Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates

Abstract

AbstractPharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded twoPseudomonassp. isolates: a fast-growingPseudomonassp. that degraded 200 mg/L of paracetamol in approximately 10 hours while excreting a dark brown component to the medium, and a slow-growingPseudomonassp. that degraded paracetamol without obvious intermediates in more than 90 days. EachPseudomonassp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and thePseudomonasspp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs.HighlightsParacetamol was fully degraded by activated sludge from hospital wastewater.Low paracetamol concentrations were removed by a diverse microbial community.Pseudomonassp. dominated cultures with high paracetamol concentration.Uncharacterized amidases are probably involved in degrading paracetamol in WWTPs.Deaminases and dioxygenases might be degrading paracetamol transformation products.