Research On Mutagenesis Breeding Of Lactobacillus Bulgaricus And D-Lactic Acid Production By Simultaneous Saccharification Of Corn Stover

As one of the most abundant lignocellulosic resources,straw is mainly composed of cellulose,hemicellulose and lignin.After hydrolysis of cellulose,reducing sugar can be produced for fermentation.For example,straw is used as raw material for bio-refinery ethanol,butyric acid and lactic acid and other chemicals that are the main direction of high value utilization of straw.D-lactic acid is a conformation of lactic acid and is widely used in the fields of medicine,industry and perfume synthesis,with the rise of the poly lactic acid industry,the demand for D-lactic acid has increased year by year.In the process of straw bio-refining D-lactic acid,there are problems of low yield,more by-products,and low saccharification rate of straw.In order to solve the above problems,strain WB01,which the L-lactate dehydrogenase was knocked out plasma mutagenesis technique and UV mutagenesis technology as the main treat motheds,D-lactic acid high-yield mutants was successfully bred.Subsequently,sulfamic acid?NH₂SO₃H?was used as the straw pretreatment reagent,NaCl as the auxiliary treatment reagent,A new green high-efficiency corn straw pretreatment process was established.Finally,at last the D-lactic acid fermentation process from corn stover synchronous saccharification was established by pretreated corn stover as fermentation medium.1.The optimal growth condition of bacteria WB01 was:growth temperature 37?,rotation speed 120 rmp,pH value 6.5;the optimal fermentation conditions was:bactericidal amount 5%,fermentation temperature 42?,initial glucose concentration 5%,D-lactic acid production under these conditions was 36.9 g/L.2.The conditions of room temperature plasma and UV mutage nesis:The treatment time was 60 s,the irradiation distance was 3 mm,the ultraviolet irradiation time was 60 s,and the irradiation vertical distance was 15 cm,and the UV wavelength was 254nm.After two cycless of mutagenesis,two positive mutant strains were screened,and the D-lactic acid yield increased by 27.1% and 25.5% respectively.After re-mutation,three positive mutants were obtained and D-lactic acid increased by 46.3%,44.2% and 46.3% respectively.After 6 generations of stable genetic detection,W209 could maintain stable inheritance.The D-lactic acid production was stable at 54 g/L under the conditions of temperature 42?,inoculation volume 5%,and initial sugar concentration 7%.3.By using corn stover as raw material,sulfamic acid?NH₂SO₃H?as sulfonating reagent and NaCl as the compounding reagent for corn stover pretreatment,the optimal pretreatment process was established:was 4%NH₂SO₃H and 3%NaCl.The treatment time was 60 min,the treatment temperature was 110?,the cellulase addition amount was 30FPU/g,the enzymatic hydrolysis was 72 h,the glucose yield was 80.94 mmol/L,and the glucose conversion rate was 71.92%.The conversion rate of corn stover glucose was increased by 36.76%,75.74% and 242.43%.The structure changes of pretreated corn stover were by scanning electron microscopy?SEM?,X-ray?XRD?and Fourier transform infrared spectroscopy?FTIR?.The results showed that the surface of corn stover became loose and the pores increased,the crystallinity decreased and recrystallization occurred.The absorption peak of the cellulose zone increased and the absorption peak of the hemicellulose zone became weak.4.In order to improve cycle times of W209,we studied the process flow of sodium alginate immobilized lactic acid bacteria with sodium alginate.The results showed that0.75%sodium alginate,3%CaCl₂,voltage was 600 V,pump speed was 1.96 ml/min,and the vibration frequency was 80 Hz.The immobilized microcapsules maintained the good activity of Lactobacillus bulgaricus.After continuous fermentation for two times.The acid production of microencapsulated Lactobacillus bulgaricus after 2 consecutive fermentations reached 59.6 g/L and 56.4 g/L respectively.