*4.6. Optimal Conditions for the Cultivation of Bacillus strains*

To determine the optimal cultivation temperature, the strains were incubated at 20.0, 25.0, 30.0, and 35.0 ◦C. Czapek medium for bacteria was used as a basis [63]. Carbon sources sucrose, glucose, molasses, and glycerol were added in the test media. In the study of carbon sources nitrogen sodium nitrate served as the unchanged nutrition component. In determining the optimal sources of nitrogen nutrition peptone, NaNO3, yeast and corn extracts were tested with glucose as the only (constant) carbon source. To choose the optimal acidity of the medium, the strains were grown on a liquid medium, g/L KCl—0.5, MgSO4 × 7H2O—0.5, K2HPO4 × 3H2O—1.0, CaCO3—3.0, FeSO4 × 7H2O—0.01, corn extract—2.0, molasses—20.0 at optimum temperatures. By adding lactic acid or alkali (4N NaOH solution), the medium pH was adjusted to 3.0, 6.0, 8.0, and 10.0 using a Sartorius PB-11 pH-meter (Goettingen, Germany). To determine the optimal cultivation time, samples for analysis were taken after 8, 16, 24, 36, 48, and 72 h from the start of cultivation. All experiments were replicated three times. For all experiments, a liquid culture was obtained by the method of periodic cultivation. Incubation was carried out in thermostat cell cultivation systems (180 rpm) "New Brunswick Scientific Excella E25" (Enfield, CT, USA) for 48 h. Periodic cultivation was carried out in conical flasks (350 mL) with a nutrient medium volume of 100 mL and preliminary introduction of stock culture (2% of the nutrient medium volume). The stock culture was obtained by introducing agar blocks with the studied strains into conical flasks and subsequent cultivation.

At the end of cultivation, the number of bacterial cells was determined by the Koch method in all experiments on MPA [63]. The grown colonies were counted with the Color Qcount, Spiral Biotech, 530 (Canton, MA, USA) system for the automatic counting colonies.

In our research we used Unique Scientific Facility "New generation technological line for developing microbiological plant protection products" of Federal Research Center of Biological Plant Protection, Krasnodar, Russia (http://ckp-rf.ru/%E2%84%96671367, accessed on 21 August 2019).
