Sampling for metabolome analysis of microorganisms

In the present work we investigated the most commonly applied methods used for sampling of microorganisms in the field of metabolomics in order to unravel potential sources of error previously ignored but of utmost importance for accurate metabolome analysis. To broaden the significance of our study, we investigated different Gram-negative and Gram-positive bacteria, i.e., Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Gluconobacter oxydans, Pseudomonas putida, and Zymononas mobilis, and analyzed metabolites from different catabolic and anabolic intracellular pathways. Quenching of cells with cold methanol prior to cell separation and extraction led to drastic loss (>60%) of all metabolites tested due to unspecific leakage. Using fast filtration, Gram-negative bacteria also revealed a significant loss (>80%) when inappropriate washing solutions with low ionic strength were applied. Adapting the ionic strength of the washing solution to that of the cultivation medium could almost completely avoid this problem. Gram-positive strains did not show significant leakage independent of the washing solution. Fast filtration with sampling times of several seconds prior to extraction appears to be a suitable approach for metabolites with relatively high intracellular level and low turnover such as amino acids or TCA cycle intermediates. Comparison of metabolite levels in the culture supernatant and the cell interior revealed that the common assumption of whole broth quenching protocols attributing the metabolites found exclusively to the intracellular pools may not be valid in many cases. In such cases a differential approach correcting for medium-contained metabolites is required.