Study On The Breeding Of Yeast Strains In Fuel Ethanol Production From Sweet Sorghum Stalks

Sweet sorghum is C4 plant, twice to eight times much as efficiency of photosynthesis of C3 plant. It has the characters of high yield and accumulating rapidly of sugar; It also can be used as food supplies and feedstuff,and resists bad growth conditions as drought, water logging, salinity and leanness. Producing fuel ethanol by fermentation from sweet sorghum will be an efficient way to solve the problem of energy resources.This study did a research on breeding of strains in solid-fermentation of sweet sorghum stalk including hexose and fermenting conditions; And using the sweet sorghum after fermenting as raw materials for the producing of feed, lay technical basis for the reusing of fermented leavings; The breeding of strains in fermentation of cellulose hydrolysis product xylose from sweet sorghum stalk after fermenting is a big problem, aiming at this problem, cloned five key enzyme gene of xylose metabolic pathway, and expressed TAL with activity. The research content as follows:1) Saccharomyces cerevisiae was selected as original strain and mutated by radiation of 60Co-γ, got H12, H13, H15 strain by three levels screening. Through the solid-state fermenting test of sweet sorghum stalk, H13 strain was ascertained to be the best.2) We did a research on fermentation condition of H13 strain in solid-state fermentation of sweet sorghum stalk, the result is: 300g sweet sorghum stalk, inoculation size of thalli 5‰, 68% of water content, 36℃of culture temperature, 60h of fermentati on time; Through determining, the ethanol yield reached 6.4g/100g, the reducing sugar yield of matrix was 0.31%.3) Using the sweet sorghum after fermenting as raw materials, adopting different combination of mold and the yeast H13, detertmined the best fermentation combination of Trichoderma koningii and yeast H13, and optimizated the fermentation conditions of the best strains combination. The result as follows: 150g sweet sorghum stalk with 15% bean cake and 2% (NH₄)₂SO₄, inoculums size 12%, water content 60%, , culture temperature 32℃, fermentation time 4 days, the raw protein is raised up to 21.7% from 16.5%, increasing amplitude for 31.5%, the raw fibre is taken to 12.5% from 15.8%, decreasing amplitude for 20.9%. 4) Total RNA was extracted from Pichia stipitis and Saccharomyces cerevisiae. Gene XYL1, XYL2, XKS1, TAL1, TKL1 were amplified by RT-PCR using the total RNA as template, and cloned into cloning pGM-T, transformed into E coli DH5α, then got recombined cell. DNA sequencing indicates that the five cloned genes was identical with the reported sequence on gene library, the identities is above 99%, the identities of sequence and amino acid of TAL1 were 99.90% and 100%.5) Composing expression vector pAUR123-TAL1, DNA sequencing indicated that the sequence of TAL1 in pAUR123-TAL1 was totally the same to the sequence of TAL1 in pGM-TAL1. Transform recombined vector pAUR123-TAL1 into Saccharomyces cerevisiae through electroporation transformation and TAL1 expressed in recombined Saccharomyces cerevisiae.