SOME NUTRITIONAL CHARACTERISTICS OF PREDOMINANTCULTURABLE RUMINAL BACTERIA

M. P. BRYANT AND I. M. ROBINSONAnimal Husbandry Research Division,

U. S. Department of Agriculture, Beltsville, Maryland

Received for publication March 31, 1962

ABSTRACT

BRYANT, M. P. (U.S. Department of Agri-culture, Beltsville, Md.) AND I. M. ROBINSON.Some nutritional characteristics of predominantculturable ruminal bacteria. J. Bacteriol. 84:605-614. 1962.-The effect of enzymatic hydrolysateof casein, NH4+, a mixture of volatile fattyacids (acetic, n-valeric, isovaleric, 2-methyl-butyric, and isobutyric), hemin, and ruminalfluid on growth of 89 freshly isolated strains ofpredominant culturable ruminal bacteria wasstudied, using basal media containing glucose,cellobiose, or maltose as energy source, minerals,cysteine, and S= as reducing agents, and H2CO3-HCO - buffer. Of these strains, 13% (four mor-phological groups) grew poorly or not at all indefined medium plus casein hydrolysate; 6%(one morphological group) required caseinhydrolysate; 56% (four morphological groups)grew with either NH4+ or casein hydrolysateas the main source of nitrogen; and NH4+, butnot casein hydrolysate, was essential for 25%of the strains (five morphological groups).The volatile fatty acid mixture excluding ace-tate was essential for 19% of the strains (fivemorphological groups), and this mixture andacetate were necessary for good growth of 23%of the strains (one morphological group) whencasein hydrolysate was excluded from the me-dium; 30% of the strains (one morphologicalgroup) required hemin. Similar studies are re-ported on 35 old laboratory strains of ruminalbacteria, most of which were previously identi-fied. The results indicate that most strains ofruminal bacteria can be grown in defined media,and suggest the relative importance of NH4+and volatile fatty acids and the relative lackof importance of organic nitrogen compoundssuch as amino acids in the nutrition of thesebacteria.

Previous studies indicated that some strainsof bacteria known to function in the rumen

could be grown in defined media. These bac-teria include many strains of Streptococcus bovtis(Niven, Washburn, and White, 1948), anaerobiclactobacilli (Wasserman, Seeley, and Loosli,1953; Huhtanen, 1955; Gibbons and Doetsch,1959; Phillipson, Dobson, and Blackburn,1959), Bacteroides succinogenes (Bryant, Robin-son, and Chu, 1959), and the genus Rumino-coccus Sijpesteijn (1948) (Fletcher, 1956; Alli-son, Bryant, and Doetsch, 1958; Ayers, 1958;Bryant and Robinson, 1961 a, b); two strainsof the genus Butyrivibrio Bryant and Small(1956b) (Gill and King, 1958; Gordon and Moore,1961); and one strain each of Lachnospira multi-parus Bryant and Small (1956a) (Emery,Smith, and Fai To, 1957) and Bacteroides rumi-nicola subsp. brevis Bryant et al. (1958).

Results indicated that some strains of thepredominant anaerobic species had rather pe-culiar growth requirements. One or more of thevolatile fatty acids, such as n-valeric, isovaleric,2-methylbutyric, or isobutyric acids, were essen-tial for growth of some species (Bryant andDoetsch, 1955; Allison et al., 1958, 1962a; Weg-ner and Foster, 1957, 1960). Some of these strainsrequired and fixed amounts of NH4+ approxi-mately equal to or greater than the amount ofcellular nitrogen produced during growth,regardless of the amount of amino acid andpeptide nitrogen present in the medium, anddid not require organic nitrogen compounds forgrowth (Bryant and Robinson, 1961b). Strainsof other species were shown to fix large amountsof NH4+ during growth in media containinglarge amounts of amino acids (Phillipson et al.,1959; Gill and King, 1958).Attempts have not yet been made to study

the nutritional requirements of many of themore numerous species, and some were shown torequire unknown growth factors (Fletcher,1956; Ayers, 1958; Bryant et al., 1958; Bryantand Robinson, 1961a).The present survey was initiated to obtain a

better knowledge of the extent of growth re-

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quirements among the predominant anaerobicruminal bacteria for volatile fatty acids, NH4+,B-vitamins, and other factors as a preliminaryto more detailed studies on individual species.Early in the study it was found that the "rumenfluid" factor required by B. ruminicola subsp.ruminicola could be replaced by hemin.

MATERIALS AND METHODS

The methods and medium used for enumera-

tion and isolation of bacteria were those ofBryant and Robinson (1961c). The freshlyisolated strains studied were isolated from twosamples of rumen contents obtained by stomachtube from a 1,300-lb Holstein cow fed once a

day on 11.3 lb of alfalfa hay and 7.5 lb of grainmixture (15.4% crude protein). The sampleswere collected 6 and 22 hr after feeding, andcolony counts after 3 days of incubation indi-cated 5.2 and 2.2 billion bacteria per g, respec-

tively.The methods used to maintain strains of both

freshly isolated and previously described strainswere those of Bryant and Robinson (1961a),except that the slant medium was modified tocontain 0.5% of Trypticase (BBL) and 0.05%(each) glucose, cellobiose, and soluble starch.References to the characteristics of the 35

previously identified strains included in thestudy were given by Bryant (1959).The freshly isolated strains were placed in

morphological groups on the basis of Gramstains and wet mounts as described by Bryantand Burkey (1953), except that the slant me-

dium was modified as indicated above. Colonytypes were recorded but were of only limited use

in grouping strains. Group A included gram-

negative, nonmotile rods similar to B. succino-

genes, B. ruminicola, or Bacteroides amylophilusHamlin and Hungate (1956). Group B includedgram-variable to gram-positive nonmotile rodssimilar to Eubacterium ruminantium Bryant(1959). Group C included gram-variable, non-

motile, pleomorphic coccus-shaped to shortrod-shaped organisms that occurred as singlesand short chains, and all strains produced yellowcolonies. Group D included gram-variable coccisimilar to the genus Ruminococcus. Group E,typical of L. multiparus, included gram-variable,motile, curved rods that produced a woollycolony. Group F included gram-negative, motile,curved rods similar to Selenomonas. Group G

included all gram-negative, motile, more or lesscurved rods other than those placed in groupsF and H. Almost all of these were similar toButyritibrio. Group H included Borrelia sp.Bryant (1952), and group I included gram-negative, motile, lancet- to spindle-shaped or-ganisms that occurred in chains.

In studies on the growth requirements offreshly isolated strains, the basal mediumcontained 0.3% glucose (except that cellobiosewas substituted in the case of ruminococci andmaltose in the case of strains similar to B.amylophilus), 0.0001% resazurin, 0.4% Na2CO3,0.025% (each) cysteine *HCl *H20 and Na2S.9H20, 0.09% (each) KH2PO4 and NaCl, 0.002%(each) CaCl2 and MgCl2-6H20, 0.001% MnCl2.4H20, 0.001% CoCl2-6H20, 0.2 mg per 100 ml(each) of thiamine. HCl, Ca-D-pantothenate,nicotinamide, riboflavine, and pyridoxal, 0.01mg per 100 ml of p-aminobenzoic acid, 0.005mg per 100 ml (each) of biotin, folic acid, andDL-thioctic acid, 0.002 mg per 100 ml of co-balamin, and was equilibrated with oxygen-freeC02. All ingredients except cysteine, Na2S,and Na2CO3 were mixed, adjusted to pH 6.5with H2S04, and autoclaved at 15 psi for 15min. Sterile, C02-equilibrated Na2CO3 solutionwas added and the medium was added to tubes(13 by 100 mm) in 2.8-ml amounts, using anaero-bic methods and C02 as previously described(Bryant and Robinson, 1961a); 2 ml of the mix-tures shown in Table 1, plus 0.2 ml of a solutionof cysteine and Na2S (added just before inocu-lation), brought the medium constituents tofinal concentration.The mixtures A-H (Table 1) were prepared

aseptically with sterile solutions of the ingre-dients, were made to volume with sterile water,boiled briefly under C02 gas to remove dissolved02, and equilibrated with C02 while cooling to50 C or lower before being added to the basalmedium.The solutions from which the mixtures were

prepared were as follows. The vitamin and(NH4)2S04 solutions were prepared in appro-priate concentrations in distilled and deionizedwater. The volatile fatty acids were adjustedto pH 8 with NaOH solution and mixed. Hemin(Nutritional Biochemical Corp.) was dissolvedin 0.02% NaOH. The casein hydrolysate wasadjusted to pH 12 with 10% NaOH, aerated toremove most of the NH3, and neutralized with

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TABLE 1. Mixtures added to the basal medium usedto determine certain nutritional requirements offreshly isolated strains of predominant

ruminal bacteria

Mixtures added tomedium

Ingredients

A B C D E G H

(NH4)2SO4 (0.09%)............ + + + + - + +Volatile fatty acidsa........... + - + + - -

Casein hydrolysate + acetateb - + + + + +Hemin (0.2 mg/100 ml)........ +++++ + -

Clarified ruminal fluid (20%,v/v)........... - - -

a Consisted of 0.306% sodium acetate- 3H20,0.0017% sodium isobutyrate, and 0.0019% (each)sodium valerate, isovalerate, and DL-a-methyl-n-butyrate.

b Consisted of 0.2% (w/v) enzymatic hydroly-sate of "vitamin-free" casein (Nutritional Bio-chemical Corp.). This amount of the productalso contained approximately 0.34% acetate,calculated as sodium acetate-3H20.

5 N H2SO4. All of these solutions were autoclavedat 15 psi for 5 min and stored in the refrigerator.Clarified ruminal fluid was prepared as pre-viously described (Bryant and Robinson,1961a), as was the solution of cysteine and Na2S(Bryant and Robinson, 1961c). Menadione(USP) was dissolved in absolute ethanol anddiluted with sterile water.Media used to determine some growth re-

quirements of the previously described strainswere the same as the above, except that heminwas not included and the vitamin mix wassterilized separately; the latter was added tothe mixtures (Table 1) but was deleted frommedium H. Also, medium F, which was thesame as medium D but with the vitamin mixdeleted, was included.The inoculation procedure and methods of

determining growth were as previously de-scribed (Bryant and Robinson, 1961b), exceptthat medium H was used as the inoculum me-dium, wa.shed cells were prepared after onlyone transfer of strains on this medium, and theinoculum was 0.1 ml of washed cells adjustedto an optical density (OD) of 0.30. All strainswere inoculated into duplicate tubes of media;the same batch of basal medium to which thevarious mixtures were added was used for any

one culture. The results obtained with manystrains were checked with a second batch ofmedia. B. succinogenes S85 was used as a controlto determine whether each batch of media wassatisfactory. This organism was particularlysatisfactory for this purpose because of itsrequirement for a branched- and a straight-chainvolatile fatty acid, biotin, and NH4+, and be-cause of its sensitivity to cultural conditionssuch as pH and Eh (Bryant et al., 1959).The effects of certain components of the media

on growth of the strains (Tables 2 and 3) wereestimated by comparing growth, both maximalOD and incubation time for maximal OD, incertain media. Casein refers to the effect ofcasein hydrolysate in the presence of volatilefatty acids, including acetic acid; this effectwas estimated by comparing growth in mediaB and D. Casein + acetate - VFA refers tothe effect of casein hydrolysate plus acetate inthe absence of the other volatile fatty acids,and was estimated by comparing growth inmedia A and C. VFA refers to the effect of thevolatile fatty acids, other than acetate, in thepresence of casein hydrolysate and acetate,and was estimated by comparing growth inmedia C and D; and VFA - casein refers to theeffect of the volatile fatty acids, including ace-tate, in the absence of casein hydrolysate, andwas estimated by comparing growth in mediaA and B. Comparison of growth in media G andD showed the effect of unknown factors inruminal fluid; E and D, the effect of NH4+;D and F, the effect of B-vitamins; and G and H,the effect of hemin. The clarified ruminal fluidcontained only enough hemin or hemin-replacingfactors to allow a small amount of growth (10 to20% of maximum) of hemin-requiring strains.The effect of each of the components, acetate,

the volatile fatty acids other than acetate, andcasein hydrolysate, on growth of selected strainsin the presence or absence of one or both of theother components was studied by use of eightmedia designated as the cva series. Medium ocontained none of the three ingredients; mediac, v, and a contained casein hydrolysate, thevolatile fatty acids other than acetate, andacetate, respectively, in the same concentrationsas shown in Table 1. Media containing two ofthe ingredients were designated cv, ca, and va,and medium eva contained all three ingredients.The casein hydrolysate was "vitamin-free"

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Casitone (Difco); acetate was not added nor

was it treated for removal of NH4+. The basalmedium was identical with that describedabove but with vitamins, hemin, and (NH4)2SO4added before it was autoclaved. Methods ofinoculation were as indicated above except thatcells for the inoculum were grown in medium cva.

RESULTS

Nutrition of previously identified species. Allof the strains studied (Table 2) either did notgrow or grew very slowly on medium F. Thisindicated that one or more of the vitamins inthe mixture was essential or very highly stimula-tory to all strains which grew well in medium D.This medium contained defined ingredients ex-

cept for the "vitamin-free" casein hydrolysate.Strains which failed to grow well in this mediumor other media which did not contain ruminalfluid included one strain each of Butyrivibriosp. and two strains of unidentified, anaerobic,motile, lancet-shaped rods that occurred in

chains. The latter strains gave growth responsessimilar to Butyritibrio sp. (Table 2).Eubacterium species. One or more of the vola-

tile acids, other than acetate, and NH4+ were

essential for growth of the species E. ruminan-tium (Table 2). By use of media of the cva seriesand strain B1C23, it was shown that acetate hadno effect on growth except for a slight stimulationwhen casein hydrolysate was deleted.The strain of Eubacterium sp. Bryant (1959)

has a strict requirement for NH4+. Casein hy-drolysate plus acetate was stimulatory but one or

more of the volatile acids, including acetate,replaced this stimulatory effect. Further studieswith media of the cva series showed that acetatewas highly stimulatory, casein hydrolysate re-

placed most but not all of this stimulatory effect,and the volatile acids other than acetate had no

effect on growth.Selenomonas ruminantium. Results in Table 2

show that all of four strains grew well in mediawithout casein hydrolysate or NH4+ but varied

TABLE 2. Effect of rumen fluid, NH4+, casein hydrolysate, and volatile fatty acids on growth of species ofanaerobic ruminal bacteria

Effect of:Growth

Species Strain no. (OD Rumi- Casein Similar strainsin medium Rumi- CaenF

iilrstanD nal NH4W Casein + VFA VFA-

fluid acetate- caseinVFA

Eubacterium ruminantium... B4 61 (24)a +b E + 0 E E GA195, B1C23Eubacterium sp.......... B17 40 (24) + E + +++ - +++Selenomonas ruminantium. .. HD4 66 (24) - - + + ++ ++ PC18

GA192 42 (17) - - + 0 E E GA31Bacteroides ruminicola subsp.ruminicola ... ... 23c 119 (19) - + ++ +++ + ++ GA20C

B. ruminicola subsp. brevis.. B14 59 (24) + - - +++ - +++GA33 110 (21) - + E E ++ 0 GA103, 118B

Butyrivibrio fibrisolvens .... A38 53 (45) + +++ ++ 0 E EDl 60 (21) + + ++ ++ - -

49 69 (17) - - ++ +++ - +1 43 (49) ++ - E E + 0

Butyrivibrio sp.............. 28 0 (168) E 0 0 0 0 0Lachnospira multiparus ...... D32 90 (16) - - - ++ - ++ 40Peptostreptococcus elsdenii. . B159 73 (48) + _ E E - 0 T81Succinivibrio dextrinosolvens 24 53 (69) ++ +++ E E - 0Lactobacillus sp.. GAl 78 (40) ++ I ++++++ - - GA19, 440, 229Lactobacillus sp. (+R3). T185 75 (16) - I ++ ++ - - R62L. lactis (anaerobic variety). B62 37 (51) ++ I + + - + T112

a Figures in parentheses refer to the hours of incubation for maximal OD.b + ++, and +++ = moderately to very highly stimulatory; E = essential;- = no effect; 0 =

little or no growth in the media compared; and I = inhibitory.C These strains required hemin for growth.

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IA N-

00

x

C

I

3

0a:

114v

120

100

80

60

40

20

0c

-strainGA20f,

g srain

-/23

1 1 1 1 A0.4 0.8 1.2HEMIN-pG PER ML

FIG. 1. Growth response (means of two experi-mients) of two strains of Bacteroides ruminicolasubsp. ruittinicola to different levels of hemin inmediuni D.

in their response to the volatile fatty acids,one or more of these acids other than acetatebeing essential for growth of two strains andquite stimulatory to the others. Further studieson strains HD4 and GA192 showed that caseinhydrolysate was moderately stimulatory andacetate was stimulatory but only in media notcontaining casein hydrolysate.

Bacteroides ruminicola. Previous studies (Bry-ant et al., 1958) showed that B. ruminicolasubsp. ruminicola, the more numerous of the twosubspecies found in the rumen, required agrowth factor present in ruminal fluid but notpresent in Trypticase or yeast extract; thepresent strains (23 and GA20) failed to grow inthe media from which ruminal fluid was de-leted. In view of the recent studies showinghemin to be a grow-th factor for other anaerobes(Gibbons and MIacdonald, 1960), this compoundwas tested and shown to support excellent growthof both strains in medium D (Fig. 1). Heminwas, therefore, incorporated into media A-Gin studying the response of these strains to theother materials.

Studies on these two strains and four hemin-independent strains of B. ruminicola subsp.brevis showed that all strains grew well in certainof the media not containing ruminal fluid, andNH4+ either had no effect or moderately stimu-lated growth (Table 2). None of the strains grewwell except in the media containing either thevolatile fatty acids, including acetate, or caseinhydrolysate and acetate. Casein hydrolysatewas essential for three strains; and, with one

exception (strain BA4), casein hydrolysate andvolatile fatty acid(s) more or less stimulatedgrowth when added to media with only thesesingle components deleted.By use of media of the cva series, it was found

that volatile fatty acid(s), other than acetate,and acetate were both involved in replacing(strain B14) or partially replacing (strain 23)the stimulatory effect of casein hydrolysate andin stimulating growth above that obtained withcasein hydrolysate alone (strain GA33). Theacetate, but not the other volatile fatty acids,showed the latter effect in strain 23. Figure 2shows some of these results.

Succinimonas amylolytica Bryant et al. (1958).The growth of this organism (strain B24) wasvery similar to that of strain B14, B. ruminicola(Fig. 2), except that no growth occurred unlessboth acetate and the other volatile fatty acidsor casein hydrolysate were present; acetate stimu-lated growth well above that obtained with

120 CVA STRAIN GA33

CACV°x soM0

x0 400

0 V A VA

0 20 40 60 80HR

100STRAINB,4

VA

080o0 ~ fx ~ ~ AA

0 40 IIV0

0 20 40 60 80HR

FIG. 2. Growth response of two strains of Bac-teroides ruininicola in defined basal medium withno additions (0), with casein hydrolysate (C), witha mixture of isobutyrate, isovalerate, 2-methylbu-tyric and n-valerate (V), with acetate (A), and withvarious combinations of these ingredients.

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casein hydrolysate alone. The effect of NH4+was not determined.

Butyrivibrio fibrisolvens. The four strains ofthis species studied (Table 2) showed great varia-tion in growth as affected by ruminal fluid,NH4+, casein hydrolysate, and volatile fattyacids. By use of media of the cva series, bothacetate and other volatile acid(s) were found tobe stimulatory for strain 1, which requiredcasein hydrolysate. Strain 49 was stimulated bycasein hydrolysate and acetate, and growth was

better with both ingredients present. The othervolatile acid(s) had little effect. Growth ofstrain Dl was highly stimulated by casein hy-drolysate; volatile acids, including acetate, hadno effect. Strain A38, which was very highlystimulated by NH4+ and required volatilefatty acid(s), was stimulated by casein hydroly-sate but acetate had no effect.

Lachnospira multiparus. The two strainsstudied (Table 2) grew about equally well inmedia containing either casein hydrolysate or

acetate; ruminal fluid, NH4+, and volatile fattyacids other than acetate had little or no effect ongrowth.

Peptostreptococcus elsdenii Gutierrez et al.(1959). The two strains required factors in caseinhydrolysate, and ruminal fluid was moderatelystimulatory. Acetate was shown to be slightlystimulatory. The other volatile fatty acids andNH4+ had no effect on growth.

Succinivibrio dextrinosolvens Bryant and Small(1956a). A factor(s) present in casein hydroly-sate was essential for growth of the one strainof this species studied (Table 2). It differed fromall other strains which required casein hydroly-sate, in that NH4+ was highly stimulatory.Volatile fatty acids and acetate had no observ-able effect on growth.

Anaerobic lactobacilli. The eight strains ofthree groups studied were similar in beingmoderately inhibited by NH4+ but differed inthe degree of stimulation due to ruminal fluidand casein hydrolysate (Table 2).

In media of the cva series, it was shown thatboth casein hydrolysate and acetate stimulatedgrowth of Lactobacillus lactis B62, anaerobicvariety Mann and Oxford (1954). The effects ofthese components were additive; the other vola-tile fatty acids had no effect. However, whilecasein hydrolysate greatly stimulated growthof strains GAl and T185 (Table 2), as indicated

by the much better growth in media C and Dthan in media A and B, this effect was not seenin media of the cva series; e.g., media o and va,similar to media A and B, allowed growth (OD0.69 to 0.71 in 21 hr) about as good as mediaca and cva, similar to media C and D. Theonly apparent differences in media A or B (Table1, except that hemin was deleted) and mediao and va were that, in the latter media, (NH4)2-S04 and the vitamin mix were autoclaved withother ingredients of the basal medium, heminwas included, and inocula were prepared at dif-ferent times and from medium not containingruminal fluid. Further studies showed that therewas no difference in growth of strains B62,T185, and GAl in media va and cva preparedas usual or sterilized by filtration. Whetherhemin or the methods of growing cells for inoc-ulum were involved in the differing growthresponses in media not containing casein hydrol-ysate was not studied. No other strains ofbacteria showed these great differences.Some nutritional characteristics of freshly iso-

lated strains. A summary of some of the resultsof the nutritional and morphological study ofthe 89 freshly isolated strains is shown in Table3. In many cases, the nutritional patterns ofmorphological groups fit with the nutritionalpatterns of previously identified strains withsimilar morphology. Previous results on strainsof B. succinogenes and the genus Ruminococcus(Allison et al., 1962a) and the present results onE. ruminantium and one strain of B. fibrisolvensindicate that they required NH4+ and volatilefatty acid(s) other than acetate but little or noorganic nitrogen for growth. Among the freshisolates, all four strains similar morphologicallyto E. ruminantium (group B), the one strainsimilar morphologically to the genus Rumino-coccus (group D), two of the group G strainssimilar in morphology to Butyrivibrio, sevenof the group A strains similar in morphology toB. succinogenes, and all of three strains of mor-phological group C showed these requirements.

Eight strains (group E) were similar in mor-phology and colony type to L. multiparus andwere all nutritionally similar to this species.Presumably, the volatile fatty acid stimulationwhen casein hydrolysate was deleted was due toacetate, as indicated above with the knownstrains of L. multiparus.The one strain of morphological group F,

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TABLE 3. Effect of rumen fluid, NH4+, casein hydrolysate, and volatile fatty acids on growth of representativefreshly isolated strains of ruminal bacteria and their growth in the complete semidefined medium D

Effect of:

Strain no.a Morphological Growth (OD X O Caseingroup medium Db id N4 Caseina+ 'A VARuminal NH4+ Casein actae VFA cVFsAinflumina acetate- casein

VFA

4 (2) A 42 (51) ++C ++ - ++96 (3)d A 122 (21) - - ++ +++ + ++95 (2) A 95 (28) + - +++ E ++ E85 (17)d A 110 (26) - - +++ E ++ E62 (4)d A 110 (18) - - E E + 070 (4)e A 55 (20) - E - + - +34 (6) A 69 (28) + E - 0 E E6 (0) A 9 (42) + 0 0 0 0 01 (3) B 78 (26) - E ++ 0 E E

23 (2) C 69 (28) + E - 0 E E54 (0) D 53 (43) + E - 0 E E77 (7) E 100 (19) - + + ++ - ++18 (0) F 52 (18) - - ++ + ++ +29 (12) G 85 (20) - - ++ + - +21 (1) G 41 (98) +++ E - 0 E E38 (8) G 8 (47) + 0 0 0 0 059 (0) H 0 (168) E 0 0 0 0 064 (0) I 0 (170) E 0 0 0 0 0

a Figures in parentheses refer to the number of other strains studied which were similar in responses.bFigures in parentheses refer to hours of incubation for maximal OD.c See footnote b, Table 2.d These strains required hemin for growth.a Maltose but not glucose or cellobiose was utilized as the energy source.

similar to S. ruminantium, showed nutritionalpatterns similar to those of the known strains ofS. ruminantium except that the volatile fattyacid(s) was less stimulatory.The nutritional results on the first five strains

shown in Table 3, plus their morphology, sug-gest that they are identical with or closely re-lated to B. ruminicola. There were considerabledifferences in nutritional responses within bothof these groups but strains within each groupshowed similarities to strains of the other. Itis suggested that the requirement for hemindoes not correlate with other nutritional variableswithin the species.Of 24 strains morphologically similar to the

genus Butyrivibrio (group G, Table 3), 13 (seestrain 29) were similar in nutritional charac-teristics to strain 49 of B. fibrisolvens. Twostrains (see strain 21, Table 3) grew very poorlyexcept in the medium containing ruminal fluidbut showed the requirement for NH4+ andvolatile fatty acids similar to that of strain

A38 of B. fibrisolvens. Nine strains, representedby strain 38 of Table 3, failed to grow well inany of the media.Growth of all of the freshly isolated strains

that grew poorly or not at all in media D or G(Table 1) was compared in medium D and inmedium D with menadione (0.2 ug per ml),maltose (0.2%), and cellobiose (0.2%). Onlyfive strains responded to the added ingredients,and all of these were of morphological group A(gram-negative, nonmotile rods). Further studieswith single deletions showed that all of thesestrains required maltose and could not utilizeglucose or cellobiose for growth. With maltoseadded to the media in place of glucose, it wasfound that all of these strains required NH4+,and casein hydrolysate and volatile fatty acidswere only slightly stimulatory (strain 70, Table3). In further studies of strain 70, growth inmedium o was equal to that in medium eva(OD 0.71 in 16 hr), indicating that casein hy-drolysate and the volatile fatty acids, including

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acetate, had little if any effect. The morphology,lack of motility, the absence of a ruminal fluidgrowth requirement, and utilization of maltosebut not glucose or cellobiose as energy source inthese five strains suggest that they are relatedto the species B. amylophilus (Hamlin and Hun-gate, 1956).

DISCUSSION

This study, together with previous results,indicates that many strains of most of the con-siderable variety of anaerobic bacterial species,which are among the predominant bacteriaobtainable in pure culture from ruminal contents,can be grown in relatively simple chemicallydefined media or in medium in which "vitamin-free" casein hydrolysate is the only ingredientnot defined. From results shown in Table 3,it was calculated that 88% of strains freshlyisolated from the rumen grew well in the lattermedium and less than 7% of these required fac-tors in casein hydrolysate. Species grown forthe first time in defined or semidefined mediainclude at least two species of anaerobic lacto-bacilli, Eubacterium sp., E. ruminrantium, P.elsdenii, S. ruminantium, S. dextrinosolvens, S.amylolytica, and a species similar to B. amyloph-ilusThe present results, together with previous

studies, suggest that the ability to synthesizecellular nitrogen compounds from NH4+ is ofconsiderable survival value in the ruminal en-vironments which have selected most speciesof ruminal bacteria. Most of the known speciesand 82% of the freshly isolated strains (Table 3)grew well in media containing NH4+ and a smallamount of cysteine (1.4 ,umoles per ml) as themain sources of nitrogen. It seems doubtfulthat eysteine is required by many of these spe-cies. Gilroy (1957) concluded that many ruminalbacteria have simple nitrogen requirements,and many strains of species not included in thepresent study are known to be capable of utiliz-ing NH4+ as the main if not the only nitrogensource (e.g., see Wasserman et al., 1953; Gibbonsand Doetsch, 1959; Phillipson et al., 1959;Bryant and Robinson, 1961b). S. bovis, the mostcommonly found species of the genus found inruminal contents, was reported to have the leastcomplex nitrogen requirements of any species ofStreptococcus studied (Niven et al., 1948); NH4+satisfies the nitrogen requirements of many

strains (see Prescott, 1961, for references).The anaerobic' ruminal lactobacilli appear tohave much less complex nitrogen requirementsthan lactobacilli from many other habitats.

It was previously suggested that the ability toutilize efficiently exogenous nitrogen sourcessuch as amino acids is of limited survival valuein the microhabitats of the ruminal cellulolyticbacteria and that they have lost this ability(Allison et al., 1962a; Bryant and Robinson,1961b). It now seems also to apply to ruminalspecies attacking many other substrates. Strainsof at least seven species (Tables 2 and 3) and25% of the fresh isolates required NH4+ forgood growth; recent work indicates that strainsof these species, with the exception of S. dextrino-solvens, fix a large amount of NH4+ and littleprotein hydrolysate-C14 when grown in thepresence of both protein hydrolysate and NH4+(Allison et al., 1962a; Bryant and Robinson,Proc. 8th Intern. Congr. Microbiol., in press).Also, B. ruminicola and some strains of B.fibrisolvens, which do not require NH4+, fixvery little protein hydrolysate-C'4.The results substantiate those of Wegner

and Foster (1957), indicating that a large num-ber of ruminal bacteria require one or more ofthe volatile fatty acids, n-valeric, isovaleric,isobutyric, and 2-methylbutyric, for growth.These organisms represented 19% of the strainsand five different morphological groups amongthe fresh isolates (Table 3), and included strainsof E. ruminantium, S. ruminantium, and B.fibrisolvens which were not previously knownto have this requirement. Another indicationof the significance of these volatile fatty acidsin the nutrition of ruminal bacteria is the factthat strains of four species were more or lessstimulated by the acid mixture. Recent studiesindicate that certain of these acids are pre-cursors of certain amino acids and longer chainedfatty acids and aldehydes in some of the species(Allison et al., 1962a, b; Wegner and Foster,1961).The present results also indicate that acetate

is important in the nutrition of many species ofruminal bacteria. Whether the difference instimulation of some strains by acetate in mediawith and without casein hydrolysate ("vitamin-free" Casitone) was due to the presence ofacetate as a contaminant or the presence ofacetate-replacing factors such as certain amino

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NUTRITION OF RUMINAL BACTERIA

acids or long chain fatty acids in the caseinhydrolysate is not known. Lipoic acid was addedto these media.The fact that the strains of B. ruminicola

subsp. ruminicola and 31% of the fresh isolatesrequired hemin for growth indicates the impor-tance of hemin or hemin-replacing factors ingrowth of ruminal bacteria. Recent work (Cald-well, White, and Bryant, 1962) indicates thatcertain porphyrins, such as deutroporphyrin,mesoporphyrin, hematophorphyrin, protopor-phyrin IX, uroporphyrinogen, and copropor-phyrinogen, will replace hemin for growth ofB. ruminicola subsp. ruminicola in iron-containingmedia, but many other materials, includingchelating agents, porphyrins, and known inter-mediates in porphyrin synthesis, would not re-place hemin. Both the above subspecies andheme-independent B. ruminicola subsp. brevtiscontain a cytochrome of the b type, which ap-pears to be involved in electron transport forfumarate reduction to succinate by reduceddiphosphopyridine nucleotide (White, Bryant,and Caldwell, Proc. 8th Intern. Congr. Micro-biol., in press).The present results suggest that there are not

great differences in nutritional requirementsbetween freshly isolated strains and strains ofruminal bacteria maintained in pure cultureunder the conditions used in our laboratory forperiods of 4 to 10 years.

After this manuscript was submitted, KennethPittman of our laboratory showed that methio-nine replaced the casein hydrolysate requirementof strain GA33, B. ruminicola, and the greatstimulation of casein hydrolysate for strain 23 ina medium similar to medium D but with eithercysteine and S= serving as reducing agent.

LITERATURE CITED

ALLISON, M. J., M. P. BRYANT, AND R. N.DOETSCH. 1958. A volatile fatty acid growthfactor for cellulolytic cocci of the bovinerumen. Science 128:474-475.

ALLISON, M. J., M. P. BRYANT, AND R. N.DOETSCH. 1962a. Studies on the metabolicfunction of branched-chain volatile fattyacids, growth factors for ruminococci. I. In-corporation of isovalerate into leucine. J.Bacteriol. 83:523-532.

ALLISON, M. J., M. P. BRYANT, I. KATZ, AND M.KEENEY. 1962b. Studies on the metabolic func-tion of branched-chain volatile fatty acids.II. Biosynthesis of higher branched-chain

fatty acids and aldehydes. J. Bacteriol.83:1084-1093.

AYERS, W. A. 1958. Nutrition and physiology ofRuminococcus flavefaciens. J. Bacteriol.76:504-509.

BRYANT, M. P. 1952. The isolation and charac-teristics of a spirochete from the bovinerumen. J. Bacteriol. 64:325-335.

BRYANT, M. P. 1959. Bacterial species of the ru-men. Bacteriol. Rev. 23:125-153.

BRYANT, M. P., AND L. A. BURKEY. 1953. Culturalmethods and some characteristics of some ofthe more numerous groups of bacteria in thebovine rumen. J. Dairy Sci. 36:205-217.

BRYANT, M. P., AND R. N. DOETSCH. 1955. Factorsnecessary for the growth of Bacteroides suc-cinogenes in the volatile acid fraction of rumenfluid. J. Dairy Sci. 38:340-350.

BRYANT, M. P., AND I. M. ROBINSON. 1961a. Somenutritional requirements of the genus Ru-minococcus. Appl. Microbiol. 9:91-95.

BRYANT, M. P., AND I. M. ROBINSON. 1961b. Stud-ies on the nitrogen requirements of some ru-minal cellulolytic bacteria. Appl. Microbiol.9:96-103.

BRYANT, M. P., AND I. M. ROBINSON. 1961c. Animproved non-selective culture medium forruminal bacteria and its use in determiningdiurnal variation in numbers of bacteria inthe rumen. J. Dairy Sci. 44:1446-1456.

BRYANT, M. P., I. M. ROBINSON, AND H. CHU.1959. Observations on the nutrition of Bac-teroides succinogenes-a ruminal cellulolyticbacterium. J. Dairy Sci. 42:1831-1847.

BRYANT, M. P., AND N. SMALL. 1956a. The an-aerobic monotrichous butyric acid-producingcurved rod-shaped bacteria of the rumen.J. Bacteriol. 72:16-21.

BRYANT, M. P., AND N. SMALL. 1956b. Character-istics of two new genera of anaerobic curvedrods isolated from the rumen of cattle. J.Bacteriol. 72:22-26.

BRYANT, M. P., N. SMALL, C. BOUMA, AND H.CHU. 1958. Bacteroides ruminicola, sp. nov. andSuccinimonas amylolytica, gen. nov.-speciesof succinic acid-producing anaerobic bacteriaof the bovine rumen. J. Bacteriol. 76:15-23.

CALDWELL, D. R., D. C. WHITE, AND M. P. BRY-ANT. 1962. Specificity of the heme require-ment for growth of Bacteroides ruminicola-a ruminal saccharolytic bacterium. J. DairySci. 45:690.

EMERY, R. S., C. K. SMITH, AND L. FAI To. 1957.Utilization of inorganic sulfate by rumen

microorganisms. II. The ability of singlestrains of rumen bacteria to utilize inorganicsulfate. Appl. Microbiol. 5:363-366.

613VOL. 84, 1962

on Septem

ber 29, 2020 by guesthttp://jb.asm

.org/D

ownloaded from

BRYANT AND ROBINSON

FLETCHER, D. W. 1956. Studies on the growth re-quirements of a cellulolytic coccus from thebovine rumen. Thesis, State College of Wash-ington, Pullman.

GIBBONS, R. J., AND R. N. DOETSCH. 1959. Physi-ological study of an obligately anaerobicureolytic bacterium. J. Bacteriol. 77:417-428.

GIBBONS, R. J., AND J. B. MACDONALD. 1960. He-min and vitamin K compounds as requiredfactors for the cultivation of certain strainsof Bacteroides melaninogenicus. J. Bacteriol.80:164-170.

GILL, J. W., AND K. W. KING. 1958. Nutritionalcharacteristics of a Butyrivibrio. J. Bacteriol.75:666-673.

GILROY, J. J. 1957. Nitrogen requirements forgrowth of bovine rumen bacteria. Thesis,University of Maryland, College Park.

GORDON, G. R., AND W. E. C. MOORE. 1961.Growth stimulation of Butyrivibrio by mucin.J. Dairy Sci. 44:1772-1773.

GUTIERREZ, J., R. E. DAVIS, I. L. LINDAHL, ANDE. J. WARWICK. 1959. Bacterial changes inthe rumen during the onset of feedlot bloat ofcattle. Appl. Microbiol. 7:16-22.

HAMLIN, L. J., AND R. E. HUNGATE. 1956. Cultureand physiology of a starch-digesting bac-terium (Bacteroides amylophilus n. sp.) fromthe bovine rumen. J. Bacteriol. 72:548-554.

HUHTANEN, C. N. 1955. Pantethine and caseinhydrolysate in the growth of certain lacto-bacilli. Proc. Soc. Exptl. Biol. Med. 88:311-312.

MANN, S. O., AND A. E. OXFORD. 1954. Studiesof some presumptive lactobacilli isolated fromthe rumens of young calves. J. Gen. Micro-biol. 11:83-90.

NIVEN, C. F., JR., M. WASHBURN, AND J. WHITE.1948. Nutrition of Streptococcus bovis. J. Bac-teriol. 55:601-606.

PHILLIPSON, A. T., M. J. DOBSON, AND T. H.BLACKBURN. 1959. Assimilation of ammonianitrogen by rumen bacteria. Nature 183:402-404.

PRESCOTT, J. M. 1961. Utilization of sulfur com-pounds by Streptococcus bovis. J. Bacteriol.82:724-728.

SIJPESTEIJN, A. K. 1948. Cellulose-decomposingbacteria from the rumen of cattle. Thesis,Leiden University, Leiden.

WASSERMAN, R. H., H. W. SEELEY, AND J. K.LoOSLI. 1953. The physiology and nutritionof a rumen Lactobacillus. J. Animal Sci.12:935-936.

WEGNER, G. H., AND E. M. FOSTER. 1957. Fattyacid requirements of certain rumen bacteria.Bacteriol. Proc., p. 24.

WEGNER, G. H., AND E. M. FOSTER. 1960. Fattyacid requirements of certain rumen bacteria.J. Dairy Sci. 43:566-568.

WEGNER, G. H., AND E. M. FOSTER. 1961. Incor-poration of isobutyrate-1-C'4 and valerate-1-C14 into phospholipid by Bacteroides succino-genes, a cellulolytic bacterium from therumen. Bacteriol. Proc., p. 170.

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