Valorization of agro-waste for sustainable production and encapsulation of Kosakonia pseudosacchari TL13: Towards advanced bioinoculants

Kosakonia pseudosacchari TL13 is a recently isolated rhizobacterium that exerts multiple beneficial effects on plants and represents a promising candidate as plant probiotic. Plant growth-promoting bacteria are key components of climate-smart, quality-oriented agriculture and environmental protection; however, cost-effective and eco-friendly approaches are needed to enable their broader application. This study focused on the development of a sustainable process for the production, stabilization and encapsulation of high titers of viable K. pseudosacchari TL13 cells using citrus waste as renewable substrate. A Plackett-Burman preliminary screening design identified the nitrogen source as primary factor enhancing biomass yield while exopolysaccharide biosynthesis was promoted under nitrogen-limiting conditions. These results were confirmed in controlled bioreactor experiments with hydrolysed citrus waste achieving up to 2.7±0.04 x 10 10 CFU/mL, the highest concentration reached to date. The exopolysaccharide was explored as a natural preservative during freeze-drying and storage of broth samples to simplify downstream procedures and enable co-delivery of viable cells and secreted bioactive compounds that may promote plant health. Membrane emulsification was considered as alternative to traditional encapsulation methods for its mild operating conditions and suitability for shear-sensitive ingredients. Both whole broth and biomass samples generated uniform capsules with >95% encapsulation efficiency, without compromising bacterial viability and structural integrity. Finally, whole genome sequencing revealed a set of carbohydrate-active enzymes that highlight the strain’s metabolic potential to utilize complex agro-industrial by-products further promoting a more efficient use of renewable resources. Overall, these findings demonstrate a comprehensive approach for the production and delivery of K. pseudosacchari TL13 as a next-generation bioinoculant towards a greener future. • Green process for promoting use of K. pseudosacchari TL13 as biostimulant • Valorization of citrus waste through conversion to high viable cell and EPS yields • Simplified and improved formulation process by using naturally secreted EPS • Membrane emulsification is suitable and efficient for cell and broth encapsulation • The strain’s gene pool shows potential for efficient use of renewable resources