Cellulolytic bacteria in buffalo rumen

The influence of three different feeds, wheat straw, sorghum and berseem, on total and cellulolytic bacterial counts in the buffalo rumen at different time intervals from 0 to 8 h after feeding was studied. Berseem feeding supported maximum growth of rumen bacteria in general and cellulolytic bacteria in particular. Wheat straw supported the poorest growth.
The types of cellulolytic bacteria recovered from the rumen of adult buffaloes were Ruminococcus albus, R. flavefaciens, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Clostridium lochheadii, Cl. longisporum and other Clostridium spp. Cellulolytic cocci were present in smaller numbers than rod forms in the rumen of wheat-straw-fed buffaloes, whereas the cocci outnumbered rod forms in sorghum-and berseem-fed buffaloes.     

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.     

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.     

Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture.

A total of 10 strains of rumen bacteria, Selenomonas ruminantium HD4, Megasphaera elsdenii B159, Butyrivibrio fibrisolvens A38, Streptococcus bovis JB1, Lactobacillus vitulinus GA1, Bacteroides ruminicola B14, B. ruminicola GA33, Ruminococcus albus 7, Ruminococcus flavefaciens C94, and Bacteroides succinogenes S85, were grown in energy-limiteH of the medium reservoir was lowered approximately 0.3 pH units, and the energy source concentration remaining in the culture vessel, optical density, cell mass, and pH were determined. A low pH appeared to have a detrimental effect on cell yields. Large variations were seen among strains in both the magnitude of yield depressions at lower pH values and in the pH at which the culture washed out. Lactate analysis indicated ta are discussed in relation to the effect of pH on the efficiency of protein synthesis in the rumen and rumen microbial ecology.     

Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents.

Four sheep were fed an alfalfa hay diet. Rumen content samples were collected three hours after feeding in order to total microorganism population (TP), solid attached population (SAP) and solid attached firmly population (SAFP). Fibrolytic specific activities (xylanase, CMCase and beta-glycosidases) were estimated by the amount of reducing sugars or p-nitrophenol released from the appropriate substrate. The distribution of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens) was quantified by dot-blot hybridisation using specific 16S-rRNA-targeting probes. Specific activities of polysaccharidase enzymes were higher in SAP than in TP, and in SAFP than in SAP. The sum of RNA of the three cellulolytic bacterial species represented on average 9% of the total bacterial RNA, and increased after filtration. In all samples, the relative population size of F. succinogenes was higher than that of R. albus and of R. flavefaciens. These results demonstrate that the most active enzymes are secreted by the particle-associated microorganisms. The differences in composition of the microflora between the solid and liquid phase suggest that bacteria are not equally distributed throughout the rumen content: the cellulolytic species are present in a higher proportion in the solid phase of rumen contents.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

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.     

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.     

Interactions between rumen bacterial strains during the degradation and utilization of the monosaccharides of barley straw cell-walls

Pure cultures and pair-combinations of strains representative of the rumen cellulolytic species Ruminococcus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisovens were grown on cell-wall materials from barley straw. Of the pure cultures, R. flavefaciens solubilized straw most rapidly. The presence of B. fibrisolvens , which was unable to degrade straw extensively in pure culture, increased the solubilization of dry matter by R. flavefaciens and the solubilization of cell-wall carbohydrates by both R. flavefaciens and F. succinogenes. During fermentation, both R. flavefaciens and F. succinogenes released bound glucose and free and bound arabinose and xylose into solution. The accumulation of these sugars, especially arabinose and xylose, was greatly reduced in co-cultures containing B. fibrisolvens , suggesting that significant interspecies cross feeding of the products of hemicellulose hydrolysis (particularly soluble bound xylose released by F. succinogenes ) occurs during straw degradation by mixed cultures containing this species.     

Dominant bacterial communities in the rumen of Gayals (Bos frontalis), Yaks (Bos grunniens) and Yunnan Yellow Cattle (Bos taurs) revealed by denaturing gradient gel electrophoresis

The dominant rumen bacteria in Gayals, Yaks and Yunnan Yellow Cattle were investigated using PCR-DGGE approach. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis 16S rDNA sequences in DGGE profiles were combined to reveal the dominant bacterial communities and compared the differences between those cattle species. DGGE profiles revealed that Gayals had the most abundant dominant bacteria and the lowest similarity of intraspecies between individuals than other two cattle species. A total of 45 sequences were examined and sequence similarity analysis revealed that Gayals had the most sequences appeared to uncultured bacteria, accounting for 85.0% of the total sequences, Yaks and Yunnan Yellow Cattle had 44.4 and 68.8% uncultured bacterial sequences, respectively. According to phylogenetic analysis, the rumen dominant bacteria of Gayals were mainly phylogenetically placed within phyla firmicutes and bacteroidetes, and the known bacteria were mainly belonged to the genera Lachnospiraceae bacterium, Ruminococcus flavefaciens and Clostridium celerecrescens. Moreover, the dominant bacteria of Yaks were also mainly belonged to phyla firmicutes and bacteroidetes, and the known dominant bacteria were including Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Schwartzia succinivorans and Clostridiales bacterium, most of them are common rumen bacteria. In addition, the dominant bacteria in Yunnan Yellow Cattle were belonged to phyla firmicutes, bacteroidetes and Actinobacteria, and the known dominant bacteria containing Prevotella sp., Staphylococci lentus, Staphylococcus xylosus and Corynebacterium casei. Present study first detected Staphylococcus lentus and Staphylococcus xylosus in the rumen of cattle.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

The effect of ammonia treatment on the solubilization of straw and the growth of cellulolytic rumen bacteria

Pre-treatment of straw with anhydrous ammonia increased its susceptibility to solubilization by the predominant cellulolytic bacteria from the rumen, Bacteroides succinogenes, Ruminococcus albus and R. flavefaciens. Ammonia treatment also increased the production of microbial protein and fermentation products by all three species. Scanning electron microscope observations of straw during digestion suggested that the attack of straw by these bacteria was accompanied by the formation of substantial numbers of adherent microcolonies.     

Degradation of isolated grass mesophyll, epidermis and fibre cell walls in the rumen and by cellulolytic rumen bacteria in axenic culture

The degradation of cell walls of mesophyll, epidermis and fibre cells isolated from leaves of perennial and Italian ryegrass within the sheep rumen or by selected strains of rumen bacteria in vitro , was followed by estimation of dry matter loss, or loss of neutral sugar residues. Primary cell walls (mesophyll and epidermis) were fully degraded within 12 h in the rumen, while the more heavily lignified fibre cell walls showed only a 40% loss of dry matter over the same period. Neutral sugar residues were lost at a common rate from walls of all three cell types. Incubation of cell walls with cellulolytic bacteria showed that the extent to which cell walls were attacked was constantly ordered (epidermis > mesophyll > fibre). The rate of degradation of cell walls was less in axenic culture than within the rumen. Greatest weight losses were produced by Ruminococcus albus , followed by Bacteroides succinogenes , with Ruminococcus flavefaciens effecting the least change, regardless of the nature of the cell wall provided as a substrate. Xylose was more readily lost from primary cell walls than glucose during the early stages of attack, but both were lost at a common rate from fibre cell walls. Dry matter losses produced by the hemicellulolytic strain, Bacteroides ruminocola , were limited even after extended incubation. Electron microscopy indicated that R. albus was less commonly attached to cell walls than were the other cellulolytic strains, although evidence of capsular material was present. Bacteroides succinogenes was seen with an extensive capsule which enveloped clusters of cells, forming micro-colonies in association with the plant cell wall. Vesicle-like structures, commonly associated with the cellulolytic bacteria R. albus and B. succinogenes , were found on comparatively few occasions in this study.     

Competition for cellulose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions.

Three predominant ruminal cellulolytic bacteria (Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, and Ruminococcus albus 7) were grown in different binary combinations to determine the outcome of competition in either cellulose-excess batch culture or in cellulose-limited continuous culture. Relative populations of each species were estimated by using signature membrane-associated fatty acids and/or 16S rRNA-targeted oligonucleotide probes. Both F. succinogenes and R. flavefaciens coexisted in cellulose-excess batch culture with similar population sizes (58 and 42%, respectively; standard error, 12%). By contrast, under cellulose limitation R. flavefaciens predominated (> 96% of total cell mass) in coculture with F. succinogenes, regardless of whether the two strains were inoculated simultaneously or whether R. flavefaciens was inoculated into an established culture of F. succinogenes. The predominance of R. flavefaciens over F. succinogenes under cellulose limitation is in accord with the former's more rapid adherence to cellulose and its higher affinity for cellodextrin products of cellulose hydrolysis. In batch cocultures of F. succinogenes and R. albus, the populations of the two species were similar. However, under cellulose limitation, F. succinogenes was the predominant strain (approximately 80% of cell mass) in cultures simultaneously coinoculated with R. albus. The results from batch cocultures of R. flavefaciens and R. albus were not consistent within or among trials: some experiments yielded monocultures of R. albus (suggesting production of an inhibitory agent by R. albus), while others contained substantial populations of both species. Under cellulose limitation, R. flavefaciens predominated over R. albus (85 and 15%, respectively), as would be expected by the former's greater adherence to cellulose. The retention of R. albus in the cellulose-limited coculture may result from a combination of its ability to utilize glucose (which is not utilizable by R. flavefaciens), its demonstrated ability to adapt under selective pressure in the chemostat to utilization of lower concentrations of cellobiose, a major product of cellulose hydrolysis, and its possible production of an inhibitory agent.     

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.

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cereal supplementation modified the fibrolytic activity but not the structure of the cellulolytic bacterial community associated with rumen solid digesta

4 ruminally cannulated cows were fed a forage diet (93% hay + 7% straw) and a mixed diet (33 % hay + 7% straw + 40% barley) in a 2 x 2 crossover experimental design. In sacco degradation of forage, fibrolytic activities (polysaccharidases and glycosidases) of the solid-associated bacteria (SAB), and distribution of the 3 main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, Ruminococcus flavefaciens) were determined for both diets. Barley supplementation decreased the hay degradation rate and mainly the polysaccharidase activities of the SAB (30% on average). The sum of rRNA of the 3 cellulolytic bacterial species represented on average 17% of the total bacterial signal and R. albus was the dominant cellulolytic bacterial species of the 3 studied. Barley supplementation did not modify the proportion of the 3 cellulolytic bacteria attached to plant particles. The negative effect of barley on the ruminal hay degradation rate is due to a decrease in fibrolytic activity of the SAB, and not to a modification of the balance of the three cellulolytic bacterial species examined.     

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.     

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.     

Synergism of rumen microbial hydrolases during degradation of plant polymers

In isolated mixture of exocellular enzymes of rumen bacteria Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and rumen fungus Neocallimastix frontalis, specific activities of cellulases, hemicellulases and glycosidases were determined. The highest specific activities were shown mostly for proteins of N. frontalis.