JOURNAL OF BACrERIOLOGY, Jan., 1967, p. 456-463 Vol. 93, No. 1Copyright © 1967 American Society for Microbiology Printed In US.A.

Function of Growth Factors for RumenMicroorganisms

I. Nutritional Characteristics of Selenomonas ruminantiumSHIRO KANEGASAKI AND HAJIME TAKAHASHI

Department of Agricultural Chemistry, Faculty ofAgriculture, Tohoku University, Sendai, Japan

Received for publication 19 October 1966

ABSTRACTNutritional characteristics of Selenomonas ruminantium var. lactilytica isolated

from a sheep rumen were studied. The organism required for growth the additionof a clarified rumen fluid to a Trypticase-yeast extract medium with either lactateor glucose as an energy source. The requirement for rumen fluid was found to besatisfied by volatile fatty acids in glucose media and by biotin in lactate media.Straight-chain saturated fatty acids with C3 to C10 carbon skeleton had been foundto be effective. Among them, n-valerate was most effective at the lowest concentra-tion. An abnormal morphology was observed with n-valerate-deficient glucosemedia. n-Valerate was essential in glucose media, and it was stimulatory in lactatemedia. Fermentation products from glucose were lactate, propionate, and acetate,and fermentation products from lactate were propionate and acetate. When cellswere grown in a glucose medium containing n-valerate-C'4, the label was present incell fractions. Almost all of the activity was found in lipid materials.

It is well known that some rumen bacteria re-quire rumen fluid for growth, and that rumen fluidcannot entirely be replaced by yeast extract orTrypticase (7). Sometimes rumen fluid can bereplaced by a mixture of volatile fatty acids. Inmost of the cases so far reported, replacementhas been by branched-chain acids, sometimes incombination with straight-chain acids (1, 8). Therequirement, however, has been mainly studiedwith cellulolytic rumen anaerobes, and there havebeen few reports dealing with noncellulolyticorganisms.

Bryant and Robinson (3) reported that growthof several strains of Selenomonas ruminantiumrequired or was stimulated by a mixture of vola-tile fatty acids which contained isobutyrate,n-valerate, isovalerate, and DL-a-methyl butyrate.Acetate was stimulatory only in the absence ofcasein hydrolysate in the culture medium.Hobson, Mann, and Smith (4) mentioned thatacetate was stimulatory for growth of a strain ofS. ruminantium even in the presence of caseinhydrolysate. The addition of other volatile fattyacids including both straight- and branched-chainacids was not stimulatory, and was even inhibi-tory in some cases.The present work was carried out to establish

the nutritional characteristics of a freshly isolatedstrain of S. ruminantium var. lactilytica.

MATERIALS AND METHODS

Media. Three kinds of basal media were used un-less otherwise stated: a Trypticase-yeast extract me-dium containing 0.2% each of Trypticase (BBL) andyeast extract (Daigo Eiyo Kagaku, Tokyo, Japan); ayeast extract medium containing 0.2% yeast extract;a casein hydrolysate-vitamins-valerate medium con-tained 0.25% vitamin-free casein acid hydrolysate(Nissui Seiyaku, Tokyo, Japan), a vitamin mixture,and 0.0019% n-valerate. As an energy source, 0.5%glucose, 2.5% (v/v) sodium DL-lactate (50%), or 1%(v/v) glycerol (85%) was added to the above media.The composition of the vitamin mixture (mg/100 ml)was as follows: thiamine-HCI, Ca pantothenate,nicotinamide, riboflavine, and pyridoxal-HCl, 0.2each; p-aminobenzoic acid (PABA), 0.1; biotin, 0.01;folic and DL-thioctic acids, 0.005 each; and cobalamin0.002 (final concentrations).The medium used for the isolation of organisms

was the lactate-Trypticase-yeast extract medium sup-plemented with 20% (v/v) clarified rumen fluid pre-pared as described by Allison et al. (1) and 1.2% agar.

Mineral salts added to the above media were essen-tially similar to those of Bryant and Robinson (3),except that 0.8% Na2CO3 was included instead of0.4%. As a reducing agent, 0.075% cysteine-HCI wasused.

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Isolation of the organism. Rumen contents fromwhich the organism was isolated were obtained viarumen fistula from a sheep which was fed once a dayon hay and concentrates. The contents were collectedbefore feeding, filtered through two sheets of cheesecloth, and centrifuged at 100 X g for 5 min to obtainthe precipitate. The precipitate was washed twicewith the mineral salts medium containing cysteine.Finally, the precipitate was diluted appropriately withthe above medium, and S. ruminantium var. lactilyticawas isolated by a roll-tube method. Almost all colonieswhich appeared in the roll tubes had the characteristicmorphology of S. ruminantium.

Culture of the organism. The organism was cul-tured anaerobically according to the method ofHungate (6) in various media. All incubations were at37 C andpH 6.8. The organism was precultured for 14hr in a lactate-rumen fluid medium of essentially thesame composition as the isolation medium except thatthe agar was omitted. Cells were harvested and washedtwice with the salts solution. The optical density of theresulting suspension was adjusted to 0.2 at 660 m, and0.2 ml of the suspension was used as inoculum for 10ml of medium. Bacterial growth was followed bymeasuring optical density in a Hitachi-FPW 4 color-imeter (Hitachi Seisakujo, Tokyo, Japan) equippedwith a 66 filter (660 mu).

Analysis of fermentation products. Analysis fororganic acids produced was performed by silicic acidcolumn chromatography according to the method ofBelasco (2).

Isotopic experiment. n-Valerate-l-C'4 (5 ,c; finalconcentration = 0.0031%) was added to 100 ml of theglucose-yeast extract medium. After inoculation andincubation, cells were harvested, washed twice withwater, and fractionated by the method of Wegner andFoster (8). A portion of the protein fraction wasextracted twice with chloroform-methanol (1:3), atroom temperature for 30 min and at 80 C for 1 hr.The solvent-soluble fraction was obtained after cen-trifugation. The other portion of the protein fractionwas digested with pronase (200 ,ug/ml; Kaken Kagaku,Tokyo, Japan) for 24 hr at pH 8.0. The solubilizedmaterial was collected after centrifugation. Radio-activity of each fraction was counted with a gas-flowwindowless counter (Erigaku Kenkyujo, Tokyo,Japan).

Chemicals. n-Valerate-J-C'4 was purchased fromCalbiochem. All other chemicals were commercialpreparations of analytical grade.

Electron microscopy. Cells were harvested andwashed twice with water. They were then placed on ancollodion film, dried at room temperature, andshadowed with chromium. Electron micrographs ofthe cells were prepared with a Hitachi HW-1 electronmicroscope (Hitachi Seisakujo, Tokyo, Japan).

RESULTS

Growth response ofS. ruminantium to n-valeratein glucose medium. S. ruminantium can utilizeglucose, glycerol, or lactate as an energy source.The Trypticase-yeast extract medium supple-mented with either glycerol or lactate did not sup-

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FIG. 1. Growth response of Selenomonas ruminan-tium in the glucose medium. The following additionswere made to the glucose-Trypticase-yeast extract me-

dium: clarified rumen fluid (10%, v/v) (R), 0.306%Na acetate, 0.0017% Na isobutyrate, 0.0019% each ofNa n-valerate and Na isovalerate (VFA's), and no addi-tion (NO) for Fig. la. The volatile fatty acid mixture as

described above but with acetate omitted (VFA's),0.0017% Na isobutyrate (iso-But), 0.0019% each ofNa n-valerate (n-Val) and Na isovalerate (iso-Val) forFig. lb.

port its rapid growth. However, abnormal typeof growth was observed in the glucose medium(Fig. la). When 10 to 20% (v/v) of the clarifiedrumen fluid was added to the medium, normalgrowth was obtained with glucose, glycerol, orlactate. In the glucose medium, the clarifiedrumen fluid could be replaced by a mixture ofvolatile fatty acids consisting of acetate, isobu-tyrate, n-valerate, and isovalerate (Fig. la).Further experiments revealed that n-valerate was

required for growth of this organism as shownin Fig. lb.

Vitamin requirements of S. ruminantium. Vita-min requirements were studied with casein hy-drolysate as a nitrogen source. It was found thatPABA was essential and biotin was stimulatoryin the glucose medium (Fig. 2). The slower rate

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minantium in the glucose medium. The basal medium(Basal) was the glucose-casein hydrolysate-vitamins-valerate medium. The following deletions were madefrom the basal medium: biotin (-Biotin), PABA(-PABA), n-valerate (-n-Val), and all vitaminsexcept biotin and PABA (Biot-PABA-n-Val). Thecontrol medium (Control) was the glucose-yeast ex-tract medium supplemented with 0.0019% n-valerate.

of growth in the glucose-casein hydrolysate-vita-mins-valerate medium (basal) as compared withthe glucose-yeast extract medium supplementedwith n-valerate (control) suggests an unidentifiedstimulatory factor(s) in yeast extract. The addi-tion of L-tryptophan, adenine, guanine, uracil,and xanthine to the casein hydrolysate-vitamins-valerate medium, however, was without effect.

Figure 3a shows that rumen fluid could bereplaced by the vitamin mixture but not with fattyacids in the lactate-Trypticase-yeast extract me-dium. Biotin was the sole effective vitamin in themixture (Fig. 3b). As shown later in Fig. 4, biotinappears to be essential for growth of this orga-nism in the lactate medium. The sluggish growthobserved with the lactate-Trypticase-yeast extractmedium, might be explained by a small amount ofbiotin contained in this medium. Probably theorganism has a high requirement for biotin.

Stimulatory effect of n-valerate in lactate me-

dium. The omission of Trypticase from the lac-tate-Trypticase-yeast extract medium resulted ina poor growth as shown in Fig. 4. The addition ofn-valerate to the yeast extract medium, however,had a marked stimulatory effect. The growth wasalmost equal to that with the Trypticase-yeastextract medium (Fig. 4). This result suggeststhat Trypticase might contain n-valerate-like ma-terial. This is in accord with the observation that

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FIG. 3. Growth response of Selenomonas ruminan-tium in the lactate medium. The basal medium was thelactate-Trypticase-yeast extract medium. The composi-tion of the volatile fatty acid mixture was the same asthat in Fig. la. The vitamin mixture had the same com-position as described in Materials and Methods. Theadditions made to the basal medium were clarifiedrumen fluid (R), vitamin mixture (Vit mix), volatilefatty acids (VFA's), and no addition (NO) for Fig. 3a.For Fig. 3b, all experimental conditions were the sameas above except that the following deletions were madefrom the vitamin mixture: PABA (-PABA) andbiotin (- Biotin) . The single deletion experiments exceptbiotin gave essentially the same growth response as-PABA.

abnormal growth occurs either in the glucose-yeast extract medium with a low level of addedn-valerate or in the glucose-Trypticase-yeast ex-tract medium without added n-valerate (Fig. la).Although biotin was essential for growth and

no other vitamins tested were stimulatory (Fig. 4)in the lactate medium, growth in the semisyn-thetic medium (lactate-casein hydrolysate-vita-mins-valerate medium) was poor as comparedwith that in the complex media (lactate-Trypti-case-yeast extract medium supplemented withbiotin or lactate-yeast extract medium supple-mented with biotin and n-valerate; Fig. 4). Further

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C Vit-n-Vol

C0 -Biotin

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HOURSFIG. 4. Stimulatory effect ofn-valerate in the lactate

medium. The experimental conditions were the same asin Fig. 3a and b. T- Y- Biot represents the growth in thelactate-Trypticase-yeast extract medium supplementedwithl biotin (0.01 mg/100 ml). The following deletionsand additions were made: minus Trypticase (- Tryptic),minus Trypticase plus n-valerate (Y. Biot . n-Va!), minusTrypticase and yeast extract plus casein hydrolysate,the vitamin mixture, and n-valerate, i.e., the caseinhydrolysate-vitamins-valerate medium (C- Vit-n-Val),and minus biotin from the preceding medium (-Biotin).

studies are needed to establish the requirements.Again, the addition of tryptophan and nucleicacid bases to the casein hydrolysate-vitamins-

valerate medium had no growth-promotingeffect. The requirement for growth factors in theglycerol medium was similar to that in the lactatemedium. Every nutritional experiment presentedhere was performed at least three times. Consistentand reproducible results were obtained.

Relation between cell morphology and nutritionalconditions. An electron micrograph (Fig. 5) illus-trates the normal morphology of cells grown inthe glucose-Trypticase-yeast extract mediumsupplemented with n-valerate (0.0019%). How-ever, the omission of n-valerate resulted in ab-normal growth as shown in Fig. la. Figures 7aand b show the appearance of the cells at 24 hr ina medium not supplemented with n-valerate. Thecells were much longer and sometimes showed aghostlike appearance. Although some normalcells were observed in these micrographs, almostall cells at the initial maximal growth at 6 hrshowed elongated appearance. As shown inFig. 6, cells grown in the lactate medium ap-peared to be smaller than those grown in the glu-cose medium. Thus far, no abnormal morphologyhas been found in lactate media.

Specificity ofrequiredfatty acids for growth. Toknow the specificity for the n-valerate require-ment, various fatty acids were added individuallyand growth of the bacterium was recorded. It wasfound that any straight-chain saturated mono-carboxylic acid with 3 to 10 carbons couldsupport growth at certain concentrations in placeof n-valerate in the glucose-yeast extract medium.

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FIG. 5. Electron micrograph of cells grown in the glucose-Trypticase-yeast extract medium supplemented withn-valerate (24-hr culture). X 12,S00.

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FIG. 6. Cells grown in the lactate-Trypticase-yeast extract medium supplemented with biotin (24-hr culture).X 12,500.

However, as can be seen in Fig. 8, n-valerate wasmost effective at the lowest concentration. Acetatewas not effective at any concentration examined.Pyruvate, DL-lactate, DL-malate, fumarate, suc-

cinate, L-asparagine, L-glutamine, and putrescine,each at a concentration of 0.3 %, and DL-meva-lonic acid, at a concentration of 0.02%, were

without effect. Pentadecanoic acid (C15-saturatedacid), which had been reported as a substitutefor n-valerate in growth of Bacteroides succino-genes (8), was without effect for this bacterium atconcentrations ranging from 0.0002 to 0.2%.

Incorporation of n-valerate-l-C14 into cell frac-tions. The distribution of radioactivity in cellfractions is shown in Table 1. Most of the assimi-lated C'4 was located in protein and lipid frac-tions. The cold and hot acid-soluble fractions hadlittle radioactivity. Approximately 80 to 90% ofthe radioactivity iu the protein fraction was ex-

tractable with chloroform-methanol dan was notdigestible with pronase (Table 1). It is concluded,

therefore, that most of the incorporated n-val-erate existed as lipid materials within the cells.

Fermentation products from glucose and lactate.Amounts of 100 ml each of the glucose-Trypti-case-yeast extract medium or the lactate-Trypti-case-yeast extract medium supplemented withsufficient amounts of both n-valerate (0.0019%)and biotin (0.01 mg/100 ml) were inoculatedand incubated. A portion of the culture (3 ml)was withdrawn at intervals with a syringe andwas analyzed for fermentation products. The timecourse of the acid production is shown in Fig. 9.This organism produced lactate, propionate, andacetate from glucose and propionate and acetatefrom lactate.

DIscussIoNThe present data clearly demonstrated that

the strain of S. ruminantium var. lactilytica re-puired any of C3 to C1o n-saturated fatty acids atcertain concentrations when grown in a medium

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FIG. 7. Cells grown in the glucose-Trypticase-yeast extract medium not supplemented with n-valerate (24-hrculture). (a) X 5,000. (b) X 7,500.

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CONCENTRATION OF FATTY ACIDS (%)FIG. 8. Growth ofSelenomonas ruminantium in re-

sponse to the concentration of n-saturated fatty acids.Various amounts offatty acids as indicated were addedto the glucose-yeast extract medium. The growth wasfollowed at 3-hr intervals, and the maximal growth rates,i.e., the maximal increments in optical density per 3 hr,were plotted versus the concentrations of n-saturatedfatty acids. No growth was observed in the medium sup-

plemented with 0.2% propionate.

containing glucose. Among them, n-valerate wasmost effective at the lowest concentration. Thoughn-valerate or n-caproate has been known as agrowth factor of some strains of B. succinogenesand Borrelia sp. isolated from rumen contents,these organisms also required a branched-chainfatty acid in addition to n-valerate or n-caproate(8).Bryant and Robinson (3) reported that several

strains of S. ruminantium required variably a mix-ture of volatile fatty acids which contained n-val-

erate, although they did not identify which acidwas essential for growth. According to Hobsonet al. (4), a strain of S. rwninantium did notrequire any of volatile fatty acids except acetate.The discrepancy might be explained by the fol-lowing observation. The organism grown in theglucose-Trypticase-yeast extract medium in theabsence of added n-valerate, which produced theabnormal growth curve shown in Fig. 1, wastransferred to the new glucose-Trypticase-yeastextract medium. After repeated transfers, therequirement for fatty acids was examined, and itwas found that the organism thus selected nolonger required added n-valerate. No such selec-tion was observed when the organism was storedin the lactate-Trypticase-yeast extract mediumsupplemented with rumen fluid.Of some interest is the finding that this organ-

ism produces propionic acid from lactate orglucose, and this acid is one of the effective acidsin supporting growth in the glucose medium.n-Valerate, however, was essential for growth inthe glucose medium but was stimulatory in thelactate medium. This might be due to the vigorousformation of propionate in the lactate medium,as was shown in Fig. 9.A number of bacteria isolated from rumen con-

tents require B vitamins, especially biotin andPABA (7). The strain studied here also showedthe requirement for biotin and PABA. However,biotin was essential when the organism was cul-tured in the lactate medium, and was stimulatorywhen the glucose medium was used. PABA, onthe contrary, was essential in the glucose medium,but was not effective in the lactate medium. Thenutritional requirements of this organism in rela-tion to the available energy sources are sum-marized in Table 2.

It has been reported by many workers thatmost of the ruminal bacteria require CO2 for

TABLE 1. Distribution of radioactivity in cell fractions of Selenomonas ruminantium grown in mediumcontainintg n-valerate-l-C"4

Total radioactivityCell fractions

Counts per min Per cent

Whole cells.562,000 100Cold trichloroacetic acid-soluble.31,000 5.5Ethyl alcohol-ether soluble.310,000 55.1Hot trichloroacetic acid-soluble.1,000 0.0Residual (protein fraction).200,000 35.6 100

Chloroform-methanol-soluble.155,000 27.6 77.5Chloroform-methanol-insoluble.33,000 5.9 16.5

Solubilized by pronase.17,000 3.0 8.5Not solubilized.183,000 32.6 91.5

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la

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FIG. 9. Growth and fermentation products of Selenomonas ruminantium in glucose (a) and in lactate (b)media. The following symbols were used: formation or utilization of lactate (L), formation of propionate (P),and acetate (A).

TABLE 2. Nutritional requirements of Selenomonasruminantium as related to energy source

Energy Essential requirement(s) Stimulatorysource for growth for growth

Glucose n-Valerate and PABA BiotinLactate Biotin n-ValerateGlycerol Biotin n-Valerate

growth (6). The S. ruminantium strain used inthis study also required CO2 for growth, both inglucose and in lactate media. In view of the re-

ported mechanism of propionate formation inS. ruminantium (M. J. B. Paynter, M.S. Thesis,Sheffield Univ., Sheffield, England), which in-volves succinate as an intermediate, the fixationofCO2 might play an important role in the metab-olism of lactate or glycerol, and thus a largeramount of biotin might be needed under theseconditions. Why PABA is essential in the glucosemedium and not in the lactate medium is un-

resolved.The isotopic studies presented here demon-

strated that n-valerate was incorporated exclu-sively into lipid materials. Wegner and Foster(8) found that n-valerate was used mainly forthe synthesis of n-C1s and C15 fatty acids andaldehydes, and they suggested that odd-numberedprecursors yielded products with odd-numberedfatty acids, and that those with even numbersgave rise to even-numbered products. The syn-thesis of longer fatty acids was supposed to takeplace with precursors as starting blocks and sub-sequent additions of two carbon units to thecarboxyl end of the molecules, as was described

by Horning et al. (5). The analysis of labeledlipid materials with n-valerate is in progress.

ACKNOWLEDGMENTSWe are grateful to G. Tamura of the University of

Tokyo for his generous supply of mevalonic acid. Wealso acknowledge T. Sato's help in the preparation ofelectron micrographs.

LITERATURE CITED1. ALLISON, M. J., M. P. BRYANT, AND R. N.

DOETSCH. 1962. Studies on the metabolic func-tion of branched-chain volatile fatty acids,growth factors for ruminococci. I. Incorpora-tion of isovalerate into leucine. J. Bacteriol.83:523-532.

2. BELASCO, I. J. 1954. Composition of urea and pro-tein meals as nitrogen sources for rumen micro-organisms: the production of volatile fatty acids.J. Animal Sci. 13:748-757.

3. BRYANT, M. P., AND I. M. ROBINSON. 1962. Somenutritional characteristics of predominant cul-turable ruminal bacteria. J. Bacteriol. 84:605-614.

4. HOBSON, P. N., S. 0. MANN, AND W. SMITH. 1963.Growth factors for Selenomonas ruminantium.Nature 198:213.

5. HORNING, M. G., D. B. MARTIN, A. KARMEN, ANDP. R. VAGELOS. 1961. Fatty acid synthesis inadipose tissue. II. Enzymatic synthesis ofbranched-chain and odd-numbered fatty acids.J. Biol. Chem. 236:669-672.

6. HUNGATE, R. E. 1950. The anaerobic mesophiliccellulolytic bacteria. Bacteriol. Rev. 14:1-63.

7. HUNGATE, R. E., M. P. BRYANT, AND R. A. MAH.1964. The rumen bacteria and protozoa. Ann.Rev. Microbiol. 18:131-166.

8. WEGNER, G. H., AND E. M. FOSTER, 1963. Incorpo-ration of isobutyrate and valerate into cellularplasmalogen by Bacteroides succinogenes. J.Bacteriol. 85:53-61.

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