Effect of Substrate on the Fatty Acid Composition of Hydrocarbon-utilizing Microorganisms1

Transcription

1 JOURNAL OF BACTERIOLOGY, Dec. 1967, p Copyright 1967 American Society for Microbiology Vol. 94, No. 6 Printed in U.S.A. Effect of Substrate on the Fatty Acid Composition of Hydrocarbon-utilizing Microorganisms1 K R. DUNLAP A J. J. PERRY Department of Microbiology, North Carolina State University, Raleigh, North Carolina Received for publication 18 September 1967 The fatty acid pattern in three hydrocarbon-utilizing bacteria during growth on various substrates was examined. The predominant fatty acids in acetate-grown cells were C16, C16:1, Ci8:1, and Br-C1g and the major fatty acids in propane-grown cells were C15, C17, C17:1, C18:1, and Br-C18. When one organism (Mycobacterium sp. strain OFS) was grown on the n-alkanes from C13 to C17, the major fatty acid in the cells was of the same chain length as the substrate. Studies on the incorporation of acetate into the cellular fatty acids of microorganisms growing on C15 and C17 n-alkanes suggest that the oxidative products of the substrate are incorporated into the cellular fatty acids without degradation to acetate. Most microorganisms that grow on saturated hydrocarbons utilize these substrates as a sole source of carbon and energy. Consequently, the products of hydrocarbon degradation must serve as building blocks for all constituents of the microbial cell, including an efficient system for the assimilation of these exceedingly hydrophobic molecules (7). The solubility of hydrocarbons in lipid suggests that cellular lipoidal material might be involved in this process. This would be analogous to the role played by phosphotidylinositol in the assimilation of relatively insoluble sulfur by Thiobacillus thiooxidans (18). Microbes utilizing the more insoluble long-chain n-alkanes, i.e., n- tetradecane and n-pentadecane, as the sole source of carbon and energy could convert the monoterminally oxidized substrates directly to cellular lipids without further degradation. The synthesis of excess amounts of fatty acid and lipoidal material by microorganisms utilizing long-chain n-alkanes for growth under laboratory conditions is well documented: ester formation by Micrococcus cerificans (19), mono- and diterminal fatty acid excretion by Myobacterium 7E1C (10), and the production of waxes and glycerides by Nocardia (2). The control of lipid synthesis by long-chain fatty acids and the direct incorporation of fatty acids into cells of Lactobacillus plantarum has been reported (3). This investigation is concerned with a comparative study of the fatty acids and chloroformmethanol extractable lipids in cells after growth 1 Paper number 2474 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N.C on various hydrocarbon and nonhydrocarbon substrates. The qualitative and quantitative effect of the chain length of the growth substrate on the cellular lipids was examined along with the influence of long-chain n-alkanes on the synthesis of fatty acids from acetate. MATERIALS A METHODS Microorganisms. The microorganisms utilized in this study were originally isolated in the laboratory of the late J. W. Foster at the University of Texas. The organisms are designated as follows: JOB5, a Brevibacterium sp. Ooyama and Foster (15); OFS, a Mycobacterium sp.; and OC2A, a Nocardia sp. (A. S Kester, Ph.D. Thesis, Univ. of Texas, 1961). Media and growth conditions. Organisms were cultured on the L-salts medium of Leadbetter and Foster (11) supplemented with the appropriate carbon source. was added at 0.2% and propane (sulfur-free and 99.9% pure, purchased from Phillips Petroleum Co., Bartlesville, Okla.) was added by replacing 50% of the air in a closed container with the gaseous alkane. The C13-C17 carbon n-alkanes (obtained from Humphrey Chemical Co., North Haven, Conn.) were added directly to sterilized media at 0.66% (v/v). Microorganisms were grown on propane or acetate in 2-liter flasks containing 500 ml of medium and inoculated with 50 ml of a logphase culture growing on propane. Cells were grown on the longer n-alkanes by inoculating 300 ml of medium in a 1-liter flask with 2 ml of a heavy cell suspension washed from a propane-grown slant. Flasks were incubated on a rotary shaker at 27 C. After maximal growth (3 to 4 days), cells were collected by centrifugation and were washed thoroughly with distilled water. Cells were harvested after growth on longer-chain n-alkanes by centrifugation and 1919

2 1920 DUNLAP A PERRY J. BACTERIOL. were washed with a small amount of petroleum ether (bp 30 to 60 C) to remove residual growth substrate. Preparation of fatty acid methyl esters. Cellular fatty acids were converted to the corresponding methyl ester directly from wet cell preparations by transesterification. Wet cells equivalent to 50 to 100 mg (dry weight) were suspended in 3 to 4 ml of a mixture of nine parts of 14% BF3 in methanol (Applied Science Laboratories, State College, Pa.) and one part of benzene (14). -grown and propanegrown cells were heated for 6 hr, and cells grown on higher n-alkanes were heated for 12 hr in a 60 C water bath in glass-stoppered tubes. Very little volume change occurred, indicating that the concentration of BFa-methanol remained constant. After tooling, 6 to 8 ml of water was added and the mixture was extracted twice with 10-ml portions of petroleum ether. The ether extracts were combined and washed with 10 ml of water and concentrated to 1 ml. This esterification method was compared with two other methods (12, 13) requiring preliminary saponification, and it resulted in the same qualitative fatty acid pattern with a better quantitative yield. Gas-liquid chromatography. One of the columns used in this study was copper tubing ( inch X 6 feet) packed with 15% diethyleneglycol succinate (DEGS) on 60/70 mesh Gas Chrom P having 700 theoretical plates as measured with methyl palmitate. The other column was copper tubing ( inch X 7 feet) packed with 15% Apiezon L on 60/70 mesh Gas Chrom P containing 200 theoretical plates as measured with methyl palmitate. The column packing materials were purchased from Applied Science Laboratories, State College, Pa. The DEGS column was operated at 170 C and the Apiezon column at 250 C. The carrier gas was helium at a flow rate of 75 ml/min for both columns. The injection port temperature was 270 C and the detector was maintained at 290 C. The chromatographic system was an F & M model 810 equipped with a hydrogen flame ionization detector. Identification and quantitation of fatty acid methyl esters. The relative retention times of various standard fatty acid methyl esters (Applied Science Laboratories) on both DEGS and Apiezon columns were compared with the retention time of methyl palmitate. The relative retention time of knowns to methyl palmitate was designated the RR16 and was used in the identification of unknowns. Unsaturated compounds were identified by the shift in their position relative to saturated compounds on the DEGS column as compared with the Apiezon column (6). The identification was confirmed by following the disappearance of the corresponding peak on bromination (5). The linear relationship between the logarithm of the relative retention time and the carbon number (6) was also used for identification purposes. The area of each chromatographic peak was determined by triangulation according to the method of James (6) and Horning et al. (4). Extractable lipid determination. After growth on the appropriate medium, cells were harvested, washed, and lyophilized. A sample of weighed cells was shaken with 10 ml of chloroform-methanol (2:1, v/v) for 12 hr. The suspension was centrifuged to a clear supernatant fluid, and the insoluble material was separated and dried to constant weight. -2-_4C incorporation. A 2% inoculum from a log-phase culture of strain OFS (propane grown) was added to duplicate flasks containing 100 ml of L-salts medium with propane, n-pentadecane, or n-heptadecane as substrate. Sodium acetate-2-14c (2.5,c; specific activity 25.5,uc/mmole) was added to each flask, and unlabeled carrier sodium acetate was added to one of the duplicates at a concentration of 0.02%. The flasks were incubated on a rotary shaker at 27 C for 72 hr, were harvested by centrifugation, and washed. After lyophilization, a tared sample of cells was shaken for 24 hr with 100 ml of chloroform-methanol (2: 1, v/v). The chloroform-methanol was separated and evaporated on a steam bath and the residue was partitioned between 50 ml of diethyl ether and 50 ml of water. The ether phase was decanted and taken to dryness in a stream of nitrogen. This resultant material was taken up in 5 ml of ether and analyzed. The total lipid content of the ether extract was determined by a modification of the method of Rosen (17). A sample was evaporated in a stream of nitrogen and 1 ml of dichromate reagent was added, followed by heating at 85 C for 15 min. The solution was cooled and diluted with 35 ml of water. The optical densities of a reagent blank and sample were read at 350 m,i against a water blank. The sample was quantified by comparison with a standard curve prepared with various levels of palmitic acid. The incorporation of 14Clabeled material into the total lipid was determined by placing an appropriate volume of sample in a glass counting vial and evaporating in a nitrogen stream. Scintillation fluid (12 ml) was added and the sample was counted in a Mark I analyzer (Nuclear Chicago Corp., Des Plaines, Ill.). RESULTS The three organisms, Mycobacterium sp. (OFS), Brevibacterium sp. (JOB5), and Nocardia sp. (OC2A), were grown on propane and on acetate as the sole source of carbon and energy, and the cellular fatty acids were subjected to chromatographic analysis. The results are presented in Table 1. The percentages of odd-numbered normal fatty acids C15, C15:1, C17, and C17:1 were highest in the three organisms when the cells were grown on propane. All three organisms contained low levels of C16 and C18:1 fatty acids after growth on propane. A reversal of the pattern was obtained in the same organisms when acetate served as growth substrate. The even-numbered C16 and Clg:l fatty acids predominated in the acetategrown cells with small or undetectable amounts of C15, C15:1, C17, and C17:1 fatty acid present. The level of Br-C1g (branched n-nonadecane) and C16.1 was markedly lower in strains OFS and OC2A when grown on propane than the level of these fatty acids in acetate-grown cells. The com-

3 VOL. 94, 1967 EFFECT OF SUBSTRATE ON FATrY ACID COMPOSITION 1921 TABLE 1. Fatty acid distribution patterns in three hydrocarbonutilizing microorganisms after growth on acetate and propanea Fatty acid C14 Cl1 C15:1 C16 C16:1 Br-C,60 C17 C17:1 Cis C18:1 Br-Cis Br-C,, Nocardia sp. OC2A 1.4 b Mycobacterium Sp. OFS Brevibacterium sp. JOBS Propane Propane Pro Pra I Recorded as percentage of the total fatty acids present. The organisms were grown on a rotary shaker at 27 C for 72 hr. The substrate was added at 0.2% for acetate (Na) and 50:50 (v/v) for propane. b -none detected. I Br-branched-chain fatty acid. ponent (RR16 = 1.54) identified as Br-C8 was present in increased amounts in all three organisms after growth on propane. Regardless of growth substrate, the relative level of unsaturation in the total fatty acids was virtually the same in strain OFS (48% on acetate versus 45% on propane) and strain JOB5 (49% versus 52%), but there was somewhat less unsaturation in strain OC2A fatty acids after growth on propane (45% versus 54%). One strain, OFS, was grown on the n-alkanes from C13 through C17 as the sole source of carbon and energy. The patterns of fatty acids demonstrated by chromatographic analysis are presented in Table 2; also shown are results with propane and acetate as substrate. The results with C13 to C16 n-alkanes demonstrate that cells that corresponded to the chain length of the growth substrate contained fatty acid in greatest quantity. Cells grown on the C17 n-alkane were an exception and resembled propane-grown cells. Cells grown on C14 and C18 n-alkanes had a strikingly low content of odd-numbered fatty acids, whereas C1i-, C15-, and C17-grown cells had an abundance of odd-numbered fatty acids. C15- grown cells contained 97% nonbranched odd carbon number fatty acids of which 75% was pentadecanoic acid, while C17-grown cells contained 73% odd-number fatty acids of which only 25% was heptadecanoic acid. The incorporation of added acetate-2-14c into the lipids of strain OFS during growth on propane, n-pentadecane, and n-heptadecane is shown in Table 3. The contribution of labeled acetate to the total fatty acids in C15- and C17-grown cells was markedly lower than in cels utilizing propane as the carbon and energy source. There was a 100-fold or greater incorporation of acetate-2-14c into propane-grown cells than in the cells grown on the longer-chain n-alkane. The results confirm that there is a much higher lipid content in cells grown on the longer-chain n-alkanes. The results of the chloroform-methanol extractable lipid analysis on cells after growth on acetate and propane are presented in Table 4. Strains JOB5 and OC2A contained approximately 25% more extractable lipid after growth on propane than was found in acetate-grown cells. Strain OFS contained less extractable lipid after growth on propane. The total cell yield in JOB5 and OC2A was significantly lower with propane as substrate than observed with acetate. OFS grew equally well on both substrates. DISCUSSION A comparison of the fatty acids in acetategrown and propane-grown cells of the three strains OFS, OC2A, and JOB5 (Table 1) confirmed the presence of a higher level of unbranched odd carbon number fatty acids in propane-grown cells. Strains OC2A, OFS, and JOB5 contained 76, 71, and 91% even carbon number fatty acids, respectively. Propane-grown cells had 69, 66, and 44% odd carbon number fatty acids. The a-oxidation of propane to propionate, followed by two carbon additions, could be the origin of the high level of these odd-number fatty acids. Kaneda (8) found that incorporation of propionate into the growth medium of Bacillus subtilis resulted in a relative increase in C15 and C17 fatty acids of this organism. The branchedchain fatty acids might also arise from the incorporation of propionate into fatty acids as described by Woodward (20). The presence of high levels of C16 and Cl8 saturated and unsaturated fatty acids in acetate-grown cells has been demonstrated repeatedly in other organisms (1, 9). The major fatty acid in cells of strain OFS grown on C13 to C17 n-alkanes are the saturated and unsaturated n-fatty acids homologous to the growth substrate (Table 2). Whereas, the major fatty acids in acetate-grown cells are C18 and C1g, the C13-grown cells predominate in C13 and C1B fatty acids. A marked difference in pattern occurs with cells grown on C14, C15, and C16 substrates where 39, 83, and 78% of the fatty acid in the cell corresponds to the monoterminal oxidation product of the growth substrate. C17-grown cells contained 46% fatty acid of this chain length, al-

4 1922 DUNLAP A PERRY J. BACTERIOL. TABLE 2. Fatty acid composition of Mycobacterium sp. (Strain OFS) after growth on various n-alkanesa Fatty acid Growth substrate C13 C14 C15 C16 C17 Propane C C C C C b C15: C C16: C C17: C18: C19 Br-C1g Recorded as per cent of the total fatty acids present. The cells were grown for 72 hr at 27 C on a rotary shaker. The substrate was added at 0.66%. b = none detected. TABLE 3. Incorporation of acetate-2-14c ilnto the lipids of Mycobacterium sp. (Strain OFS) during growth on hydrocarbon substratesa Carrier weghyo Lipid Specific Growth substrate acetate cellsgo content activity mg % CPM/,sg Propane n-pentadecane n-heptadecane a Cells grown for 72 hr on L-salts + 50/50 (v/v) propane or 0.66% C15 and C17, 100 ml of medium per 500-ml flask. Specific activity of acetate-2-14c, 2.5,curies/0.1 Asmole. Carrier sodium acetate was added at 0.02%. though the overall pattern was similar to that found in propane-grown cells C is incorporated into the fatty acids of strain OFS during growth on propane in much higher (> 130 X) specific activity (Table 3) than during growth on C15 and C17 n-alkanes. This is a measure of the synthesis of fatty acids from acetate under these conditions and indicates that the greater part of the fatty acids in C15- and C17-grown cells derives from the substrate without degradation to the acetate level. Growth on propane was about double when 200,ug/ml of carrier acetate was incorporated into the growth medium, and the per cent lipid was not changed signifi- TABLE 4. Total chloroform-methanol extractable lipid in three hydrocarbon-utilizing organisms after growth on acetate (Na) and propane Strain Gro;t1h weight of Lipid sbtae cells rnga %/, dry wt Brevibacterium sp. (JOB5) Propane Nocardia sp. (OC2A) Propane Mycobacterium sp. (OFS) Propane cantly. The total amount of lipoidal material in C15 and C17 n-alkane grown cells was much higher than in propane-grown cells. Such levels of lipid in Mycobacteria (1) and Nocardia (16) have previously been reported. Since C]5 n-alkane grown strain OFS had 82% of the total fatty acid as C15 saturated or unsaturated fatty acids (Table 2), the suggestion would be that the substrate is incorporated directly after a monoterminal oxidation, and acetate-2-14c is not competitively incorporated into the fatty acids of cells. Results with L. plantarum (3) demonstrated that the incorporation of long-chain fatty acids in growth media repressed the synthesis of fatty acids from acetate. The low specific activity in C17-grown cells would suggest that the C15 fatty acid in this organism is synthesized by a 3-oxidation of C17, and Ci8i1

5 VOL. 94, 1967 EFFECT OF SUBSTRATE ON FATTY ACID COMPOSITION 1923 fatty acids are not synthesized from the exogenous acetate-2-l4c. The chloroform-methanol extractable lipid in the three strains (Table 4) demonstrates that propane-grown cells of OC2A and JOB5 contain more lipid than acetate-grown cells, although the cell yield is greater on acetate. There was less lipid in strain OFS after growth on propane, and the cell yield of OFS on propane more nearly approached the yield on acetate. LITERATURE CITED 1. ASSELINEAU, J The bacterial lipids, p Holden-Day, Inc., San Francisco. 2. DAVIS, J. B Microbial incorporation of fatty acids derived from n-alkanes into glycerides and waxes. Appl. Microbiol. 12: HEERSON, T. O., A J. J. McNEiLL The control of fatty acid synthesis in Lactobacillus plantiarum. Biochem. Biophys. Res. Commun. 25: HORNING, E. C., E. H. AHRENS, JR., S. R. LIPSKY, F. H. MATTSON, J. F. MEAD, D. A. TURNER, A W. H. GOLDWATER Quantitative analysis of fatty acids by gas-liquid chromatography. J. Lipid Res. 5: JAMES, A. T., A A. J. P. MARTIN Gasliquid chromatography: the separation and identification of methyl esters of saturated and unsaturated acids from formic acid to n-octadecanoic acid. Biochem. J. 63: JAMES, A. T Qualitative and quantitative determination of the fatty acids by gas-liquid chromatography. Methods Biochem. Analy. 8: JOHNSON, M. J Utilization of hydrocarbons by microorganisms. Chem. Ind. (London), p KANEDA, T Biosynthesis of branchedchain fatty acids. IV. Factors affecting relative abundance of fatty acids produced by Bacillus subtilis. Can. J. Microbiol. 12: KATES, M Bacterial lipids, p In R. Paoletti and D. Kritchevsky [ed]., Advances in lipid research, vol. 2. Academic Press, Inc., New York. 10. KESTER, A. S., A J. W. FOSTER Diterminal oxidation of long-chain alkanes by bacteria. J. Bacteriol. 85: LEADBETTER, E. R., A J. W. FOSTER Studies on some methane-utilizing bacteria. Arch. Mikrobiol. 30: METCALF, L. D., A A. A. SCHMITZ The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33: METCALF, L. D., A. A. SCHMITZ, A J. R. PELKA Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem. 38: MORRISON, W. R., A L. M. SMITH Preparation of fatty acid methyl esters and dimethyl acetals from lipids with boron fluoride methanol. J. Lipid Res. 5: OOYAMA, J., A J. W. FOSTER Bacterial oxidation of cycloparaffinic hydrocarbons. Antonie van Leeuwenhoek J. Microbiol. Serol. 31: RAYMO, R. L., A J. B. DAVIS n-alkane utilization and lipid formation by a Nocardia. Appl. Microbiol. 8: ROSEN, H Quantitative measurement of the amine buffer: 2 amino-2-hydroxymethyl- 1, 3-propanediol. Ann. N.Y. Acad. Sci. 92: SCHAEFFER, W. I., A W. W. UMBREIT Phosphotidylinositol as a wetting agent in sulfur oxidation by Thiobacillus thiooxidans. J. Bacteriol. 85: STEWART, J. E., R. E. KALLIO, D. P. STEVENSON, A. C. JoNEs, A D. 0. SCHISSLER Bacterial hydrocarbon oxidation. I. Oxidation of n-hexadecane. J. Bacteriol. 78: WOODwARD, R. B Neuere entwicklungen in der chemie der naturstoffe. Angew. Chem. 68:13-20.