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研究生:林語蓁
研究生(外文):Yu-Chen Lin
論文名稱:重金屬對棲息於台灣紅樹林落葉的破囊壺菌之形態及生理上的影響
論文名稱(外文):Morphological and Physiological Effects of Heavy Metals on Thraustochytrids Isolated from Fallen Mangrove Leaves in Taiwan
指導教授:彭家禮彭家禮引用關係
指導教授(外文):Ka-Lai Pang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:135
中文關鍵詞:破囊壺菌紅樹林重金屬
外文關鍵詞:Thraustochytridsmangroveheavy metals
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摘要
本論文主要目的是探討銅、鋅、鉛三種常見於台灣金屬汙染流域的重金屬對三種分別分離自台南二仁溪出海口紅樹林、台南鹽田紅樹林生態區及苗栗竹南紅樹林之落葉的破囊壺菌,依序為Schizochytrium sp.EJ56、Schizochytrium sp.YT90、Schizochytrium sp.CN75。在濃度漸增的重金屬壓力下,其生長及形態所受到的影響,並以原子吸收光譜偵測重金屬,探討在破囊壺菌吸附或吸收的情況。
由破囊壺菌生長的結果顯示,鋅在64 ppm時,就使Schizochytrium sp.EJ56、Schizochytrium sp.CN75的生長達到約60% 的抑制率,銅是在128 ppm時,對Schizochytrium sp.YT90、Schizochytrium sp.CN75兩株菌的生長達到60%的抑制率,而鉛則是到512 ppm才對三株菌達到60%以上的生長抑制率。 從本結果推論,重金屬對破囊壺菌的生長而言,毒性是: 鋅> 銅> 鉛。 以破囊壺菌對重金屬的耐受力而言,Schizochytrium sp.CN75在銅、鋅兩種金屬的壓力下有最高的耐受力,Schizochytrium sp.YT90在鉛金屬的壓力下有最高的耐受力,而整體耐受力最好的菌株則是Schizochytrium sp.CN75,因此推論自靠近工業區的河流 (二仁溪) 所分離出之破囊壺菌 (Schizochytrium sp.EJ56) 面對重金屬壓力時的反應較為敏感。
以掃描式電子顯微鏡觀察破囊壺菌的細胞壁在重金屬壓力下之形態變化,結果顯示在逐漸上升的重金屬濃度中,破囊壺菌出現細胞壁破洞,細胞表面皺縮,無法維持細胞的圓形輪廓之情形,並且在最高之金屬濃度時,細胞有溶裂情形,呈現破碎、不完整之顆粒。 實驗結果佐以光學顯微鏡所拍攝之照片比對,發現在控制組時,細胞壁呈現完整的圓圈,圍住細胞;而到了最高濃度的重金屬,在SEM照片出現細胞溶裂情形時,光學顯微鏡下的細胞壁已無法呈現完整圓形,甚至看見細胞壁消失的情形。
以定量的重金屬濃度測試破囊壺菌在生長之前和之後,對環境中重金屬之減少率,結果顯示環境中的銅、鋅和鉛金屬在破囊壺菌的生長前後,有減少的情形,減少率為Pb2+ > Zn2+ > Cu2+,減少最顯著的濃度範圍則是在1 ppm~256 ppm之間。然而破囊壺菌對銅、鋅和鉛金屬減少的機制未明,究竟是吸收或吸附亦或兩者皆有,仍待後續研究才能證實。
由本研究結果顯示當破囊壺菌遇到高濃度的重金屬壓力,其生長率會下降、停止,甚至死亡,而破囊壺菌身為營養階層中的基礎生物,若其族群因為污染而遭到滅亡則會經由食物網的交互作用,衝擊到紅樹林生態甚至海域生態。 故在此力薦輕重工業業者在排放重金屬廢水之時,宜先詳細規劃排放前之處理措施,及政府對於河川整治、復育之重要性和急迫性。
Abstract
The objectives of this thesis are: (1) to investigate the growth and morphological effects of increasing heavy metal stress on three strains of thraustochytrids isolated from fallen mangrove leaves collected in Kaohsiung Erjen river estuary, Tainan Yan-Tian ecological park mangrove and Miaoli Chu-Nan mangrove, caused by three heavy metals commonly exist in Taiwan’s metal-polluted river: copper, zinc and lead, and (2) to trace the fate of the selected heavy metal ions using atomic absorption spectrophotometer (AAS).
Experiment on growth indicated that EC60 of Schizochytrium sp. EJ56 and Schizochytrium sp.CN75 was 64 ppm Zn2+, while EC60 of Schizochytrium sp.YT90 and Schizochytrium sp.CN75 was 128 ppm Cu2+ and EC60 of all strains was 512 ppm Pb2+. These results suggest that the toxicity of heavy metals on growth of thraustochytrids is in the order: Zn2+> Cu2+> Pb2+. Schizochytrium sp.CN75 was most tolerant to Cu2+ and Zn2+ while Schizochytrium sp.YT90 grew best in the presence of lead. Among all the isolates tested, Schizochytrium sp.CN75 was the most tolerant to the selected heavy metal ions. These suggest that thraustochytrids isolated from the river near the industry park (Schizochytrium sp.EJ56) may be more sensitive when facing to heavy metal stress.
Observation of morphological changes on thraustochytrids under elevated heavy metal pressures under SEM (scanning electron microscope), heavy metals led to the formation of holes on cell wall, shrinkadge of cells and deformation of cell. At highest metal concentrations, cell lysis was observed and intracellular content leaked out. Under differential interference contrast microscope, perfectly round cells were observed in control and deformed cells were seen at the highest metal concentration.
We detected heavy metal reduction by treating quantitative concentrations of heavy metals before and after thraustochytrids growing. The appearance of copper, zinc and lead reduction indicated that thraustochytrids can reduce in vitro heavy metals during their growth and the reduction rate was: Pb2+ > Zn2+ > Cu2+. The most significant reduction range were between 1 ppm and 256 ppm. The mechanism of heavy metal reduction by thraustochytrids remains unknow. We look forward follow-up studies to find out if the missing metals were adsorpted or absorpted by thraustochytrids, or both pathways contribute to the results.
The results in the current study clearly shown that in the presence of high concentrations of heavy metal ions, growth of thraustochytrids would be retarded, eventually causing death. More effort should be done on the effects of heavy metal ions on the ecology of thraustochytrids, an important decomposer in marine environment.
目錄
第一章、 前言
1-1 分類……………………………………………………1
1-2 形態與特性……………………………………………2
1-3 生態角色………………………………………………3
1-4 重金屬…………………………………………………4
1-5 研究目的………………………………………………5
第二章、 材料與方法
1. 實驗菌株…………………………………………………7
2. 實驗方法…………………………………………………9
2-1 GYP固態培養基製備…………………………………9
2-2 GYP液態培養基製備…………………………………9
2-3 斜板培養機製備……………………………………10
3. 實驗菌株取得
3-1 菌種分離……………………………………………10
3-2 菌株純化……………………………………………10
3-3菌株-80 ℃保種……………………………………11
第三章、 銅、鋅、鉛對破囊壺菌的生長影響
一、 前言…………………………………………………12
二、 材料與方法
1. 實驗菌株………………………………………13
2. 重金屬溶液……………………………………13
3. 實驗設計
3-1. 條件設定……………………………………15
3-2. 實驗方法……………………………………15
三、 結果
1. 銅離子對三種破囊壺菌的生長影響…………17
2. 鋅離子對三種破囊壺菌的生長影響…………19
3. 鉛離子對三種破囊壺菌的生長影響…………21
四、 討論…………………………………………………22
第四章、 銅、鋅、鉛對破囊壺菌的形態影響
一、 前言…………………………………………………27
二、 材料與方法
1. 實驗菌液準備……………………………………28
2. SEM樣品前處理的藥品配製
2-1. 0.2 M cacodylate buffer (pH 7.2)……28
2-2. 固定液 (fixative)………………………28
3. 以共軛焦(DIC)顯微鏡觀察細胞形態…………29
4. 實驗設計
4-1. 樣品處理條件設定…………………………29
4-2. 實驗方法……………………………………30
三、 結果
1. 不同前處理條件下之結果………………………32
2. 銅離子對Schizochytrium sp.EJ56的影響…34
3. 銅離子對Schizochytrium sp.YT90的影響…39
4. 銅離子對Schizochytrium sp.CN75的影響…44
5. 鋅離子對Schizochytrium sp.EJ56的影響…49
6. 鋅離子對Schizochytrium sp.YT90的影響…54
7. 鋅離子對Schizochytrium sp.CN75的影響…59
8. 鉛離子對Schizochytrium sp.EJ56的影響…64
9. 鉛離子對Schizochytrium sp.YT90的影響…68
10. 鉛離子對Schizochytrium sp.CN75的影響…72
四、 討論…………………………………………………76
第五章、 破囊壺菌對銅和鋅的吸收與吸附
一、 前言…………………………………………………81
二、 材料與方法
1. 實驗方法………………………………………82
2. 實驗溶液配製…………………………………82
3. 儀器分析………………………………………83
4. 實驗設計
4-1. 錐形瓶瓶壁吸附測試……………………83
4-2. 三株破囊壺菌對銅、鋅、鉛的影響……84
三、 結果…………………………………………………85
四、 討論…………………………………………………96
第六章、 結論…………………………………………………………98
第七章、 參考文獻……………………………………………………99
第八章、 附錄
附錄一、實驗用藥品………………………………………111
附錄二、實驗用器材………………………………………112
附圖一、錐形瓶壁對銅的吸附情形………………………114
附圖二、錐形瓶壁對鋅的吸附情形………………………115
附圖三、錐形瓶壁對鉛的吸附情形………………………116

表目錄
表一、實驗菌株之採樣點環境條件……………………………………7
表二、GYP培養基的成分………………………………………………9
表三、破囊壺菌在不同濃度二價銅離子培養下生長之生長率………18
表四、破囊壺菌在不同濃度二價鋅離子培養下生長之生長率………20
表五、破囊壺菌在不同濃度二價鉛離子培養下生長之生長率………22
表六、SEM 樣品處理流程…………………………………………28



圖目錄
圖一、二仁溪、曾文溪、中港溪之水體分析…………………………8
圖二、Schizochytrium sp.EJ56在B-1條件下處理後的SEM照片…33
圖三、Schizochytrium sp.EJ56在A-2條件下處理後的SEM照片…33
圖四、在0 ppm 銅離子下的Schizochytrium sp.EJ56……………35
圖五、在1 ppm 銅離子下的Schizochytrium sp.EJ56……………35
圖六、在16 ppm 銅離子下的Schizochytrium sp.EJ56……………35
圖七、在64 ppm 銅離子下的Schizochytrium sp.EJ56……………36
圖八、在96 ppm 銅離子下的Schizochytrium sp.EJ56……………36
圖九、在128 ppm 銅離子下的Schizochytrium sp.EJ56……………36
圖十、在256 ppm 銅離子下的Schizochytrium sp.EJ56……………37
圖十一、在384 ppm 銅離子下的Schizochytrium sp.EJ56…………37
圖十二、在512 ppm 銅離子下的Schizochytrium sp.EJ56…………37
圖十三、在768 ppm 銅離子下的Schizochytrium sp.EJ56………38
圖十四、在0 ppm 銅離子下的Schizochytrium sp.YT90…………40
圖十五、在1 ppm 銅離子下的Schizochytrium sp.YT90…………40
圖十六、在16 ppm 銅離子下的Schizochytrium sp.YT90…………40
圖十七、在64 ppm 銅離子下的Schizochytrium sp.YT90…………41
圖十八、在96 ppm 銅離子下的Schizochytrium sp.YT90…………41
圖十九、在128 ppm 銅離子下的Schizochytrium sp.YT90…………41
圖二十、在256 ppm 銅離子下的Schizochytrium sp.YT90………42
圖二十一、在384 ppm 銅離子下的Schizochytrium sp.YT90………42
圖二十二、在512 ppm 銅離子下的Schizochytrium sp.YT90………42
圖二十三、在768 ppm 銅離子下的Schizochytrium sp.YT90………43
圖二十四、在0 ppm 銅離子下的Schizochytrium sp.CN75………45
圖二十五、在1 ppm 銅離子下的Schizochytrium sp.CN75………45
圖二十六、在16 ppm 銅離子下的Schizochytrium sp.CN75………45
圖二十七、在64 ppm 銅離子下的Schizochytrium sp.CN75………46
圖二十八、在96 ppm 銅離子下的Schizochytrium sp.CN75………46
圖二十九、在128 ppm 銅離子下的Schizochytrium sp.CN75………46
圖三十、在256 ppm 銅離子下的Schizochytrium sp.CN75………47
圖三十一、在384 ppm 銅離子下的Schizochytrium sp.CN75………47
圖三十二、在512 ppm 銅離子下的Schizochytrium sp.CN75………47
圖三十三、在768 ppm 銅離子下的Schizochytrium sp.CN75………48
圖三十四、在0 ppm 鋅離子下的Schizochytrium sp.EJ56………50
圖三十五、在1 ppm 鋅離子下的Schizochytrium sp.EJ56………50
圖三十六、在16 ppm 鋅離子下的Schizochytrium sp.EJ56………50
圖三十七、在64 ppm 鋅離子下的Schizochytrium sp.EJ56………51
圖三十八、在256 ppm 鋅離子下的Schizochytrium sp.EJ56………51
圖三十九、在512 ppm 鋅離子下的Schizochytrium sp.EJ56………51
圖四十、在640 ppm 鋅離子下的Schizochytrium sp.EJ56………52
圖四十一、在768 ppm 鋅離子下的Schizochytrium sp.EJ56………52
圖四十二、在896 ppm 鋅離子下的Schizochytrium sp.EJ56………52
圖四十三、在1024 ppm 鋅離子下的Schizochytrium sp.EJ56……53
圖四十四、在0 ppm 鋅離子下的Schizochytrium sp.YT90………55
圖四十五、在1 ppm 鋅離子下的Schizochytrium sp.YT90………55
圖四十六、在16 ppm 鋅離子下的Schizochytrium sp.YT90………55
圖四十七、在64 ppm 鋅離子下的Schizochytrium sp.YT90………56
圖四十八、在256 ppm 鋅離子下的Schizochytrium sp.YT90………56
圖四十九、在512 ppm 鋅離子下的Schizochytrium sp.YT90………56
圖五十、在640 ppm 鋅離子下的Schizochytrium sp.YT90………57
圖五十一、在768 ppm 鋅離子下的Schizochytrium sp.YT90………57
圖五十二、在896 ppm 鋅離子下的Schizochytrium sp.YT90………57
圖五十三、在1024 ppm 鋅離子下的Schizochytrium sp.YT90……58
圖五十四、在0 ppm 鋅離子下的Schizochytrium sp.CN75………60
圖五十五、在1 ppm 鋅離子下的Schizochytrium sp.CN75………60
圖五十六、在16 ppm 鋅離子下的Schizochytrium sp.CN75………60
圖五十七、在64 ppm 鋅離子下的Schizochytrium sp.CN75………61
圖五十八、在256 ppm 鋅離子下的Schizochytrium sp.CN75………61
圖五十九、在512 ppm 鋅離子下的Schizochytrium sp.CN75………61
圖六十、在640 ppm 鋅離子下的Schizochytrium sp.CN75………62
圖六十一、在768 ppm 鋅離子下的Schizochytrium sp.CN75………62
圖六十二、在896 ppm 鋅離子下的Schizochytrium sp.CN75………62
圖六十三、在1024 ppm 鋅離子下的Schizochytrium sp.CN75……63
圖六十四、在0 ppm 鉛離子下的Schizochytrium sp.EJ56………65
圖六十五、在1 ppm 鉛離子下的Schizochytrium sp.EJ56………65
圖六十六、在16 ppm 鉛離子下的Schizochytrium sp.EJ56………65
圖六十七、在64 ppm 鉛離子下的Schizochytrium sp.EJ56………66
圖六十八、在128 ppm 鉛離子下的Schizochytrium sp.EJ56………66
圖六十九、在256 ppm 鉛離子下的Schizochytrium sp.EJ56………66
圖七十、在512 ppm 鉛離子下的Schizochytrium sp.EJ56………67
圖七十一、在768 ppm 鉛離子下的Schizochytrium sp.EJ56………67
圖七十二、在1024 ppm 鉛離子下的Schizochytrium sp.EJ56……67
圖七十三、在0 ppm 鉛離子下的Schizochytrium sp.YT90………69
圖七十四、在1 ppm 鉛離子下的Schizochytrium sp.YT90………69
圖七十五、在16 ppm 鉛離子下的Schizochytrium sp.YT90………69
圖七十六、在64 ppm 鉛離子下的Schizochytrium sp.YT90………70
圖七十七、在128 ppm 鉛離子下的Schizochytrium sp.YT90………70
圖七十八、在256 ppm 鉛離子下的Schizochytrium sp.YT90………70
圖七十九、在512 ppm 鉛離子下的Schizochytrium sp.YT90………71
圖八十、在768 ppm 鉛離子下的Schizochytrium sp.YT90………71
圖八十一、在1024 ppm 鉛離子下的Schizochytrium sp.YT90……71
圖八十二、在0 ppm 鉛離子下的Schizochytrium sp.CN75………73
圖八十三、在1 ppm 鉛離子下的Schizochytrium sp.CN75………73
圖八十四、在16 ppm 鉛離子下的Schizochytrium sp.CN75………73
圖八十五、在64 ppm 鉛離子下的Schizochytrium sp.CN75………74
圖八十六、在128 ppm 鉛離子下的Schizochytrium sp.CN75………74
圖八十七、在256 ppm 鉛離子下的Schizochytrium sp.CN75………74
圖八十八、在512 ppm 鉛離子下的Schizochytrium sp.CN75………75
圖八十九、在768 ppm 鉛離子下的Schizochytrium sp.CN75………75
圖九十、在1024 ppm 鉛離子下的Schizochytrium sp.CN75……75
圖九十一、重金屬誘發ROS及組織損傷之作用機制 (Stohs and Bagchi, 1995) …………………………………………………………79
圖九十二、Schizochytrium sp.EJ56對不同濃度銅金屬的減少率…87
圖九十三、Schizochytrium sp.YT90對不同濃度銅金屬的減少率…88
圖九十四、Schizochytrium sp.CN75對不同濃度銅金屬的減少率…89
圖九十五、Schizochytrium sp.EJ56對不同濃度鋅金屬的減少率…90
圖九十六、Schizochytrium sp.YT90對不同濃度鋅金屬的減少率…91
圖九十七、Schizochytrium sp.CN75對不同濃度鋅金屬的減少率…92
圖九十八、Schizochytrium sp.EJ56對不同濃度鉛金屬的減少率…93
圖九十九、Schizochytrium sp.YT90對不同濃度鉛金屬的減少率…94
圖一百、Schizochytrium sp.CN75對不同濃度鉛金屬的減少率…95
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