STUDIES ON PRODUCTION OF ETHANOL FROM KANS GRASS BIOMASS USING SEQUENTIAL-CO-CULTURE SYSTEM

Abstract

The renewable and environment friendly source of energy is now mandatory for the sustainable development of a society. The energy demand is increasing worldwide and the natural fossil fuel resource is depleting. Therefore alternative fuel source is required to bridge the gap between the demand and supply. An unconventional source of energy molecule, ethanol is gaining importance as liquid transportation fuel. It is considered as clean fuel runs in zero carbon cycle. It is produced from sugar via fermentation. The most abundant and cheap source of sugars is lignocellulosic biomass. Ethanol has traditionally been produced from sugar cane and sugar beet juice or from various starch-containing raw materials like corn or wheat. But there is potential conflict between land use for food (and feed) production and energy feedstock production. The utilization of lignocellulosic biomass for ethanol production overcomes this conflict and is less expensive than the conventional agricultural feedstock. Lignocellulosic rawmaterials are grown in all parts of the world. Among the various lignocellulosic raw materials perennial grasses (C4 plant) are promising because of high yields, low costs, and good suitability for low-quality land with almost no requirement of water supply for its growth, availability throughout the year and no net green house gas production. Kans grass (Saccharum spontaneum) possessing all these properties wasused as the lignocellulosic raw material for the present research work. Two main technical problems have been identified from the literature for lignocellulosic biomass to ethanol conversion process: (1) efficient release of soluble sugars from the polymeric structure of lignocellulosic biomass with minimum or no toxics generations and (2) utilization of maximum sugar potential for ethanol production. In the present study both of these problems have been tried to address successfully. Few preliminary experiments were conducted to obtain important parameters (mentioned in Section A of the Results and Discussion) followed by a novel fractionating hydrolysis process for fermentable sugar production (Section B) and finally a sequential-co-culture system was developed to utilize both xylose and glucose sugars in a single reactor (Section C). Kans grass, a novel raw material was examined for compositional analysis for identification of its sugar potential and found as (% dry weight basis): cellulose 43.78±0.4; hemicellulose 24.22±0.5; acid insoluble lignin 23.45±0.3; acid soluble lignin 2.85±0.4 and ash 4.62±0.2. The total carbohydrate content (i.e cellulose and hemicellulose content) of Kans grass was found as ii 68 % on dry weight basis. This justified the selection of Kans grass as raw material for ethanol production. Toinvestigate the effect of acid concentration, biomass loading and reaction time for releasingreducing sugars from Kans grass a 23 rotatable central composite design was adopted for designing the experiments and response surface methodology was used to optimize the process.The optimum acid concentration, biomass loading and reaction time were found to be 61.10%(w/w), 10.80% (w/v) and 45 min respectively. The batch processing of all the experiments werecarried out at normal boiling temperature of water under standard atmospheric pressure. Underthese conditions significantly high total reducing sugar yield (83.5 % w/w)was obtained ontotal carbohydrate content basis. To utilize hemicellulose fraction of Kans grass single step dilute acid treatment was applied and its fermentation to ethanol was carried out by Pichia stipitis. 0.204 g/g total reducing sugar was obtained under experimental conditions in which 0.172 g/g was the pentose sugars. After conditioning fermentation was conducted and found that 74% of xylose wasconverted to ethanol with a yield of 0.429 g/g and productivity of 0.231 g/L h.The appropriate mathematical models for cell and ethanol production rate have been identified to explain theoretically the bioconversion of Kans grass hemicellulose acid hydrolysateto ethanol and validated statistically. During hydrolysis of lignocellulosic biomass various degradation compounds are generated like furfural, hydroxymethyl furfural and phenolic compounds. These compounds are known to be inhibitory and affect the growth of microorganisms. The experiments were designed to identify the tolerable concentration of these compounds. It was found that only 9 % and 7 % reduction in specific growth rate of P. stipitis was occurred with 0.4 g/L furfural and 0.5 g/L vanillin concentration respectively. Similarly 8 % and 7 % reduction in specific growth rate of Z. mobilis was observed with 1 g/L HmF and 1.0 g/L vanillin concentration respectively. These concentrations of inhibitors were considered as tolerable to the respective microorganisms. Further experiment was designed to obtain critical level of ethanol concentration for P. stipitisandZ. mobilisgrowth. Appropriate model was used to obtain maximum concentration of ethanol over which the growth of microorganisms ceased. It was found that 37 g/L and 104 g/L ethanol concentration was the maximum over which no growth of P. stipitis and Z. mobilis was observed respectively. iii A novel single vessel multi-step fractionating hydrolysis process was developed in the laboratory.The main objective of the investigation was oriented towards the maximum soluble sugar extraction with minimum toxic compounds generation from KGB. The sequential addition of increased sulfuric acid concentration from 1 to 35 % v/v (total nine steps) along with direct steam insertion at 100°C (i.e. at atmospheric pressure) to the reaction system for 30 min each step was successfully used to extract 95.3% of the total reducing sugars (TRS) available in the KGB in the form of carbohydrate polymer. After analyzing the hydrolysate of the entire process steps two main sugar streams were generated as xylose rich fraction (XRF) and glucose rich fraction (GRF). The fermentation media prepared using these sugar streams without any detoxification process contained significantly low concentration of toxics due to moderate temperature and short exposure time. Fermentation of XRF and GRF media were conducted by P. stipitis and Z. mobilis respectively. The specific growth rate (μ), yield (Yp/s) and specific productivity (qp) of ethanol was found as 0.121 h-1, 0.427 g/g and 0.731 g/g/h in XRF media and 0.173 h-1, 0.443 g/g and 1.179 g/g/h respectively in synthetic media. Similarly μ, Yp/s and qpwas found as 0.243 h-1, 0.476 g/g and 3.587 g/g/h in GRF and 0.338 h-1, 0.494 g/g and 4.805g/g/h in synthetic media respectively. Sequential-co-culture technique was investigated in this experiment for the production of ethanol using XRF and GRF as generated from the aforementioned investigation. The consortium of P. stipitis and Z. mobilis was used to develop a suitable sequential-co-culture system. The P. stipitis cells and respective fermentation media (XRF)were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media (GRF) were fed to the same vessel. Different strategies have been followed and experiments were conductedinitially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentationmedia and Kans grass hydrolysate media to compare the kinetic parameters. The sequentialaddition of cultures with their respective media and imposed process conditions, showed better utilizationof total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic conditionforZ.mobilis fermentation were found significant. The average ethanol yield (Yp/s) and overall volumetricproductivity (rpo) were found as 0.453 g/g and 1.580 g/L/h respectively for Kans grass hydrolysate mediaand 0.474 g/g and 2.901 g/L/h respectively for synthetic fermentation media. The significance of the present study revealed that the novel process of fractionating hydrolysis of KGB generated toxics in very low quantity and no detoxification process was involved before fermentation. Further the developed sequential-co-culture system of P. stipitis and Z. iv mobiliswas found suitable and successfully used for bioconversion of xylose and glucose in a single bioreactor system.