Enhancing Light-Driven Biological Nitrogen Fixation in Azotobacter vinelandii Using Carbonized Polymer Dots

Biological nitrogen fixation (BNF) is a vital natural process that sustains the global nitrogen cycle and agricultural productivity. However, its efficiency is inherently constrained by the high energy demand and oxygen sensitivity of the nitrogenase enzyme complex. This study investigated the use of carbonized polymer dots (CPDs) as photoactive nanomaterials to enhance BNF in Azotobacter vinelandii through light-driven photocatalytic processes. CPDs were synthesized using a one-post hydrothermal methods and characterized with spectroscopic and microscopic techniques that verified successful synthesis, nanoscale morphology, and favorable optical properties for visible-light activation. Biological evaluation demonstrated that CPDs were biocompatible with A. vinelandii and can closely associate with bacterial cells. Ion-chromatography-mass spectrometry showed that CPD-treated cultures exhibited enhanced ammonium production under light irradiation, with 3.125 µg/mL CPDs having the highest increase over 8 hours from the untreated control of 1.84-fold. To investigate the underlying mechanism, intracellular reactive oxygen species (ROS), NADH/NAD+, and ATP levels were evaluated. Photoactivated CPDs induced only a modest increase in ROS while enhancing NADH utilization and increasing intracellular ATP levels, suggesting improved electron transfer and energy availability for nitrogenase activity. These findings all support a mechanism in which photoexcited CPDs facilitate electron transfer to bacterial metabolism, promoting more efficient nitrogen fixation under visible-light exposure. Overall, this study demonstrates that CPDs are effective photoactive nanomaterials for enhancing BNF in A. vinelandii and provides mechanistic insight into nano-bio interactions that may contribute to the development of sustainable, nanomaterial-assisted nitrogen fixation.