BIOETHANOL PRODUCTION FROM LIGNOCELLULOSIC BIOMASS HYDROLYSATE BY CO-FERMENTATION

Abstract

Kans grass biomass is one of the most promising lignocellulosic biomass available throughout the year in large quantities. Kans grass biomass have up to 60% fermentable sugars in the form of glucose and xylose entrapped as cellulose (glucose polymer) and hemi-cellulose (xylose polymer). These sugars can be extracted from Kans grass biomass by acid hydrolysis process in the form of glucose rich fraction and xylose rich fraction. Zymomonas mobilis (bacteria) is used for glucose fermentation whereas Pichia stipitis (yeast) was used for xylose fermentation. However, the concentration of glucose and xylose obtained in hydrolysate by acid hydrolysis process can vary which in turn effects the fermentation process. So, the effect of initial glucose and xylose concentration on growth pattern and ethanol productivity of Zymomonas mobilis and Pichia stipitis, respectively, was studied using synthetic sugars at flask level. The microorganisms were grown to determined optimum as well as inhibitory concentration of sugars and kinetic parameters were determined. The optimum glucose and xylose concentration was found to be 160 g/L and 60 g/L for Zymomonas mobilis and Pichia stipitis, respectively. 100 g/L glucose resulted into maximum ethanol productivity of 2.06 g/L/h after 24 h of fermentation with maximum ethanol production of 49.63 g/L while 60 g/L xylose resulted into maximum ethanol productivity of 2.06 g/L/h after 24 h of fermentation with maximum ethanol production of 49.63 g/L. Inhibitory glucose concentration for Zymomonas mobilis was 400 g/L and for Pichia stipitis, it was 100 g/L xylose. At these sugar concentrations, either the growth of microorganisms slows down or arrested. The studies were also tried on bioreactor to verify the scalability of processes and its suitability for lignocellulosic biomass hydrolysate. An in-situ distillation strategy is employed to reduce the effect of ethanol concentration on P. stipitis during the sequential fermentation process with Z. mobilis. Kans grass biomass hydrolysate obtained through fractional hydrolysis technique was used for this study. First, the sequential fermentation was performed with synthetic media without ethanol removal and compared with hydrolysate media. The process resulted in low xylose utilization due to the inhibitory effect of ethanol produced from glucose fermentation as well as nutrient depletion. So, the in-situ distillation strategy was introduced to reduce the ethanol concentration below the tolerance level of P. stipitis before its inoculation, along with addition of fresh salts. Further, the kinetic performance of P. stipitis was improved during batch-wise fermentation by optimizing the agitation speed to 400 rpm and this speed was incorporated in the sequential fermentation. This modified sequential fermentation technique with an initial glucose and xylose concentration of 60 and 40 g/L resulted in an overall ethanol concentration and productivity of 45.73 g/L and 1.45 g/L/h respectively, from Kans grass biomass hydrolysate. The overall ethanol productivity, by employing this strategy, enhanced by two-fold compared to the un-optimized sequential fermentation of Kans grass biomass hydrolysate. This strategy can be applied in large-scale bioreactor studies for the fermentation of LCB hydrolysate containing glucose and xylose as mixed sugars. Sequential fed-batch fermentation was performed using a high cell density inoculum of Zymomonas mobilis and Pichia stipitis to shorten the lag phase and improve ethanol productivity from glucose-xylose mixtures. The process is performed in a single bioreactor in batch-wise, fed-batch and sequential fed-batch mode using initial high cell density inoculum of both the micro-organisms. In fed-batch fermentation, pulse of concentrated sugar (glucose or xylose) was added to bioreactor at fixed intervals to maintain a constant sugar level and prevent substrate inhibition. The fed-batch process resulted in an ethanol yield and productivity of 0.48 g/g and 7.25 g/L/h from glucose and 0.49 g/g and 1.67 g/L/h xylose, respectively. Further, the sequential fed-batch fermentation of glucose-xylose mixtures is performed with ethanol removal strategy after glucose exhaustion. The process resulted in an ethanol yield and productivity of 0.49 g/g and 2.84 g/L/h, respectively, with a total sugar (glucose + xylose) consumption of 209.29 g/L in a short time of 36 h. The current process is great alternative for the conventional co-fermentation process to ferment lignocellulosic biomass hydrolysate with a high sugar (glucose + xylose) content in less time to produce bioethanol. Lab scale bioreactors gives good ethanol yield and productivity and it can be further scaled up on pre-pilot scale level bioreactors. So, bioethanol production from glucose and xylose has been performed on 20L scale in 50L bioreactor using Zymomonas mobilis and Pichia stipitis. 60 g/L glucose resulted into maximum ethanol productivity of 1.23 ± 0.15 g/L/h after 24 h of fermentation with maximum ethanol production of 29.46 ± 3.71 while 60 g/L xylose resulted into maximum ethanol productivity of 1.16 ± 0.01 g/L/h after 24 h of fermentation with maximum ethanol production of 27.73 ± 0.12 g/L.