臺灣博碩士論文加值系統

本研究將先前分離出具同時硝化脫硝能力之菌株THU，利用DNA序列鑑定及G+C content 比對確認菌株THU屬於Ochrobactrum菌屬，再利用DNA-DNA hybridization比對發現THU與Ochrobactrum相似度僅84.6 %，因此THU應為一株新種菌且THU發現時為一好氧脫硝菌株，故將THU命名為 Ochrobactrum aerodenitrificant。
Ochrobactrum aerodenitrificant在NH4+ 濃度為 150 mg/L以下時，其NH4+去除率可達到96.7 %。當NH4+濃度達到200 mg/L 以上時，O. aerodenitrificant會受到NH4+ 抑制此時之NH4+ 去除率在1-50 % 之間，且在此條件下菌株之比生長速率僅0-0.50 hr-1，因此O. aerodenitrificant在高NH4+ 濃度時會受到抑制。
本研究利用O. aerodenitrificant結合SBMBR系統進行批次實驗與反應槽之操作，改變五種基質條件找尋O. aerodenitrificant在反應槽中最適合操作條件，發現在C/N ratio=7 和高鹼度的條件下，能連續操作110天且能維持高的比SND速率及SCOD去除率。而此條件之最佳之NH4+去除率及SCOD去除率分別為98.1 % 和91.3 %。

The isolate was identified as a strain of Ochrobactrum using 16S rDNA sequence, G+C content and DNA-DNA hybridization (the similarity was 84.6 % between strain THU and Ochrobactrum genus), strain THU should be new specie of Ochrobactrum genus. Proposed name for strain THU, Ochrobactrum aerodenitrificant with hading denitrification capacity under aerobic, initially.
When NH4+ concentration was lower than 150 mg/L, the NH4+ removal rate could achieve 96.7 % by O. aerodenitrificant. NH4+ removal rate was between 1-50 % and the specific growth rate only 0-0.50 hr-1 of O. aerodenitrificant under high NH4+ concentration (> 200 mg/L). Thus, O. aerodenitrificant would be inhibited under high ammonia concentration.
Combination of O. aerodenitrificant and SBMBR system removes ammonia from synthetic wastewater for batch testing and reactor operating. This system could maintain high NH4+removal rate and SCOD removal rate for 110 days continuously under C/N ration=7 and alkalinity=1000 mg-CaCO3/L operation conditions. The best NH4+removal rate and SCOD removal rate are 98.1 % and 91.3 %.

目 錄
第一章 前 言……………………………………………………… 1
第二章 文獻回顧……………………………………………………… 3
2.1 氮循環………………………………………………………… 3
2.2 廢水處理……………………………………………………… 5
2.2.1 硝化反應…………………………………………………… 5
2.2.2 脫硝反應…………………………………………………...… 8
2.3 同時硝化與脫硝反應……………………………………….… 9
2.4 薄膜反應槽…………………………………………………..… 16
2.4.1 MBR之形式………………………………………………… 16
2.4.2 MBR之優點………………………………………………… 19
2.4.3 循序批分式生物薄膜反應槽……………………………..…21
2.4.4 結合具SND能力之菌株與循序批分生物薄膜反應槽法…25
2.4.5 即時控制系統監測反應之參數變化情形………………..…26
2.5 研究目的……………………………………………………….. 29
第三章 實驗方法與設計………………………………………………30
3.1 實驗流程……………………………………………………….. 30
3.2 菌種來源……………………………………………………..… 30
3.3 菌種之活化與培養…………………………………………..… 32
3.3.1 菌種之活化………………………………………………..…32
3.3.2 菌種之培養………………………………………………..…32
3.3.3 低限液態培養基…………………………………………..…33
3.4 SND菌株的生理特性再測試……………………………………34
3.4.1 溫度、pH、碳源和碳氮比……………………………………37
3.4.2 基質濃度之影響…………………………………………..…37
3.4.3 絕對厭氧測試……………………………………………..…38
3.4.4 菌株16S rDNA Sequence之鑑定……………………………38
3.4.4.1 菌種染色體DNA之抽取……………………………..… 38
3.4.4.2 洋菜膠體水平電泳……………………………………… 40
3.4.4.3 聚合酶連鍞反應………………………………………… 41
3.4.4.4 序列比對………………………………………………… 43
3.4.5 Lab-VIEW(Laboratory Virtual Instrument Engineering Workbench)電腦即時監測……………………………...… 43
3.4.6 BNP(Biochemical Nitrogen Potential)反應測試………….… 46
3.5 THU菌株固定化於薄膜反應槽之同時硝化脫硝反應……..… 49
3.5.1 反應槽之操作與架設…………………………………..……49
3.5.2 反應槽操作……………………………………………..……50
3.6 實驗分析項目與方法……………………………………..…… 53
第四章 結果與討論………………………………………………… 61
4.1 研究菌株之活化………………………………………………61
4.2 菌株生理特性再測試…………………………………………66
4.2.1 氨氮濃度對菌株THU硝化能力之影響………………… 66
4.2.2 溶氧對THU菌株之影響………………………………… 69
4.2.3 休眠菌株之硝化反應測試……………………………..… 73
4.2.4 碳氮比對菌株之影響…………………………………..… 76
4.3 菌種鑑定………………………………………………………79
4.3.1 16S rDNA gene序列分析………………………………..…79
4.3.2 研究菌株和Ochrobactrum及Brucella之生化特性探討比較……………………………………………………….…81
4.4 薄膜反應槽的批次實驗…………………………………...… 86
4.4.1 Phase 1高碳氮比及高鹼度下之反應…………………..… 88
4.4.2 Phase 2最佳碳氮比及高鹼度下之反應………………..… 89
4.4.3 Phase 3、Phase 4和Phase 5於最佳碳氮比及低鹼度下之反應………………………………………………………..…91
4.5 SND反應時之氧化還原電位及pH變化即時監測……..……94
4.5.1 高碳氮比及高鹼度下之反應………………………………94
4.5.2 最佳碳氮比及高鹼度下之反應……………………………97
4.5.3 最佳碳氮比及低鹼度下之反應……………………………99
4.6 SBMBR系統連續操作之SND反應………………...……… 101
4.6.1 高碳氮比及高鹼度下之反應……………………..……… 101
4.6.2 最佳碳氮比及高鹼度下之反應…………………..……… 104
4.6.3 最佳碳氮比及低鹼度下之反應…………………..……… 107
4.7 SND反應之Nernst equation…………………………….…… 110
4.8 菌株THU進行SND反應時之質量 平衡方程式…………114
第五章 結論與建議………………………………………………..…116
5.1 結論………………………………………………………….…118
5.2 建議………………………………………………………….…118
參考文獻………………………………………………..……….…… 120
附錄……………………………………………………………………129
附錄一 THU菌株 16S rRNA gene 序列定序結果……………….129
表 目 錄
Table 2-1. Reported bacteria that had enzymes of AMO and HAO…….. 7
Table 2-2. Reported heterotrophic nitrifying bacteria that had enzymes of
HAO………………………………………………………….. 7
Table 2-3. Bacteria that maybe have abilities of heterotrophic nitrification
and aerobic denitrification…………………………………... 11
Table 2-4. Table 2-4. Application of MBR system for treating nitrogen wastewater and reclamination water………………..……..… 19
Table 3-1. The composition of solution A…………………..………..... 34
Table 3-2. The composition of solution B……………………..………. 34
Table 3-3. The composition of solution C……………………...…….... 34
Table 3-4. The carbon, nitrogen concentration and alkalinity in the
MA……………………………………….……………….… 36
Table 3-5. The components of TE buffer………………………...……. 40
Table 3-6.The component of stock 5X TBE buffer……………………. 41
Table 3-7. The components and their concentration used in PCR…...…42
Table 3-8. The heating program of PCR…………………………….… 42
Table 3-9. Concentration of influent of SBMBR…………………...…. 52
Table 3-10. The operation condition of SBMBR…………………….... 52
Table 3-11. The analytical methods………………………………….... 60
Table 4-1. The removal rate of nitrogen and SCOD by strain THU after subculture for 3 month…………………………………….... 66
Table 4-2 Similarity of 16S rRNA gene of strain THU to Ochrobactrum sp. and Brucella sp………………………………………….. 81
Table 4-3 Comparison of biochemical characteristics between strain THU, Ochrobactrum and Brucella………………………………… 84
Table 4-4 Comparison of utilization of different substrates between strain THU, Ochrobactrum and Brucella………………………….. 85
Table 4-5 Comparison of three times operation condition of this study……………………………………………………… 111
圖 目 錄
Fig 2-1 The conversion cycle of nitrogen…………………………… 4
Fig 2-2. The pathways of simultaneous nitrification and denitrification occur in T. pantotropha……………… …………………… 12
Fig 2-3. The comparison of traditional nitrification-denitrification process
and simultaneous nitrification-denitrification process……… 13
Fig 2-4. Types of MBR: Side-stream membrane reactor (left) and Submerged membrane reactor (right)……………………….. 23
Fig 2-5. The real-time monitoring of ORP profile during nitrification and
denitrification in traditional wastewater treatment………...… 27
Fig 3-1 Flow chart of experimental design…………………………... 31
Fig 3-2 The Lab VIEW real-time monitoring system for the reaction… 45
Fig 3-3 The BNP test system………………………………………… 48
Fig 3-4. Schematic diagram of the Lab-SBMBR system……………. 50
Fig 3-5 The gas chromatographic spectrum of N2 standard…………. 57
Fig 4-1. SND reaction of strain THU after immediate retrieval from deep-freezing and subculturing……………………………… 62
Fig. 4-2 SND reaction and growing condition by strain THU after acclimating one month………………………………………. 64
Fig. 4-3 The nitrification of strain THU with different NH4+
Concentration………………………………………………..… 69
Fig. 4-4 The growth rate of strain THU with different NH4+
Concentration………………………………………………….. 69
Fig. 4-5 The nitrification efficiency and growth rate of strain THU by
using NH4+ as sole nitrogen compounds under aerobic condition.. 70
Fig. 4-6 The denitrification efficiency and growth rate of strain THU by
using NO2- as sole nitrogen compounds under aerobic condition... 71
Fig. 4-7 The denitrification efficiency and growth rate of strain THU by
using NO2- as sole nitrogen compounds under aerobic condition... 71
Fig. 4-8 The nitrification, denitrification efficiency and growth rate of
strain THU after culturing 5 days under anaerobic condition…. 74
Fig. 4-9 Nitrification by strain THU at resting stage…………………... 75
Fig 4-10 Specific growth rate of strain THU with different C/N ratio.... 78
Fig 4-11 NH4+ removal rate by strain THU under different C/N ratio.... 79
Fig 4-12 Phylogenetic tree of Ochrobactrum and Brucella constructed
from 16S rRNA gene sequences showing the position of strain
THU……………………………………………………………. 82
Fig 4-13 Effluent of nitrogen concentration, SCOD and alkalinity from
SBMBR under C/N=12 and alkalinity=1000 mg-CaCO3/L
conditions………….…………………………………………... 89
Fig 4-14 Effluent of nitrogen concentration, SCOD and alkalinity from
SBMBR under C/N=7 and alkalinity=1000 mg-CaCO3/L conditions……………………………………………………… 90
Fig 4-15 Effluent of nitrogen concentration, SCOD and alkalinity from
SBMBR under C/N=7 and alkalinity=300 mg-CaCO3/L
conditions……………………………………………………… 92
Fig 4-16 Changing of ammonia concentration and growth rate under
C/N=7 and alkalinity=0, 100 mg-CaCO3/L condition…………. 94
Fig 4-17 Real-time monitoring for the SND process with ORP and pH
under C/N ratio=12 and ALK=1000 mg-CaCO3/L……………. 95
Fig 4-18 Real-time monitoring for the SND process with ORP and pH
under C/N ratio=7and ALK=1000 mg-CaCO3/L……………… 98
Fig 4-19 Real-time monitoring for the SND process with ORP and pH
under C/N ratio=7 and ALK=300 mg-CaCO3/L………………100
Fig 4-20. Daily profiles of SCOD, DO concentration and pH of SBMBR
operating under C/N ratio=12 and ALK=1000 mg-CaCO3/L.. 102
Fig 4-21 Daily profiles of NH4+, NO2- and NO3- concentration of
SBMBR operating under C/N ratio=12 and ALK=1000
mg-CaCO3/L………………………………………………….. 104
Fig 4-22 Daily profiles of SCOD, DO concentration and pH of SBMBR
operating under C/N ratio=7 and ALK=1000 mg-CaCO3/L…..106
Fig 4-23 Daily profiles of NH4+, NO2- and NO3- concentration of
SBMBR operating under C/N ratio=7 and ALK=300
mg-CaCO3/L………………………………………………….. 108
Fig 4-24 Daily profiles of SCOD, DO concentration and pH of SBMBR
operating under C/N ratio=7 and ALK=300 mg-CaCO3/L…... 109
Fig 4-25 Daily profiles of NH4+, NO2- and NO3- concentration of
SBMBR operating under C/N ratio=7 and ALK=300
mg-CaCO3/L………………………………………………….. 110
Fig 4-26 Monitoring concentration of products during SND reaction
under different operation condition…………………………... 114
Fig 4-27 Gas production by strain THU during SND process………... 115

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