The tremendous progress in sequencing technologies has made DNA sequencing routine for microbiome studies. Additionally, advances in mass spectrometric techniques have extended conventional proteomics into the field of microbial ecology. However, systematic studies that provide a better understanding of the complementary nature of these 'omics' approaches, particularly for complex environments such as wastewater treatment sludge, are urgently needed. Here, we describe a comparative metaomics study on aerobic granular sludge from three different wastewater treatment plants. For this, we employed metaproteomics, whole metagenome, and 16S rRNA amplicon sequencing to study the same granule material with uniform size. We furthermore compare the taxonomic profiles using the Genome Taxonomy Database (GTDB) to enhance the comparability between the different approaches. Though the major taxonomies were consistently identified in the different aerobic granular sludge samples, the taxonomic composition obtained by the different omics techniques varied significantly at the lower taxonomic levels, which impacts the interpretation of the nutrient removal processes. Nevertheless, as demonstrated by metaproteomics, the genera that were consistently identified in all techniques cover the majority of the protein biomass. The established metaomics data and the contig classification pipeline are publicly available, which provides a valuable resource for further studies on metabolic processes in aerobic granular sludge.

Natural habitats of microorganisms are dynamic environments with non-continuous supply of carbon and energy sources, in which intermediate storage of substrates can increase competitiveness. Plasticicumulans acidivorans are polyhydroxybutyrate (PHB) accumulating bacteria enriched from activated sludge using carbon feast-famine cycles as selective pressure. Despite growing slowly, P. acidivorans outcompetes other bacteria by quickly taking up acetate and storing it intracellularly as PHB to later use it for growth. As soon as acetate is depleted, these bacteria immediately ‘switch’ their metabolism from PHB production to consumption entailing a very interesting regulatory challenge as parallel activity could lead to significant losses (futile cycling). While the stoichiometry for both feast and famine phases has been extensively described in literature, the switch regulation is not yet fully understood. To elucidate the responsible regulatory processes, an enrichment of P. acidivorans was studied using targeted intracellular metabolite analysis over time, with emphasis on the feast to famine switch. In combination with extracellular rates, the measured intracellular metabolite pools are used to design a labelling experiment to obtain actual intracellular fluxes (dynamic 13C flux analysis). Here the challenge is to create an isotopically non-stationary state (usually mediated by changing the substrate’s isotopic composition) to study the metabolic response in the transition from presence-to-absence of substrate.In this way, we aim to unravel the responsible regulatory mechanism governing the metabolic switch from storage-to-consumption and use this knowledge not only to understand its ecological relevance, but to also propose novel metabolic strategies for microbial cell factory design.