Extracellular Microbial Metabolomics: The State of the Art

Abstract

Microorganisms produce and secrete many primary and secondary metabolites to the surrounding environment during their growth. Therefore, extracellular metabolites provide important information about the changes in microbial metabolism due to different environmental cues. The determination of these metabolites is also comparatively easier than the extraction and analysis of intracellular metabolites as there is no need for cell rupture. Many analytical methods are already available and have been used for the analysis of extracellular metabolites from microorganisms over the last two decades. Here, we review the applications and benefits of extracellular metabolite analysis. We also discuss different sample preparation protocols available in the literature for both types (e.g., metabolites in solution and in gas) of extracellular microbial metabolites. Lastly, we evaluate the authenticity of using extracellular metabolomics data in the metabolic modelling of different industrially important microorganisms.

Keywords: analytical instruments; culture medium; data integration; exometabolome; extracellular metabolites; intracellular metabolites; metabolic modelling; metabolite footprinting; sample preparation.

Figure 1

Discriminant function analysis (DFA) to visualize exometabolomics data. Extracellular metabolites from deliberately contaminated and control samples from microalgal fermentation were analyzed using Gas-chromatography and mass spectrometry (GC-MS) [42]. The data from 56 samples were log transformed prior to performing DFA and three distinct clusters were observed. Here, black circles represent the samples from flasks contaminated with Pseudomonas aeruginosa, red circles show the samples contaminated by Bacillus subtilis, and light green and dark green circles present the samples collected from contaminated flasks at time 0 and non-contaminated flasks, respectively. This figure was reproduced from Sue et al. with the authors’ permission [42].

Figure 2

Extracellular sample preparation, handling, and storage. After centrifugation or fast filtration, culture media containing extracellular metabolites are usually stored at a low temperature and under dark conditions. Sometime, organic solvents are added to denature active enzymes. For some metabolites, specific extraction procedures need to be followed before being analyzed by appropriate instruments. Prior to analysis, extracellular samples are often freeze-dried to concentrate the level of metabolites.

Figure 3

Overview of extracellular metabolite analysis. Different technical methods, such as solid-phase extraction (SPE), solid-phase micro extraction (SPME), head space (HS) analysis, and HS-SPME, are used for the preparation of extracellular samples. Sample preparation protocols depend on the type of metabolite. Here, GC-MS = gas-chromatography coupled to mass spectrometry; LC-MS = liquid-chromatography coupled to mass spectrometry; NMR = nuclear magnetic resonance spectroscopy; HPLC = high pressure liquid chromatography; FTIR = Fourier transform infra-red spectroscopy.