BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC MATERIAL OF KANS GRASS

Abstract

World's energy demand has been increasing constantly with increasing in population and better life standard that resulted in the more use of energy in form of oil, coal and petroleum gas. This is causing depletion of natural fossil fuel reserves that result in increase of crude oil prices as well as emission of green house gasses. Bioethanol is one of the promising unconventional sources of bioenergy that can be produced from agricultural and other feedstocks including plant material, crop residue and industrial lignocellulosic waste Kans grass (Saccharurn sponteneum), a variety of switch- grass is a perennial energy crop growing in waste land and has good potential to be used for energy production. Kans grass grows fast, does not require any maintenance and once planted it can supply biomass through out the year. Therefore, in the present study efforts were made to utilize Kans grass biomass for ethanol production by using different steps including pretreatment, hydrolysis and fermentation. Two pretreatment procedures: dilute acid and alkali were utilized for hemicellulosic sugars as well as lignin removal respectively. An effort was also done for the crude cellulase enzyme production from Trichoderma reesei and it was further used for the depolymerization of total reducing sugars of pretreated Kans grass biomass. The solubilized sugars obtained after acid pretreatment as well as enzymatic hydrolysis were further used for the ethanol production by Pichia stipitis as well as Saccharomyces cerevisiae. An adaptive strain to toxic compounds was also developed. Kans grass, a novel substrate, was evaluated for composition analysis such as holocellulose, lignin, ash, moisture and other contents. A high holocellulose (cellulose and hemicellulose together) content (64.73%) make it suitable to utilize as sugars source The Moisture and ash content of Kans grass biomass were calculated to be 4.70±0.55 and 2.10±0.2% respectively while lignin content was estimated as 25.15±0.55%. The other components were found to be 3.32±0.25 % which may contain different extractives such as proteins and /or fatty acids. Hence Kans grass may be used as a potential substrate due to its availability throughout the year with out making burden on the food land. Kans grass (5% w/v) was treated with different concentrations of .H2SO4 (0.5-2% v/v) at moderate temperatures (100-120°C) for various length of reaction time (30-120 minutes). The total reducing sugars concentration in liquid residue was found to be in the range of 2.60-8.15 g/1 at 100°C. When the pretreatment temperature was increased to 120°C the total reducing sugars concentration was also enhanced and was found to be in 1 the range of 7.23-11.66 g/l. Different toxic compounds (furfural and acetic acid) concentrations were also determined (which may inhibit the microbial growth during fermentation) in acid hydrolysate which were observed to be in the range of 1.17-2.34 and 0.12-1.8 g/1 for furfural and acetic acid respectively at all pretreatment conditions. This range of toxic compounds were minimized to 0.32-1.23 g/l (furfural) and 0.05-0.87 g/1 (acetic acid) after detoxification process by using Ca(OH)2. Pre-treatment condition corresponding to 2% H2SO4 concentration at 120°C and reaction time of 90 minutes was found to be the optimum condition that gives maximum total reducing sugar (TRS) concentration (11.66 g/1) in the acid-hydrolysate. However the sugar yield% was observed to be 17.2%. The Kans grass biomass after acid pretreatment was also analyzed for composition change as well as configurational change (SEM analysis). Further this hydrolysate which is composed of mixture of sugars (majorly pentose) was utilized as carbon source for hexose as well as pentose utilizing yeast to produce ethanol. NaOH pretreatment of Kans grass (5% w/v) was carried out with low concentrations of NaOH (0.5- 2%) for different duration of time (30-120°C) at variable temperatures (100-120). At 120°C this range of lignin removal was observed to be 14.25-79.30%. It was also observed that at 120°C with -reaction time of 120 minutes for all concentrations of NaOH (0.5-2%), more than 70% (70.13-73.23%) delignification was observed. The maximum lignin removal (79.31%) was observed at 90 minutes of duration at 120°C with 2% NaOH. In comparison of the H2SO4 pretreatment, NaOH assisted pretreatment was found to be more effective in terms of removal of the lignin which was observed to be 79.3 1 %, however it was .found to be 28.03% after H2SO4 pretreatment. Thus, NaOH pretreatment favors more lignin removal whereas H2SO4 pretreatment may be more suitable for hemicellulosic sugars liberation. In . some studies including sorghum straw and cotton stalks 65-77% of lignin removal was observed with NaOH pretreatment. Hence, NaOH pretreatment with Kans grass improved the delignification. T reesei (NCIM 1052) was used for CMCase production with different carbon sources (cellulose, glucose, lactose and xylose), pH (4, 5 and 6), and temperatures (25, 28 and 30°C) and for variable duration of time (0-8 days). 28°C was found to be the optimum temperature with for maximum CMCase production (1.46 U/ml) with cellulose as substrate at pH 5. This crude preparation of enzymes also composed of xylanase (6.6 U/ml) activity with total cellulase activity of 1.14 U/ml. Saccharification of H2SO4 treated biomass with 20 FPU/gdb crude enzymes was studied with the biomass loading of 1, 2, 2.5, 5 and 6% (w/v) in 0.05 M citrate buffer at ii . 50°C. The best condition for saccharification was observed at 2% biomass (dilute acid pretreated) loading after 84 hours with 69.08 mg/gdb TRS formation. The maximum recovery of xylose sugars (14.13 mg/gdb) was also observed at 2% biomass loading. When same experiment was conducted with pure (commercial) cellulase preparation on acid pretreated biomass (20 FPU/gdb with biomass loading in the range of (1-6%) the TRS released was found to be maximum at 2% biomass loading (TRS, 63.21 mg/gdb). When untreated biomass (prior to acid treatment) was directly treated with enzyme preparation, the corresponding maximum TRS released was found to be 34.67 and 31.82 mg/gdb under same condition with crude and pure enzyme respectively with negligible amount of xylose released. It was observed that TRS released for untreated biomass was found to be much less as compared to acid pretreated biomass, that may be due to presence of lignin component in untreated biomass which acts as obstacle in enzymatic action, however this lignin was observed to be minimized after H2SO4 pretreatment and that resulted comparatively more TRS after enzymatic hydrolysis. As NaOH pretreatment of Kans grass biomass, at 120°C (all NaOH concentration and residence time) resulted in removal of more than approximately 50% of lignin, hence NaOH pretreated biomass from these conditions were utilized for further saccharification with 20 FPU/ gdb of crude enzyme mixture (derived from T. reesei with 1.14 FPU total cellulase activity, 1.46 U/ml of CMCase and 6.6 U/ml of xylanase activity) in citrate buffer for 96 hours released 350mg/gdb (63% w/w) TRS under 5% biomass loading for NaOH pretreated biomass (0.5% for 120 minutes). Hence, in comparison of dilute H2SO4 pretreatment, NaOH pretreated biomass was observed to be more efficient for enzymatic hydrolysis. An adaptive strain of P. stipitis was developed, adapted against inhibitors by sequentially transferring and growing the cell in the ' media containing ' increase concentration of non detoxified acid hydrolysate (20, 40, 60. and 80%) with supplementation of other media components. An adaptive strain that tolerated 60% non detoxified acid hydrolysate showed better ethanol yield (0.28 g/g) as comparison to wild strain (0.23 g/g) in detoxified acid hydrolysate media. The hydrolysate (11.66 g/1) obtained after optimized condition of dilute acid pretreatment was evaluated for the bioethanol production by increasing the sugars concentration to 60 and 90 g/l using adaptive P. stipitis. At 60 g/l the maximum ethanol yield was found to be 0.41 g/g after 72 hours whereas at 90 g/1 it was 0.38 for same duration of time. The sugars hydrolysate obtained after enzymatic hydrolysis (with- crude enzyme) of dilute acid pretreated biomass was supplied for fermentation by S. cerevisiae and P. stipitis, 111 the ethanol yield was found to be 0.46 g/g and 0.43 g/g respectively after 28 hours. An ethanol yield of 0.44 g/g was also observed after 24 hours when hydrolysate obtained (after enzymatic hydrolysis) from NaOH pretreated biomass was fermented by using P. stipitis. Additionally, after fermentation with S. cerevisiae the ethanol yield was observed to be 0.38 g/g after 32 hours. This yield is comparable to the other studies carried out with bioethanol formation. From the above study it can be concluded that Kans grass biomass which is a weedy plant, may be used as substrate for renewable energy production due to higher carbohydrate content. Dilute H2SO4 pretreatment of Kans grass biomass is more favourable for liberation of maximum soluble - sugars. An improved delignification was observed during NaOH pretreatment and after enzymatic saccharification it resulted the liberation of more sugars in comparison of dilute H2SO4 pretreated biomass. Further, crude cellulase enzyme produced from T. reesei in present study is equally efficient that of commercial enzyme. As enzyme production cost contributes 40% of total cost of bioethanol synthesis process. This higher cost may be due to involved enzyme purification steps. Thus, utilization of crude enzyme (as produced in the present study) may cut down this cost in some extend. A toxic compound tolerate strain was also developed which showed much improved bioethanol yield as well as shorter fermentation of time in comparison .to that of wild strain.