An Introduction to the use of Fatty Acid Analysis for the Characterisation of Bacteria and Fungi

From the Manual of the IMI Training Course 'Modern Techniques in the Identification of Bacteria and Filamentous Fungi'

The Principles of Fatty Acid Analysis

Like all cellular life forms, bacteria and fungi possess a cytoplasmic membrane as a component of their cell envelope, the composition of which is roughly 50% lipid. The membrane lipids are a diverse group of molecules which are frequently used as markers for the classification and identification of microorganisms. In particular, the amphipathic lipids (possessing hydrophilic and hydrophobic regions) have great relevance to microbial systematics, both as complete molecules and when broken down to their constituent parts. Amphipathic lipids such as glycoplipids and phospoglycerides consist of a polar "head" and nonpolar hydrocarbon "tails" and are generally arranged to form a lipid-bilayer. The nonpolar tails are generally esters of longchain fatty acids.

The fatty acid composition of microorganisms has been used extensively to aid microbial characterisation. In general, the fatty acid profile of most bacteria ranges from n-C9 to n-C20. The fatty acids in most Gram-positive bacteria are located in the cytoplasmic membrane, though some, like the mycobacteria and related genera have long chain lipids, known as mycolic acids (ß-hydroxy fatty acids) which are found in an outermembrane-like structure. Gram-negative bacteria possess fatty acids in the cytoplasmic membrane though they can also be found in the cell wall fraction, generally in the lipopolysaccharide (LPS). In common with most eukaryotic cells, fungal fatty acids composition is much simpler than the prokaryotes, n-C16 and n-C18 acids predominate. These are commonly found as components of more complex cytoplasmic membrane lipids such as acylglycerols and glycerophospholipids, they can also be found in the "free" form, though the significance of this remains unclear.

Microbial fatty acids can be found in a variety of forms. The basic types of acids consists of; straight chain saturated, mono- and polyunsaturated acids, methyl- branched acids and those with cyclopropane rings or with hydroxy groups. Methyl-branched fatty acids of the iso- and anteiso- configurations occur predominantly in Gram-positive organisms. Though there are Gram-negative exceptions such as members of the genus Xanthomonas. In general, cyclopropylated and hydroxylated fatty acids are more commonly found in Gram-negative bacteria. The hydroxylated acids are of particular importance as they are generally components of the lipopolysaccharides, and can be highly diagnostic in determining species identity, particularly among non-fluorescent pseudomonads. Polyunsaturated fatty acids generally do not occur in prokaryotes though they do have significance in fungal characterisation.

The analysis of fatty acids can be broken down into five steps:

  1. Cell culture under standard conditions.
  2. Release of fatty acids from the cell surface (both cytoplasmic and outer membranes) by saponification.
  3. Methylation of the fatty acids to increase volatility.
  4. Analysis by gas chromatography, frequently using capillary columns to enhance sensitivity and resolution.
  5. Exploitation of the fatty acid profile for classification or identification. Identification can occur by comparing the fatty acid profile of an unknown to those in the literature or by computer library matching. Classification can involve statistical analysis using fatty acid profiles from related or similar organisms.

Further details of microbial lipids and fatty acids in particular can be found in the reference list below.

General References on Fatty Acid Analysis

  • Evershed, R.P. (1992). Gas Chromatography of Lipids. In Lipid Analysis: A Practical Approach. pp. 113-151. (Eds. R.J. Hamilton & S. Hamilton). Oxford, IRL Press.
  • Gunstone, F.D. (1992). Introduction. In Lipid Analysis: A Practical Approach. pp. 1-12. (Eds. R.J. Hamilton & S. Hamilton). Oxford, IRL Press.
  • Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics. Methods in Microbiology, 19: 161-207.
  • Stead, D.E., Sellwood, J.E., Wilson, J. & Viney, I. (1992). Evaluation of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria. Journal of Applied Biochemistry, 72: 315-321.
  • Tornabene, T.G. (1985). Lipid analysis and the relationship to chemotaxonomy. Methods in Microbiology, 18: 209-234.
  • Welch, D.F. (1991). Applications of cellular fatty acid analysis. Clinical Microbiology Reviews, 4: 422-438.

The Use of Fatty Acid Analysis in Bacterial Characterisation

  • Bendinger, B., Kroppenstedt, R.M., Klatte, S. & Altendorf, K. (1992). Chemotaxonomic differentiation of coryneform bacteria isolated from biofilters. International Journal of Systematic Bacteriology, 42: 474-486.
  • Casano, F., Wells, J. & van der Zwet, T. (1988). Fatty acid profiles of Erwinia amylovora as influenced by growth medium, physiological age and experimental conditions. Journal of Phytopathology, 121: 267-274.
  • Caudales, R., Wells, J.M., Antoine, A.D. & Butterfield, J.E. (1995). Fatty acid composition of symbiotic cyanobacteria from different host plant (Azolla) species: evidence for coevolution of host and symbiont. International Journal of Systematic Bacteriology, 45: 364-370.
  • Chase, A.R., Stall, R.E., Hodge, N.C. & Jones, J.B. (1992). Characterization of Xanthomonas campestris strains from aroids using physiological, pathological, and fatty acid analyses. Phytopathology, 82: 754-759.
  • Decallonne, J., Delmee, M., Wauthoz, P., El-Lioui, M. & Lambert, R. (1991). A rapid procedure for the identification of lactic acid bacteria based on the gas chromatographic analysis of the cellular fatty acids. Journal of Food Protection, 54: 217-224.
  • Esnard, J., Potter, T.L. & Zuckerman, B.M.(1994). Differentiation of six strains of Bacillus thuringiensis by hydrolyzable fatty acid composition. Journal of Agricultural and Food Chemistry, 42: 1251-1255.
  • Faucher, E., Paradis, E., Goyer, C., Hodge, N.C., Hogue, R., Stall, R.E. & Beaulieu, C. (1995). Characterization of streptomycetes causing deep-pitted scab of potato in Quebec, Canada. International Journal of Systematic Bacteriology, 45: 222-225
  • Graham, P.H., Sadowsky, M.J., Tighe, S.W., Thompson, J.A., Date, R.A., Howieson, J.G. & Thomas, R. (1995). Differences among strains of Bradyrhizobium in fatty acid-methyl ester analysis. Canadian Journal of Microbiology, 41: 1038-1042.
  • Gudmestad, N.C., Henningson, P.J. & Bugbee, W.M. (1988). Cellular fatty acid comparison of strains of Corynebacterium michiganense subsp. sepedonicum from potato and sugar beet. Canadian Journal of Microbiology, 34: 716-722.
  • Haak, S.K., Garchow, H., Odelson, D.A., Forney, L.J. & Klug, M.J. (1994). Accuracy, reproducibility, and interpretation of fatty acid methyl ester profiles of model bacterial communities. Applied and Environmental Microbiology, 60: 2483-2493.
  • Havelaar, A.H., Schets, F.M., van Silfhout, A., Jansen, W.H., Wieten, G. & van der Kooij, D. (1992). Typing of Aeromonas strains from patients with diarrhoea and from drinking water. Journal of Applied Bacteriology, 72: 435-444.
  • Helander, I.M. & Haikara, A. (1995). Cellular fatty acyl and alkenyl residues in Meagsphaera and Pectinatus species: contrasting profiles and detection of beer spoilage. Microbiology, 141: 1131-1137.
  • Henningson, P.J. & Gudmestad, N.C. (1991). Fatty acid analysis of phytopathogenic coryneform bacteria. Journal of General Microbiology, 137: 427-440.
  • Janse, J.D. (1991). Pathovar discrimination within Pseudomonas syringae subsp. savastanoi using whole cell fatty acids and pathogenicity as criteria. Systematic and Applied Microbiology, 14: 79-84.
  • Janse, J.D. & Smits, P.H. (1990). Whole cell fatty acid patterns of Xenorhabdus species. Letters in Applied Microbiology, 10: 131-135.
  • Jarvis, B.D.W. & Tighe, S.W. (1994). Rapid identification of Rhizobium species based on cellular fatty acid analysis. Plant and Soil, 161: 31-41.
  • Krausse, R. & Ullmann, U. (1991). A modified procedure for the identification of anaerobic bacteria by high performance liquid chromatography - quantitative analysis of short-chain fatty acids. Zentralblatt fur Bakteriologie, 276: 1-8.
  • Livesley, M.A., Thompson, I.P., Gern, L. & Nuttall, P.A. (1993). Analysis of intra-specific variation in the fatty acid profiles of Borrelia burgdorferi. Journal of General Microbiology, 139: 2197-2201.
  • MacKenzie, S.L., Lapp, M.S. & Child, J.J. (1979). Fatty acid composition of Rhizobium spp. Canadian Journal of Microbiology, 25: 68-74.
  • Minato, H., Ishibashi, S. & Hamaoka, T. (1988). Cellular fatty acid and sugar composition of representative strains of rumen bacteria. Journal of General and Applied Microbiology, 34: 303-319.
  • Mouahid, M., Hinz, K.H., Engelhard, E., Mutters, R. & Mannheim, W. (1992). Characterization of Haemophilus paragallinarum by analysis of whole cell carbohydrates, fatty acids and phospholipids. Avian Pathology, 21: 127-136.
  • Persson, P. & Sletten, A. (1995). Fatty acid analysis for the identification of Erwinia carotovora subsp. atroseptica and E. carotovora subsp. carotovora. Bulletin OEPP/EPPO Bulletin, 25:151-156.
  • Romalde, J.L., Magarinos, B., Turnbull, K.D., Baya, A.M., Barja, J.M. & Toranzo, A.E. (1995). Fatty acid profiles of "Pasteurella" piscicida: comparison with other fish pathogenic gram-negative bacteria. Archives of Microbiology, 163: 211-216
  • Roy, M.A. (1988). Use of fatty acids for the identification of phytopathogenic bacteria. Plant Disease, 72: 460.
  • Saddler, G.S., O'Donnell, A.G., Goodfellow, M. & Minnikin, D.E. (1987). SIMCA pattern recognition in the analysis of streptomycete fatty acids. Journal of General Microbiology, 133: 1137-1147.
  • Saxegaard, F., Andersen, O., & Jantzen, E. (1983). Wild animal mycobacterial isolates: characterization by cellular fatty acid composition and polar lipid patterns. Acta Veterinaria Scandinavica, 24: 225-237.
  • Seifert, H.S., Bohnel, H., Bunge, J., Erbrecht, A., Kuenheim, U. & Weck, H. (1979). A new method to classify pathogenic anaerobes, the statistical evaluation of quantitative determined patterns from metabolic fatty acids. Zentralblatt fur Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene, Erste Abteilung Originale 1, 243A: 82-101.
  • Siegel, J.P., Smith, A.R., Maddox, J.V. & Novak, R.J. (1993). Use of cellular fatty acid analysis to characterize commercial brands of Bacillus thuringiensis var. israelensis. Journal of the American Mosquito Control Association, 9: 330-334.
  • Sincoweay, H., Miyagawa, E. & Kume, T. (1981). Cellular fatty acid composition in staphylococci isolated from bovine milk. National Institute of Animal Health Quarterly, 21: 14-20.
  • Stead, D.E. (1989). Grouping of Xanthomonas campestris pathovars of cereals and grasses by fatty acid profiling. Bulletin OEPP/EPPO Bulletin, 19: 57-68.
  • Stead, D.E. (1992). Grouping of plant-pathogenic and some other Pseudomonas spp. by using cellular fatty acid profiles. International Journal of Systematic Bacteriology, 42: 281-295.
  • Suzuki, K.I. & Komagata, K. (1983). Taxonomic significance of cellular fatty acid composition in some coryneform bacteria. International Journal of Systematic Bacteriology, 33: 188-200.
  • Tas, A.C., Wieten, G., de Waart, J., Berwald, L. & van der Greef, J. (1988). Characterisation of Salmonella and possible interfering strains using GC profiling and factor analysis. Journal of Microbiological Methods, 8: 333-345.
  • Vauterin, L., Yang, P., Hoste, B., Pot, B., Swings, J. & Kersters, K. (1992). Taxonomy of xanthomonads from cereals and grasses based on SDS-PAGE of proteins, fatty acid analysis and DNA hybridisation. Journal of General Microbiology, 138: 1467-1477.
  • Wells, J.M., Butterfield, J.E. & Revear, L.G. (1993). Identification of bacteria associated with postharvest diseases of fruits and vegetables by cellular fatty acid composition: an expert system for personal computers. Phytopathology, 83: 445-455.
  • Wells, J.J., Zwet, T. van der, Butterfield, J.E. & Van der Zwet, T. (1994). Differentiation of Erwinia species in the 'Herbicola' group by class analysis of cellular fatty acids. Journal of Phytopathology, 140: 39-48.
  • Wells, J.M., Zwet, T. van der, Hale, C.N. & Van der Zwet, T. (1994). Differentiation of Erwinia species in the 'Amylovora' group by class analysis of cellular fatty acids. Journal of Phytopathology, 140: 31-38.
  • Weibgen, U., Russa, R., Yokota, A. & Mayer, H. (1993). Taxonomic significance of the lipopolysaccharide composition of the three biovars of Agrobacterium tumefaciens. Systematic and Applied Microbiology, 16: 177-182.
  • Wong, P.K. & Chung, W.K. (1993). Fatty acid analysis of bacterial foaming in the activated sludge process. Microbios, 75: 227-232.Xanthomas albilineans. Systematic and Applied Microbiology, 16: 420-426.
  • Yang, P., Vauterin, L., Vancanneyt, M., Swings, J. & Kersters, K. (1993). Application of fatty acid methyl esters for the taxonomic analysis of the genus Xanthomonas. Systematic and Applied Microbiology, 16: 47-71.
  • Zelles, L. & Bai, Q.Y. (1994). Fatty acid patterns of phospholipids and lipopolysaccharides in environmental samples. Chemosphere, 28: 391-411.
  • Zelles, L., Bai, Q.Y., Rackwitz, R., Chadwick, D., & Beese, F. (1995). Determination of phospholipid- and lipopolysaccharide-derived fatty acids as an estimate of microbial biomass and community structures in soils. Biology and Fertility of Soils, 19: 115-123.

The Use of Fatty Acid Analysis in Fungal Characterisation

  • Amano, N., Shinmen, Y., Akimoto, K., Kawashima, H., Amachi, T., Shimizu, S. & Yamada, H. (1992). Chemotaxonomic significance of fatty acid composition in the genus Mortierella (Zygomycetes, Mortierellaceae). Mycotaxon, 64: 257-265.
  • Bentivenga, S.P. & Morton, J.B. (1994). Stability and heritability of fatty acid methyl ester profiles of glomalean endomycorrhizal fungi. Mycological Research, 98: 1419-1426.
  • Jabaji-Hare, S. (1988). Lipid and fatty acid profiles of some vesicular-arbuscular mycorrhizal fungi: contribution to taxonomy. Mycologia, 80: 622-629.
  • Johnk, J.S. & Jones, R.K. (1994). Comparison of whole-cell fatty acid compositions in intraspecific groups of Rhizoctonia solani AG-1. Phytopathology, 84: 271-275.
  • Johnk, J.S. & Jones, R.K. (1992). Determination of whole-cell fatty acids in isolates of Rhizoctonia solani AG-1 IA. Phytopathology, 82: 68-72.
  • Martinez, A.T., Barrasa, J.M., Prieto, A. & Blanco, M.N. (1991). Fatty acid composition and taxonomic status of Ganoderma australe from southern Chile. Mycological Research, 95: 782-784.
  • Marumo, K. & Aoki, Y. (1990). Discriminant analysis of cellular fatty acids of Candida species, Torulosis glabrata and Cryptococcus neoformans determined by gas-liquid chromatography. Journal of Clinical Microbiology, 28: 1509-1513.
  • Muller, M.M., & Hallaksela, A.M. (1994). Variation in combined fatty acid and sterol profiles of Ascocoryne, Nectria, and Neobulgaria-strains isolated from Norway spruce. European Journal of Forest Pathology, 24: 11-19.
  • Muller, M.M., Kantola, R. & Kitunen, V. (1994) Combining sterol and fatty acid profiles for the characterization of fungi. Mycological Research, 98: 593-603.
  • Paulsrud, J.R., Queener, S.F., Bartlett, M.S. & Smith, J.W. (1993). Total cellular fatty acid composition of cultured Pneumocystis carinii. Journal of Clinical Microbiology, 31: 1899-1902.
  • Sancholle, M. & Dalpe, Y. (1993). Taxonomic relevance of fatty acids of arbuscular mycorrhizal fungi and related species. Mycotaxon, 49: 187-193.