Development of hybrid yeast strains for the production of bioethanol from lignocellulosic biomass

Abstract

In recent years, lignocellulosic biomass is considered as a potential feedstock for the production of bioethanol because of its abundant availability, low cost and renewable sources. However, the lack of efficient microorganism to ferment pentose and hexose sugars released from lignocellulosic materials is one of the main factors limiting the utilization of lignocellulose for actual bioethanol production. Further, commercially during fermentation, yeast cells are subjected to multiple stresses that affect the bioethanol production. Therefore, the main focus of the present research involves the development of hybrid yeast strains that are capable of fermenting hexose and pentose sugar components of lignocellulosic biomass even under the stress conditions. Various hybrid yeast strains were prepared by the fusion of protoplasts of S. cerevisiae and a variety of xylose fermenting yeasts such as Pichia stipitis, Pachysolen tannophilus and Candida shehatae. Among the various fusants, fusant RPR39 comprising of S. cerevisiae and P. tannophilus was found to be the most efficient strain giving maximum ethanol concentration of 76.8 gL-1, ethanol productivity of 1.06 gL-1h-1 and ethanol yield of 0.458 gg-1 by fermentation of glucose-xylose mixture. The fusant RPR39 was further subjected to sequential mutagenesis for improvement of its various important properties such as stability and stress tolerance using various mutagenic agents. The mutants were evaluated for their tolerance to ethanol, temperature, fermentation inhibitors and stability. Among these mutants, mutant RPRT90 exhibited high ethanol tolerance, inhibitor tolerance and reasonably good thermotolerance. The mutant RPRT90 showed improved ethanol production (73.6 gL-1) from glucose-xylose mixture with higher ethanol yield (0.461 gg-1), productivity (1.05 gL-1h-1) and sugar conversion (86.2 %) compared to fusant RPR39. The strain has shown its efficiency towards ethanol production under various stress conditions during fermentation of glucose-xylose mixture (3:1 ratio). Under the combined effect of thermal (39oC) and inhibitor stress (0.25 gL-1 vanillin, 0.5 gL-1 furfural and 4 gL-1 acetic acid), the mutant produced ethanol with a yield of 0.379 gg-1, while under combined effect of ethanol (5 %, v/v) and inhibitor stress, the ethanol yield was 0.431 gg-1. Even, under the synergistic effect of thermal (39oC), ethanol (5%, v/v) and inhibitor stress, the strain has shown to be active achieving good ethanol yield (0.3 gg-1) productivity (0.59 gL-1h-1). Further, the developed yeast strain has been successfully applied to produce bioethanol from the locally available lignocellulosic biomass, Ipomoea carnea and Lantana camara. Under the optimum conditions of biomass conversion steps, the mutant hybrid strain has shown
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encouraging results in fermenting the mixed hydrolysates obtained from both I. carnea and L. camara. The fermentation of I. carnea mixed hydrolysate containing the detoxified acid hydrolysate (18.69 gL-1 sugar) and enzymatically hydrolysed cellulosic hydrolysate (48.10 gL-1 sugars) produced 27.2 gL-1 ethanol, with ethanol yield and productivity of 0.456 gg-1 and 0.971 gL-1h-1. The ethanol produced from the mixture of undetoxified acid hydrolysate and enzymatic hydrolysate was found as 23.01 gL-1, with the ethanol yield and productivity of 0.415 gg-1 and 0.821 gL-1h-1. Thus, it has been established that the developed strain RPRT90 is an efficient strain that may pave the way to produce bioethanol from lignocellulosic biomass economically at an industrial scale. Furthermore, a comparable ethanol yield and productivity of 0.434 gg-1 and 0.412 gg-1 were achieved with detoxified and undetoxified hydrolysates derived from L. camara biomass by fermentation using RPRT90 strain.