Methane emissions and microbial dynamics in ruminants

PhD student: Rajan Dhakal


Thesis defended: 11 March 2024

Ruminants are commonly associated with the production of enteric methane (CH4) emissions due to the anaerobic fermentation that occurs in the rumen. Ruminant agriculture has contributed to global warming, which is a growing concern in society. In recent years, ruminants and the rumen microbiome have emerged as the main focus of scientific research to reduce CH4 emissions. To effectively screen and evaluate potential mitigation strategies for CH4 emissions from ruminants, a rapid, accurate, and precise scientific technique is necessary. The in vitro simulation of the rumen is a promising approach that offers speed and accuracy, although there are some limitations to consider.

This PhD study explores anti-methanogenic strategies and develops in vitro rumen fermentation techniques that simulate the real conditions of the in vivo rumen to reduce the climate impact of ruminant production.

The specific objectives of the PhD study were as follows:

  1. To compare the differences and similarities in the prokaryotic composition and structure between in vivo and in vitro rumen systems (Manuscript I).
  2. To study the temporal dynamics of volatile fatty acid (VFAs), CH4 production, and microbiome composition in the in vitro rumen fermentation systems (Manuscript II).
  3. To evaluate the efficacy of different strategies, including lytic polysaccharide monooxygenase (LPMO) treatment and dose-dependent feed additives, to reduce CH4 production, improve nutrient utilization, and optimize rumen fermentation in the in vitro system (Manuscript III and IV).

An in vitro rumen fermentation series (at least three for each project) was carried out to meet the aforementioned objectives. An anaerobic buffer was prepared with macro- and micro-minerals during each fermentation. Rumen fluid from a fistulated heifers owned by the Large Animal Hospital at the University of Copenhagen and licensed according to Danish law (authorisation nr.2012-15-2934-00648) was used as an inoculation source for in vitro rumen fermentation. Heifers were fasted for 12 h before each collection to maintain uniformity in rumen fluid. Maize silage was used as the basal feed in all projects. The ANKOM system was used to measure gas production, and a gas bag was attached to collect the gas produced during fermentation. Fermentation was stopped based on the experimental design. Filtration was then carried out to calculate the percentage of degraded dry matter, and the filtrate was collected and stored until further analysis of the VFAs and microbiome. GC-FID was used to quantify the CH4 in the total gas and VFAs composition. Amplicon sequences were used to assess each project's microbial composition and structure.

The first study (Manuscript I) showed that the prokaryotic community and structure of rumen fluid before and after fermentation remained similar even after 48 h of incubation, and the in vitro procedure mimicked the in vivo conditions. During in vitro rumen fermentation (Manuscript II), the first 12 h and 36 h are critical for understanding the microbial, metabolite, and fermentation dynamics. The total gas production and degraded dry matter can be independently used to estimate the CH4 emissions on a temporal basis. Bacteriodetes, Campilobacterota, Firmicutes, Proteobacteria, and Spirochaeota showed significant temporal variations. The results of the third study (Manuscript III) showed that pretreatment of maize silage with the LPMO enzyme reduced CH4 production and increased the production of VFAs compared to untreated maize silage. In this study, Bacteroidetes, Firmicutes, Kiritimatiellaeota, Lentisphaerae, Sprirochates, Patescibacteria, and Proteobacteria were strongly correlated with fermentation end-products. The results from study four (Manuscript IV) showed that rumen fermentation pathways (methanogenesis, propionigenesis, and reductive acetogenesis) can be manipulated using inhibitors and electron donors. Di-bromoethanesulfonate decreased CH4 production by more than 95%, decreasing VFAs production without reducing the percentage of degraded dry matter. Increasing the dose of p-hydrocinnamic acid decreased the degraded dry matter (dDM), total gas production (TGP), and CH4 yields. Sodium fumarate dibasic decreased CH4 production and increased total VFAs and propionic acid levels. In all studies (Manuscript I, II, III, and IV), alpha diversity showed similar values for both Shannon and observed indices, and the core microbiomes were Bacteroidetes, Proteobacteria, and Firmicutes taxa. 

The in vitro rumen fermentation technique allowed for a high degree of species diversity, closely resembling the rumen in vivo. Multiple time points are crucial for understanding the temporal microbial dynamics and rumen fermentation parameters, and methane can be predicted on a temporal basis. Pretreatment of MS with LPMO increased tVFA and decreased CH4 production. Rumen fermentation pathways, especially methanogenesis, reductive acetogenesis, and propionigenesis, can be manipulated using different targeted compounds. Despite different treatments, the core bacterial taxa were Bacteroidetes, Proteobacteria, and Firmicutes.