Pilot Study On Beauveria Bassiana Conidia Production Using Air Phase Pulsation Solid State Fermentor

Beauveria bassiana is a well known entomopathogenic fungus with a wide spectrum of insects, including both important agricultural pests and insect vectors of microbial pathogens of men, and thus have been intensively studied to develop mycoinsecticides for integrated-pest-management (IPM).Conidia are often the means for dispersal and transmission of entomopathogenic fungi; they must come into contact with, germinate on, and then penetrate the insect cuticle. So it is crucial to massly produce conidia for commercialization of fungal insecticide. Recently, production of B. bassiana conidia is mainly based on liquid-solid two-phase state fermentation, by which hyphae and blastospore is produced in liquid state fermentation, and then inoculated to solid matrix to produce aerial conidia. However, the solid state fermentation generally adopted the open-pan culture, which was easily contaminated, difficult to control the ferment parameters, and the conidia quality differed in different batches. Great attention has been paid on improvement and development of novel solid state fermentor to overcome the shortage of traditional fermentation. In our previous work, a novel solid state fermentation instrument for fungal conidia production—air phase pulsation solid state fermentor was developed according to the growth features of entomopathogenic fungi. Using this fermentor, solid substrates can be cultured in a container separating from outside environment and avoid being contamination. To evaluate the the value of the instrument in production of conidia of entomopathogenic fungi, we investigate the pilot production B. bassiana conidia using the solid state fermentor in this paper. For the purpose of improving the conidia yield and quality, the fermentation process was optimized by analyzing fermentation parameters, regulating water activity of liquid fermentation and controlling drying modes of solid fermentation product. In addition, the effect of light on conidiation in B. bassiana was investigated.The main results as follow:1. Optimization solid state fermentation conditions The solid matrix temperature is a very important factor for a ccumulating biomass of B. bassiana during solid state fermentation. It is favor to accumulate biomass when the matrix temperatures during lag phase, exponential phase and stationary phase were controlled at at 24-26℃, 27-28℃and 28-29℃, respectively, and the maximum biomass was reached after 120hr fermentation, which was 279.32 mg mycelium/g dry substrate. At this optimization temperature, the effects of water content, substrate thickness, proportion of liquid-solid substrate on the solid state fermentation was optimized using the method of orthogonal design (L₉ (3⁴) ). The result suggested that all three factors remarkably influenced the growth of B. bassiana in solid state fermentation, and the order of the three factors according to influence extent is substrate thickness > water content > proportion of liquid-solid substrate. However, there was no significant difference among these three factors each other. Statistical analysis suggested that the optimized parameter of solid state fermentation is 8 cm of substrate thickness, 0.5 L/kg of proportion of liquid-solid substrate and 55-60 % of water content.The conditions of sporulation for solid substrate was investigated and the result indicated that the highest conidial yield was reached under the condition of 25°C of the environment temperature and 63 % of relative humidity, which was 1.57×10¹⁰ conidia/g dry weight substrate and increased by 35.34 % compared to that of other solid state fermentation in our country (1.16×10¹⁰ conidia/g dry weight substrate).2. Regulate water activity to improve conidia qualityIn our previous work, appropriately reducing the water activity of media during liquid fermentation obviously improved physiological state of the mycelium and blastospore and increased biomass accumulation. By inoculating the liquid seed to solid meidia to produce aerial conidia, it was showed that the conidial yield was also increased and the conidia quality was improved. To further evaluate the feasibility of the ferment technology in practice, here it was investigated using liquid fermentor and the air phase pulsation solid state fermentor. The result indicated that adjusting the water activity to 0.96 during logarithmic growth period (at 12 hr after inoculation) also increased the biomass accumularion, and the maximum biomass (0.0166 g/mL) was higher that in fermentation with nature water activity. Then the improved liquid seed was inoculated to the solid substrate for fermentation, the biomass and conidia yield were increased by 6.6 % and 8.6 % compared to that inoculated by liquid seed with natural water activity. The quality of the improved conidia was tested and the resulted suggested that germination of was accelerated and the median germination time was shortened by 0.57 hr, the stress resistance and stockage stability were obvirously enhanced compared the normal conidia. In addition, the bioassay showed that the virulence of the improved conidia was increased and the median lethal time to Myzus persicae at the concentration of 1×10⁷ conidia/ mL was shortened by 14.47 hr compared to the normal conidia. The results demonstrated suggested that it is feasible to improve aerial conidia by inoculating the liquid seed which was reduced water activity duing liquid state fermentation, to solid meidia to fermentate.3. Drying method affect the B. bassiana conidia qualityDrying solid state fermentation is important for shortening production cycle, harvesting conidia and improvingconidia quality. Here the effects of several drying modes of ferment product on conidia water content, comtamination rate with other microbes, and conidial viability, stress resistance and virulence were evaluated. It was demonstrated that drying at 35℃for 5 hr was the optimized drying mode, by which the water content of conidia was decreased from 23.07 % to 7.44%. The dried conidia remain a very high viability with 98.33 % of the ultimate germination rate 9.60 hr of the median germination time, which was equivalent to fresh conidia. The comtamination rate of the dried ferment product is only 3.80 %, which was lower than requirement of fungal insecticides in China (< 5%). The stress resistance of the dried conidia was obviously higher than the fresh conidia. Bioassay of Myzus persicae showed that the virulence of conidia was not influenced by drying. The results demonstrated above suggested that the drying mode of solid ferment product not only shortened production cycle, but also improved conidia quality.4. Light influence B. bassiana conidiationLight is an important environment fator influencing conidiation in fungal kingdom. For the sake of furtherly optimizing coniditins of the solid state fermentation, the effect of light on conidiation of B. bassiana was studied. Incubation in total darkness leaded to continued vegetative growth and lack of reproductive structures. In contrast, growth of the fungus in continuous illumination resulted in prolific formation of conidiophores bearing abundant mature conidia. Different effect of light color on coidial yield was found and the result showed that blue light among the single color lights was most suitable for stimulating conidiation in B. bassiana. Sporulation was photoinduced in competent mycelia by a single pulse of blue light and colonies were competent only after they had grown at least 96 hr under total darkness. However, light pulse given after 144 hr of growth caused obviously decreased conidia production. 3 min of light pulse was enough for stimulating conidiation in competent mycelia, but prolonged light exposure over 3 min resulted in decreased conidia yield.All the results demonstrated in this study provide important parameters for industrialization of B. bassiana insectcides.