Modulating nitrogenase activity of encapsulated diazotrophs through oxygen-regulated microenvironments and carbon availability in alginate hydrogel beads

Abstract Aims Biological nitrogen fixation (BNF) by free-living diazotrophs has the potential to supplement nitrogen inputs in agricultural soils, but nitrogenase activity is highly sensitive to oxygen and environmental fluctuations. This study evaluated whether alginate hydrogel beads can function as soil-applied microenvironments that regulate oxygen diffusion and support sustained nitrogenase activity of free-living diazotrophic bacteria. Methods Hydrogel beads were prepared by crosslinking sodium alginate with divalent and trivalent cations (Ca 2 ⁺, Sr 2 ⁺, Zn 2 ⁺, Ni 2 ⁺, Cu 2 ⁺, and Al 3 ⁺) to modify matrix structure and diffusion properties. The diazotrophs Azospirillum brasilense Sp7 and Herbaspirillum seropedicae Z152 were encapsulated within the beads. Oxygen concentrations, encapsulation efficiency, bacterial viability, and biodegradability in soil were evaluated. Nitrogenase activity was quantified using the acetylene reduction assay, and the effect of incorporating simple carbon sources into the beads was also examined. Results All alginate matrices generated oxygen-reduced microenvironments, with bulk-equivalent dissolved oxygen concentrations ranging from 59.9 ± 16.4 to 215.0 ± 42.8 nmol L⁻ 1 . Ca 2 ⁺- and Sr 2 ⁺-alginate beads showed the highest encapsulation efficiency and maintained the greatest bacterial viability. Nitrogenase activity within Ca 2 ⁺-alginate beads reached 1.76 ± 0.31 and 2.25 ± 0.39 for A. brasilense and 2.25 ± 0.39 nmol C 2 H 4 mg protein⁻ 1 h⁻ 1 for H. seropedicae , comparable to semi-solid culture conditions. Carbon supplementation increased nitrogenase activity of H. seropedicae but reduced activity in A. brasilense , indicating species-specific responses to the encapsulated environment. Soil incubation showed that the beads degraded by more than 50% within 120 days. Conclusions Alginate hydrogel beads can create localized oxygen-regulated microhabitats that support nitrogenase activity of free-living diazotrophs. These biodegradable matrices function as synthetic microenvironments that can sustain biological nitrogen fixation and may represent a strategy to enhance biological nitrogen inputs in non-leguminous cropping systems.