Enhanced rhizobial survival through mitigation of cytotoxicity and oxidative stress by alginate-functionalized graphene oxide aerogels

Abstract

Graphene-based nanomaterials possess exceptional physicochemical properties and are increasingly used in environmental and agricultural applications. However, direct application of graphene oxide (GO) remains limited due to its cytotoxic interactions with rhizobacteria. In this study, a sodium alginate-electrochemically synthesized graphene oxide (SA-EGO) aerogel was developed as a biocompatible carrier for Bradyrhizobium japonicum (CB1809), providing evidence that alginate functionalization mitigates EGO-associated toxicity. Structural characterization using SEM, XRD, XPS, FTIR, and BET analysis confirmed the successful formation of a porous SA-EGO aerogel network. The performance of SA-EGO aerogel was evaluated alongside EGO, sodium alginate (SA), peat, and control across concentrations (5, 10, and 50 µg mL −1 ) and incubation periods (1, 6, and 12 days). Across all treatments, SA-EGO aerogel consistently improved relative viable count, metabolic activity, and membrane integrity across all tested concentrations and incubation periods. Rhizobial relative viable counts reached 66%, 73%, and 92% at 5, 10, and 50 µg mL −1 , respectively, on day 6, and remained above 63% after 12 days of incubation. Live/Dead staining, XTT metabolic activity assay, and glutathione (GSH) measurements, together with SEM observations, indicated that SA-EGO aerogel mitigated EGO-associated cytotoxicity by maintaining membrane integrity, cellular metabolic activity, and oxidative stress homeostasis. The improved performance of SA-EGO aerogel was associated with a protective alginate network, which likely reduced direct interactions between EGO nanosheets and rhizobial cells while preserving the beneficial physicochemical properties of EGO. Overall, these findings indicate that alginate functionalization improves biocompatibility towards rhizobia, highlighting its potential for nano-enabled microbial delivery systems in sustainable agriculture.