Lipid Metabolism of Rumen Ciliates Bacteriaaem.asm.org/content/11/3/260.full.pdfLipid Metabolism of RumenCiliates and Bacteria II. Uptake of Fatty Acids and Lipid Analysis of Isotricha

Lipid Metabolism of Rumen Ciliates and BacteriaII. Uptake of Fatty Acids and Lipid Analysis of Isotricha intestinalis and

Rumen Bacteria with Further Information on Entodinium simplex

P. P. WILLIAMS, J. GUTIERREZ, AND R. E. DAVIS

Animal Husbandry Research Division, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Mliaryland

Received for publication 16 January 1963

ABSTRACT

WILLIAMS, P. P. (U.S. Department of Agriculture,Beltsville, Md.), J. GUTIERREZ, AND R. E. DAVIS. Lipidmetabolism of rumen ciliates and bacteria. II. Uptake offatty acids and lipid analysis of Isotricha intestinalis andrumen bacteria with further information on Entodiniumsimplex. Appl. Microbiol. 11:260-264. 1963.-The totallipid and free fatty acid contents of Isotricha intestinalis,Entodinium simplex, and the rumen bacterial flora of therespective protozoa were determined. Warburg mano-metric 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. Rumenbacteria provided fatty acids produced lower manometricgaseous increases when compared with the protozoa.Volatile fatty acids were produced by I. intestinalisand rumen bacteria with tributyrin, but not with tri-palmitin. Sodium oleate gave little volatile fatty acidresponse with I. intestinalis or rumen bacteria. Washedsuspensions of I. intestinalis and rumen bacteria concen-trated C14-labeled oleic, palmitic, stearic, and linoleicacids within the cells during short incubation periods.Autoradiographs demonstrated the conversion of C14-labeled oleic, palmitic, stearic, linoleic, and acetic acids inthe rumen protozoa and bacterial cells.

Saturation of C'4 oleate to stearate has been previouslydemonstrated in Isotricha prostoma (Gutierrez et al., 1962).Other long-chain fatty acids have been reported to stimu-late gas production by I. prostoma, and Entodinium simplexand I. prostoma have been shown to concentrate C14-labeled fatty acids. The major fatty acid present in thelipid fraction of I. prostoma was found to be palmitic acid,and the approximate chloroform-methanol (2:1) extracta-ble lipid content was 7.69 %.

This report describes further work on the respirationand metabolism experiments of fatty acids by the rumenciliates, E. simplex and I. intestinalis, and gives a compara-tive study of their respective bacterial flora with informa-tion on the protozoal lipid contents.

MATERIALS AND METHODS

Suspensions of I. intestinalis and E. simplex were ob-tained from the ruminal ingesta of 6-month-old calvesreared in isolation, previously inoculated with the respec-tive single species of protozoa. The sedimentation methodfor preparation of washed suspensions of I. intestinalis hasbeen previously described (Gutierrez, 1955). Mineralbuffer 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 rumeningesta, filtering the sample through one layer of cheese-cloth, and adding one-third liquid volume by the additionof mineral buffer solution to the rumen liquor in separa-tory funnels at 39 C. Excess plant debris floated to thesurface of the separatory funnel, and the bottom fluid con-taining entodinia was placed in vertical glass columns (Wil-liams et al., 1961). The remaining heavy plant debris set-tled rapidly to the bottom of the columns and was removed.The solution was then centrifuged at 1,230 relative cen-trifugal force (RCF) for 5 min. The protozoa were thenwashed in samples of mineral buffer solution in rubber-stoppered tubes until free of most bacteria and small plantfragments.Rumen bacterial suspensions from calves harboring

either I. intestinalis or E. simplex were prepared by col-lecting ingesta, filtered through one layer of cheeseclothand dispensed into 500-ml separatory funnels at 39 C.Plant debris floated to the surface and the lower fractionwas centrifuged at 1,230 RCF for 10 min to remove pro-tozoa and plant debris. Supernatants were pooled andrecentrifuged at 10,400 RCF for 15 min. The bacterialpellets were resuspended in mineral buffer solution con-taining 0.05 % (w/v) cysteine HCI. Two additional re-centrifugations were made, and, in the latter centrifuga-tion, the bacterial cells were pooled and standardized. Theconcentrated bacterial suspensions used in manometricand isotope studies gave optical density (OD) readings of0.20 to 0.29 at 600 m,u with a Spectronic 20 (Bausch andLomb Optical Co., Rochester, N.Y.) when 0.25-ml sam-ples of the bacteria were diluted in 9.75 ml of mineralbuffer solutions. Rumen bacteria from a calf harboring

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UPTAKE OF FATTY ACIDS BY RUMEN CILIATES

only I. intestinalis were designated Ii, and rumen bacteriafrom calves harboring only E. simplex were designated Es.

MIethods for the quantitative analysis of the lipid con-

tents of I. intestinalis, E. simplex, and their respectivebacterial populations have been previously described, as

well as fatty acid chromatographic techniques, autoradio-graphs, fatty acid isotope uptake, and extraction pro-

cedures (Gutierrez et al., 1962). Radioisotope incorpora-tions were determined with a windowless gas-flow countertube and a Nuclear-Chicago scaler. Manometric fatty acidgaseous quantities from various substrates were deter-mined with the conventional Warburg apparatus.A modification of the Chiriboga and Roy (1962) tech-

nique was used for the detection of decarboxylation sodiumacetate-i-C14 by I. intestinalis and the protozoan's respec-

tive bacterial population. I. intestinalis (6 mg dry weight)and 15 mg of Ii rumen bacteria, each suspended in 1.0 mlof ani anaerobic mineral buffer solution, were incubatedwith 0.5 Ac of C14 sodium acetate at 39 C for 90 min inChiriboga and Roy (1962) tubes. Incorporation of C14sodium acetate into long-chain fatty acids or volatile fattyacid fractions was determined as follows. I. intestinalis(25 mg dry weight) and Ii rumen bacteria (32.0 mg dryweight) were each suspended in a total volume of 5.0 ml ofmineral buffer solution containing 1 ic of C14 sodium ace-

tate. C14 sodium acetate controls without microorganisms,and the experimental vessels with protozoa or bacteria,wereincubated at 39 C for 90 min. Chloroform-methanol (2: 1)extraction removed free fatty acids from the microbialcellular materials. Long-chain fatty acid and volatile fattyacid chromatograms, as well as autoradiograms, were pre-

pared with the free fatty acid extracts. The solvent usedfor separation of the volatile fatty acids, which were con-

TABLE 1. Total lipid determinations

Cellular Chloroform- KCI urifiedSample ~~dry methanol lipid KClpuiedSample 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 bacterialpopulation ........... 919 108 (11.75) 63 (6.85)

E. simplex bacterialpopulation ........... 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 bac-teria from the triglycerides, tributyrin and tripalmitin, in0.4 %O (w/v) concentrations with total volumes of 80.0 mlwas determined. Experimental flasks contained I. intes-tinalis (255 mg dry weight) and Ii rumen bacteria (276 mgdry weight) with substrates. Control flasks, containingprotozoa or bacteria without substrate, and other flasks,containing substrates with filtered I. intestinalis super-

natant or Ii rumen bacteria centrifuged supernatant, were

provided. Both control and experimental flasks were incu-bated for 180 min at 39 C. Suspensions of I. intestinalis(105 mg dry weight) and Ii rumen bacteria (150 mg dryweight) were each incubated for 120 min at 39 C with0.25 %O (v/v) neutralized sodium oleate and mineral buffersolution in total volumes of 20 ml. The 20-ml samplesfrom both control and experimental flasks were steam-distilled and titrated for amounts of acidity in the volatilefractions.Dry weights of various fractions of rumen ingesta were

determined by sampling a growing 7- to 9-month-old calfharboring only the rumen ciliate I. intestinalis. Rumensamples were collected and filtered through two layers ofcheesecloth. The unfiltered plant debris was resuspendedin distilled water, refiltered, and dried at 70 C. The pooledfiltrates were incubated at 39 C in separatory funnels untilfiltered plant debris floated to the surface and the protozoasettled out, leaving a middle layer of rumen liquor. Thethree 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, sedi-mented in a glass column, washed with distilled water, anddried to constant weight. Plant debris was resuspended indistilled water, recentrifuged, and dried. All supernatantsused to collect bacteria were pooled and centrifuged twiceat 18,400 RCF for 15 min. The pooled supernatants andthe washed bacteria were dried separately to constantweight at 70 C.

RESULTS

Total lipid determinations were made on washed sus-

pensions of I. intestinalis, E. simplex, Ii rumen bacteria,and Es rumen bacteria (Table 1). Lower lipid fraction

TABLE 2. Identified fatty acids from lipid fractions

Identified fatty acids*Species

Stearic Palmitic Oleic Myristic Linoleic Lauric

Entodinium simplex .X X- Trace, unsaturated - -Isotricha intestinalis .Trace X Trace Unsaturated TraceEs bacteria .x x - Trace, unsaturated X TraceIi bacteria .......x X. - Trace, unsaturated X Trace

* Unknown RF values were similar to the known values. X = present.

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values were noted when aqueous KCl was used to purifythe chloroform-methanol lipid extracts. I. intestinalis gavea higher lipid content than E. simplex, 9.11 % as comparedwith 6.32 %, and Ii bacteria gave values very similar tothose of Es bacteria (6.85 to 6.70 %).

Free fatty acids, which were eluted by chloroform-methanol (2:1), and fatty acids, which were released frompurified lipid by saponification, acidification, and extrac-tion with petroleum ether, were chromatographed. Knownfatty acids were run in parallel with unknown prepara-tions, and their RF values were compared (Table 2).Stearate and palmitate were prevalent in the free fattyacid fractions eluted from rumen bacteria Ii and Es, andE. simplex. Stearate was not observed in the free fattyacid fractions of I. intestinalis; however, palmitate was inhigh concentration. Fatty acids removed from purifiedlipid fractions showed palmitate prevalent in all themicro)bial lipid extracts. Unsaturated fatty acids were pres-ent in trace amounts, primarily possessing RF values simi-

TABLE 3. C'4 fatty acid RF values of free and bound fatty acids frommicrobial lipid extracts

Species

Cellular fractionIsotricha intesti-

nalis

I. intestinalis (li)rumen bacteria

SupernatantI. intestinalis

li rumen bacteria

C14 substrate

Stearic-l-C14Oleic-l-C"4Linoleic-l-C'4Palmitic-l-CM4Stearic-l-C"4Oleic-l-C"4Linoleic-l-C14Palmitic-l-C"4

Stearic-l-C"Oleic-l-C'4Linoleic-l-C"4Palmitic-l-C14Stearic-l-C14Oleic-l-C"4Linoleic-1-C14Palmitic-1l_CI4

Known C14 compounds and theirRp values* with acetic acid-

paraffin solvent

Pal-Stearic mitic-(0.33) oleic

(0.47)

0.35

0.310.300.300.28

0.36

0.46

0.42

0.47

Lino-leic

(0.82)

0.820.82

0.79

0.82

0.810.82

Uni-denti-fiedfattyacid

(<0.33)

0.180.220.200.250.190.200.17

0.20

0.17

* Shown in parentheses after compounds.

TABLE 4. Mean C14 fatty acid uptake by Isotricha intestinalis andrumen bacteria (60 min incubation at 39 C)

Substrate I. intestinalis Ru,men (Ii) bacteria

Palmitic-1-C14 ................. 946* 427*Stearic-1-C'4 ................... 193 572Oleic-1-C'4 ..................... 690 842Linoleic-1-Ci4 .................. 244 156

* Results expressed as counts per min per 50-,uliter sample.

lar to myristate, and I. intestinalis showed unsaturatedproperties in spots similar to oleate and linoleate RF values.

I. intestinalis and Ii rumen bacteria were incubated for150 min in mineral buffer solution with 0.5 ,uc each of thefollowing 1-C'4-labeled compounds: stearic, linoleic, oleic,and palmitic (purchased from Applied Science Labora-tory, State College, Pa.). Autoradiographs were preparedfrom the lipid-extractable preparations from both super-natants and cellular materials (Table 3).Rumen bacteria incorporated stearic-l-C'4 as stearic

and an unidentified fatty acid (RF 0.19). Oleic-1-C'4 wasconverted to compounds identified as stearic, a trace ofpalmitic-oleic, and an unidentified fatty acid (RF 0.20).Linoleic-1-C'4 was hydrogenated to stearic, incorporatedinto an unidentified fatty acid (RF 0.17), and remained intrace amounts as linoleic. Palmitic-1-C'4 radioactivity waspresent in palmitic-oleic and stearic. Supernatants fromcentrifuged bacteria incubated with fatty acid isotopesindicated that some alterations of long-chain fatty acidsmay occur extracellularly with bacterial enzymes.

I. intestinalis converted the radioactivity of stearic-1-C14to a fatty acid having an RF value of 0.18. Oleic-1-C14radioactivity showed saturation, unsaturation, and in-corporation of oleic-1-C14 into stearic, linoleic, and anunidentified fatty acid (RF 0.22). Linoleic-1-C'4 was in-corporated into the cellular makeup of I. intestinalis aslinoleic and an unidentified fatty acid (RF 0.20). Palmitic-1-C'4 radioactivity was converted to an unidentified fattyacid with an RF value of 0.25. I. intestinalis supernatantfluid showed no activity, with the exception of a traceam.ount of residual linoleic substrate. Absence of radio-activity, and the nonconversion of linoleic-1-C'4 in thesupernatants free of protozoa, indicated little bacterialactivity in the I. intestinalis suspensions.

Results from C'4 Millipore filter experiments with I. in-testinalis (60 mg dry weight) and Ii rumen bacteria (55mg dry weight), each in a total of 10.0 ml of 0.5 uc of sub-strate, are shown in Table 4.

C14 uptake was noted in all four substrates in both I.intestinalis and the rumeni bacteria removed from theruminal inge-ta of an animal harboring only I. intestinalis.Manometric gaseous measurements with rumen ciliates

and bacteria are shown in Table 5. Washed suspensions ofI. intestinalis and E. simplex (8 to 18 mg dry weight) andrumen bacteria (32 to 52 mg dry weight), each suspendedin a total of 2.0 ml of anaerobic mineral buffer solution,were incubated at 39 C, usually with 0.15 %c (v/v) neu-tralized fatty acids and other substrates. I. intestinalisshowed most stimulation by sodium acetate, sodium ole-ate, and tributyrin. With and without sodium oleate, theratio of hydrogen to carbon dioxide produced by I. intes-tinalis was approximately 1:1. E. simplex showed somestimulation with tributyrin and little stimulation withsodium acetate and sodium oleate. Glycerol was negativefor both I. intestinralis and E. simplex. Rumen bacteria Iiand Es gave similar results on the various substrates. With

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and without sodium oleate and Ii rumen bacteria, theratio of hydrogen to CO2 and other gases was approxi-mately 1:9. Overall, the rumen bacterial suspensions wereless active on the substrates than the rumen ciliates, al-though they were two to three times more concentratedon a dry-weight basis than were the protozoa. The ratiosof H2:CO2 for I. intestinalis (1:1) and rumen bacteria(1:9), with and without sodium oleate during mano-metric incubations, remained unchanged. This indicatedthat the fatty acid substrate stimulated endogenous gasproduction and was not due simply to release of CO2from the buffered solution or decarboxylation of thesubstrate.

Conversion of C14 sodium acetate to C1402 was noted inboth suspensions of I. intestinalis and Ii rumen bacteria.Release of carbon dioxide by acidification and absorptionby potassium hydroxide showed radioactivity above con-trols of 368 counts/min for I. intestinalis and 424 counts/min for Ii rumen bacteria. C14 sodium acetate incubated

TABLE 5. Manontetric gaseous production by rumen ciliates and theirrespective bacteria on fatty acids and other substrates*

I. intestinalis E. simplexSubstrate I. intestinalis E. simplex bacterial bacterial

population population

Tributyrin....... 171 54 33 30Glycerol ......... 2 0 19 10Acetate .......... 138 28 11 0Valerate ......... 0 NDt 21 34Laurate ......... 14 0 64 21Myristate ....... 5 60 13 36Palmitate ....... 24 18 28 46Stearate ......... 10 23 68 35Oleate ........... 249 32 14 45Linoleate........ 37 52 44 60Arachidate ...... 31 17 0 0

* Calculated mean pressure differences from endogenous andexperimental vessels per 70 min (mm Brodie solution). Meanpressure magnitudes in the endogenous vessels per 70 min were,for I. intestinalis, 180-mm pressure and, for E. simplex, Ii, and Esbacteria, 100-mm pressure each.

t Not determined.

TABLE 6. Fractionation of rumen ingesta (per 500 ml)

Sample Dry wt range from Mean dry wt at7 to 9 months* 9 montbst

g g

Unfiltered plant debrist. 47.00-130.00 123.00Filtered plant debris.6.72-8.00 7.50Supernatant .0.33-7.27 5.60Rumen bacteria . 0.78-1.10 1.00Isotricha intestinalis .00.60-1.70 1.05

* Weight of successive sampling of rumen ingesta 2 hr afterthe morning feeding from a growing 7- to 9-month-old isolatedcalf harboring only the rumen ciliate, I. intestinalis.

t Mean dry weight calculated from five consecutive 1-daysamplings.

t Plant debris collected from cheesecloth filtration of rumeningesta to obtain 500 ml of filtrate.

with I. intestinalis and Ii rumen bacteria, and extractedwith chloroform-methanol (2:1), showed the followingresults. C14 radioactivity was observed in the volatilealiphatic acid autoradiograms. Ii rumen bacteria showedC14 sodium acetate in trace amounts at an RF value of 0.33,and an unknown spot at RF 0.11. I. intestinalis showedonly a trace amount of acetate at RF 0.33. The long-chainfatty acid autoradiograms showed no C14 activity in theC-12 to C-18 fatty acids.Steam distillation experiments with I. intestinalis and

1i rumen bacteria indicated an increase in volatile fattyacid production with tributyrin. I. intestinalis showed anincrease of 0.320 meq and Ii rumen bacteria showed anincrease of 0.230 meq over the tributyrin controls. Tripal-mitin, as a substrate, showed no increase in volatile fattyacids. Insolubility both with tributyrin and tripalmitinwas a factor which may have influenced the amounts oftitratable acidities. I. intestinalis and Ii rumen bacteriawith sodium oleate produced volatile fatty acids in theamounts of 0.056 meq and less than 0.010 meq, respec-tively, over the endogenous controls.Rumen ingesta dry weight values from the animal har-

boring I. intestinalis are given in Table 6. Fluctuation ofdry weights in the unfiltered plant debris and supernatantwere noted, whereas filtered plant debris values were rela-tively constant. The protozoan dry weight range showedmore fluctuation than the bacterial population within the7- to 9-month growth period of the calf. The bacterial andprotozoal population dry weights were equivalent at 9months, with each being approximately 1 g per 500 ml ofrumen liquor.

DISCUSSION

Among the rumen ciliates, the holotrich, I. prostoma,and an oligotrich, E. simplex, have been shown to in-corporate the C'4 fatty acids, oleic, palmitic, and stearic,during short incubation periods (Gutierrez et al., 1962).Similarities have been noted between I. prostoma and I.intestinalis in that both hydrogenate oleic acid to stearicacid, and gas production is stimulated by tributyrin,sodium oleate, and sodium acetate. I. prostoma and I.intestinalis also have similar lipid contents (7 to 9 %), withpalmitic acid being the main aliphatic component. E.simplex and mixtures of rumen bacteria differ from theholotrichs in containing higher concentrations of stearicacid in the free fatty fractions and less lipid. They alsodiffer in their endogenous gas production stimulation (perdry weight) by aliphatic compounds; with rumen bacteria,some hydrogenation appeared to occur extracellularly.Steam-distillation experiments showed that both I. intes-tinalis and rumen bacteria readily converted the triglyc-eride, tributyrin, to volatile fatty acids, and sodiumoleate stimulated volatile fatty acid production by I.intestinalis more than with rumen bacteria.

Comparison of mixtures of rumen bacteria from calvesharboring either I. intestinalis or E. simplex indicated

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they were very similar in their response to fatty acidswhen measured manometrically. Large populations ofbacterial-feeding holotrichs or oligotrichs may not alterthe ability of bacterial flora to utilize aliphatic compounds.A comparison of rumen bacteria and I. intestinalis showedthat both metabolize sodium acetate with the productionof CO2. The rumen bacteria apparently differ from I.intestinalis in their ability to assimilate C14 acetate into acomponent extractable with chloroform-methanol (2: 1).

Ingestion of associated bacteria has been demonstratedfor several species of ruminal ciliates (Gutierrez, 1958;Gutierrez and Davis, 1959). Rumen bacteria capable ofutilizing neutralized volatile fatty acids, such as sodiumacetate, and incorporating nonvolatile aliphatic acids intocomplex lipoidal materials may ultimately be ingestedinto protozoal protoplasm, particularly with holotrichs.Allison, Bryant, and Doetsch (1959) have shown conver-sion of isovalerate-1-C14 to leucine by the rumen bacteriumRuminococcus flavefaciens. However, much of the iso-valerate remained unchanged in the cellular lipid fraction.Some rumen bacteria require for growth a critical molarratio of straight and branched-chain fatty acids (2: 1 ratioof n-valeric acid to isovaleric acid; Bryant and Doetsch,1955). Manometric studies have shown that the fungus,Emericellopsis salmosynnemata, is stimulated by all fattyacids tested, with the exception of oxalic acid which provedto be inhibitory. Acetate was very stimulatory to thefungal respiratory system (Fleischman and Pisano, 1962).Warner (1962) compared the approximate volumes (,U3)

of individual cells of rumen microorganisms. Isotricha andEntodinium were, respectively, 1,000,000 and 10,000 timesgreater in total volume than bacteria. From size compari-son of different microbial species, rumen ciliates appear tocontribute significantly to the over-all microbial proto-plasmic activity in the rumen, especially when isolatedcalves faunated with I. intestinalis showed protozoal popu-lations equivalent to bacterial populations on a dry-weightbasis. Rumen ciliates hydrogenate fatty acids and may

contribute significantly in metabolizing volatile and non-volatile aliphatic compounds, and in the formation ofcomplex lipoidal materials. The degree of hydrogenation offatty acids in rumen ingesta may be influenced by the par-ticular species of holotrich or oligotrich present in highconcentrations. Also, the total number of protozoa com-peting for the aliphatic substrate with the rumen bacteriamay affect the amount of hydrogenation, depending onwhether they are, or are not, predaceous on rumen bac-teria.

LITERATURE CITED

ALLISON, M. J., M. P. BRYANT, AND R. N. DOETSCH. 1959. Con-version of isovalerate to leucine by Ruminococcus flavefaciens.Arch. Biochem. Biophys. 84:246-247.

BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG. 1955. A manual ofpaper chromatography and paper electrophoresis. AcademicPress, Inc., New York.

BRYANT, M. P., AND R. N. DOETSCH. 1955. Factors necessary forthe growth of Bacterioides succinogenes in the volatile acidfraction of rumen fluid. J. Dairy Sci. 38:340-350.

CHIRIBOGA, J., AND D. N. Roy. 1962. Decarboxylation of com-pounds labelled with carbon-14. Nature 193:684-685.

FLEISCHMAN, A. I., AND M. A. PISANO. 1962. Studies on the respira-tion of Emericellopsis salmosynnemata: the oxidation of fattyacids. Bacteriol. Proc., p. 127.

GUTIERREZ, J. 1955. Experiments on the culture and physiology ofholotrichs from the bovine rumen. Biochem. J. 60:516-522.

GUTIERREZ, J. 1958. Observations on bacterial feeding by therumen ciliates Isotricha prostoma. J. Protozool. 5:122-126.

GUTIERREZ, J., AND R. E. DAVIS. 1959. Bacterial ingestion by therumen ciliates Entodinium and Diplodinium. J. Protozool.6:222-226.

GUTIERREZ, J., P. P. WILLIAMS, R. E. DAVIS, AND E. J. WARWICK.1962. Lipid metabolism of rumen ciliates and bacteria. I.Uptake of fatty acids by Isotricha prostoma and Entodiniumsimplex. Appl. Microbiol. 10:548-551.

WARNER, A. C. I. 1962. Some factors influencing the rumen micro-bial population. J. Gen. Microbiol. 28:128-146.

WILLIAMS, P. P., R. E. DAVIS, R. N. DOETSCH, AND J. GUTIERREZ.1961. Physiological studies of the rumen protozoan Ophryo-scolex caudatus Eberlein. Appl. Microbiol. 9:405-409.

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