第五章 結與建議
5.2 建議
1. 堆肥過程中可加入鎂鹽和磷鹽，使得過程中所產生氨以形成 strvite crystals 型態沈澱下來，透過此方式氨的揮發量將大大的降低，有助於 大幅增加堆肥總氨氮含量，以乾基形式來換算將達到一般堆肥的 3-5 倍。
2. 可以嘗試利用兩個反應槽連接的設計，將第一個反應槽之出流氣體導入 至第二反應槽，增加第二反應槽在高溫狀態下持續時間，以便達到 class A水準，提升堆肥成品的品質。
3. 由實驗的經驗得知 TVS 分析容易受到採樣誤差及樣品代表性之影響，
以二氧化碳產率對堆肥反應有較佳代表性，因此若以 TVS 指標判斷堆 肥成效時，最好配合二氧化碳產率及累積量加以佐證。
4. 影響堆肥實驗因子眾多，隨著基質的改變而有不同的操作影響參數，在 進行因子最佳化實驗前，可以先進行因子設計法篩選出較顯著因子，再 決定利用何種實驗設計方法得到最佳化操作參數。
5. DGGE 電泳圖實驗結果可進一步將不同株菌加以定序，比對出菌種名稱 以利後續的研究。
6. 吸附實驗所用之腐熟堆肥為未經硏磨之堆肥，如能將堆肥進一步硏磨 後，粒徑縮小表面積增加，預期可以達到更佳的吸附效率。
7. 將吸附重金屬後的堆肥作 XPS, SEM, XRD, FTIR 以及 EDS 等分
析，可以進一步瞭解腐熟堆肥吸附重金屬的內部反應機制。
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附錄一 分子生物實驗步驟 Genomic DNA 萃取步驟
1. Add 800µl of Extraction Solution into 15 ml polypropylene centrifuge tube, add < 1 g soil sample to the solution, vortex vigorously for 30s.
* Do not use polyethylene tube, which cannot be used in microwave oven.
2. Loose the cap and stand the tube in a glass beaker and put into the microwave oven. Heat the sample for 10 s.
*If treat mutliple samples at a time, adjust heating time until extraction solution is foaming without popping out.
3. Vortex vigorously for 30s-1min, then centrifuge at 3,500g for 5 min at RT.
4. Transfer the supernatant into 1.5 ml microcentrifuge tube.
Optional: If RNA-free DNA is desired, add 4 ul RNaseA solution (100mg/ml ) and incubate at RT for 5 min.
5. Add 1/3 volume of Precipitation Solution, mix by inverting the tube, incubate on ice for 5 min.
*The solution will become cloudy, becouse of precipitation of detergen and proteins.
6. Centrifuge at top speed (12-14,000 rpm) for 5 min at RT.
7. Collect the supernatant into a new microcentrifuge tube without
disturbing the pellet.
8. Add 0.5 volume of Binding Solution and one volume of Ethanol, mix throughly by pippeting.
*For Example: To 400 µl lysate, add 200µl Binding Solution and 400µl Ethanol.
9. Transfer 650 µl of the mixture including any precipitate into Spin Column with collection tube and spin for 1 min and discard the flow-through.
10. Repeat step 9 with remaining mixture.
11. Optional : Discard the flow-through, wash with 500 µl of Binding Buffer.
*Apply this step to remove excess fumic acid for high humic acid soil, which will make eluted DNA brownish.
12. Discard the flow-through, wash twice with 700 µl of Wash Solution.
13. Discard the flow-through, centrifuge for 3 min at top speed to remove ethanol.
14. Place the column in a new microcentrifuge tube, and incubate the spin column at 50-60℃oven for 5 mins to evaporate all the ethanol before eluting the DNA.
15. Add preheated 60-70℃of 100-200 ul Elution Solution or H2O or TE (pH must between 7.0-8.5 ), wait for 1-2 min, to elute the DNA by
centrifugation for 1 min, and store the DNA at -20℃.
PCR 步驟
1. 依照表一中指定的劑量分別加入 200 µl eppendrop 中 2. 混合均勻後離心 10 秒
3. 放入 PCR 機器中依表二程序進行
表一 PCR 藥劑添加劑量表
Item Quantity (µl) DNA template 2
Primer-f-GC 1.5 Primer-r 1.5 PCR master mix 10
d2H2O 35
Total 50
表二 PCR 反應程序表
Procedure
Cycle 1 (1x) Step 1 94OC for 6:00 Cycle 2 (35X) Step 1 94OC for 1:00 Step 2 55OC for 1:00 Step 3 72OC for 1:10 Cycle 3 (1X) Step 1 72OC for 6:00 Cycle 4 (1X) Step 1 4OC for ∞
DGGE 製膠步驟
1. 將大片玻璃置於下層，放上間隔板後再將小片玻璃放上，形成玻璃板組。
2. 將玻璃板組用兩個鑄膠夾夾緊後至於鑄膠台。在兩片玻璃之間放入排列 卡，並旋鬆、旋緊鑄膠夾，直到玻璃片、間隔板及鑄膠夾皆在同一平面 上為止。
3. 配 置 所 需 的 膠 體 濃 度 （ 如 0% 與 100%， 參 照 表 三 ）， 將 40%
acrylamide、TAE buffer、formamide、urea 及去離子水先混合均勻（不 可用 vertex 避免產生氣泡），備用之。
4. 將鑄膠器上的兩個注射筒放置適當位置。將 10% APS 及 TEMED 分 別加入步驟 3 配好的膠體液並混合均勻後，立刻用注射器吸取然後將注 射器架設在鑄膠器上。
5. 以鑄膠器上之注射筒將膠體溶液注入步驟 1 中之玻璃片空隙，需緩慢旋 轉以避免產生氣泡，膠體注射完成後，插入齒模，靜置膠體凝固 (約 2 小時)。
DGGE 電泳：
1. 先將1X TAE buffer 預熱至約 60℃。
2. 取適當量之 PCR product 並加入適當量之 6X loading buffer 後離心 之，備用。
3. 將步驟 1 中 buffer 倒入電泳槽中，並將凝固後的膠體放入。
4. 將步驟 2 的 PCR product loading 至膠體上的 well 內。調整適合的 DGGE 操作溫度、電壓及時間。
表三 DGGE 製膠劑量添加表
80% Denaturing solution
10% Gel (100-300bp) 8% Gel (200-400b)p
25ml 20ml 25ml 20ml
40% Acrylamide 6.25ml 5ml 5ml 4ml
50x TAE 0.5ml 0.4ml 0.5ml 0.4ml Formamide 8ml 6.4ml 8ml 6.4ml
Urea 8.4g 6.72g 8.4g 6.72g
d2H2O To 25ml To 20ml To 25ml To 20ml 10% APS 250μl 200μl 250μl 200μl
TEMED 10μl 8μl 10μl 8μl
40% Denaturing solution
10% Gel (100-300bp) 8% Gel (200-400b)p
25ml 20ml 25ml 20ml
40% Acrylamide 6.25ml 5ml 5ml 4ml
50x TAE 0.5ml 0.4ml 0.5ml 0.4ml Formamide 4ml 3.2ml 4ml 3.2ml
Urea 4.24g 3.36g 4.24g 3.36g
d2H2O To 25ml To 20ml To 25ml To 20ml 10% APS 250μl 200μl 250μl 200μl
TEMED 10μl 8μl 10μl 8μl
附錄二 反應槽操作對策表
發生情況 解決方法 (處理及應變步驟) 改善後恢復情形
反應槽內置攪拌器停止轉動 打開電控箱檢查線路是否擠壓到 攪拌器正常運轉
反應槽高溫期冷凝管下方集水瓶水量過少 檢查管線是否漏氣或抽氣流量是否正確 收集水量恢復正常
二氧化碳加上氧氣數值不等於 20.9 左右 二氧化碳或氧氣偵測器異常請廠商檢修 二氧化碳分析儀恢復
正常 堆肥產氣含水率過高影響二氧化碳分析儀分析
結果 加裝一組 50C 恆溫冷凝器去除水分 二氧化碳分析結果正
常
二氧化碳分析儀校正偏移值大於 20% 通知廠商校正歸零 二氧化碳分析儀恢復
正常
電腦介面卡訊號不穩定 Channel 上外加大電阻並將不用 channel 用電線封死避免干擾 訊號恢復穩定
電腦介面卡沒有訊號 檢查電腦內部卡片或傳輸線是否鬆脫 訊號恢復正常
Thermal couple 訊號定時發生
大幅度異常 Thermal couple 老舊更換新的 thermal couple 訊號恢復正常
電子磅秤測值明顯異常 線路接觸不良請廠商更換維修 秤值正常
電子磅秤不插電即無法使用 電池故障請廠商更換 功能恢復正常
附錄三 實驗原始數據總灠
中央合成設計第一組 MR: 50%, MC: 40%
Time
(hr) Temperature
(OC) pH Electrical conductivity
(ms/cm) Moisture content
(%) CO2 evolution rate
(g CO2/Kg VS-hr) TVS loss
(%) Carbon decomposition rate (%)
0 26 6.39 0.36 42 0.00 0.00 0.00
13 34 6.32 0.51 42 2.02 -0.08 0.98
21 48 6.33 0.52 42 7.39 2.78 7.65
29 40 5.79 0.64 42 5.00 5.23 11.50
37 42 6.74 0.59 42 9.93 5.86 16.03
45 57 8.45 0.62 42 11.56 9.06 24.89
53 52 8.67 0.82 40 12.56 8.50 32.03
61 57 9.02 0.88 39 18.00 11.76 40.84
69 45 9.05 0.73 40 1.55 15.84 46.78
77 42 8.78 0.80 43 5.61 22.69 48.57
97 38 8.94 0.55 38 3.22 19.85 56.35
107 40 8.81 0.61 35 3.03 18.06 59.26
120 30 8.84 0.70 33 0.71 18.13 60.83
中央合成設計第二組 MR: 71%, MC: 44%
Time (hr)
Temperature
(OC) pH Electrical conductivity (ms/cm)
Moisture content (%)
CO2 evolution rate (g CO2/Kg VS-hr)
TVS loss (%)
Carbon decomposition rate (%)
中央合成設計第三組 MR: 50%, MC: 70%
Time
(hr) Temperature
(OC) pH Electrical conductivity
(ms/cm) Moisture content
(%) CO2 evolution rate
(g CO2/Kg VS-hr) TVS loss
(%) Carbon decomposition rate (%)
中央合成設計第四組 MR: 50%, MC: 55%
Time
(hr) Temperature
(OC) pH Electrical conductivity
(ms/cm) Moisture content
(%) CO2 evolution rate
(g CO2/Kg VS-hr) TVS loss
(%) Carbon decomposition rate (%)
中央合成設計第五組 MR: 29%, MC: 66%
Time
(hr) Temperature
(OC) pH Electrical conductivity
(OC) pH Electrical conductivity