Summary & Conclusion

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1 Summary & Conclusion

2 CHAPTER 6 SUMMARY & CONCLUSIONS Concerns regarding the soaring cost of gasoline and the depleting petroleum reserves have led to an urge for a sustainable alternative to gasoline such as biofuels. Among several types of biofuels (viz. bioethanol, biohydrogen, biodiesel, biobutanol), bioethanol is most widely accepted round the globe. Till date bioethanol is commercially produced from sugary and starchy materials (first generation bioethanol) such as sugarcane juice and corn grains. However, the upcoming shortage of these food materials in future due to their diverging utilization as fuel cannot be overlooked. Lignocellulosics, the most abundant and renewable organic resource available on the earth provide an abundant raw material, which on efficient utilization could be a potent solution to the problem of energy security. Lignocellulosics are mainly comprised of cellulose (a homopolymer of D-glucose units), hemicellulose (a heteropolymer of several pentose and hexose sugars) and lignin (a polymer of phenyl propanoid units). However, the recalcitrancy of lignin and the crystalline nature of structural carbohydrate hinders its fullest utilization for bioethanol production. Therefore, a structure disruption i.e., pretreatment of lignocellulosics is necessary to make the substrate amenable for the hydrolysis. The pretreated substrate will be then hydrolyzed enzymatically and subsequently be fermented to ethanol, which will serve as biofuel. In the present study, an attempt has been made to develop a bioethanol production process using Prosopis juliflora, a weed of tropical and subtropical vegetation of Indian subcontinent as raw material. SUMMARY Pretreatment of lignocellulosic substrate Various pretreatment strategies i.e., alkali treatment, acid treatment, chlorite treatment and biological treatments were employed and compared for their amenability to enzymatic hydrolysis. Among them chlorite treatment was observed to have maximum hydrolysis efficiency (> 90%), although the process 243

3 was suffered from the problem of hemicellulose loss. Therefore, a biphasic pretreatment i.e., acid hydrolysis followed by chlorite treatment was adopted, which resulted in a sequential removal of hemicellulose (~100%) and lignin (~80%). Further to enhance the efficiency of the process both the pretreatment steps were optimized using one factor at a time (OFAT) and response surface method (RSM). The optimal pretreatment conditions for acid hydrolysis of P. juliflora were 10% substrate consistency, 3% sulfuric acid, 120 C and 45 minutes. While the optimized chlorite treatment conditions were 4% substrate consistency, 4% sodium chlorite, 120 C and 30 minutes. Under these optimal conditions, the acid hydrolysis of P. juliflora resulted in a release of mg/g dry substrate. While the chlorite treatment caused 81.6 % delignification. Further, the acid hydrolysis and the chlorite treatment were translated to 3.0 and 1.0 Kg level. At higher scales no significant variations in the process efficacy proves the scalability of the process. Detoxification of acid hydrolysate Though dilute acid hydrolysis is a fast and convenient method, yet it is hampered by its non-selectivity and the degradation of sugars. Under stringent conditions (low ph and high temperature), the pentose and hexose sugars present in the hydrolysates were degraded to generate furfurals and hydroxyl methyl furfurals, while the acid on reaction with acid labile lignin generated phenolic moieties. All these compounds i.e., furfural, HMF and phenolics are inhibitory to the fermenting microorganisms. Therefore prior to the fermentation of the sugars present in the hydrolysate, the removal of these inhibitory compounds are necessary. In the present study, several strategies such as neutralization, overliming, activated charcoal, ion-exchange resin treatment and enzymatic treatment were evaluated for the detoxification of acid hydrolysate of P. juliflora. Among these detoxification methods, activated charcoal adsorption (2.0% w/v) was observed to be the most efficient strategy to remove all the inhibitors. The scale up of detoxification of the hydrolysate was carried out upto 20 L scale with a non-significant variation in the detoxification efficiency. 244

4 Enzymatic hydrolysis of delignified substrates The enzymatic hydrolysis of delignified substrate was carried out using both one factor at a time (OFAT) approach and response surface methodology (RSM). Among different parameters studied, the maximum saccharification (>90%; 40 g/l) of delignified substrate was achieved when the saccharification was carried out with 22 IU FPase, 68 U β-glucosidase, 1% Tween 80, 10 mm Cu ++ at 5% substrate consistency, ph 5.0, 150 rpm and 50 C. Since higher sugar concentration in the hydrolysates is a prerequisite for the production of cost efficient bioethanol production, therefore an attempt to increase the sugar concentration using high solid enzymatic saccharification was made. In the present study, the strategy of fed-batch enzymatic saccharification to achieve the high solid consistency has been adopted. Using the fed-batch enzymatic saccharification approach a 20% substrate consistency could be achieved, which brought about a sugar concentration of ~127 g/l with a saccharification yield of 64%, which was ~50% higher than the sugar released under batch operation (80.78 g/l). Moreover, the comparison of the accuracy of the model prediction validated that a well designed fed-batch approach could be used to allow an STR reactor capable of handling delignified substrate at approximately below than 10% insoluble substrate with a goal to achieve 20% cumulative insoluble solids. Both the batch and fed-batch enzymatic hydrolysis were scaled up successfully from the laboratory flask level to 20 L level in a 30L bioreactor. Fermentation of hydrolysates and distillation of ethanol A total of 20 xylose utilizing yeast strains were screened for their pentose to ethanol conversion efficiency and among them Pichia stipitis NCIM 3499 resulted in maximum production of ethanol (7.02 g/l) in 40 h of incubation. The fermentation conditions for ethanol production from detoxified acid hydrolysate were optimized for various physical and nutritional parameters. The maximum ethanol concentration (~7 g/l) with highest productivity 0.43 g/l/h was achieved at 30 C, ph 5.5, 150 rpm, 40% DO, 80% inoculum (10 h old), 1.2 % soybean meal and 0.8% casamino acid. The optimization of fermentation conditions for ethanol production from detoxified acid hydrolysate resulted in a decline of fermentation time from 40h to 16h, which thereby improved the 245

5 efficiency approximately 2.5 fold. Further the fermentation of detoxified hydrolysate was scaled upto 20 L level successfully with an ethanol yield of 0.35 g/g and productivity of 0.43 g/l/h. On the other hand, the fermentation of enzymatic hydrolysate was carried out with hexose fermenting yeast, Saccharomyces cerevisiae HAU. Under optimal conditions (35 C, ph 6.0, 200 rpm, 40% DO, 6% inoculum (8h old) and 1.6% soybean meal), the enzymatic hydrolysate containing 120 g/l sugars resulted in g/l ethanol. The ethanol production from enzymatic hydrolysate under the optimized conditions reduced the fermentation time from 68 h to 12 h and increased the ethanol productivity by ~ 5.66 folds. Moreover, the fermentation of enzymatic hydrolysate was scaled up successfully to 20 L scale with an ethanol yield and productivity of 0.45 g/g and 4.52 g/l/h, respectively. The ethanol thus recovered after fermentation of enzymatic hydrolysate was distilled using a fractional distillation apparatus fitted with a spirally coiled condenser. The distillation was carried out in two batches i.e., the distillate from first batch was further distilled in second batch. The concentration of ethanol in first batch was 30%, which after a second batch of distillation was further improved to 70%. Mass balance The material balance for 1.0 Kg of P. juliflora was evaluated for the complete bioethanol production process. It has been estimated that from 1.0 Kg of Prosopis juliflora, approximately 86% conversion of holocellulose to sugars (~590 g) was achieved, which subsequently produce ~250 g of ethanol with 84% of the theoretical ethanol yield. The results showed that the process has an overall efficiency of >80% in all the steps i.e., pretreatment, saccharification and fermentation. The high efficiency of the process steps makes this process industrially competitive. CONCLUSION In the present study, bioethanol production process from a novel lignocellulosic weed i.e., P. juliflora was developed. In order to fraction the lignin and hemicellulose from the lignocellulosic substrate, a biphasic pretreatment (acid hydrolysis followed by chlorite treatment) was adopted, which offers the possibility of producing cellulosic material that eventually will be highly 246

6 amenable for enzymatic hydrolysis. A detoxification strategy (activated charcoal adsorption) was also developed to eliminate the fermentation inhibitors from the hemicellulosic hydrolysates. The delignified substrate was then hydrolyzed enzymatically and the sugar concentration in the hydrolysate was improved using fed-batch enzymatic hydrolysis strategy. Moreover the kinetic analysis and modeling of both batch and fed-batch enzymatic saccharification was also studied. The fermentation of detoxified acid hydrolysate and enzymatic hydrolysate was carried out with P. stipitis and S. cerevisiae, respectively. The optimization of fermentation conditions of acid and enzymatic hydrolysates brought about an improvement of ~2.5 and ~5.66 fold, respectively, in the ethanol productivities. Further a mass balance sheet for the ethanol production process was made which revealed more than 80% efficiency of the complete process, which signifies the industrial competitiveness of the process. It's extraordinary how inventive one can be with ethanol right now Daniel Yergin 247