Lipid Metabolism of Rumen Ciliates and Bacteria

WILLIAMS, P. P. (U.S. Department of Agriculture, Beltsville, Md.), J. GUTIERREZ, AND R. E. DAVIS. Lipid metabolism of rumen ciliates and bacteria. II. Uptake of fatty acids and lipid analysis of Isotricha intestinalis and rumen bacteria with further information on Entodinium simplex. Appl. Microbiol. 11:260-264. 1963.-The total lipid and free fatty acid contents of Isotricha intestinalis, Entodinium simplex, and the rumen bacterial flora of the respective protozoa were determined. Warburg manometric data showed that the sodium salts of tributyrin, oleic, and acetic acids stimulated gas production in I. intestinalis, whereas tributyrin was stimulatory with E. simplex and less active with oleic and acetic acids. Rumen bacteria provided fatty acids produced lower manometric gaseous increases when compared with the protozoa. Volatile fatty acids were produced by I. intestinalis and rumen bacteria with tributyrin, but not with tripalmitin. Sodium oleate gave little volatile fatty acid response with I. intestinalis or rumen bacteria. Washed suspensions of I. intestinalis and rumen bacteria concentrated C14-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Autoradiographs demonstrated the conversion of C14labeled oleic, palmitic, stearic, linoleic, and acetic acids in the rumen protozoa and bacterial cells. Saturation of C'4 oleate to stearate has been previously demonstrated in Isotricha prostoma (Gutierrez et al., 1962). Other long-chain fatty acids have been reported to stimulate gas production by I. prostoma, and Entodinium simplex and I. prostoma have been shown to concentrate C14labeled fatty acids. The major fatty acid present in the lipid fraction of I. prostoma was found to be palmitic acid, and the approximate chloroform-methanol (2:1) extractable lipid content was 7.69 %. This report describes further work on the respiration and metabolism experiments of fatty acids by the rumen ciliates, E. simplex and I. intestinalis, and gives a comparative study of their respective bacterial flora with information on the protozoal lipid contents. MATERIALS AND METHODS Suspensions of I. intestinalis and E. simplex were obtained from the ruminal ingesta of 6-month-old calves reared in isolation, previously inoculated with the respective single species of protozoa. The sedimentation method for preparation of washed suspensions of I. intestinalis has been previously described (Gutierrez, 1955). Mineral buffer solution used in the experiments contained (w/v): 0.1 % NaHCO3, 0.5 % NaCl, 0.01 % MgSO4, 0.01 % CaCl, and 0.1 % KH2PO4, under 5 % CO2-95 % N2 at 39 C. E. simplex suspensions were prepared by removing rumen ingesta, filtering the sample through one layer of cheesecloth, and adding one-third liquid volume by the addition of mineral buffer solution to the rumen liquor in separatory funnels at 39 C. Excess plant debris floated to the surface of the separatory funnel, and the bottom fluid containing entodinia was placed in vertical glass columns (Williams et al., 1961). The remaining heavy plant debris settled rapidly to the bottom of the columns and was removed. The solution was then centrifuged at 1,230 relative centrifugal force (RCF) for 5 min. The protozoa were then washed in samples of mineral buffer solution in rubberstoppered tubes until free of most bacteria and small plant fragments. Rumen bacterial suspensions from calves harboring either I. intestinalis or E. simplex were prepared by collecting ingesta, filtered through one layer of cheesecloth and dispensed into 500-ml separatory funnels at 39 C. Plant debris floated to the surface and the lower fraction was centrifuged at 1,230 RCF for 10 min to remove protozoa and plant debris. Supernatants were pooled and recentrifuged at 10,400 RCF for 15 min. The bacterial pellets were resuspended in mineral buffer solution containing 0.05 % (w/v) cysteine HCI. Two additional recentrifugations were made, and, in the latter centrifugation, the bacterial cells were pooled and standardized. The concentrated bacterial suspensions used in manometric and isotope studies gave optical density (OD) readings of 0.20 to 0.29 at 600 m,u with a Spectronic 20 (Bausch and Lomb Optical Co., Rochester, N.Y.) when 0.25-ml samples of the bacteria were diluted in 9.75 ml of mineral buffer solutions. Rumen bacteria from a calf harboring 260 on A uust 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom UPTAKE OF FATTY ACIDS BY RUMEN CILIATES only I. intestinalis were designated Ii, and rumen bacteria from calves harboring only E. simplex were designated Es. MIethods for the quantitative analysis of the lipid contents of I. intestinalis, E. simplex, and their respective bacterial populations have been previously described, as well as fatty acid chromatographic techniques, autoradiographs, fatty acid isotope uptake, and extraction procedures (Gutierrez et al., 1962). Radioisotope incorporations were determined with a windowless gas-flow counter tube and a Nuclear-Chicago scaler. Manometric fatty acid gaseous quantities from various substrates were determined with the conventional Warburg apparatus. A modification of the Chiriboga and Roy (1962) technique was used for the detection of decarboxylation sodium acetate-i-C14 by I. intestinalis and the protozoan's respective bacterial population. I. intestinalis (6 mg dry weight) and 15 mg of Ii rumen bacteria, each suspended in 1.0 ml of ani anaerobic mineral buffer solution, were incubated with 0.5 Ac of C14 sodium acetate at 39 C for 90 min in Chiriboga and Roy (1962) tubes. Incorporation of C14 sodium acetate into long-chain fatty acids or volatile fatty acid fractions was determined as follows. I. intestinalis (25 mg dry weight) and Ii rumen bacteria (32.0 mg dry weight) were each suspended in a total volume of 5.0 ml of mineral buffer solution containing 1 ic of C14 sodium acetate. C14 sodium acetate controls without microorganisms, and the experimental vessels with protozoa or bacteria,were incubated at 39 C for 90 min. Chloroform-methanol (2: 1) extraction removed free fatty acids from the microbial cellular materials. Long-chain fatty acid and volatile fatty acid chromatograms, as well as autoradiograms, were prepared with the free fatty acid extracts. The solvent used for separation of the volatile fatty acids, which were conTABLE 1. Total lipid determinations Cellular ChloroformKCI urified Sample ~~dry methanol lipid KClpuied Sample weight extract lipid mg mg dry wt mg dry wt Isotricha intestinalis. 6,191 627 (10.61)* 564 (9.11) Entodinium simplex.. 1,726 216 (12.51) 109 (6.32) I. intestinalis bacterial population ........... 919 108 (11.75) 63 (6.85) E. simplex bacterial population ........... 1,700 171 (10.06) 114 (6.70) * Figure in parentheses is the per cent. verted to ethylamine salts, was 1 %O NH40H-95 ethanol (Block, Durrum, and Zweig, 1955). Volatile fatty acid production by rumen ciliates and bacteria from the triglycerides, tributyrin and tripalmitin, in 0.4 %O (w/v) concentrations with total volumes of 80.0 ml was determined. Experimental flasks contained I. intestinalis (255 mg dry weight) and Ii rumen bacteria (276 mg dry weight) with substrates. Control flasks, containing protozoa or bacteria without substrate, and other flasks, containing substrates with filtered I. intestinalis supernatant or Ii rumen bacteria centrifuged supernatant, were provided. Both control and experimental flasks were incubated for 180 min at 39 C. Suspensions of I. intestinalis (105 mg dry weight) and Ii rumen bacteria (150 mg dry weight) were each incubated for 120 min at 39 C with 0.25 %O (v/v) neutralized sodium oleate and mineral buffer solution in total volumes of 20 ml. The 20-ml samples from both control and experimental flasks were steamdistilled and titrated for amounts of acidity in the volatile fractions. Dry weights of various fractions of rumen ingesta were determined by sampling a growing 7to 9-month-old calf harboring only the rumen ciliate I. intestinalis. Rumen samples were collected and filtered through two layers of cheesecloth. The unfiltered plant debris was resuspended in distilled water, refiltered, and dried at 70 C. The pooled filtrates were incubated at 39 C in separatory funnels until filtered plant debris floated to the surface and the protozoa settled out, leaving a middle layer of rumen liquor. The three fractions, plant debris, protozoa, and rumen liquor, were centrifuged separately at 1,230 RCF for 5 min. I. intestinalis, from the protozoan layer and rumen liquor, were suspended in anaerobic mineral buffer solution, sedimented in a glass column, washed with distilled water, and dried to constant weight. Plant debris was resuspended in distilled water, recentrifuged, and dried. All supernatants used to collect bacteria were pooled and centrifuged twice at 18,400 RCF for 15 min. The pooled supernatants and the washed bacteria were dried separately to constant