3D Printed Biocatalytic Silica Hydrogel Flow-Through Reactor For Atrazine Degradation

Abstract

One of the most heavily used herbicides in the world, atrazine, provides a serious environmental challenge that we face presently. Atrazine has been consistently applied to farms due to its proven ability to remove broadleaf weeds, allowing for increased yields of corn crops, which is necessary to feed an ever-growing world population. However advantageous the use of atrazine is, toxic effects have been identified when humans ingest atrazine. Also, the high mobility of atrazine during run-off events after application to fields allows atrazine to be easily incorporated into water systems around agricultural land, creating a large-scale health and environmental problem as the increased atrazine concentrations negatively impact human health when ingested as well as ecological disturbances when affecting local algal communities. The presented work investigates the application of 3D printing as an approach to solving this significant problem. We hypothesize that with direct-write 3D printing of biologically active, printed materials to perform the bioremediation of atrazine, may enhance bioremediation capacity compared to conventional methods by utilizing the near limitless rapid design flexibility intrinsic to 3D printing to allow fabrication of structures with high surface area to volume ratio (SA:V), yielding lower diffusion length scales that allow improved encapsulated biocatalyst usage. We introduce a novel 3D printing method to produce application specific complex 3D geometries from a sol-gel based silica material with encapsulated biocatalysts. To produce a bioactive material with the incorporation of biocatalysts, silica hydrogel formed through a sol-gel process was used as the ink base. E. coli genetically engineered to overexpress the AtzA enzyme, which degrades the toxic herbicide atrazine to the non-toxic compound hydroxyatrazine, were encapsulated within the silica-based ink. This process leverages the strong mechanical properties, high chemical transport properties, and biocompatibility of the silica base material along with the full material customization, precision in spatial deposition, and design flexibility intrinsic to the 3D printing process to overcome obstacles that hinder the use of bioactive materials within conventional 3D printers (material constraints and biologically deadly processing). The developed 3D printer ink was characterized in terms of gelation kinetics, mechanical properties, cell distribution, and degradation capability. Results confirmed that the 3D printed AtzA biocatalysts sustained biodegradation ability through the removal of atrazine and production of hydroxyatrazine through batch reactor experiments. High SA:V geometries produced through 3D printing also showed improved degradation efficiency by encapsulated biocatalysts. This allowed for an advantage over previously presented work because by providing high SA:V structures, the atrazine did not have to diffuse over long length scales until it was biotransformed within a bacterial cell. Structures with low SA:V were shown to decrease in degradation efficiency because as the atrazine concentration gradient decreased, only the cells closer to the surface would perform the biotransformation of atrazine, the cells located more centrally would not contribute to the degradation. Therefore, with a decrease in diffusion length to all encapsulated biocatalysts, the overall function of the encapsulated population as the concentration of atrazine dropped would be improved over past methods. Additionally, a flow-through bioreactor was designed, simulated, and experimentally tested. ANSYS reaction-flow simulations were completed to determine experimental flow rates necessary to positively identify atrazine degradation in the flow-through bioreactor. Finally, atrazine degradation was proven in flow-through experiments at an inlet flowrate of 1 ml/min. Observed atrazine degradation equated to 15 ± 5% of overall inlet concentration atrazine. Through this work, we have shown as a proof of concept that 3D printed silica-encapsulated biocatalysts sustain the function to degrade an environmental pollutant. This work may be expanded further via the incorporation of multiple types of biocatalysts encapsulated in an organized pattern (multiple different 3D printer inks printed in a designed pattern) that enhances biotransformation and transport of products between the multiple biocatalysts. In addition, this work may be applied to advance fields where complex geometries of encapsulated biocatalysts are necessitated, which may include the fields of pharmaceutical, medical, environmental, and materials science.