Preparation, Process Optimization And Scale Up For Wettable Powder Formulations Of Paenibacillus Polymyxa And Bacillus Marinus

Wettable powder is the main type of microbial pesticide formulations in China. However, fewer studies were reported about the formulation, process optimization and scale up in microbial pesticide. Paenibacillus polymyxa fine granular (PPFG) formulation, with independent intellectual property rights in China, is the first microbial pesticide developed by Paenibacillus strain. PPFG can control many soilborne diseases; however, the large-scale application of PPFG is limited because of its inconveniences. In addition, although there is little study about microbial pesticide with marine microorganisms as biocontrol agents, Bacillus marinus B-9987 has broad application prospects as microbial pesticide. So studies about the preparation and industrial production of P. polymyxa and B. marinus microbial pesticide should be carried out urgently.With P. polymyxa and B. marinus as study objectives, formulation, milling, spray-drying of microbial pesticide wettable powder were investigated systematically in this paper, and antifungal substance of P. polymyxa was also investigated. Industrial production processes of P. polymyxa wettable powder and pilot production processes of B. marinus wettable powder were established together. The results in the paper not only provided technical support for the industrialization of P. polymyxa and B. marinus wettable powder, but also provided reference for other microbial pesticide.Suspensibility is an important property of wettable powder and poor suspensibility of P. polymyxa wettable powder had hindered its industrial production in larger scale. It was showed that jet milling and surfactant A could synergistically improve the suspensibility of P. polymyxa wettable powder M-1000-P1 (prepared by spray-drying the mixture of carrier M-1000 and P. polymyxa HY96-2 broth from factory). On the other hand, no obvious adverse effect of jet milling on P. polymyxa population in powder was observed. Besides, surfactant A has better biocompatibility than those of EFW(?), D-425(?), sodium dodecyl benzene sulfonate or tea saponin. When jet milling powder was added with surfactant A at 5%(w:w), its suspensibility was improved from 58.3% to 81.1%.When P. polymyxa wettable powder was spray-dried at small (maximum water evaporation of 5kg/h), medium (maximum water evaporation of 50kg/h) or large dryer (maximum water evaporation of 800kg/h) with inlet temperature 210～230℃and outlet temperature 80～90℃, bacteria survival rate above 75% and moisture content below 5% were obtained. It implies that the spray-drying process of P. polymyxa at small and medium dryer can be scaled up to production scale. On the other hand, jet milling process at laboratory scale (5kg/h) was successfully scaled up to production scale (50kg/h) under the same condition (air pressure was 0.8Mpa and grinding chamber was at room temperature). Thus, the preparation process of P. polymyxa wettable powder in production scale was established. The product has good quality properties with bacteria population of 2.1×109CFU/g, moisture content of 3%, wetting time of 95 seconds, suspensibility of 76.1% and fineness that all particles could pass through a 325-mush sieve. All the properties meet the requirements of pesticide wettable powder in China. P. polymyxa wettable powder had good control efficacies on tomato bacteria wilt (91.66%), watermelon fusarium wilt (83.75%), angular leaf spot of cucumber (81.14%) and watermelon anthracnose (77.01%), respectively, which indicates that P. polymyxa wettable powder is a microbial pesticide with broad control spectrum to plant diseases.Furthermore, P. polymyxa polysaccharide extract (PPPE) has obvious suspensibility improving effect on carrier kaolin clay, M-1000 and N-1000. In addition, PPPE has protective effect for P. polymyxa spores from heat and ultraviolet (UV). Based on the above results, a strategy, improving the suspensibility of P. polymyxa wettable powder and heat or ultraviolet stability of P. polymyxa spore by PPPE, was developed. Using the suspensibility improving effect of PPPE and wettability improving effect of adjuvant S, the components of P. polymyxa wettable powder were rescreened and further optimized with mixed experiment design. Then, a new formulation and preparation process were developed which had low producing cost because milling was no longer needed. The new product has eligible wettability (99 seconds of wetting time) and suspensibility (71.7%).Fusaricidin is a kind of lipopeptide produced by many P. polymyxa strains. Fusaricidin A was firstly obtained from a commercial strain—P. polymyxa HY96-2. Fusaricidin A has good control effect on gray mold of cucumber:250μg/ml and 62.5μg/ml of fusaricidin A had control effect of 95.0% and 63.3%, which were better than those of pyrimethanil (one chemical pesticide as control). The results of fusaricidin A indicate that the inhibition activity or antibiotic content of P. polymyxa formulation could be considered as a new product quality control indicator.B. marinus B-9987 formulation with carrier of N-1000 had better suspensibility and B-9987 formulation with carrier of kaolin clay had worse wettability. Organosilicon had better biocompatibility with B. marinus B-9987 and could improve the suspensibility of B. marinus B-9987 powder formulation (N as carrier). The spray-drying process at laboratory scale could be scaled up to pilot scale. When B. marinus wettable powder was spray-dried at medium dryer with inlet temperature at 230℃and outlet temperature 80～90℃, bacteria survival rate above 70% and moisture content below 5% were obtained. Then, pilot production process of B-9987 wettable powder was established and the product had a eligible quality performance with bacteria population of 1.5×1010CFU/g, moisture content of 3.5%, wetting time of 56 seconds, suspensibility of 84.4% and fineness that all particles could pass through a 325-mush sieve. The field incidence control efficacies of B. marinus powder on tomato bacterial wilt and melon anthracnose were 74.42% and 63.31%, respectively, which suggests that B. marinus wettable powder has broad application prospect in plant disease control.