Otimização dos parâmetros para fermentação líquida submersa de Clonostachys rosea

Abstract

The potential of Clonostachys rosea to act as a biocontrol agent for diseases and pests – as well as to promote plant growth – is well known. However, one of the major challenges for the development of products based on this antagonist is its mass production. Currently, its production is carried out through solid-state fermentation, a labor-intensive process that consumes too much space and time. The use of liquid fermentation can help overcome this challenge. The objective of this study is to optimize different combinations between variables that interfere with the fermentation process in a submerged medium in order to increase the concentration of conidia produced in the culture medium. The numbers of colony-forming units (CFU), microsclerotia and dry fungal biomass were also evaluated. The effectiveness of the propagules of C. rosea produced in liquid fermentation in the control of Botrytis cinerea, Sclerotinia sclerotiorum and Bemisia tabaci was evaluated. Initially, an assay was carried out using the Plackett-Burman experimental design, studying the interaction between six variables of the culture medium, which are C:N ratio, total carbon of the medium, pH, initial inoculum and concentrations of Ca and Fe. Subsequently, the optimization of the culture conditions was performed with Rotational Central Composite Design (RCCD), using three nitrogen sources (yeast extracts, cottonseed meal and urea), and varying the pH during the fermentation. The first two steps were performed in 250 mL Erlenmeyers with three basal deflectors, in which 90 mL of sterilized different media and 10 mL of inoculum were added. The final fermentation step was carried out in a 3L bench bioreactor (Eppendorf, 115 BioFlo, Germany). The highest concentration of conidia obtained from submerged liquid fermentation was 7.87 x 108 conidia mL-1 at 120 hours of fermentation in the medium with: C:N 50:1, Ca 1 g L-1, Fe 100 mg L-1, pH 6, total carbon 24 g L-1 and initial inoculum 1x105 conidia mL-1. In the solid fermentation using rice grains, the maximum value obtained after 30 days of fermentation was 1.87 x 109 g-1. The submerged fermentation of C. rosea carried out in a bench bioreactor resulted in a lower production of conidia when compared to the production obtained in the previous steps (1.49 x 107 conidia mL-1 after 120 hours of fermentation). C. rosea produced by submerged fermentation and solid-state fermentation controlled all pathogens and pest evaluated in the same way.