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Evaluation of quenching and extraction procedures for performing metabolomics in Acidithiobacillus ferrooxidans

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Abstract

Introduction

Acidithiobacillus ferrooxidans has a central role in the microbial community metabolism that drives production of acid mine drainage (AMD), a major environmental concern. Metabolomic profiling can offer insight into how At. ferrooxidans contributes to these processes.

Objective

The unique biology of some organisms means that protocols for metabolomic profiling need to be species-specific. Current protocols have largely been optimized for neutrophilic model organisms and, presently, no protocol exists for studying acidophilic extremophiles such as At. ferrooxidans. An appropriate protocol was developed and applied to investigate At. ferrooxidans’ metabolomic capabilities in relation to the colonization of AMD sites.

Methods

We quantified the overall effectiveness of three quenching solutions in combination with three extraction solutions, quantifying the amount of metabolite leakage, number of metabolites extracted and degradation of C13 labeled standards. We then used this method to quantify how the At. ferrooxidans metabolome differed between early and late stages in the logarithmic growth phase to investigate infer how the metabolism of the organism changes as it colonizes the AMD environment.

Results and discussion

An acidic methanol:water based quenching solution with ammonium formate salt used in conjunction with an isopropanol:methanol:water extraction solution produced the smallest amount of leakage, extracted the largest number of metabolites, and was most effective in recovering known standards. When this protocol was applied to the metabolomic fingerprinting of At. ferrooxidans in the beginning and end of its logarithmic growth phase, there was a clear separation in the metabolome at each growth point. Overall, 3% of the metabolome differed significantly.

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Acknowledgements

The authors thank Eric Gauthier and Jason Tennessen for reviewing an MSc thesis version of this manuscript.

Funding

This study was funded by Grant 230113 from the Canada Research Chairs Program to Thomas Merritt and a Northern Ontario Heritage Fund Corporation Internship position to Marney Doran.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, P3E 2C6, Canada

    Marney L. Doran, Alexandra Methé & Thomas J. S. Merritt

  2. Department of Biology, Laurentian University, Sudbury, ON, P3E 2C6, Canada

    Nadia Mykytczuk & Arielle Bieniek

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  1. Marney L. Doran
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  2. Nadia Mykytczuk
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  3. Arielle Bieniek
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  5. Thomas J. S. Merritt
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Correspondence to Thomas J. S. Merritt.

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11306_2017_1298_MOESM1_ESM.tiff

Supplemental Figure 1: The natural logarithm of the ferric iron concentrations versus time as determined by spectrophotometric analysis of iron production when At. ferrooxidans is grown in TK media. The assumption being that the exponential phase of iron production matches the exponential phase of At. ferrooxidans growth (TIFF 24 KB)

Supplemental Figure 2: Mean of technical replicates (n = 3) of the cell supernatant (TIFF 23 KB)

11306_2017_1298_MOESM3_ESM.tiff

Supplemental Figure 3: Mean of technical replicates (n = 3) of cell supernatant and AmFm quenching solution (TIFF 21 KB)

11306_2017_1298_MOESM4_ESM.tiff

Supplemental Figure 4: Mean of the technical replicates (n = 3) of cell supernatant and TK quenching solution (TIFF 20 KB)

11306_2017_1298_MOESM5_ESM.tiff

Supplemental Figure 5: Mean of technical replicates (n = 3) of cell supernatant and physiological saline quenching solution (TIFF 20 KB)

Supplemental Figure 6: Mean of technical replicates (n = 3) of the IMW extraction (TIFF 28 KB)

Supplemental Figure 7: Mean of technical replicates (n = 3) of the CMW extraction (TIFF 26 KB)

Supplemental Figure 8: Mean of technical replicates (n = 3) of the AAW extraction (TIFF 27 KB)

11306_2017_1298_MOESM9_ESM.tiff

Supplemental Figure 9: Mean of technical variation (n = 3) in fourteen 13C labeled standards in three extraction solutions (TIFF 33 KB)

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Doran, M.L., Mykytczuk, N., Bieniek, A. et al. Evaluation of quenching and extraction procedures for performing metabolomics in Acidithiobacillus ferrooxidans . Metabolomics 13, 156 (2017). https://doi.org/10.1007/s11306-017-1298-4

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