| Summary: | 碩士 === 國立成功大學 === 環境工程學系碩博士班 === 101 === To understand the regulation of gene expression in the perturbation environments, a label-free quantitative proteomic approach called E-NSAF was developed in this study. The concept of E-NSAF is using a selected housekeeping gene, which can maintain the stable expressed level under different experimental conditions (elongation factor is selected in this study), as an internal standard to normalize the protein abundance factor (NSAF). In this study, this approach is applied to investigate metaproteome of methanogens in the thermophilic biofilm reactor during treating terephthalate (TA) at different loading conditions.
In the anaerobic biological treatment processes, methanogens are the key microorganisms that are responsible for the final step of degradation of organic compounds. Therefore, to achieve stable and efficient operation in the anaerobic bioreactor, the further understanding of the metabolic pathway and regulatory mechanisms within the methanogens is highly desirable. In this study, thermophilic biofilm reactor was operated under two loading stages (3 vs 0.25 kg-TA/m3-day), and monitored the performance of bioreactor. After that, protein extracted from the bioreactor while reached the high removal rate of terephthalate. Totally, the 2-D nano HPLC combined with Obitrap mass spectrometry detected expression of 667, 453, and 323 coding genes of Methanosaeta thermophila, Methanosaeta concilii, and Methanolinea tarda corresponding to a detected rate of 39%, 16%, and 16%, respectively of total protein coding genes. Among these, 322, 132, and 83 proteins were detected at both loadings. These proteins contained approximately 90%, 67%, and 70% of total relative abundance. The results indicated that the perturbation of loading have great impact on M. concilii and M. tarda other than predominant methanogens M. thermophila. The classification of KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway revealed that about 30-40% of abundance is involved in methanogenesis, and higher abundance in M. thermophila than M. concilii and M. tarda (higher by 10%). However, M. concilii and M. tarda have great impact (increased more than 10 %) within the increasing loading quite different from M. thermophila (less than 1% difference). Further analysis proteins involved in methanogenesis of M. thermophila shows the up-regulated (over 2-fold) of acetyl-coenzymeA synthetase (acs), which catalyzed the first step of acetate degradation, even if the less influence during the increasing loading. Nevertheless, the second step of aceticlastic methanogenesis was not up-regulated obviously (less than 2-fold). The possible explanation is that the acetyl-CoA forming from acetate degradation was used to cell growth instead of methanogenesis. Remarkably, it has been long determined that Methanosaeta are obligatory acetoclastic methanogens, the only substrate for methane formation and energy biosynthesis is acetate. Surprisingly, the entire suite of proteins detected that involved in hydrogenotrophic methanogenesis pathway were found in M. thermophila. Even though expressed at lower abundance compared to aceticlastic methanogenesis, acetate utilized still the main energy source rather than the CO2-reducing pathway. M. concilii, which is in the same genus of Methanosaeta, shows differentially functional expression from M. thermophila. Almost two-fold up-regulated in the entire aceticlastic methanogenesis within the loading increased, but the incomplete hydrogenotrophic methanogenesis pathway (lack of 3-4 essential enzymes) was observed. The hydrogenotrophic methanogens, Methanolinea tarda, almost all of the proteins were up-regulated more than 2-fold when the loading increased, implying the ability of methane generation by using CO2/H2 will improve as the loading increased. Besides, the adapted strategies of methanogens such as S-layer formation, stress resistance proteins, and regulated the membrane transport were observed in this study, indicated that methanogens encountered several environmental challenges in the terephthalate-degrading system during the changing loading. Totally, we provide the new insight into the biological processes and ecological functions of methanogens in the complex ecosystem. The findings obtained in this study can provide the further ecological understanding of methanogens under the perturbation environments.
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