Kwanruthai Malairuang

The screening of exopolysaccharides (EPS) produced by 52 isolates of endophytic and basidiomycete fungi was studied on two different media, PDB and PYGM. There were five isolates that could produce dried exopolysaccharide of more than 4 g/L (S. commune LF01962, LF01001, LF01581, Pycnoporus sp. MMCR00271.1, Pestalotiopsis sp. PP0005). The molecular weights of these exopolymers were found to be in the range of 2.5–500 kDa. These five exopolysaccharides, produced by five different fungal isolates, showed non-cytotoxic activity against NCTC clone 929 and HDFn cell lines. The selected fungal isolate of S. commune LF01962 was used for further optimization of different medium compositions affecting exopolysaccharide production using statistical methods. Among four conditions tested in the first step (xylose + peptone, glucose + (NH₄)₂HPO₄, fructose + peptone, and mannose + yeast extract), mannose + yeast extract resulted in the highest exopolysaccharide production of 5.10 ± 2.00 g/L. In the second step using Plackett–Burman design, the optimal medium for S. commune exopolysaccharide production was found to consist of 40 g/L glucose, 5 g/L mannose, 20 g/L (NH₄)₂HPO₄, 5 g/L yeast extract, 3 g/L monosodium glutamate, 0.5 g/L KH₂PO₄, 0.5 g/L K₂HPO₄, 0.2 g/L MgSO₄, 1 mL/L trace elements, and 3 mL/L vitamin solution, which resulted in 8.16 g/L exopolysaccharide production. Exopolysaccharide production in a 5 L bioreactor using small pellets as seed inoculum was found to produce 18.28 g/L exopolysaccharide.

Vip3A (vegetative insecticidal protein) is a representative member of the Vip3 family, which is widely used for lepidopteran pest control. This Vip3A protein, a non-growth-associated protein, is an effective bioinsecticide against insect pests, but there is relatively little information about its production processes at large scales. Hence, the effects of environmental factors on Vip3A production by Bacillus thuringiensis Bt294 (antifoam agents, shaking speeds, agitation and aeration rates), as well as controlling physical conditions such as the lowest point of dissolved oxygen and controlling of culture pH, were observed in shaking flasks and bioreactors. The results showed that antifoam agents, flask types and shaking speeds had significant effects on Vip3A and biomass production. Cultivation without pH control and DO control in 5 L bioreactors at lower agitation and aeration rates, which was not favorable for biomass production, resulted in a high Vip3A protein production of 5645.67 mg/L. The scale-up studies of the Vip3A protein production in a pilot-scale 750 L bioreactor gave 3750.0 mg/L. Therefore, this study demonstrated the significant effects of agitation, aeration rates and culture pH on Vip3A production by B. thuringiensis Bt294. Balancing of physical conditions was necessary for obtaining the highest yield of Vip3A by slowing down the production rate of biomass. Moreover, this Vip3A protein has high potential as a bioinsecticide for lepidopteran pest control in organic crops. This information will be important for significantly increasing the Vip3A protein concentration by the bacterium and will be useful for field application at a lower cost.