多層複合濾料水質淨化系統(Multi-Soil-Layering, MSL)對水質的淨化效果主要來自於複合濾料包(Soil mixture bag, SMB)，SMB由土壤、鐵粒、碳粉與木屑所組成，土壤提供微生物生長所需之微量元素同時也具有生物分解、過濾與吸附能力，鐵粒用以提升磷酸鹽吸附能力，碳粉可供微生物生長並吸附水中有機物，木屑做為脫硝反應中的電子提供者。MSL透過物理、化學及生物機制淨化水質，而SMB中的微生物更是與有機物去除密切相關且微生物族群易受到環境條件產生變化，但過去研究鮮少針對MSL內部微生物多樣性與變化進行了解，或僅以實驗室規模進行討論。
澎湖成功水庫MSL模場已運作3年，用於處理優養化且有機碳濃度高的水庫原水，根據過往研究成果其淨化效果大多來自生物分解，為了解用於處理現地處理優養化水庫的MSL與其前處理系統BioNet中細菌族群結構與分佈，以及MSL中環境因子與水質參數、細菌族群變化的關聯性，本研究自111年10月起至111年3月止，期間每週監測進出流水質，並於試驗前後期採集各一次BioNet擔體和MSL中SMB土包樣品。樣本經萃取與PCR擴增後使用Illumina ＭiSeq定序平台及P300雙邊定序試劑對16S rRNA基因(V3-V4) 高變異區間以次世代高通量定序(Next Generation Sequencing, NGS)取得微生物體資訊。
本研究使用Bray-Curtis距離進行排序與相似性分析，透過主座標分析(Principal co-ordinates analysis, PCoA)以及非度量多維度分析(Non-Metric Multi-Dimensional Scaling, NMDS)區分不同樣本菌群之間的相似性，PCoA 及NMDS結果顯示樣本中微生物族群組成與多樣性變動與取樣深度及季節有顯著的關聯性，因MSL的水質淨化效果亦與深度及季節有關，初步研判微生物菌群變動與生物淨化功能變化有關。長時間運轉後MSL系統中Shannon index顯著提升，進流所帶來的營養物質有助於增加微生物族群多樣性，促進MSL中微生物生長與繁殖。為釐清環境因子對微生物族群變化的影響，可藉由典範對應分析(Redundancy Analysis, RDA)了解樣本、物種及環境因子之間的相關性，RDA結果顯示土壤環境因子中的Moisture、OC、TN及NH4-N與微生物族群變動最為相關，進流水中及SMB中可利用的有機質將有利於Bacteroidetes、Firmicutes及Actinobacteria生長，試驗後期土壤中的氨態氮及總氮與Patescibacteria、Nitrospirae以及Planctomycetes變化正相關，說明硝化菌馴養四個月後可明顯增加且SMB有利於水中有機氮進行氨氧化與硝化。次世代高通量定序結果顯示，去除有機物、氮系及磷系物質相關功能微生物包括分解複雜有機物相關細菌、氨氧化細菌(AOB)、亞硝酸鹽氧化細菌(NOB)、脫硝菌(DNB)、脫硝及異化硝酸鹽還原成銨菌(DN-DRNAB)、異營硝化及好氧脫硝菌(HN-ADB)、磷蓄積菌(PAO)、脫硝磷蓄積菌(DNPAO)，生物處理為BioNet及MSL系統去除有機物、氮系物質以及吸附固磷的重要途徑，且去除效率降低主要受季節水溫下降影響，MSL較BioNet更具有轉化複雜有機物能力，其將可提供脫硝菌利用並增強脫硝性能。FAPROTAX用以預測淨水系統中微生物功能，根據FAPROTAX分析結果，好氧代謝為MSL去除有機物的主要機制，且上層SMB為此機制的主要運作區塊。低 HLR (<1,000 L/m2/d)、高溶氧、中高溫、高有機碳是 適合快速馴養 MSL 微生物並提升 MSL 的 TOC 及氮系物質去除效果的較佳條件。MSL系統於試驗前期硝化途徑可能依賴異營硝化，試驗後期異營硝化菌下降而自營硝化菌Nitrospira上升，研判MSL硝化途徑可能依賴Comammox。BioNet系統及MSL系統中均具有好氧及厭氧脫硝菌，厭氧脫硝受抑制之主要原因為低溫環境、碳源限制及高DO濃度等，MSL上層SMB中能進行好氧脫硝的HN-ADB可能為MSL系統重要脫氮途徑。MSL系統硝化能力將隨馴養時間增加而穩定提升，硝化菌經過4個月馴養後將顯著提升，而脫硝菌經過一個月即有顯著提升，研判本試驗環境不利於 MSL 系統 AOB 與 NOB 的養成。PAO及DNPAO並非MSL系統主要除磷途徑，磷系物質的去除更多來自於攔除、吸附及螯合等物理機制。

The carbon source, dissolved oxygen, and temperature are the critical factors for the stable growth of microorganisms and the control of the redox environment in the system to realize the removal of nitrogen and organic matter from Multi-Soil-Layering (MSL). Biodegradation is the primary removal mechanism of MSL. By studying the microbial composition and diversity of the pilot-scale MSL model plant, the purification efficiency of the eutrophic raw water can be improved. In this study, microbial changes in the MSL under the seasonal variation of raw water were investigated, as well as microbial composition and diversity between different depths. Illumina Miseq High-throughput sequencing analysis showed that water content, organic carbon, total nitrogen, and ammonia nitrogen in MSL were most associated with changes in the microbial community. Aerobic metabolism is the primary mechanism for MSL to remove organic matter, and the upper SMB is the preceding operating block of this mechanism. In nitrification, it is speculated that MSL mainly depends on Comammox, and denitrification may be principally contributed by aerobic denitrification. This study implies that Low HLR (<1,000 L/m2/d), high dissolved oxygen, medium-high temperature, and high organic carbon are the best conditions for rapid domestication of MSL microorganisms and improved removal efficiency of TOC and nitrogen from MSL. The nitrifying bacteria will increase significantly after four months of domestication, while the denitrifying bacteria must only be domesticated for one month. It is suggested that the environment is not conducive to developing AOB and NOB in the MSL system.

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