Rumen Methanogenesis, Rumen Fermentation, and Microbial Community Response to Nitroethane, 2-Nitroethanol, and 2-Nitro-1-Propanol: An In Vitro Study

Simple Summary The present study comparatively investigates the inhibitory difference of nitroethane (NE), 2-nitroethanol (NEOH), and 2-nitro-1-propanol (NPOH) on in vitro rumen fermentation, microbial populations, and coenzyme activities associated with methanogenesis. The results showed that both NE and NEOH were more effective in reducing ruminal methane (CH4) production than NPOH. This work provides evidence that NE, NEOH, and NPOH were able to inhibit methanogen population and dramatically decrease methyl-coenzyme M reductase gene expression and the content of coenzymes F420 and F430 with different magnitudes in order to reduce ruminal CH4 production. Abstract Nitroethane (NE), 2-nitroethanol (NEOH), and 2-nitro-1-propanol (NPOH) were comparatively examined to determine their inhibitory actions on rumen fermentation and methanogenesis in vitro. Fermentation characteristics, CH4 and total gas production, and coenzyme contents were determined at 6, 12, 24, 48, and 72 h incubation time, and the populations of ruminal microbiota were analyzed by real-time PCR at 72 h incubation time. The addition of NE, NEOH, and NPOH slowed down in vitro rumen fermentation and reduced the proportion of molar CH4 by 96.7%, 96.7%, and 41.7%, respectively (p < 0.01). The content of coenzymes F420 and F430 and the relative expression of the mcrA gene declined with the supplementation of NE, NEOH, and NPOH in comparison with the control (p < 0.01). The addition of NE, NEOH, and NPOH decreased total volatile fatty acids (VFAs) and acetate (p < 0.05), but had no effect on propionate concentration (p > 0.05). Real-time PCR results showed that the relative abundance of total methanogens, Methanobacteriales, Methanococcales, and Fibrobacter succinogenes were reduced by NE, NEOH, and NPOH (p < 0.05). In addition, the nitro-degradation rates in culture fluids were ranked as NEOH (−0.088) > NE (−0.069) > NPOH (−0.054). In brief, the results firstly provided evidence that NE, NEOH, and NPOH were able to decrease methanogen abundance and dramatically decrease mcrA gene expression and coenzyme F420 and F430 contents with different magnitudes to reduce ruminal CH4 production.


Introduction
Nitrocompounds are classified into aromatic compounds containing nitro groups in the aromatic ring and aliphatic-aromatic compounds containing nitro groups only in the aliphatic side chain. Among the naturally occurring aliphatic nitrocompounds, 3-nitro-1-propanol (3-NPOH) and 3-nitro-1-propionic

Materials and Methods
The donor animals and experimental procedures were approved by the requirements of Beijing Municipal Council on Animal Care according to the protocol of CAU20171014-1.

In Vitro Experiment
In vitro fermentations in anaerobic glass bottles (volume capacity of 120 mL) incubated with rumen fluids were performed following the previous description of Zhang and Yang [11]. The treatments included the control (no additive treatment), 10 mmol/L of NE, 10 mmol/L of NEOH, and 10 mmol/L of NPOH. Corn meal and alfalfa hay (500 mg; 80:20, w/w) were used as the fermentation substrates.
Rumen fluids were collected before morning feeding from each rumen-cannulated donor cow into a pre-warmed thermos flask at 39 • C. After filtering through 4 layers of cheesecloth and mixed in equal proportion, 25 mL of rumen fluids were incubated into anaerobic glass bottles with 50 mL buffered medium (pH 6.8) [12]. The batch cultures were performed at 39 • C in both automated and manual systems. In the automated system, five bottles per treatment were connected to the gas inlets of an automated gas recording system (AGRS) and continuously incubated for 72 h to continuously record cumulative gas production (GP). In the manual system, five bottles per treatment were connected to pre-emptied air bags to collect fermentation gas samples and removed at 6, 12, 24, 48, and 72 h of incubation. The batch cultures were repeated and completed in three consecutive runs. One milliliter of gas sample was drawn out of the air bags using a syringe to measure the CH 4 concentration according to the gas chromatographic method.

Sampling
After 6, 12, 24, 48, and 72 h of incubation in the manual system, the contents of each bottle were filtered through a nylon bag (8 × 12 cm; 42 µm pore size) and dried at 105 • C to determine the in vitro dry matter disappearance (IVDMD). Then, the culture fluids (6 × 1.0 mL) were sampled into DNase-free polypropylene tubes and stored at −80 • C for later analysis of VFA, nitrocompounds, microbial populations, mcrA (methyl coenzyme-M reductase subunit A) gene expression, coenzyme F 420 content, and coenzyme F 430 content.

Measurement of VFA, Coenzyme, and Nitrocompound Contents
The culture fluids (1.0 mL) from each of the 5 aforementioned time stamps were mixed with 300 µL metaphosphoric acid solution (25%, w/v) for 30 min and centrifuged at 10,000× g for 15 min at 4 • C. Supernatants (0.5 mL) were injected into gas chromatography to determine the concentrations of acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate [11].
Following the description of Reuter et al. [13], coenzyme F 420 was determined and expressed as fluorescence intensity. Assays were performed at 37 • C anaerobically in the dark. Culture fluid samples (1.0 mL) were stirred continuously and boiled at 95 • C in water bath for 30 min. Fluid aliquots were then centrifuged at 10,000× g for 10 min, and a volume of 500 µL from supernatants was mixed with 1 mL of isopropanol. Subsequently, the mixture was precipitated for 2 h and centrifuged at 10,000× g again for 15 min. Finally, the fluorescence intensity of the supernatants was measured at 420 nm by the fluorescence spectrophotometer (Thermo Fisher Scientific Co., Ltd., shanghai, China). Coenzyme F 430 content was examined via the ultraviolet/visible spectrum by determining the loss of absorbance [14]. Briefly, the culture fluids were quenched with equal volumes of methanol and centrifuged aerobically at 6153× g for 20 min in dim light. The precipitate was discarded and the supernatants were determined colorimetrically using a spectrophotometer at 430 nm (Laspec Technology Co., Ltd., shanghai, China). Coenzyme F 430 content was expressed as the relative absorbance of coenzyme F 430 at 430 nm.
The contents of NE, NEOH, and NPOH were determined colorimetrically by a spectrophotometer (Laspec Technology Co., Ltd., shanghai, China) [15]. The culture fluids (1 mL) were firstly centrifuged at 10,000× g for 15 min. Supernatants (50 µL) were then diluted with 2 mL of distilled water and mixed with 100 µL of NaOH (0.65 M) and 100 µL of diazotized p-nitroaniline. Finally, the absorbances of the culture fluids were measured at a wavelength of 405 nm.

Microbial Population Analysis with Real-Time PCR
Total DNA of culture fluid (1 mL) was extracted with the FastDNA kit and FastPrep instrument (Tiangen®Biotech, Beijing, China) by a bead-beating method as described by Denman and McSweeney [18]. According to the real-time PCR method [18], enumeration of total bacteria, total methanogens, Methanobacteriales, Methanococcales, Methanomicrobiales, protozoa, fungi, Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes was measured using the Bio-Rad Multicolor Real-Time PCR Detection System (Bio-rad Company, CA, USA) with qReal Master Mix SYBR®Green (Tiangen®Biotech, Beijing, China). The primer sets for the detection and enumeration of the microbial populations were described by Denman and McSweeney [18], Zhou et al. [19], and Yu et al. [20]. The abundance of the microbial population was expressed as a proportion of total estimated rumen bacterial (16S rDNA) according to the following equation: relative quantification = 2 −(CT target -CT total bacteria) , where CT represents the threshold cycle.

Data Calculation and Statistical Analysis
Cumulative gas production data from the AGRS were fitted according to an exponential model as described by France [21]. In addition, the average gas production rate (AGPR) (mL/h) and hydrogen recovery (2Hrec) were also calculated following the description by Zhang et al. [22].
The data for gas production, IVDMD, fermentation gas composition, coenzyme contents, and VFAs were subjected to analysis of variance with the MIXED model procedure of SAS (Statistical Analysis for Windows, SAS Institute Inc., Cary, NC, USA). The model was applied as Y ijk = µ + R i + N j + T k + (N × T) jk + e ijk " where Y ijk is the dependent variable, µ represents the overall mean, R i is the effect of the experimental run, N j is the effect of the nitrocompound treatment, T k is the effect of the incubation time, N × T are the interactions between the nitrocompounds and the incubation time, and e ijk is the residual. The data for microbial abundance analysis were applied according to the model Y ijk = µ + R i + N j + e ijk , where Y ijk is the dependent variable, µ represents the overall mean, R i is the effect of the experimental run, N j is the effect of the nitrocompound treatment, and e ijk is the residual. Least square means and standard error (SEM) were calculated, and treatment differences were estimated using a multiple comparisons test (Tukey/Kramer). Correlation analyses between variables were performed using the CORR procedure of SAS. Significance was declared at p < 0.05.

IVDMD and Gas Production Kinetics
As the incubation time increased, both IVDMD ( Figure 1a) and gas production ( Figure 1b) continuously increased. Addition of NE, NEOH, and NPOH slowed down the fermentation process and caused IVDMD to decline (Figure 1a, p < 0.01).
Regarding the kinetics of gas production, NE, NEOH, and NPOH addition decreased asymptotic gas production (A) ( Table 1, p < 0.01), while NE and NEOH addition increased the fractional gas production rate (c, h −1 ). Neither NE nor NEOH addition altered AGPR, but NPOH decreased AGPR compared to the control. Table 1. Effect of NE, NEOH, and NPOH addition (10 mmol/L) to culture fluids on gas production kinetics and fermentation gas composition during 72 h incubation. a-d Means with different superscripts within a row are significantly different (p < 0.05); 1 A: asymptotic gas production; c: fractional gas production rate; T 1/2 : the time when half of A occurred; AGPR: average gas production rate. 2 NE: nitroethane; NEOH: 2-nitroalcohol; NPOH: 2-nitro-1-propanol. 3 Interaction effect between treatment and incubation time.

Fermentation Gas Composition
H 2 accumulation in the NE, NEOH, and NPOH groups was far greater than in the control group (Table 1, p < 0.01). The molar proportions of CO 2 were increased (5.7%, 5.2%, and 5.4%) by NE, NEOH, and NPOH compared to the control, whereas the molar proportions of CH 4 were notably decreased (96.7%, 96.7%, and 41.7%, Table 1). An interaction between the nitrocompound treatment and the incubation time was observed for gas composition (p < 0.01). The molar proportion of CH 4 constantly increased with increasing incubation time in the control and NPOH groups, but it was at a pretty low level in both the NE and NEOH groups (p < 0.01, Figure 2a). In contrast, molar H 2 production in the NE and NEOH groups continuously increased with increasing incubation time and was always far greater than the control and NPOH throughout the incubation (p < 0.01, Figure 2b). In addition, as the incubation time increased, the molar proportion of CO 2 gradually decreased in all groups, and it was greater in the nitrocompound-treated cultures than the control (p < 0.01, Figure 2c).
Animals 2020, 6 of 15 in all groups, and it was greater in the nitrocompound-treated cultures than the control (p < 0.01, Figure 2c).

Coenzymes Related to CH4 Production
Compared to the control, both coenzyme F420 fluorescence intensity and F430 ultraviolet (UV) absorbance declined in the NE, NEOH, and NPOH groups ( Table 2, p < 0.01). In addition, NE, NEOH, and NPOH addition decreased mcrA gene expression by 83.1%, 79.7%, and 53.5%, respectively. Figure 2. Effect of NE, NEOH, and NPOH addition to culture fluids on CH 4 (a), H 2 (b), and CO 2 (c) production of grain-rich feed incubated with rumen fluids obtained from lactating dairy cows. NE: nitroethane; NEOH: 2-nitroalcohol; NPOH: 2-nitro-1-propanol. Statistical analyses showed the effects of nitrocompounds on CH 4 , H 2 , and CO 2 , with correlations significant at p < 0.01. Table 2. Effect of NE, NEOH, and NPOH addition (10 mmol/L) on coenzyme content, mcrA gene expression, and volatile fatty acid (VFA) production in fermentation fluids across different incubation times (6,12,24,48, and 72 h). An interaction between the nitrocompound treatment and the incubation time was evident for mcrA gene expression and coenzyme F 420 and F 430 contents (p < 0.01). As the incubation time increased, mcrA gene expression in the NPOH group relative to the control peaked at 12 h during the 72 h incubation, and it was continuously far lower in the NE and NEOH groups than in the control and NPOH groups (Figure 3a). Coenzyme F 420 fluorescence intensity gradually declined in both the NE and NEOH groups with increasing incubation time, and it was continuously lower in the nitrocompound-treated cultures than the control (Figure 3b). The F 430 ultraviolet (UV) absorbance continuously decreased in the NE and NEOH groups with increasing incubation time (Figure 3c), but it Animals 2020, 10, 479 7 of 15 fluctuated during 72 h incubation in the NPOH group. In addition, the coenzyme F 430 content was constantly lower in the nitrocompound-treated cultures than the control. mcrA gene expression and coenzyme F420 and F430 contents (p < 0.01). As the incubation time increased, mcrA gene expression in the NPOH group relative to the control peaked at 12 h during the 72 h incubation, and it was continuously far lower in the NE and NEOH groups than in the control and NPOH groups (Figure 3a). Coenzyme F420 fluorescence intensity gradually declined in both the NE and NEOH groups with increasing incubation time, and it was continuously lower in the nitrocompound-treated cultures than the control (Figure 3b). The F430 ultraviolet (UV) absorbance continuously decreased in the NE and NEOH groups with increasing incubation time (Figure 3c), but it fluctuated during 72 h incubation in the NPOH group. In addition, the coenzyme F430 content was constantly lower in the nitrocompound-treated cultures than the control. Statistical analyses showed that the effects of nitrocompounds were significant at p < 0.01.

Fermentation Characteristics
Concentrations of total VFAs and acetate were lower in the NE, NEOH and NPOH groups than in the control ( Table 2, p < 0.01), whereas the concentration of butyrate increased with NE addition ( Table 2, p < 0.01). NE, NEOH, and NPOH addition had no significant influence on propionate and branched-chain VFAs (BCVFAs). Compared to the control, NE, NEOH, and NPOH addition decreased 2Hrec by 30.2%, 28.3%, and 12.3%, respectively (Table 2, p < 0.01). An interaction between the nitrocompound treatment and the incubation time was observed for total VFA, acetate, propionate, and butyrate. The concentrations of total VFAs, acetate, propionate, butyrate, and BCVFA were continuously increased in all groups with increasing incubation time (Figure 4). In addition, concentrations of acetate were continuously lower in nitrocompound-treated cultures than in the control, whereas the concentrations of propionate, butyrate, and BCVFAs fluctuated during 72 h incubation.

Microbial Populations
Real-time PCR results showed that NE, NEOH, and NPOH addition decreased the relative populations of total methanogens, Methanobacteriales, Methanomicrobiales, and Fibrobacter succinogenes ( Table 3, p < 0.05). Compared to the control, the relative populations of total methanogens decreased by 49.2%, 36.9%, and 41.5% in the NE, NEOH, and NPOH groups, and the populations of Methanobacteriales decreased by 46.1%, 35.9%, and 17.9%, respectively. In addition, the relative populations of fungi tended to decrease according to nitrocompound treatment (p = 0.09). Compared with control, the populations of R. albus increased by 50.0% and 50.0% in the NEOH and NPOH groups, and the populations of R. flavefaciens decreased by 84.5% and 70.7% in the NE and NEOH groups. However, the populations of protozoa were not affected (p = 0.29).
( Table 2, p < 0.01). NE, NEOH, and NPOH addition had no significant influence on propionate and branched-chain VFAs (BCVFAs). Compared to the control, NE, NEOH, and NPOH addition decreased 2Hrec by 30.2%, 28.3%, and 12.3%, respectively (Table 2, p < 0.01). An interaction between the nitrocompound treatment and the incubation time was observed for total VFA, acetate, propionate, and butyrate. The concentrations of total VFAs, acetate, propionate, butyrate, and BCVFA were continuously increased in all groups with increasing incubation time (Figure 4). In addition, concentrations of acetate were continuously lower in nitrocompound-treated cultures than in the control, whereas the concentrations of propionate, butyrate, and BCVFAs fluctuated during 72 h incubation. Statistical analyses showed that the effects of nitrocompounds on acetate and butyrate were significant at p < 0.01, but the effects of nitrocompounds on propionate and branched-chain VFAs were not significant.

Microbial Populations
Real-time PCR results showed that NE, NEOH, and NPOH addition decreased the relative populations of total methanogens, Methanobacteriales, Methanomicrobiales, and Fibrobacter succinogenes ( Table 3, p < 0.05). Compared to the control, the relative populations of total methanogens decreased

Disappearance of Nitrocompounds
Concentrations of NE, NEOH, and NPOH decreased with increasing incubation time ( Figure 5). The disappearance of nitrocompounds was fitted to a linear model, and the nitrocompound degradation rate was ranked as NEOH (−1.

Effects of Nitrocompounds on IVDMD and Gas Production
A majority of CH4 inhibition strategies tend to compromise fermentative efficiency, resulting in the reduction of certain digestive processes [11,23]. In the present study, the addition of NE, NEOH, and NPOH caused notable reductions of IVDMD, gas production, and total VFAs, indicating that the activity of the microbes responsible for the degradation of substrates was inhibited by these nitrocompounds. The inclusive level of nitrocompounds was determined according to previous studies [11,24], however, a remarkable reduction in total VFA production occurred in the NE,

Effects of Nitrocompounds on IVDMD and Gas Production
A majority of CH 4 inhibition strategies tend to compromise fermentative efficiency, resulting in the reduction of certain digestive processes [11,23]. In the present study, the addition of NE, NEOH, and NPOH caused notable reductions of IVDMD, gas production, and total VFAs, indicating that the activity of the microbes responsible for the degradation of substrates was inhibited by these nitrocompounds. The inclusive level of nitrocompounds was determined according to previous studies [11,24], however, a remarkable reduction in total VFA production occurred in the NE, NEOH, and NPOH groups, indicating that the fermentative bacterial population may be sensitive to a 10 mM dose level of nitrocompounds.

Effects of Nitrocompounds on CH 4 Production
The antimethanogenic activity of NE, NEOH, and NPOH was previously observed. For instance, more than 90% of CH 4 production was inhibited by NE, NEOH, and NPOH at a concentration of 9~24 mM after 24 h incubation [5,24]. The addition of NE, NEOH, and NPOH in the present study caused reductions in CH 4 production of 96.7%, 97.2%, and 39.5%, respectively, which agreed with the results of earlier studies [24,25]. In addition, both NE and NEOH were more effective in reducing ruminal CH 4 production than NPOH.
The nitro group is strongly electron-withdrawing. Because of this property, nitrocompounds may be able to serve as electron acceptors within ruminal microbes to reduce CH 4 production. Early studies done by Anderson et al. [26] revealed that nitrocompounds could serve as electron acceptors within ruminal microbes to inhibit CH 4 production. However, nitrocompounds also directly inhibit ruminal methanogenesis [27]. The first author in a review summarized the anti-methanogenic roles of nitrocompounds and their potential inhibitory action modes in terms of VFA production, hydrogen accumulation, formate oxidation and ferredoxin-linked hydrogenase activity [28]. Notable accumulation of H 2 in NE-, NEOH-, and NPOH-treated cultures and CH 4 production inhibition were observed in the present study. Similarly, Anderson et al. [25] also found that accumulations of H 2 were higher in nitrocompound-supplemented cultures than in controls after 24 h of incubation. In the unperturbed rumen, H 2 is usually present at approximately 1 µmol/L (0.1 kPa); however, it often increases to concentrations that inhibit hydrogenase activity (1 kPa), while ruminal CH 4 production is inhibited due to declined H 2 consumption by methanogens [23]. Considering that hydrogenases are reversible enzymes that can catalyze either the production or oxidation of H 2 , the authors in the present study speculated that nitrocompounds may have inhibited H 2 -oxidation hydrogenase activity as well, which was in accordance with Angermaier and Simon [29], who reported that NEOH inhibited ferredoxin-linked hydrogenase uptake activity. Likewise, Anderson et al. [30] also noted that NE, NEOH, NPOH, and NPA each inhibited oxidation of H 2 or formate when these reductants were added in excess (60 mM) to mixed cultures of rumen microbial populations, thereby implicating a possible mechanism of activity against ruminal methanogenesis.
Consistent with the different levels of CH 4 inhibition efficiency, NE and NEOH were shown to be nearly equally effective in promoting H 2 accumulation in vitro, with both of them promoting H 2 accumulation more effectively than NPOH. The molar proportions of H 2 in total fermentation gas production were 9.8%, 10.5%, and 2.0% in the NE, NEOH, and NPOH groups, respectively. However, the decreasing extent of 2Hrec in the present study was far less than that of CH 4 production. The fate of the remaining H 2 is not known with certainty, however, the consumption of reducing equivalents may occur during anabolic processes, including cell growth, intracellular polyhydroxyalkonoate, or extracellular polysaccharide production [25].

Effects of Nitrocompounds on VFA Production
In order to compensate for the disruption of electron flow to ruminal methanogenesis, some CH 4 inhibitors, such as sodium sulfite, organic halides, and monensin, cause notable increases in propionate production during fermentation, which is frequently accompanied by decreased acetate [23]. However, NE, NEOH, and NPOH addition in the present study had little effect on propionate produced by ruminal populations. Therefore, the results indicate that the reducing equivalents produced during ruminal fermentation were not necessarily directed toward increased production of propionate. In addition, the concentrations of acetate in the present study were decreased in the nitrocompound-treated cultures, along with the reduction of CH 4 production. It is well recognized that acetate accompanies H 2 production, and the latter can be used for CH 4 formation by methanogenic archaea, with CH 4 being positively associated with the acetate to propionate ratio [31].

Effects of Nitrocompounds on Methane Production and Related Changes in Microbial Populations and Coenzyme Contents
Methanogens are a unique group of ruminal microbes that generate CH 4 as a stoichiometric end-product of their metabolism. Methanogen populations are generally closely associated with CH 4 production [32]. In the present study, NE, NEOH, and NPOH addition decreased total methanogens by 49.2%, 36.9%, and 41.5%, respectively, and the authors speculated that NE, NEOH, and NPOH likely exerted a direct inhibition of ruminal methanogenesis via direct suppression of methanogens.
Methanobacteriales are the predominant populations and constitute the major portion of methanogen community in ruminants, being the second most prevalent archaea in the rumen ecology [33]. Presently, both the relative abundances of Methanobacteriaceae and Methanomicrobiales were reduced with NE, NEOH, and NPOH addition, but different sensitivity responses to these nitrocompounds were Rumen protozoa constitute only a small portion of ruminal microorganisms, however, they play important roles in feed degradation and making energy and protein available to the hosts. The RT-PCR results showed no apparent decrease in the protozoal population in the NE-, NEOH-and NPOH-treated cultures, indicating that there were no adverse effects of the nitrocompounds on the rumen protozoa populations.
Few studies have determined microbial responses to nitrocompounds. Anderson et al. [26] noted that 3-NPOH and NPA modestly inhibited total culturable anaerobes from bovine rumens, but inhibited microbes were not characterized. Rumens harbor different types of bacteria, which are most actively involved in plant fiber degradation. It is well recognized that the depression of cellulolysis can decrease the rate and extent of neutral detergent fiber digestion [34], and the inhibition to cellulolytic microorganisms (R. flavefaciens, F. succinogenes) by nitrocompounds may explain why decreased IVDMD was observed in the cultures supplemented with NE, NEOH, or NPOH. Fibrolytic bacteria families, including R. flavefaciens, R. albus, and F. succinogenes, have vbeen shown to release many fibrolytic enzymes and promote H 2 , acetate and formic acid production for methanogen utilization [35]. Real-time PCR results showed that the relative populations of the methanogens R. Flavefaciens and F. succinogenes decreased with nitrocompound addition, suggesting that the mutual-aid interaction between methanogens and fibrolytic bacteria might be one reason why R. Flavefaciens and F. succinogenes decreased along with methanogen inhibition by NE, NEOH, and NPOH. The rumen protozoa produce fermentation end-products similar to those made by the bacteria, particularly acetate, butyrate, and H 2 . They utilize large amounts of starch at one time and can store it in their bodies. The corn-rich substrate applied in the present study may have been adequate to maintain the growth of protozoa, which may explain why the nitrocompounds had no negative effect on the abundance of protozoa. In the present study, the abundance of R. albus increased with NEOH and NPOH addition, while anaerobic rumen fungi decreased with nitrocompound supplementation. This phenomenon could be attributed to the antagonistic association between ruminal fungi and cellulolytic bacteria [36]. In summary, although nitrocompounds could change the relative abundance of some microbial populations, the differences in diversity and metabolic activity in response to NE, NEOH, and NPOH need further investigation in order to determine the maximal inhibitory effect on CH 4 production with minimal adverse effects on rumen fermentation.
The hydrogenotrophic, methylotrophic, and acetoclastic pathways are the three major pathways for ruminal CH 4 production. The biochemical reactions and enzyme profiles involved in methanogenesis are well identified and described [37]. Methyl-coenzyme M reductase (mcr) is a key enzyme responsible for catalyzing the CH 4 -producing step in the process of methanogenesis. As a gene encoding the alpha-subunit of mcr, mcrA was evolutionarily highly conserved, probably due to functional constraints on the catalytic activity of mcr [35]. Recently, the determination of mcrA gene expression was accepted for mcr activity measurement [16]. A positive correlation between decreased mcrA gene expression and decreased molar CH 4 proportions was found in the present study, suggesting that NE, NEOH, and NPOH inhibited CH 4 production by decreasing mcr activity. In addition, the activity of mcr is dependent mainly on the unique nickel-containing tetrapyrrole known as coenzyme F 430 [38]. As reported by Gunsalus and Wolfe [39], coenzyme F 430 is a yellow nonfluorescent compound released from mcr, with an absorption maximum at 430 nm on its UV-Vis absorption spectrum. In the present study, NE, NEOH, and NPOH addition also reduced the content of coenzyme F 430 . Coenzyme F 420 is of importance for methanogenesis and can act as an indicator for methanogenic activity [8,40]. The coenzyme F 420 content in this study also decreased in the nitrocompound-treated cultures and was accompanied by CH 4 reduction, with the anti-methanogenic activity ranked as NEOH > NE > NPOH. A possible reason for these results might be due to the toxic action of nitrocompounds to methanogens [24,27]; this needs further clarification.

Degradation of NE, NEOH, and NPOH
Decreased concentrations of NE, NEOH, and NPOH were observed with increasing incubation time in all of the nitrocompound-treated cultures, thus confirming the presence of competent nitrocompound-degrading microbes within the incubations. A previous study by Anderson et al. [26] revealed that most of the rumen microorganisms tolerated nonlethal concentrations of naturally occurring nitrocompounds. In addition, the degradation rate of nitrocompounds can be enhanced via exposure to nitropropionic acid-containing forages. This phenomenon could be ascribed the improvement of nitro-degrading activity or enrichment in numbers of nitro-degrading microorganisms. Until now, Denitrobacterium detoxificans was recognized as a unique nitrocompound-reducing microbe which oxidizes reducing substrates, including H 2 , formate, and lactate, to reduce nitrocompounds to their respective amines and minor nitrites [41]. Consequently, D. detoxificans has the potential to outcompete ruminal methanogens for available reductants [15]. In the present study, the nitrocompound disappearance rate was ranked as NEOH (−1.20) > NE (−0.80) > NPOH (−0.78), suggesting that the rumen microbes presented divergent metabolic capabilities regarding nitrocompound degradation, thus partially explaining why NE, NEOH, and NPOH showed different competition for methanogenesis-produced reductants.

Conclusions
Along with a dramatic increase in H 2 accumulation, both NE and NEOH were shown to be more effectivene in inhibiting methanogenesis than NPOH. Although nitrocompound addition decreased acetate and total VFA production, it had no negative effect on propionate. In addition, NE, NEOH, and NPOH addition decreased the population abundance of total methanogens, Methanobacteriales, and Methanomicrobiales, also causing decreases in mcrA gene expression and coenzyme F 420 and F 430 contents. The results provided evidence that NE, NEOH, and NPOH could reduce methanogen populations and dramatically decrease mcrA gene expression and coenzyme F 420 and F 430 contents with different magnitudes to reduce overall ruminal CH 4 production.