Fatty+Acid+Methyl+Ester+Analysis


 * Fatty Acid Methyl Ester Analysis **
 * (FAME/PFLA) **
 * John W. Dille **
 * Applied & Environmental Microbiology **

categorized by merely perusing surface detail. This is especially true when considering microorganisms. Tens of thousands of species of microorganisms share a handful of phenotypic traits. Bacteria may appear as rods, spheres or spirals. The body shape of such organisms simply does not offer enough information for phylogenetic analysis. Scientists have wrestled with this conundrum for over 300 years, since the discovery of microorganisms in 1675 by Anton van Leeuwenhoek. During this time many novel methods have been developed to highlight and analyze characteristics that distinguish between species. The purpose of this document is to highlight one such method, Fatty Acid Methyl Ester Analysis (FAME). ||
 * ** Introduction ** ||
 * It is in our nature to organize and categorize the chaos around us. Alas, not all mysteries unravel easily and not all objects can be


 * Concept **

all cells providing an isolated, conditioned environment in which the extremely specific chemical reactions of life may take place. The plasma membrane itself is made up of a bilayer of countless individual phospholipid subunits, with the occasional receptor protein sandwiched in. The polar head groups of the phospholipids face aqueous environments while the hydrophobic fatty acid tails face inward. [3 ] An interesting and particularly useful revelation was made about the individual phospholipids that make up the membrane. While all species possess a plasma membrane, the fatty acid tails of the phospholipid subunits possess a variety of chemical make-ups between species. Hundreds of different fatty acids have been detected in bacterial species and more are likely to be discovered. [2] Therefore, each individual species possesses a certain fatty acid fingerprint that can be detected. || || material using an extraction technique. Next the phospholipid head group and the fatty acid tails are isolated from one another by transesterification. At this point there will be a variety of fatty acids specific to the organism are present in solution. The solution is passed through a gas chromatograph. The gas chromatograph produces a series of peaks relative to the retention times of the fatty acids in the column. Every species will produce its own unique GC fingerprint. [2] || || others. This statistical approach allows researchers to determine how closely related the organisms are related phylogenetically. This is often referred to as a cluster analysis. || ||
 * The plasma membrane is a cellular characteristic that all living organisms possess. This membrane is a sheath that surrounds
 * The FAME technique is rather straight forward. A researcher separates the plasma membrane from the rest of the cellular
 * Once all samples have been processed the distinguishing characteristics for each organism may be compared with that of the

**
 * Methods
 * Lipid Extraction **

Such samples might include soil, humic material, animal integument, sand, and any number of confounding chemicals. These items must be separated from the phospholipids that we wish to study. Lipid extraction serves two purposes; cellular components are separated from debris, and cells are broken open or dissolved so that cellular components themselves may be isolated from one another. || || chemical stressors that serve to dissolve the bonds between cellular components and between cellular components and debris. This chemical process is usually performed using chloroform (moderately hydrophobic), and methanol (fully miscible). These chemicals serve to separate cellular components and debris according to hydrophobicity. During the exposure to these chemicals the sample is often shaken vigorously for an extended amount of time which serves to mechanically break cells open and shake bonds apart. Phospholipids are somewhat hardy and their extraction procedure can last much longer than something like a DNA extraction. [2] || || settle for 24 hours or the sample may be centrifuged for a shorter period of time. Centrifugation serves to settle the heavier cellular components to the bottom of the sample tube. When the sample finally settles and all chemicals are allowed to phase out the phospholipids will obey their nature and settle out where the organic phase meets the aqueous phase. The polar phospholipid head group is attracted to the methanol, while the fatty acid tails are attracted to the more hydrophobic chloroform layer. [1] || ||
 * Many times when a researcher wishes to analyze a sample that sample is far from clean.
 * During the lipid extraction the sample is subjected to two actions. The sample is subjected to
 * Once the shaking procedure has finished, two hours, the sample may either be allowed to

containing the phospholipids are decanted. It is important to note that the phospholipids are not the only chemical compounds that will be attracted to the area where the phases meet. There are likely to be many lipid compounds that are not phospholipids present. Therefore the phospholipids must be isolated. This isolation is accomplished by liquid column chromatography. The sample is added to the top of a bonded phase column and an eluent is added to “drag” the sample through the column. As the sample moves, the different lipids will separate out based on their chemical structure. [4] || || fatty acid carries a negative charge when it is detached from its phospholipid head group will quickly bind to the inside of the chromatograph column and never release their bond. In order to rectify this problem a methyl group is added to the polar end of the fatty acid in an ingenious method called transesterification. Transesterification is a chemical reaction in which the oxygen in an ester trades one of its current carbon neighbors for another. With respect to FAME the carbon that is removed is the carbon that connects the fatty acid to the phospholipid head group and the carbon it is replaced by is a methyl group. Hence, the terms methanolysis and Fatty Acid Methyl Ester suddenly make a lot more sense. These fatty acid methyl esters may now be injected into a gas chromatograph and their retention times may be represented graphically and analyzed by any number of statistical methods. [2] ||
 * Phospholipid Extraction **
 * With the debris and cellular components pelleted at the bottom of the tube the organic and aqueous phase
 * Transesterfication/Methanolysis **
 * As we will soon see fatty acids are processed using a gas chromatograph. Yet the oxygen group of a


 * [[image:phosphatidate.gif width="380" height="374"]] || [[image:figure03-20.jpg]] || [[image:triglyceride.jpg width="426" height="114"]] ||


 * ** Sources ** ||


 * Alef K, Nannipieri P. 1998. Methods in Applied Soil Microbiology and Biochemistry. Academic Press. San Diego, Ca. 431-436 ||


 * Shutter ME, Dick RP. 2000. Comparison of Fatty Acid Methyl Ester (FAME) Methods for Characterizing Microbial Communities. Soil Science Society of America Journal. 64:1659–1668 ||


 * Singer, S.J. and G. L. Nicolson . 1972. The fluid mosaic model of the structure of cell membranes. Science. 175 720-731 ||


 * Vollhardt KPC, Schore NE. 2007. Organic Chemistry: Structure and Function. Fifth Edition. Freeman. New York, NY. 1212-1213. ||


 * Weaver RW, et al. 1994. Methods of Soil Analysis: Part 2 – Microbiological and Biochemical processes. Soil Science Society of America. Madison, WI. 551-553 ||