Breeding Of High-yield Lactic Acid Strains By Heavy Ion Beam Irradiation And Its Fermentation Technology

Lactic acid is an important versatile organic acid,and mainly produced by microbial fermentation.As global demand for lactic acid is increasing year by year,creation of high-yield microbial strains plays an important role on the innovation development for lactic acid industry.Because of the significant resistance characteristic to saline-alkali land,sweet sorghum has become an important energy crop,which can be planted on saline-alkali land in the western region in China,and sweet sorghum stalks are rich in fermentable sugar,which can be used by microbial strain as cheap raw materials.Therefore,based on heavy ion beam mutation platform of Heavy Ion Research Facility in Lanzhou,this paper carried out breeding of lactic acid bacteria with improved tolerability via the ¹²C⁶⁺ion beam(energy=80 Me V/μ¹²C⁶⁺and LET=40kev/um),and developed relative process control strategies,aimed at producing lactic acid with low-cost using sweet sorghum as a feedstock.The specific research results are as follows:（1）Simulating sugar composition of acid-hydrolyzed sweet sorghum juice,A69strain（obtained by two rounds of heavy ion mutagenesis）was firstly used to produce L-lactic acid using glucose,fructose and mixed sugars（glucose:fructose=1:1）.The results showed that A69 strain had a good ability to produce L-lactic acid using glucose and fructose as raw material,further indicating that A69 strain can use sweet sorghum juice to produce L-lactic acid;Secondly,when using purification strategy,L-lactic acid production efficiency increased by 15%and 17%under low sugar concentration and high sugar concentration of sweet sorghum juice,comparing to the results of original squeezing sweet sorghum juice;Comparing key enzyme activities of glycolytic pathway,the results showed that when using purified sweet sorghum juice as raw material,A69 strain exhibited significantly improved enzyme activities of hexokinase（HK）,fructose phosphate kinase（PFK）,pyruvate kinase（PK）,and lactate dehydrogenase（LDH）,thus accelerating metabolic flow from glucose to lactic acid in A69 strain and further promoting L-lactic acid efficient accumulation.In order to further reduce the cost,optimized yellow bean cake powder was used（25 g/L）,and A69strain can accumulate 114.6 g/L L-lactic acid and productivity rate of 2.61 g/L/h in a 5L bioreactor when using purified sweet sorghum juice and yellow bean cake powder as raw material.（2）Based on the strategies of weakening free radicals,effect of antioxidant vitamin C addition on L-lactic acid fermentation by A69 strain was studied.The optimal addition time of vitamin C was 0 h,and L-lactic acid accumulation increased by 16%when compared with the results without vitamin C addition.The added concentration of vitamin C（75 mmol/L）was optimized,resulting in an increase of L-lactic acid by31%.By testing the total antioxidant enzyme activities,addition of vitamin C can effectively alleviate the damage of membrane and biological macromolecules by intracellular reactive oxygen species in A69 strain,and also improved key enzyme activities of glycolytic pathway,hereby promoting the efficient accumulation of L-lactic acid.Finally,A69 strain can accumulate 118.8 g/L L-lactic acid and productivity rate of 3.71 g/L/h in a 1 L bioreactor when using purified sweet sorghum juice and yellow bean cake powder as raw material.（3）Based on the combination strategy of heavy ion mutagenesis and pressure enrichment,highly tolerant strains G-1 and G-10 were obtained.The shake flask fermentation results showed that L-lactic acid accumulation of G-1 and G-10 was significantly higher than that of A69 strain under the conditions of both high glucose and low glucose concentration,which increased by 20%and 13.89%,12.21%and10.32%,respectively.The key enzyme activities of glycolytic pathway of G-1 and G-10 were also higher than that of A69 strain.In a 5 L bioreactor,G-1 strain can accumulate 126.1 g/L L-lactic acid and productivity rate of 3.5 g/L/h.The microbial droplet culture system was used to further improve the tolerant of G-1 strain via domesticating with different Na Cl concentration.Finally,improved tolerant strain GS-7,GS-8 and GS-10 were obtained,and L-lactic acid accumulation ability of GS-7,GS-8 and GS-10 were increased by 7.83%,10.5%and 15.88%comparing to that of G-1strain,respectively.In a 5 L bioreactor,GS-10 strain accumulated 129 g/L L-lactic acid and productivity rate of 4.03 g/L/h when using purified sweet sorghum juice and yellow bean cake powder as raw material.