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Critical evaluation of solid waste sample processing for DNA-based microbial community analysis

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Abstract

Landfills represent a unique microbial ecosystem and play a significant role in global biogeochemical processes. The study of complex ecosystems such as landfills using DNA-based techniques can be advantageous since they allow for analysis of uncultured organisms and offer higher resolution in measuring demographic and metabolic (functional) diversity. However, sample acquisition and processing from refuse is challenging due to material heterogeneity. Decomposed refuse was used to evaluate the effect of seven sample processing methods on Bacteria and Archaea community structure using T-RFLP. Bias was assessed using measured richness and by comparing community structure using multi-dimensional scaling (MDS). Generally, direct methods were found to be most biased while indirect methods (i.e., removal of cellular material from the refuse matrix before DNA extraction) were least biased. An indirect method using PO4 buffer gave consistently high bacterial and archaeal richness and also resulted in 28 and 34% recovery of R. albus and M. formicicum spiked into refuse, respectively. However, the highest recovery of less abundant T-RFs was achieved using multiple processing methods. Results indicate differences in measured T-RF diversity from studies of landfill ecosystems could be caused by methodological (i.e., processing method) variation rather than refuse heterogeneity or true divergence in community structure.

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Acknowledgements

This work was funded by Waste Management, Inc. We would like to thank Dr. Jose Barcena and the N.C. State University Biotechnology Training and Education Center for use of the real-time PCR machine, Dr. Stefan Franzen (N.C. State Chemistry Dept.) for use of the fluorometer and Dr. Kenneth Pollock (N.C. State Zoology Dept.) for his kind review of the approach used to measure bias, and Xia He for help with statistical analysis. Bryan Staley’s work was partially funded through a scholarship from the Environmental Research & Education Foundation and a fellowship from N.C. Beautiful.

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  1. Environmental Research and Education Foundation, 3301 Benson Drive, Suite 301, Raleigh, NC, 27609, USA

    Bryan F. Staley

  2. Department of Civil and Environmental Engineering, American University of Beirut, Beirut, Lebanon

    Pascal E. Saikaly

  3. Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, NC, USA

    Francis L. de los Reyes III & Morton A. Barlaz

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  1. Bryan F. Staley
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  2. Pascal E. Saikaly
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  3. Francis L. de los Reyes III
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Correspondence to Bryan F. Staley.

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Staley, B.F., Saikaly, P.E., de los Reyes, F.L. et al. Critical evaluation of solid waste sample processing for DNA-based microbial community analysis. Biodegradation 22, 189–204 (2011). https://doi.org/10.1007/s10532-010-9387-3

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