臺灣博碩士論文加值系統

摘要

矽藻是自然界中種類最多的單細胞藻類，至今約 200 屬 10 萬多種。本實驗室藻種來自墾丁、台中港與澎湖，以 18S ribosomal DNA V4 region (18S rDNA V4 region) 分子鑑定，搭配光學顯微鏡和掃描式電子顯微鏡 ( SEM ) 型態鑑定，至今尚有 4 種矽藻樣本未確定分類，分別為 AQ1、AQ11、AQ15、PNP2。於本研究鑑定結果中，AQ1 為 Nitzschia sp.，但因相似度太低，判斷可能為一新鑑定的藻種，AQ11 為Psammoneis pseudojaponica，AQ15 為 Navicula. lanceolata，PNP2 只能鑑定至 Navicula sp.，另外加上其他十種藻種做 4oC 冷藏保存 (cold storage) 週期測試並分析其蛋白質成分，篩選出以應用於水產養殖業之藻種，此外，再以 AQ1 和 AQ11 進行 f/2 培養基調整與放大培養體積的測試及分析其脂質含量，以應用於未來大規模培養藻種條件。

在 4oC 冷藏保存 (cold storage) 週期測試上，在保存期限之內取出回復原來溫度培養，除了 AQ15 (Navicula lanceolata) 和 TPC5 (Thalassiosira lundiana) ，其餘藻種冷藏一週後皆可看到矽藻回復培養生長，保存週期超過 8 週以上的藻種為 AQ1 (Nitzschia sp.)、TPC14 (Odontella sinensis)、TPC11 (Skeletonema tropicum)，保存週期介於 4-8 週的藻種為 PNP2 (Navicula sp.)、TPC7 (Cyclotella meneghiniana)、TPC8 (Chaetoceros sp.)、TPC10 (Thalassiosira minima)，低於四週的藻種為 TPC13 (Chaetoceros sp.)、AQ15 (Navicula lanceolata)、TPC3 (Rhizosolenia setigera)、AQ11 (Psammoneis pseudojaponica)、TPC5 (Thalassiosira lundiana)、TPC6 (Thalassiosira tenera)。少部分藻種冷藏後會有萎縮的現象，但延長培養時間後可回復原來的大小，再藉冷藏週期測試及分析其蛋白質成分，篩選出適用於水產養殖業之藻種，另外，如果將藻種以冷藏於一個月後回復正常溫度培養一週再放回 4oC 的方式來繼代，可延長保存期限至少1~2 個月。而以 AQ1 和 AQ11 進行f/2培養基調整與放大培養體積的測試結果顯示，其脂質變化量，發現當 AQ1 培養體積放大為 5 倍時，脂質量含量為 2 倍，而同培養基濃度及同培養體積時，AQ11 含脂質量約為 AQ1 之 9.5 倍。

Abstract

Diatoms are the majority group of unicellular algae in nature. There are more than 100,000 species and approximately 200 genera so far discovered. Our laboratory collected diatoms and seawater from Kenting, the Port of Taichung and the Penghu islands. Almost all had their 18S ribosomal DNA (18S rDNA) sequence compared in BLAST and an optical microscope and Scanning Electron Microscopy (SEM) were also used to observe diatom shell patterns and frustule microstructure for further proof of identification. However, an additional four unclassified diatoms named AQ1, AQ11, AQ15 and PNP2 diatoms where then later classified. In this study, we determined that the Nitzschia genus is the closest in results to AQ1, but there was a newly identified sequence added to the BLAST data base, due to low similarity with existing Nitzschia diatoms, AQ11 was identified as Psammoneis pseudojaponica, AQ15 was identified as Navicula lanceolata, and PNP2 was identified as Navicula sp. They were then added to another 10 diatoms species to test the retention period of cold storage in 4oC. We then analyzed their total protein content to select the diatom species that could be applied in commercial aquaculture. In addition, we also tested f/2 medium adjustments and working volume amplification by using the above mentioned AQ1 and AQ11 and analyzed their total lipid content in order to test for possible future scale up conditions.
In order to culture them after different periods of time in the 4oC cold storage experiment, the diatoms were unfrozen and taken back the original temperatures. After one week of culturing following cold storage, they could all grow normally except for AQ15 and TPC5. For more than eight weeks of cold storage, the diatoms that could still be cultured were AQ1 (Nitzschia sp.), TPC14 (Odontella sinensis), and TPC11 (Skeletonema tropicum); for between 4-8 weeks, the diatoms that could be cultured were PNP2 (Navicula sp.)、TPC7 (Cyclotella meneghiniana), TPC8 (Chaetoceros sp.), and TPC10 (Thalassiosira minima); for less than four weeks, the diatoms that could be cultured were TPC13 (Chaetoceros sp.), AQ15 (Navicula lanceolata), TPC3 (Rhizosolenia setigera), AQ11 (Psammoneis pseudojaponica),TPC5 (Thalassiosira lundiana), and TPC6 (Thalassiosira tenera). According to their retention period in cold storage at 4oC, the total protein content for selected diatom species was analyzed to determine their viable application in commercial aquaculture. Under this subculture method revised, it was estimated that the shelf life of diatoms could be extended for at least one to two months under cold storage conditions (4oC) where the diatoms could be brought back to normal temperatures in order to re-culture them for a week; the f/2 medium adjustment and working volume amplification were also tested by using the above mentioned AQ1 and AQ11, and the results showed that the total lipid content of AQ1 increased 2X when the culture volume was amplified 5X and that the total lipid content of AQ11 was 9.5X more than in AQ1 when in the same culture medium and with the same volume.

目錄

摘要..............................................................................................................................i
Abstract…………………………………………………………………………...…ii
目錄……………………………………………………………………………...…..iv
表目次…………………………………………………………………………..…...vi
圖目次…………………………………………………………………………….....vi
第一章 前言…………………………………………………………………………1
1.1. 矽藻簡介……………………………………………………………………1
1.2. 矽藻的保存………………………………………………………………....4
1.3. 矽藻的應用…………………………………………………………………5
1.4. 研究動機與目的 …………………………………………………………..6
第二章 材料與方法…………………………………………………………………7
2.1 實驗材料.......................................................................................................7
2.1.1. 藥品與試劑…………………………………………………………….7
2.1.2. 儀器及裝置…………………………………………………………….8
2.1.3. 培養基配置………………………………………………………….…9
2.1.4. 矽藻來源……………………………………………………………….9
2.2 實驗方法…………………………………………………………………..10
2.2.1. 矽藻的培養……………………………………………………………10
2.2.2. 矽藻密度和大小測量………………………………………………....10
2.2.3. Genomic DNA 抽取……………………………………………………11
2.2.4. DNA 定序………………………………………………………………11
2.2.5. BLAST 序列分析……………………………………………………...12
2.2.6. P-distance……………………………………………………………….12
2.2.7. 矽殼製備……………………………………………………………….12
2.2.8. 場發射掃描式電子顯微鏡觀察 (Field-Emission Scanning Electron
Microscope, FE-SEM)………………………………………………...13
2.2.9. 穿透式電子顯微鏡觀察 (Transmission Electron Microscope, TEM)..14
2.2.10. 冷藏保存實驗 (1):每週取出回復培養………………………………14
2.2.11. 冷藏保存實驗 (2):每月繼代一次……………………………………15
2.2.12. 冷凍乾燥………………………………………………………………15
2.2.13. 脂質含量分析…………………………………………………………15
2.2.14. 蛋白質含量分析………………………………………………………16

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2.2.15. 矽藻藻種放大培養及收集……………………………………………16
2.2.16. f/2培養基調整與放大培養體積之測試……………………………...16
第三章 結果…………………………………………………………………………17
3.1 野外分離的矽藻的鑑定……………………………………………………17
3.2 冷藏保存……………………………………………………………………23
3.3 f/2 培養基調整與放大培養體積之測試…………………………………..30
第四章 討論…………………………………………………………………………31
4.1 藻種的鑑定………………………………………………………………...31
4.2 藻種的保存………………………………………………………………...33
4.3 藻種的放大培養…………………………………………………………...34
第五章 結論…………………………………………………………………………35
參考文獻……………………………………………………………………………..36
圖表結果……………………………………………………………………………..42
附錄…………………………………………………………………………………..82























v
表目次

表一、18S rDNA BLAST 分析所得最接近物種
表二、 18S rDNA V4 region BLAST 分析所得最接近物種
表三、兩種冷藏保存方法之結果比較
表四、藻種樣本每週回復培養冷藏保存超過 8 週之細胞大小變化
表五、藻種樣本每週回復培養冷藏保存介於 4-8 週之細胞大小變化
表六、藻種樣本每週回復培養冷藏保存低於 4 週之細胞大小變化
表七、藻種樣本每週回復培養冷藏保存超過 8 週之細胞乾重變化
表八、藻種樣本每週回復培養冷藏保存介於 4-8 週之細胞乾重變化
表九、藻種樣本每週回復培養冷藏保存低於 4 週之細胞乾重變化
表十、藻種樣本每月拿出繼代培養冷藏保存超過 6 個月之細胞大小變化
表十二、藻種樣本每月拿出繼代培養冷藏保存介於 3-6 個月之細胞大小變化
表十二、藻種樣本每月拿出繼代培養冷藏保存低於 3 個月之細胞大小變化
表十三、藻種樣本每週回復培養冷藏保存超過 6 個月之細胞乾重變化
表十四、藻種樣本每週回復培養冷藏保存介於 3-6 個月之細胞乾重變化
表十五、藻種樣本每週回復培養冷藏保存低於 3 個月之細胞乾重變化
表十六、TPC8 (Chaetoceros sp.) 蛋白質含量分析
表十七、TPC14 (Odontella sinensis) 蛋白質含量分析
表十八、f/2 培養基調整與放大培養體積之測試所得細胞數
表十九、f/2 培養基調整與放大培養體積之測試所得細胞乾種
表二十、脂質於細胞乾種中之含量

圖目次

Fig. 1鑑定藻種之 V4 reiogn 及 18S rDNA 之 PCR產物電泳
Fig. 2 AQ1 V4 and 18S rDNA sequence
Fig. 3 AQ11 V4 and 18S rDNA sequence
Fig. 4 AQ15 V4 and 18S rDNA sequence
Fig. 5 PNP2 V4 and 18S rDNA sequence
Fig. 6 四種鑑定矽藻之光學圖
Fig. 7 AQ1 之 SEM 圖
Fig. 8 AQ1 之TEM圖
Fig. 9 AQ11 之 SEM 圖

vi
Fig. 10 AQ11 之 TEM 圖
Fig. 11 AQ15 之 SEM 圖
Fig. 12 AQ15 之 TEM 圖
Fig. 13 PNP2 之 SEM 圖
Fig. 14 PNP2之 TEM圖
Fig. 15 藻種樣本冷藏後每週回復培養及每月拿出繼代放回之細胞數變化
Fig. 16 藻種樣本冷藏後每週回復培養及每月拿出繼代放回之比生長速率變化Fig. 17 藻種樣本冷藏後每週回復培養及每月拿出繼代放回之細胞數與螢光值變化
Fig. 18 AQ1 和 AQ11 在各 f/2 medium 與 5f medium 之 working volume 500 ml及 2500 ml中，比較不同體積不同濃度下，細胞生長曲線之差異
Fig. 19 AQ1 和 AQ11 在各 f/2 medium 與 5f medium 之 working volume 500 ml及 2500 ml中，比較不同體積不同濃度下，培養 15 天後，細胞密度的差異
Fig. 20 AQ1 和 AQ11 在各 f/2 medium 與 5f medium 之 working volume 500 ml及 2500 ml中，比較不同體積不同濃度下，比生長速率的差異。





















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