Dual advantage of Klebsiella oxytoca layered biofilm in fixing aerial nitrogen to rice plant roots, arresting soil arsenic availability and translocation

Arsenic (As) contamination of paddy soils threatens food security across South and Southeast Asia, while nitrogen limitation concurrently curtails rice productivity. Here we demonstrate that Klebsiella oxytoca M5a1, a diazotrophic, biofilm-forming rhizobacterium, exerts a dual protective function on rice (Oryza sativa IR64): (i) fixing aerial nitrogen and translocating ammonium to the root rhizoplane via a stratified biofilm architecture, and (ii) immobilising soil arsenic within its extracellular polymeric substance (EPS) matrix, attenuating root-to-grain translocation. Greenhouse pot experiments with graded arsenic treatments (0–50 mg kg−1) under biofilm (+BF) and non-biofilm (−BF) conditions showed that biofilm-inoculated plants maintained shoot nitrogen at 1.72%–1.88% across all arsenic doses, versus a decline to 0.92% in non-biofilm plants at 50 mg kg−1 As. Acetylene reduction assay confirmed biofilm-associated nitrogenase activity at 484.7 ± 27.7 nmol C₂H₄ g−1 root h−1, ~4-fold above non-biofilm controls. ToF-SIMS depth profiling resolved a stratified NH₄⁺ gradient across biofilm layers, suggesting interfacial ammonium enrichment at the root epidermal contact zone, consistent with directed transfer. EPS polysaccharide content escalated from ~47 to ~732 µg mg−1 under 50 mg kg−1 arsenic stress, sequestering arsenic in residual and Fe/Mn-oxide-bound soil fractions and reducing grain arsenic by 45.5%. Oxidative stress markers and growth parameters corroborated the bioprotective phenotype. These findings establish K. oxytoca layered biofilm as a tractable bioinoculant conferring simultaneous nitrogen nutrition and arsenic phytoprotection in contaminated paddy agroecosystems.