Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

The transcriptome response of the ruminal methanogen Methanobrevibacter ruminantium strain M1 to the inhibitor lauric acid.

OBJECTIVE: Lauric acid (C12) is a medium-chain fatty acid that inhibits growth and production of the greenhouse gas methane by rumen methanogens such as Methanobrevibacter ruminantium. To understand the inhibitory mechanism of C12, a transcriptome analysis was performed in M. ruminantium strain M1 (DSM 1093) using RNA-Seq. RESULTS: Pure cell cultures in the exponential growth phase were treated with 0.4 mg/ml C12, dissolved in dimethyl sulfoxide (DMSO), for 1 h and transcriptomic changes were compared to DMSO-only treated cells (final DMSO concentration 0.2%). Exposure to C12 resulted in differential expression of 163 of the 2280 genes in the M1 genome (maximum log2-fold change 6.6). Remarkably, C12 hardly affected the expression of genes involved in methanogenesis. Instead, most affected genes encode cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), and proteins involved in detoxification or DNA-repair processes. Enrichment analysis on the genes regulated in the C12-treated group showed a significant enrichment for categories 'cell surface' and 'mobile elements' (activated by C12), and for the categories 'regulation' and 'protein fate' (represssed). These results are useful to generate and test specific hypotheses on the mechanism how C12 affects rumen methanogens.

Development of a Modified-Release Formulation of Lovastatin Targeted to Intestinal Methanogens Implicated in Irritable Bowel Syndrome With Constipation.

There is growing evidence that methane production, predominantly by Methanobrevibacter smithii, in the intestines is a cause of constipation, pain, and bloating in irritable bowel syndrome with constipation (IBS-C). M smithii resides primarily in the large intestine but can also colonize the small intestine. In vitro studies found that the prodrug lactone form of lovastatin, found in cholesterol-lowering drugs, inhibited methane production in stool samples from patients with IBS-C. However, the cholesterol-lowering lovastatin ß-hydroxyacid was ineffective at inhibiting methane production in this system. A considerable amount of lovastatin is converted to hydroxyacid in the stomach and is absorbed. It was hypothesized that galenic innovations could protect lovastatin from the stomach and allow release in 2 strategic locations, the duodenum and the ileocecal region, to reach M smithii. The desired release profile was achieved by developing an oral dosage form containing lovastatin and coated with 2 different enteric polymers that enabled a pH-dependent "dual pulse" drug release. Combinations of the 2 coated tablets were encapsulated together to deliver the desired amount of lovastatin to the targeted intestinal locations. The capsules have been tested in vitro and in vivo and show promise in treating IBS-C.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Dietary pea fiber increases diversity of colonic methanogens of pigs with a shift from Methanobrevibacter to Methanomassiliicoccus-like genus and change in numbers of three hydrogenotrophs.

BACKGROUND: Pea fiber (PF) is a potential fibrous supplement in swine production. The influence of dietary PF on microbial community in the colon of pigs remains largely unexplored. Methanogens in the hindgut of monogastric animals play important roles in degradation of dietary fibers and efficient removal of microbial metabolic end product H2. Understanding the impact of dietary PF on the structure of colonic methanogens may help understand the mechanisms of microbe-mediated physiological functions of PF. This study investigated the influence of PF on the diversity and quantity and/or activity of colonic methanongens of piglets and finishing pigs. Four archaeal 16S rRNA clone libraries were constructed for piglets and finishers fed with control (Piglet-C and Finisher-C) or PF diet (Piglet-P and Finisher-P). RESULTS: There were 195, 190, 194 and 196 clones obtained from the library Piglet-C, Piglet-P, Finisher-C and Finisher-P, respectively, with corresponding 12, 11, 11 and 16 OTUs (operational taxonomic units). Significant differences of Shannon Index among the four libraries were found (P < 0.05). Libshuff analysis showed that the archaeal community structure among the four libraries were significantly different (P < 0.0001). The predominant methanogens shifted from Methanobrevibacter to Methanobrevibacter and Methanomassiliicoccus-like genus as a result of dietary PF. Supplementation of PF significantly increased the copy numbers of mcrA and dsrA genes (P < 0.05). CONCLUSIONS: Alteration of methanogenic community structure may lead to functional transition from utilization of H2/CO2 to employment of both H2/CO2 and methanol/CO2. Quantification of three functional genes (mcrA, dsrA and fhs) of methanogens, sulfate-reducing bacteria (SRB) and acetogens revealed that dietary PF also increased the activity of methanogens and SRB,probably associated with increased proportion of Methanomassiliicoccus luminyensis-species. Further study is required to examine the interaction between specific methanogens and SRB during fermentation of dietary PF.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

Effect of dietary fiber on the methanogen community in the hindgut of Lantang gilts.

The primary objective of this study was to investigate the effect of dietary fiber on methanogenic diversity and community composition in the hindgut of indigenous Chinese Lantang gilts to explain the unexpected findings reported earlier that Lantang gilts fed low-fiber diet (LFD) produced more methane than those fed high-fiber diet (HFD). In total, 12 Lantang gilts (58.7±0.37 kg) were randomly divided into two dietary groups (six replicates (pigs) per group) and fed either LFD (NDF=201.46 g/kg) or HFD (NDF=329.70 g/kg). Wheat bran was the main source of fiber for the LFD, whereas ground rice hull (mixture of rice hull and rice bran) was used for the HFD. Results showed that the methanogens in the hindgut of Lantang gilts belonged to four known species (Methanobrevibacter ruminantium, Methanobrevibacter wolinii, Methanosphaera stadtmanae and Methanobrevibacter smithii), with about 89% of the methanogens belonging to the genus Methanobrevibacter. The 16S ribosomal RNA (rRNA) gene copies of Methanobrevibacter were more than three times higher (P0.05) was observed in 16S rRNA gene copies of Fibrobacter succinogenes between the two dietary groups, and 18S rRNA gene copies of anaerobic fungi in gilts fed LFD were lower than (P<0.05) those fed HFD. To better explain the effect of different fiber source on the methanogen community, a follow-up in vitro fermentation using a factorial design comprised of two inocula (prepared from hindgut content of gilts fed two diets differing in their dietary fiber)×four substrates (LFD, HFD, wheat bran, ground rice hull) was conducted. Results of the in vitro fermentation confirmed that the predominant methanogens belonged to the genus of Methanobrevibacter, and about 23% methanogens was found to be distantly related (90%) to Thermogymnomonas acidicola. In vitro fermentation also seems to suggest that fiber source did change the methanogens community. Although the density of Methanobrevibacter species was positively correlated with CH4 production in both in vivo (P<0.01, r=0.737) and in vitro trials (P<0.05, r=0.854), which could partly explain the higher methane production from gilts fed LFD compared with those in the HFD group. Further investigation is needed to explain how the rice hull affected the methanogens and inhibited CH4 emission from gilts fed HFD.

Rumen microbial abundance and fermentation profile during severe subacute ruminal acidosis and its modulation by plant derived alkaloids in vitro.

Rumen microbiota have important metabolic functions for the host animal. This study aimed at characterizing changes in rumen microbial abundances and fermentation profiles using a severe subacute ruminal acidosis (SARA) in vitro model, and to evaluate a potential modulatory role of plant derived alkaloids (PDA), containing quaternary benzophenanthridine and protopine alkaloids, of which sanguinarine and chelerythrine were the major bioactive compounds. Induction of severe SARA strongly affected the rumen microbial composition and fermentation variables without suppressing the abundance of total bacteria. Protozoa and fungi were more sensitive to the low ruminal pH condition than bacteria. Induction of severe SARA clearly depressed degradation of fiber (P < 0.001), which came along with a decreased relative abundance of fibrolytic Ruminococcus albus and Fibrobacter succinogenes (P < 0.001). Under severe SARA conditions, the genus Prevotella, Lactobacillus group, Megasphaera elsdenii, and Entodinium spp. (P < 0.001) were more abundant, whereas Ruminobacter amylophilus was less abundant. SARA largely suppressed methane formation (-70%, P < 0.001), although total methanogenic 16S rRNA gene abundance was not affected. According to principal component analysis, Methanobrevibacter spp. correlated to methane concentration. Addition of PDA modulated ruminal fermentation under normal conditions such as enhanced (P < 0.05) concentration of total SCFA, propionate and valerate, and increased (P < 0.05) degradation of crude protein compared with the unsupplemented control diet. Our results indicate strong shifts in the microbial community during severe SARA compared to normal conditions. Supplementation of PDA positively modulates ruminal fermentation under normal ruminal pH conditions.

Influence of pH and the degree of protonation on the inhibitory effect of fatty acids in the ruminal methanogen Methanobrevibacter ruminantium strain M1.

AIMS: To investigate the relationship between the protonation of medium-chain fatty acids (MCFA) and their inhibitory effect on a ruminal methanogen species. METHODS AND RESULTS: Cell suspensions of Methanobrevibacter ruminantium M1 in 1 mg dry matter (DM) ml(-1) were supplemented with lauric acid (C12 ) and myristic acid (C14 ) at a concentration of 8 µg ml(-1) with different pH levels of the potassium-free buffer, where the calculated degrees of protonation of C12 and C14 varied from 0·3 to 50% and from 1 to 76% respectively. Methane formation, ATP efflux, potassium leakage and cell viability were monitored 15, 30 and 45 min after the reaction started. Declining methane formation rate, increasing ATP efflux and potassium leakage, and decreasing survival of M. ruminantium were observed with increasing degrees of protonation, i.e. with decreasing pH. CONCLUSIONS: The inhibition of methanogenesis by C12 and C14 is more efficient at a pH of 5-6 as compared to pH 7. SIGNIFICANCE AND IMPACT OF THE STUDY: Methane mitigation strategies in ruminants which use supplementation of feed with MCFA such as C12 and C14 may be more effective in a low rumen pH environment. This finding is helpful in designing diets to effectively decrease methane emissions by ruminants.

The effect of saturated fatty acids on methanogenesis and cell viability of Methanobrevibacter ruminantium.

Saturated fatty acids (SFAs) are known to suppress ruminal methanogenesis, but the underlying mechanisms are not well known. In the present study, inhibition of methane formation, cell membrane permeability (potassium efflux), and survival rate (LIVE/DEAD staining) of pure ruminal Methanobrevibacter ruminantium (DSM 1093) cell suspensions were tested for a number of SFAs. Methane production rate was not influenced by low concentrations of lauric (C12; 1 µg/mL), myristic (C14; 1 and 5 µg/mL), or palmitic (C16; 3 and 5 µg/mL) acids, while higher concentrations were inhibitory. C12 and C14 were most inhibitory. Stearic acid (C18), tested at 10-80 µg/mL and ineffective at 37°C, decreased methane production rate by half or more at 50°C and ≥50 µg/mL. Potassium efflux was triggered by SFAs (C12 = C14 > C16 > C18 = control), corroborating data on methane inhibition. Moreover, the exposure to C12 and C14 decreased cell viability to close to zero, while 40% of control cells remained alive after 24 h. Generally, tested SFAs inhibited methanogenesis, increased cell membrane permeability, and decreased survival of M. ruminantium in a dose- and time-dependent way. These results give new insights into how the methane suppressing effect of SFAs could be mediated in methanogens.

Simultaneous methanogenesis and oxygen reduction by Methanobrevibacter cuticularis at low oxygen fluxes.

Methanogenic Archaea are often encountered in habitats that are not entirely anoxic in space or time. Recent biochemical and genomic studies have revealed the capacity of methanogens to reduce molecular oxygen. O(2) reduction by Methanobrevibacter species was investigated. Cell suspensions incubated in agar tubes under a headspace of H(2)-CO(2) and increasing concentrations of O(2) formed a distinct growth band, which coincided with the oxic-anoxic interface and indicated that the influx of O(2) into the band was balanced by its consumption. However, in batch cultures methanogenesis ceased as soon as traces of O(2) were added. Focusing on Methanobrevibacter cuticularis, a species colonizing the microoxic gut epithelium of termites, a diffusion-limited setup was used that allowed the exposure of dense cell suspensions to controlled O(2) fluxes. Here, Methanobrevibacter cuticularis was capable of simultaneous CH(4) production and O(2) consumption. Low O(2) fluxes (10% of the CH(4) production rate) had virtually no influence on methanogenesis [4.5 micromol CH(4) (mg dry wt)(-1) h(-1)], whereas higher O(2) fluxes (up to 30% of the initial CH(4) production rate) caused a reversible decrease in methanogenesis, which was accompanied by a reversible, partial conversion of coenzyme F(420) to factor F(390). The maximum O(2) reduction rate [4.8 micromol O(2) (mg dry wt)(-1) h(-1)] that could be maintained over extended time periods (>30 min) was similar to the CH(4) production rate under anoxic conditions.

Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane.

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.