Table 2.

Summary of optimized extraction methods for bacterial metabolomics studies.

Bacteria	Approach	Optimal extraction method	Findings	Ref.
Gram-negative
E. coli	Untargeted	(−40oC) methanol	(i) Highest detection of metabolites spots (80–99) compared to perchloric acid, alkaline, hot ethanol, methanol/chloroform, and hot methanol.	[25]
E. coli	Untargeted	(−48°C) methanol plus freeze-thaw	(i) Highest recovery of peaks from methanol extraction method compared to other methods.	[71]
E. coli	Untargeted	Acidic acetonitrile-methanol	(i) Extraction minimizes the loss of high-energy metabolites and their conversion into low-energy derivatives.	[94]
E. coli	Untargeted	(−40°C) methanol: chloroform (1 : 1)	(i) (−40°C) methanol: chloroform (1 : 1) extracts higher concentration of metabolites compared to (−40°C) methanol.	[24]
E. coli	Untargeted	Buffered hot water (95°C)	(i) Buffered hot water showed the best reproducibility with smallest detection limits that enable estimation of true in vivo enzymes as exemplified for fructose 1,6-biphosphate, and citrate synthase.	[101]
C. crescentus	Untargeted	(−20°C, 80%) methanol: water (8 : 2) with freeze-thaw cycles	(i) High recovery of polar metabolites, CoA and CoA thioester derivatives, citric acid, and some nucleotides.	[59]
E. coli, P. aeruginosa, S. typhimurium, and MSSA	Untargeted	Bead milling in (−80°C) methanol: water (9 : 1)	(i) Higher yield of metabolites with more efficient dispersal of cell pellet.	[53]
P. taiwanensis VLB120	Targeted	Pressure driven fast filtration approach followed by boiling ethanol: water (75 : 25, v/v) at 70°C	(i) Detection of 107 metabolites and quantification of 94 metabolites including nucleotides, amino acids, central carbon metabolism intermediates, and redox cofactors.	[60]
E. coli	Targeted	40 : 40 : 20 methanol: acetonitrile: H2O with 0.1% formic acid	(i) 106°C metabolites were confidently detected and 21 isotope-labelled metabolites were quantified.	[38]
Gram-positive
MRSA	Untargeted	(−20°C, 60%) ethanol	(i) High efficiency and reproducibility in extracting some polar compounds such as nucleotides and phosphorylated sugar.	[36]
			(ii) Successfully characterized 210 of well-defined compounds.
S. aureus	Untargeted	(−20°C, 60%) ethanol plus glass bead with two cycles in homogenizer	(i) Produce the most useful outcome for a global metabolomics analysis with detection of higher concentration and highest number of metabolites.	[49]
B. subtilis	Untargeted	Two-step extraction method, first with 60% cold ethanol and second with cold water with freeze-thaw	(i) Detection of highest metabolite amounts with a good EC-value.	[56]
S. aureus	Untargeted	Bead beating in a cold (−20°C) methanol: chloroform: water (3 : 1: 1)	(i) Fast and reproducible, allows direct comparison between different bacterial growth states.	[72]
B. licheniformis	Untargeted	Bead milling in liquid nitrogen	(i) Identification of 116 metabolites.	[87]
			(ii) More types of amino acids with high concentrations were identified compared to liquid nitrogen grinding.
Streptomyces ZYJ-6	Targeted	Suspension in 50% (v/v) methanol and three cycles of freeze-thaw	(i) 44 of most highly abundant intracellular metabolites were found and quantified.	[66]