BIODETOXIFICATION OF LIGNOCELLULOSIC HYDROLYSATE LIQUOR TO IMPROVE BIOFUEL PRODUCTION

Abstract

Biochemical conversion of lignocellulosic feedstock to biofuels and other valuable chemicals through a sugar-platform process involves pretreatment and hydrolysis steps, which facilitate the conversion of polysaccharides to simple sugars. A major drawback of acid pretreatment and hydrolysis process is formation of lignocellulose-derived by-products, such as furfural and 5-hydroxy-2-methylfurfural (HMF), which inhibit microbial growth during fermentation. Hence, an efficient and environment friendly technique for removal of these inhibitors is essential. We report isolation of a bacterium from soil having excellent capability to degrade furfural and HMF present in lignocellulosic hydrolysate liquor. This isolated bacterium was identified as Bordetella sp. BTIITR. Strain BTIITR was observed to be Gram stain-negative, coccobacillus and non-motile. Various biological and biochemical tests were performed to compare the characteristics of the isolated strains with the reported species of Bordetella. The isolated bacterium, Bordetella sp. BTIITR, was able to degrade 100 % of furfural and 80 % of HMF from simulated hydrolysate liquor in 16 h of incubation period. For the actual sugarcane bagasse hydrolysate liquor, it was able to remove 100 % of furfural, 94 % of HMF and 82 % of acetic acid in 16 h of incubation period. The isolated bacterium also demonstrated remarkable selectivity for HMF and furfural over the sugars present in hydrolysate liquor. Sugars were not consumed significantly before furfural and HMF were degraded to a low threshold concentration. This substrate priority of Bordetella sp. BTIITR for HMF and furfural makes it a potential organism for biodetoxification of inhibitory compounds present in the lignocellulosic hydrolysate liquor. To the best of our knowledge, this is the first study that illustrates the capability of Bordetella sp. to degrade the furan derivatives. The above study was further extended by immobilizing Bordetella sp. BTIITR within chitosan beads for detoxification of hydrolysate liquors. We hypothesized that the immobilized cells will enable significant process benefits over the free cells. The immobilized cells were observed to degrade 93 % HMF + 100 % furfural and 86 % HMF + 100 % furfural in 20 h at pH 8 and temperature of 40 0C from simulated and sugarcane bagasse hydrolysate liquors, respectively. As a significant improvement over the free cells, the immobilized cells were able to detoxify lignocellulosic hydrolysate liquor at broader range of operating pH, temperature ii and inhibitor concentration. Besides, the immobilized cells were successfully reused for seven consecutive cycles of operations with nearly constant degradation efficiency. Furthermore, a packed bed reactor (PBR) was designed and developed with the cell-immobilized chitosan beads. The idea was to employ a bioreactor for continuous detoxification of lignocellulosic hydrolysate for longer period of time, which may potentially lead to an industrially viable detoxification technology. Indeed, the PBR was observed to successfully emulate the performance of the batch operations. The height of the bed and the flow rate were optimized and it was observed that a bed height of 37.5 cm and feed flow rate 4 ml/min were most suitable for detoxification. 86.3 % of total furan was removed from the simulated hydrolysate. To demonstrate the benefit of the bioreactor, experiments were also conducted with continuous flow of sugarcane bagasse hydrolysate liquor for 15 h and a satisfactory degradation of furan inhibitors was observed. A simplified plug flow reactor model was also employed to perform kinetic studies of detoxification reaction within the PBR. It was observed that the degradation reaction followed first order kinetics in the PBR with apparent rate constants of 0.049 min-1 and 0.043 min-1 for HMF and furfural degradation, respectively. Finally, advantage of detoxification was established by ethanol formation using Pichia stipitis as the fermenting microorganism. Both detoxified simulated and detoxified bagasse hydrolysate liquors demonstrated significantly improved growth of fermenting microorganism and resulted in improved production of ethanol in comparison to the undetoxified hydrolysate liquor. Fermentation results revealed 1.6 times higher ethanol production and 2.6 times higher volumetric productivity from the detoxified sugarcane bagasse hydrolysate liquor in comparison to the undetoxified sugarcane bagasse hydrolysate liquor. Concentration of ethanol was recorded as 8.4 gL-1 and 5.2 gL-1 for detoxified bagasse and undetoxified bagasse hydrolysate liquors, respectively. Overall, the substrate priority of the isolated strain and the efficacy of detoxification can potentially lead to a translational technology for efficient degradation of fermentation inhibitors, which subsequently promises to improve the overall economics of ethanol formation from lignocellulosic biomass.