Detection of Gel-Forming Polymers via Calcium Crosslinking, Applied to the Screening of Extracellular Polymeric Substances Extracted from Biological Aggregates

The valorization of biological aggregates through the extraction of hydrogel-forming polymers can enhance the economics and sustainability of various processes in which bacteria are involved in organic waste transformation, such as wastewater treatment. Achieving these goals requires the development of a method capable of detecting the presence of gel-forming polymers in complex mixtures containing biopolymers that are most often unknown and uncharacterized. A miniaturized screening method capable of detecting gelation via ionic crosslinking using only 1 to 3 mg of the tested samples (commercial molecules or extracellular polymeric substances, EPSs) is proposed. The method consists of calculating a percentage of reactivity (%R) through UV-vis spectra and determining the percentage of gel volume (%Vg) formed after the addition of calcium. Both factors were combined to give a gelling factor (GF), and the test was applied to pure commercial molecules (BSA, DNA, alginate (ALV), and a mixture of them), allowing the classification of the following solutions according to their gel-forming capacity: GF_(ALV) > GF_(ALV+DNA) > GF_{(BSA+ALV+DNA)} > GF_(BSA+ALV) > GF_(DNA) > GF_(BSA+DNA) > GF_(BSA). As a relevant tool for screening hydrogel-forming solutions, the method was applied to the EPS extracted from aerobic granular sludge. The EPS (0.5% <i>w</i>/<i>v</i>) had a GF of 0.16 ± 0.03, equivalent to approximately half of the GF of ALV (0.38 ± 0.02 at 0.5% <i>w</i>/<i>v</i>). The developed test pushes the limits of the existing gel-detection techniques because it allows for quicker, less consuming, and more informative gelation detection through the use of simple methods that do not require sophisticated equipment.