Identification of protein-degraders in an anaerobic digester by protein stable isotope probing and metagenomics

Presence of carbohydrates hampers protein degradation in anaerobic digesters. To understand this phenomenon, we used proteogenomics to identify the active protein-degraders in the presence of low and high carbohydrates concentrations. Active metabolic pathways of the identified protein-degraders were investigated using proteomics with ¹³C-protein substrates (protein stable isotope probing). Results showed that 1) Acinetobacter was the active protein-degraders under both protein-fed and protein-glucose mixture-fed conditions, 2) the relative abundance of Acinetobacter was not affected by the presence of carbohydrates, 3) the incorporation of the ¹³C-labelled protein substrate was predominantly observed in outer membrane-bound proteins and porin proteins, which are associated with proteinases or the transportation of amino acids across the cell wall. The Acinetobacter metabolic model and the incubation conditions suggested that glucose and proteins were degraded through anaerobic respiration. The negative impact of carbohydrates on protein biodegradation was attributed to Acinetobacter's preference for carbohydrates. This work highlights that efficient degradation of protein and carbohydrate mixtures in anaerobic digesters requires a staged or time-phased approach and enrichment of active protein-degraders, offering a new direction for process optimization in anaerobic digestion systems. KEY POINTS: • Acinetobacter identified for the first time as main anaerobic protein-degrader • Metabolic model revealed protein degradation via anaerobic respiration • Metabolic pathway analysis indicated SO₄^2- or Fe³⁺ as terminal electron acceptors.