為了解在不同環境下，微生物代謝功能表現的差異，本研究建立一項免標定蛋白質體的分析方法(label-free proteomic)。此方法主要是修改蛋白豐富度因子(NSAF)，導入一個housekeeping gene當作內標(本研究使用elongation factor)，將不同的樣本的基因表現作正規化，使得樣本與樣本之間的基因表現水平可以進行比較。本研究中運用這個方法來探討高溫厭氧生物膜反應槽中，甲烷菌在不同負荷下之蛋白質體表現。
在厭氧生物處理系統中，甲烷菌扮演重要的角色，負責有機物降解的最後一個步驟，使反應槽能操作在穩定狀態下，並將有機物轉換成甲烷與二氧化碳等最終產物。因此，了解甲烷菌在不同環境下代謝路徑及調控機制的變化是很重要的，相關知識將有助於提昇厭氧生物處理系統的效率。在本研究中，分別將高溫厭氧生物膜反應槽操作在3及 0.25 kg-TA/m3-day的負荷時，監測反應槽操作效率並萃取蛋白，進行二維奈米級高效液相層析儀串聯式質譜儀的分析，分別從系統中主要之甲烷菌Methanosaeta thermophila、Methanosaeta concilii及Methanolinea tarda，測得667、453及323個實際表現蛋白的身分，其中有322、132及83個蛋白重複出現在兩個負荷的樣本中，這些蛋白佔各菌總蛋白豐富度約90%、67%及70%，顯示出M. thermophila對於負荷的改變影響較小，M. concilii及M. tarda則較大。根據KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway的分類分析，顯示出約有30-40%是參與甲烷化路徑，在M. thermophila約比M. concilii及M. tarda高出約10%的蛋白豐富度，但在不同負荷下對M. concilii及M. tarda參與甲烷化之蛋白豐富度有明顯影響(大於10%)，而對 M. thermophila的影響卻很小(小於1%)。更進一步檢視參與甲烷化路徑之中的各個基因表現，結果發現雖然負荷變化對M. thermophila的基因表現影響不顯著，但在負荷提升時，代謝醋酸第一個步驟之酵素(acetyl-coenzymeA synthetase, acs)，有豐富度明顯增加趨勢(增加大於兩倍)，此酵素之功能為將醋酸(acetate)轉換為乙醯輔酶A(acetyl-CoA)，但在嗜醋酸型甲烷化第二步驟之酵素(acetyl-CoA decarbonylase/synthase, cdh)則無明顯向上調節，推測當負荷提升時增加代謝醋酸產生之乙醯輔酶A用來細胞生長而非提升甲烷化效率。值得注意的是Methanosaeta 在過去被認為只能利用醋酸產生甲烷，然而在我們的系統中M. thermophila卻發現整套利用二氧化碳產生甲烷的酵素被表現出來，雖然所佔之豐富度較嗜醋酸型甲烷化低，顯示能量來源還是以醋酸代謝為主，但仍可能提供另一個產生能量來源的路徑，並有助於調節反應槽內的氫分壓。在Methanosaeta屬底下之另一嗜醋酸型甲烷菌種 M. concilii，則與M. thermophila很不一樣的功能性表現。當負荷降低時，參與甲烷化蛋白之豐富度約降為高負荷之一半，而在代謝輔酶及維他命的功能中卻有顯著的向上調節的趨勢(約17%)。更進一步檢視，發現主要是由硫胺素(Thiamine)的代謝及葉酸(Folate)的合成有關蛋白的提升所造成。與M. thermophila有很大的不同是在參與嗜醋酸型甲烷化之蛋白表現則幾乎沒有差異，且負荷提升時使豐富度增加，而在嗜氫型甲烷化之路徑則缺少3~4個關鍵酵素被偵測到，使得代謝路徑不完整。在嗜氫型甲烷菌M. tarda的分析結果也顯示出負荷對其能量代謝之影響，與M. concilii較為相似，但在功能未知的蛋白中，卻也出現了相當大的差異，這也指出當環境產生變化時甲烷菌的基因表現還有一些未知的調節機制，參與嗜氫型甲烷化之蛋白也隨著負荷上升時而表現量提升。此外，還有一些蛋白表現的調節在環境改變之下扮演著重要的角色，像是S-layer的形成，可以保護細胞減少外在環境變化的影響。膜蛋白運輸可以傳輸細胞生長時必須之無機離子及胺基酸等物質的調控，也可將毒性物質輸出，使細胞免於受到傷害。還有一些抵抗壓力之蛋白的表現也顯示出甲烷菌生長在本系統中需面對許多的環境衝擊。整合上述之研究成果，我們所運用之研究方法成功地提供了甲烷菌在反應槽中實際功能表現的資訊，更進一步了解甲烷菌在負荷改變下之代謝路徑及調節機制。

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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