Monitoring of methanogenic population dynamics of anaerobic digestion treating swine wastewater and biological conversion of refractory organic materials

DoctorGlobal swine production has increased more than 3.5-fold during the past 40 years. Associated with these increased swine herds are large quantities of swine manure. The anaerobic digestion process is a biological treatment method that is widely used to treat wastewater containing highly concentrated organic compounds such as swine wastewater. In this study, I demonstrated links between reactor operating conditions, physical and chemical performance parameters, and microbial population dynamics. The DGGE analysis provides us with information to recognize the population shift occurring in the microbial community with different time and substrates, and to deduce the probable major populations present in the community. In chapter 3, the DGGE bands corresponding methanogenic species Methanoculleus bourgensis, Methanoculleus receptaculii, Methanospirillum hungatei, methanobrevibacter sp.AbM4, Methanobrevibacter gottschalkii, Methanobacterium oryzae, Methanosphaera stadtmanae, Methanosaeta concilii, and Methanosarcina siciliae were identified with strong intensity during the methanogenesis. A more-detailed and quantitative analysis with higher resolution by other molecular approaches, such as FISH or real time quantitative PCR (QPCR), might lead to a better understanding of the roles by these methanogenic species. Therefore, the quantitative approaches by QPCR were carried out along with the primer and probe sets design of dominant methanogens under lab scale anaerobic condition.The ten primers and probes sets for group- or species-specific detection of methanogenic species using a QPCR were designed. The target-specificity of the ten primers and probes sets were evaluated by online analysis of a potential false-positive and false negative result and then empirically verified by testing the pure culture DNAs. Results show good agreement between the expected and observed specificity of the sets for their target groups. The ten primers and probes sets were highly specific to their target groups. Target groups were amplified and detected in a wide dynamic range of 101 to 109 template copies by corresponding primer and probe set and the linearity of the quantifications was demonstrated over a range of eight to nine log steps. The accuracy of quantification method was successfully validated by cross-comparing the results quantified using all corresponding primer and probe sets. Quantitative with real-time QPCR and qualitative analysis with DGGE showed that the M. bourgensis and M. AbM4 were the major methanogenic groups in the anaerobic digestion of swine wastewater containing highly concentrated organic materials and ammonia. Although the substrate had highly concentrated acetate, the dominant methanogens were not aceticlastic mathanogens, but hydrogenotrophic methanogens. In chapter 4, qualitative with real-time QPCR and qualitative analysis with DGGE showed that the M. concilii was the major methanogenic group in the pilot plant anaerobic digestion of swine wastewater containing highly concentrated organic maerials and ammonia. Although this methanogenic 16S rRNA gene concentration decreased until 1.0 turnover time, after then the gene copy number was maintained with constant, about 50% of total methanogenic amounts. Two different methanogens with this aceticlastic methanogen, M. bourgensis and M. AbM4 were observed as another major methanogens, which belonged to hydrogenotrophic methanogen. According to the DGGE result, the identified methanogenic profiles were similar with lab scale methanogenic DGGE result. However the QPCR data was different with lab scale quantitative data. That is, the absolute quantification concentration of 16S rRNA gene of major methanogens in pilot plant system was lower than that of lab scale methanogen. Also, most important thing is that the most dominant methanogenic species was different in two different sized reactor systems. In case of lab scale anaerobic digestion system, the major methanogenic speciese were hydrogenotrophic methanogen such as M. bourgensis and M. AbM4. On the other hands, in case of pilot plant anaerobic digestion system, the dominant occupied methanogenic species was aceticlastic methanogen such as M. concilii. The distinctions of dominant methanogenic species between lab scale process and pilot scale process were resulted in performance efficiency.In chapter 5, to observe the degradable activity by white rot mycelia, we selected the most useful mycelia among three white rot fungi in previous studies. Among P. ostreatus, H. erinaceum and G. lucidum, G. lucidum was shown the most fast growth activity in solid-state swine wastewater cultivation. Compared with other mycelia, the growth rate of G. lucidum calculated by the RSA model was higher than that of other mycelia. The RSA model estimated a maximal radial extension rate (10.59 mm/d) under conditions at 5 g COD/L concentration, pH 5 and 29.7 oC. Therefore, the liquid cultivation of G. lucidum carried out in 500 ml Erlenmeyer flasks containing 300 ml of effluent of pilot plant methanogenic reactor at the optimum condition resulting from solid culture RSM.According to the results, humic substances were bio-degradable material using myceliaon the contrary, fibers were not degraded by mycelia. It was thought that this profile comes from mycelial properties. Because mycelia was composed of various poly-saccharides such as α and β glucan. Therefore, more and more poly-saccharides were produced with increase of mycelia growth and then, the amounts of fibers were also increase during incubation times. Because the treatment of major biorefractory humic substances generates valuable products, this investigation of the extracellular mineralization of humic substances by mushroom mycelia was tested as a novel bioconversion technology for swine waste