STUDIES ON BIOCONVERSION OF LIGNOCELLULOSIC BIOMASS TO BIOFUEL

Abstract

In today’s world, fossil fuel (petroleum, natural gas, and coal) is one of the major sources of energy. The global demand for energy is growing each year along with population growth. However, climate changes caused by the use of fossil fuels and its limited availability and sustainability are driving for search of a sustainable supply of energy. The sustainability problem of fossil fuels and the emission of greenhouse gases open the doors for scientists to search for a sustainable and relatively safer supply of energy to the world. The aim of this research was to isolate, identify and characterize novel bacteria having lignocellulosic potentials (lignin, cellulose, and hemicellulose depolymerizing ability) from termite guts. The performance and the capabilities of the microbes were evaluated on lignocellulose biomass (wheat straw, rice straw and sorghum straw) after isolation and application of microbes on the model compounds. The delignification and hydrolysis efficiencies of the bacteria were evaluated simultaneously and separately. The polysaccharide hydrolysis efficiencies of the microorganisms were also evaluated by combining microbial hydrolysis with NaOH pretreatments, Organosolv pretreatment and microwave assisted NaOH pretreatment. The microorganisms were screened and identified by subsequent plate culturing and 16S rRNA gene sequencing. The growth conditions for the isolates were optimized experimentally. Using the optimized conditions, the delignification capabilities, polysaccharide hydrolysis capabilities and efficiencies were investigated experimentally in simultaneously and separately system. The compositional analysis of the biomass before and after pretreatment processes were studied to observe and understand the changes in composition and to know the extent of degradation due to the pretreatment process. The pretreated and raw biomass were characterized by field emission- electron microscopy (FE-SEM), X-ray diffractometer (powdered XRD) and Fourier transform infrared spectroscopy (FTIR). The extent of polysaccharide hydrolysis was investigated by measuring the amount of reducing sugars released per unit time. The reducing sugars were measured by 3, 5 dinitrosalycilic acid method. In this study, two different bacteria strains, Bacillus sp. BMP01 and Ochrobactrum oryzae BMP03 strain were isolated and identified from termite guts which have lignocellulose degrading capabilities. Bacillus sp. BMP01 strain has capabilities to hydrolyze carboxymethylcellulose and xylan to glucose and xylose, respectively. This strain showed high xylanase activity (about 0.21 U/mL) and carboxymethyl cellulase activity (about 0.25 U/mL). Ochrobactrum oryzae BMP03 v strain showed laccase activity, which indicates its ability to depolymerize lignin. This result demonstrated that the guts of termites comprise of lignocellulose degrading bacteria that can be cultured and grown in a bioreactor and can be used for the production of vital chemicals like bioethanol. Biological conversion of rice straw, wheat straw and sorghum straw were studied by applying the isolates. The maximum lignin removal was observed after the 14th day of rice straw biotreatment in separate delignification and hydrolysis process (about 53.74% lignin removal). About 69.96% of total reducing sugars were obtained after the 14th day of hydrolysis of biodilignified rice straw. In simultaneous delignification and hydrolysis process, about 58.67 % of total reducing sugars were obtained after the 13th day biotreatment. The lignin degrading bacteria preserves polysaccharides (cellulose and hemicellulose). Biodelignification followed by microbial hydrolysis of wheat straw increased the amount of total reducing sugars and biofuels yield. It was shown that the production of total reducing sugars in separate hydrolysis system by Bacillus sp. BMP01 strain achieved 439 mg / g at 16th days of hydrolysis time, which is 9.45% higher than the simultaneous system. About 44.47% lignin was degraded by Ochrobactrum oryzae BMP03 strain after 16th days of biotreatment. This also contributed to increase in cellulose content by 22.38% and hemicellulose content by 18.64% after biodelignification. The simultaneous system converted 368 mg of reducing sugars/ g of wheat straw. About 64.29% of lignin from sorghum straw was removed after 14th day of biotreatment while 48.45% increase in cellulose and 22% increase in hemicellulose after biotreatment were found. About 69.81% of total reducing sugars were obtained after the 14th day of hydrolysis of biodilignified sorghum straw. In simultaneous delignification and hydrolysis process, about 54.52 % of total reducing sugars were converted after 14th day biotreatment. Separate biodelignification and hydrolysis have an advantage over the simultaneous system in terms of hydrolysis efficiency and vice versa in terms of biotreatment time. The study showed the possibilities of biological conversion of lignocellulose biomass to its monomers by bacteria from termite guts. The combination of sodium hydroxide pretreatment with microbial hydrolysis has significant capacity for biofuel production. Pretreatment in 3% and 7% sodium hydroxide concentration at 80 0C were effective in removing 67.13% and 71.29% of lignin, respectively from rice straw. 70.56% and 71.33% of cellulose were preserved after 3% and 7% pretreatments, respectively due to solubilization of high amount of lignin and hemicellulose. 88.27% and 88.40% of all the vi polysaccharides were converted to glucose and xylose after subjecting to microbial hydrolysis. In case of wheat straw, 10% NaOH pretreatments on wheat straw biomass leads to effective cellulose release (72.67%) and solubilization of significant amount of hemicellulose (55.55%) and lignin (69.5%). Maximum conversion of polysaccharides (83.68%) to glucose and xylose were observed at 10% NaOH pretreated wheat straw after subjecting to microbial hydrolysis. In case of sorghum straw, the maximum amount of lignin removal (78.20%) were observed after 5% sodium hydroxide pretreatment and also maximum polysaccharide releases (76.29%) were observed. About 820 mg of reducing sugars /g of biomass were released (91.27% conversion) after 13th day of hydrolysis. Microbial sources from termite showed great potential to replace commercial enzymes. Generally, the study revealed the enhancement of bioethanol production by coupling alkali pretreatment with microbial hydrolysis and fermentation. Organosolv pretreatment of rice straw, wheat straw and sorghum straw were studied for maximum cellulose release and lignin solubilization and minimum hemicellulose decomposition at pretreatment temperature from 60-1000C, treatment time from 10-50 minutes and acid concentrations from 50%-90%. In the case of rice straw pretreatment, optimized condition was achieved at 780C, 69.5% acid concentration and pretreatment time of 32 minutes. At this condition about 73.73% of lignin was removed, 74.65% of cellulose released and 52.66% hemicellulose preserved in the residues. Exposure time has found to be more influential factor than acid concentration and temperature during rice straw depolymerization. Hydrolysis of the pretreated straw resulted in conversion efficiency of 62.09% (515 mg/g of rice straw) using Bacillus sp. BMP01. Fermentation of hydrolyzed straw resulted in 0.38 g of ethanol/g of reducing sugars after 96 hours of fermentation (Ethanol yield of 74.46%). In the case of wheat straw pretreatment, optimum pretreatment condition was achieved at temperature of 790C, 69.5% acid concentration and 29 minutes of exposure time. At this pretreatment condition, 69.42% lignin was removed (5.55% remained in the residue), 74.13% cellulose was released and 13.54% hemicellulose was preserved (67.26% of original hemicellulose was preserved). About 70.22% of conversion efficiency (684 mg/g) was achieved after 10th day of hydrolysis. Ethanol yield of 0.417 g/g of total reducing sugars was obtained by fermentation of the hydrolyzed straw. This study revealed that Organosolv pretreatment followed by bacterial hydrolysis could be a potential alternative for the vii conversion of rice straw, wheat straw, sorghum straw and also with possible extension to other agricultural residues to enhance reducing sugars yields and ethanol yields. Response surface methodology (RSM) based on central composite design (CCD) was used to design experiments and to optimize experimental conditions for efficient depolymerization of rice straw, wheat straw and sorghum straw using microwave assisted NaOH pretreatment method. Temperature from 1200C -2000C, NaOH concentration from 0.5%-2.5 %, w/v, and pretreatment time from 5- 25 minutes were considered at five different levels. In the case of rice straw, NaOH concentration is found to be the most influential factor to ensure efficient depolymerization of the straw. At optimal pretreatment condition (temperature 1600C, time 15 minutes and NaOH concentration 1.5%, w/v), 71.53% of lignin removal (6.02% lignin retained in the residue), 78.51% of cellulose release, and 9.99% hemicellulose were obtained. A significant amount of reducing sugars (736 mg/ g of rice straw) was hydrolyzed after bacterial hydrolysis (Bacillus sp. BMP01) of the microwave assisted NaOH pretreated sample. In the case of wheat straw, the pretreatment condition at 1600C, 1.5% NaOH concentration and 15 minutes preserved high cellulose and removed high amount of lignin. At this point 38.34% of hemicellulose and 74.15% of cellulose were preserved in the solid residue and 69.49% of lignin was solubilized. 718 mg/g of reducing sugars was released after hydrolysis pretreated wheat straw by Bacillus sp. BMP01. Ethanol yield of 0.348 g/g of total reducing sugars was obtained after 96 hours of fermentation. This study demonstrated that the combination of microbial hydrolysis and microwave assisted NaOH pretreatment enhanced ethanol yield. Overall, this study demonstrated the possibility of lignocellulose biomass conversion by coupling various pretreatment methods with microbial hydrolysis for enhancing bioethanol yield. Moreover, it demonstrated that the guts of termites comprise of lignocellulose degrading bacteria that can be cultured and grown in a bioreactor and can be used for the production of vital chemicals like bioethanol. This proofs the possibilities of microbial hydrolysis in replacing enzymatic hydrolysis in lignocellulose biomass conversion to bioethanol.