Optimizacija postupka dobijanja mikrobne biomase i formulacije mikrobiološkog preparata sa potencijalnim biopesticidnim i fitostimulatornim dejstvom

Abstract

Microbial fertilizers represent an alternative approach in agriculture that is supposed to replace agrochemicals that have a negative effect on the environment and human health. These formulations have the potential to stimulate plant growth, assimilate minerals from the soil and increase plant resistance to pathogens. Bacillus strain has numerous properties that make it suitable for incorporation into biofertilizers with the aim of stimulating plant growth and control the disease. Therefore, this doctoral dissertation aimed to optimize the production of B. subtilis NCIM 2063 biomass and to formulate the microbiological preparation with potential biopesticidal and phytostimulatory effects. First, the influence of process factors on bacterial growth was examined in shake-flasks. The highest biomass yield (6.2 g/L) was achieved in a medium with 10 g/L glucose and at an OTR value of 10 mol/m3h when the specific growth rate and generation time were 0.27 h-1 and 2.54 h, respectively. Such a result can be achieved in just 24 h. By applying the design of the experiments, the response surface methods, and the Deringer’s desirability function the growth conditions in shake-flasks were further optimized. Statistically significant models were proposed for each of the tested response values, which led to the definition of the conditions for achieving maximum viability and sporulation (T=33 °C, OTR=10 mol/m3h and glucose concentration 4.89 g/L). Under the given conditions, the maximum concentration of vegetative cells and spores is predicted to be 9.66 LOG (CFU/ml) and 9.19 LOG (CFU/ml), respectively. The process was scaled up to the bioreactor level by using the oxygen mass transfer coefficient as the main criteria. An empirical correlation for calculation of the mixing speed was defined and scale-up effect was monitored by parallel cultivation in a shake-flask and in a bioreactor under the same conditions. After 48 h of cultivation 9.51 ± 0.09 LOG (CFU/ml) of cells and 9.08 ± 0.06 LOG (CFU/ml) of spores were achieved in shake-flasks, while in the bioreactor the total number of cells was 9.66 ± 0.13 LOG (CFU ml), of which 9.11 ± 0.19 LOG (CFU/ml) spores. A microbiological powder preparation was formulated using spray drying method, and the response surface models were developed. In order to obtain a microbiological preparation of maximum viability and yield, and with a minimum amount of residual moisture, spray drying conditions were optimized by applying the Deringer function of desirability. As a unique solution with a combined desirability function D=0.874, the following was proposed: an inlet temperature of 130 °C, maltodextrin concentration of 50 g/L and a feed flow rate of 6.1 ml/min. There was a minimum deviation of experimental and software predicted values. Obtained microbiological preparation showed a high survival rate of encapsulated bacteria after storage at room temperature for 6 months and 1 year period. and pronounced antifungal and phytostimulatory effect.