Most experiments used in this meta-analysis reported valerate production. In batch cultures, the participation of valerate as a [H] sink slightly decreased from 4.8 to 4.5% between predicted 0 and 100% methanogenesis inhibition (not shown). In continuous cultures, valerate increased its participation as a [H] sink from 7.9 to 10.7% of total [2H]_produced between predicted 0 and 100% methanogenesis inhibition (not shown). Caproate is an end product of fermentation even more reduced than valerate. Caproate was reported only in the study by Anderson et al. (2010) in batch culture, and tended to increased its participation from 2.2 to 4.8% of total [2H]_produced between predicted 0 and 100% methanogenesis inhibition (not shown). Formate was reported in only 6 batch culture experiments, and at predicted 100% methanogenesis inhibition it accounted for an average of 16% of [H] in CH₄ in the corresponding control treatments (not shown). This figure is close to the missing [H] in CH₄ found in formate in recent experiments, which agree with the estimation obtained by Hungate et al. (1970) of 18% ruminal CH₄ formed from formate (S. Muetzel, pers.com). Immig (1996) reported no alterations in major fermentation products when formate was added to methanogenesis-inhibited incubations, suggesting that formate accumulated and was not further metabolized. In the continuous culture experiment by Slyter (1979), there was a slight formate accumulation when methanogenesis was inhibited using dichloroacetamide which accounted for little [2H]_incorporated. Metabolic hydrogen incorporation into succinate, ethanol and lactate accounted for little [2H]_incorporated both in batch and continuous cultures (not shown). It is important that formate and other atypical reduced end products of fermentation, apart from H₂, as well as the more reduced VFA valerate and caproate, are reported in experiments in which methanogenesis is inhibited.

Czerkawski (1986) proposed that inhibiting methanogenesis could favor microbial biomass production as an alternative [H] sink. Chalupa (1977) suggested that [H] incorporated into excess NADH was redirected to fatty acids synthesis and fermentation end products such as lactate and ethanol, although the latter sinks were not quantitatively important in the experiments that reported them in the present analysis (not shown).

Microbial biomass production depends on the rates and efficiencies of ATP generation (catabolism) and utilization for growth (anabolism). Inhibiting methanogenesis decreases the system's reducing potential through greater availability of reducing equivalents in electron donors such as H₂ (Sauer and Teather, 1987) and NADH (Hino and Russell, 1985). If anabolic processes that incorporate reducing equivalents, such as fixation of NH⁺₄ into carbon chains for amino acids synthesis, and synthesis of fatty acids, were kinetically limited by the availability of [H] (or were close to thermodynamic equilibrium), increased availability of [H] consequence of inhibiting methanogenesis could stimulate amino acids and fatty acids synthesis and therefore microbial growth. Stimulation of microbial anabolism could then increase the importance of microbial biomass as a [H] sink through two mechanisms: (i) Greater microbial biomass production; (ii) A more reduced microbial biomass composition: if microbial content of lipids increased, that would mean that more [H] was incorporated per gram of microbial organic matter produced.

Inhibiting methanogenesis could also have consequences on microbial ATP generation. In anaerobic systems like the rumen, part of the negative Gibbs energy change associated to fermentation is used to generate ATP through substrate level and electron transport-linked phosphorylation. An inhibition of FH, as in most batch culture experiments in the present meta-analysis, should decrease ATP generation. Apart from being affected by FH, ATP generation can be affected by the fermentation profile. It has been shown in experiments with defined cultures that the presence of methanogens alters the pattern of fermentation of bacteria and fungi. A shift from acetate, ethanol, H₂ and CO₂ produced by Ruminococcus albus in pure culture, to acetate and CH₄ in co-culture with a methanogen, increased ATP generation by R. albus and added the ATP generated by the methanogen (Wolin et al., 1997). Similarly, ruminal anaerobic fungi decreased the production of ethanol and lactate, and increased the production of acetate, when grown with methanogens, increasing ATP generation (Bauchop and Mountfort, 1981; Marvin-Sikkema et al., 1990).

However, when inhibiting methanogenesis in mixed cultures, accumulation of ethanol seems of small importance compared to pure cultures of ethanol producers. In the present meta-analysis, there was an increase in [H] incorporated into propionate production in batch cultures when methanogenesis was inhibited. This agrees with greater production of succinate and propionate by R. flavefaciens or Selenomonas ruminantium, respectively, when grown in pure culture than when co-cultured with methanogens (Wolin et al., 1997). It is important to understand what could be the consequences of an acetate to propionate shift on microbial ATP generation.

Propionate is produced via succinate (randomizing pathway) or acrylate (non-randomizing pathway) in the rumen. Reduction of fumarate to succinate is associated with ATP generation through a transmembrane electrochemical gradient (Russell and Wallace, 1997), but the acrylate pathway does not seem to be associated with ATP generation (Thauer et al., 1977). The proportions of extra propionate produced through the succinate and acrylate pathways when inhibiting methanogenesis are unknown, and therefore constitute an uncertainty of the consequences of an acetate to propionate shift upon ATP generation. Furthermore, the stoichiometry of ATP generation per pair of reducing equivalents incorporated in the reduction of fumarate to succinate is not clearly established as measurements have disagreed (Reddy and Peck, 1978; Kröger and Winkler, 1981).

Generation of ATP in propionate's randomizing pathway is also affected by other metabolic steps. Oxaloacetate (or malate) formation from phosphoenolpyruvate (PEP) entails the utilization of PEP high-energy phosphate bond, which is then not used to generate ATP, but when catalyzed by PEP carboxykinase, PEP carboxylation can still generate ATP or GTP without additional energy input (Atwal and Sauer, 1974). Also, when the entire randomizing pathway takes place in a single cell (i.e., without interspecies transfer of succinate), carboxylation of pyruvate to form oxaloacetate can occur coupled to methylmalonyl-CoA decarboxylation to propionyl-CoA, making oxaloacetate formation from pyruvate energetically neutral (Deborde and Boyaval, 2000).

Therefore, our knowledge about ATP generation by the mixed ruminal microbiota is incomplete due to the difficulties to determine ATP generation associated to propionate production. For the moment, it seems that we will have to rely on the few existing empirical results on the response of microbial growth to methanogenesis inhibition rather than on a deeper mechanistic understanding on the consequences of methanogenesis inhibition on microbial ATP generation and utilization. Improved microbial growth efficiency allowed obtaining increased microbial biomass with methanogenesis inhibition even with decreased true organic matter digestibility (Ungerfeld et al., 2007). Other in vitro work also found stimulation of microbial growth by methanogenesis inhibition (van Nevel et al., 1969; Guo et al., 2009). Nolan et al. (2010) reported a numerical increase of 27% in microbial protein production in sheep whose CH₄ production was decreased by 23%. However, other work has found no effect or even a decrease in microbial growth with methanogenesis inhibition (van Nevel et al., 1969, 1971; Russell and Martin, 1984; Lee et al., 2009). Apart from biomass accretion, a more reduced microbial biomass composition could account for part of the “missing [H].” Changes in microbial biomass amount and composition, shifts in anabolic pathways, and ultimately ATP generation and utilization, as a response to methanogenesis inhibition, need to be studied.

If some acetate produced when methanogenesis is inhibited was originated from reductive acetogenesis, part of acetate production would be an unaccounted [H] sink. In the typical ruminal fermentation, methanogens drop H₂ pressure to a level at which reductive acetogenesis has been estimated to be thermodynamically unfeasible (Kohn and Boston, 2000) and as a result the process does not occur (le Van et al., 1998). However, reductive acetogenesis has been shown to occur in batch cultures if methanogenesis is inhibited and reductive acetogens are added (Nollet et al., 1997; le Van et al., 1998). Native reductive acetogens do inhabit the rumen even when methanogenesis is not inhibited (Leedle and Greening, 1988; Henderson et al., 2010), but they seem to rely on substrates other than CO₂ and H₂ (Joblin, 1999). It is possible that reductive acetogenesis could over time become a [H] sink in continuous culture and in vivo when methanogenesis is inhibited. It is unknown whether or not some reductive acetogens can adapt to methanogenesis-inhibited continuous culture conditions to conduct reductive acetogenesis. In the experiments used for this meta-analysis, acetate produced through fermentation of carbohydrates would be indistinguishable from acetate produced from reductive acetogenesis, if the latter occurred. Therefore, decreases observed in acetate production with methanogenesis inhibition (data not shown) are not evidence against the possibility of reductive acetogenesis.

Redirection of [H] toward propionate production when methanogenesis was inhibited was more favored in batch than in continuous cultures. Perhaps, important propionate producers do not survive well, or at least are not stimulated as much by methanogenesis inhibition, in continuous compared to batch culture. Decline in microbial diversity over time in continuous cultures has been reported (Johnson et al., 2009). For example, Broudiscou et al. (2014) reported that the effect of H₂ pressure on propionate production per mol of FH in batch cultures depended on the source of inoculum, which were continuous cultures incubated with different substrates. Because they studied the effect of added H₂ on short-term, 6 h batch incubations, the presence of sufficient numbers of propionate producers in their continuous culture inocula may have determined the response of propionate to H₂. In fact, the greatest response in propionate to headspace H₂ in the study by Broudiscou et al. (2014) came from the inoculum obtained from a continuous culture fed a roughage substrate which produced more propionate per mole of FH than two other continuous cultures fed different high concentrate substrates.

It is possible that some propionate or succinate producers do not adapt well to continuous cultures to take advantage of the favorable thermodynamic conditions for propionate production occurring when methanogenesis is inhibited. Differences between batch and continuous cultures have been reported for some succinate-producing bacteria with regard to their response to methanogenesis inhibition. For example, inhibiting methanogenesis resulted in an increase in Fibrobacter succinogenes in ruminal batch cultures (Guo et al., 2007, 2008; Goel et al., 2009), but no changes in continuous cultures (Goel et al., 2009), although lack of effects in batch cultures has also been reported (González et al., 2006). R. flavefaciens was either decreased (Goel et al., 2009) or unaffected (González et al., 2006; Guo et al., 2007, 2008) by inhibiting methanogenesis in batch cultures, and decreased in continuous cultures (Goel et al., 2009).

An alternative explanation to greater response in propionate in batch than in continuous culture could be based on thermodynamics. Most batch culture systems operate with pressurized tubes or bottles, allowing greater H₂ pressure at equal H₂ molar percentage in comparison to continuous cultures, where gas is collected at nearly atmospheric pressure. Elevation of H₂ pressure favors a shift from acetate to propionate production (Janssen, 2010). Based on this technical difference between most batch and continuous cultures, it could be expected that batch cultures favor more a shift to propionate compared to continuous cultures. Indeed, there was a negative quadratic relationship between the estimated final H₂ pressure in batch cultures and the acetate to propionate molar ratio (R² = 0.62; P < 0.001; not shown). Theoretically, batch incubation systems that allow the release of gas to the atmosphere (Wang et al., 2013) should better mimic the in vivo situation from a thermodynamics point of view.

Despite most batch culture systems resulting in supra-atmospheric total gas pressures, inhibition of methanogenesis resulted in greater H₂ accumulation in batch than in continuous culture. This could suggest some adaptation over time of continuous cultures to incorporate part of the accumulated H₂ into other pathways not modeled in the present analysis e.g., reductive acetogenesis. In agreement, a progressive decrease in H₂ accumulation in batch cultures from 24 to 48 and 72 h of incubation was observed when methanogenesis was inhibited with 2-bromoethanesulphonic acid (Lee et al., 2009). Still, mid- and long-term H₂ accumulation has been reported to occur in continuous culture with high-concentrate substrates even in the absence of methanogenesis inhibitors (Broudiscou et al., 2014), as well as in various methanogenesis inhibition in vivo experiments (Trei et al., 1972; Clapperton, 1974; Kung et al., 2003; van Zijderveld et al., 2011; Mitsumori et al., 2012), and incubations of rumen contents from animals administered methanogenesis inhibitors for long periods (Czerkawski and Breckenridge, 1975). Therefore, it seems that, although there can be some long term adaptation of microbiota to incorporate [H] into alternative pathways in the methanogenesis-inhibited ruminal fermentation, this is not complete and consistent, and H₂ remains as an end product of fermentation (with the possible exception of the use of some lipids as methanogenesis inhibitors, as discussed below).

It is difficult to conclude on whether the batch or continuous culture system might better reflect changes in fermentation occurring in vivo when methanogenesis is inhibited. If there is some long-term adaptation of microbiota to incorporate H₂ into other pathways, the continuous culture systems may better represent the in vivo situation in comparison to batch culture. On the other hand, the slight overall increase in propionate production observed in batch culture may better reflect the increase in propionate concentration or molar proportion of most experiments when methanogenesis is inhibited in vivo (Clapperton, 1974; Davies et al., 1982; McCrabb et al., 1997; Abecia et al., 2012, 2013; Mitsumori et al., 2012), although actual VFA production, which includes VFA removed by both absorption and passage from the rumen, as well as VFA incorporated into microbial fatty acids and amino acids (Kristensen, 2001), was not quantified in those in vivo studies. Simultaneous in vivo measurement of VFA and gases actual production when inhibiting methanogenesis would be necessary to conclude which type of in vitro system, if any, can better mimic the in vivo situation.

Both in batch and continuous culture, redirection of [H] toward H₂ was greater with more concentrate in the substrate, and in batch culture there was greater H₂ accumulation with greater DMD (not shown). Bauchop (1967) found a 2-fold increase in H₂ accumulation when inhibiting methanogenesis with chlorinated CH₄ analogs in the absence of added substrate, whereas H₂ accumulation was much greater if ruminal solids or formate were incubated. Czerkawski and Breckenridge (1975) reported that when CH₄ production was inhibited, H₂ accumulation was much greater 2 h after the morning feeding, in an actively fermenting rumen, compared to pre-feeding levels. Perhaps, that could be an indication that incorporation of [H] into sinks other than H₂ when fermentation is very active might be limited by the activity of alternative [H]-incorporating pathways i.e., enzyme kinetics. In steers not subjected to methanogenesis-inhibition treatments, Rooke et al. (2014) determined that animals fed a high concentrate diet produced gas with a greater ratio of H₂ to CH₄ compared to those fed a mixed diet, and suggested that rapid H₂ production immediately after feeding can result in methanogens being overloaded in their capacity to utilize H₂. This effect would likely accentuate in the methanogenesis-inhibited fermentation. Slowly degradable diets and uniform feed intake might then be strategies to decrease H₂ accumulation when methanogenesis is inhibited. In that regard, Swainson et al. (2011) suggested that there was a greater spillover of H₂ from fermentation in sheep fed twice a day than 8 times a day. Elevated H₂ associated to rapid fermentation may also been a consequence of methanogens being inhibited by the low ruminal pH associated with the consumption of high concentrate diets (Janssen, 2010).

Greater increase in [2H]_Pr was obtained by inhibiting methanogenesis at lower pH in batch, but not in continuous culture (not shown). Even though the range in pH was greater in continuous than in batch cultures, the mean (Table 1) and median final pH of batch cultures were numerically lower than pH of continuous cultures. Perhaps better buffered continuous cultures precluded detecting interactions between methanogenesis inhibition and pH. With the exception of the Slyter (1979) experiment in continuous culture in which the pH of the buffer was varied in some treatments, for the rest of the treatment means in the batch and continuous cultures databases, pH was a response to treatments, rather than a treatment itself. Controlled experiments of methanogenesis inhibition at different pH would have more power to detect the interaction between methanogenesis inhibition and pH on [H] sinks.

Perhaps the most interesting result related to variation in [H] sinks among experiments was the lack of detection of H₂ accumulation with strong methanogenesis inhibition with linoleic and linolenic acids (O'Brien et al., 2013). Other in vitro batch (van Nevel and Demeyer, 1981) and continuous (Czerkawski and Clapperton, 1984; Machmüller et al., 1998) culture (Table S4), and in vivo (Clapperton, 1974; Czerkawski and Clapperton, 1984) experiments with linseed oil, which is rich in linolenic acid, also found minimal or no H₂ accumulation. On the other hand, Marty and Demeyer (1973) reported considerable H₂ accumulation when inhibiting CH₄ production in batch cultures with linseed oil. Czerkawski and Clapperton (1984) indicated that linseed oil stimulated microbial growth, which would be an alternative [H] sink to CH₄ and H₂, although the opposite result was reported by van Nevel et al. (1969).

It is interesting that linoleic and linolenic acid and linseed oil were strongly propionogenic with mixed (O'Brien et al., 2013) or chemically defined (van Nevel and Demeyer, 1981), but not with roughage (Fievez et al., 2007; O'Brien et al., 2013), substrates (Table S4). Indeed, between about 25 and 50% of [H] spared from methanogenesis was redirected toward propionate production with mixed (O'Brien et al., 2013) and chemically defined substrates (van Nevel and Demeyer, 1981) when methanogenesis was inhibited with linoleic or linolenic acids or linseed oil, which is numerically much higher than [H] redirection to propionate with most of the rest of the inhibitors (Table S4). In continuous culture, linseed supplementation stimulated propionate production disregarding the level of concentrate inclusion (Machmüller et al., 1998). Clapperton (1974) and Yang et al. (2009) reported increases in ruminal propionate concentration in vivo with linseed supplementation of mixed diets. Stimulation of propionate production as an alternative [H] sink could therefore partially explain lack of or small H₂ accumulation when methanogenesis was inhibited using linoleic and linolenic acid with mixed and chemically defined substrates, although this explanation seems less likely with roughages. Other unsaturated oils such as some fish (Fievez et al., 2003) and algae (Fievez et al., 2007) oils in batch culture, and sunflower seed (Machmüller et al., 1998) and monolaurin (Klevenhusen et al., 2009) in continuous culture, were also associated with small H₂ production and were propionogenic.

Research is needed to understand how linolenic acid and linseed oil promoted propionate production, and how that seems to be substrate-dependent. Linseed oil supplementation increased propionate concentration in the rumen, but succinate-producing F. succinogenes and R. flavefaciens numbers were decreased (Yang et al., 2009). In a batch culture experiment, both linoleic and linolenic acid stimulated propionate production and inhibited F. succinogenes, but effects on R. flavefaciens were less clear (Zhang et al., 2008). These results are in agreement with pure culture results in which both linoleic and linolenic acid were inhibitory to F. succinogenes and R. flavefaciens; however, other succinate and propionate producers such as R. amylophilus, Prevotella spp., Mitsuokella multiacidus, S. ruminantium, Veilonella parvula, and Anaerovibrio lipolytica were insensitive to linoleic and linolenic acids at the concentration tested (Maia et al., 2007). Greater toxicity of linoleic and linolenic acids to cellulolytic bacteria and fungi than to other species (Maia et al., 2007), might then explain why linoleic and linolenic acid stimulated propionate more with a mixed than with a roughage substrate (O'Brien et al., 2013). The response of non-cellulolytic succinate and propionate producers in mixed cultures to linoleic and linolenic acid, and linseed oil, would need to be studied.

Unsaturated fatty acids may also decrease H₂ accumulation through biohydrogenation. It is true that the decrease of CH₄ production through competition for [H] by fatty acids biohydrogenation is considered to be small (Nagaraja et al., 1997). However, because H₂ accumulation when methanogenesis is inhibited is smaller than CH₄ production in the typical, methanogenesis-uninhibited ruminal fermentation, the relative biohydrogenation effect on H₂ accumulation would be more important than on CH₄. Furthermore, the effect of biohydrogenation on H₂ would be stoichiometrically greater than on CH₄ by a 4 to 1 ratio. For example, linolenic acid at 1.25 ml/l decreased CH₄ production by 60 and 70% with a mixed and a roughage substrate, respectively (O'Brien et al., 2013). Complete biohydrogenation of linolenic acid at 1.25 ml/l to stearic acid would have incorporated 14 and 18% of [2H]_CH4 in the control treatment (calculations not shown). This is greater than the average percentage of [H] diverted from CH₄ to H₂ in batch cultures at predicted 100% methanogenesis inhibition (calculation from Table 3), and thus, theoretically, it could explain lack of H₂ accumulation. It should be considered, however, that 1.25 ml/l of linolenic acid represented about 11% of the substrate incubated and resulted in decreased DMD (O'Brien et al., 2013); less [2H]_produced, would then also contribute to explain lack of detection of H₂.

Unsaturated fatty acids have been shown to inhibit lipolysis and biohydrogenation (Noble et al., 1974; Beam et al., 2000; Boeckaert et al., 2007, 2008; Toral et al., 2012), but absolute [H] incorporated of into fatty acids double bonds might increase with oil supplementation, even if the proportion of reduced double bonds relative to the total double bonds available in fatty acids was decreased. The net result would depend on the balance between the limiting [H]-incorporating substrate (fatty acids double bonds) and the effect of the oil supplement on enzyme activity (inhibition of lipases, isomerases and reductases).

The classification of methanogenesis inhibitors into chemical inhibitors, ionophores, oils, and nitrate/nitrocompounds, was inaccurate to describe the effects of the different inhibitors on fermentation shifts. For example, two fish oils differed in the extent they caused H₂ accumulation and stimulated propionate production when inhibiting methanogenesis (Fievez et al., 2003). It has been known that fatty acids differ on their effects on CH₄ production, with polyunsaturated and medium-chain free fatty acids exerting the greatest inhibitory effects on methanogenesis (Beauchemin et al., 2008; Martin et al., 2010); from this analysis, it appears that fatty acids also differ regarding the consequences on [H] incorporation into other pathways when methanogenesis is inhibited. Also, a hexadecatrienoic acid from algae origin (Ungerfeld et al., 2005) caused considerable more H₂ accumulation than a commercial algae product (Fievez et al., 2007). Content of algal secondary compounds such as isoprenoids and small halogenated compounds varies among algae lineages and is associated with the extent of methanogenesis inhibition algae exert in vitro (Machado et al., 2014); the type and content of secondary compounds can contribute to explain differences among algae and algae-based products on their effects on shifts in [H] sinks when methanogenesis is inhibited.

Nitrate caused somewhat less H₂ accumulation than did nitrocompounds (not shown). Reduction of NO⁻₃ to NH⁺₄ is both thermodynamically and stoichiometrically very favorable (Thauer et al., 1977). Nitrate reduction would then outcompete H₂ formation for [H], likely explaining the relatively small effect of NO⁻₃ on H₂ accumulation; however, in situ reduction of the nitro moiety in nitrocompounds (Anderson et al., 1993) could be thought to act similarly, and therefore nitrocompounds would be expected to cause equally small H₂ accumulation as NO⁻₃.

Inhibiting methanogenesis decreased FH in batch cultures. In contrast to this overall response, nitrocompounds increased FH in the experiment by Anderson et al. (2010). In other in vitro and in vivo experiments with nitrocompounds, total VFA concentration was generally not decreased (Anderson et al., 2003, 2006; Brown et al., 2011; Martínez-Fernández et al., 2014), perhaps because nitrocompounds might be partly metabolized to VFA. Nitrocompounds such as 3-nitropropionate may perhaps stimulate propionate production because they were partly converted to propionate. However, 3-carbon units 2-nitropropanol decreased propionate production, and on the other hand 2-carbon unit compounds nitroethane and nitroethanol increased propionate production (Anderson et al., 2003, 2010).

Martínez-Fernández et al. (2014) reported no effects of nitrocompounds on in situ apparent digestibility. Inhibiting methanogenesis in vivo using 3-nitrooxypropanol resulted in a quadratic tendency to increase dry matter and organic matter digestibility at the highest dose (Romero-Perez et al., 2014). A complete understanding of the effects of nitrocompounds on [H] sinks in the rumen is of much interest to applied animal production.