Evaluation of quenching and extraction procedures for performing metabolomics in <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

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.