Vol. 58, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 99-1050099-2240/92/010099-07$02.00/0Copyright © 1992, American Society for Microbiology

Comparative Growth Rates of Various Rumen Bacteria in ClarifiedRumen Fluid from Cows and Sheep Fed Different Diets

N. 0. VAN GYLSWYK,* K. WEJDEMAR, AND K. KULANDER

Department ofAnimal Nutrition and Management, Swedish University of Agricultural Sciences,Kungsangen Research Station, S-753 23 Uppsala, Sweden

Received 5 August 1991/Accepted 22 October 1991

Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluidsupplemented with glucose, bicarbonate, and reducing agent (cysteine and sulfide). Growth rates were

determined in a series of experiments. Strains of species most abundant in the rumen grew more rapidly thanstrains of less abundant bacteria. Ammonia, amino acids, and peptides increased growth rates to some extent,but the greatest stimulatory effect for less abundant bacteria was provided by other factors, present in yeastextract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release wasslow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undeterminednature probably play an important role in determining the predominance of different bacterial species in therumen.

Over the past 40 years, the principal bacterial speciesinhabiting the rumen have been identified and studied (3, 5).Numbers of different species can fluctuate considerablywhen ruminants are fed different diets, but it appears thatBacteroides ruminicola is usually the major species whennormal production diets are fed (1). Butyrivibriofibrisolvensis also found among the predominant species (5) and canprobably be regarded as the second most abundant species inthe rumen. The predominance of B. ruminicola and then ofB. fibrisolvens was again confirmed recently in the case ofdairy cows fed grass silage-based diets (7). Specific reasonsfor the predominance of these two species have not beenadvanced. It was decided to measure the growth rates inrumen fluid of previously isolated strains (7) of B. rumini-cola, B. fibrisolvens, and several other species to seewhether rates of growth were in any way related to numbersin the rumen. Glucose was added to the rumen fluid as theenergy source, cysteine and sulfide together served as thereducing agent, and bicarbonate and CO2 served as thebuffer system. In all experiments, parallel runs were madewith the same "medium" supplemented with yeast extractto indicate possible deficiencies of growth factors in therumen fluid.

MATERIALS AND METHODS

Animals and feed. Rumen-fistulated Swedish Landracewethers and lactating cows of the Swedish Red and Whitebreed were fed as shown in Table 1. The times of sampling ofrumen fluid relative to the morning feed are given in subse-quent tables.

Bacteria. Strains of B. ruminicola, B. fibrisolvens, and agram-negative, chain-forming, butyrate-producing coccuswere isolated from nonspecific medium used for growingtotal culturable bacteria; Eubacterium cellulosolvens andRuminococcus albus were isolated from cellulose-containingmedium; Ruminococcus flavefaciens (also cellulolytic) wasisolated from cellobiose-containing medium; and Selenomo-nas ruminantium and Megasphaera elsdenii (both ferment

* Corresponding author.

lactate) were isolated from medium containing lactate as thesole carbon energy source (7).

Culture media. Bacteria were maintained on slopes of agarmedium containing the following (per liter): K2HPO4, 0.45 g;KH2PO4, 0.45 g; NaCl, 0.90 g; (NH4)2SO4, 0.90 g; CaCl2(anhydrous), 0.09 g; MgSO4 - 7H20, 0.18 g; NaHCO3, 6.37g; agar, 15 g; glucose, 5 g; yeast extract (Difco), 5 g;cysteine. HCl H20, 0.25 g; Na2S (hydrated), 0.25 g; indigocarmine, 0.005 g; and rumen fluid (centrifuged at 1,500 x gfor 20 min), 400 ml. The gas phase consisted of 02-free CO2.The medium used for growing inocula was the same butwithout yeast extract. The yeast extract was omitted tominimize carryover of growth factors to medium used foroptical density (OD) measurements. Both media were heatsterilized. Yeast extract used in the experiments was alwaystaken from the same batch. The amino acid and peptidecontents of the yeast extract were determined by high-performance liquid chromatography on unhydrolyzed andacid-hydrolyzed samples.Rumen fluid was the main constituent of the culture

medium used for measuring growth rates. It was sampled bysuction tube through the rumen fistula and passed through adouble layer of cheesecloth. It was centrifuged for 1 h atabout 26,000 x and then at about 50,000 x g for 30 min. Inall experiments, the following were added to the rumen fluid(final concentration in grams per liter): glucose, 5; NaHCO3,6.37; cysteine HCl H20, 0.25; and Na2S (hydrated), 0.25.Other additions depended on the treatment. The gas phasewas O2-free CO2. Rumen fluid was sterilized by means ofdisposable Millipore filters, initially with a pore diameter of0.22 ,um but later with a pore diameter of 0.45 ,um which alsoproved to be effective (i.e., no case of contamination de-tected) and allowed for greater ease in filtering. In experi-ments 1 to 5, sterile, concentrated solutions of glucose,bicarbonate, and either yeast extract or an equivalent vol-ume of sterile, deionized water were added to rumen fluid,with water constituting between 20 and 30% (depending onthe experiment) of the total volume. In subsequent experi-ments, these and other substances [(NH4)2SO4 and caseinhydrolysate] were added dry to portions of rumen fluid anddissolved before filter sterilization. In all experiments, cys-

99

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

100 VAN GYLSWYK ET AL.

TABLE 1. Amounts of concentrate, silage, hay, and straw fed to fistulated sheep and fistulated lactating cows

Approx mean daily intake of dry matter (DM),Period of Daily ration (kg) crude protein (CP), neutral detergent fiber (NDF),

Expt Animal(s) adaption and metabolizable energy (ME)no. to diet

(wk) Concentrate Grass Grass Barley DM (kg) CP (kg) NDF (kg) ME (MJ)silage hay straw

1 Sheep ia 1 1.0b 1.0 2 0.3 0.6 212 Sheep la 3 1.0b 1.0 2 0.3 0.6 213 Cow 999 1 9.1c 4.8 2.2 13 1.8 4.4 157

Cow 966 1 9.6d 5.1 13 3.2 4.3 1604 Sheep 1 4 1.Ob 1.0 2 0.3 0.6 215 Sheep 2 >10 0.3b 0.5 1 0.1 0.3 86-8 Cow 981 >4 8.0b 7.0 2.0 11 1.9 3.0 1419 Cow 980 4 11.2e 5.1 2.2 16 1.6 4.8 20510 Cows 97, 101, 981 4 11.2e 5.1 2.2 16 1.6 4.8 205a Frothy bloat.b Standard concentrate C: barley, 32%; molassed beet pulp, 14%; soybean meal, 10o; rapeseed meal, 10%; fat, 2%.c Concentrate A: barley, 17.2%; oats, 17.2%; molassed beet pulp, 6.6%; wood molasses, 1.5%; sugar cane molasses, 31.6%; soybean meal, 2.5%; rapeseed

meal, 5.6%; rapeseed, 12.7%; maize gluten meal, 5.1%;.d Concentrate B: barley, 14.0%; oats, 14.0%; maize, 35.3%; molassed beet pulp, 5.3%; wood molasses, 1.2%; soybean meal, 2.0%; rapeseed meal, 4.5%;

rapeseed meal (heat treated), 11.8%; rapeseed, 9.4%; urea, 2.4%.e Concentrate E: barley, 43.8%; maize, 10.4%; sugar cane molasses, 26.0%; rapeseed, 15.6%.

teine hydrochloride and sodium sulfide were added togetherin a single, concentrated (50X) alkaline solution just beforeinoculation. The pH of the media was approximately 6.7.The final volume in tubes used for OD measurements wasusually 4 ml. Glucose was used as the energy source in allthe experiments because all strains fermented glucose (7).Some of the strains may prefer other substrates, but this wasnot tested.

Lysate preparations from mixed rumen microorganisms.Growth factor preparations were obtained from samples ofrumen fluid taken on the same day and from the same animalas the rumen fluid used for growth rate studies on purecultures of rumen bacteria.For experiment 4, rumen fluid was sampled from sheep 1

(Table 1) at 3 h after feeding, strained through two layers ofcheesecloth, and centrifuged at 1,500 x g for 20 min at 2°C.The supernatant (SN1) which contained many bacteria wasthen centrifuged at ca. 50,000 x g for 15 min at 2°C. Thepellet was suspended in deionized water and made up toone-sixth of the volume of SN1. The mixture was incubatedfor 1 h at 38°C. After centrifugation, the supernatant (SN2)was filter sterilized and added to sterile rumen fluid to formone-sixth of the final volume.

In experiment 5, rumen fluid was obtained from sheep 2(Table 1) at 3 h after feeding and a lysate preparation wasobtained in the same way as described above. The onlydifference was that the suspended pellet was incubated at38°C for 5 h instead of 1 h. In addition, the pellet obtainedafter centrifugation at low speed, which was very fluid andwhich contained bacteria, protozoa, and small feed particles,was compacted by centrifugation at high speed. It was thensuspended in deionized water and made up to one-sixth ofthe volume originally centrifuged. This whole fraction wasalso incubated for 5 h at 38°C, after which it was againcentrifuged at high speed. The supernatant was filter steril-ized and added to rumen fluid to form one-sixth of the finalvolume.A different approach was made in experiment 9. Rumen

fluid was taken from cow 980 (Table 1) before and 3 h afterfeeding. Each sample was strained through cheesecloth anddivided into four portions. Portion 1 was centrifuged imme-diately at high speed (50,000 x g, 30 min, 2°C). Portions 2, 3,

and 4 were incubated at 38°C for 2, 4, and 7 h, respectively,before they were also centrifuged. After centrifugation, thesupernatants were stored at -18°C. Before use, the fourdifferent portions were thawed, centrifuged again in thesame way to remove small amounts of particulate matter,and filter sterilized, and the filtrate was used as the growthmedium.Maintenance of bacteria and inocula. Agar slopes contain-

ing yeast extract were inoculated and incubated until goodgrowth had occurred. The slopes were stored at ca. -80°C.When required, the slopes were thawed and the bacteriawere transferred to 5-ml agar slopes, free of yeast extract,which were then incubated for about 16 h. Bacterial growthon each slope was diluted with 2 ml of anaerobic diluent andsuspended. Portions of 0.2 ml were added to 4 ml of mediumcontained in glass tubes. All transfers of bacteria and addi-tions of solutions to sterile bottles or tubes were done withsterile syringes, with the needles piercing butyl rubber serumcaps.OD measurements and expression of results. ODs were read

on a spectrophotometer in culture tubes of 13.5-mm internaldiameter at 600 nm. The spectrophotometer was fitted with aclamp to hold the tubes in a fixed position. The tubes wereread immediately after inoculation (to). They were incubatedin a water bath at 38°C and read at hourly intervals immedi-ately after thorough shaking. All tests were done in dupli-cate, and the data given in Tables 2 to 5 were calculated fromthe means. The data were calculated from the greatestchange in OD between two consecutive readings (1-h inter-val between readings) within 6 h after inoculation.

RESULTS

Twenty-two strains of B. ruminicola-like bacteria werechosen randomly from those isolated earlier (7) and wereexamined for possible use in the current tests. Fifteen of thestrains grew rapidly on slopes of agar medium (containingyeast extract) and showed good growth within 16 h. Nine ofthese strains morphologically resembled B. ruminicolasubsp. brevis in that the cells were mostly coccoid (5), whilethe remaining six strains morphologically resembled B.ruminicola subsp. ruminicola, which consisted predomi-

APPL. ENVIRON. MICROBIOL.

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

COMPARATIVE GROWTH RATES OF VARIOUS RUMEN BACTERIA 101

nantly of short to long rods (5). Of the 22 strains, 7 grewmuch slower and required up to 48 h to produce visiblegrowth. Of these, three strains appeared B. ruminicolasubsp. brevis-like and the remainder appeared B. ruminicolasubsp. ruminicola-like. The three strains chosen for use inthe experiments were fast growers. Strains TCl-1 and TN1-5resembled B. ruminicola subsp. brevis, and strain TC18resembled B. ruminicola subsp. ruminicola. In subsequentgrowth tests, it was found that the latter strain grew consid-erably slower than either of the other two strains. Noselection was applied in choosing strains of other species.Examples of curves depicting the growth of bacterial

strains of six species in rumen fluid supplemented withglucose, bicarbonate, and reducing agent, both in the ab-sence and presence of yeast extract, are shown in Fig. 1.

Table 2 gives the maximum growth rates for differentstrains of B. ruminicola and B. fibrisolvens as well as strainsof four other species in rumen fluid taken before and atdifferent times after the morning feed from a sheep and cowsfed a variety of diets (Table 1). Fast-growing strains of B.ruminicola (strains TN1-5 and TCl-1) grew more rapidlythan any of the other strains. The next most rapidly growingstrains were the fast-growing strains of B. fibrisolvens(strains TC1-14, TC33, and TC1-4). Strains of the otherspecies all had low maximum growth rates. The slow-growing strains of B. ruminicola and B. fibrisolvens (TC18and TV1-4, respectively) received relatively little stimulationfrom the addition of yeast extract, which suggested that theirinherent growth rates were indeed low. Growth of all strainsof B. ruminicola were little affected by the addition of yeastextract. Despite the different sources of rumen fluid, growthrates of the different species, relative to each other, showedthe same tendencies. Rumen fluid taken 3 or 3.5 h after themorning feed was somewhat superior to that taken beforefeeding, although this was not apparent in all cases. Rumenfluid sampled 7 or 8 h after feeding was usually inferior tothat obtained before feeding, although again there wereexceptions.

In the foregoing experiments, concentrations of essential,or growth-stimulating, factors in the rumen fluid would havediminished rapidly with growth of the bacteria. An attemptwas therefore made to supply factors of the type released inthe rumen by adding filter-sterilized fluid from preparationsof lysed rumen microbes to clarified, filter-sterilized, rumenfluid. Incubation of mixed rumen microbes without addedsubstrate would presumably lead to the rapid depletion ofeasily fermentable energy sources and, as long as growthoccurred, also to the depletion of other factors that areessential or stimulatory for the growth of a variety ofmicrobes. However, starvation of rumen bacteria results inrapid lysis (2). Thus, in incubated preparations free of addedenergy sources, lysis may lead to the release of growthfactors at a rate equal to or greater than would normallyoccur in the rumen. Some experiments were done to obtainan indication of the rate of release of such factors byincubating various preparations of rumen solids for differentperiods and adding the filter-sterilized fluid from such lysatesto clarified sterilized rumen fluid in proportions that couldsimulate the supply of growth factors from lysing microbesin the rumen (see Materials and Methods). Results forfast-growing strains of B. ruminicola and B. fibrisolvens areshown in Table 3.Lysate from a fraction consisting largely of bacteria and

preincubated for 1 h had some stimulatory effect in rumenfluid sampled before and 3 h after the morning feed (exper-iment 4). In experiment 5, a similar preparation, incubated

B. ruminicola TCl-l

0 2 4Time (h)

B. fibrisolvens TC33

6

S. ruminant L14

0 2 4Time (h)

0 2 4 6Time (h)

E. cellulosolvens Alc

<I

1]

C6

R. flavefaciens CB7

:3

0-0

I -

2 4Time (h)

6

2 4Time (h)

6

M. elsdenii TD1-14

_ <.0 2 4

Time (h)6

FIG. 1. Examples of typical growth curves of strains of rumenbacteria in filter-sterilized rumen fluid supplemented with glucose,bicarbonate, and reducing agent (cysteine and sulfide) in the absence( ) and presence (-) of yeast extract.

for 5 h, had a somewhat greater stimulatory effect on thegrowth of B. fibrisolvens TC1-14 in rumen fluid sampled 3 hafter feeding, but a lysate prepared from a mixture ofmicrobes and small feed particles increased the maximumgrowth rate of the B. fibrisolvens strain almost to that of theB. ruminicola strain, the growth of which was little affectedby the additions.

In experiment 9, separate portions of cheesecloth-strainedrumen fluid, which contained microbes and small feed par-ticles, were either not incubated before centrifugation or

were incubated for 2, 4, or 7 h before centrifugation and filtersterilization. These were then used as the growth media.Preincubation of the rumen fluid resulted in only minorincreases in the maximum growth rates of the B. ruminicolastrain. Increases were much greater for the B. fibrisolvensstrain, but, interestingly, the greatest increases occurred

VOL. 58, 1992

a I I

O-

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

102 VAN GYLSWYK ET AL.

TABLE 2. Growth rates of rumen bacteria in filter-sterilized rumen fluid sampled before and after feeding from a sheep and cowsfed different diets

Time of sampling of Maximum growth rates (AOD)b of bacteriaExpt no. Strain of bacteria rumen fluid' relative to in rumen fluid

the morning feed No addition + Yeast extractc

1 B. ruminicola TN1-5 Before 0.75 1.033.5 h after 0.73 0.93

B. fibrisolvens TC1-14 Before 0.21 0.973.5 h after 0.18 0.92

2 B. ruminicola TC1-1 Before 0.56 0.743.5 h after 0.56 0.76

B. ruminicola TC18 Before 0.10 0.233.5 h after 0.08 0.14

B. fibrisolvens TC33 Before 0.17 0.823.5 h after 0.20 0.68

B. fibrisolvens TV1-4 Before 0.08 0.173.5 h after 0.10 0.13

3Cow 966 B. ruminicola TC1-1 Before 0.44 0.58

8 h after 0.52 0.63B. fibrisolvens TC1-4 Before 0.25 0.36

8 h after 0.16 0.33Cow 999 B. ruminicola TC1-1 Before 0.43 0.69

8 h after 0.39 0.70B. fibrisolvens TC1-4 Before 0.24 0.49

8 h after 0.15 0.416 B. ruminicola TC1-1 Before 0.45 0.73

3 h after 0.56 0.737.h after 0.32 0.74

B. fibrisolvens TC33 Before 0.13 0.903 h after 0.27 0.877 h after 0.11 0.78

E. cellulosolvens Alc Before 0.03 0.483 h after 0.07 0.497 h after 0.09 0.48

R. flavefaciens CB7 Before 0.09 0.123 h after 0.08 0.087 h after 0.05 0.14

S. ruminantium L14 Before 0.05 0.783 h after 0.10 0.787 h after 0.06 0.72

M. elsdenii TD1-14 Before 0.01 0.193 h after 0.03 0.097 h after 0.01 0.20

aIn all cases, rumen fluid was supplemented with glucose (0.5%), NaHCO3 (0.64%), and reducing agent (cysteine. HCI H20 and hydrated Na2S, 0.025%each).

b Growth rates represent the steepest portion of the growth curves.Concentration of yeast extract was 0.5%.

with a preincubation period of 2 h, both with rumen fluidsampled before and that sampled 3 h after feeding. Thissuggested that initial lysis may have been succeeded bygrowth of the microbes with a concomitant uptake of growthfactors during preincubation in the rumen fluid in which themicrobial population was not concentrated, as was the casein experiments 4 and 5. In all three experiments, the B.ruminicola strains attained maximum growth rates thatwere, on average, 80% of those obtained with yeast extract,while the average for the highest growth rates reached ineach of the three experiments by B. fibrisolvens strains wasonly 47% of the average for corresponding values obtainedwhen yeast extract was added. Thus, it seemed that, witheither relatively low or relatively high concentrations ofgrowth factors of the type released in the rumen, B. rumini-cola has a decided competitive advantage over B. fibrisol-vens in being less dependent on such factors.Ammonia, amino acids, and peptides would be among the

nutrients released from feed particles and from lysing mi-crobes in the rumen. The effect of adding 6.8 mM ammoniaas ammonium sulfate is shown in Table 4. In experiment 7,strains of both B. ruminicola and B. fibrisolvens respondedto the addition of ammonia to rumen fluid sampled 7 h afterfeeding, with maximal growth rates increased by more than50%. Strains of E. cellulosolvens and S. ruminantium did notrespond. Rumen fluid used in experiment 8 was obtainedfrom the same cow on the same day as that used inexperiment 7. In this case (experiment 8), the ammoniaconcentration in the rumen fluid was determined and foundto be 2.9 mM before, and 8.6 mM 3 h after, feeding. Theconcentration of ammonia was clearly growth rate limitingfor B. ruminicola TCl-1 in the rumen fluid sampled beforefeeding and less so for some of the other strains. Growth ofB. ruminicola strains was again least affected by the additionof yeast extract. It was also noticeable that rumen fluid taken3 h after feeding generally supported more rapid growth than

APPL. ENVIRON. MICROBIOL.

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

COMPARATIVE GROWTH RATES OF VARIOUS RUMEN BACTERIA 103

TABLE 3. Effects of adding growth factors from lysed rumen microbes on the growth of B. ruminicola and B. fibrisolvens in rumen fluidtaken before and after the morning feed from two sheep fed different diets and from a lactating cowa

Time of sampling of rumen Maximum growth rates (AOD) ofExpt Strain of bacteria fluid relative to the Type of lysate bacteria in rumen fluidno. feed prepn addedbmorningfeedNo addition + Yeast extract

4 B. ruminicola TN1-5 Before None 0.59 0.79Before A 0.64 0.783 h after None 0.61 0.723 h after A 0.61 0.71

B. fibrisolvens TC1-14 Before None 0.14 0.74Before A 0.21 0.773 h after None 0.30 0.773 h after A 0.35 0.80

5 B. ruminicola TN1-5 3 h after None 0.50 0.713 h after B 0.54 0.713 h after C 0.58 0.73

B.fibrisolvens TC1-14 3 h after None 0.11 0.643 h after B 0.25 0.763 h after C 0.52 0.88

9 B. ruminicola TC1-1 Before None 0.54 0.71Before D 0.56 0.68Before E 0.59 0.69Before F 0.62 0.703 h after None 0.64 0.763 h after D 0.65 0.753 h after E 0.67 0.803 h after F 0.62 0.80

B. fibrisolvens TC33 Before None 0.08 0.66Before D 0.32 0.94Before E 0.29 0.87Before F 0.25 0.863 h after None 0.17 0.963 h after D 0.40 1.013 h after E 0.31 0.963 h after F 0.31 0.98

a See Table 2, footnotes a, b, and c.b Lysate preparations (see Materials and Methods) were as follows. (A) The bacterial fraction of rumen fluid sampled 3 h after feeding was incubated for 1 h

at 38°C after which it was centrifuged, and the filter-sterilized supernatant served as a concentrated source of growth factors. (B) This was prepared as describedfor preparation A except that the incubation was for 5 h. (C) The whole fraction of'rumen fluid sampled 3 h after feeding was incubated for 5 h at 38°C after whichit was centrifuged, and the filter-sterilized supernatant was used as a concentrated source of growth factors. (D, E, and F) Cheesecloth-strained rumen fluid wasincubated at 38°C for 2 h (D), 4 h (E), and 7 h (F), after which it was centrifuged, and the filter-sterilized supernatant was used as the growth medium.

that taken before feeding, irrespective of the effects ofammonia.The yeast extract was examined for free amino acids and

peptide content which could, in part, have been responsiblefor growth stimulation. Free amino acids were found toconstitute 40% (by weight) of the yeast extract, while totalamino acids constituted 62%, indicating a peptide contentof 22%. Acid-hydrolyzed and enzyme-hydrolyzed caseinas sources of free amino acids and peptides, respectively,were used to investigate to what exte'nt they were responsi-ble for growth stimulation. According to th'e product speci-fication'(Sigma Chemical Co.), the acid hydrolysate wasvirtually free of vitamins and growth' factors while thevitamin content of the enzymatic hydrolysate was statedltobe very low (6).The effect on growth rates of strains of various species of

rumen bacteria in rumen fluid- supplemented with acid-hydrolyzed and enzyme-hydrolyzed casein were examinedin experiment 10. The ammonia content of the rumen fluid(pooled in equal proportion from three different cows fedthe same diet) was 3.18 mM. To prevent possible deficien-cie's of ammonia affecting responses to amino acids orpeptides, anl treatments, except the zero treatment, weresupplemented' with 10 mM ammonia as (NH4)2SO4. The

concentrations of the casein hydrolysates included in thetreatments were calculated to contribute about the sameamount of amino acids as would be contributed by 0.5%yeast extract. The results obtained in experiment 10 aregiven in Table 5.

Inclusion of ammonia alone increased the growth rates ofB. ruminicola and R. flavefaciens strains. The further addi-tion of free amino acids (as acid hydrolysate of casein)increased the growth rates of all the strains, with relativelysmall increases for B. ruminicola, E. cellulosolvens, and R.flavefaciens. Except for R. flavefaciens and the chain-forming coccus, the peptides (as enzymatic hydrolysate ofcasein) had a markedly greater stimulatory effect. However,yeast extract remained superior in all cases, but the B.ruminicola strain grew almost as rapidly with the peptidesource as with yeast extract.

Differences between maximum growth rates obtained inunsupplemented rumen fluid and fluid supplemented withonly yeast extract were tested for significance in the case ofthose species for which sufficient data were available,namely, B. ruminicola (23 pairs), B. fibrisolvens (21 pairs),E. cellulosolvens (6 pairs), S. ruminantium (6 pairs), andM. elsdenii (5 pairs). All differences were significant at aprobability well in excess of 99% (t test). It is interesting to

VOL. 58, 1992

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

104 VAN GYLSWYK ET AL.

TABLE 4. Relative growth rates of different rumen bacteria in rumen fluid taken before and after feeding from a lactating cow andsupplemented with ammonia or yeast extract'

Expt Time of sampling of rumen Maximum growth rates (AOD) of bacteria in rumen fluidExPt Bacteria examined fluid relative to theno. morning feed No addition + Ammonia + Yeast extract

7 B. ruminicola TCl-1 7 h after 0.32 0.50 0.66B. fibrisolvens TC33 0.13 0.24 0.70E. cellulosolvens Alc 0.09 0.09 0.51S. ruminantium L14 0.07 0.07 0.74

8 B. ruminicola TCl-1 Before 0.36 0.53 0.603 h after 0.52 0.52 0.76

B. ruminicola TC18 Before 0.19 0.23 0.243 h after 0.23 0.23 0.22

B. fibrisolvens TC33 Before 0.10 0.14 0.883 h after 0.22 0.28 0.97

E. cellulosolvens Alc Before 0.02 0.02 0.473 h after 0.06 0.06 0.49

S. ruminantium L14 Before 0.03 0.04 0.963 h after 0.07 0.07 0.97

M. elsdenii TD1-14 Before 0.02 0.02 0.183 h after 0.02 0.03 0.17

aSee Table 2, footnotes a, b, and c.

note that, while growth of B. ruminicola was least affectedby the addition of yeast extract, the response to its additionwas meaningful. When differences in response to yeastextract by strains of B. ruminicola and B. fibrisolvens werepaired in the case of tests performed under identical condi-tions (21 pairs), they were also found to be significant (t test,p > 99%).

DISCUSSION

Ideally, a comparison of growth rates of bacteria should beexpressed in a parameter such as specific growth rate.However, this requires estimations of bacterial numbers (4),which was not considered feasible in view of the pleomor-phic nature of, for instance, B. ruminicola or the variabilityin chain formation as in the case of R. flavefaciens. Thus, fora cursory survey of the growth of a number of bacterialspecies in rumen fluid from a variety of animals fed differentdiets, it was deemed adequate to determine the slope of thesteepest portion of growth curves (approximating the loga-rithmic growth phase) from OD measurements as an approx-imate index of growth.Of the bacteria examined, those species that occur in

greatest numbers in the rumen grew most rapidly in rumen

fluid taken at different times of day from cows and sheep feda variety of diets. The higher growth rates of the dominantspecies may reflect a competitive advantage in that they are

related to higher numbers in the rumen. In the experimentsconducted in this study, energy, supplied as glucose, was notlimiting. Ammonia, amino acids, and peptides had some

stimulatory effect, but it appeared that other constituents,present in yeast extract, were primarily responsible for theslow growth of bacteria normally present in lower numbersin the rumen. It appears that rumen fluid from animals fednormal types of diets, such as those used in this study, ischaracteristically deficient in one or more growth factorsthat would allow minority species to grow more rapidly.Thus, the proportions of different species in the rumen may,at least to some extent, be directly dependent on theconcentrations of certain growth factors. These concentra-tions appear to be remarkably constant for a variety of diets.Although the content of growth-stimulating factors in rumenfluid varied somewhat with the time of sampling relative tothe morning feed, it did not reach concentrations thatequaled those present in 0.5% yeast extract. In general,rumen fluid sampled about 3 h after feeding was superior tothat sampled before, or 7 to 8 h after, feeding.

TABLE 5. Effects of ammonia alone or together with an acid hydrolysate of casein, an enzymatic hydrolysate of casein, or yeastextract on maximum growth rates of different species of rumen bacteria in pooled rumen fluid drawn before

the morning feed from three lactating cows (experiment 10)'

Maximum growth rates (AOD) of bacteria in rumen fluidStrains of bacteria

No addition + Ammonia + Ammonia plus acid + Ammonia plus enzymatic + Ammonia plushydrolysate of casein hydrolysate of casein yeast extract

B. ruminicola TC1-1 0.46 0.57 0.59 0.65 0.69B.fibrisolvens TC33 0.06 0.05 0.14 0.23 0.75E. cellulosolvens Alc 0.04 0.04 0.06 0.21 0.51R. flavefaciens CB7 0.05 0.07 0.09 0.08 0.08S. ruminantium L14 0.08 0.19 0.37 0.96Chain-forming coccus TV2-6 0.03 0.04 0.11 0.10 0.22

aSee Table 2, footnotes a, b, and c.

APPL. ENVIRON. MICROBIOL.

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from

COMPARATIVE GROWTH RATES OF VARIOUS RUMEN BACTERIA 105

The question of the supply of growth-limiting factors tothe bacteria in the rumen hinges largely on the rate of releaseof these factors from feed components and lysing, or living,microbes. The results of some preliminary experiments inthis study in which growth rates of B. ruminicola and B.fibrisolvens strains were compared suggest that the releaseof such factors occurs at a rate too low to allow maximalgrowth of B. fibrisolvens. Strains of this species require atleast one of the vitamins, biotin, pyridoxine, and folic acid,while the vitamin requirements of B. ruminicola strainsappear to be minimal (1). The growth factor requirements ofa number of rumen bacteria are fairly well known and haverecently been summarized (8). They include a number ofvitamins, particularly B vitamins, of which yeast extract is awell-known source. Perhaps the supply of certain B vitaminsis consistently too low in the rumen to allow optimal growthof certain species, although the total rumen contents maycontain much higher amounts (3).Work is currently under way to identify factors that could

limit the growth of the minority bacteria.

ACKNOWLEDGMENTS

We thank Erik Lindgren for constant support and encouragement,David Eaker of the Biomedical Centre, Uppsala University, for the

analysis of yeast extract, and Janicka Nilsson of our analyticallaboratory for the ammonia determinations.

REFERENCES

1. Bryant, M. P. 1974. Nutritional features and ecology of predom-inant anaerobic bacteria of the intestinal tract. Am. J. Clin. Nutr.27:1313-1319.

2. Hespell, R. B. 1979. Efficiency of growth by ruminal bacteria.Fed. Proc. 38:2707-2712.

3. Hungate, R. E. 1966. The rumen and its microbes. AcademicPress, Inc., New York.

4. Neidhardt, F. C., J. L. Ingraham, and M. Schaechter. 1990.Physiology of the bacterial cell-a molecular approach, p. 197-225. Sinauer Associates, Inc., Sunderland, Mass.

5. Stewart, C. S., and M. P. Bryant. 1988. The rumen bacteria, p.21-75. In P. N. Hobson (ed.), The rumen microbial ecosystem,Elsevier Applied Science, London.

6. Sigma Chemical Co. Personal communication.7. van Gylswyk, N. 0. 1990. Enumeration and presumptive identi-

fication of some functional groups of bacteria in the rumen ofdairy cows fed grass silage-based diets. FEMS Microbiol. Ecol.73:243-253.

8. Wolin, M. J., and T. L. Miller. 1988. Microbe-microbe interac-tions, p. 343-359. In P. N. Hobson (ed.), The rumen microbialecosystem. Elsevier Applied Science, London.

VOL. 58, 1992

on May 2, 2018 by guest

http://aem.asm

.org/D

ownloaded from