Study On The Breeding Of High Lactic Acid Producing Strain By Microwave Mutagenesis And The Fermentation Producation Of Lactic Acid From Starch Wastewater

To solve the problems in the treatment and resource recovery of the starch industry wastewater and the mutation breeding of microogranism, this study focuses on the microwave mutation breeding technology of lactic acid bacteria with high L-lactic acid yielding ability, based on molecular biology and microwave irradiation technology. The technology of breeding lactic acid bacteria with high lactic acid yielding ability by microwave mutation was developed, the ralted experimental device was designed and developed, breeding of strain with high lactica acid yielding ability was developed; the mutant strain was characterized by physiological, biochemical and molecular biological methods, the process and mechanism of mutation of lactic acid yielding baceria by microwave was discussed, major influencing factors and rule in microwave field for the strain with high lactic acid yielding ability was investigated. Meanwhile, the technology of fermentation production of L-lactic acid by using wastewater of starch industry was studied, activated carbon fibers (ACF) was introduced into the frementation system as carrier immobilizing lactic acid bacteria, modifying method of HNO3-Fe(Ⅲ) of ACF was consructed. 315 anion exchange resin was used to separate the lactic acid form the fermentation broth.Lactobacillus casei subsp, X1-12, was used as the starter strain for mutation, a microwave irradiation breeding device with continuous power and adjustable water circulating cooling was designed to counteract the heating effect produced by the microwave irradiation. Under a microwave power of 400W and irradiation length of 3min, a mutated strain W4-3-9 with high-yielding L-lactic acid was obtained by screening. Compared with the starting strain X1-12,the L-lactic acid production of W4-3-9 was increased by 58.0% to a concentration of 115.8g/L.The strain maintained the ability to produce a high L-lactic acid level after 10 generations. Cell surface morphology and DNA structures of parental and mutated strains were observed by atomic force microscopy (AFM). Amplified fragment length polymorphism (AFLP) DNA fingerprint experiments were conducted on X1-12 and W4-3-9, polyacrylamide gel electrophoresis suggested difference in AFLP band pattern between the mutated and non-mutated strains, and the order of different band of d21 were checked, BLAST sequence similarity index in GenBank was conducted, the results revealed that catalytic site of lactate dehydrogenase (DHL) was changed due to the microwave induced mutation.Mutated Strain W4-3-9, which had high L-lactic acid yield, was applied in the technology of producing L-lactic acid from starch industry wastewater. The results showed that when the ratio of corn steep water and waste water was 3:7, glucose was 70g/L, initial pH was 6.8, inoculation was 5%, after anaerobic fermentation at 37℃for 72h, the yield of L-lactic acid was 62.28 g/L, and 45% of the COD of wastewater was removed, the removal of NH4+-N was 75%, B/C was increased, biodegradability.was also improved.After separating microorganism in starch wastewater with plate dilution method, four main kinds of microbe including coccus, bacillus, molds, yeast were found by the preliminary identification. Sterilization condition of microwave irradiation for starch wastewater culture medium was studied. When microwave irradiation power reached 500W, microwave irradiation time got 100s, micro-organisms were killed totally, the total number of micro-organisms was 0 cfu/mL. The loss ratio of total sugar, reducing sugar,and protein nutrition in starch wastewater culture medium are 11.76%,18.77%,7.42% , is better than the result of high-pressure steam sterilization.315 anion exchange resin was used to separate the lactic acid form the fermentation broth, after adsorption isotherm experiment and the optimization experiment, the optimal conditions were determined as follows: flow rate was 1.5BV/h, pH was 1.9, eluent was deionized water, elution rate was 1BV/h. based on the realization of the separation of lactic acid from acetic acid, citric acid, pyruvic acid and glucose, the purity was improved from72.4% to 85.3%. From adsorption to elution, the extract rate of lactic acid was 79.2%.Effect of activated carbon fibers (ACF) carrier on immobilization of lactic acid was studied by biomass immobilization fermentation. ACF surface was modified by HCl, H2SO4, HNO3 and Fe3+ with high concentration, and ACF before and after modification was characterized by XPS and the thickness of the biofilm was observed by scanning electron microscope (SEM). After HNO3-Fe(Ⅲ) co-modification, the thickness of single biofilm was 40μm. The acidic oxygen-containing functional group increased on the surface of ACF, the positive charge also increased, after modification, the surface property of ACF improved the immobilization of lactic acid bacteria. When the ACF carrier immobilizing lactic acid was used to ferment, the fermentation time reduced to 48h, compared with non-carrier, the yield of L-lactic acid increased from 62.2g/L to 68.6 g/L, providing good conditions for the separation of later lactic acid broth.Results showed that starch industry wastewater resource recovery and harmlessness can be realized by using wastewater from corn starch as fermentation medium, ACF as lactic acid bacteria carrier, separating lactic acid from fermentation broth by anion exchange resin adsorption, which brought both good economic and environmental benefits.