Enzyme-functionalized alginate microparticles enable anaerobic culture under ambient oxygen

Methods for culturing oxygen-sensitive cells and organisms under anaerobic conditions are vital to biotechnology research. Here, we report a biomaterial-based platform for anaerobic culture that consists of glucose oxidase (GOX) functionalized alginate microparticles (ALG-GOX), which are designed to deplete dissolved [O₂ ] through enzymatic activity. ALG-GOX microparticles were synthesized via a water-in-oil emulsion and had a size of 132.0 ± 51.4 µm. Despite having a low storage modulus, the microparticles remained stable under aqueous conditions due to covalent crosslinking through amide bonds. Enzyme activity was tunable based on the loaded GOX concentration, with a maximum activity of 3.6 ± 0.3 units/mg of microparticles being achieved at an initial loading concentration of 5 mg/mL of GOX in alginate precursor solution. High enzyme activity in ALG-GOX microparticles resulted in rapid oxygen depletion, producing a suitable environment for anaerobic culture. Microparticles loaded with both GOX and catalase (ALG-GOX-CAT) to reduce H₂ O₂ buildup exhibited sustained activity for potential long-term anaerobic culture. ALG-GOX-CAT microparticles were highly effective for the anaerobic culture of Bacteroides thetaiotaomicron, with 10 mg/mL of ALG-GOX-CAT microparticles supporting the same level of growth in an aerobic environment compared to an anaerobic chamber after 16 h (8.70 ± 0.96 and 10.03 ± 1.03 million CFU, respectively; N.S. p = 0.07). These microparticles could be a valuable tool for research and development in biotechnology.