Enzyme‐functionalized alginate microparticles enable anaerobic culture under ambient oxygen

Abstract

AbstractMethods 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 H2O2 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.