Coupling pH-Responsive Dyes to Agarose Hydrogels for Monitoring Metabolic States of Encapsulated Phototrophic Microbial Consortia

The interaction of microbial consortia within biofilms leads to emergent properties such as high resistance to environmental fluctuations, efficient biocatalytic performance, and stable metabolic states. However, the mechanisms governing these interactions are hard to capture and are not fully understood. Agarose has proven to be a good mimic of the extracellular polymeric substance, which is the polymeric matrix supporting the connectivity of microbial consortia within biofilms. We aimed at modifying polymeric agarose to generate <i>in situ</i> sensing functionalities for monitoring metabolic states within the encapsulated microbial consortia. For that, agarose was end-on chemically modified to couple the pH-responsive FAM dye. After reconstitution of hydrogels out of the functionalized agarose, a stable covalent coupling of the dyes was demonstrated using fluorescence recovery after photobleaching, showing a reduction of free FAM dye from 51% using common water washing procedures to no measurable amount of free dye using our washing procedure. Furthermore, the ability to monitor relevant pH ranges between 6 and 8 was experimentally demonstrated in the hydrogels by laser scanning microscopy. Furthermore, the application of the functional agarose hydrogels was shown in cell cultures with chemoheterotrophic and phototrophic microbial strains (<i>Pseudomonas taiwanensis</i> and <i>Synechocystis sp.</i>). The monitoring of pH changes of microbial consortia dependent on their metabolic performance over up to 3 days was proven, paving the road to the utilization of such functional agarose matrices to study metabolic interactions in complex microbial consortia.