Applied Microbiology and Biotechnology

, Volume 103, Issue 10, pp 4177–4192 | Cite as

Advancing biomarkers for anaerobic o-xylene biodegradation via metagenomic analysis of a methanogenic consortium

  • Karen Rossmassler
  • Christopher D. Snow
  • Dora Taggart
  • Casey Brown
  • Susan K. De LongEmail author
Applied microbial and cell physiology


Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.


Metagenomics Methanogenic Biomarkers Fumarate-adding enzymes O-xylene Enzyme homology modeling 



Thanks to Elizabeth Edwards and Fei Luo for supplying the enrichment culture and for assistance with culturing. Thanks to Jan Leach for the use of her qPCR machine while ours was repaired, and to Jillian Lang for assistance with their machine. Thanks to Diana Marcela Nuñez Hernandez for conducting some gas chromatography measurements. Thanks to Jennifer Steyaert for conducting preliminary protein structural modeling.


This project was funded by NSF CBET 1438660.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9762_MOESM1_ESM.pdf (947 kb)
ESM 1 (PDF 947 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringColorado State UniversityFort CollinsUSA
  2. 2.Division of Pulmonary Sciences and Critical Care MedicineUniversity of Colorado DenverAuroraUSA
  3. 3.Department of Chemical and Biological EngineeringColorado State UniversityFort CollinsUSA
  4. 4.Microbial Insights, Inc.KnoxvilleUSA

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