Optimization of Fermentation Process of Atrazine-degradation Strain DNS32and Preparation of Bacteria Preparations

Pesticide is an important agricultural means of production to ensure high-yield, which has brought huge economic benefits to the human, while excessive pesticide use lead to negative impact of the environment. Atrazine, as a herbicide, has been, and it will lead to serious pollution of the ecological environment once it sinks into the soil, air and water due to the characteristics of long residual, low bio-degradability, high speed of migration and biological toxicity. Microbial remediation is considered as the most effective means of the remediation. The current studies mainly focus on bacterium biological properties, degradation characteristics and mechanisms. Few studies focus on atrazine degradation of the bacterial of the actual production of fermentation.This study based on the practical application of atrazine-degradation strains, and made the atrazine microbial remediation technology is beyond theoretical studies. The strain DNS32used in the present investigation showed phylogenic relationships with Acinetobacter sp. which was isolated from long atrazine contaminated soil in Wuchang, Heilongjiang Province. The cheap agricultural products such as corn flour and soybean meal were the fermentation substrate of DNS32. The paper adopted One-factor-at-a-time experiment, the Plackett-Burman method, response surface method and orthogonal test to study the strain seed culture medium composition, production and fermentation conditions, collection of the carrier materials of bacteria preparations and storage conditions, and test the efficiency of biodegradation. The main results were listed as follows:1) Optimized test seed medium components (corn flour, soybean powder, CaCO3, KH2PO4, MgSO4·7H2O, NaCl) of atrazine degradation strain DNS32was used by Plackett-Burman and central composite experimental design. And through statistical screening and analysis and model prediction, the composition of the seed culture medium was determined as follows:corn meal39.49g·L-1, soybean powder,25.50g·L-1, K2HPO4,3.27g·L-1, CaCO33.00g·L-1,MgSO4·7H2O and NaCl0.20g·L-1.The result of the test also showed that the biomass (7.19×108CFU·mL-1) of seed could be cultivated through24h cultivation and inoculum size0.2%(v/v)(the bacterium suspension cultivated through12h cultivation by LB medium)by the seed culture medium.2) Optimization of fermentation conditions test through single factor test and Box-Behnken size, fermentation temperature. After statistical analysis and modeling forecasts, determine the shake flask (in the250ml triangular flask) fermentation process as following:corn flour9.96g, soybean powder6.04g, nutrient solution10mL, distilled water30mL, inoculum size6.19mL (the seed medium diluted concentration of4.2×108CFU·mL-1), fermentation temperature25℃, initial pH7.24, fermentation time48h, and the forecast fermentation production of bacteria was3.27×109CFU·g-1; the actual fermentation production was3.11×109CFU·g-1.3) Screening on solid carrier material of atrazine-degradation bacteria DNS32and testing storage conditions. Optimization of three types of carrier materials kaolin, attapulgite, humic acid mixed dosage by using the orthogonal experimental method and the results as following:kaolin1g, attapulgite0.5g and humic acid0.5g, stored at room temperature (25℃) of dark place, and the viable survival was60.86%after45days storage. The result also showed that the best protection under the anti-ultraviolet test, and the optimum storage temperature was the room temperature (25℃).4) In the actual remediation test of bacteria preparations of strain DNS32, atrazine which was soluble in acetone was sprayed on soil and the concentration of contaminated soil sample was20mg·Kg-1. The contaminated soil sample added bacteria preparations with different treatments was placed for28d in a incubator (a constant temperature30℃) avoiding light. The result showed that the optimal dosage of the actual bacteria preparations is5‰(Ratio of the quality of bacteria preparations of strain DNS32and contaminated soil) and the degradation rate of which was the best showed93.31%after28days.