Detection And Application Of Nitrite-dependent Methanotroph In Rumen

The methane produced by ruminants not only aggravates greenhouse effect,but also leads to a loss of 2%to 12%feed energy.Studies have shown that nitrate supplementation in ruminants could effectively reduce methane emission.Nitrate can compete with CO₂for hydrogen to reduce methane production and be used as non-protein nitrogen for ruminants.However,nitrate supplementation can lead to nitrite poisoning in rumen and ruminants,and there are no effective measures to solve this problem.Nitrite-dependent methanotroph（NC10 phylum bacteria）were existed in freshwater sludge environments,which can use methane to reduce nitrite.In the previous work,it has been found that there are NC10phylum bacteria in rumen,which were not detected in the enrichment system of ruminant nitrate-dependent methanotroph.This result indicated that there may be other methanotrophs that can more effectively use methane and nitrite.Experiment 1 Diversity of nitrite-dependent methanotroph in rumen fluidTo investigate the organisms performing the nitrite-dependent anaerobic methane oxidation（N-DAMO）in rumen,the enrichment culture system was established by inoculating with dairy goat rumen fluid,supplying with Na NO₂ as the electron acceptor and methane as the sole carbon source.The results showed that it inhibited the bacterial growth of Bacteroidetes and Firmicutes,promoted the bacterial growth of Proteobacteria and Acfinobacteria after 6 months of enrichment culture.Proteobacteria became predominant in the N-DAMO enrichment system and Pseudomonas aeruginosa（P.aeruginosa）became the dominant OTU in the system.Experiment 2 Isolation,identification and functional verification of P.aeruginosa NWAFUP1Based on the results of experiment 1,the single strain was isolated and cultured by using the traditional isolation and pure culture technique of microorganisms.The isolated strain was identified as P.aeruginosa in N-DAMO enrichment culture system by morphology and molecular biology,and named P.aeruginosa NWAFUP1.Then it was inoculated into the system which was provided methane as the sole carbon source and 4 m M KNO₃ or 2 m M Na NO₂ as the main electron acceptor.The results showed that P.aeruginosa NWAFUP1 was found to have the ability of finishing the whole denitrifying anaerobic methane oxidizing process by itself（P<0.05）,and 2%O₂ and acetate-propionate had different effects on the DAMO function of P.aeruginosa NWAFUP1,but did not lead to the disappearance of DAMO function.Experiment 3 Effects of P.aeruginosa NWAFUP1,nitrate in vitro on rumen fermentationTen mmol/L KNO₃alone,P.aeruginosa NWAFUP1 alone or both of them were added to rumen fluid to study the effects of P.aeruginosa NWAFUP1 on nitrite accumulation induced by nitrate supplementation,fermentation performance and methane emission in rumen.In vitro rumen fermentation revealed that supplementation with P.aeruginosa NWAFUP1 can reduce methane emissions（P<0.05）.Compared with nitrate supplementation alone,The addition of both can significantly increase the digestibility of neutral detergent fiber and acid detergent fiber,the concentration of isobutyric and isovaleric,fermentation efficiency,and significantly decrease the concentration of NH₃-N（P<0.05）,alleviated both nitrite accumulation and inhibition of propionic acid synthesis caused by nitrate supplementation.Overall,in the rumen enrichment culture system with methane as the sole carbon source and nitrite as the electron acceptor,P.aeruginosa（the dominant OTU）which can use methane as the sole carbon source,nitrate or nitrite as the electron acceptor to oxidize methane to carbon dioxide,nitrate and nitrite were reduced to nitrogen.In addition,P.aeruginosa still had the function of nitrate-dependent anaerobic methane oxidation in the presence of 2%O₂ and acetate-propionate.P.aeruginosa supplementation not only reduced methane emissions,but also alleviated the inhibition of nitrite accumulation and propionic acid synthesis caused by the addition of nitrate in vitro.