Breeding And Fermentation Process Optimization Of Lturin A Producing Strain

Iturin A, one of the Bacillus lipopeptides, is heptapeptide linked to aβ-amino fatty acid chain with a length of 14 to 17 carbons. The amino acid sequence of iturin A is Asn-Tyr-Asn-Gln-Pro-Asn-Ser. Iturin A exhibits the antifungal activity and it can be served as a versatile weapon for plant disease biocontrol.In this study, strain 3-10 with a broad anti-fungal activity was isolated from soil and identified as Bacillus subtilis according to morphological and physiological characteristics and nucleotide sequence of 16S rRNA. The antifungal compound was identified as iturin A according to the physicochemical property and LC-MS. In pot tests, strain 3-10 could promote the growth of the plant and biocontrol the watermelon fusarium wilt. These results highlighted the strain 3-10 had the huge potential to be applied as growth promotion and biocontrol agent.A microtiter plate-based bioassay method was established for highthroughput screening the high-yield iturin A mutants. In this study, strain 3-10 was treated by Ultraviolet (UV), Diethyl sulfate (DES) or Nitro so guanidine (NTG) and the mutant strains were initially selected from LB medium (solid) containing Cu2+, thiourea and crude iturin A. Then the mutants were firstly screened through high-throughput cultivation method in 96-well microtiter plate, and then the high-yield mutants were further screened by fermentation and HPLC detection. the mutant UDI-2 with iturin A production improved by 26.8% of strain 3-10 was obtained. More work was required for strain breeding.The fermentation medium and cultivation conditions were optimized for B. subtilis 3-10 in flasks. The optimum fermentation medium contains (g/L):glucose 20, soybean meal 90, K2HPO4·3H2O 0.5, MgSO4·7H2O 0.5, MnSO4·H2O 0.005. The optimum cultivation conditions:temperature 28℃, initial pH 7.5, inoculum age 10-12 h, the inoculum amount 2%(v/v), the broth content 30 mL/250 mL flask. Under the optimum fermentation medium and conditions, the biomass of B. subtilis 3-10 was 1.24×1010 CFU/mL, the iturin A yield was improved by 7.48 fold than the KMB medium in flasks and reached 6.10 g/L. In 3 L-scale bioreactor, the biomass of B. subtilis 3-10 was 1.51x1010 CFU/mL, the yield of iturin A was 6.53 g/L.1% of glucose was added to the culture at 12th hour, the yield of iturin A was improved by 12.4% compared to CK.It was found that B. subtilis 3-10 could also produce poly-y-glutamic acid (y-PGA) in MK medium. In this study, rapeseed meal was used as basic substrate under solid state fermentation (SSF) for B. subtil is 3-10 co-producing iturin A andγ-PGA. In SSF, theγ-PGA production achieved 51.3 g/kg-wet weight culture, and the number of viable cells reached 8.67×109 CFU/g-wet weight culture, the iturin A production was 8.65 g/kg-wet weight culture. The SSF culture had both biocontrol and growth-promoting effects on the plant growth. Therefore, the rapeseed meal, a low-cost byproduct, was improved through fermentation by B. subtilis 3-10 and served as a more efficient bio-organic fertilizer. The new fertilizer could replace the chemical fertilizers and pesticides abused in the agricultural production and protect environment against agricultural pollution.