The overall goal of this protocol is to identify fatty acids from bacteria and to identify the position of methyl branching and double bonds. This method can help answer key questions in the bacteriology field such as the role of fatty acids in the way bacteria adapt to their environment. The main advantage of this technique is that prolification of fatty acids is not required.
The implications of this technique extend toward bacterial identification because of the importance of fatty acid composition in bacterial taxonomy. Though this method was developed on the bacteria Bacillus cereus, it is also very relevant to study other species containing branch chain fatty acids. Generally, in development to this method, we struggled.
Because current identification method for fatty acids do not identify methyl branching in double bond position. To begin this procedure, prepare a lawn of the bacteria by spreading 100 microliters of an overnight culture of the strain, incubated at 30 degrees Celsius in LB over the surface of the plate of LB agar medium. Incubate the plate overnight at 30 degrees Celsius.
In order to obtain lipid fatty acids, harvest bacterial cells by scraping colonies from the agar plate. And transferring approximately 40 milligrams into a ten milliliter glass tube with a screw cap and PTFE seal. To perform transesterification, add five milliliters of 0.2 molar potassium hydroxide in methanol to the fresh bacterial cells.
Vortex, and incubate at 37 degrees Celsius for one hour. Following this, add one milliliter of one molar ascetic acid to lower the pH to seven. Now, add three milliliters of hexane and vortex one minute to extract the fatty acid methyl esters or FAMEs.
Transfer the upper phase into clean tubes and concentrate by evaporation at room temperature under a continuous flow of nitrogen to obtain approximately 200 microliters of FAME extract. Then, transfer the extracts into GC vials with inserts. Inject the extracts into a gas chromatography mass spectrometry, or GC-MS system equipped with a capillary column.
Set the injection port temperature to 250 degrees Celsius. Hold the oven temperature at 50 degrees Celsius for one minute. Increase to 190 degrees Celsius at a rate of 20 degrees Celsius per minute and increase to a final temperature of 230 degrees Celsius at a rate of two degrees Celsius per minute.
For the MS, record the mass spectra by electron ionization, or EI, at 70 electron volts, and set the acquisition of the total ion current between 50 and 400 atomic mass units, or AMU. Dissolve a sample of the dried FAME extract in one milliliter of dry dichloromethane. Then, add the extract solution and 0.2 milliliters of 3-pyridinemethanol to 0.1 milliliters of one molar potassium tert-butoxide in tetrahydrofuran.
Heat the solution at 40 degrees Celsius for 30 minutes in a closed vial. After cooling the solution to room temperature, add two milliliters of purified deionized water and four milliliters of hexane. Mix the solution for one minute with a vortex mixer.
After allowing the phases to separate, collect the upper organic phase. Next, add anhydrous sodium sulfate until the organic phase is clear. Transfer the organic phase into a clean tube, then evaporate the organic phase under a stream of nitrogen until reaching a volume of approximately 200 microliters.
Add 250 milligrams of 2-amino-2-methyl-1-propanol to sample of dried FAME extract. Flush the tube with nitrogen and close it with a stopper. Then, heat the solution at 190 degrees Celsius overnight.
After cooling the solution to room temperature, add three milliliters of dichloromethane and five milliliters of purified deionized water. Vortex the mixture and allow the phases to separate. Then, remove the aqueous phase.
Following this, wash the organic phase with five milliliters of water. Add anhydrous sodium sulfate until the organic phase is clear. After transferring the organic phase to a new test tube, evaporate it under a stream of nitrogen until reaching a volume of approximately 200 microliters.
The I16 picolinal derivative mass spectrum confirms methyl branching. The gap of 28 AMU between ions at 304 and 332 corresponds to fragments created before and after the branched carbon. The diagnostic ions, 113 and 126, characteristic of DMOX derivatives, are shown here.
The molecular ion 307 is characteristic of the 16:1 isomer, and a gap of 12 AMU between 196 and 208 indicates a double bond at carbon. The gap of 12 AMU is no longer observed when the double bond is before carbon 7. The intense 153 ion, characteristic of a double bond at carbon 5, and the 307 molecular ion in the DMOX derivative mass spectrum identified this compound as n16 one delta five.
The 307 ion of the DMOX derivative and the 345 ion of the picolinal derivative indicate a 16:1 fatty acid isomer. A gap of 28 AMU indicates the branching position, and a gap of 12 AMU for DMOX and 26 AMU for picolinal ester identifies the double bond. Fatty acids and corresponding diagnostic ions identified in Bacillus cereus are listed here.
The diagnostic ions identify the methyl branching and double bond positions on the carbon chain for the DMOX and picolinal ester derivatives. Once mastered, this technique can be done in two days, if it is performed properly. Following this procedure, fatty acid can be quantified by GC-MS and the release of the FAME derivatives.
After it's development, this technique paved the way for researchers in the field of bacteriology to explore bacterial diversity and mechanisms of bacterial adaptations. After watching this video, you should have a good understanding of how to study fatty acids in bacteria including methyl branched and unsaturated fatty acids. Don't forget that working with reagents can be extremely hazardous, and precautions such as working with a laboratory fume hood and wearing gloves should always be taken while performing this procedure.
