Vol. 60, No. 10
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1994, p. 3697-3703
0099-2240/94/$04.00+0 Copyright C 1994, American Society for Microbiology
The Use of 16S rRNA-Targeted Oligonucleotide Probes To Study Competition between Ruminal Fibrolytic Bacteria: Pure-Culture Studies with Cellulose and Alkaline Peroxide-Treated Wheat Straw AGNES A. ODENYO,'t RODERICK I. MACKIE,' DAVID A. STAHL,2t AND BRYAN A. WHITE"* Department ofAnimal Sciences' and Departments of Veterinary Pathobiology, Microbiology, and Civil Engineering,2 University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received 31 January 1994/Accepted 22 July 1994
Specific oligonucleotide probes targeted to sites on the 16S rRNA of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, and Fibrobacter succinogenes S85 and a domain Bacteria probe were used to study bacterial interactions during the fermentation of cellulose and alkaline hydrogen peroxide-treated wheat straw in monocultures, dicultures, and tricultures. Results showed that R. albus 8 inhibited the growth of R. flavefaciens FD-1 when grown as a diculture with cellulose or alkaline hydrogen peroxide-treated wheat straw as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85 grown on cellulose or alkaline hydrogen peroxide-treated wheat straw, competition was not detected. R. flavefaciens FD-1 outcompeted F. succinogenes S85 when cellulose was used as the carbon source. In tricultures with cellulose as the carbon source, R. flavefaciens FD-1 was inhibited, R. albus 8 appeared to dominate during the early phase of degradation (12 to 48 h), while F. succinogenes S85 became predominant during the later phase of degradation (60 to 70 h). When alkaline hydrogen peroxide-treated wheat straw was used as a growth substrate, F. succinogenes S85 showed better growth than either R. albus 8 or R.,flavefaciens FD-1. However, R.flavefaciens FD-1 was present in small numbers throughout the incubation period, unlike the growth patterns when cellulose was the carbon source. Ruminant animals are able to use plant fiber containing cellulose and xylan as feed because of their symbiotic association with the microorganisms in their rumens. The members of the complex microbiota of the rumen interact and compete for survival in this large community. The major fibrolytic bacteria in the rumen are Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes (14). Competitive and cooperative interactions between fibrolytic microorganisms may affect the degradation of fibrous feed and hence the energy provided to the animal (5). Knowledge concerning these interactions is essential to our further understanding of fiber degradation in the rumen. Classical methods for enumerating microorganisms, based primarily on pure-culture isolation, have numerous limitations. Thus, an accurate method for the identification and quantification of fibrolytic microorganisms is needed for the study of microbial interactions within the rumen or other complex community. Because of the limitations of culture-based techniques, most studies relating to interactions between fibrolytic microorganisms have concentrated on the measurement of degradation rates and end product formation, rather than actual measurement of microbial population dynamics during the incubation (5). Nevertheless, these studies clearly show that microbial interactions can result in positive effects on fermentation rate and extent (5). Recently, the different rRNAs have been used
to identify and quantify natural microbial populations, and, as such, they represent a powerful strategy which can be applied to the study of bacterial population dynamics (1, 7, 11-13). In
the accompanying paper, we described the design and use of oligonucleotide probes for the genus Rumincoccus and their use in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes when grown on the soluble substrate cellobiose (10). In the present work, we have extended the use of these 16S rRNA-targeted oligonucleotide hybridization probes for F. succinogenes, R. albus, and R. flavefaciens to an in vitro study of the microbial interactions between the predominant fibrolytic ruminal bacteria when the insoluble substrates cellulose and alkaline hydrogen peroxide-treated wheat straw were used as carbon sources. (A preliminary report of this work has been presented previously [lOa].) MATERUILS AND METHODS Bacteria. F. succinogenes S85, R albus 8, and R. flavefaciens FD-1 were from our culture collection in the Department of Animal Sciences. Cultures were maintained in complex medium with 0.12% (wt/vol) acid-swollen cellulose for several weeks before the growth experiments were performed (9). RNA concentrations of the inocula (Table 1) were used to correct for the amounts of each bacterium in the culture and normalize inocula. Oligonucleotide synthesis and labeling. Oligonucleotide probes were labeled and purified as described previously (10). The following four probes were used, with the numbers corresponding to the complementary positions in Escherichia coli 16S rRNA: RAL196 (5'-GTC ATG CGG CITT CGT TAT-3'), positions 196 to 213; RFL196 (5'-AGG ATG CCC 1TC AAT TAT-3'), positions 196 to 213; F. succinogenes S85
* Corresponding author. Mailing address: Department of Animal Sciences, University of Illinois, 436 Animal Sciences Laboratory, 1207 West Gregory Drive, Urbana, IL 61801. Phone: (217) 333-2091. Fax: (217) 333-8804. Electronic mail address:
[email protected]. t Present address: Department of Biology, Egerton University, Njoro, Kenya. t Present address: Department of Civil Engineering, Technological Institute, Northwestern University, Evanston, IL 60208.
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FIG. 1. Bacterial growth (0), measured by protein concentration, and substrate disappearance (I) of cellulose-grown cultures. (A) Diculture of R. albus 8 and R. flavefaciens FD-1; (B) diculture of R. albus 8 and F. succinogenes S85; (C) diculture of R. flavefaciens FD-1 and F. succinogenes S85; (D) triculture of R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85.
subsp. probe SUB1 (13), and the domain Bacteria probe EUB 338 (5'-GCT GCC TCC CGT AGG AGT-3'), positions 338 to 355. A standard curve was generated by using purified 16S rRNA from E. coli and dilutions of rRNA of the target bacteria as described previously (10). Extraction of total RNA. rRNA was extracted from 10 ml of cellulose-grown cultures by using the zirconium bead method described previously (10). A single-step method of RNA isolation with acid guanidinium thiocyanate-phenol-chloroform (3, 4) was used for extraction of RNA from cultures grown with alkaline hydrogen peroxide-treated wheat straw as the substrate. Immediately after centrifugation, the pellet was TABLE 1. Bacterial cell numbers and total RNA from cellulose-grown inocula Bacterium'
1010 No. of cells
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placed in a -20°C freezer for 1 to 2 h. The pellet was then removed, and 4 M guanidinium thiocynate, 25 mM sodium acetate, 5% sarcosyl, and 0.1 M 2-mercaptoethanol (pH 7.0) were added prior to thawing. The samples were transferred to a 15-ml Corex tube (baked at 250°C overnight); 0.1 ml of 2 M sodium acetate, 1 ml of phenol, and 0.2 ml of chloroformisoamyl alcohol (24:1) were added sequentially; and the samples were subjected to thorough mixing. They were then incubated on ice for 15 min and centrifuged at 13,800 x g for 20 min at 4°C. The aqueous phase was removed, 1 volume of isopropanol was added, and the samples were incubated at -20°C for 1 h to precipitate the RNA. The RNA pellet was washed with 75% ethanol and then suspended in 50 pul of RNase-free double-distilled H20. Oligonucleotide probe hybridization. Different dilutions of RNA isolated from reference bacteria and cultures were blotted on a nylon membrane and used in hybridizations as described previously (10). Quantification of rRNA blots. The amount of bound probe was converted to micrograms of 16S rRNA to make a standard curve as described previously (10). It should be noted here that when used to quantitate microorganisms, the specific/total rRNA ratio is only an approximate estimate of relative cell
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FIG. 2. Slot blot quantitation of relative 16S rRNA levels of cellulose-grown cultures by using R. albus probe RAL196, R. flavefaciens probe RFL196, and F. succinogenes probe SUB1. (A) R. albus 8 (aI) and R flavefaciens FD-1 (a); (B) R. albus 8 (E) and F. succinogenes S85 (U); (C) R. flavefaciens FD-1 (l) and F. succinogenes S85 (U); (D) R. albus 8 (l), R. flavefaciens FD-1 (U), and F. succinogenes S85 (A).
numbers, since cellular rRNA content varies with growth rate. All the experiments were repeated two or three times, and the mean amount of 16S rRNA (in micrograms) was used. These data were reproducible under the conditions used. Cellulose-grown and alkaline hydrogen peroxide-treated wheat straw-grown cultures. In the cellulose studies, complex medium (9) with 0.12% (wt/vol) acid-swollen cellulose was used. The bacteria were grown in a 10-ml volume. Cultures were grown without agitation and were mixed by inversion twice daily. For each time course study, replicate tubes were inoculated with 0.5 ml of a 96-h (optical density at 600 nm, 1.0) cellulose-grown culture which had been subcultured twice. One tube was removed at different time points for total RNA extractions and protein determinations. The entire 10-ml culture, supernatant plus the remaining cellulose, was used in the extractions. For protein determinations, the Coomassie blue dye-binding method, with bovine serum albumin as the standard, was used (2). Alkaline hydrogen peroxide-treated wheat straw previously ground through a 0.5-mm screen was weighed into 25-ml Balch tubes, and anaerobically prepared complex medium was added to a total volume of 10 ml (9). Treated wheat straw was used at 0.4% (wt/vol). The tubes were inoculated with 0.5 ml of
cellulose-grown culture (optical density at 600 nm, 1.0) which had been subcultured twice. Cultures were grown without agitation and were mixed by inversion twice daily. The tubes were incubated at 39°C for 8 days. One tube from each treatment category was removed each day for extraction of total RNA (see above) and for protein determinations (see above). Both the supernatant and the remaining solid substrate were included in the extraction. Substrate disappearance. The phenol-sulfuric acid procedure (6) was used to determine substrate disappearance from cellulose-grown cultures. Samples (0.5 ml) were centrifuged, the pellet was suspended in 0.5 ml of 10 mM NaPO4 (pH 6.8), and then 1 j.l of mutanolysin (5,000 U ml-'; Sigma Chemical Co., St. Louis, Mo.) was added; this was followed by incubation at 55°C for 5 min. The sample was centrifuged, the pellet was suspended in 1 ml of 2% cetyltrimethylammonium bromide in 0.5 M sulfuric acid, and the mixture was boiled for 30 minutes. The sample was again pelleted and suspended in 10 ml of double-distilled H20. The sample (0.15 ml) was then mixed with an equal volume of 4% (wt/vol) phenol followed by 0.75 ml of stock sulfuric acid (6), and the optical density at 485 nm was determined. Glucose was used as the standard for quantitation of carbohydrate concentration.
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FIG. 3. Bacterial growth (0), measured by protein concentration, and substrate disappearance (0) of alkaline hydrogen peroxide-treated wheat straw-grown cultures. (A) Diculture of R. albus 8 and R. flavefaciens FD-1; (B) diculture of R. albus 8 and F. succinogenes S85; (C) diculture of R. flavefaciens FD-1 and F. succinogenes S85; (D) triculture of R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85.
Substrate disappearance from the alkaline hydrogen peroxide-treated wheat straw was measured by determining dry matter disappearance by the method of Odenyo et al. (9).
RESULTS
Quantitation of fibrolytic bacteria in cellulose-grown cultures. The cellulose in monocultures of R. flavefaciens FD-1
completely degraded in 4 days (data not shown), whereas by the end of 7 days, cultures incubated with R. albus 8 or F. succinogenes S85 still had some cellulose remaining (9.0 and 16.7%, respectively). When a diculture of R. albus 8 and R. flavefaciens FD-1 was grown on cellulose, visual examination of the cellulose discs revealed no yellow pigmentation (visual indication of the presence of R. flavefaciens) while the discs in the R. fiavefaciens FD-1 monoculture had an intense yellow color. The diculture of R. albus 8 and R. flavefaciens FD-1 degraded 87.5% of the cellulose after 7 days (Fig. 1A). The diculture of R. albus 8 and F. succinogenes S85 degraded all of the cellulose (Fig. 1B). The cellulose discs in the diculture of R. flavefaciens FD-1 and F. succinogenes S85 had an intense yellow pigmentation throughout the incubation period, and 95.8% of the cellulose was degraded (Fig. 1C). The triculture was
of R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 degraded all the cellulose (Fig. 1D). Quantitation of fibrolytic bacteria in these defined mixed cellulose-grown cultures was determined by using hybridization of oligonucleotide probes to total extracted RNA. Hybridization of specific oligonucleotide probes to rRNA extracted from diculture of R. albus 8 and R. flavefaciens FD-1 grown on cellulose showed that R. albus 8 outcompeted R. flavefaciens FD-1 (Fig. 2A). The population was composed mostly of R. albus 8 cells, and, as with the experiments with cellobiose (10), R. flavefaciens FD-1 was barely detectable in the culture after only 12 h of incubation and was undetectable after 48 h. To confirm the total absence of R. flavefaciens FD-1 in the culture, the amount of RNA per blot was doubled. This resulted in a very weak signal for R. flavefaciens FD-1, which eventually disappeared (after 48 h, [data not shown]). In dicultures containing R. albus 8 and F. succinogenes S85, the relative proportions of the two bacteria were similar, but, as with the cellobiose cultures (12), there seemed to be a slightly higher proportion of F. succinogenes S85 than R. albus 8 once the substrate was depleted (Fig. 2B). However, in dicultures containing R. flavefaciens FD-1 and F. succinogenes S85, the results (Fig. 2C) showed that R. flavefaciens FD-1 predomi-
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FIG. 4. Slot blot quantitation of relative 16S rRNA levels of alkaline hydrogen peroxide-treated wheat straw-grown cultures with R. albus probe RAL196, R. flavefaciens probe RFL196, and F. succinogenes probe SUB1. (A) R. albus 8 (O) and R. flavefaciens FD-1 (-); (B) R. albus 8 (O) and F. succinogenes S85 (-); (C) R. flavefaciens FD-1 (O) and F. succinogenes S85 (-); (D) R. albus 8 (O), R. flavefaciens FD-1 (-), and F. succinogenes S85 (A).
nated. R flavefaciens FD-1 maintained high population levels, whereas the levels of F. succinogenes S85 were low until substrate was depleted. When these three bacteria were grown in a triculture, R. flavefaciens FD-1 was again barely detectable in the culture after only 12 h of incubation and was undetectable after 48 h (Fig. 2D). Quantitation of fibrolytic bacteria in alkaline hydrogen peroxide-treated wheat straw-grown cultures. The growth of bacteria cultured alone and in di- and tricultures on the alkaline hydrogen-treated wheat straw was generally slow compared with growth on cellulose (Fig. 3). Spectrophotometric detection of total RNA yielded unsatisfactory results. However, even though total RNA concentrations were very low, 16S rRNA levels could be detected by hybridization with the EUB338 kingdom Eubacteria probe or species target oligonucleotide probes. As in the cellulose studies, quantitation of fibrolytic bacteria in defined dicultures grown on alkaline hydrogen-treated wheat straw medium was determined by using hybridization of oligonucleotide probes to total extracted RNA. Similar to the experiments with cellobiose (10) or cellulose as the carbon source, hybridization of specific oligonucleotide probes to rRNA extracted from dicultures of R albus 8 and R flavefaciens FD-1 grown on alkaline hydrogen-treated wheat straw showed that R. albus 8 outcompeted
R flavefaciens FD-1 (Fig. 4A). However, in contrast to the cellobiose (10) and cellulose studies, the growth dynamics differed in that R. flavefaciens FD-1 showed evidence of growth in the first 8 h after inoculation, followed by a slow decline in the population levels until 48 h, when it was again undetectable by the methods used. In dicultures containing R. albus 8 and F. succinogenes S85, the relative proportions of the two bacteria were again similar (Fig. 4B). However, in marked contrast to the cellulose-grown dicultures containing R. flavefaciens FD-1 and F. succinogenes S85, in alkaline hydrogen peroxide-treated wheat straw dicultures containing R. flavefaciens FD-1 and F. succinogenes S85 the relative proportions of the two bacteria were remarkably similar until the substrate was depleted (Fig. 3C and 4C). When these three bacteria were grown in a triculture, R flavefaciens FD-1 again grew poorly but nonetheless was detectable after 70 h (Fig. 4D). However, in contrast to the dicultures containing R. albus 8 and F. succinogenes S85, the competition in the triculture between R. albus 8 and F. succinogenes S85 again demonstrated different population dynamics (Fig. 4D). These experiments showed that R. albus 8 population levels were similar to those of R. flavefaciens FD-1 and differed little, while F. succinogenes S85 predominated after 24 h of incubation.
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DISCUSSION
Many studies (5) have demonstrated the degradative abilities of cellulolytic bacteria and sygnergism when these bacteria are grown together on different substrates. However, the proportions of these bacteria in relation to each other while degrading the substrates synergistically or nonsynergistically have never been determined. The major reason for this is that there has been no accurate method for doing so. The present work has clearly demonstrated that it is possible to use the 16S
rRNA-targeted oligonucleotide hybridization probes to enumerate and study competition between microorganisms in vitro. It is possible to apply these techniques to the study of the competitive abilities of these microorganisms from natural environments as complex as the rumen. When compared with conventional methods, this approach has several advantages. For example, it eliminates the difficulties involved in isolation and enumeration of cellulolytic bacteria which adhere to the substrate, and, because it works well with frozen samples, many samples can be collected and then analyzed at the same time. This work therefore presents a powerful method for studying competition between ruminal fibrolytic bacteria. This is an area in which the classical methods of isolation, identification, and quantification have proved laborious and unreliable. It should be noted that the culture conditions used in this study were chosen on the basis of culture conditions commonly used in our laboratory and were not intended to yield optimal plant cell hydrolysis by monocultures of any of the three bacteria used. Rather, these conditions support growth of all three organisms and allowed us to evaluate the usefulness of 16S rRNA-targeted oligonucleotide probes in studying competition between ruminal fibrolytic bacteria. The cellulose competition studies showed similar population dynamics to those reported for cellobiose cocultures (10). R. flavefaciens FD-1 outcompeted F. succinogenes S85 on both cellobiose and cellulose, and the relative proportions of R. albus 8 and F. succinogenes S85 in cellulose cultures were again similar. Further, in these cellulose-grown cultures, R. flavefaciens FD-1 was inhibited by the bacteriocin-like substance(s) produced by R. albus 8 (10). The population dynamics in alkaline hydrogen peroxide-treated wheat straw cultures differed only in the diculture of R. flavefaciens FD-1 and F. succinogenes S85 and in the culture containing all three bacteria. In the alkaline hydrogen peroxide-treated wheat straw diculture containing R. flavefaciens FD-1 and F. succinogenes S85, the relative proportions of the two bacteria were remarkably similar, whereas in cellobiose-grown (10) and cellulose-grown cultures, R. flavefaciens FD-1 clearly outcompeted F. succinogenes S85. The reason for this difference in competition may be that F. succinogenes can degrade the more-complex substrate more efficiently or completely (8), or it may involve other as yet unidentified factors. More interestingly, when the three bacteria were grown in a triculture, R. flavefaciens FD-1 grew poorly but was still detectable after 70 h. These population dynamics also suggested that R. albus 8 did not grow well in this triculture. However, it is clear that when R. albus 8 and R. flavefaciens FD-1 are grown together on cellobiose or cellulose, the growth of R. flavefaciens FD-1 is inhibited by a substance(s) produced by growing cells of R. albus 8. Therefore, further experiments are needed to examine factors affecting the production of this antagonistic compound. Like other methods, the use of 16S rRNA-targeted oligonucleotide probes for studying competition or any other form of interaction among bacteria has several limitations. The method
requires a large database of 16S rRNA sequences for alignment and comparison during the probe design. Sequencing and
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building a large database requires a significant input of time and expense. Further, the method may detect both viable and nonviable cells and therefore may not give accurate numbers when used in an environment with high populations of nonviable cells. This could be overcome by the combined use of viable and total direct microscopic counts or the use of a vital stain. However, there could still be problems with the use of solid substrate and particulate matter. Because of such limitations, this method may be suitable only for apparent estimates of the population numbers, and further development is needed before application to the complex ecosystem of the rumen. Despite the limitations, this work has demonstrated that the use of oligonucleotide-specific hybridization probes to quantitate 16S rRNA of a particular bacteria in defined mixed cultures grown on insoluble substrates is very attractive method for studies of competition between ruminal fibrolytic bacteria. ACKNOWLEDGMENTS This study was supported by U.S. Department of Agriculture grant 35-038 and by the Agricultural Experimental Station of the University of Illinois to B.A.W. and by grants from the Environmental Protection Agency and U.S. Department of Agriculture to D.A.S. A.A.O. is the recipient of an USAID IDAT Project for Graduate Training Fellowship. REFERENCES 1. Amann, R. I., L. Krumholz, and D. A. Stahl. 1990. Fluorescent oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol.
172:762-770. 2. Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal. Biochem. 72:248-254. 3. Bremer, H., and P. P. Dennis. 1987. Modulation of chemical composition and other parameters of the cell by growth rate, p.
1527-1542. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 4. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 5. Dehority, B. A. 1993. Microbial ecology of cell wall fermentation, p. 425-453. In H. J. Jung, R. Hatfield, and P. Weimer (ed.), Forage cell wall structure and digestibility. American Society for Agronomy, Inc. Crop Science Society of America, Inc., and Soil Science Society of America, Inc., Madison, Wis. 6. Dubois, M., K. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Biochem. 28:350-353. 7. Hahn, D., M. J. C. Starrenburg, and A. D. L. Akkermans. 1990. Oligonucleotide probes that hybridize with rRNA as a tool to study Frankia strains in root nodules. Appl. Environ. Microbiol. 56:1342-1346. 8. Odenyo, A. A. 1992. Molecular ecology of plant cell wall hydrolysis by mixed cultures of Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, and Fibrobacter succinogenes S85. Ph.D. thesis. University of Illinois at Urbana-Champaign. 9. Odenyo, A. A., R. I. Mackie, G. C. Fahey, Jr., and B. A. White. 1991. Degradation of wheat straw and alkaline hydrogen peroxidetreated wheat straw by Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1. J. Anim. Sci. 69:819-126. 10. Odenyo, A. A., R. I. Mackie, D. A. Stahl, and B. A. White. 1994. The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production. Appl. Environ. Microbiol. 60:3688-3696. 10a.Odenyo, A. A., R. I. Mackie, and B. A. White. 1992. The use of 16S rRNA targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria, p. 396. Abstr. 92nd Gen. Meet.
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Am. Soc. Microbiol. 1992. American Society for Microbiology, Washington, D.C. 11. Pace, N. R., D. A. Stahl, D. J. Lane, and G. J. Olsen. 1985. Analyzing natural microbial populations by rRNA sequences. ASM News 51:4-12. 12. Stahl, D. A. 1986. Evolution, ecology, and diagnostics: unity in variety. BiofFechnology 4:623-628.
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13. Stahl, D. A., B. Flesher, H. R. Mansfield, and L. Montgomery. 1988. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54:1079-1084. 14. Stewart, C. S., and M. P. Bryant. 1988. The rumen bacteria, p. 21-76. In P. N. Hobson (ed.), The rumen microbial ecosystem. Elsevier Science Publishing Co., New York.
