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Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase ( mcr ) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated. Rumen methanogenic archaea are major sources of methane emissions and potential targets for methane mitigation strategies. Poulsen et al. now show that dietary rapeseed oil (RSO) supplementation can reduce the abundance of methanogenic Thermoplasmata archaea inhabiting the bovine rumen.

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 °C) and all other methanongens (84-86 °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.

This study investigates the effects of supplementing a control diet (CON) with chitosan (CHI) or ivy fruit saponins (IVY) as natural feed additives. Both additives had similar abilities to decrease rumen methanogenesis (–42% and –40%, respectively) using different mechanisms: due to its antimicrobial and nutritional properties CHI promoted a shift in the fermentation pattern towards propionate production which explained about two thirds of the decrease in methanogenesis. This shift was achieved by a simplification of the structure in the bacterial community and a substitution of fibrolytic (Firmicutes and Fibrobacteres) by amylolytic bacteria (Bacteroidetes and Proteobacteria) which led to greater amylase activity, lactate and microbial protein yield with no detrimental effect on feed digestibility. Contrarily, IVY had negligible nutritional properties promoting minor changes in the fermentation pattern and on the bacterial community. Instead, IVY modified the structure of the methanogen community and decreased its diversity. This specific antimicrobial effect of IVY against methanogens was considered its main antimethanogenic mechanism. IVY had however a negative impact on microbial protein synthesis. Therefore, CHI and IVY should be further investigated in vivo to determine the optimum doses which maintain low methanogenesis but prevent negative effects on the rumen fermentation and animal metabolism. Rumen function is generally suboptimal leading to loses in the form of methane and nitrogen, analysis of the rumen microbiome is vital to understand the mode of action of new feed additives to improve rumen function. Graphical Abstract Figure. Rumen function is generally suboptimal leading to loses in the form of methane and nitrogen, analysis of the rumen microbiome is vital to understand the mode of action of new feed additives to improve rumen function.

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 (P L  = 0.0033) and Methanimicrococcus (P L  = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (P L  = 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 (P Q  = 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.

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 H 2 . 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 H 2 /CO 2 to employment of both H 2 /CO 2 and methanol/CO 2 . 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.

The anaerobic treatment of saline effluents using halophilic and halotolerant microbial consortia is of major interest. Inhibition of anaerobic digestion is known to occur at high salt content. However, it seems that the suitable adaptation of an anaerobic sludge makes possible the treatment of saline wastewater. In this study, a non-saline anaerobic sludge was inoculated in two anaerobic batch reactors operating with a different substrate (distillery vinasse and ethanol) and subjected to increasing NaCl concentrations. The performance of the digesters appeared to be highly dependent on the nature of the substrate, and a similar level of inhibition (i.e. around 90% of the specific loading rate and specific methanogenic activity) was stated at 10 g l-1 of NaCl with distillery vinasse and 60 g l-1 of NaCl with ethanol. The characterization of the microflora and its adaptation to increasing NaCl conditions were also investigated using molecular tools based on the analysis of genomic 16S rDNA. The microbial communities revealed a high diversity that could be maintained in both reactors despite the increase in NaCl concentrations.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Four experiments were conducted to investigate the effects of ractopamine hydrochloride (RAC) on ruminal fermentation and proteolysis. In Exp. 1, in vitro gas and VFA production was measured in flasks incubated with 0, 0.226, 2.26, 22.6, and 226.0 mg of RAC/L of buffered ruminal fluid. Ractopamine hydrochloride had a quadratic effect on in vitro gas production (P < 0.05; 177, 181, 185, 190, and 170 mL for 0, 0.226, 2.26, 22.6, and 226.0 mg, respectively). Total VFA production was not significantly changed with RAC (P > 0.50). In Exp. 2, IVDMD was measured with tubes incubated with 0, 0.226, 2.26, or 22.6 mg of RAC/L of buffered ruminal fluid with 4 substrate combinations: corn, corn plus soybean meal, corn plus urea, and corn plus soybean meal plus urea. Dry matter disappearance was measured after 2, 4, 6, 8, or 12 h of fermentation. There was an interaction between RAC and substrate (P < 0.01), with more degradable forms of nitrogen eliciting greater IVDMD from RAC. Significant main effects also were detected for RAC, substrate, and hour (P < 0.001). In Exp. 3, AA and ammonia were measured in tubes treated with 0 or 2.26 mg of RAC/L of buffered ruminal fluid. Tubes were incubated for 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, or 240 min. There were decreases in ammonia and AA concentrations with RAC (P < 0.001). Experiment 4 used 16 ruminally fistulated Holstein steers in a 2 x 2 x 2 factorial arrangement of treatments. Factors consisted of grain processing method (steam-flaked or dry-rolled corn), concentration of dried distillers grains (DG) with solubles (0 or 25% DG, DM basis), and concentration of RAC (0 or 200 mg/d). Ruminal ammonia concentrations were less when RAC was fed in combination with dry-rolled corn, but not when RAC was fed in conjunction with steam-flaked corn (grain processing x RAC, P < 0.01). Addition of RAC, steam-flaked corn, and DG all resulted in reduced ruminal ammonia concentrations (P < 0.01). Amino acid concentrations were decreased when RAC was added to diets with DG but were unchanged in diets without added DG (DG x RAC, P < 0.05). Changes in ruminal ammonia and AA concentrations with RAC supplementation are dependent on grain processing and the addition of DG to finishing diets. Results from these studies suggest that RAC affects fermentation by ruminal microflora. Effects of the interactions between RAC and protein source, grain processing, and DG on proteolysis could have important implications when formulating diets for cattle supplemented with RAC.

During periods of drainage, both water stress and oxygen can cause damage to indigenous methanogens. In the present study, we evaluated the tolerance of seven methanogenic strains (Methanobrevibacter arboriphilicus, Methanobacterium formicicum, Methanococcus vannielii, Methanospirillum hungatei, Methanoculleus olentangyi, Methanoplanus limicola, and Methanosarcina mazei) to long-term exposure to air/nitrogen and drying. We found that these methanogenic strains except for M. limicola and M. olentangyi in pre-dried cells offered more tenacious resistance to desiccation and oxygen exposure than those in enriched liquid cultures. In the case of M. formicicum, the liquid culture of this strain could remain viable when mixed well with fresh or sterile soil, but not when cultured without soil, or with agar slurry. These results suggest that indigenous methanogens localize within soil compartments to protect themselves from the damage caused by gradual drying under an oxic atmosphere.

Two genes encoding esterases EstA and EstB of Picrophilus torridus were identified by the means of genome analysis and were subsequently cloned in Escherichia coli. PTO 0988, which is encoding EstA, consists of 579 bp, whereas PTO 1141, encoding EstB, is composed of 696 bp, corresponding to 192 aa and 231 aa, respectively. Sequence comparison revealed that both biocatalysts have low sequence identities (14 and 16%) compared to previously characterized enzymes. Detailed analysis suggests that EstA and EstB are the first esterases from thermoacidophiles not classified as members of the HSL family. Furthermore, the subunits with an apparent molecular mass of 22 and 27 kDa of the homotrimeric EstA and EstB, respectively, represent the smallest esterase subunits from thermophilic microorganisms reported to date. The recombinant esterases were purified by Ni²⁺ affinity chromatography, and the activity of the purified esterases was measured over a wide pH (pH 4.5-8.5) and temperature range (10-90°C). Highest activity of the esterases was measured at 70°C (EstA) and 55°C (EstB) with short pNP-esters as preferred substrates. In addition, esters of the non-steroidal anti-inflammatory drugs naproxen, ketoprofen, and ibuprofen are hydrolyzed by both EstA and EstB. Extreme thermostability was measured for both enzymes at temperatures as high as 90°C. The determined half-life (t ₁/₂) at 90°C was 21 and 10 h for EstA and EstB, respectively. Remarkable preservation of esterase activity in the presence of detergents, urea, and commonly used organic solvents complete the exceptional phenotype of EstA and EstB.