Methane is a potent greenhouse gas and also represents a loss of gross feed energy for the ruminant, hence the increasing interest in strategies to manipulate CH₄ production (Johnson and Johnson, 1995; Gerber et al., 2013). Archaea produce CH₄ in the rumen mainly by reducing C₁ compounds with H₂ (among others substrates) thus maintaining a low hydrogen partial pressure (Janssen, 2010). Although methanogens are the main H₂ consumers, due to the thermodynamically favorable pathway of CH₄ formation, there are rumen bacteria, which are also able to use H₂ as substrate thus generating alternative end products (Leng, 2014). Thus, when CH₄ production is decreased, [H] might be redirected into alternative sinks nutritionally useful for the ruminant (Ungerfeld, 2015a). However, an excessive increase of the H₂ partial pressure in the rumen after methane inhibition might have detrimental effects on rumen function (Wolin et al., 1997). Therefore, management of H₂ accumulation in the rumen is an important strategy to consider when inhibiting CH₄ emissions from livestock.

A recent published study (Martinez-Fernandez et al., 2016), using chloroform as an antimethanogenic compound, has examined the [H] flow and rumen microbial community responses in cattle fed with hay or supplemented with concentrate. It showed that a CH₄ reduction of about 30% had no detrimental effects on rumen function in cattle. With both diets a decrease in CH₄ production was accompanied by an increase in H₂ expelled. Interestingly, the animals fed with the concentrate diet eructated relatively more H₂ than the hay diet suggesting inefficient redirection of [H] into other fermentation end products. The main changes observed in rumen fermentation metabolites with both diets were a shift toward propionate and branched-chain fatty acids; an accumulation of formate, particularly with the concentrate diet; and an increase in the concentration of amino acids, organic and nucleic acids. However, although a redirection of [H] was observed, it was predicted that dietary supplements would be needed to increase the capture of excess H₂ via alternative energy yielding metabolic pathways (Martinez-Fernandez et al., 2016). Failure to capture the excess H₂ released from the system should be considered as an energy loss to the animal.

Microbial metabolic processes, such as reductive acetogenesis, propionogenesis, reduction of nitrate and sulfate, formate formation, and an increase of microbial biomass production when CH₄ is inhibited, have been identified as nutritionally useful [H] sinks in the rumen (Newbold et al., 2005; van Zijderveld et al., 2010; Leng, 2014; Gagen et al., 2015; Ungerfeld, 2015b). Another [H] sink which has gained little attention is the reduction of phenolic compounds by rumen microorganisms. Phenolic compounds, such as flavonoids, are present in many of the forage plants consumed by ruminants. Rumen bacteria, in some cases are capable of transforming these compounds into energy yielding products for the host (Murdiati et al., 1992; McSweeney et al., 2001). Flavonoids undergoing microbial degradation in the rumen generally form phloroglucinol as an intermediate metabolite (Supplementary Figure 1). Several studies have shown that specific rumen bacteria are able to reduce phloroglucinol using H₂ or formate as electron donors thus yielding acetate as the terminal product (Tsai and Jones, 1975; Patel et al., 1981; Krumholz and Bryant, 1986). However, there is scarce information about the use of unconventional dietary treatments that may stimulate rumen microorganisms capable of capturing the [H] available when CH₄ is inhibited in vivo.

The aims of the present study were to analyze the effect of phloroglucinol on CH₄ production, [H] flows and subsequent responses in rumen fermentation and microbial community composition in cattle when methanogenesis is inhibited. It was hypothesized that less H₂ would be expelled by the chloroform + phloroglucinol-treated animals due to a shift in fermentation toward acetate by the reduction of the phenolic compound.

The experimental protocol complied with the Australian Code for the Care and Use of Animals for Scientific Purposes (eighth edition, 2013) and was approved by the local Animal Experimentation and Ethics Committee (A08/2014).

Inhibition of methanogenesis in ruminants using halogenated compounds has previously shown a redirection of [H] toward propionate and branched-chain fatty acids with increased H₂ eructation from the rumen (Mitsumori et al., 2012; Martinez-Fernandez et al., 2016). Likewise, the administration of chloroform to animals in this trial produced a similar response, thus providing a model system for testing the ability to alter rumen metabolic pathways through the capture of excess H₂. A number of alternative [H] sinks have been identified for when methanogenesis is inhibited in ruminant livestock such as: acetogenesis (Joblin, 1999; Fonty et al., 2007; Gagen et al., 2015), propionogenesis (Newbold et al., 2005), nitrate reduction to ammonia (Morvan et al., 1996; Anderson et al., 2003; van Zijderveld et al., 2010; Latham et al., 2016), sulfate reduction to hydrogen sulfite (van Zijderveld et al., 2010), formate formation (Leng, 2014), and increases of microbial biomass production (Ungerfeld, 2015b). However, there is scarce information about the amount of [H] redirected to these metabolic processes or emitted as H₂ gas under in vivo conditions. While the rumen microbiota can adapt to inhibition of CH₄ formation by redirecting a proportion of [H] into energy-yielding metabolites, an important proportion of the unutilized [H] accumulates and is eructated as H₂ gas, which still represents an energy loss to the animal. This study demonstrates for the first time in vivo that under methanogenesis inhibition, H₂ gas accumulation can be reduced by redirecting [H] toward alternative sinks through the nutritional stimulation of microbial groups that generate metabolites of value to the host. Therefore, the use of dietary treatments to promote key rumen microbial groups, may be a practical way of capturing [H] that would normally be lost as H₂ gas when a strategy of directly inhibiting methanogens in the rumen is employed to reduce CH₄ formation.

Interestingly, an increase in daily weight gain was observed for the chloroform + phloroglucinol treated animals, indicating that the [H] redirection toward an alternative sink facilitated the utilization of a substrate, which is not normally regarded as an energy-yielding nutrient for the host. Although the increase in weight gain is a promising result, it should be interpreted with caution due to the relatively short length of the trial and small number of animals used. A recent trial with dairy cattle using 3-nitrooxypropanol to reduce CH₄ emissions produced an 80% increase on the average body weight gain over the experimental period (Hristov et al., 2015). While this most likely indicates a shift in metabolism to end products of greater nutritional or metabolic value, the authors also observed a 64-fold increase in hydrogen emissions, suggesting that further efficiencies could be gained. However, to confirm this hypothesis further in vivo research on rumen digestibility and metabolism should be carried out.

In relation to the redirection of [H], the inclusion in the rumen of the phenolic substrate phloroglucinol resulted in greater acetate formation with less H₂ expelled per mole of CH₄ decreased, and decreased formate concentration in the rumen. This is in agreement with in vitro studies (Patel et al., 1981; Krumholz and Bryant, 1986) that have shown that rumen microorganisms were able to metabolize phloroglucinol to acetate by consuming H₂ or formate as reducing agents. It should be noted that the formula for calculation of the [H] used in SCFA formation (Martinez-Fernandez et al., 2016) does not account for the acetate produced through reductive processes, such as acetogenesis or reduction of phenolic compounds, which most likely explains why there was no difference in the calculated flow of [H] into these organic acids between the treatments. Chloroform + phloroglucinol treated animals expelled per day 1.42 mol of H₂ less than the chloroform only treated group, which is lower than the predicted moles of H₂ (2.18) used to degrade the amount of phloroglucinol provided per day (1 molecule phloroglucinol + 1 molecule H₂ = 2 molecules acetate + 2 molecules carbon dioxide (Tsai et al., 1976; Conradt et al., 2016) (Supplementary Figure 1). This discrepancy might indicate that phloroglucinol was not totally metabolized, however, near complete degradation of similar quantities of phenolic compounds have been reported previously (Murdiati et al., 1992). It is more likely that both formate and H₂ were used to reduce phloroglucinol based on the lower formate concentrations in the chloroform + phloroglucinol treated animals (0.83 vs 11.3 mM) compared with chloroform only. Formate is produced during normal rumen fermentation and is mainly consumed by methanogens to produce CH₄ (Hungate et al., 1970; Asanuma et al., 1998). Similar to the current study, an increase in formate has been reported previously in steers which were treated with chloroform alone to reduce methanogenesis, particularly with concentrate-supplemented diets (Martinez-Fernandez et al., 2016). Our results support the hypothesis that increased formate accumulation represents a response by the rumen microbiota to control H₂ partial pressures in the rumen, by acting as a hydrogen sink (Leng, 2014; Ungerfeld, 2015b; Martinez-Fernandez et al., 2016).

Members of the Synergistetes phylum are sensitive to H₂ partial pressures within the rumen and generally are reduced when the H₂ partial pressure increases or methanogenesis is restricted (Denman et al., 2015; Wallace et al., 2015; Martinez-Fernandez et al., 2016). Similar reductions in the Synergistetes phylum were observed in this study with the chloroform-only treatment and increased H₂ partial pressures. The addition of phloroglucinol and redirection of [H] led to lower partial pressures of H₂ and probably resulted in increases in the Synergistetes phylum further supporting their significance as a key indicator group around hydrogen transactions.

Of particular interest, was the greater increase in formate concentration when the animals were exposed to longer periods of chloroform, allowing for subsequent increases in the acetate:propionate ratio, presumably driven by the decrease in H₂ partial pressures. Fibrolytic Ruminococcus species are able to alter their fermentative pathways to provide alternative reducing equivalent sinks, including the production of formate and ethanol to relieve the inhibition of the reoxidation of NADH (Miller and Wolin, 1974; Shi et al., 1997). With the higher partial pressures of H₂ observed in the rumen for the chloroform treated animals, the shift to increased formate might be linked with increases of Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, and M. ruminantium species. Methanogen species capable of utilizing formate are also able to produce formate from CO₂ and H₂ under conditions of high H₂ partial pressures and when inhibited with halogenated hydrocarbons (Thiele and Zeikus, 1988; Bleicher and Winter, 1994). The observed increase in the relative abundance of M. ruminantium over the predominant rumen methanogen M. gottschalkii, which is incapable of formate utilization, likely reflects the activation of this mechanism. By converting H₂ and CO₂ to formate while H₂ is in excess and methanogenesis is impeded may ensure survival until conditions become more favorable for producing CH₄, thus providing M. ruminantium with a competitive advantage over non-formate utilizing methanogens. The addition of phloroglucinol altered these conditions by redirecting [H] away from formate and reducing the H₂ partial pressure which allowed M. gottschalkii to outcompete M. ruminantium and once again become the dominant methanogen species in this system. The changes in the microbiota illustrate the ability of the rumen to approach a new equilibrium by adapting to the altered environmental conditions. Of course, this also means that these changes might not correspond to a fully adapted and stable rumen microbiome as is illustrated by the continued shift in the microbiome for the chloroform only group between days 21 and 37. Longer trial periods would need to be studied to fully answer if the rumen has reached a stable microbiota.

Phloroglucinol is a phenolic compound, formed through the degradation of complex plant molecules such as flavonoids, which are commonly present in the diet of the grazing ruminant (Tsai et al., 1976). It is also present at high levels in brown algae (Ecklonia cava) which is widely harvested for its medicinal effects (Park et al., 2012). In the rumen, microorganisms degrade this compound predominately to produce acetate and CO₂ as end-products. Our findings are in accordance with previous in vitro studies (Tsai et al., 1976; Patel et al., 1981; Krumholz and Bryant, 1986), which observed this increase in acetic acid concentration when phloroglucinol was metabolized by rumen microorganisms. Several rumen bacteria have been identified as phloroglucinol degraders and were classified as Eubacterium oxidoreducens, Streptococcus bovis, and Coprococcus spp. (Tsai et al., 1976; Patel et al., 1981; Krumholz and Bryant, 1986). Krumholz and Bryant (1986) reported that E. oxidoreducens required H₂ or formate to degrade phloroglucinol to acetate. The analysis of the rumen microbiome in this study did not identify E. oxidoreducens but did reveal several OTUs assigned to Coprococcus spp. increased within the chloroform + phloroglucinol treated animals, suggesting the genus is a significant contributor to the metabolism of this phenolic compound. Tsai et al. (1976) found that Coprococcus spp. were able to degrade 1 molecule of phloroglucinol to 2 molecules of acetic acid and 2 molecules of carbon dioxide. A more detailed study by Patel et al. (1981) showed that phloroglucinol is initially reduced to dihydrophloroglucinol by Coprococcus spp. using NADPH as the electron donor. This would help alleviate the increase in NADPH in the rumen generated by the inhibition of methanogenesis through its oxidation to NADP⁺ (Chalupa, 1977). Furthermore, a recently published study (Conradt et al., 2016) identified three phloroglucinol reductases belonging to the family of NADPH dehydrogenases/reductases involved in the anaerobic degradation of phloroglucinol which involves hydrolytic ring cleavage to 3-hydroxy-5-oxohexanoic and then acetate formation. However, to confirm our hypothesis, further studies using metagenomics and transcriptomic approaches should be carried out to evaluate the functional genes in Coprococcus spp. associated with the NADPH-dependent reduction of phloroglucinol and the redirection of H₂ in the rumen. Furthermore, as the limitations of the trial did not allow for the inclusion of a phloroglucinol only treatment, we cannot ascertain if these effects may have also been possible in the presence of functional methanogenesis and lower H₂ partial pressures. Indeed, a calculated ΔG° of -158 kJ/mol phloroglucinol to acetate indicates this reaction is likely to be very favorable thermodynamically and may even occur in the presence of hydrogenotrophic methane formation (Kaiser and Hanselmann, 1982; Krumholz and Bryant, 1986).

The Coprococcus genus is also involved in other important metabolic pathways in the rumen. Recently, Shabat et al. (2016) found a greater proportion of Coprococcus species (Coprococcus catus) in the rumen of dairy cows with lower CH₄ emissions and higher feed efficiencies. In the more feed efficient cows there were an increase in abundance of genes aligned to the acrylate pathway, which involves the conversion of lactate to propionate, and were annotated as C. catus. In general, the acrylate pathway rather than the succinate pathway for propionate production was more dominant in the efficient animals (Shabat et al., 2016). Other metabolic pathways in which Coprococcus spp. are also involved, is the degradation of nitrotoxins in the rumen (Majak and Cheng, 1981) through reductive processes. These and our findings suggest that Coprococcus genus is involved in key metabolic pathways in the rumen, and they should be considered when developing strategies to promote microorganisms which might improve the rumen efficiency and metabolize plant toxins in ruminants.

The present study in cattle demonstrated that under anti-methanogenesis conditions the addition of phloroglucinol into the rumen can stimulate microbial groups which utilize H₂ and formate as reductants in the metabolism of phloroglucinol to acetate and thus decrease the partial pressure of H₂ in the rumen and the amount of this gas eructated by the animal. This demonstrates that the negative impact of H₂ accumulation in the rumen under suppression of methanogenesis can be ameliorated by the provision of novel compounds that have nutritional value for the animal when degraded by reductive processes.

CM, SD, and GM-F conceived and designed the experiments and analytical approaches. GM-F performed the animal trial. GM-F and JC analyzed the biological samples. SD and GM-F analyzed the data. GM-F, CM, and SD wrote the manuscript. All authors agree to be accountable for all aspects of the work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.