Optimizing initial inoculum ratios to enhance metabolite production and biocontrol activity in Bacillus subtilis and Pseudomonas fluorescens co-cultures.

Microbial co-cultivation has emerged as a promising strategy for stimulating metabolic interactions that enhance the synthesis of bioactive compounds. However, the extent to which the initial inoculum ratio influences the metabolic behavior and biocontrol potential of bacterial consortia remains largely unexplored. Here, we evaluated the effects of different initial inoculum ratios of Bacillus subtilis and Pseudomonas fluorescens on the production of volatile organic compounds (VOCs), chitinase, β-1,3-glucanase, siderophores, and antifungal activity against economically important phytopathogenic fungi. The initial inoculum ratio markedly influenced both the quantity and composition of the metabolites produced. The consortium containing 75% B. subtilis and 25% P. fluorescens generated nearly three times more VOCs than the consortium containing 25% B. subtilis and 75% P. fluorescens and the P. fluorescens monoculture. This consortium also exhibited the highest chitinase activity, siderophore production, and antifungal performance against several phytopathogens, whereas β-1,3-glucanase activity varied according to consortium composition. Moreover, shifts in inoculum ratio reshaped the VOC chemical profile, indicating that metabolic interactions between the two bacterial species depend on their initial population balance. These findings demonstrate that the initial inoculum ratio is a critical driver of microbial interactions and metabolic output in bacterial co-cultures. Manipulating consortium composition therefore represents a simple and effective strategy to optimize metabolite production and improve the performance of microbial biocontrol agents for sustainable agriculture.