Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/17/2007
Publication Date: 10/5/2007
Citation: Pryor, S., Gibson, D.M., Hay, A., Gossett, J., Walker, L. 2007. Optimization of Spore and Antifungal Lipopeptide Production during the Solid State Fermentation of Bacillus subtilis. Applied Biochemistry and Biotechnology. 143:63-79. Interpretive Summary: The biological control agent, Bacillus subtilis, produces environmentally resistant spores and a spectrum of antifungal compounds that are presumed to be important in biological control. Despite the available research on B. subtilis, little has been done to examine different combinations of factors in solid surface fermentation and their impact on overall antifungal activity and spore production. This paper describes the use of a two-stage optimization consisting of both factorial design and a compositie design to model B. subtilis fermentation. Optimized fermentation conditions were determined with response surface models fitted for both antifungal activity of the biological control product and final spore concentration. Using these approaches have allowed a quantitative assessment of the effects of multiple fermentation conditions on producing a B. subtilis product with both a high concentration of antifungal lipopeptides and spores. This information should be useful in developing an optimal biological control product.
Technical Abstract: Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-liter solid state fermentation (SSF) bioreactors for the production of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 26-1 fractional factorial design followed by a central composite face-centered (CCF) design. Initial SSF parameters included in the optimization were: aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature. CCF design parameters included incubator temperature, aeration rate, and initial moisture content. Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive to substrate moisture content with highest levels of each response variable occurring at maximum moisture levels. While maximum antifungal activity was seen in a limited area of the design space, spore production was fairly robust with near-maximum levels occurring over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in spore production over non-optimized conditions.