Exploring the microbial community and nitrogen removal effiency for ANAMMOX system

傳統生物除氮程序是利用微生物的硝化和脫氮能力淨化水中含氮污染物質，因具有高效率、低費用等優點，故長久以來被廣泛應用於廢水處理系統。然而，利用生物處理氨氮廢水實例中，近年來以厭氧氧氨氧化程序(ANaerobic AMMonium OXidation, ANAMMOX程序)最受重視，此程序具有高效能除氮能力，且毋須額外添加有機碳源，其產生之污泥量更是遠少於傳統除氮程序所產生之污泥量，具節省成本等優點。現今國外有很多對於ANAMMOX之相關研究及將厭氧氨氧化微生物應用於實場的例子，但國內外的生物處理系統操作條件及厭氧氨氧化菌種類等環境因素有所差異，為此，本研究著手針對台灣地區厭氧氨氧化微生物進行調查。 本研究首先採集台灣地區可能具有厭氧氨氧化菌的環境樣本，並由實驗證實確具具有厭氧氨氧化微生物族群存在於樣本中，且其存在的數量亦有所不同。經由調查結果顯示，存在台灣地區環境樣本中的厭氧氨氧化微生物種類大多為Candidatus Brocadia sp.，數量可達104 ~ 108 copies/μg DNA。由於垃圾滲出水污泥樣本中存在的厭氧氨氧化微生物數量最高，因此，本研究後續試驗將以垃圾滲出水污泥樣本作為菌種來源，進行厭氧氨氧化微生物相關試驗。 選定菌種來源後，利用500 mL批次反應槽進行培養條件試驗，實驗證實培養條件可成功馴養厭氧氨氧化微生物。進一步提升批次反應槽體積至1000 mL，亦成功馴養厭氧氨氧化微生物Candidatus Anammoxoglobus propionicus，其數量最高可達2.87 ± 0.74 × 1010 copies/μg DNA，且此系統之氨氮去除率達92％，亞硝酸鹽氮去除率達100％，硝酸鹽氮無生成率，反而約有去除率15％。而15 L膜薄反應槽進行大量厭氧氨氧化微生物馴養方面，成功馴養出厭氧氨氧化微生物Candidatus Brocadia sp.，包含Candidatus Brocadia sp. 40、Candidatus Brocadia anammoxcidans及Candidatus Brocadia sinica三類菌種，數量最高可達3.23 ± 0.01 × 108 copies/μg DNA，目前系統最大氮負荷可達0.7 g-N/L/day，氨氮及亞硝酸鹽氮總去除率達96％。於氮負荷140 mg-N/L/d條件下，單一批次中，系統於進流基質後5小時，其氨氮及亞硝酸鹽氮去除率達95％，且anammox bacteria基因表現與產氣速率呈正比，而SAA為0.94 ± 0.24 g N2-N (g VSS)-1d-1。 本研究所建立兩個不同的ANAMMOX系統，其菌種植種來源相同，使用的培養基成份相同，可培養出不同的厭氧氨氧化微生物種類，存在於AR系統中為Candidatus Anammoxoglobus propionicus，而存在MS系統中則為Candidatus Brocadia sp.。由於本研究所馴養之厭氧氨氧化微生物Candidatus Anammoxoglobus propionicus顯少被文獻所提及，且其存在之系統具有良好的除氮效能，因此特針對AR系統進行與厭氧氨氧化微生物共存之微生物族群分析。研究顯示，系統中與其共存之微生物以屬於Chloroflexi門的絲狀菌族群為主，另有Acidobacteria、Actinobacteria、Azospirillum brasilense、Ignavibacteria、Nitrosomonas sp.、Nitrospirae bacteria、Pseudomonas stutzeri、Rubrivivax gelatinosus等微生物族群存在，其中厭氧氨氧化微生物存在比例已達89 ± 15％，而AOB及NOB族群總比例為10％，由此顯示系統中以厭氧氨氧化微生物為主要族群。此外，本研究特針對Candidatus Anammoxoglobus propionicus設計專一性引子組Amx368F/Apr1023R。經由實驗證實此引子組專一性佳，可順利量測AR系統中Candidatus Anammoxoglobus propionicus的數量變化。藉由Real-Time PCR分析結果可知，系統中的Candidatus Anammoxoglobus propionicus數量已達108 copies/μg DNA以上，其氨氮及亞硝酸鹽氮去除率仍維持95％以上，且其數量愈高，系統產氣量愈多。綜合本研究成果已順利推測AR系統中微生物與氮化物轉換的關係，及各微生物族群的互動模式。In traditional biological wastewater treatment, nitrification/denitrification processes have been widely used for removing nitrogen compounds due to its high efficiency and low operating cost. On the other hand, anaerobic ammonium oxidation (ANAMMOX) is a new biological wastewater treatment technology developed in recent years. The ANAMMOX process is markedly superiority for the treatment of nitrogenous wastewater, being associated with a significant savings of aeration energy as well as obviating the need for organic carbon and producing less sludge compared with the conventional nitrification and denitrification processes. Full scale plants have been implemented for ANAMMOX process abroad. However, the operating of biological treatment system, anammox bacteria strains and other environmental factors at Taiwan and abroad could be different. Therefore, the possible anammox bacterial diversity in Taiwan is worth being investigated. In this study, sediments were collected from the natural ecosystems for identifying anammox microorganism. The cell counts of anammox bacteria from the natural ecosystems were significantly different. The major anammox bacteria strain in the sediments from Taiwan was Candidatus Brocadia sp. and the cell counts of anammox bacteria was 104 ~ 108 copies/μg DNA. Because of the highest cell number of anammox bacteria was identified from a particular landfill-leachate treatment plan, sludge from this site was selected as seeding for this study. First, batch incubations for the enrichment of anammox bacteria were successfully achieved using 500 mL serum bottles. Then, a laboratory-scale reactor (1000 mL, labelled as AR reactor) was used to increase the biomass. In this 1000 mL reactor, the existence of anammox bacterial community was identified as Candidatus Anammoxoglobus propionicus, its cell counts was 2.87 ± 0.74 × 1010 copies/μg DNA. Results showed that the system could keep an average removal efficiency of ammonium and nitrite between 90% and 100% stably and 15% of nitrate was removed. Finally, an anammox membrane bioreactor (Anammox-MSBR, 15 L, labelled as MS reactor) was prepared and the bacterial community structure within the reactor was expolred. In this reactor, the existence of anammox bacterial community included Candidatus Brocadia sp. 40、Candidatus Brocadia anammoxcidans and Candidatus Brocadia sinica, the cell counts of anammox bacteria was 3.23 ± 0.01 × 108 copies/μg DNA, the nitrogen loading rate was 0.7 g-N/L/day and the removal efficiency of ammonium and nitrite was 96%, SAA was 0.94 ± 0.24 g N2-N (g VSS)-1d-1. In this study, two different ANAMMOX systems (AR and MS) were established using same seeding and mineral medium, but the final outcomes of anammox bacterial community were different, Candidatus Anammoxoglobus propionicus existed in AR reactor and Candidatus Brocadia sp. existed in MS system. The rarely found anammox microorganism Ca. Anammoxoglobus propionicus is an anammox species that have been reported once in the reference. The high nitrogen removal rate was attained in AR reactor and bacterial community structure and interaction of co-existing microorganisms on this anammox system were studied as well. The results showed that the main co-existing microorganism was Chloroflexi. Other microbial communities in this system included Acidobacteria, Actinobacteria, Azospirillum brasilense, Ignavibacteria, Nitrosomonas sp., Nitrospirae bacteria, Pseudomonas stutzeri, Rubrivivax gelatinosus. Fluorescent in situ hybridization (FISH) analysis showed that ratio of anammox bacteria to total eubacteria reached 89 ± 15%, while the ratio of AOB and NOB was 10%. In addition, a specific real-time PCR primers targeting the 16S rRNA of Ca. Anammoxoglobus propionicus was designed and tested in this thesis to quantify this specific microorganism in AR reactor. In this reactor, the cell concentrations of anammox bacteria reached 108 copies/μg DNA, the nitrogen removal rate remained above 95%. Overall, the results showed the community structure and interaction of co-existing microorganisms as well as their effects on this anammox system performance and various microbial communities in interactive mode.第一章 前言 1 1-1 研究緣起 1 1-2 研究目的 2 第二章 文獻回顧 3 2-1 含氮污染物處理方法 3 2-1-1 含氮污染物來源與其影響 3 2-1-2 含氮污染物生物處理方法 6 2-2 厭氧氨氧化微生物 13 2-2-1 厭氧氨氧化菌存在之環境 13 2-2-2 厭氧氨氧化菌種類 14 2-2-3 厭氧氨氧化菌生理特性 17 2-2-4 厭氧氨氧化菌生長特性 25 2-2-5 厭氧氨氧化菌代謝機制 30 2-2-6 厭氧氨氧化微生物活性分析 33 2-2-7 與厭氧氨氧化微生物共生之微生物 33 2-3 台灣地區厭氧氨氧化微生物相關研究 35 2-4 文獻閱讀心得及研究方向擬定 37 第三章 材料與方法 40 3-1 實驗架構及實驗流程 40 3-2 實驗設備 41 3-3 實驗方法 42 3-3-1 環境樣本及系統植種來源 42 3-3-2 分子生物技術 42 3-3-3 水質及氣體組成分析方法 52 3-3-4 電子顯微鏡觀察 54 3-3-5 人工合成基質 55 3-3-6 薄膜生物反應槽之設計 56 3-3-7 厭氧氨氧化微生物活性分析 57 第四章 結果與討論 60 4-1 台灣地區厭氧氨氧化微生物族群調查 60 4-1-1 Anammox bacteria指紋譜建立 60 4-1-2 Anammox bacteria數量分析 65 4-1-3 台灣地區之厭氧氨氧化微生物與文獻相較 66 4-2 厭氧氨氧化微生物之馴養 67 4-2-1 500 mL批次培養情形 67 4-2-2 1000 mL批次培養情形 70 4-2-3 15 L 薄膜生物反應槽培養情形 76 4-3 薄膜生物反應槽除氮效能測試及ANAMMOX活性表現分析 80 4-3-1 薄膜生物反應槽除氮效能測試 80 4-3-2 Anammox bacteria活性表現 90 4-3-3 SAA分析 93 4-4 不同ANAMMOX系統之微生物組成與除氮效能分析比較 95 4-5 AR系統中與厭氧氨氧化微生物共存之微生物族群分析 100 4-5-1 Clone試驗分析AR系統之微生物組成 100 4-5-2 螢光原位雜合技術分析AR系統之微生物組成 108 4-6 Candidatus Anammoxoglobus propionicus的檢測技術建立 109 4-6-1 AR系統中Candidatus Anammoxoglobus propionicus存在情形 109 4-6-2 Candidatus Anammoxoglobus propionicus專一性引子組設計 114 4-6-3 Candidatus Anammoxoglobus propionicus實測情形 116 4-7 AR系統綜合討論 120 第五章 結論 127 5-1 結論 127 5-2 建議 130 第六章 參考文獻 13