Choline has a marked effect on methanogenesis. The results of the preliminary experiment indicated that, although some methyl compounds did enrich MMC, this did not allow MMC to out-compete other methanogens to the point where they were the only methanogen remaining. In fact, the study showed that choline, after at first (day 1) promoting the rumen methanogen population and CH4 formation at high supplementation level, eventually led to a near complete cessation of the methanogenic activity. Methanogens were negatively affected already from day 2 onwards, as shown by the decline of CH4 production. In order to ensure that the influence on CH4 was due to choline itself, two different chemical forms of choline – ChCl and ChHCO3 – were supplemented. The maximal level of CH4 reduction achieved at 200 mM was nearly the same with ChCl (98%) and ChHCO3 (97%).
The present study henceforth sought to answer how these phenomena can be explained using results from ruminal fermentation and rumen microbiome composition. According to the results of the hNMR analysis, more than 90% of the choline was utilized (Table 2) and likely converted to TMA and acetaldehyde by choline TMA-lyase13. The substantial increase found in TMA concentration in the incubation with both forms of choline along with CH4 mitigation might therefore be an indicator of inhibition of methanogenesis. The supplementation of 200 mM TMA did not reduce CH4 production. Therefore, it is likely that the acetaldehyde as end product of choline metabolism may play an active role leading to the CH4 mitigation, and choline may act as a selector. The group of bacteria possessing choline TMA-lyase would be the first to benefit from acetaldehyde. The choline TMA lyase and its activating enzyme have been identified in the differentially abundant mOTUs clusters, including members of the Anaerovoraceae and Olsenella umbonata enriched by ChCl supplementation and Enterococcus avium, Alkaliphilus spp., Proteus mirabilis/vulgaris and unknown Lachnotalea spp. enriched by ChHCO3. Accordingly, the two forms of choline stimulated entirely different species capable of degrading choline13. E. avium, Proteus mirabilis, unknown Alkaliphilus and unknown Lachnotalea could be the species metabolizing choline in the ChHCO3 group, and Olsenella umbonata and unknown Anaerovoraceae those metabolizing choline in the ChCl group (Fig. 5). All but Lachnotalea possess eut gene clusters associated with choline utilization via microcompartment13–14. Inside microcompartments choline can be metabolized to acetaldehyde and ammonia, the acetaldehyde may subsequently be converted to ethanol and acetate15. All species can produce acetate, but only a subset can produce propionate and butyrate (Table S3). The two Enterococcus species dominant with the ChHCO3 treatment are also predicted to be able to metabolize ethanolamine. The organisms that can cleave choline to TMA and acetaldehyde are presented in Fig. 5 along with the average abundance of each species in each condition. Acetaldehyde enters the central carbon metabolism and thus can lead to the production of lactate, succinate, ethanol and formate, which are alternative H2-sinks, and influence the downstream microbial crosstalk. Figure 5 illustrates simplified pathways of ruminal degradation of choline and of certain structural carbohydrates (xylan, cellulose, pectin) as well as of the production of ethanol, formate, succinate and VFA along with the predicted capability of species of high abundance and species of interest.
Choline affects ruminal NH3 formation, which can potentially inhibit CH4 formation. Theoretically even the very high level of 200 mM choline chloride could be used in livestock nutrition in the European Union, because the responsible organization, the European Food Safety Authority16, has not set a limit for this particular supplement in feed in their regulations. However, the NH3 concentration in the incubation liquid found at 200 mM choline supplementation by far exceeded the critical level for NH3 toxicity of > 110 mM17. The increase in NH3 is likely to come from choline metabolism, but the total amount of NH3-N produced exceeds that added as choline-N, so NH3 is likely also produced from sources other than choline.
The high level of NH3 produced by choline treatment may also contribute to the lowering of CH4 production. At the physiological ruminal pH of 6.5 or lower, almost all NH3 exists in the form of the NH4+ ion18. NH3 can pass through the cell membrane and requires cellular H+ to form NH4+. In methanogens this may divert H+ away from methanogenesis19. When methanogen cultures were inhibited with 400 mM NH4Cl, the cytoplasmic NH3 concentration ranged from 100 mM to above 200 mM19. The ammonia concentration in the choline supplemented Rusitec incubation liquid was well above 400 mM in the current study, which suggests that NH3 may be acting to cause CH4 inhibition.
In a previous study20, there was increased production of N containing microbial compounds, likely due to the improvement in efficiency of synthesis of such compounds when methanogenesis was inhibited, despite decreased OM digestibility. Also, some species identified in the present study are able to ferment amino acids and produce NH3 from them, while some are able to reduce nitrate to NH3, or catabolize ethanolamine to produce NH321. A particularly important role in explaining the NH3 excess in the present study could be attributed to the presence of M. elsdenii (ref_mOTU_v25_01516), its high abundance correlates positively with NH3 concentration (r = 0.801, P < 0.001). This species has been observed to produce NH3 nearly as fast as obligatory amino acid fermenting bacteria22. In the present study, the N retained by NH3 exceeded that of the N input from the feeds provided with the nylon bags, which suggest microbial N fixation may have occurred from the N2 gas used to keep Rusitec anaerobic23. This phenomenon has been described previously24. It is possible that the inhibition of methanogenesis may increase nitrogenase activity, as was found under rice paddy conditions25. However, nitrogenase requires 16 moles of ATP to fix one mole of N226, which makes it inferior in a competitive environment such as the rumen. It is possible to power the nitrogenase by a proton membrane potential via a FixABC membrane complex27, but this operon was only predicted to be present in Proteus mirabilis which does not harbour a nitrogenase reductase complex. Instead, nitrogenase reductase has been identified in Prevotella bryantii and Lachnospira multipara28, and predicted in M. elsdenii, i.e., members of the Lachnospiraceae, Lachnotalea spp. and Anaerovoracaceae spp. Therefore, it is unlikely the N fixation could be channelled by FixABC and the plausibility of N fixation with high choline supplementation requires further study.
Ethanol was among the metabolites most strongly associated with reduced CH4 production. Its high concentration in the incubation liquid suggests that this compound is an important alternative H2-sink29, which could have been a consequence from microbiome adaptation to the absence of methanogenesis. Unlike other alternative H2-sinks such as succinate and lactate that are readily converted to propionate by bacteria, ethanol might have been primarily utilized by the methanogens30–31. Therefore, the lack of methanogens likely led to the accumulation of ethanol.
Alternative electron acceptors, for example sulphate (SO42− + 4 H2 + H+ → HS− + 4H2O, ∆G= -234 kJ), nitrate (NO3− + H2 → NO2− + H2O, ∆G= -161 kJ) and nitrite (NO2− + 3 H2 + 2 H+ → NH4+ + 2H2O, ∆G= -519 kJ) are thermodynamically more favourable than methanogenesis (CO2 + 4 H2 → CH4 + 2 H2O, ∆G= -134 kJ)32. This makes sulphate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) effective in H competition with methanogens, when sufficient substrate is present33 − 34. Both SRB and NRB were detected in the choline supplemented treatments (Fig. 5). Predicted SRB include M. elsdenii, Prevotella spp., Alkaliphilus spp., Prevotella bryantii, Proteus mirabilis35 and Lachnotalea spp. Predicted NRB include Alkaliphilus spp., Proteus mirabilis and Denitrobacterium detoxificans36. Among these organisms, M. eldsenii and D. detoxificans were negatively correlated with CH4 production in the present experiment.
Furthermore, D. detoxificans gains energy by oxidizing nitrogenous compounds such as nitroethane, 2-nitroalcohol and 3-nitro-1-propionate36. These nitrogenous compounds can be accumulated in forages, in particular legumes such as the alfalfa used in the basal diet in the present study and be made available within the rumen37. The N compounds mentioned are known to act as methanogen inhibitors38, and thus may have contributed to the CH4 inhibition observed in the present study.
Although lactate and succinate were not detected in high concentrations in the incubation liquid of the current study, the high abundance of lactate producing lactic acid bacteria (LAB), along with lactate consuming M. elsdenii and its negative correlation to CH4 production suggest strongly that lactate were a prominent H2-sink39 in the present study as well. Both lactate and succinate are intermediate metabolites that can be readily converted to propionate40 and the production of propionate could compete with CH4 production41. However, a significant decrease of propionate was observed in the choline treatments. In previous batch cultures, inhibition of CH4 was accompanied by metabolic H redirected from acetate to propionate; however, continuous cultures like Rusitec behave differently: when CH4 was mitigated by > 50%, generally no overall metabolic H redirection to propionate or butyrate had been observed29,42. It was speculated29 that the H was diverted to other H2-sink or microbial cell mass. This means that lactate may not have been primarily converted to propionate in the present study. Lactate can also be oxidized to pyruvate by an NAD-independent lactate dehydrogenase43 connected to electron bifurcation from electron-transferring flavoprotein Etf, butyryl-CoA dehydrogenase Bcd and ferredoxin/flavodoxin-NAD+ reductase Rnf complex44–45. All of these enzymes are present in M. elsdenii, which may then use the pyruvate to increase microbial cell mass39.
The production of CH4 from H2 by methanogens prevents H2 accumulation and thereby avoids inhibition of fermentation of nutrients via negative feedback loops, especially of fibre where most H2 is produced. Therefore, the inhibition of CH4 production by choline metabolism was expected to have a negative effect on ruminal nutrient degradation as observed earlier in continuous culture experiments29. Hydrolysis of plant structural carbohydrates xylan, cellulose and pectin releases hexoses, which are metabolized via the glycolytic pathways to produces pyruvate, a branching point to acetate, propionate, butyrate, lactate, formate or ethanol production42. The individual steps in the glycolytic pathway are not affected by the increased H2 partial pressure, but the regeneration of NAD+ required for glycolysis is negatively impacted46, and organisms may be driven to use alternative H2-incorporating reactions, such as succinate, lactate and ethanol production which directly regenerates NAD+. In this way, the microbiome is able to adapt, and the surviving microbiome likely harbours alternative H2 incorporating pathways such as lactate or succinate-mediated propionate production31,45. This allows fermentation to continue but at a reduced capacity47.
Assuming the H2 concentration between liquid phase and gas phase is in equilibrium according to Henry’s law, both ChCl and ChHCO3 in fact increased H2 partial pressure (7.1-fold and 16.9-fold H2 accumulation compared to control, respectively), which governs the Gibbs free energy (∆G) of VFA production41. Furthermore, the ∆G must be greater than the minimum amount of energy required for ATP production for the reaction to be viable in bacteria. Therefore, the surviving microbiome after choline treatment is likely capable of decoupling energy production from H2 partial pressure by various means, including usage of alternative H2-sinks.
The richness of the microbiome in the control group after 15 days of operation indicates that Rusitec is a good simulation system for the rumen prokaryotes. The microbiome revealed a decline of Euryarchaeota, i.e., the methanogens, in the choline treatment groups, which corresponds to the reduced CH4 formation. Some methanogens have syntrophic interaction with specific H2 producers via adhesins48. The reduced alpha diversity suggests that syntrophic interaction may have been broken, which could contribute to the reduced CH4 production. All of the most differentially abundant taxa found in the present study are either able to utilize H2 and produce metabolites such as lactate and ethanol, or they are able to make use of the alternate H2-sink metabolites produced by other bacteria. It is unknown whether the use of alternative H2 sinks is the result of CH4 reduction, or a contributor to CH4 reduction.