Postprandial variations in the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents

The diurnal variations in the specific activities of polysaccharide-degrading enzymes after feeding were monitored in adherent and non-adherent microbial populations separated from bovine rumen liquor and digesta solids. There were marked differences in the activity profiles of the enzymes within the subpopulations. Enzymes involved in the degradation of soluble carbohydrates were more active in the non-adherent populations, and in the liquor phase subpopulation activities increased in the 1–2 h post-feed period. The muralytic enzymes were most active in the adherent population. Specific activities increased by up to 20-fold over the 24 h period, with an initial five-fold increase occurring between 8 h and 12 h after feeding. Enzyme levels in the three non-adherent populations were similar at the end of the postprandial period. In the population recovered from the liquid associated with the digesta particles, however, the activities did not increase until the latter stages of the period, whereas in the non-adherent population from the digesta solids the activities varied little during the diurnal cycle. The numbers of micro-organisms associated with the digesta solids were similar at 2 h and 20 h after feeding; the variations in enzyme levels did not occur as a result of a population increase but were due to increased activities in an established population. The plant cell wall structural polysaccharides were degraded at different rates. There was no appreciable cellulose digestion during the first 8 h of the postprandial period and although hemicellulosic constituents were removed continuously the rate of loss of both polymers was increased in the later stages of the diurnal cycle when enzyme activities were maximal.     

Postprandial variations in the activity of polysaccharide-degrading enzymes of fluid- and particle-associated ruminal microbial populations

Effect of an algicidal product fromOscillatoria late-virens and of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) on growth, photosynthesis, and mouse toxicity inMicrocystis PCC 7820 was examined. Their lethal concentrations abolished photosystem (PS)-II reactions and eventually bleached and detoxified the cyanobacterium. Although loss of protein, chlorophyll, and toxicity were also induced by sublethal antibiotic doses, photosynthetic activities remained unchanged and developed antibiotic tolerance. These effects could be duplicated in natural conditions, implying utility of the natural algicide in control of toxic cyanobacteria.     

The distribution of polysaccharide-degrading enzymes in the bovine rumen digesta ecosystem

The location and level of activity of the principal polysaccharidases and glycoside hydrolases involved in the degradation of plant structural and storage polysaccharides were monitored in microbial populations isolated from liquid and particulate phases of bovine rumen digesta. The three principal subpopulations, and their constituent subgroups studied, all contained polysaccharide depolymerizing enzymes; however, the specific activities of the enzymes that degraded the plant cell wall structural polymers were highest within the adherent particle-associated populations. Separate functional groups of organisms could be recongnized in the particle-associated population by their distinctive enzyme profiles.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4–8 times (β-d-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass ( Lolium perenne ) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes and fibre breakdown in the rumen ecosystem.

The effect of ciliate protozoa on the activity of polysaccharide-degrading enzymes in microbial populations from the digesta solids and liquor fractions of rumen contents was examined after the refaunation of ciliate-free sheep with an A-type rumen protozoal population. Although the culturable rumen bacterial population was reduced after refaunation the number of fibrolytic micro-organisms detected was higher; the xylanolytic bacterial population and numbers of fungal zoospores were increased after refaunation. The proportion of propionic acid was lower in the refaunated animals, whereas the concentration of ammonia and the acidic metabolites acetate, butyrate and valerate were all increased. The range of enzyme activities present in the digesta subpopulations were the same in defaunated and refaunated animals. The activities of the polysaccharide-degrading enzymes, however, were increased in the microbial populations associated with the digesta solids after refaunation, and at 16 h after feeding the activities were 4-8 times (beta-D-xylosidase 20 times) higher than the levels detected in the adherent population from defaunated sheep. The protozoa, either directly through their own enzymes or indirectly as a consequence of their effects on the population size and activity of the other fibrolytic micro-organisms present, have an important role in determining the level of activity of polysaccharide-degrading enzymes in the rumen ecosystem. Although the extent of ryegrass (Lolium perenne) hay digestion was similar after 24 h in the absence or presence of protozoa, the initial ruminal degradation was higher in refaunated sheep.     

Extraction of PCR-quality plant and microbial DNA from total rumen contents

DNA from rumen digesta has several diagnostic applications such as studying microbial community dynamics, transgene/DNA stability, and population typing of various rumen bacteria. Several DNA extraction procedures are described in the literature for rumen digesta, which describe the removal of tannins, polysaccharides, and other PCR inhibitors. Some of these protocols are time-consuming and impractical when handling a large number of samples routinely. Here we describe a rapid method for the extraction of PCR-quality plant and microbial DNA from total rumen contents that is based on modifications in the cetyltrimethylammonium bromide procedure followed by cleanup using a Qiagen column. This procedure is highly reproducible and relatively short, once the initial grinding of the samples is performed, and it consistently yields PCR-quality DNA.     

Improved protocol for high-quality Co-extraction of DNA and RNA from rumen digesta

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.     

Changes in microbial community structure, methanogenesis and rumen fermentation in response to saponin-rich fractions from different plant materials

Aims:  Investigation of the effects of saponin-rich fractions on rumen fermentation, methane production and the microbial community.
Methods and Results:  Saponins were extracted from Carduus , Sesbania and Knautia leaves and fenugreek seeds. Two levels of saponin-rich fractions with a substrate were incubated using the Hohenheim gas method. Methane was measured using an infrared-based methane analyser and microbial communities using quantitative PCR. On addition of saponin-rich fractions, methane and short-chain fatty acid production was not affected. The protozoal counts decreased by 10–39%. Sesbania saponins decreased methanogen population by 78%. Decrease in ruminal fungal population (20–60%) and increase in Fibrobacter succinogenes (21–45%) and Ruminococcus flavefaciens (23–40%) were observed.
Conclusions:  The saponins evaluated possessed anti-protozoal activity; however, this activity did not lead to methane reduction. Fenugreek saponins seemed to have potential for increasing rumen efficiency. The saponins altered the microbial community towards proliferation of fibre-degrading bacteria and inhibition of fungal population.
Significance and Impact of the Study:  The uni-directional relationship between protozoal numbers and methanogenesis, as affected by saponins, is not obligatory. All saponins might not hold promise for decreasing methane production from ruminants.     

In depth analysis of rumen microbial and carbohydrate-active enzymes profile in Indian crossbred cattle

Rumen houses a plethora of symbiotic microorganisms empowering the host to hydrolyze plant lignocellulose. In this study, NGS based metagenomic approach coupled with bioinformatic analysis was employed to gain an insight into the deconstruction of lignocellulose by carbohydrate-active enzymes (CAZymes) in Indian crossbred Holstein-Friesian cattle. Cattle rumen metagenomic DNA was sequenced using Illumina-MiSeq and 1.9 gigabases of data generated with an average read length of 871 bp. Analysis of the assembled sequences by Pfam-based Carbohydrate-active enzyme Analysis Toolkit identified 17,164 putative protein-encoding CAZymes belonging to different families of glycoside hydrolases (7574), glycosyltransferases (5185), carbohydrate-binding modules (2418), carbohydrate esterases (1516), auxiliary activities (434) and polysaccharide lyases (37). Phylogenetic analysis of putative CAZymes revealed that a significant proportion of CAZymes were contributed by bacteria belonging to the phylum Bacteroidetes (40%), Firmicutes (30%) and Proteobacteria (10%). The comparative analysis of HF cross rumen metagenome with other herbivore metagenomes indicated that Indian crossbred cattle rumen is endowed with a battery of CAZymes that may play a central role in lignocellulose deconstruction. The extensive catalog of enzymes reported in our study that hydrolyzes plant lignocellulose biomass, can be further explored for the better feed utilization in ruminants and also for different industrial applications.     

Production of polysaccharide-degrading enzymes by saprophytic fungi from glyphosate-treated flax and their involvement in retting

The fungi present on glyphosate-treated flax plants were isolated. Cladosporium herbarum, Epicoccum nigrum, Botrytis cinerea and yeasts occurred most frequently immediately after glyphosate treatment but as retting progressed the frequency of occurrence of Fusarium culmorum, Alternaria alternata and a Phoma sp. increased. Many of the fungi isolated from retting flax were also present as epiphytes on healthy flax stems. Glyphosate was shown to be fungitoxic in vitro but it had only a very slight effect on fungi colonising the flax. The application of sucrose and urea to flax 1 wk after glyphosate treatment resulted in more rapid fungal colonisation of the stems, but did not significantly enhance retting. When grown on sterilised flax stem sections, fungi known to be saprophytic on flax produced polysaccharide-degrading enzymes. All seven fungi tested produced polygalacturonase, pectin-lyase and xylanase. The greatest cellulase activity was present in stem tissues inoculated with F. culmorum and the Phoma sp. while no cellulase was detected in tissue inoculated with B. cinerea, a Mucor sp. or a Penicillium sp. Extracts from flax inoculated with the cellulolytic fungi caused the solubilisation of native cellulose. Pectinases, xylanase and cellulase were also detected in naturally-colonised senescing and dead flax stems. Stems which had been treated with a sucrose solution tended to contain the greatest enzyme activity.     

Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations

In the recent years, the exploration of bioactive phytochemicals as natural feed additives has been of great interest among nutritionists and rumen microbiologists to modify the rumen fermentation favorably such as defaunation, inhibition of methanogenesis, improvement in protein metabolism, and increasing conjugated linoleic acid content in ruminant derived foods. Many phytochemicals such as saponins, essential oils, tannins and flavonoids from a wide range of plants have been identified, which have potential values for rumen manipulation and enhancing animal productivity as alternatives to chemical feed additives. However, their effectiveness in ruminant production has not been proved to be consistent and conclusive. This review discusses the effects of phytochemicals such as saponins, tannins and essential oils on the rumen microbial populations, i.e., bacteria, protozoa, fungi and archaea with highlighting molecular diversity of microbial community in the rumen. There are contrasting reports of the effects of these phytoadditives on the rumen fermentation and rumen microbes probably depending upon the interactions among the chemical structures and levels of phytochemicals used, nutrient composition of diets and microbial components in the rumen. The study of chemical structure–activity relationships is required to exploit the phytochemicals for obtaining target responses without adversely affecting beneficial microbial populations. A greater understanding of the modulatory effects of phytochemicals on the rumen microbial populations together with fermentation will allow a better management of the rumen ecosystem and a practical application of this feed additive technology in livestock production.     

Comparative studies of microbial populations in the rumen,duodenum, ileum and faeces of lactating dairy cows

Aims: Understanding factors that influence the composition of microbial populations of the digestive system of dairy cattle will be key in regulating these populations to improve animal performance. Although rumen microbes are well studied, little is known of the dynamics and role of microbial populations in the small intestine of cows. Comparisons of fingerprints of microbial populations were used to investigate the effects of gastrointestinal (GI) segment and animal on community structure. Methods and Results: Samples from four lactating dairy cows with ruminal, duodenal and ileal cannulae were collected. Terminal‐restriction fragment length polymorphism (T‐RFLP) comparisons of small subunit rRNA genes revealed differences in microbial populations between GI segments (P < 0·05). No significant differences in either methanogen populations or microbial community profiles between animals were observed. Quantitative PCR was used to assay relative changes in methanogen numbers compared to procaryote rRNA gene numbers, and direct microscopic counts were used to enumerate total procaryote numbers of the duodenal and ileal samples. Conclusions: T‐RFLP comparisons illustrate significant changes in microbial diversity as digesta passes from one segment to another. Direct counts indicate that microbial numbers are reduced by eight orders of magnitude from the rumen, through the abomasum, and into the duodenum (from c. 10¹² to c. 3·6 × 10⁴ cells per ml). Quantitative PCR analyses of rRNA genes indicate that methanogens are present in the duodenum and ileum. Significance and Impact of the Study: The contribution of microbial populations of the small intestine to the nutrition and health of cattle is seldom addressed but warrants further investigation.     

Nonstarch polysaccharide-degrading enzymes alter the microbial community and the fermentation patterns of barley cultivars and wheat products in an in vitro model of the porcine gastrointestinal tract

An in vitro experiment was carried out to assess how nonstarch polysaccharide (NSP)-degrading enzymes influence the fermentation of dietary fiber in the pig large intestine. Seven wheat and barley products and cultivars with differing carbohydrate fractions were hydrolyzed using pepsin and pancreatin in the presence or not of NSP-degrading enzymes (xylanase and β-glucanase) and the filter retentate was subsequently fermented with sow fecal bacteria. Dry matter, starch, crude protein and β-glucan digestibilities during hydrolysis were measured. Fermentation kinetics of the hydrolyzed ingredients were modelled. Short-chain fatty acids (SCFA) production and molar ratio were compared after 12, 24 and 72 h. Microbial communities were analyzed after 72 h of fermentation using terminal restriction fragment length polymorphism. The results showed an increase of nutrient digestibility (P<0.001), whereas fermentability and SCFA production decreased (P<0.001) with addition of the enzyme. SCFA and bacterial community profiles also indicated a shift from propionate to acetate and an increase in cellulolytic Ruminococcus- and xylanolytic Clostridium-like bacteria. This is explained by the increase in slowly fermentable insoluble carbohydrate and the lower proportion of rapidly fermentable β-glucan and starch in the retentate when grains were incubated with NSP-degrading enzymes. Shifts were also different for the four barley varieties investigated, showing that the efficiency of the enzymes depends on the structure of the carbohydrate fractions in cereal products and cultivars.     

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.