A new lioprotector medium for lyophilization of non-symbiotic nitrogen-fixing bacteria promising for the biofertilizers production

The aim of the study was to develop a protective medium for lyophilization of a strain of non-symbiotic nitrogen-fixing bacteria isolated by the authors from agricultural soils of the Krasnoyarsk Region and identified as Azotobacter sp. The strain was incubated on Sabouraud's modified nutrient medium at 25 °C for 7 days. After incubation, bacterial cells were suspended in protective media and lyophilized using a Bio-Rus-4SFD lyophilizer. Three protective media were used: standard gelatin-sucrose agar (sucrose 10 %, gelatin 1.5, agar 0.01 %), recommended by the All-Russian Collection of Microorganisms; gelatin-sucrose agar with the addition of 1 % ascorbic acid as an antioxidant; peptone-sucrose-glycerol mixture developed by the authors (distilled water 90 ml, glycerol 10 ml, sucrose 10 g, peptone 3.2 g). Bacterial cells suspended in distilled water without lyoprotectors were used as controls. The viability of lyophilized cells was assessed by their ability to form microcolonies on the agar culture medium using phase contrast microscopy. The viability of bacterial cells after lyophilization in distilled water (control) was 8.04 %; after lyophilization in standard gelatin-sucrose agar - 9.92 %; after lyophilization in a peptone-sucrose-glycerol mixture developed by the authors - 12.89 %. The difference between these protective media in terms of cell viability is statistically significant at the level of P 2). No viable cells were found after lyophilization in gelatin-sucrose agar with the addition of 1% ascorbic acid. Thus, the protective medium developed by us provides an increase in the proportion of viable cells during lyophilization by 1.3 times in comparison with standard gelatin-sucrose agar.