Metagenomics and proteomics profiling of extracellular polymeric substances from municipal waste sludge and their application for soil and water bioremediation

Abstract

This study assessed the components of anaerobically digested sludge, activated sludge, and microbial and extracellular polymeric substance (EPS) enzymes to identify the mechanisms underlying nitrogen removal and soil regeneration. 16S rRNA gene amplicon-based sequencing was used to determine the microbial community composition and the related National Center for Biotechnology Information (NCBI) protein database was used to construct a conventional library from the observed community. EPS components were identified using gel-free proteomic (Liquid Chromatography with tandem mass spectrometry-LC/MS/MS) methods. Alginate-like EPS from aerobically activated sludge have strong potential for soil aggregation and water-holding capacity, whereas total EPS from anaerobic sludge have significant potential for ammonia removal under salt stress. Fourier transform infrared spectroscopy (FTIR) revealed that both EPS may contain proteins, carbohydrates, humic compounds, uronic acid, and DNA and determined the presence of O？H, N？H, C？N, Cdouble bondO, and C？H functional groups. These results demonstrate that the overall enzyme activity may be inactivated at 30 g L-1 of salinity. An annotation found in Kyoto Encyclopedia of Genes and Genomes (KEGG)- KEGG Automatic Annotation Server (KAAS) revealed that the top two metabolic activities in the EPS generated from the anaerobic sludge were methane and nitrogen metabolism. Therefore, we focused on the nitrogen metabolism reference map 00910. EPS from the anaerobically digested sludge exhibited nitrate reductase, nitrite reductase, and dehydrogenase activities. Assimilatory nitrate reduction, denitrification, nitrification, and anammox removed ammonia biochemically. The influence of microbial extracellular metabolites on water-holding capacity and soil aggregation was also investigated. The KAAS-KEGG annotation server was used to identify the main enzymes in the activated sludge-derived alginate-like extracellular EPS (ALE-EPS) samples. These include hydrolases, oxidoreductases, lyases, ligases, and transporters, which contribute to soil fertility and stability. This study improves our understanding of the overall microbial community structure and the associated biochemical processes, which are related to distinct functional genes or enzymes involved in nitrogen removal and soil aggregation. In contrast to conventional methods, microbial association with proteomics can be used to investigate ecological relationships, establishments, key player species, and microbial responses to environmental changes. Linking the metagenome to off-gel proteomics and bioinformatics solves the problem of analyzing metabolic pathways in complex environmental samples in a cost-effective manner.