Rumen Methanogenic Genotypes Differ in Abundance According to Host Residual Feed Intake Phenotype and Diet Type

Methane is an undesirable end product of rumen fermentative activity because of associated environmental impacts and reduced host feed efficiency. Our study characterized the rumen microbial methanogenic community in beef cattle divergently selected for phenotypic residual feed intake (RFI) while offered a high-forage (HF) diet followed by a low-forage (LF) diet. Rumen fluid was collected from 14 high-RFI (HRFI) and 14 low-RFI (LRFI) animals at the end of both dietary periods. 16S rRNA gene clone libraries were used, and methanogen-specific tag-encoded pyrosequencing was carried out on the samples. We found that Methanobrevibacter spp. are the dominant methanogens in the rumen, with Methanobrevibacter smithii being the most abundant species. Differences in the abundance of Methanobrevibacter smithii and Methanosphaera stadtmanae genotypes were detected in the rumen of animals offered the LF compared to the HF diet while the abundance of Methanobrevibacter smithii genotypes was different between HRFI and LRFI animals irrespective of diet. Our results demonstrate that while a core group of methanogen operational taxonomic units (OTUs) exist across diet and phenotype, significant differences were observed in the distribution of genotypes within those OTUs. These changes in genotype abundance may contribute to the observed differences in methane emissions between efficient and inefficient animals.     

Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes

A model rumen system, dual-flow continuous culture fermenters, was evaluated by two comparative criteria in two experiments using ribosomal (r)RNA-targeted DNA probes to compare key microbial groups in samples. The initial experiment measured temporal changes in population structure during adaptation of ruminal microbial populations in fermenters over 240 h. The fermenter inoculum contained 34.9% Bacteria, 60.1% Eukarya and 6.8% Archaea measured as a fraction of total small subunit (SSU) rRNA quantified using a universal probe. The cellulolytic bacterial genus Fibrobacter comprised 9.5% of total SSU rRNA in the inoculum. After 240 h of fermenter operation, the average abundance was 80.9% Bacteria, 6.1% Eukarya, 5.1% Archaea and Fibrobacter genus accounted for 6.6% of the total SSU rRNA. Divergence between ruminal and fermenter population structure was evaluated in the second experiment and samples were classified as ruminal, inoculum or fermenter (96, 120, 144 and 168 h of fermenter operation). Fermenter samples had higher relative abundances of Bacteria (84.5%) and Archaea (2.1%) and lower relative abundances of Eukarya (1.8%) than ruminal samples (average 48.0% Bacteria, 1.3% Archaea and 61.5% Eukarya). The relative abundance of Fibrobacter was similar in all samples, averaging 2.5%. The ruminal and fermenter samples had similar proportions of F. succinogenes and F. succinogenes subgroup 3 (as a percentage of Fibrobacter SSU rRNA). Fibrobacter succinogenes subgroup 1 and F. intestinalis proportions of Fibrobacter were lower in fermenter samples (8.2% and 0.7% respectively) than in ruminal samples (28.4% and 2.2% respectively). Fermenters were able to maintain a core prokaryotic community structure similar to the native microbial community in the rumen. Although protozoa populations were lost, maintenance of Fibrobacter and archaeal populations indicated that the model system supported a functional community structure similar to the rumen. This model rumen system may serve as a suitable tool for studying aspects of ruminal microbial ecology and may resolve some of the relationships between microbial community structure and function by providing control of experimental conditions.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

C.S. MCSWEENEY, B. PALMER, R. BUNCH AND D.O. KRAUSE. 2001 .
Aims: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function.
Methods and Results: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides - Porphyromonas - Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly.
Conclusions: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis.
Significance and Impact of the Study: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Influence of the composition of the cellulolytic flora on the development of hydrogenotrophic microorganisms, hydrogen utilization, and methane production in the rumens of gnotobiotically reared lambs

We investigated the influence of the composition of the fibrolytic microbial community on the development and activities of hydrogen-utilizing microorganisms in the rumens of gnotobiotically reared lambs. Two groups of lambs were reared. The first group was inoculated with Fibrobacter succinogenes, a non-H(2)-producing species, as the main cellulolytic organism, and the second group was inoculated with Ruminococcus albus, Ruminococcus flavefaciens, and anaerobic fungi that produce hydrogen. The development of hydrogenotrophic bacterial communities, i.e., acetogens, fumarate and sulfate reducers, was monitored in the absence of methanogens and after inoculation of methanogens. Hydrogen production and utilization and methane production were measured in rumen content samples incubated in vitro in the presence of exogenous hydrogen (supplemented with fumarate or not supplemented with fumarate) or in the presence of ground alfalfa hay as a degradable substrate. Our results show that methane production was clearly reduced when the dominant fibrolytic species was a non-H(2)-producing species, such as Fibrobacter succinogenes, without significantly impairing fiber degradation and fermentations in the rumen. The addition of fumarate to the rumen contents stimulated H(2) utilization only by the ruminal microbiota inoculated with F. succinogenes, suggesting that these communities could play an important role in fumarate reduction in vivo.     

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.     

Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR

A set of PCR primers was designed and validated for specific detection and quantification of Prevotella ruminicola, Prevotella albensis, Prevotella bryantii, Fibrobacter succinogenes, Selenomonas ruminantium-Mitsuokella multiacida, Streptococcus bovis, Ruminococcus flavefaciens, Ruminobacter amylophilus, Eubacterium ruminantium, Treponema bryantii, Succinivibrio dextrinosolvens, and Anaerovibrio lipolytica. By using these primers and the real-time PCR technique, the corresponding species in the rumens of cows for which the diet was switched from hay to grain were quantitatively monitored. The dynamics of two fibrolytic bacteria, F. succinogenes and R. flavefaciens, were in agreement with those of earlier, culture-based experiments. The quantity of F. succinogenes DNA, predominant in animals on the hay diet, fell 20-fold on the third day of the switch to a grain diet and further declined on day 28, with a 57-fold reduction in DNA. The R. flavefaciens DNA concentration on day 3 declined to approximately 10% of its initial value in animals on the hay diet and remained at this level on day 28. During the transition period (day 3), the quantities of two ruminal prevotella DNAs increased considerably: that of P. ruminicola increased 7-fold and that of P. bryantii increased 263-fold. On day 28, the quantity of P. ruminicola DNA decreased 3-fold, while P. bryantii DNA was still elevated 10-fold in comparison with the level found in animals on the initial hay diet. The DNA specific for another xylanolytic bacterium, E. ruminantium, dropped 14-fold during the diet switch and was maintained at this level on day 28. The concentration of a rumen spirochete, T. bryantii, decreased less profoundly and stabilized with a sevenfold decline by day 28. The variations in A. lipolytica DNA were not statistically significant. After an initial slight increase in S. dextrinosolvens DNA on day 3, this DNA was not detected at the end of the experiment. S. bovis DNA displayed a 67-fold increase during the transition period on day 3. However, on day 28, it actually declined in comparison with the level in animals on the hay ration. The amount of S. ruminantium-M. multiacida DNA also increased eightfold following the diet switch, but stabilized with only a twofold increase on day 28. The real-time PCR technique also uncovered differential amplification of rumen bacterial templates with the set of universal bacterial primers. This observation may explain why some predominant rumen bacteria have not been detected in PCR-generated 16S ribosomal DNA libraries.     

Dominance of <Emphasis Type="Italic">Prevotella</Emphasis> and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR

Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising <0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.     

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive

AIM: To examine the effect of concentrate and yeast additive on the number of cellulolytic bacteria in the rumen of sheep. METHODS AND RESULTS: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens were quantified using real-time PCR (targeting 16S rDNA) in parallel to cellulolytic flora enumeration with cultural techniques. Whatever the conditions tested, R. flavefaciens was slightly more abundant than F. succinogenes, with both species outnumbering R. albus. Before feeding, the shift from hay to hay plus concentrate diet had no effect on rumen pH and on the number of the three specie; while after feeding, the concentrate-supplemented diet induced a decrease (-1 log) of the number of the three species concomitant with the rumen acidification. Overall, the presence of the live yeast resulted in a significant increase (two- to fourfold) of the Ruminococci. CONCLUSION: The use of real-time PCR allowed us to show changes in the number of cellulolytic bacterial species in vivo in response to diet shift and additives that could not be as easily evidenced by classical microbial methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the understanding of the negative impact of readily fermentable carbohydrates on rumen cellulolysis and the beneficial effect of yeast on rumen fermentation.     

Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria.

Molecular beacons are fluorescent probes developed for solution rather than membrane hybridization. We have investigated the utility of these probes to study rumen microbial ecology. Two cellulolytic species, Ruminococcus albus and Fibrobacter succinogenes, were tested. Membrane and solution hybridizations gave similar results in competition experiments with cocultures of R. albus 8 and F. succinogenes S85.     

Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens

Competitive PCR assays were developed for the enumeration of the rumen cellulolytic bacterial species: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. The assays, targeting species-specific regions of 16S rDNA, were evaluated using DNA from pure culture and rumen digesta spiked with the relevant cellulolytic species. Minimum detection levels for F. succinogenes, R. albus and R. flavefaciens were 1-10 cells in pure culture and 10(3-4) cells per ml in mixed culture. The assays were reproducible and 11-13% inter- and intra-assay variations were observed. Enumeration of the cellulolytic species in the rumen and alimentary tract of sheep found F. succinogenes dominant (10(7) per ml of rumen digesta) compared to the Ruminococcus spp. (10(4-6) per ml). The population size of the three species did not change after the proportion of dietary alfalfa hay was increased. All three species were detected in the rumen, omasum, caecum, colon and rectum. Numbers of the cellulolytic species at these sites varied within and between animals.     

Impact of feed efficiency and diet on adaptive variations in the bacterial community in the rumen fluid of cattle

Limited knowledge of the structure and activities of the ruminal bacterial community prevents the understanding of the effect of population dynamics on functional bacterial groups and on host productivity. This study aimed to identify particular bacteria associated with host feed efficiency in steers with differing diets and residual feed intake (RFI) using culture-independent methods: PCR-denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR analysis. PCR-DGGE profiles were generated from the ruminal fluid of 55 steers fed a low-energy-density diet and then switched to a high-energy-density diet. Bacterial profile comparisons by multivariate statistical analysis showed a trend only for RFI-related clusters on the high-energy diet. When steers (n = 19) belonging to the same RFI group under both diets were used to identify specific bacterial phylotypes related to feed efficiency traits, correlations were detected between dry matter intake, average daily gain, and copy numbers of the 16S rRNA gene of Succinivibrio sp. in low-RFI (efficient) steers, whereas correlations between Robinsoniella sp. and RFI (P < 0.05) were observed for high-RFI (inefficient) animals. Eubacterium sp. differed significantly (P < 0.05) between RFI groups that were only on the high-energy diet. Our work provides a comprehensive framework to understand how particular bacterial phylotypes contribute to differences in feed efficiency and ultimately influence host productivity, which may either depend on or be independent from diet factors.     

Microbial and Carbohydrate Active Enzyme profile of buffalo rumen metagenome and their alteration in response to variation in the diet

Rumen microbiome represents rich source of enzymes degrading complex plant polysaccharides. We describe here analysis of Carbohydrate Active Enzymes (CAZymes) from 3.5 gigabase sequences of metagenomic data from rumen samples of Mehsani buffaloes fed on different proportions of green or dry roughages to concentrate ration. A total of 2597 contigs encoding putative CAZymes were identified by CAZyme Analysis Toolkit (CAT). The phylogenetic analysis of these contigs by MG-RAST revealed predominance of Bacteroidetes, followed by Firmicutes, Proteobacteria, and Actinobacteria phyla. Moreover, a higher abundance of oligosaccharide degrading and debranching enzymes in buffalo rumen metagenome and that of cellulases and hemicellulases in termite hindgut was observed when we compared glycoside hydrolase (GH) profile of buffalo rumen metagenome with cow rumen, termite hindgut and chicken caecum metagenome. Further, comparison of microbial profile of green or dry roughage fed animals showed significantly higher abundance (p-value < 0.05) of various polysaccharide degrading bacterial genera like Fibrobacter, Prevotella, Bacteroides, Clostridium and Ruminococcus in green roughage fed animals. In addition, we found a significantly higher abundance (p-value < 0.05) of enzymes associated with pectin digestion such as pectin lyase (PL) 1, PL10 and GH28 in green roughage fed animals. Our study outlines CAZyme profile of buffalo rumen metagenome and provides a scope to study the role of abundant enzyme families (oligosaccharide degrading and debranching enzymes) in digestion of coarse feed.     

Activity and properties of fumarate reductase in ruminal bacteria

Fumarate-reducing bacteria were sought from the main ruminal bacteria. Fibrobacter succinogenes, Selenomonas ruminantium subsp. ruminantium, Selenomonas ruminantium subsp. lactilytica, and Veillonella parvula reduced fumarate by using H(2) as an electron donor. Ruminococcus albus, Prevotella ruminicola, and Anaerovibrio lipolytica consumed fumarate, although they did not oxidize H(2). Of these bacteria, V. parvula, two strains of Selenomonas, and F. succinogenes had a high capacity to reduce fumarate. In all the fumarate-reducing bacteria examined, fumarate reductase existed in the membrane fraction. Based on the activity per cell mass and the affinity of fumarate reductase to fumarate, these bacteria were divided into two groups, which corresponded to the capacity to use H(2): A group of bacteria with higher activity and affinity were able to use H(2) as an electron donor for fumarate reduction. The bacteria in this group should gain an advantage over the bacteria in another group in fumarate reduction in the rumen. Cellulose digestion by R. albus was improved by fumarate reduction by S. lactilytica as a result of an increased growth of R. albus, which may have been caused by the fact that S. lactilytica immediately consumed H(2) produced by R. albus. Thus fumarate reduction may play an important role in keeping a low partial pressure of H(2) in the rumen.     

Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum.

Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet

AIM: To examine the effect of sulfur-containing compounds on the growth of anaerobic rumen fungi and the fibrolytic rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in pure culture and within the cattle rumen. METHODS AND RESULTS: The effect of two reduced sulfur compounds, 3-mercaptopropionic acid (MPA) or 3-mercapto-1-propanesulfonic acid as the sole S source on growth of pure fibroyltic fungal and bacterial cultures showed that these compounds were capable of sustaining growth. An in vivo trial was then conducted to determine the effect of sulfur supplements (MPA and sodium sulfate) on microbial population dynamics in cattle fed the roughage Dichanthium aristatum. Real-time PCR showed significant increases in fibrolytic bacterial and fungal populations when cattle were supplemented with these compounds. Sulfate supplementation leads to an increase in dry matter intake without a change in whole tract dry matter digestibility. CONCLUSIONS: Supplementation of low S-containing diets with either sodium sulfate or MPA stimulates microbial growth with an increase in rumen microbial protein supply to the animal. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the use of real-time PCR monitoring, a better understanding of the effect of S supplementation on discrete microbial populations within the rumen is provided.     

Changes in Rumen Microbial Community Composition during Adaption to an In Vitro System and the Impact of Different Forages

This study examined ruminal microbial community composition alterations during initial adaption to and following incubation in a rumen simulation system (Rusitec) using grass or corn silage as substrates. Samples were collected from fermenter liquids at 0, 2, 4, 12, 24, and 48 h and from feed residues at 0, 24, and 48 h after initiation of incubation (period 1) and on day 13 (period 2). Microbial DNA was extracted and real-time qPCR was used to quantify differences in the abundance of protozoa, methanogens, total bacteria, Fibrobacter succinogenes, Ruminococcus albus, Ruminobacter amylophilus, Prevotella bryantii, Selenomonas ruminantium, and Clostridium aminophilum. We found that forage source and sampling time significantly influenced the ruminal microbial community. The gene copy numbers of most microbial species (except C. aminophilum) decreased in period 1; however, adaption continued through period 2 for several species. The addition of fresh substrate in period 2 led to increasing copy numbers of all microbial species during the first 2–4 h in the fermenter liquid except protozoa, which showed a postprandial decrease. Corn silage enhanced the growth of R. amylophilus and F. succinogenes, and grass silage enhanced R. albus, P. bryantii, and C. aminophilum. No effect of forage source was detected on total bacteria, protozoa, S. ruminantium, or methanogens or on total gas production, although grass silage enhanced methane production. This study showed that the Rusitec provides a stable system after an adaption phase that should last longer than 48 h, and that the forage source influenced several microbial species.     

Enzymes associated with metabolism of xylose and other pentoses by Prevotella (Bacteroides) ruminicola strains, Selenomonas ruminantium D, and Fibrobacter succinogenes S85.

Prevotella (Bacteroides) ruminicola strains B(1)4 and S23 and Selenomonas ruminantium strain D used xylose as the sole source of carbohydrate for growth, whereas Fibrobacter succinogenes was unable to metabolize xylose. Prevotella ruminicola strain B(1)4 exhibited transport activity for xylose. In contrast, F. succinogenes lacked typical xylose uptake activity but did exhibit low binding potential for the sugar. Prevotella ruminicola strains B(1)4 and S23 as well as S. ruminantium D showed low xylose isomerase activities but higher xylulokinase activities, using assays that gave high activities for these enzymes in Escherichia coli. Xylose isomerase appeared to be produced constitutively in these ruminal bacteria, but xylulokinase was induced to varying degrees with xylose as the source of carbohydrate. Fibrobacter succinogenes lacked xylose isomerase and xylulokinase. All three species of ruminal bacteria possessed transketolase, xylulose-5-phosphate epimerase, and ribose-5-phosphate isomerase activities. Neither P. ruminicola B(1)4 nor F. succinogenes S85 showed significant phosphoketolase activity. The data indicate that F. succinogenes is unable to either actively uptake or metabolize xylose as a result of the absence of functional xylose permease, xylose isomerase, and xylulokinase activities, although it and both P. ruminicola and S. ruminantium possess the essential enzymes of the nonoxidative branch of the pentose phosphate cycle.     

Interactions between anaerobic cellulolytic bacteria and fungi in the presence of Methanobrevibacter smithii

A mixed inoculum of cellulolytic rumen bacteria depressed straw degradation by a mixed culture of cellulolytic fungi grown in the presence of Methanobrevibacter smithii. The inhibitory effect appeared to be caused by Ruminococcus albus strain JI and R. flavefaciens strain 007. Ruminococcus albus strain J1 also depressed straw degradation by the fungi, but R. albus strain SY3 and three strains of Bacteroides (Fibrobacter) succinogenes tested showed little or no inhibitory activity. It seems that some ruminococci show competitive or antagonistic activity towards certain rumen fungi.     

Detection of novel Fibrobacter populations in landfill sites and determination of their relative abundance via quantitative PCR

Members of the bacterial genus Fibrobacter have long been considered important components of the anaerobic cellulolytic community in the herbivore gut, but their presence and activity in other environments is largely unknown. In this study, a specific polymerase chain reaction (PCR) primer set, targeting the 16S rRNA gene of Fibrobacter spp., was applied to community DNA from five landfill sites followed by temporal thermal gel electrophoresis (TTGE) analysis of cloned amplification products. Phylogenetic analysis of clone sequences indicated the presence of novel clusters closely related to the genus Fibrobacter . There are two named species, Fibrobacter succinogenes and F. intestinalis , and only two of the 58 sequenced clones were identified with them, and both were F. succin ogenes. The clone sequences from landfill were recovered in five distinct clusters within the Fibrobacter lineage, and four of these were novel. Quantitative PCR (qPCR) assays of reverse-transcribed community RNA from landfill leachates and rumen fluid samples indicated that the abundance of Fibrobacter spp. relative to total bacteria varied from 0.2% to 40% in landfill, and 21% to 32% in the rumen, and these data demonstrate that fibrobacters can be a significant component of the microbial community in landfill ecosystems. This is the first evidence for Fibrobacter spp. outside the gut ecosystem, and as the only cultivated representatives of this group are actively cellulolytic, their diversity and abundance points to a possible role in cellulose hydrolysis in landfill, and perhaps other anaerobic environments also.