C. J. Newbold

Microbial ecosystem and methanogenesis in ruminants

Ruminant production is under increased public scrutiny in terms of the importance of cattle and other ruminants as major producers of the greenhouse gas methane. Methanogenesis is performed by methanogenic archaea, a specialised group of microbes present in several anaerobic environments including the rumen. In the rumen, methanogens utilise predominantly H2 and CO2 as substrates to produce methane, filling an important functional niche in the ecosystem. However, in addition to methanogens, other microbes also have an influence on methane production either because they are involved in hydrogen (H2) metabolism or because they affect the numbers of methanogens or other members of the microbiota. This study explores the relationship between some of these microbes and methanogenesis and highlights some functional groups that could play a role in decreasing methane emissions. Dihydrogen (H2 from this point on) is the key element that drives methane production in the rumen. Among H2 producers, protozoa have a prominent position, which is strengthened by their close physical association with methanogens, which favours H2 transfer from one to the other. A strong positive interaction was found between protozoal numbers and methane emissions, and because this group is possibly not essential for rumen function, protozoa might be a target for methane mitigation. An important function that is associated with production of H2 is the degradation of fibrous plant material. However, not all members of the rumen fibrolytic community produce H2. Increasing the proportion of non-H2 producing fibrolytic microorganisms might decrease methane production without affecting forage degradability. Alternative pathways that use electron acceptors other than CO2 to oxidise H2 also exist in the rumen. Bacteria with this type of metabolism normally occupy a distinct ecological niche and are not dominant members of the microbiota; however, their numbers can increase if the right potential electron acceptor is present in the diet. Nitrate is an alternative electron sinks that can promote the growth of particular bacteria able to compete with methanogens. Because of the toxicity of the intermediate product, nitrite, the use of nitrate has not been fully explored, but in adapted animals, nitrite does not accumulate and nitrate supplementation may be an alternative under some dietary conditions that deserves to be further studied. In conclusion, methanogens in the rumen co-exist with other microbes, which have contrasting activities. A better understanding of these populations and the pathways that compete with methanogenesis may provide novel targets for emissions abatement in ruminant production.

Mode of action of the yeast Saccharomyces cerevisiae as a feed additive for ruminants

Two suggested modes of action of yeast in stimulating rumen fermentation were investigated. The first, that yeast respiratory activity protects anaerobic rumen bacteria from damage by O2, was tested using different strains of yeast that had previously been shown to have differing abilities to increase the viable count of rumen bacteria. Saccharomyces cerevisiae NCYC 240, NCYC 1026, and the commercial product Yea-Sacc, added to rumen fluid in vitro at 1.3 mg/ml, increased the rate of O2 disappearance by between 46 and 89%. The same three preparations also stimulated bacterial numbers in an in vitro fermenter (Rusitec). S. cerevisiae NCYC 694 and NCYC 1088, which had no influence on the viable count in Rusitec, also had no effect on O2 uptake. Respiration-deficient (RD) mutants of S. cerevisiae NCYC 240 and NCYC 1026 were enriched by repeated culturing in the presence of ethidium bromide. S. cerevisiae NCYC 240 and NCYC 1026 stimulated the total and cellulolytic bacterial populations in Rusitec, while the corresponding RD mutants did not. Rigorous precautions to exclude air from Rusitec resulted in S. cerevisiae NCYC 240 no longer stimulating total bacterial numbers, although it still increased numbers of cellulolytic bacteria. The second hypothesis, that yeast provides malic and other dicarboxylic acids which stimulate the growth of some rumen bacteria, was examined by comparing the effects of yeast and malic acid on rumen fermentation in sheep. Three mature sheep were given 0.85 kg barley/d plus 0.55 kg chopped ryegrass hay/d either unsupplemented, or supplemented with 4 g S. cerevisiae NCYC 240/d or 100 mg L-malic acid/d either mixed with the diet or in aqueous solution infused continuously into the rumen. Yeast increased the total viable count of bacteria (P < 0.05) whereas malic acid did not, and no other effect of the treatments reached statistical significance. It was concluded, therefore, that the stimulation of rumen bacteria by S. cerevisiae is at least partly dependent on its respiratory activity, and is not mediated by malic acid.

Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism

ABSTRACT A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

The importance of methanogens associated with ciliate protozoa in ruminal methane production in vitro

The importance of methanogenic bacteria associated with ciliate protozoa was estimated either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum, a co‐culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. Methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium, was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum. Methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.

Influence of foliage from African multipurpose trees on activity of rumen protozoa and bacteria

Samples and extracts of foliage from African multipurpose trees were screened for their effects on rumen protozoa and bacteria with a view to predicting their safety as feed supplements and for identifying species with potential antiprotozoal activity. The species tested were Acacia aneura, Chamaecytisus palmensis, Brachychiton populneum, Flindersia maculosa, Sesbania sesban, Leucaena leucocephala and Vernonia amyedalina. Antimicrobial effects were mild except for S. sesban, which was highly toxic to rumen protozoa in vitro, and A. aneura, which was toxic to rumen bacteria. The antiprotozoal factor in S. sesban was apparently associated with the fraction of the plant containing saponins. When S. sesban was fed to sheep, protozoal numbers fell by 60% after 4 d, but the population recovered after a further 10 d. In vitro experiments demonstrated that washed protozoa from later times were no more resistant to S. sesban than on initial exposure, suggesting that other micro-organisms, probably the bacteria, adapted to detoxify the antiprotozoal agent. Thus S. sesban may be useful in suppressing protozoa and thereby improving protein flow from the rumen, but only if the bacterial metabolism of the antiprotozoal factor can be avoided.

Effects of Yucca schidigera extract on fermentation and degradation of steroidal saponins in the rumen simulation technique (RUSITEC)

Abstract A rumen simulation technique (RUSITEC) apparatus with eight 940xa0ml fermentation vessels was used to study the effects of the steroidal saponins in Yucca schidigera extract (YE) on ruminal microbial activity and saponin degradation. The YE contained approximately 4.4% (w/w) saponin, as smilagenin equivalents, and was included at 0 (control) or 0.5xa0mgxa0ml −1 ( n =4) in the McDougalls buffer infused continuously into the vessels (dilution rate=0.75 day −1 ). Each vessel received 5xa0g chopped alfalfa hay and 5xa0g concentrate (as-fed basis) daily for 22xa0days. Ammonia concentrations were lower ( P P >0.05) by YE, but molar proportions of iso-butyric and iso-valeric acids were lower ( P −1 did not affect ( P >0.05) dry matter disappearance (DMD) from hay or from concentrate, nor did it affect total gas or methane production, or bacterial numbers (total or cellulolytic populations) in homogenates prepared from fermenter vessel liquid and feed particles. Protozoal numbers in the homogenates were substantially reduced ( P −1 ), protease activity was increased ( P 2 were unaffected ( P >0.05) and activity against Ala 5 was reduced by 25% ( P >0.05). When the homogenates from control and YE-supplemented (0.5xa0mgxa0ml −1 ) vessels were used to inoculate roll tubes containing 0 or 5xa0mgxa0ml −1 of YE, fewer colonies developed ( P −1 altered proteolytic activity and reduced protozoal numbers, but did not affect DMD or bacterial activity, and did not induce resistance to YE at a concentration of 5xa0mgxa0ml −1 .

Influence of peptides, amino acids and urea on microbial activity in the rumen of sheep receiving grass hay and on the growth of rumen bacteria in vitro

Soto, R. C., Muhammed, S. A., Newbold, J., Stewart, C. S., Wallace, R. J. (1994). Influence of peptides, amino acids and urea on microbial activity in the rumen of sheep receiving grass hay and on the growth of rumen bacteria in vitro. Animal Feed Science and Technology, 49 (1-2), 151-161.

Chemical composition and degradation characteristics of foliage of some African multipurpose trees.

Samples of foliage from multipurpose leguminous trees (MPT) which had been selected as potential feed supplements for ruminants were examined for their chemical composition and in situ degradation characteristics, and were compared with alfalfa (Medicago sativa) hay and teff (Eragrostis abyssinica) straw. Organic matter (OM), acid detergent fibre (ADF), neutral detergent fibre (NDF), nitrogen, neutral detergent nitrogen, acid detergent lignin (ADL), soluble phenolics, NDF-bound proanthocyanidins and in vitro digestibility were determined in Acacia angustissima, Chamaecytisus palmensis (Tagasaste), Leucaena leucocephala, two cultivars of Sesbania sesban and Vernonia amygdalina (bitter leaf). The MPT all had a nutrient content, particularly in terms of N (up to 39.5 g available N per kg dry matter (DM)) similar to alfalfa hay, which would be suitable for supplementing teff straw, which had a high fibre, but low N (4.0 g available N per kg DM) content. In situ nylon bag digestion and in vitro gas production analyses were carried out to assess microbial degradation characteristics. The MPT were highly degradable in situ, however gas production in vitro decreased as the MPT:teff straw ratio increased in A. angustissima, indicating that antimicrobial components were present in this species. None of the chemical estimations were correlated with antimicrobial properties. It is concluded that some of the MPT tested may prove to be useful dietary supplements for ruminants receiving poor quality forages like teff straw, as has been found in other studies. However, chemical analysis alone will be of limited value in predicting the nutritive value of a new MPT which contains antimicrobial components or material toxic to the animal itself.

Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen

ABSTRACT Excessive NH3 production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH3 production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH3 production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH3 production from Trypticase varied from 1.8 to 19.7 nmol mg of protein−1 min−1 depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH3 production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH3 at >250 nmol min−1 mg of protein−1, depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH3 production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

Effect of adding acetogenic bacteria on methane production by mixed rumen microorganisms

Six reductive acetogenic bacteria from a variety of ruminal and non-ruminal environments were investigated for their ability to prevent the accumulation of methane when added to rumen fluid incubated in vitro. Acetitomaculum ruminis, Eubacterium limosum strains ATCC 10825 and ATCC 8486, Ruminococcus productus ATCC 35244, and two acetogenic bacteria, Ser 5 and Ser 8 isolated from 20 h old lambs, were grown in media containing glucose. The bacteria retained the ability to produce acetate from H2 when incubated in the absence of sugar, while no acetate was produced in the absence of H2. When cultures of the acetogens were added to incubations of mixed rumen microorganisms in vitro, providing between 0.05 and 0.13 mg of acetogen protein/ml, methane production decreased by about 5% after 24 h with E. limosum ATCC 8486 and Ser 5, while the other bacteria had no effect on methane production. Increasing the concentration of E. limosum ten-fold did not cause a further decrease in methane production. When E. limosum ATCC 8486 and Ser 5 were added to cultures of mixed ruminal microorganisms in the presence of 2-bromoethanesulfonic acid, which inhibited methane formation and caused H2 to accumulate, both bacteria caused substantial increases in acetate production and decreased H2 formation. It was concluded that although acetogenic bacteria can utilise H2 and CO2 to form acetate in the rumen when methanogenesis is inhibited, even large concentrations of acetogenic bacteria cannot compete for H2 with methanogenic archaea under normal circumstances. # 1999 Elsevier Science B.V. All rights reserved.