature on the rate of enzymatic hydrolysis was shown in fig.3. It can be seen that temperature had no significant effects on YRS when over 80 C. The YRS was high at the temperature 90 C. Therefore 90 C was selected for the alkali pretreatment. Figure 4. Effect of microwave exposure time on enzymatic hydrolysis (other conditions: 10% NaOH loading, 4:1 LSR, 90 C) E. Enzymatic hydrolysis The rate of enzymatic hydrolysis at different enzyme loadings was shown in fig.5. It can be seen that the enzyme loading had significant influence on YRS and also in the rate of enzymatic hydrolysis. The rate of enzymatic hydrolysis of microwave heating pretreated BPS was higher than the rate of convection heat pretreatment BPS. According to the experimental results, when enzyme loading was 30 FPU/g of solid, the YRS% of convection heat pretreated BPS was only 65% compared with 84% for microwave heating pretreated BPS and comparing the time taken for hydrolysis of both the Figure 3. Effect of temperature on enzymatic hydrolysis (other conditions : 10% NaOH loading, 3:1 LSR, 8 min microwave irradiation time) Figure 5. Enzymatic hydrolysis of microwave assisted alkali pretreated BPS with different enzyme loading. (other conditions: NaOH loading 10%, 3:1 LSR, 90 C and 8 min of microwave irradiation time) D. Effect of microwave exposure time It was found from the fig.4 that there was an optimum time at which maximum YRS was obtained. Further increase in time had no significant effect on enzymatic hydrolysis. Figure 6. Enzymatic hydrolysis of alkali pretreated BPS using convection mode of heating with different enzyme loading. (other conditions: NaOH loading 10%, 3:1 LSR, 90 C and 1.5 h of convection mode of heating) BPS samples, it was also found that the microwave heated BPS took much less time i.e., 80 to 90 h to reach the V2-69

4 maximum YRS% than the convection heat pretreated BPS which took h to reach maximum YRS% as shown in fig. 5 & 6. It is also evident from the fig.5 that there was only marginal difference about 2% between the final YRS % of 20 FPU/g of solid and 30 FPU/g of solid enzyme loadings and after 80 h of hydrolysis the difference between the YRS% was only 4%. It indicated that the enzymatic digestibility of BPS could be enhanced by microwave heating even with low enzyme loading and lesser time of enzymatic hydrolysis. F. Residual cellulase activity The residual cellulase activity in the liquid phase after 110 h of enzymatic hydrolysis was determined and shown in fig. 7. The residual cellulase activity of microwave pretreated BPS samples was higher than that of the convectional heat treated BPS. As mentioned in [17], the cellulase enzyme could be irreversibly adsorbed by lignin, which indicated that higher lignin content in the pretreated solid would result in more loss of free cellulase activity during enzymatic hydrolysis. Thus delignification was also increased during the pretreatment of BPS by using the microwave irradiation when compared to convectional heating mode. Table 1 shows the % retention of residual cellulase activity for different enzyme loadings and it was observed that the percentage retention of cellulase activity was 77.5% at 20 FPU/g of solid enzyme loading but it was only 71.6% for high enzyme loading of 30 FPU/g of solid. This loss in % retention of residual cellulase activity may be due to high YRS obtained at 30 FPU/g of solid enzyme loading. The high residual cellulase activity present in the liquid phase could be reused for second batch of the enzymatic hydrolysis to reduce the enzyme loading. Figure 7. Residual cellulase activity in the liquid phase after 110 h of enzymatic hydrolysis TABLE I. PERCENTAGE RESIDUAL CELLULASE ACTIVITY Enzyme loading FPU/g of solid % Residual Cellulase Activity Retention Microwave heat pretreated BPS Convection heat pretreated BPS G. Discussion The potential of using microwave as an efficient heating method in the pretreatment of BPS to increase its enzymatic digestibility was shown. The YRS was higher in microwave heating than convectional heating mode of pretreatment, The YRS% of convectional heat pretreated BPS was only 65% but it was 84% for microwave preheated BPS after 110 h of enzymatic hydrolysis. Volumetric and selective heating of lignocelluloses by microwave, facilitates the disruption of their recalcitrant structures more efficiently [10]. Even though the increase in YRS was marginal i.e., 19%, the pretreatment time of microwave heating was only 8-10 min which was much lesser when compared to pretreatment time of 1.5 h for convectional heat pretreatment. Microwave heat pretreated BPS, requires low enzyme loading of 20 FPU/g of solid and also results in high % retention of residual cellulase activity (77%) for the similar YRS%, thus increasing the scope of reusing the cellulase enzymes. As given in [18], enzyme loading can amount to as much as 60% of the process cost of lignocellulosic biomass conversion to bioethanol. Minimum pretreatment time, low cellulase enzyme loading and high residual cellulose activity lead to conclude that the production cost of ethanol could be significantly reduced by using microwave assisted alkali pretreatment of BPS. At the same time, power requirement and increase in capital costs are inevitable in microwave pretreatment method. Therefore, an economic evaluation considering the total process from biomass to sugars is needed for further comparison. However, the optimal pretreatment conditions need to be compromised with energy input and cost saving in large scale processing of BPS using microwave assisted alkali pretreatment. IV. CONCLUSION BPS can be a potential lignocellulosic biomass resource for the bioethanol production. Microwave assisted alkali pretreatment could enhance the enzymatic digestibility of BPS. The experimental results showed that when BPS was pretreated using microwave heating with 10% NaOH, 4:1 liquid-solid ratio, at temperature 90 C and pretreatment time of 8 min, the YRS% reached its maximum 84% after 110 h of enzymatic hydrolysis at 30 FPU/g of solid enzyme loading. Considering the rate of enzymatic hydrolysis and % retention of the residual cellualse activity, the enzymatic hydrolysis could be done with even 90 h of hydrolysis and with 20 V2-70

5 FPU/g of solid for microwave assisted alkali pretreatment process. However, it should be noted that a critical assessment of cost analysis is needed because the capital investment and operation cost of microwave heating is higher than convectional heating mode. Conclusively, the results of this study can serve for further optimization of bioethanol production process from lignocellulosic biomass. ACKNOWLEDGMENT S.C thanks Dr.S.Kumaran, Assistant Professor of Biotechnology, Periyar Maniammai University for his constructive suggestions and guidance. REFERENCES [1] F.W.Bai, W.A.Anderson and M.Moo-Young, Ethanol fermentation technologies from sugar and starch feedstocks, Biotechnol adv, vol. 26, 2008, pp , doi: /j.biotechadv [2] F.Talebinia, D.Karakasher and I.Angelidaki, Production of bioethanol from wheat straw: an overview on pretreatment, hydrolysis and fermentation, Bioresour Technol, vol. 101, 2010, pp , doi: /j.biortech [1] Y.Lin and S.Tanaka, Ethanol fermentation from biomass resources: current state and prospects, Appl. Microbiol. Biotechnol., vol. 69(6), 2006, pp , doi: /j.biortech [2] O.J.Sanchez and C.A.Cardona, Trends in biotechnological production of fuel ethanol from different feedstocks, Bioresor Technol, vol. 99, 2008, pp , doi: /j.biortech [3] K.Li, S.Fu, Y.Zhan and L.A.Lucia, Analysis of the chemical composition and morphological structure of Banana pseudostem, Bioresources, vol.5(2), 2010, pp [4] M.Chen, J.Zhao and L.Xia, Comparison of four different chemical pretreatments of corn stover for enhancing enzymatic digestibility, Biomass Bioenerg, vol 33, 2009, pp [5] B.Yang, and CE.Wyman, Pretreatment the key to unlocking low cost cellulosic ethanol, Biofuels Bioprod Bioref, vol.2, 2008, pp [6] Y.Sun and J.Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review, Bioresour Technol, vol. 83, 2002, pp [7] P.Alvira, E.Tomas-Pejo, M.Ballestros and M.J.Negro, Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review, Bioresour Technol, vol. 101, 2010, pp [8] D.Mohapatra, S.Mishra and N.Sutar, Banana and its by-product utilization: an overview, J.Sci Ind Res, vol 69, May. 2010, pp [9] M.J.Taherzadeh and K.Karimi, Enzyme based hydrolysis processes for ethanol from lignocellulosic materials- a review, Bioresources, vol. 2(4), 2007, pp [10] A.T.W.M.Hendriks and G.Zeeman, Pretreatment to enhance the digestibility of lignocellulosic biomass, Bioreosur Technol, vol.100, 2009, pp [11] Z.Hu and Z.Wen, Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment, Biochem. Eng. J., vol. 38, 2008, pp [3] XB.Zhao, F.Peng, K.Cheng and D.Liu, Enhancement of enzymatic digestibiltiy of sugarcane baggasse by alkali peracetic acid pretreatment, Enzyme Microb. Technol. vol. 44, 2009, [12] G.L.Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. vol. 31(3), 1959, [13] T.K.Ghose, Measurement of cellulsae activities, Pure Appl. Chem, vol. 59, 1987, [14] XB.Zhao, L.Wang and DH.Liu, Peracetic acid pretreatment of sugarcane bagasse for enzymatic hydrolysis: a continued work, J Chem Technol Biotechnol, vol. 83, 2008, pp [15] R.Yao, B.Qi, S.Deng, N.Liu, S.Peng and Q.Cui, Use of surfactants in enzymatic hydrolysis of rice straw and lactic acid production from rice straw by simultaneous saccharification and fermentation, Bioresources, vol. 2(3), 2007, pp V2-71