Quantification Of Methanogens And Manipulation Of Microbial Community In The Rumen

Methane production in the rumen represents a loss of energy for the host animal, and, in addition, methane eructated by ruminants may contribute to a greenhouse effect or global warming. Scientists have been recently paying much attention to ruminal methanogens and methanogenesis with the global concerning on greenhouse gas emission. This study, divided into four parts, was carried out to establish molecular techniques to analyze the metanogens in the rumen, and to investigate the relationships among methanogenesis and methanogens and microbial community structure. In Part One, molecular techniques were established to monitor the change of methanogens and other microorganisms in the rumen. In Part Two, three known methnogen-inhibitors were selected to study their influence on ruminal methanogens and microbial populations. Tea saponin was used in Part Three to study rumen methanogenesis and methanogens. Part Four was conducted to study the relationship between methanogens and other microbial population in forage-rich diets in vitro.Part One:Establishing Real Time PCR technique to monitor ruminal methanogens and microbial populationReal Time PCR technique was conducted to monitor the microbial populations including methanogens, fungi, F. succinogenes and R. flavefaciens, and activity of methanogens as well. Absolute quantification of methanogens was established. There was significant correlation on the quantification of Methanobrevibacter ruminantium by real time PCR (y) and counting (x):y= 1.0152x-3×108(r=0.9753; P<0.01). Then expression of mcrA gene was analyzed to predict the activity of methanogens. BES significantly reduced the mcrA gene expression in whole period of incubation, while lumazine began to reduce the gene expression after 6 h of incubation. Relative quantification was also established to detect the methanogens, fungi, F.succinogenes and R.flavefaciens in mixed culture when treated with BES. All standard curves have high R2 and their amplification efficiency were very close (98.4-99.9%).Real time PCR could be used to quantify ruminal microbial population. In practice, absolute and relative quantification could be adopted according to different research purpose.Part Two:Use of inhibitors to manipulate methanogens and microbial population Manipulation of inhibitors on methanogens and microbial population in mixed culture was investigated. After 24 h of incubation, BES, Lumazine and Mevinolin had different modes of effects on ruminal methanogenesis, methanogens and expression of mcrA gene. BES significantly reduced methane production, methanogens and expression of mcrA gene. Lumazine reduced methane production and methanogens, while Mevinolin had no significant effect. There was difference between present and active methanogens revealed by the DGGE profiles of DNA amplicons from 16S rDNA and 16S rRNA. Protozoa was increased by BES, Lumazine and Mevinolin. DGGE showed that there are big differences between present and active bacteria. BES and Lumazine had greater effect on the community of active bacteria.BES and Lumazine, which act on the pathway of methanogenesis, had greater effect on methanogenesis, methanogens and microbial community in mixed culture compared to Mevinolin.Part Three:Manipulation of methanogens and microbial population by tea saponinsTo explore the mechanism of tea saponin on reducing methnaogenesis, effect of tea saponin on ruminal microbial community, methanogens and pure culture of Methanobrevibacter ruminantium was investigated. Protozoa was significantly reduced by 51.2 and 22.9%(P<0.05) by the addition of 0.2 and 0.4 mg/ml tea saponin, respectively. Tea saponin did not have obvious effect on methanogens, and there was inconsistency between methanogenesis and methanogens. As the dominant methanogens in the rumen, Methanobrevibacter ruminantium was adopted to explore its response to tea saponin. There was no inhibited effect of tea saponin on Methanobrevibacter ruminantium. However, expression of mcrA gene in mixed culture was reduced by 76% with addition of 0.4 mg/ml tea saponin.Tea saponin inhibited protozoa and then reduced methane production, while it did not have significant effect on methanogens. It is speculated that tea saponin inhibited protozoa and reduce hydrogen production, and then reduced the activity of methanogens. Expression of mcrA gene is a more reasonable parameter to predict the change of methane production in the complex ecosystem compared to methanogenic population.Part Four:Manipulation of methanogens and microbial population in forage-rich dietDifferent sources and levels of neutral detergent fibers (NDFs) were adopted to simulate forage-rich diet in vitro. Different sources of NDF significantly influenced the methanogenesis and methanogens. As showed by DGGE, methanogens' community structures of NDFs extracted from alfalfa and corn silage were more similar to each other. After incubation of 24 h, NDF source, level and their interaction had significant effects on methanogens. There was negative correlation between methanogens and methanogenesis. Level of NDF had minor effect on the community structure of methanogens, but had strong effect on bacteria community structure and the similarity index was only 46-72% as showed by DGGE.There were negative correlation of methanogenesis and methanogens, F.succinogenes and R.flavefaciens, while positive correlation existed between methanogens and fungi.In summary, when considering the change of methanogenesis, methanogen number and mcrA gene expression should be taking into account in the ruminal ecosystem. There is significant correlation between methanogen and protozoa population, while positive correlation exists in methanogen and fibrolytic microorganisms.