Survival of beneficial microbes in liquid bioformulation and optimization of different carrier materials using RSM technique

Soil salinity in rice cultivation areas is considered a severely limiting factor that adversely affects the quantity and quality of rice production in wetlands. Recently, the alternative use of salt-tolerant plant growth-promoting rhizobacteria (PGPR) inhabiting extreme saline conditions has gained remarkable attention and had positive effects on soil and crops. Therefore, a study has been initiated to develop a liquid biofertilizer formulation from locally isolated multi-strain salt-tolerant PGPR strains such as Bacillus tequilensis and Bacillus aryabhattai, using glycerol (5 mM), trehalose (10 mM), and polyvinylpyrrolidone (PVP) at 1% as additives to prolong the shelf-life of the bacteria. After 3 months of incubation, the bacterial population in the trehalose-supplemented mixed strain was highest at 9.73×107 CFU/mL, followed by UPMRE6 and UPMRB9 at 9.40×107 CFU/mL and 8.50×107 CFU/mL respectively. The results showed that the optimal trehalose concentration successfully prolonged the shelf-life of bacteria with minimal cell loss. Validation of quadratic optimization by response surface methodology revealed that the cell density of the mixed strain was 4.278×107 log CFU/mL after 24 h. The precision ratio was 99.7% higher than the predicted value in the minimized medium formulation: 0.267 g/mL trehalose, 1% glycerol, at 120 rpm agitation using the data analysis tools of Design Expert software. The population study confirmed the better and longer survival of salt-tolerant PGPR fortified with 10 mM trehalose, which was considered the best liquid biofertilizer formulation. Moreover, the optimized trehalose-glycerol liquid formulation can be used commercially as it is cost-effective.