Quantification of Bacterial Fatty Acids by Extraction and Methylation

Materials and Reagents

1. Cell culture for lipid analysis. When analyzing bacterial cultures expressing a thioesterase, it is advisable to wait until the late stationary phase (8-24 h for E. coli) to perform the extraction to allow sufficient time for product accumulation (Lennen et al., 2013)
   
2. Methyl heptadecanoate (should be  ≥ 99% purity) (e.g. Sigma-Aldrich, catalog number: 51633 )
   
3. GC sample vials (VWR International, catalog number: 46610-722 ) (ensure you purchase vials compatible with any autosampler you are using)
   
4. Compressed nitrogen gas/regulator/tubing
   
5. Reverse osmosis water
   
6. Methanol
   
7. Absolute (100%) ethanol
   
8. High resolution gas chromatography grade hexane (99.9% pure mixture of hexane isomers)
   
9. Glacial acetic acid (Thermo Fisher Scientific, catalog number: A38-212 )
   
10. Deionized water
    
11. Anhydrous 1.25 M HCl in methanol (Sigma-Aldrich, catalog number: 17935 )
    
12. Sodium bicarbonate
    
13. 0.5 M Sodium methoxide in methanol (Sigma-Aldrich, catalog number: 403067 )
    
14. Chloroform
    
15. Appropriate internal standards (e.g. non-native, odd chain fatty acids). Some possibilities include:
    1. Heptadecanoic acid (C17:0) (99% + pure)
       
    2. Pentadecanoic acid (C15:0) (99% + pure)
       
    3. 1,2-dipentadecanoyl-sn-glycero-3-phosphoethanolamine in chloroform (99% + pure) (Avanti Polar Lipids,  catalog number: 850704 )
       
16. External standards (the methyl esters of fatty acids you wish to quantify at a known concentration generally these may be obtained from a commercial source). A useful external standard mixture is sold by Supelco through Sigma-Aldrich (catalog number: 18918-1AMP )
    
17. Antifoam 204 (Sigma-Aldrich, catalog number: A8311 )

Equipment

1. 5 ml glass pipettes
   
2. 10 ml Glass centrifuge tubes (Thermo Fisher Scientific, Kimble^TM, catalog number: 73785-10 ) with fluroropolymer lined caps (Thermo Fisher Scientific, Kimble^TM, catalog number: 73802-15415 )
   
3. Gloves
   
4. Goggles
   
5. Appropriate personal protective equipment according to local regulations
   
6. Chemical fume hood
   
7. Centrifuge
   
8. Vortex mixer
   
9. Vacuum source/aspiration equipment
   
10. Lyophilizer
    
11. Water bath
    
12. Mass spectrometer or flame ionization detector
    
13. Agilent 7890 GC with a 30 m x 0.25 mm HP 5-ms capillary column

Procedure