Using stable isotope labeling approach and two dimensional correlation spectroscopy to explore the turnover cycles of different carbon structures in extracellular polymeric substances.

Extracellular polymeric substances (EPS) from activated sludge comprise many organic constituents with polysaccharides and proteins as the main components of two different functionalities. Despite a number of previous EPS studies, a fundamental question remained unanswered, namely, whether the different EPS components would have the same turnover cycle (i.e., formation/dissolution) in biological wastewater treatment systems. In this study, we employed a stable isotope labeling approach based on isotope-enriched substrates (i.e., ¹³ C-glucose and ¹⁵ NH ₄ Cl) to examine the potential discrepancies in the turnover cycles among different major EPS constituents. Our results, based on substrate consumption in a batch bioreactor, evidenced the existence of differences in carbon and nitrogen cycles within bulk EPS with an earlier replenishment of organic carbon relative to organic nitrogen. The changes in the 13C nuclear magnetic resonance (13C NMR) spectra of EPS with operation clarified the relative differences in the turnover periods among several identified EPS structures with different chemical functionalities. Two-dimensional correlation spectroscopy (2D-COS) on the 13C NMR spectra further showed that the substrate-assimilated carbon functional groups appear to preferably formed within bulk EPS in the order of O-alkyl carbons > amides > α amino acids > aliphatic carbons. This study provides a novel insight into the dissimilar formation rates of different EPS structures after substrate assimilation. This isotope labeling approach can be further applied to determine the mass balance among the substrate, biomass, and bound/soluble EPS within activated sludge systems.
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