Metagenomic Studies Of Microbial Community In Lower Respiratory Tract Of A Patient With Pneumonia And Solid-state Biogas Reactor

The appearance of metagenomics enlarges the view of microbiology research.With the advantage of new technology, metagenomics has been applied widely in various studies, such as ocean, soil, hot spring, oral cavity, gastrointestinal tract, energy, environmental remediation, agriculture and biophylaxis. Normally, lower respiratory tract is relatively closed and sterile, thus bacterial invasion would be harmful or even fatal. But due to the restriction of sampling, recent research of lower respiratory tract microbiology is not in-depth enough. Moreover, other metagenomic researches of extracorporeal enclosed environment, such as methane fermenting, are also at initial stage.This dissertation focuses on samples from lower respiratory tract of patients with pneumonia and biogas fermentation plant. We optimized the methods of DNA extraction. Through high throughput sequencing and bioinformatics analysis, we studied the microfloras of these two enclosed environment and found several important but rarely reported species.In the metagenomic research of lower respiratory tract, we collected microflora from the deep part of patient’s lower respiratory tract by bronchoalveolar lavage, which effectively reduced the interference of microflora from upper respiratory tract and oral cavity. Using reverse hybrid selection, we captured and removed a proportion of human DNA. It is proved by Real-time PCR and pyrosequencing platform that this method is effective, for it not only preserves the characteristics of abundant microflora but also nicely covers low or middle abundance microflora. Then bioinformatic analysis was performed and20species were found which are related to respiratory tract severe infection. Moreover, we reported two potentially pathogenic species in lower respiratory tract, Microbacterium laevaniformans and Borrelia garinii. Through gene function annotation we detected four drug resistant genes and six virulence factors. Our results were corresponding to the symptom of the patient, which provides foundation for quickly clinical diagnosis and pharmacy.In the research of biogas anaerobic fermentation, we compared different DNA extraction methods and made optimization. We noticed that different cell lysis methods and lysis level had obvious influence on the subsequent flora analysis. Through pyrosequencing platform and bioinformatic analysis, characteristic of microflora in biogas anaerobic fermentation was obtained. The dominant genera mainly participate in the degradation of protein, fat, cellulose and other molecular polymer. We detected both aceticlastic and hydrogenotrophic pathways in this system. Abundant Psychrobacter species, which produce low temperature-adaptive lipases, and Anaerococcus species, which have weak fermentation capabilities, were identified for the first time in biogas fermentation. These results provided basis for further improvement of methane yield in low temperature by bioengineering technology.