Pilot submerged cultivation of <i>Trichoderma asperellum</i> RNCIM F-1323 micromycete

Abstract

The study is aimed at developing a pilot industrial method for producing a biofungicide based on the fungus of Trichoderma genus. The cultivation was carried out by submerging in a pilot industrial bioreactor line with an inoculation bioreactor having a total volume of 10 litres and a working bioreactor with a total volume of 100 litres. Czapek’s modified nutrient medium was used with yeast extract and molasses replacing sucrose, g/L: molasses – 20; yeast extract – 7; NaNO 3 – 2; K 2 HPO 4 – 1; MgSO 4 – 0.5; KCl – 0.5; FeSO 4 – 0.01. Cultivation was carried out maintaining the following parameters: temperature – 27±0.5 o С ; culture medium mixing speed – 600 rpm; aeration intensity – 1 L of air per 1 L of culture medium per min. The inoculum was grown on a nutrient medium of a similar composition in rocking flasks with a total and a working volume of 750 and 100 mL, respectively. Inoculated flasks were incubated using an Innova 40R shaker incubator (New Brunswick, USA) at 200 rpm for 22–24 h until a dry biomass index of 6.5–7.5 g/L was achieved. Conidia concentration was calculated using a Goryaev camera. Liquid cultivation was selected due to its relatively short process time. In addition, unlike solid-phase, the submerged cultivation provides for the accumulation of numerous secondary metabolites having antagonistic activity against phytopathogens in the finished form of the preparation. The object of the study was the Trichoderma asperellum RNCIM F-1323 strain with antagonistic activity against many phytopathogens. From a technical and economic point of view, the culture transfer to an inoculation bioreactor in an amount of 5 % vol. was established to be justified as a result of the experiments. The effect of various time values for cultivation in inoculation bioreactor on the growth rate of micromycetes in a working bioreactor was studied. The optimal time for transfer to a working bioreactor was determined to be within 20–24 h. At the end of the cultivation process, the conidia concentration was equal to 1.9·108 conidia/mL.