Unlocking the potential of Rhodopseudomonas palustris as a biofertilizer: Soil factors and microbial partnerships...

Overreliance on synthetic nitrogen fertilizers poses environmental risks and undermines agricultural sustainability. Free-living nitrogen fixation (FNF) by diazotrophs is a promising alternative. Rhodopseudomonas palustris (R. palustris), a metabolically versatile phototrophic diazotroph, shows strong potential for nonsymbiotic nitrogen fixation; however, its contribution to soil nitrogen input and ecological mechanisms remains unclear. This study evaluates the FNF performance of R. palustris in nutrient-poor, acidic lateritic soil and fertile, near-neutral phaeozem using ¹⁵N₂ isotope tracing, microbial community profiling, and network analysis. In sterilized soils, R. palustris accounts for >99.8% of nifH transcripts, resulting in net nitrogen fixation increases of 7.31-mg N·kg^-1 in lateritic soil and 23.87-mg N·kg^-1 in phaeozem. Inoculation markedly enhances FNF in native soils, with low-dose inoculation in phaeozem reaching a peak net increase of 147.01-mg N·kg^-1, an 11.96-fold increase over the control. This enhancement results from cooperative networks of diazotrophic Cyanobacteria and heterotrophs, which account for 94.1% of the variation in FNF. In lateritic soil, high-dose inoculation yields 7.94-mg N·kg^-1, attributed to improved diazotroph survival and function under stress. Path analysis confirms that R. palustris exerts the strongest positive direct effect on FNF (β = 0.786, P < 0.001), primarily owing to its abundance and nifH transcript copy number. These findings reveal distinct soil-specific modes of regulation: under strong edaphic constraints (low pH, low nutrients, limited C supply), FNF enhancement depends mainly on establishing an active diazotrophic population, whereas in fertile soil, low-dose inoculation can promote cooperative microbial processes that increase net nitrogen input. These findings provide a mechanistic baseline for designing subsequent field evaluations under fertilization or reduced-fertilization regimes.