Fungal biopesticide production by solid-state fermentation : growth and sporulation of Coniothyrium minitans

The use of an inert carrier impregnated with a chemically defined medium might be attractive in industrial processes. This system can be used as alternative for agricultural substrates such as grains in solid-state fermentation for the production of fungal biopesticides. During recent years, the use of fungal spores for the biological control of plant pests and diseases has received increasing interest. Coniothyrium minitans is a biocontrol agent of Sclerotinia sclerotiorum a widespread plant pathogen affecting more than 360 plant species. As with other biopesticides, large quantities of effective spores of C. minitans are needed. An SSF process seems the preferred mass production method for this fungus. However, rational design and operation of an SSF-process for mass production of fungal conidia are hampered by several factors. One of them is the lack of knowledge about the physiology and the kinetics of fungal growth and sporulation in SSF.

In this thesis, research on the physiology and kinetics of growth and sporulation of C. minitans in SSF is described. The aim was to determine the optimal substrate composition for spore production and to determine the kinetic parameters and stoichiometry of the bioconversion reactions. The quantity of biomass is an essential variable in characterizing the optimal growth and sporulation conditions. However, direct measurement of biomass is almost impossible in SSF since fungi penetrate into and bind tightly to the solid substrate. Therefore, various indirect methods to estimate the amount of biomass, being respiration measurements and several biochemical analyses, were first evaluated for C. minitans .

Secondly, the influence of the main medium components, carbon and nitrogen, was studied in more detail using chemically defined media, which facilitate reproducible studies. Several nitrogen sources in combination with glucose or starch were evaluated for their influence on sporulation of C. minitans. The medium with the combination of nitrogen and carbon source giving the best results was further optimized with respect to spore quantity using statistically based experimental designs. These designs are more efficient than varying one factor at a time. This optimization strategy allowed the spore production to be increased by a factor 7 from 4*10 ⁹to almost 3*10 ¹⁰spores per Petri dish of 9-cm diameter. These numbers correspondes with a spore production of 2*10 ⁸to 3*10 ¹²spores.kg ^-1medium.

Thirdly, the stoichiometry and kinetics of mycelium and spore production were studied on defined media with different concentrations of starch, urea and trace elements. By means of elemental balances the stoichiometry of growth and sporulation was established. Based on the kinetics, the process costs for producing spores were roughly calculated. It was shown that fermentor costs form the major part of the production costs.

In all these laboratory studies the use of a chemically defined medium was very useful. It facilitates reproducible and detailed physiological and kinetic studies in SSF, which will eventually be the basis for efficient process development, control strategies and reactor design. The use of an inert carrier impregnated with a chemically defined medium, as alternative for agricultural substrates in SSF, might be attractive in industrial processes too. The industrial potential of inert carriers impregnated with chemically defined medium for the production of spores of C. minitans and other (high-added-value) products is finally discussed.