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

View ORCID ProfileAna B. Rios-Miguel, Garrett J. Smith, Geert Cremers, Theo van Alen, View ORCID ProfileMike S.M. Jetten, View ORCID ProfileHuub J. M. Op den Camp, View ORCID ProfileCornelia U. Welte
doi: https://doi.org/10.1101/2022.05.05.490616
Ana B. Rios-Miguel
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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  • ORCID record for Ana B. Rios-Miguel
  • For correspondence: a.riosmiguel@science.ru.nl c.welte@science.ru.nl
Garrett J. Smith
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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Geert Cremers
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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Theo van Alen
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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Mike S.M. Jetten
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
bSoehngen Institute of Anaerobic Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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Huub J. M. Op den Camp
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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Cornelia U. Welte
aDepartment of Microbiology, Radboud University, Radboud Institute for Biological and Environmental Sciences, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
bSoehngen Institute of Anaerobic Microbiology, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
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  • ORCID record for Cornelia U. Welte
  • For correspondence: a.riosmiguel@science.ru.nl c.welte@science.ru.nl
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Abstract

Pharmaceuticals 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 two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 hours while excreting a dark brown component to the medium, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. 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 the Pseudomonas spp. 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.

Highlights

  • Paracetamol was fully degraded by activated sludge from hospital wastewater.

  • Low paracetamol concentrations were removed by a diverse microbial community.

  • Pseudomonas sp. 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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • https://doi.org/10.17026/dans-xwd-fbj5

  • https://dataview.ncbi.nlm.nih.gov/object/PRJNA831879?reviewer=fts2mvskca35vkaq72pg16nota

  • List of abbreviations used

    WWTP
    wastewater treatment plant
    MBR
    membrane bioreactor
    GAC
    granular activated carbon
    APAP
    N-acetyl-p-aminophenol or paracetamol
    4-AP
    4-aminophenol
    HQ
    hydroquinone
    HRT
    hydraulic retention time
    SRT
    solid retention time
    Pfast
    Pseudomonas sp. isolate growing fast on APAP as sole carbon source
    Pslow
    Pseudomonas sp. isolate growing slow on APAP as sole carbon source.
    HGT
    horizontal gene transfer
    MAG
    metagenome-assembled genome
    TPM
    transcripts per million
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    Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates
    Ana B. Rios-Miguel, Garrett J. Smith, Geert Cremers, Theo van Alen, Mike S.M. Jetten, Huub J. M. Op den Camp, Cornelia U. Welte
    bioRxiv 2022.05.05.490616; doi: https://doi.org/10.1101/2022.05.05.490616
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    Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates
    Ana B. Rios-Miguel, Garrett J. Smith, Geert Cremers, Theo van Alen, Mike S.M. Jetten, Huub J. M. Op den Camp, Cornelia U. Welte
    bioRxiv 2022.05.05.490616; doi: https://doi.org/10.1101/2022.05.05.490616

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