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.

Assessment of Bacterial Community Composition of Anaerobic Granular Sludge in Response to Short-Term Uranium Exposure.

The effect of 10-50 µM uranium (U(VI)) on the bacterial community of anaerobic granular sludge was investigated by 24-h exposure tests, after which the bacterial community was analyzed by high-throughput sequencing. The specific U(VI) reducing activity of the anaerobic granular sludge ranged between 3.1 to 19.7 µM U(VI) g-1(VSS) h-1, independently of the initial U(VI) concentration. Alpha diversity revealed that microbial richness and diversity was the highest for anaerobic granular sludge upon 10 µM uranium exposure. Compared with the original biomass, the phylum of Euryarchaeota was significantly affected, whereas the Bacteroidetes, Firmicutes, and Synergistetes phyla were only slightly affected. However, the abundance of Chloroflexi and Proteobacteria phyla clearly increased after 24 h uranium exposure. Based on the genus level analysis, significant differences appeared in the bacterial abundance after uranium exposure. The proportions of Pseudomonas, Acinetobacter, Parabacteroides, Brevundimonas, Sulfurovum, and Trichococcus increased significantly, while the abundance of Paludibacter and Erysipelotrichaceae incertae sedis decreased dramatically. This study shows a dynamic diversification of the bacterial composition as a response to a short time (24 h) U(VI) exposure (10-50 µM).

Bioactive fractions from the pasture legume Biserrula pelecinus L. have an anti-methanogenic effect against key rumen methanogens.

Methanogenic archaea (methanogens) are common inhabitants of the mammalian intestinal tract. In ruminants, they are responsible for producing abundant amounts of methane during digestion of food, but selected bioactive plants and compounds may inhibit this activity. Recently, we have identified that, Biserrula pelecinus L. (biserrula) is one such plant and the current study investigated the specific anti-methanogenic activity of the plant. Bioassay-guided extraction and fractionation, coupled with in vitro fermentation batch culture were used to select the most bioactive fractions of biserrula. The four fractions were then tested against five species of methanogens grown in pure culture. Fraction bioactivity was assessed by measuring methane production and amplification of the methanogen mcrA gene. Treatments that showed bioactivity were subcultured in fresh broth without the bioactive fraction to distinguish between static and cidal effects. All four fractions were active against pure cultures, but the F2 fraction was the most consistent inhibitor of both methane production and cell growth, affecting four species of methanogens and also producing equivocal-cidal effects on the methanogens. Other fractions had selective activity affecting only some methanogens, or reducing either methane production or methanogenic cell growth. In conclusion, the anti-methanogenic activity of biserrula can be linked to compounds contained in selected bioactive fractions, with the F2 fraction strongly affecting key rumen methanogens. Further study is required to identify the specific plant compounds in biserrula that are responsible for the anti-methanogenic activity. These findings will help devise novel strategies to control methanogen populations and activity in the rumen, and consequently contribute in reducing greenhouse gas emissions from ruminants.

Effects and mode of action of chitosan and ivy fruit saponins on the microbiome, fermentation and methanogenesis in the rumen simulation technique.

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.

Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.

In clinical trials investigating human health and in the analysis of microbial communities in cultures and natural environments, it is a substantial challenge to differentiate between living, potentially active communities and dead cells. The DNA-intercalating dye propidium monoazide (PMA) enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. In the present study, we evaluated for the first time a PMA treatment for methanogenic archaea in cultures and particle-rich environmental samples. Using microscopic analyses, we confirmed the applicability of the LIVE/DEAD(®) BacLight™ kit to methanogenic archaea and demonstrated the maintenance of intact cell membranes of methanogens in the presence of PMA. Although strain-specific differences in the efficiency of PMA treatment to methanogenic archaea were observed, we developed an optimal procedure using 130 µM PMA and 5min of photo-activation with blue LED light. The results showed that the effectiveness of the PMA treatment strongly depends on the texture of the sediment/soil: silt and clay-rich sediments represent a challenge at all concentrations, whereas successful suppression of DNA from dead cells with compromised membranes was possible for low particle loads of sandy soil (total suspended solids (TSS)≤200 mg mL(-1)). Conclusively, we present two strategies to overcome the problem of insufficient light activation of PMA caused by the turbidity effect (shielding) in particle-rich environmental samples by (i) dilution of the particle-rich sample and (ii) detachment of the cells and the free DNA from the sediment prior to a PMA treatment. Both strategies promise to be usable options for distinguishing living cells and free DNA in complex environmental samples.

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen.

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.

Effects of ractopamine hydrochloride are not confined to mammalian tissue: evidence for direct effects of ractopamine hydrochloride supplementation on fermentation by ruminal microorganisms.

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.

Effect of nitrogen limitation on the biodegradability and toxicity of nitro- and aminophenol isomers to methanogenesis.

Monosubstituted nitro- and aminophenol isomers exhibited limited biodegradability under methanogenic conditions when supplied as the sole source of carbon and energy. This was examined by supplying to the same sediment samples, each isomer of nitro- and aminophenol as a sole source of added carbon under either N-supplemented or N-deprived methanogenic conditions. The results demonstrated that under N-supplemented conditions, only 2-NP (NP = nitrophenol), 4-NP and 4-AP (AP = aminophenol) were stoichiometrically mineralized, 2-AP, 3-AP, and the 3-AP metabolite formed from 3-NP reduction were persistent over the 51-week incubation period. In addition, NP isomers inhibited initial rates of methanogenesis, while all AP amended cultures exhibited no significant inhibition in the rate of methanogenesis. Under N-deprived conditions, 2-NP, 2-AP and 4-AP were mineralized, while 3-NP, 4-NP and 3-AP were persistent over the 51-week incubation period. Although all NP isomers were still metabolized through the corresponding AP isomer, the deprivation of nitrogen significantly depressed both the rate and extent of methanogenesis. In general, nitrogen supplemented cultures produced 25% more methane than the nitrogen limited cultures, and the initial rates of methanogenesis were four times greater. While these data showed that under N-deprived conditions methanogenesis was inhibited to a greater extent by these compounds, it also suggests that N-deprived conditions may have facilitated the establishment of a 2-AP metabolizing consortium.