Studies On The Basic Technics Of L-Lactic Acid Fermentation From Surcose And Cane Molasses

According to the strain breeding theory,L-lactic acid producing mutantsadapting to fermentation of surcose and cane molasses are selected and breeded by applying ultraviolet light to mutagenize R3038.The studies mainlyon determination of contents of fermentation broth,breeding mutants, the optimal germ and fermentation culture medium and fermentation conditionsof mutants, as well as on the treatment of cane molasses.1.Malic acid and fumaric acid are the byproducts of lactic acid fermentationby Rhizopus oryzae,so it's essential to determine the contents in broth quickly and accurately,then the fermentation and product quality can be controlled.Lactic acid Determining conditions of HPLC and HS-GC are studied.Also the condition of lactic acid isomers quantitative separation by HS-GC based on L-menthol as a chiral derivation.2.The L-lactic acid concentration can achieved 79.18g/L fermented from sucrose compared to 31.53g/L fermented from cane molasses by Rhizopus oryzae 3038 provided by China Center of Industrial Culture Collection.After mutagenizing R3038 repeatly by ultraviolet light,mutants R3038-7 and R-7 were gained.The concentration of L-lactic acid achieved 90.58g/L fermented from sucrose by R3038-7 and 52.16g/L fermented from cane molasses by R-7,the yield was increaced 14% and 65% respectively.In the broth ,99% of lactic acid produced was L-form.3.The optimal preculture medium for sucrose fermentation and production medium for cane molasses fermentation were developed by orthogonal design theory; The optimal production medium for sucrose fermentation is developed by Response surface analysis,The optimal culture conditions were confirmed by single factor experiments.(1)The optimal sucrose preculture medium(g/L):glucose 60.0,urea 2.0, KH₂PO₄ 0.2,MgSO₄·7H₂O 0.25,ZnSO₄·7H₂O 0.008.The optimal culture conditions:30℃,200r/min,loading 30mL in 250mL flask,10.0g/L CaCO₃ was added to control the pH of broth,time of the germ growing: 12h;The optimalproduction medium(g/L):sucrose 150,urea 2.2,KH₂PO₄ 0.6,MgSO₄·7H₂O 0.25,ZnSO₄·7H₂O 0.008g/L,bagasse 1.5g;The optimal fermentation conditions:30℃,200r/min,loading 50mL in 250mL flask,70.0g CaCO₃ was added to control pH of the broth, 10% preculture was inoculated.On the optimal conditions,the concentration of L-lactic acid produced in flask was 114.9 7g/L.(2) The optimal cane molasses preculture medium:sucrose in the mol- asses 50.0g/L,NH₄NO₃0.4g/L,ZnSO₄·7H₂O 0.008g/L.The optimal culture conditions:30℃,200r/mm,loading 30mL in 250mL flask,10.0g/L CaCO₃ was added to control the pH of broth,time of the germ growing:12h;The optimalproduction medium:sucrose in the molasses 80.0g/L,NH₄NO₃0.6g/L, ZnSO₄·7H₂O 0.008g/L, bagasse 1.5g;The optimal fermentation conditions:30℃,200r/min, loading 50mL in 250mL flask,70.0g CaCO₃ was added to control pH of the broth, 10% preculture was inoculated.On the optimal con ditions,the conc-entration of L-lactic acid produced in flask was 69.76g/L. 4.As the complex contents,high impurities,and some deleterious substancerestrain the fermentation of Rhizopus oryzae for L-lactic acid,cane molasses must be treated.The optimal treatment condition was confirmed after treating by different methods:H₂SO₄+active carbon+yellow potassium ferrocyanide+ NaHSO₃.The L-lactic acid concentration of fermentation brothwas increaced from 52.34g/L to 61.88g/L with the treated cane molasses,the yield was increaced 18%.