BIOETHANOL PRODUCTION BY FRACTIONAL HYDROLYSIS AND CO-CULTURE FERMENTATION

Abstract

It is widely known that transportation sector is almost entirely dependent on fossil fuels; primarily on petroleum-based fuels (liquefied petroleum gas, gasoline, compressed natural gas and diesel fuel gas). Amount of petroleum availability is depleting day by day; therefore alternatives are needed to produce liquid fuels for reducing the future effects of the shortage in supply of transportation fuels. Term biofuel refers to solid (biochar), liquid (biodiesel, bioethanol, and vegetable oil) or gaseous (biohydrogen, biosyngas, and biogas) fuels that are mainly produced from biomass. They are renewable; most common is bioethanol (petrol additive or gasoline substitute). Bioethanol has the potential to reduce both crude oil consumption, and environmental pollution. Conventional resources for ethanol production (grains majorly) compete directly with human food materials and give rise to food vs. fuel conflict. Hence, it is essential to produce ethanol from various feedstocks and not depend solely on grains and molasses. Lignocellulosic feedstocks are obtained and harvested from agricultural wastes materials as well as forest residues crops. It consists of cellulose (40-60%), hemicellulose (20-40%), and lignin 10-25% on an average. Typically cellulose and hemicelluloses part comprise 2/3rd of the total dry biomass. Carbohydrate part (cellulose and hemicellulose) of lignocellulosic biomass can be saccharified to obtain soluble sugars and further convert it into ethanol by fermentation. Major obstacles for the commercial lignocellulosic ethanol production include a. Maximum amount of fermentable sugars (hexoses and pentoses) extraction from the lignocellulosic feedstocks b. Suitable microorganisms (more tolerant toward fermentation inhibitors) and fermentation techniques to ferment maximum amount of sugars present in the lignocellulosic biomass hydrolysate for high ethanol yield and productivity, and c. Process integration requirement to minimise the total number of steps involved in overall production. Technological approach improvements and optimisation of various factors have been given priority in various studies. Nevertheless, still, there are some challenges which need to be adequately addressed in the development of a sustainable bioethanol industry. To contribute in the pool of existing knowledge, we have tried to develop a technique which can convert lignocellulosic biomass into fuel ethanol in just two process steps, with high conversion efficiency and thus hope to bring down the ethanol production cost effectively. The biomass chosen for the present work was kans grass biomass (Saccharum spontaneum), a perennial C4 plant with high amount of carbohydrate (65.5%, w/w) compared to other ii lignocellulosic biomasses which can be converted to ethanol by suitable process technologies. It grows throughout the year on marginal and wetlands. Once planted, harvesting can be done many times for many years; re-plantation and watering are not required, ensuring the steady supply of raw materials. For saccharification, a unique technique called as ‘fractional hydrolysis’ has been developed that gives us pentose and hexose sugars as separate hydrolysate fractions. Different physical and chemical parameters (preheating time, liquid:solid/biomass loading, and number of stages) have been optimised for the fractional hydrolysis process using one-variable-at-a-time (OVAT) approach. The 8-stage fractional hydrolysis process was able to recover 84.88% total reducing sugars from kans grass biomass with minimum toxics (1.27×10-2 g furfural and 3.04×10-2 g phenolics) using sulphuric acid. To validate these results, the process was extended for other cheap and easily available lignocellulosic feedstocks (wheat straw and sugarcane bagasse) and inorganic acids (hydrochloric acid, phosphoric acid, and nitric acid) up to 30% concentration (v/v). The compositional analysis of all the three selected biomasses in the present work showed high cellulose and hemicelluloses content (63-66%). The fractional hydrolysis technique has been proven independent of feedstock type and resulting in saccharification (%): kans grass 84.88, sugarcane bagasse 82.55, and wheat straw 81.66. Among the acids used, TRS recovery was very less using phosphoric acid whereas nitric acid resulted in maximum sugar recovery, but the high cost makes it economically non- feasible. The results of both HCl and H2SO4 were comparable but comparatively lower price of H2SO4 makes it as the most suitable reagent for fractional hydrolysis process resulting in maximum sugar recovery with minimum toxics. The fractional hydrolysis process was able to recover xylose and glucose sugar fractions separately and called as xylose-rich fraction (XRF) and glucose-rich fraction (GRF) respectively. The structural characterisation of raw biomass, biomass after XRF removal, and fully treated biomass showed marked differences during all the analyses. SEM images of biomass showed surface distortion in the form of cracks and pores on the surface compared to the intact surface of raw feedstocks. FESEM gave elemental composition at each stage with a high-resolution images. SPM was used to measure the roughness analyses with 3-D imaging. FTIR spectroscopic analysis of the biomass showed a decrease in the absorption peaks indicating the loss of cellulose and hemicelluloses. XRD analysis was done to measure the changes in crystallinity indices of feedstocks during the course of fractional hydrolysis. TGA provided information about the pyrolysis temperatures of cellulose, hemicelluloses, and lignin present in iii the feedstocks along with weight loss %. These results validate the efficiency of 8-stage fractional hydrolysis technique for the maximum sugar recovery with negligible toxics. Based on these results, further fermentation studies have been carried out on kans grass biomass using 8-stage fractional hydrolysis process with H2SO4. As there is no known naturally occurring microorganism that can ferment pentose and hexose sugars simultaneously with the same efficiency, hence getting the separate XRF and GRF is of tremendous advantage. Moreover, fractional hydrolysis technique merges two different steps (pretreatment and hydrolysis) into one. Also, the concentration of toxic compounds in hydrolysate was very low; therefore, after saccharification, hydrolysate fractions can be taken directly for fermentation without any detoxification, thereby cutting down the overall production cost. In the fermentation part, Z. mobilis was selected for the hexose fermentation as it gives high ethanol yield and productivity. Also, Z. mobilis is capable of producing almost a theoretical amount of ethanol from glucose, via Entner-Doudoroff pathway under anaerobic condition and tolerates high concentration of ethanol. Among xylose fermenting organisms, Candida shehatae or Scheffersomyces shehatae has been found to ferment ethanol faster compared to other organisms; also the specific rate of ethanol production was highest among pentosefermenting yeasts. The generation of two different sugar fractions as XRF and GRF from the kans grass hydrolysis enabled xylose fermentation first and thereby eliminated lower ethanol tolerance problem of the pentose- fermenting yeasts and catabolite repression of xylose by glucose consumption as the preferred carbon source for S. shehatae. It also allowed to maintain the different aeration requirements of two organisms in co-culture system (microaerobic for S. shehatae and strictly anaerobic for Z. mobilis). Moreover, a single reactor can be used for the fermentation of both the sugars. Initially, 2-step sequential co-culture fermentation was carried out using TRS concentration up to 60 g/L in XRF and 200 g/L in GRF. The process resulted in 55.95 g/L of ethanol with average yield coefficient of 0.41 and productivity of 0.65 g/L/h from the kans grass biomass hydrolysate. It was observed that upon increasing sugar concentration, sugar consumption rate decreases to a large extent. Moreover, ethanol concentration >55 g/L is very difficult to obtain even on increasing glucose up to 200 g/L. Therefore, multi-step successive glucose feeding co-culture system was developed to overcome these problems. A multi-step glucose feeding co-culture system containing S. shehatae (for xylose fermentation) and Z. mobilis (for glucose fermentation) provided high average ethanol yield, concentration and productivity compared to the previous co-culture system. The average ethanol yield coefficient iv and overall volumetric ethanol productivity were found as 0.44 and 0.79 g/L/h respectively, and 79.59 g/L of ethanol concentration (up to stage 4) was achieved by utilising sugar up to 200 g/L. The significance of the present study revealed that a novel “fractional hydrolysis” recovered the maximum amount of soluble pentose and hexose sugars (84.88% of the total reducing sugars) separately; direct from the lignocellulosic biomass with negligible toxic products generation. Moreover, a single-reactor unique approach of co-culture fermentation using Z. mobilis (for GRF fermentation) and S. shehatae (for XRF fermentation) by utilising maximum sugars present in the kans grass hydrolysate may be able to reduce the overall bioethanol production cost further with high ethanol yield (0.44) and concentration (79.59 g/L).