APPLIED
AND
ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 99-105
Vol. 58, No. 1
0099-2240/92/010099-07$02.00/0 Copyright © 1992, American Society for Microbiology
Comparative Growth Rates of Various Rumen Bacteria in Clarified Rumen Fluid from Cows and Sheep Fed Different Diets GYLSWYK,* K. WEJDEMAR, AND K. KULANDER Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Kungsangen Research Station, S-753 23 Uppsala, Sweden N.
0.
VAN
Received 5 August 1991/Accepted 22 October 1991
Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented 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 than strains 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 yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.
lactate) were isolated from medium containing lactate as the sole carbon energy source (7). Culture media. Bacteria were maintained on slopes of agar medium 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, 6.37 g; agar, 15 g; glucose, 5 g; yeast extract (Difco), 5 g; cysteine. HCl H20, 0.25 g; Na2S (hydrated), 0.25 g; indigo carmine, 0.005 g; and rumen fluid (centrifuged at 1,500 x g for 20 min), 400 ml. The gas phase consisted of 02-free CO2. The medium used for growing inocula was the same but without yeast extract. The yeast extract was omitted to minimize carryover of growth factors to medium used for optical density (OD) measurements. Both media were heat sterilized. Yeast extract used in the experiments was always taken from the same batch. The amino acid and peptide contents of the yeast extract were determined by highperformance liquid chromatography on unhydrolyzed and acid-hydrolyzed samples. Rumen fluid was the main constituent of the culture medium used for measuring growth rates. It was sampled by suction tube through the rumen fistula and passed through a double layer of cheesecloth. It was centrifuged for 1 h at about 26,000 x and then at about 50,000 x g for 30 min. In all 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 phase was O2-free CO2. Rumen fluid was sterilized by means of disposable Millipore filters, initially with a pore diameter of 0.22 ,um but later with a pore diameter of 0.45 ,um which also proved to be effective (i.e., no case of contamination detected) and allowed for greater ease in filtering. In experiments 1 to 5, sterile, concentrated solutions of glucose, bicarbonate, and either yeast extract or an equivalent volume of sterile, deionized water were added to rumen fluid, with water constituting between 20 and 30% (depending on the experiment) of the total volume. In subsequent experiments, these and other substances [(NH4)2SO4 and casein hydrolysate] were added dry to portions of rumen fluid and dissolved before filter sterilization. In all experiments, cys-
Over the past 40 years, the principal bacterial species inhabiting the rumen have been identified and studied (3, 5). Numbers of different species can fluctuate considerably when ruminants are fed different diets, but it appears that Bacteroides ruminicola is usually the major species when normal production diets are fed (1). Butyrivibriofibrisolvens is also found among the predominant species (5) and can probably be regarded as the second most abundant species in the rumen. The predominance of B. ruminicola and then of B. fibrisolvens was again confirmed recently in the case of dairy cows fed grass silage-based diets (7). Specific reasons for the predominance of these two species have not been advanced. It was decided to measure the growth rates in rumen fluid of previously isolated strains (7) of B. ruminicola, B. fibrisolvens, and several other species to see whether rates of growth were in any way related to numbers in the rumen. Glucose was added to the rumen fluid as the energy source, cysteine and sulfide together served as the reducing agent, and bicarbonate and CO2 served as the buffer system. In all experiments, parallel runs were made with the same "medium" supplemented with yeast extract to indicate possible deficiencies of growth factors in the rumen fluid.
-
MATERIALS AND METHODS Animals and feed. Rumen-fistulated Swedish Landrace wethers and lactating cows of the Swedish Red and White breed were fed as shown in Table 1. The times of sampling of rumen fluid relative to the morning feed are given in subsequent tables. Bacteria. Strains of B. ruminicola, B. fibrisolvens, and a gram-negative, chain-forming, butyrate-producing coccus were isolated from nonspecific medium used for growing total culturable bacteria; Eubacterium cellulosolvens and Ruminococcus albus were isolated from cellulose-containing medium; Ruminococcus flavefaciens (also cellulolytic) was isolated from cellobiose-containing medium; and Selenomonas ruminantium and Megasphaera elsdenii (both ferment *
Corresponding author. 99
100
APPL. ENVIRON. MICROBIOL.
VAN GYLSWYK ET AL.
TABLE 1. Amounts of concentrate, silage, hay, and straw fed to fistulated sheep and fistulated lactating cows Expt
Animal(s)
no.
1 2 3 4 5 6-8 9 10
Sheep ia Sheep la Cow 999 Cow 966 Sheep 1 Sheep 2 Cow 981 Cow 980 Cows 97, 101, 981
Period of adaption to diet (wk)
1
3 1 1 4 >10 >4 4 4
Approx mean daily intake of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy (ME)
Daily ration (kg) Concentrate
1.0b 1.0b 9.1c 9.6d 1.Ob 0.3b 8.0b
Grass
Grass
Barley
silage
hay
straw
1.0 1.0
4.8
DM (kg)
CP (kg)
NDF (kg)
ME (MJ)
2 2
0.3 0.3
0.6 0.6
21 21 157 160 21 8 141 205 205
2.2
13
1.8
4.4
11.2e
5.1 1.0 0.5 2.0 5.1
2.2
13 2 1 11 16
3.2 0.3 0.1 1.9 1.6
4.3 0.6 0.3 3.0 4.8
11.2e
5.1
2.2
16
1.6
4.8
7.0
Frothy bloat. Standard concentrate C: barley, 32%; molassed beet pulp, 14%; soybean meal, 10o; rapeseed meal, 10%; fat, 2%. 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%. a
b
c
teine hydrochloride and sodium sulfide were added together in a single, concentrated (50X) alkaline solution just before inoculation. The pH of the media was approximately 6.7. The final volume in tubes used for OD measurements was usually 4 ml. Glucose was used as the energy source in all the experiments because all strains fermented glucose (7). Some of the strains may prefer other substrates, but this was not tested. Lysate preparations from mixed rumen microorganisms. Growth factor preparations were obtained from samples of rumen fluid taken on the same day and from the same animal as the rumen fluid used for growth rate studies on pure cultures of rumen bacteria. For experiment 4, rumen fluid was sampled from sheep 1 (Table 1) at 3 h after feeding, strained through two layers of cheesecloth, and centrifuged at 1,500 x g for 20 min at 2°C. The supernatant (SN1) which contained many bacteria was then centrifuged at ca. 50,000 x g for 15 min at 2°C. The pellet was suspended in deionized water and made up to one-sixth of the volume of SN1. The mixture was incubated for 1 h at 38°C. After centrifugation, the supernatant (SN2) was filter sterilized and added to sterile rumen fluid to form one-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 was obtained in the same way as described above. The only difference was that the suspended pellet was incubated at 38°C for 5 h instead of 1 h. In addition, the pellet obtained after centrifugation at low speed, which was very fluid and which contained bacteria, protozoa, and small feed particles, was compacted by centrifugation at high speed. It was then suspended in deionized water and made up to one-sixth of the volume originally centrifuged. This whole fraction was also incubated for 5 h at 38°C, after which it was again centrifuged at high speed. The supernatant was filter sterilized and added to rumen fluid to form one-sixth of the final volume. A different approach was made in experiment 9. Rumen fluid was taken from cow 980 (Table 1) before and 3 h after feeding. Each sample was strained through cheesecloth and divided into four portions. Portion 1 was centrifuged immediately 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, the supernatants were stored at -18°C. Before use, the four different portions were thawed, centrifuged again in the same way to remove small amounts of particulate matter, and filter sterilized, and the filtrate was used as the growth medium. Maintenance of bacteria and inocula. Agar slopes containing yeast extract were inoculated and incubated until good growth had occurred. The slopes were stored at ca. -80°C. When required, the slopes were thawed and the bacteria were transferred to 5-ml agar slopes, free of yeast extract, which were then incubated for about 16 h. Bacterial growth on each slope was diluted with 2 ml of anaerobic diluent and suspended. Portions of 0.2 ml were added to 4 ml of medium contained in glass tubes. All transfers of bacteria and additions of solutions to sterile bottles or tubes were done with sterile syringes, with the needles piercing butyl rubber serum caps. OD measurements and expression of results. ODs were read on a spectrophotometer in culture tubes of 13.5-mm internal diameter at 600 nm. The spectrophotometer was fitted with a clamp to hold the tubes in a fixed position. The tubes were read immediately after inoculation (to). They were incubated in a water bath at 38°C and read at hourly intervals immediately after thorough shaking. All tests were done in duplicate, and the data given in Tables 2 to 5 were calculated from the means. The data were calculated from the greatest change in OD between two consecutive readings (1-h interval between readings) within 6 h after inoculation. RESULTS Twenty-two strains of B. ruminicola-like bacteria were chosen randomly from those isolated earlier (7) and were examined for possible use in the current tests. Fifteen of the strains grew rapidly on slopes of agar medium (containing yeast extract) and showed good growth within 16 h. Nine of these strains morphologically resembled B. ruminicola subsp. brevis in that the cells were mostly coccoid (5), while the remaining six strains morphologically resembled B. ruminicola subsp. ruminicola, which consisted predomi-
VOL. 58, 1992
COMPARATIVE GROWTH RATES OF VARIOUS RUMEN BACTERIA
nantly of short to long rods (5). Of the 22 strains, 7 grew much slower and required up to 48 h to produce visible growth. Of these, three strains appeared B. ruminicola subsp. brevis-like and the remainder appeared B. ruminicola subsp. ruminicola-like. The three strains chosen for use in the experiments were fast growers. Strains TCl-1 and TN1-5 resembled B. ruminicola subsp. brevis, and strain TC18 resembled B. ruminicola subsp. ruminicola. In subsequent growth tests, it was found that the latter strain grew considerably slower than either of the other two strains. No selection was applied in choosing strains of other species. Examples of curves depicting the growth of bacterial strains of six species in rumen fluid supplemented with glucose, bicarbonate, and reducing agent, both in the absence and presence of yeast extract, are shown in Fig. 1. Table 2 gives the maximum growth rates for different strains of B. ruminicola and B. fibrisolvens as well as strains of four other species in rumen fluid taken before and at different times after the morning feed from a sheep and cows fed a variety of diets (Table 1). Fast-growing strains of B. ruminicola (strains TN1-5 and TCl-1) grew more rapidly than any of the other strains. The next most rapidly growing strains were the fast-growing strains of B. fibrisolvens (strains TC1-14, TC33, and TC1-4). Strains of the other species all had low maximum growth rates. The slowgrowing strains of B. ruminicola and B. fibrisolvens (TC18 and TV1-4, respectively) received relatively little stimulation from the addition of yeast extract, which suggested that their inherent growth rates were indeed low. Growth of all strains of B. ruminicola were little affected by the addition of yeast extract. Despite the different sources of rumen fluid, growth rates of the different species, relative to each other, showed the same tendencies. Rumen fluid taken 3 or 3.5 h after the morning feed was somewhat superior to that taken before feeding, although this was not apparent in all cases. Rumen fluid sampled 7 or 8 h after feeding was usually inferior to that obtained before feeding, although again there were exceptions. In the foregoing experiments, concentrations of essential, or growth-stimulating, factors in the rumen fluid would have diminished rapidly with growth of the bacteria. An attempt was therefore made to supply factors of the type released in the rumen by adding filter-sterilized fluid from preparations of lysed rumen microbes to clarified, filter-sterilized, rumen fluid. Incubation of mixed rumen microbes without added substrate would presumably lead to the rapid depletion of easily fermentable energy sources and, as long as growth occurred, also to the depletion of other factors that are essential or stimulatory for the growth of a variety of microbes. However, starvation of rumen bacteria results in rapid lysis (2). Thus, in incubated preparations free of added energy sources, lysis may lead to the release of growth factors at a rate equal to or greater than would normally occur in the rumen. Some experiments were done to obtain an indication of the rate of release of such factors by incubating various preparations of rumen solids for different periods and adding the filter-sterilized fluid from such lysates to clarified sterilized rumen fluid in proportions that could simulate the supply of growth factors from lysing microbes in the rumen (see Materials and Methods). Results for fast-growing strains of B. ruminicola and B. fibrisolvens are shown in Table 3. Lysate from a fraction consisting largely of bacteria and preincubated for 1 h had some stimulatory effect in rumen fluid sampled before and 3 h after the morning feed (experiment 4). In experiment 5, a similar preparation, incubated
B.
0
ruminicola TCl-l
4 2 Time (h)
6
101
B. fibrisolvens TC33
0
S. ruminant L14
4 2 Time (h)
6
E. cellulosolvens Alc
AND
ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 99-105
Vol. 58, No. 1
0099-2240/92/010099-07$02.00/0 Copyright © 1992, American Society for Microbiology
Comparative Growth Rates of Various Rumen Bacteria in Clarified Rumen Fluid from Cows and Sheep Fed Different Diets GYLSWYK,* K. WEJDEMAR, AND K. KULANDER Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Kungsangen Research Station, S-753 23 Uppsala, Sweden N.
0.
VAN
Received 5 August 1991/Accepted 22 October 1991
Pure cultures of strains of different species of rumen bacteria were grown in filter-sterilized rumen fluid supplemented 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 than strains 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 yeast extract. Factors released from lysates of mixed rumen microbes stimulated growth, but their rate of release was slow. It was concluded that, besides energy and nitrogen sources, growth factors of an as-yet-undetermined nature probably play an important role in determining the predominance of different bacterial species in the rumen.
lactate) were isolated from medium containing lactate as the sole carbon energy source (7). Culture media. Bacteria were maintained on slopes of agar medium 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, 6.37 g; agar, 15 g; glucose, 5 g; yeast extract (Difco), 5 g; cysteine. HCl H20, 0.25 g; Na2S (hydrated), 0.25 g; indigo carmine, 0.005 g; and rumen fluid (centrifuged at 1,500 x g for 20 min), 400 ml. The gas phase consisted of 02-free CO2. The medium used for growing inocula was the same but without yeast extract. The yeast extract was omitted to minimize carryover of growth factors to medium used for optical density (OD) measurements. Both media were heat sterilized. Yeast extract used in the experiments was always taken from the same batch. The amino acid and peptide contents of the yeast extract were determined by highperformance liquid chromatography on unhydrolyzed and acid-hydrolyzed samples. Rumen fluid was the main constituent of the culture medium used for measuring growth rates. It was sampled by suction tube through the rumen fistula and passed through a double layer of cheesecloth. It was centrifuged for 1 h at about 26,000 x and then at about 50,000 x g for 30 min. In all 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 phase was O2-free CO2. Rumen fluid was sterilized by means of disposable Millipore filters, initially with a pore diameter of 0.22 ,um but later with a pore diameter of 0.45 ,um which also proved to be effective (i.e., no case of contamination detected) and allowed for greater ease in filtering. In experiments 1 to 5, sterile, concentrated solutions of glucose, bicarbonate, and either yeast extract or an equivalent volume of sterile, deionized water were added to rumen fluid, with water constituting between 20 and 30% (depending on the experiment) of the total volume. In subsequent experiments, these and other substances [(NH4)2SO4 and casein hydrolysate] were added dry to portions of rumen fluid and dissolved before filter sterilization. In all experiments, cys-
Over the past 40 years, the principal bacterial species inhabiting the rumen have been identified and studied (3, 5). Numbers of different species can fluctuate considerably when ruminants are fed different diets, but it appears that Bacteroides ruminicola is usually the major species when normal production diets are fed (1). Butyrivibriofibrisolvens is also found among the predominant species (5) and can probably be regarded as the second most abundant species in the rumen. The predominance of B. ruminicola and then of B. fibrisolvens was again confirmed recently in the case of dairy cows fed grass silage-based diets (7). Specific reasons for the predominance of these two species have not been advanced. It was decided to measure the growth rates in rumen fluid of previously isolated strains (7) of B. ruminicola, B. fibrisolvens, and several other species to see whether rates of growth were in any way related to numbers in the rumen. Glucose was added to the rumen fluid as the energy source, cysteine and sulfide together served as the reducing agent, and bicarbonate and CO2 served as the buffer system. In all experiments, parallel runs were made with the same "medium" supplemented with yeast extract to indicate possible deficiencies of growth factors in the rumen fluid.
-
MATERIALS AND METHODS Animals and feed. Rumen-fistulated Swedish Landrace wethers and lactating cows of the Swedish Red and White breed were fed as shown in Table 1. The times of sampling of rumen fluid relative to the morning feed are given in subsequent tables. Bacteria. Strains of B. ruminicola, B. fibrisolvens, and a gram-negative, chain-forming, butyrate-producing coccus were isolated from nonspecific medium used for growing total culturable bacteria; Eubacterium cellulosolvens and Ruminococcus albus were isolated from cellulose-containing medium; Ruminococcus flavefaciens (also cellulolytic) was isolated from cellobiose-containing medium; and Selenomonas ruminantium and Megasphaera elsdenii (both ferment *
Corresponding author. 99
100
APPL. ENVIRON. MICROBIOL.
VAN GYLSWYK ET AL.
TABLE 1. Amounts of concentrate, silage, hay, and straw fed to fistulated sheep and fistulated lactating cows Expt
Animal(s)
no.
1 2 3 4 5 6-8 9 10
Sheep ia Sheep la Cow 999 Cow 966 Sheep 1 Sheep 2 Cow 981 Cow 980 Cows 97, 101, 981
Period of adaption to diet (wk)
1
3 1 1 4 >10 >4 4 4
Approx mean daily intake of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and metabolizable energy (ME)
Daily ration (kg) Concentrate
1.0b 1.0b 9.1c 9.6d 1.Ob 0.3b 8.0b
Grass
Grass
Barley
silage
hay
straw
1.0 1.0
4.8
DM (kg)
CP (kg)
NDF (kg)
ME (MJ)
2 2
0.3 0.3
0.6 0.6
21 21 157 160 21 8 141 205 205
2.2
13
1.8
4.4
11.2e
5.1 1.0 0.5 2.0 5.1
2.2
13 2 1 11 16
3.2 0.3 0.1 1.9 1.6
4.3 0.6 0.3 3.0 4.8
11.2e
5.1
2.2
16
1.6
4.8
7.0
Frothy bloat. Standard concentrate C: barley, 32%; molassed beet pulp, 14%; soybean meal, 10o; rapeseed meal, 10%; fat, 2%. 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%. a
b
c
teine hydrochloride and sodium sulfide were added together in a single, concentrated (50X) alkaline solution just before inoculation. The pH of the media was approximately 6.7. The final volume in tubes used for OD measurements was usually 4 ml. Glucose was used as the energy source in all the experiments because all strains fermented glucose (7). Some of the strains may prefer other substrates, but this was not tested. Lysate preparations from mixed rumen microorganisms. Growth factor preparations were obtained from samples of rumen fluid taken on the same day and from the same animal as the rumen fluid used for growth rate studies on pure cultures of rumen bacteria. For experiment 4, rumen fluid was sampled from sheep 1 (Table 1) at 3 h after feeding, strained through two layers of cheesecloth, and centrifuged at 1,500 x g for 20 min at 2°C. The supernatant (SN1) which contained many bacteria was then centrifuged at ca. 50,000 x g for 15 min at 2°C. The pellet was suspended in deionized water and made up to one-sixth of the volume of SN1. The mixture was incubated for 1 h at 38°C. After centrifugation, the supernatant (SN2) was filter sterilized and added to sterile rumen fluid to form one-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 was obtained in the same way as described above. The only difference was that the suspended pellet was incubated at 38°C for 5 h instead of 1 h. In addition, the pellet obtained after centrifugation at low speed, which was very fluid and which contained bacteria, protozoa, and small feed particles, was compacted by centrifugation at high speed. It was then suspended in deionized water and made up to one-sixth of the volume originally centrifuged. This whole fraction was also incubated for 5 h at 38°C, after which it was again centrifuged at high speed. The supernatant was filter sterilized and added to rumen fluid to form one-sixth of the final volume. A different approach was made in experiment 9. Rumen fluid was taken from cow 980 (Table 1) before and 3 h after feeding. Each sample was strained through cheesecloth and divided into four portions. Portion 1 was centrifuged immediately 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, the supernatants were stored at -18°C. Before use, the four different portions were thawed, centrifuged again in the same way to remove small amounts of particulate matter, and filter sterilized, and the filtrate was used as the growth medium. Maintenance of bacteria and inocula. Agar slopes containing yeast extract were inoculated and incubated until good growth had occurred. The slopes were stored at ca. -80°C. When required, the slopes were thawed and the bacteria were transferred to 5-ml agar slopes, free of yeast extract, which were then incubated for about 16 h. Bacterial growth on each slope was diluted with 2 ml of anaerobic diluent and suspended. Portions of 0.2 ml were added to 4 ml of medium contained in glass tubes. All transfers of bacteria and additions of solutions to sterile bottles or tubes were done with sterile syringes, with the needles piercing butyl rubber serum caps. OD measurements and expression of results. ODs were read on a spectrophotometer in culture tubes of 13.5-mm internal diameter at 600 nm. The spectrophotometer was fitted with a clamp to hold the tubes in a fixed position. The tubes were read immediately after inoculation (to). They were incubated in a water bath at 38°C and read at hourly intervals immediately after thorough shaking. All tests were done in duplicate, and the data given in Tables 2 to 5 were calculated from the means. The data were calculated from the greatest change in OD between two consecutive readings (1-h interval between readings) within 6 h after inoculation. RESULTS Twenty-two strains of B. ruminicola-like bacteria were chosen randomly from those isolated earlier (7) and were examined for possible use in the current tests. Fifteen of the strains grew rapidly on slopes of agar medium (containing yeast extract) and showed good growth within 16 h. Nine of these strains morphologically resembled B. ruminicola subsp. brevis in that the cells were mostly coccoid (5), while the remaining six strains morphologically resembled B. ruminicola subsp. ruminicola, which consisted predomi-
VOL. 58, 1992
COMPARATIVE GROWTH RATES OF VARIOUS RUMEN BACTERIA
nantly of short to long rods (5). Of the 22 strains, 7 grew much slower and required up to 48 h to produce visible growth. Of these, three strains appeared B. ruminicola subsp. brevis-like and the remainder appeared B. ruminicola subsp. ruminicola-like. The three strains chosen for use in the experiments were fast growers. Strains TCl-1 and TN1-5 resembled B. ruminicola subsp. brevis, and strain TC18 resembled B. ruminicola subsp. ruminicola. In subsequent growth tests, it was found that the latter strain grew considerably slower than either of the other two strains. No selection was applied in choosing strains of other species. Examples of curves depicting the growth of bacterial strains of six species in rumen fluid supplemented with glucose, bicarbonate, and reducing agent, both in the absence and presence of yeast extract, are shown in Fig. 1. Table 2 gives the maximum growth rates for different strains of B. ruminicola and B. fibrisolvens as well as strains of four other species in rumen fluid taken before and at different times after the morning feed from a sheep and cows fed a variety of diets (Table 1). Fast-growing strains of B. ruminicola (strains TN1-5 and TCl-1) grew more rapidly than any of the other strains. The next most rapidly growing strains were the fast-growing strains of B. fibrisolvens (strains TC1-14, TC33, and TC1-4). Strains of the other species all had low maximum growth rates. The slowgrowing strains of B. ruminicola and B. fibrisolvens (TC18 and TV1-4, respectively) received relatively little stimulation from the addition of yeast extract, which suggested that their inherent growth rates were indeed low. Growth of all strains of B. ruminicola were little affected by the addition of yeast extract. Despite the different sources of rumen fluid, growth rates of the different species, relative to each other, showed the same tendencies. Rumen fluid taken 3 or 3.5 h after the morning feed was somewhat superior to that taken before feeding, although this was not apparent in all cases. Rumen fluid sampled 7 or 8 h after feeding was usually inferior to that obtained before feeding, although again there were exceptions. In the foregoing experiments, concentrations of essential, or growth-stimulating, factors in the rumen fluid would have diminished rapidly with growth of the bacteria. An attempt was therefore made to supply factors of the type released in the rumen by adding filter-sterilized fluid from preparations of lysed rumen microbes to clarified, filter-sterilized, rumen fluid. Incubation of mixed rumen microbes without added substrate would presumably lead to the rapid depletion of easily fermentable energy sources and, as long as growth occurred, also to the depletion of other factors that are essential or stimulatory for the growth of a variety of microbes. However, starvation of rumen bacteria results in rapid lysis (2). Thus, in incubated preparations free of added energy sources, lysis may lead to the release of growth factors at a rate equal to or greater than would normally occur in the rumen. Some experiments were done to obtain an indication of the rate of release of such factors by incubating various preparations of rumen solids for different periods and adding the filter-sterilized fluid from such lysates to clarified sterilized rumen fluid in proportions that could simulate the supply of growth factors from lysing microbes in the rumen (see Materials and Methods). Results for fast-growing strains of B. ruminicola and B. fibrisolvens are shown in Table 3. Lysate from a fraction consisting largely of bacteria and preincubated for 1 h had some stimulatory effect in rumen fluid sampled before and 3 h after the morning feed (experiment 4). In experiment 5, a similar preparation, incubated
B.
0
ruminicola TCl-l
4 2 Time (h)
6
101
B. fibrisolvens TC33
0
S. ruminant L14
4 2 Time (h)
6
E. cellulosolvens Alc
