Anal. Chem. 1996, 68, 2805-2810
Characterization of Lipid Fatty Acids in Whole-Cell Microorganisms Using in Situ Supercritical Fluid Derivatization/Extraction and Gas Chromatography/Mass Spectrometry Ahmad A. Gharaibeh and Kent J. Voorhees*
Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, Colorado 80401
In situ supercritical fluid derivatization and extraction was used as a sample preparation technique for the classification of bacteria using fatty acid profiling. Addition of a quaternary ammonium salt such as phenyltrimethylammonium hydroxide under static supercritical conditions directly to lyophilized, whole-cell bacteria in an extraction vessel resulted in the saponification of the bacterial lipids and derivatization of their fatty acids. The derivatized fatty acid methyl esters (FAMEs) were then extracted with supercritical CO2 and analyzed without additional treatment using GC/MS. Iso and anteiso C15:0 and C17:0 along with C18:0 were predominant in Gram-positive bacteria, while C16:1, C16:0, C18:1, and cyclopropyl cyC17:0 and cyC19:0 were significant in Gram-negative bacteria. Application of principal components analysis to the FAME GC/MS data resulted in the differentiation between Gram-positive and Gram-negative type bacteria. Differentiation between species among the same genera was also observed. Classical methods for the identification and classification of microorganisms are based on their biochemical, morphological, serological, and toxigenic characteristics. These methods usually require intact viable organisms and a series of tests requiring the incubation of the microorganisms. The limitations of these methods have led to the development of “analytical microbiology”,1 where analytical instrumental methods have been applied for the identification of microorganisms. In this approach, identification is based on the determination of the chemical composition of certain constituents in the microorganism, such as the profiling of lipids2,3 or the detection of a biomarker that signifies a particular microorganism.4-8 A variety of analytical techniques have been used in these analyses, including gas chromatography (GC),9-15 gas chromatography/mass spectrometry (GC/MS),16-18pyrolysis (1) Fox, A., Morgan, S. L., Larsson, L., Odham, G., Eds. Analytical Microbiology Methods: Chromatography and Mass Spectrometry; Plenum: New York, 1990. (2) Shaw, N. Adv. Appl. Microbiol. 1974, 17, 63-108. (3) Lechevalier, M. P. Crit. Rev. Microbiol. 1977, 7, 109-210. (4) Smith, C. S.; Morgan, S. L.; Parks, C.; Fox, A.; Pritchard, D. G. Anal. Chem. 1987, 59, 1410-1413. (5) Ueda, K.; Morgan, S. L.; Fox, A.; Gilbart, J.; Sonesson, A.; Larsson, L.; Odham, G. Anal. Chem. 1989, 61, 265-270. (6) Snyder, A. P.; McClennen, W. H.; Dworzanski, J. P.; Meuzelaar, H. L. C. Anal. Chem. 1990, 62, 2565-2573. (7) Watt, B. E.; Morgan, S. L.; Fox, A. J. Anal. Appl. Pyrolysis 1991, 19, 237249. (8) Voorhees, K. J.; DeLuca, S. J.; Noguerola, A. J. Anal. Appl. Pyrolysis 1992, 24, 1-21. S0003-2700(96)00076-5 CCC: $12.00
© 1996 American Chemical Society
mass spectrometry (Py-MS),19-25 pyrolysis gas chromatography/ mass spectrometry (Py-GC/MS),26-29 fast atom bombardment (FAB) mass spectrometry,30,31 and Fourier-transform infrared (FTIR) spectroscopy.32 The most common approach in the classification of bacteria through lipid profiling is the analysis of their fatty acid methyl esters (FAMEs).9-20,26,27 Commercial instruments have been introduced which correlate the fatty acid composition to bacterial type.33 One of the most successful of these instruments is based on gas chromatography. The Microbial Identification, Inc. (MIDI) approach utilizes conventional saponification of the bacterial cells and derivatization (methylation) of the fatty acids, followed by gas chromatography analysis of the fatty acid methyl esters (FAMEs). A multivariate statistical procedure is used to classify the various (9) Abel, K.; DeSchmertzing, H.; Peterson, J. I. J. Bacteriol. 1963, 85, 10391044. (10) Kaneda, T. J. Bacteriol. 1967, 93, 894-903. (11) Moss, C. W.; Lambert, M. A.; Merwin, W. H. Appl. Microbiol. 1974, 28, 80-85. (12) Moss, C. W.; Dees, S. B.; Guerrant, G. O. J. Clin. Microbiol. 1980, 12, 127130. (13) Miller, L. T. J. Clin. Microbiol. 1982, 16, 584-586. (14) Eerola, E.; Lehtonen, O.-P. J. Clin. Microbiol. 1988, 26, 1745-1753. (15) Mukwaya, G. M.; Welch, D. F. J. Clin. Microbiol. 1989, 27, 2640-2646. (16) Konda, E.; Ueta, N. J. Bacteriol. 1972, 110, 459-467. (17) Moss, C. W. J. Chromatogr. 1981, 203, 337-347. (18) Brondz, I.; Olsen, I.; Haapasalo, M.; Van Winkelhoff, A. J. J. Gen. Microbiol. 1991, 137, 1445-1452. (19) Basile, F.; Voorhees, K. J.; Hadfield, T. L. Appl. Environ. Microbiol. 1995, 61, 1534-1539. (20) DeLuca, S.; Sarver, E. W.; Harrington, P. deB.; Voorhees, K. J. Anal. Chem. 1990, 62, 1465-1472. (21) Anhalt, J. P.; Fenselau, C. Anal. Chem. 1975, 47, 219-225. (22) Tas, A. C.; De Waart, J.; Bouwman, J.; Ten Noever De Brauw, M. C.; Van Der Greef, J. J. Anal. Appl. Pyrolysis 1987, 11, 329-340. (23) Voorhees, K. J.; Durfee, S. L.; Updegraaf, D. M. J. Appl. Anal. Pyrolysis 1988, 8, 315-325. (24) Voorhees, K. J.; Durfee, S. L.; Holtzclaw, J. R.; Enke, C. G.; Bauer, M. R. J. Appl. Anal. Pyrolysis 1988, 14, 7-15. (25) DeLuca, S. J.; Sarver, E. W.; Voorhees, K. J. J. Anal. Appl. Pyrolysis 1992, 23, 1-14. (26) Holzer, G.; Bourne, T. F.; Bertsch, W. J. Chromatogr. 1989, 468, 181-190. (27) Dworzanski, J. P.; Berwald, L.; Meuzelaar, H. L. C. Appl. Environ. Microbiol. 1990, 56, 1717-1724. (28) Engman, H.; Mayfield, H. T.; Mar, T.; Bertsch, W. J. Anal. Appl. Pyrolysis 1984, 6, 137-156. (29) Smith, P. B.; Snyder, A. P. J. Anal. Appl. Pyrolysis 1992, 24, 23-38. (30) Heller, D. N.; Cotter, R. J.; Fenselau, C.; Uy, O. M. Anal. Chem. 1987, 59, 2806-2809. (31) Heller, D. N.; Murphy, C. M.; Cotter, R. J.; Fenselau, C.; Uy, O. M. Anal. Chem. 1988, 60, 2787-2791. (32) Helm, D.; Labischinski, H.; Schallelm, G.; Naumann, D. J. Gen. Microbiol. 1991, 137, 69-79. (33) Microbial Identification System Operating Manual, Version 3.0; MICROBIAL ID Inc.: Newark, DE, 1993.
Analytical Chemistry, Vol. 68, No. 17, September 1, 1996 2805
gas chromatograms. The company provides standard GC FAME distributions in five database types for over 2000 bacteria and has achieved classification to the subspecies level.33 Supercritical fluid extraction (SFE) has been extensively utilized as a sample preparation technique and as a potential alternative for traditional solvent extraction methods. Carbon dioxide has been the fluid of choice and has a solvent strength comparable to those of nonpolar organic solvents such as hexane and benzene.34 Interest in using supercritical fluids as reaction media35-38 has recently emerged, although little is known about chemical reactions in supercritical fluids. Under supercritical conditions, enhanced reaction rates at lower temperatures have been reported.39 This has triggered the coupling of derivatization reactions with supercritical fluid extraction in one step. In situ supercritical fluid derivatization/extraction offers the advantage of improved recoveries of polar and ionic compounds and eliminates the need for postextraction derivatization, which minimizes sample handling and reduces laboratory errors. A variety of derivatizing agents, such as tetramethylammonium hydroxide (TMAH), phenyltrimethylammonium hydroxide (PTAH), boron trifluoride/methanol, methyl iodide, and tetrabutylammonium hydroxide, have been used to simultaneously derivatize and extract organic acids.40-46 Examples of this approach include triglycerides in oilseeds,38 triglycerides in edible fat,45 and microbial phospholipid fatty acids from whole cells.46 The work reported in this paper demonstrates the use of supercritical carbon dioxide with PTAH for the saponification/derivatization/extraction of whole-cell bacterial lipids as their FAMEs, followed by GC/ MS analysis and statistical treatment of their FAME profiles for the characterization and differentiation of bacteria. EXPERIMENTAL SECTION Bacterial Cultures. Ten strains of bacteria were analyzed in this study: Escherichia coli strain K-12, purchased from Sigma Chemical Co. (St. Louis, MO) as lyophilized cells, and Enterobacter aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 10145), Pseudomonas fluorescens (ATCC 13525), Serratia marcescens (ATCC 13880), Bacillus cereus (ATCC 14579), Bacillus licheniformis (ATCC 14580), Bacillus subtilis (ATCC 6051), Staphylococcus aureus (ATCC 12600), and Enterococcus (Streptococcus) faecalis (ATCC 19433), obtained from the American Type Culture Collection (ATCC). All ATCC bacteria were grown as follows: about 30 g of BBL trypticase soy broth (Becton Dickinson Microbiology (34) Hawthorne, S. B. Anal. Chem. 1990, 62, 633A-642A. (35) Randolph, T. W.; Clark, D. S.; Blanch, H. W.; Prausnitz, J. M. Science 1988, 238, 387-390. (36) Evans, M. B.; Smith, M. S.; Oxford, J. M. J. Chromatogr. 1989, 479, 170175. (37) Shaw, R. W.; Brill, T. B.; Clifford, A. A.; Eckert, C. A.; Frank, E. U. Chem. Eng. News 1991, 26-39. (38) King, J. W.; France, J. E.; Snyder, J. M. Fresenius J. Anal. Chem. 1992, 244, 474-478. (39) Hills, J. W.; Hill, H. H., Jr.; Maeda, T. Anal. Chem. 1991, 63, 2152-2155. (40) Field, J. A.; Miller, D. J.; Field, T. M.; Hawthorne, S. B.; Gieger, W. Anal. Chem. 1992, 64, 3161-3167. (41) Lee, H.-B.; Peart, T. E.; Hong-You, R. L. J. Chromatogr. 1992, 605, 109113. (42) Rochette, E. A.; Harsh, J. B.; Hill, H. H., Jr. Talanta 1993, 40, 147-155. (43) Lopez-Avila, V.; Dodhiwala, N. S.; Beckert, W. F. J. Agric. Food Chem. 1993, 41, 2038-2044. (44) Croft, M. Y.; Murby, E. J.; Wells, R. J. Anal. Chem. 1994, 66, 4459-4465. (45) Berg, B. E.; Hansen, E. M.; Gjørven, S.; Greibrokk, T. J. High Resolut. Chromatogr. 1993, 16, 358-363. (46) Hawthorne, S. B.; Miller, D. J.; Nivens, D. E.; White, D. C. Anal. Chem. 1992, 64, 405-412.
2806 Analytical Chemistry, Vol. 68, No. 17, September 1, 1996
Figure 1. Conventional supercritical fluid extraction apparatus.
Systems, Cockeysville, MD) was dissolved in 1 L of deionized water in an Erlenmeyer flask and sterilized in an autoclave at 121 °C for 20 min. After the mixture cooled to room temperature, a few milligrams of a bacterial strain was suspended in the solution and the flask covered with a sterilized cheesecloth. This was then placed on a mechanical shaker inside an incubator at 30 °C for 36 h, after which the grown bacteria were centrifuged at 70009000 rpm for 10 min and collected in a 3 mL vial. The wet bacterial cells were then freeze-dried under vacuum for 24-36 h and kept frozen until used. Cellular Fatty Acid Derivatization and Extraction. A schematic diagram of the extraction apparatus is shown in Figure 1. Ten milligram samples of freeze-dried bacterial cells were placed in a 0.8 mL stainless steel extraction cell (50 mm length, 4.6 mm i.d., from Keystone Scientific, Bellefonte, PA) containing a 100 µL of 0.1 M phenyltrimethylammonium hydroxide (PTAH) in methanol. After the cell was sealed, one end of the extraction cell was connected to a Model 600 Lee Scientific SFC pump (Dionex Corp., Sunnyvale, CA) through a 2 m temperature conditioning coil of 1/16 in. o.d., 0.02 in. i.d. stainless steel tubing placed in a chromatographic oven (Hewlett Packard Model 5720A). A fused silica capillary restrictor (Polymicro Technologies, Phoenix, AZ), 10-15 cm long and 25 µm i.d., with one end sealed, was connected to the exit end of the extraction cell. The cell and contents were partially pressurized with supercritical CO2, heated to 100 °C, and then fully pressurized to 400 atm for 15 min, during which saponification and derivatization take place. After 15 min, the closed end of the restrictor was broken, and a 15 min dynamic extraction step was performed while collecting the analytes in methanol in a 3 mL vial. About 4-5 mL of liquid CO2 as measured at the pump was used in each extraction. The extract in methanol was concentrated to about 1 mL under a gentle stream of nitrogen gas and kept in the refrigerator until analyzed. Gas Chromatography/Mass Spectrometry Analysis. Fatty acid methyl ester standards and the extracts were analyzed by GC/MS using a Perkin Elmer Q-Mass 910 GC/MS system (Perkin Elmer Instruments, Norwalk, CT). A SPB-5 capillary column (Supelco, Bellefonte, PA), 30 m × 0.2 mm i.d., 0.2 µm film thickness, was used. Initial oven temperature was set at 140 °C, ramped to 230 °C at a rate of 5 °C/min, and then to 260 °C at a rate of 30 °C/min, with a final hold at 260 °C for 5 min. One microliter injections, splitless for 0.3 min, were made at an injector temperature of 280 °C. The temperature of the transfer line
Table 1. Cellular Fatty Acid Compositions of 10 Bacterial Speciesa t (min)
fatty acidb
B. cereus
B. licheniformis
B. subtilis
S. aureus
St. faecalis
En. aerogenes
E. coli
P. aeruginosa
P. fluorescens
Se. marcescens
9.12 11.48 11.51 12.8 12.95 13.62 14.73 15.29 15.71 16.75 16.95 17.27 18.73 19.01 19.05 19.13 20.28 20.36 21.82 23.31
C13:0 iC14:0 C14:0 iC15:0 aC15:0 Un Un C16:1 C16:0 iC17:0 aC17:0 cyC17:0 C18:2 C18:1 C18:1 C18:0 cyC19:0 C19:0 C20:0 C21:0
5 0 4 4 0 0 0 0 82 2 0 0 0 0 0 3 0 0 0 0
0 3 0 11 10 0 2 0 58 6 3 0 0 0 0 7 0 0 0 0
0 0 1 9 12 0 0 0 20 9 4 0 17 4 0 25 0 0 0 0
0 2 0 2 3 2 0 0 5 0 0 0 0 0 0 8 0 2 76 1
0 3 0 0 0 11 0 0 21 0 0 0 0 53 0 3 7 0 2 0
0 0 5 0 0
