High Yield Of Poly-γ-glutamic Acid From Bacillus Subtilis And Its Application

Medium and process parameters for high yield of poly -γ-glutamic acid (γ-PGA) from Bacillus subtilis CCTCC202048 were optimized under solid-state fermentation (SSF) using swine manure and dairy manure as the basis of a solid substrate, respectively. Characterization, rheological characteristics and degradation of γ-PGA were investigated. The ability and mechanism of γ-PGA that enhanced plant productivity and retained nutrients were explored. The protection of γ-PGA to Bacillus thuringiensis (Bt) from heat and UV radiation was also analyzed.Solid-state fermentation, using swine manure as the basis of a solid substrate, was carried out for high yield of γ-PGA by B. subtilis CCTCC202048. Fermentation medium and process parameters were optimized through three orthogonal array designs. The optimal medium consisted of 62.3% (w/w, dry weight basis) swine manure, 25.0% soybean cake, 5.0% wheat bran, 5.0% glutamic acid, 2.5% citric acid and 0.2% MnSO₄·H₂O. The optimal process parameters were 15.0 g medium with initial moisture content 60% and initial pH 9.0 in 250 ml flasks, inoculation at mid-log phase with a 4% inoculum level and cultivation for 48 h at 37℃. The average γ-PGA yield in triplicate was 6.0%, the ammonia content was reduced from 3.3‰ to 0.7‰, the total N, P and K kept constant, and the readily available P and K increased slightly under optimal conditions on the laboratory scale. The average γ-PGA yield in triplicate was 4.5% at compost experiment. These would lay a foundation for lessening the pollution of swine manure, increasing fertilizer efficiency and exploring a late-model organic fertilizer that retains water and nutrients.SSF medium for efficient production of γ-PGA from B. subtilis CCTCC202048 using dairy manure as the basis of a solid substrate was optimized by response surface methodology. In the first optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Wheat bran, soybean cake and glutamic acid were found to be more compatible supplement with dairy manure and positively influenced on γ-PGA production. In the second step, the concentrations of the three supplemental nutrients above were further optimized using a Box-Behnken design. The optimal medium consisted of 56.32% (w/w, dry weight basis) dairy manure, 23.35% soybean cake, 16.08% wheat bran and 4..25% glutamic acid. The optimal process parameters by orthogonal array design were 15.0 g medium with initial moisture content 60% and initial pH 9.0 in 250 ml flasks, inoculation at mid-log phase with a 8% inoculum level and cultivation for 48 h at 40℃.The average γ-PGA yield in triplicate was 4.98%, the ammonia content was reduced from 2.3‰ to 0.66‰, the total N, P and K keptconstant, and the readily available P and K increased slightly under optimal conditions on the laboratory scale. The y-PGA yield in the optimized medium was coincident with the predicted value. The average y-PGA yield in triplicate was 3.58% at compost experiment. These would make this technique promising for decontamination of dairy manure, improvement fertilizer efficiency and amelioration the ability of organic fertilizer to retain water and nutrients.The characterization of y-PGA from B. subtilis CCTCC202048 was investigated by UV, HPLC, FT-IR and NMR. The HPLC chromatograph obtained from hydrolysate of tested polymer was only one peak and was coincident with the chromatograph of glutamic acid. It indicated the polymer was composed of glutamic acid. The FT-IR chromatograph of the polymer showed no difference from that of y-PGA standard sample. It indicated the polymer was likely to be y-PGA. NMR chromatograph further proved that the polymer consisted of glutamic acid.The rheological characteristics of y-PGA solution were investigated under different concentration, temperature and pH. The y-PGA solution was a typical non-Newtonian shear-thinning fluid. With temperature raised, the viscosity of y-PGA solution decreased as exponential curve at 20-50°C. With the concentration of y-PGA solution raised, the viscosity increased as exponential curve. The viscosity was stable at pH5-ll and more sensitive to acid than alkali. Different salts at different concentration significantly effected on the viscosity of y-PGA solution. These would lay a foundation for y-PGA production from B. subtilis CCTCC202048 and application of y-PGA.y-PGA degradations, such as acidic hydrolysis, alkaline hydrolysis, hydrolytic degradation at heating, ultrasonic degradation, were studied in the paper. These degradations reduced in both y-PGA molecular weight and polydispersity. The degradation rate of y-PGA was slow in pound water sample. The degradation rates of different y-PGA concentration also showed no difference. Microorganisms did not show significant effect on the degradation of y-PGA in pound water sample. However, the degradation rate was fast in soil sample. The half-life of y-PGA in sterilized soil was 3.7 times that of in unsterilized soil. The result showed that degradation of y-PGA in the soil mainly due to microbial action. B. subtilis CCTCC202048, isolated to soil, was able to degrade y-PGA and the y-PGA depolymerase gene was also indentified by PCR. These indicated that y-PGA was an environmental-friendly polymer. These would make y-PGA promising in agriculture and environmental protection.The germination experiment about y-PGA dipping seed showed that y-PGA could enhance seed vigor. After y-PGA was used to soak pakchoi, wheat, corn and cotton seeds,their relative root length, relative bud growth, germination index, anaylase activity and catalase activity all increased to some extent.y-PGA enhanced pakchoi productivity above 30%, especially at lower nutrient levels, economized nutrient about 25% and showed the ability that retained and utilized nutrient. The suitable concentration of y-PGA was about 100-300 mg/kg at field experiment. y-PGA could chelate cation and more efficiently utilize nutrient and improve active surface area of roots, total surface area of roots and root/shoot ratio of pakchoi. y-PGA could enhance the foliar accumulation to K, Mg, P, N, also improve the foliar accumulation to Ca, Cu and Fe at lower nutrient level, while reduce Mn accumulation. y-PGA could increase the root accumulation to Mn, P, Zn and N, while decrease Cu and Mg accumulation and also reduce Fe and K accumulation at high nutrient level. y-PGA could enhance translocation of Ca, Cu, Fe, K, Mg and Zn from root to foliar. y-PGA could adjust distribution balance between foliar and root, which was the further reason that y-PGA retaind and utilized nutrient. These would make it of interest for potential application as a fertilizer synergist that retains and utilizes nutrient.In the course of spray-dried, compared to Bt formulation without y-PGA, the percentage of original crystal protein quantity remaining (OQR), the percentage of original spore viability remaining (OVR) and the percentage of original insecticidal activity remaining (OAR) of Bt formulation with 0.5% y-PGA increased 8%, 13%, 10%, respectively. After 4 h UV-irradiation, compared to Bt formulation without y-PGA, the OQR, OVR and OAR of Bt formulation with 0.5% y-PGA also increased 18%, 36%, 25%, respectively. After the sunlight for 7 d, the mortality of the Bt formulations with 0.5% y-PGA increased 24% against control without y-PGA. Under simulated rain with 50 mm, the mortality of the Bt formulations with 0.5% y-PGA increased 28% against control without y-PGA. These results would contribute to further application and dissemination of Bt biopesticide and explore a new potential application path about y-PGA.