Comparative metaproteomics demonstrates different views on the complex granular sludge microbiome

Abstract

The tremendous progress in sequencing technologies has made 16S amplicon and whole metagenome sequencing routine in microbiome studies. Furthermore, advances in mass spectrometric techniques has expanded conventional proteomics into the field of microbial ecology. Commonly referred to as metaproteomics, this approach measures the gene products (i.e., proteins) to subsequently identify the actively-expressed metabolic pathways and the protein-biomass composition of complete microbial communities. However, more systematic studies on metaproteomic and genomic approaches are urgently needed, to determine the orthogonal character of these approaches. Here we describe a deep, comparative metaproteomic study on the complex aerobic granular sludge microbiome obtained from different wastewater treatment plants. Thereby, we demonstrate the different views that can be obtained on the central nutrient-removing organisms depending on the omic approach and reference sequence databases. Furthermore, we demonstrate a homogenized Genome Taxonomy Database (GTDB) that subsequently enables a more accurate interpretation of data from different omics approaches. Ultimately, our systematic study underscores the importance of metaproteomics in the characterization of complex microbiomes; and the necessity of accurate reference sequence databases to improve the comparison between approaches and accuracy in scientific reporting.

Full-scale aerobic granular sludge for municipal wastewater treatment – granule formation, microbial succession, and process performance

“…33 However, all microbial community analysis methods suffer from biases and limitations. 74 Although amplicon sequencing is expected to approximate microbial species composition and relative abundances well, 75 the relative abundance of some taxa was likely underestimated at the expense of others. For example, the abundance of Ca.…”

“…Accumulibacter may be underestimated. 74 However, the distribution patterns of the taxa over time and between sample types, for which amplicon sequencing is widely used in water and wastewater surveys, 76 were likely captured at higher precision.…”

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal

“…Analysis by 16S rRNA gene amplicon sequencing has been described to strongly underestimate the abundance of "Ca. Accumulibacter" (Albertsen et al 2015;Kleikamp et al 2022), but it can be useful to determine flanking populations. Therefore, we gave stronger credibility to FISH and proteomic results, which showed that these two PAOs formed the majority of the microbial community.…”

Light intensity defines growth and photopigment content of a mixed culture of purple phototrophic bacteria

“…After cell lysis and protein extraction, proteins were digested with trypsin and purified by solid phase extraction. The resulting peptides were analyzed by a one-dimensional shotgun metaproteomic approach using a QE plus Orbitrap mass spectrometer ( Kleikamp et al, 2022 ). The datasets were analyzed using a taxonomy-focused UniprotKB database.…”