Using stable isotope probing and fluorescence spectroscopy to examine the roles of substrate and soluble microbial products in extracellular polymeric substance formation in activated sludge process

Abstract

In this study, we used stable isotope-labeled soluble microbial products (SMP) and substrates to explore their assimilation into the formation of new biological products (i.e., extracellular polymeric substances and biomass) in two adjacent sequencing batch reactors. The isotope labeling approach along with fluorescence spectroscopy allowed us to distinguish between refractory and labile portions of SMP constituents as well as their roles in the formation of extracellular polymeric substances (EPS). Comparison of SMP fluorescence and the specific UV absorbance values between the two reactors revealed the presence of humic-like aromatic substances in the non-consumable part of SMP, which can be ultimately released as effluent organic matter. Parallel factor analysis modeling of fluorescence spectra showed that the hydrolysis of EPS contents mostly resulted in humic-like components in SMP rather than protein-like components, which were initially abundant in EPS (˃80%). From variations in carbon and nitrogen isotopic contents in EPS and biomass, it was found that carbon-containing substrates were enriched faster than their nitrogenous counterparts. The contributions to new EPS formation reached 87.5% for carbon and 60.5% for nitrogen. Meanwhile, the isotopic tracking of the labeled SMP revealed that only 11.0% and 11.9% of carbon and 13.3% and 11.6% of nitrogen from the influent SMP were finally assimilated into EPS and biomass, respectively. In contrast, the isotopic enrichment in SMP was higher (similar to 50%) than that of EPS and biomass, indicating the low bioavailability and refractory nature of the feed SMP. This study proposed a promising approach for estimating the relative contributions of different forms of labile substrate and SMP to the formation of EPS in activated sludge processes. This approach could be suggested as a versatile method for establishing the kinetics, substrate element flow, mass balance on organic substrates and nutrients, as well as for tracking the consumption and uptake pathways of hazardous materials.