https://cyberleninka.org/article/n/1082452.pdf

Invited review: Enteric methane in dairy cattle production: Quantifying the opportunities and impact of reducing emissions

Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics

Members of the domain Archaea contribute about 0.3 to 3.3% of the microbial small subunit (16S and 18S) rRNA in the rumen (22, 39, 60). Archaea have a range of different metabolisms and are found in many habitats (6), but those known to exist in the rumen are strictly anaerobic methanogens. Yanagita et al. (59) observed that 2.8 to 4.0% of ruminal microorganisms displayed autofluorescence characteristic of F420, a methanogen cofactor, able to be seen under UV illumination during microscopy. Taken together with the small subunit rRNA abundance data, this suggests that a large part of the archaeal population is made up of methanogens. Most species of methanogens can grow using H2 and often formate as their energy sources and use the electrons derived from H2 (or formate) to reduce CO2 to CH4. Some species can grow with methyl groups, oxidizing some to CO2 to produce electrons that are used to reduce further methyl groups to methane. A few species can grow with acetate, effectively dissimilating acetate to CH4 and CO2. However, acetate is not metabolized to CH4 to any significant extent in the rumen (13). This is probably because the rate of passage of rumen contents through the rumen is greater than the growth rate of acetate-utilizing methanogens (53).

In a normally functioning rumen, proteins and polymeric carbohydrates, which usually make up the largest part of the incoming feed, are fermented by a mixed microbial community to volatile fatty acids (VFAs), NH4+, CO2, and H2. The hydrogen is metabolized by the methanogens. The VFAs are taken up by the animal across the rumen wall and serve as major carbon and energy sources for the ruminant. A part of the VFAs, undigested feed components, and microbial cells leave the rumen and enter the rest of the animal's digestive tract. The central role of H2 in the rumen fermentation (12) means that, although methanogenic archaea make up only a small part of the rumen microbial biomass, they play an important role in rumen function and animal nutrition. Efficient H2 removal leads to a nutritionally more favorable pattern of VFA formation and to an increased rate of fermentation by eliminating the inhibitory effect of H2 on the microbial fermentation (26, 53).

The rumen can be simplistically described as an open system with discontinuous solid (feed) and liquid (saliva and drinking water) inputs and multiple fractions that have different turnover rates (53). The methanogens in the rumen are found free in the rumen fluid, attached to particulate material and rumen protozoa, associated as endosymbionts within rumen protozoa, and attached to the rumen epithelium. The methanogens associated with these different fractions can be expected to have different growth rates since they will be removed from the rumen at different rates. In addition, the animal itself and the feed also influence the rate of passage of digesta through the rumen system (25). These different habitats may allow niche division among the methanogens and may explain some of the observed phylogenetic diversity of rumen archaea.

Structure of the archaeal community of the rumen.

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.

Microbial ecosystem and methanogenesis in ruminants

A new perspective on the use of plant secondary metabolites to inhibit methanogenesis in the rumen

Six rumen-fistulated Merino sheep were used in a crossover design experiment to evaluate the effects of an exogenous fibrolytic enzyme preparation (12 g/d; ENZ), delivered directly into the rumen, on diet digestibility, ruminal fermentation, and microbial protein synthesis. The enzyme contained endoglucanase and xylanase activities. Sheep were fed a mixed grass hay:concentrate (70:30; DM basis) diet at a daily rate of 46.1 g/kg of BW 0.75 . Samples of grass hay were incubated in situ in the rumen of each sheep to measure DM and NDF degradation. The supplementation with ENZ did not affect diet digestibility (P = 0.30 to 0.66), urinary excretion of purine derivatives (P = 0.34), ruminal pH (P = 0.46), or concentrations of NH 3 -N (P = 0.69) and total VFA (P = 0.97). In contrast, molar proportion of propionate were greater (P = 0.001) and acetate:propionate ratio was lower (P < 0.001) in ENZ-supplemented sheep. In addition, ENZ supplementation tended to increase (P = 0.06) numbers of cellulolytic bacteria at 4 h after feeding. Both the ruminally insoluble potentially degradable fraction of grass hay DM and its fractional rate of degradation were increased (P = 0.002 and 0.05, respectively) by ENZ treatment. Supplementation with ENZ also increased (P = 0.01 to 0.02) effective and potential degradability of grass hay DM and NDF. Ruminal fluid endoglucanase and xylanase activities were greater (P < 0.001 and 0.03, respectively) in ENZ-supplemented sheep than in control animals. It was found that ENZ supplementation did not affect either exoglucanase (P = 0.12) or amylase (P = 0.83) activity. The results indicate that supplementing ENZ directly into the rumen increased the fibrolytic activity and stimulated the growth of cellulolytic bacteria without a prefeeding feed-enzyme interaction.

Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet.

Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep.

The effects of disodium fumarate on microbial growth, CH4 production and fermentation of three diets differing in their forage content (800, 500 and 200 g/kg DM) by rumen micro-organisms in vitro were studied using batch cultures. Rumen contents were collected from four Merino sheep. Disodium fumarate was added to the incubation bottles to achieve final concentrations of 0, 4 and 8 mm-fumarate, and (15)N was used as a microbial marker. Gas production was measured at regular intervals from 0 to 120 h of incubation. Fumarate did not affect (P>0.05) any of the measured gas production parameters. In 17 h incubations, the final pH and the production of acetate and propionate were increased linearly (P 0.05) were observed for the other two diets. These results indicate that the effects of fumarate on rumen fermentation depend on the nature of the incubated substrate, the high-forage diet showing the greatest response.

/pdf/effects-of-disodium-fumarate-on-in-vitro-rumen-microbial-gr0ik9zzzr.pdf

Effects of disodium fumarate on in vitro rumen microbial growth, methane production and fermentation of diets differing in their forage:concentrate ratio.

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Batch cultures of mixed rumen micro-organisms were used to study the effects of different concentrations of malate (Rumalato(R); Norel & Nature S.A., Barcelona, Spain; composed of disodium malate-calcium malate (0.16:0.84, w/w)) on the fermentation of four cereal grains (maize, barley, wheat and sorghum). Rumen contents were collected from four Merino sheep fed lucerne hay ad libitum and supplemented with 300 g concentrate/d. Rumalato(R) was added to the incubation bottles to achieve final concentrations of 0, 4, 7 and 10 mm-malate. Gas production was measured at regular intervals up to 120 h. Malate increased (P 0.05) for maize. Malate at 4 and 7 mm increased (P<0.05) optical density of the cultures measured at 600 nm for maize, with no differences for the other substrates. The results indicate that malate may be used as a feed additive for ruminant animals fed high proportions of cereal grains, because it increased pH and propionate production and decreased CH4 production and l-lactate concentrations; however, in general, no beneficial effects of 10 compared with 7 mm-malate were observed.

/pdf/effect-of-the-addition-of-malate-on-in-vitro-rumen-3dudzeqpd3.pdf

M. J. Ranilla

Papers

Influence of direct-fed fibrolytic enzymes on diet digestibility and ruminal activity in sheep fed a grass hay-based diet.

Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep.

Effects of disodium fumarate on in vitro rumen microbial growth, methane production and fermentation of diets differing in their forage:concentrate ratio.

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of substrates with different forage:concentrate ratios

Effect of the addition of malate on in vitro rumen fermentation of cereal grains.