The Effect of Rhodopseudomonas sphaeroides to Nitrogen Source in Aquatic System

英文摘要 ................................................ IV 致謝 .................................................... V 目錄 .................................................... VI 圖目錄 .................................................. XI 表目錄 .................................................. XIV 第壹章 緒論 ............................................. 1 1.1. 研究動機 ........................................... 1 1.2. 研究目的 ........................................... 3 第貳章 文獻回顧 ......................................... 5 2.1. 前言 ............................................... 5 2.2. 光合菌簡介與應用 ................................... 11 2.3. 光合反應的固氮和能量獲得的形式 ...................... 16 2.3.1. 光合反應的固氮方式 ............................... 16 2.3.2. 光合菌的獲能形式 ................................. 17 2.4. 光合菌的營養需求 ................................... 18 2.4.1. 光合菌的碳源 ..................................... 18 2.4.2. 光合菌的氮源 ..................................... 19 2.5. 影響固氮作用的因素 ................................. 20 2.6. 光合菌除氮功能研究現況 ............................. 21 2.6.1. 光合菌之研究現況 ................................. 21 2.6.2. 現今生物性除氮方式 ............................... 23 第參章 實驗材料與分析方法 ............................... 26 3.1. 實驗材料 .......................................... 26 3.1.1. 微生物 .......................................... 26 3.1.2. 本實驗使用之培養基 ............................... 27 3.1.3. 實驗用藥品 ....................................... 28 3.2. 實驗用儀器與設備 ................................... 32 3.2.1. 儀器設備 ......................................... 32 3.2.2. 光合菌培養條件和測試儀器 ........................... 32 3.3. 光合菌的培養方法 .................................... 33 3.3.1. 全封閉式厭氣光照培養(厭氣光合菌的培養) ............. 33 3.3.2. 光合菌菌種保存 .................................... 34 3.3.3. 光合菌解凍生長 .................................... 34 3.4. 分析方法 ............................................ 34 3.4.1. 細胞固定化 ........................................ 34 3.4.2. 分光光度計測量方法 ................................. 34 3.4.3. 血球計數盤 ....................................... 35 3.4.4. 水中銨分析 ........................................ 36 3.4.5. 水中亞硝酸鹽(NO2)的分析 ........................... 38 3.4.6. 格蘭氏染色法 ..................................... 39 第肆章 實驗設計 ......................................... 42 4.1. 預備試驗 ............................................ 42 4.1.1 採樣時間與地點 ..................................... 42 4.1.2 水樣採集 ........................................... 42 4.1.3 水質分析方法 ....................................... 42 4.2. 光合菌之培養、定性、定量 ............................. 43 4.2.1 光合菌之收集及吸收波長鑑定 .......................... 43 4.2.2 標準曲線製作 ....................................... 44 4.2.3 光合菌培養環境的設計與製作 ......................... 45 4.2.4 光合菌之生長曲線製作 ............................... 46 4.3. 不同濃度的氮源對光合菌之影響 ........................ 48 4.3.1 針對不同銨氮濃度對光合菌之影響 ......................48 4.3.2 針對不同亞硝酸鹽濃度對光合菌之影響 ................. 48 4.4. 不同菌量的光合菌對氮源之影響 ........................ 49 4.4.1 針對不同光合菌菌量對銨氮混合溶液影響 ................ 49 4.4.2 針對不同光合菌菌量對亞硝酸鹽混合溶液影響 ............ 49 4.5. 應用於養殖池水之模擬試驗 ............................. 50 4.6. 實驗流程 ............................................ 54 4.7. 除氮率計算公式 ...................................... 55 第伍章 結果與討論 ......................................... 56 5.1. 預備試驗 ............................................ 56 5.2. 光合菌之培養、定性、定量 .............................59 5.2.1 R. sphaeroides 吸收光譜 ............................ 59 5.2.2 R. sphaeroides 外型 ................................ 61 5.2.3 光合菌的培養環境 ................................... 62 5.2.4 光合菌之標準曲線製作 ............................... 65 5.2.5 光合菌之生長曲線製作 ................................ 66 5.2.6 銨氮濃度及亞硝酸鹽濃度之標準曲線製作 ................. 68 5.3. 不同濃度的氮源對光合菌之影響 ......................... 70 5.3.1 針對不同銨氮濃度對光合菌之影響 ...................... 70 5.3.2 針對不同亞硝酸鹽濃度對光合菌之影響 .................. 72 5.4. 不同菌量的光合菌對氮源之影響 ......................... 74 5.4.1 針對不同光合菌菌量對銨氮溶液影響 .................... 74 5.4.2 針對不同光合菌菌量對亞硝酸鹽影響 .................... 77 5.5. 應用於養殖池水之模擬試驗 ............................ 80 5.5.1 養殖池水對光合菌去除銨氮效果影響試驗 ............... 80 5.5.2 養殖池水對光合菌去除亞硝酸鹽效果影響試驗 ........... 82 5.5.3 養殖池水中含光合菌及不含光合菌的酸鹼度變化 ......... 84 第陸章 結論與建議 ....................................... 87 6.1. 結論 .............................................. 87 6.2. 建議 ............................................... 88 第柒章 參考文獻 ......................................... 90 圖目錄 圖2-1、全面的疾病防治系統 ............................... 6 圖2-2、氮循環 ........................................... 7 圖2-3、養殖池中的共生關係 ................................. 8 圖2-4、益生菌利用周遭產物淨化環境 ......................... 10 圖2- 5、Metabolic processes of R. palustris. ............. 22 圖2- 6、 Rhodopseudomonas sphaeroides(ESM) ............... 22 圖2-7、光合菌光合作用 .................................... 23 圖3-1、血球計數盤 .........................................36 圖3-2、格蘭氏染色劑 ....................................... 41 圖4-1、對半稀釋法 ......................................... 44 圖4-2、光合反應培養系統 ................................... 45 圖4-3、不再添加營養物質密閉空間之微生物生長曲線圖 .......... 47 圖4-4、實驗室內鰻魚養殖魚缸 ............................... 53 圖4-5、光合菌對氮源影響實驗設計流程圖 ..................... 55 圖5-1、嘉義鰻魚養殖池 .................................... 57 圖5-2、R. sphaeroides 吸收光譜 ........................... 60 圖5-3、使用結晶紫染色R. sphaeroide 放大400 倍 ............ 61 圖5-4、光合菌反應器 ...................................... 63 圖5-5、光合菌反應器內部照度值及溫度比較 ................... 64 圖5-6、R. sphaeroides 定量標準曲線 ....................... 65 圖5-7、R. sphaeroides 之生長曲線與pH 變化 ................ 67 圖5-8、R. sphaeroides 接種後培養第一小時 ................. 67 圖5-9、R. sphaeroides 接種後培養第九十六小時 .............. 68 圖5-10、銨氮濃度之標準曲線 ................................. 69 圖5-11、亞硝酸鹽濃度之標準曲線 .............................. 69 圖5-12、不同濃度的銨氮對光合菌之影響 ......................... 71 圖5-13、不同濃度的銨氮對光合菌之除氮率比較 ...................... 71 圖5-14、不同濃度的亞硝酸鹽對光合菌之影響 ....................... 73 圖5-15、不同濃度的亞硝酸鹽對光合菌之除氮率比較 ..................73 圖5-16、不同菌量的光合菌對銨氮溶液影響 ......................... 75 圖5-17、不同菌量的光合菌對銨氮之除氮率比較 ..................... 76 圖5-18、不同菌量的光合菌對亞硝酸鹽影響 ......................... 78 圖5-19、不同菌量的光合菌對亞硝酸鹽之除氮率比較 ................. 78 圖5-20、不同光合菌菌量對銨氮及亞硝酸鹽影響顏色變化圖 ............ 79 圖5-21、養殖池水中去除銨氮效果影響試驗 ......................... 81 圖5-22、養殖池水中去除亞硝酸鹽效果影響試驗 ..................... 83 圖5-23、養殖池水中除氮效率 .................................... 83 圖5-24、加入光合菌及沒有加入光合菌溶液的酸鹼度變化 ............. 86 圖5-25、養殖池水對光合菌除氮效果影響試驗其顏色變化 ............. 86 表目錄 表2-1、光合菌菌體組成 ........................................ 12 表2-2、光合菌B 群維生素組成 ................................... 12 表2-3、具有氫氣之生產能力之厭氧光合菌 ......................... 15 表2-4、光合菌的分類學特徵 .................................... 16 表2-5、在各種培養條件下光合菌的生長及其獲能形式 ............... 18 表2-6、A comparison of NH4+, NO2- ,DO,BOD, H2S,pH and Temp. . 57 表2-7、A comparison of NH4+, NO2- , PO4, ΣN, ΣP ............ 58 表2-8、現今生物性除氮歸納 .................................... 25 表3-1、BCRC13100 紫色不含硫菌Rhodopseudomonas sphaeroides..... 26 表3-2、Medium 66 ............................................ 27 表3-3、紫色不含硫菌的生長的培養配方 ........................... 27 表4-1、各種菌綠素的吸收波長 .................................. 44 表4-2、不同濃度的氮源對光合菌之影響不同銨氮及亞硝酸鹽濃度配置表 51 表4-3、不同菌量的光合菌對氮源之影響不同光合菌菌量配置表 ........ 52 表4-4、應用於養殖池水之模擬試驗養殖池水之菌量及無機氮配置表..... 53 表5-1、A comparison of NH4+, NO2- ,DO,BOD, H2S,pH and Temp.,℃.57 表5-2、A comparison of NH4+, NO2- , PO4, ΣN, ΣP ............ 58 表5-3、不同濃度的銨氮對光合菌之除氮率比較表 .................... 71 表5-4、不同濃度的亞硝酸鹽對光合菌之除氮率比較表 ................ 73 表5-5、不同菌量的光合菌對銨氮之除氮率比較表 .................... 76 表5-6、不同菌量的光合菌對亞硝酸鹽之除氮率比較表 ................ 79 表5-7、養殖池水中除氮效率 ..................................... 84 表5-8、光合菌於養殖池水中pH 值變化 ............................. 85 參考文獻 Aiking, H. and Sojka, G., 1979. 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The effluent of nitrogen source is an invisible killer that could harm the aqua products. In the seawater or fresh water systems, the concentration of Ammonium(NH4+) and Nitrous acid(NO2-) higher than 2mg/L may cause the pathological change of fish. Therefore,this research was designed to study the use of Photosynthetic bacteria (PSB) to biomanipulate the concentration of inorganic nitrogen in the aquatic system. It might be helpful to the aquatic farms and aquatic environments if the increase of nitrogen concentrate is controlled efficiently. Rhodopseudomonas sphaeroides was used in this study and Microbial Pesticides methods were applied to control the content of nitrogen source in aquatic experiments. The results show that the amount of bacteria was saturated after 120 hours culture of R. sphaeroides. The saturated bacterial concentration reached to 109no./ml. In the process of culture, the pH value becomes weak alkalescence after 24 hour culture. The correlation between the amount of R.sphaeroides and the absorbance value of optical density at 660 nm is R2=0.9941. The Abs(OD660) values were utilized to measure the quantities of R. sphaeroides(Klaas,1982). Then, R. sphaeroides (108 no./ml) were added in five different concentrations of Nitrous acid in water to study the removal of Nitrous acid after 24 hours. The results show that all Nitrogen acid can be removed by R. sphaeroides completely. Next, R. sphaeroides (108 no./ml) were added in five different concentrations of Ammonium to study the removal rate of Ammonium. The removal rate of Ammonium is about 58% after 120 hours. Third, among three levels of bacterium concentration, the concentration of 109no. /ml is the best for removing Nitrous acid. Fourth, R. sphaeroides can stabilize the pH value in the water of aquaculture pond, but it has no effect on the removal of Nitrogen