In the past few decades, we have learned valuable information about the rumen ecosystem, but we are still far from unveiling the complex interplay between the different microbial groups and their functions. Microbiology and the laws of kinetics and thermodynamics can explain the majority of what we’ve learned thus far, but not all. New advances in “omics” and mixed-microbe culturing techniques are expected to improve our knowledge exponentially. This blog is inspired by my recent readings, namely “The Role of Thermodynamics in the Control of Ruminal Fermentation” by Ungerfeld and Kohn (2006).
Ruminants have a unique ability to degrade fibrous materials (“poor” quality feed) that are considered indigestible by other monogastric animals, such as pigs and poultry. The ruminants’ digestive system doesn’t have the enzymatic capacity to degrade complex fibrous structures but instead relies on diverse microbial groups residing in the rumen to break down these structural carbohydrates. This symbiotic relationship between the ruminal microbiota and the host provides, synergistically, the needed energy for both the host animal and the residing microbiota. Any external factors that lead to “dysbiosis” of this ecosystem will impair the health and productivity of the ruminant animal.
Complete oxidation of organic materials in living cells is only possible in the presence of oxygen. The end products of complete oxidation are CO2, water, and heat. Electrons generated from oxidation are transferred to O2 as the final electron “acceptor.” This process deems O2 as essential for energy generation (ATP generation) in aerobic metabolism. However, the rumen microbiota evolved as anaerobic microbes, where the presence of oxygen is not required but for the most toxic. Some facultative bacteria that tolerate oxygen do survive in the rumen and play a role in “scavenging” oxygen dissolved in water and feed, thus improving the anaerobic conditions of the rumen.
Anaerobic microbes have evolved alternative fermentative pathways to harness energy (ATP) from substrates such as cellulose without the need for oxygen. The terminal electron acceptors in the fermentation processes are the organic compounds produced in the process itself. Electron acceptors can be metabolic intermediates of fermentation (formate, lactate, fumarate, acrylyl-CoA, acetoacetyl-CoA, crotonyl-CoA, CO2,..) and fermentation end products (short-chain fatty acids, SCFA’s).
Fermentation is considered an incomplete oxidation of substrates by the microbes. It is estimated that anaerobic metabolism is up to 15 times less efficient than aerobic metabolism at generating energy. Thus, the end products of fermentation (SCFA’s) are energy-dense and, once absorbed by the digestive system of the host, constitute the primary energy source for the host animal. Gases (mainly CO2 and CH4) and heat produced by fermentation are released and deemed “unuseful.” In addition, the microbial biomass, after being washed from the rumen, digested, and absorbed in the small intestine, makes the main source of nitrogen and protein for the host. Microbial protein is the most economical source compared with exogenous feed sources such as soybean meal. Thus, maintaining optimal ruminal conditions for carbohydrate digestion can reduce the need for expensive protein supplements. It is important to note that fermentation is one type of anaerobic respiration and that other types such as nitrate, sulphate, and sulphur respiration do exist in the rumen but will not be discussed here.
The quantity and profile of SCFA’s have a direct impact on the productivity and metabolism of the host animal and are influenced by feed intake, type of feed, feeding management, type of animal, rumen microbiome (collective genome or meta-genome), and others ecological factors. Acetate is the most abundant SCFA on a molar basis followed by propionate and butyrate during high-forage feeding. However, propionate is proportionally elevated during high-grain feeding. It is important to note that propionate as an end product retains more energy than acetate. Thus, it is energetically more useful to the host animal. On the other hand, propionate fermentation, depending on the pathway, may yield less energy (ATP) for microbial growth than acetate. Therefore, fermentation “efficiency” implied by a greater propionate fermentation should not be mixed with microbial ATP generation “efficiency” (Ungerfeld, 2020).
There are very complex kinetics and thermodynamics regulations in the rumen that dictate metabolic pathways and thus end product production and inter-conversion. Classical biochemistry and microbiology studies have revealed significant differences among microbes in substrate utilization, enzymatic activities, and fermentation end products. These studies are now being integrated with new information from metagenomic, metatranscriptomic, and proteomic to enable us to interpret results and improve our understanding of the rumen environment.
There are several excellent reviews that shed light on the importance of integrating rumen thermodynamics, kinetics, and microbiology in order to improve our understanding of the rumen ecosystem. For example, “The Role of Thermodynamics in the Control of Ruminal Fermentation” by Ungerfeld and Kohn (2006) and “Metabolic Hydrogen Flows in Rumen Fermentation: Principles and Possibilities of Interventions” by Ungerfeld (2020). The readers are recommended to refer to the original articles for more information. Below, I will summarize some of the key mechanisms related to the topic discussed.
Electrons from acetate and butyrate fermentative pathways are transferred via intracellular electron carriers to form dihydrogen (H2). This process is catalyzed by H2-evolving hydrogenase enzymes, which are widely encoded by genes in fermentative microbiota. Theoretically, dihydrogen accumulation could impair fibre fermentation. However, H2 plays a role as an intercellular electron carrier where H2 generated by certain fungi, bacteria or protozoa is utilized by other microbes. Methanogens are the main H2 utilizers in the rumen (Hungate, 1967), where CO2 is reduced to CH4 as the end product. The formation of CH4 generates ATP necessary for methanogens’ growth and serves as a very important H2 “sink” in the rumen ecosystem. Methanogens have the lowest threshold for H2; therefore, they outcompete most other H2-incorporating microbes (Kohn and Boston, 2000). Methane is considered wasted energy to the animals (2-12% of ingested gross energy) (Johnson and Johnson, 1995) and a potent greenhouse gas.
Nonetheless, not all fermentative pathways are associated with a net production of metabolic hydrogen. For example, fermentation of starch to propionate as the end product is associated with a net uptake of H2. Thus, propionate production competes with CH4 as a metabolic hydrogen sink (Janssen, 2010). Hence, SCFA profile is closely associated with CH4 formation (Ungerfeld, 2020). High acetate to propionate ratio and high CH4 (% of total gas) implies a lower fermentation efficiency, but maximal ATP microbial generation. However, Hackmann et al. (2017) demonstrated in their study that a number of bacterial species encoded for “incomplete” glycolytic pathways in addition to several “alternative” pathways of carbohydrate metabolism. For example, acetate formation via the Bifidobacterium pathway was not associated with metabolic hydrogen production nor incorporation.
Suppressing methanogens by chemical agents has been shown to shift fermentation pathways towards propionate formation (Janssen 2010), which in turn is energetically valuable for glucose production in the liver of the host ruminant. The efficacy of such strategies has been variable, but recent studies have shown a potential for 3-nitrooxypropanol to reduce methane emissions in dairy cattle, for example, Melgar et al. (2020). Therefore, to optimize ruminal fermentation, for the benefit of both the microbiota and the host animal, strategies should focus on investigating non-hydrogen producing fermentation pathways and/or alternative hydrogen sinks to divert away from CH4 formation. In practice, optimizing rumen function reflects positively on farm profitability and the health of both the animal and the environment. In conclusion, there is a need for more integrative studies to maximize ruminant productivity in different feeding systems and to minimize environmental impact. These studies should comprehensively and concurrently measure SCFA, gas production, and microbial parameters in addition to performance indices. These studies should employ our recent understating of ruminal thermodynamics and recent “omics” data.
References
Hackmann, T. J., D. K. Ngugi, J. L. Firkins, and J. Tao. 2017. Genomes of rumen bacteria encode atypical pathways for fermenting hexoses to short-chain fatty acids. Environ. Microbiol. 19:4670-4683.
Hungate, R. E. 1967. Hydrogen as an intermediate in the rumen fermentation. Arch. Mikrobiol. 59:158-164.
Janssen, P. H. 2010. Influence of hydrogen on rumen methane formation and fermentation balances through microbial growth kinetics and fermentation thermodynamics. Anim. Feed Sci. Technol. 160:1-22.
Johnson, K. A. and D. E. Johnson. 1995. Methane emissions from cattle. J. Anim. Sci. 73:2483-2492.
Kohn, R. and R. Boston. 2000. The role of thermodynamics in controlling rumen metabolism. in Modelling Nutrient Utilization in Farm Animals J.P. McNamara, J. France, and D. E. Beever, ed. CAB International.
Melgar, A., K. C. Welter, K. Nedelkov, C. M. M. R. Martins, M. T. Harper, J. Oh, S. E. Räisänen, X. Chen, S. F. Cueva, S. Duval, and A. N. Hristov. 2020. Dose-response effect of 3-nitrooxypropanol on enteric methane emissions in dairy cows. J. Dairy Sci. 103:6145-6156.
Ungerfeld, E. and R. Kohn. 2006. The Role of Thermodynamics in the Control of Ruminal Fermentation. Pages 55-85 in Ruminant physiology: Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress. K. Sejrsen, T. Hvelplund, and M. O. Nielsen, ed. Wageningen Academic Publishers, Wageningen, the Netherlands.
Ungerfeld, E. M. 2020. Metabolic hydrogen flows in rumen fermentation: Principles and possibilities of interventions. Front. Microbiol. 11.
Credits: Image used under permission from Sheri Amsel (www.exploringnature.org)