Optimization of Bacillus subtilis-based fermentation of anaerobic digestate and biohazard-free application in endophyte-assisted hardening of micropropagated plantlets for increasing survivability

Anaerobic digestion of agricultural waste helps to decarbonize the energy sector while also increasing the use of stabilized residues in agriculture. Although digestate (Dg) contains amino acids, vitamins, humic acids, and micronutrients, using it directly poses an unknown risk to crops due to the presence of pathogens, heavy metals, and excessive ammonia, necessitating processing for agricultural application as fertilizer. The study aimed to minimize the undesired effects of Dg by optimizing fermentation with Bacillus subtilis CW-S and using it as a potential inoculant for endophytic-assisted hardening of micropropagated potato plantlets. The single factor test results revealed that Dg concentrations of 70%, 80%, and 90% had a greater positive impact on cell concentration than concentrations of 50%, 60%, and 100%. According to the Box-Behnken design, diluted (80%) Dg produced a higher cell concentration (4.36 × 108 CFU/mL) than undiluted (100%) (1.18 × 108 CFU/mL). In hardening experiments, Bacillus subtilis CW-S fermented digestate (FDg) had a significantly higher plantlet survival rate of 90.25% and a root length of 5.22 (cm) with greater Serendipita indica infection, whereas Dg (unprocessed) had a lower survival rate of 67.88% and a root length of 3.13 (cm) with less Serendipita indica infection. According to the findings, FDg could be employed as a non-threat bioinoculant for widespread agricultural application.