Advanced Preservation Strategies for Inoculants: A Lipid-Biophysical Approach to Bradyrhizobium japonicum Stability

Abstract

The intensive use of chemical fertilizers in soybean (Glycine max) cultivation has caused significant environmental degradation, underscoring the urgent need for sustainable alternatives. In Argentina, Bradyrhizobium japonicum E109 is widely employed as a liquid bioinoculant, yet its efficiency is limited by loss of viability during storage. This study investigated the physiological and biophysical mechanisms underlying membrane adaptation of B. japonicum E109 under storage stress and evaluated lipid supplementation as a stabilization strategy. During six months of liquid storage at 28 °C, bacterial viability (Log CFU mL−1) declined from 10.0 to 7.7, accompanied by morphological collapse and a 29% reduction in membrane fluorescence polarization, indicating increased fluidity. Fatty acid analysis revealed a drastic decrease of unsaturated 18:1 (from 80% to 40%) and a 300–400% increase in saturated 18:0, reducing the U/S ratio from 4 to 1. Spectral phasor analysis confirmed a shift in the lipid microenvironment from an ordered to a disordered state. Supplementation with 400 µM of stearic acid (18:0) restored membrane rigidity, lowered the U/S ratio to 1.5, and improved thermal tolerance. After one month of storage, 18:0-treated cultures maintained 8.0 Log CFU mL−1 and preserved viability after exposure to 37 °C, whereas controls dropped to 3.8 Log CFU mL−1. These results identify lipid remodeling as a key determinant of B. japonicum stability and demonstrate that exogenous 18:0 supplementation mimics natural adaptation, preventing membrane fluidization and enhancing inoculant shelf-life. This lipid-biophysical approach provides a rational framework for developing next generation, more resilient rhizobia formulations for sustainable agriculture.