臺灣博碩士論文加值系統

本實驗室先前利用自營性嗜酸性硫桿菌去處理沼氣中的硫化氫，結合化學反應之高氧化性及生物反應之再生循環，以提高沼氣發電可行性。自營性嗜酸性硫桿菌(Acidithiobacillus sp. CP1)為化學自營菌，生長速度非常緩慢，使整個系統效率受限於生物反應，因此希望能藉由突變而得到可快速氧化二價鐵之菌株，提昇硫化氫去除效率而得到更高純度之甲烷，作為有效能源加以利用。
本實驗利用NTG(N-methyl-N’-nitro-N-nitrosoguanidine)與Acidithiobacillus sp. CP1 作用後，篩選出具高氧化二價鐵能力之突變株Acidithiobacillus sp. A47-1，並找出其最佳化之生長條件(二價鐵離子濃度20 g L-1，通氣量1600 ml min-1，擔體大小 4.75mm )，在硫化氫濃度為2000 ppm 、鐵離子濃度為20g L-1條件下，野生株之最大硫化氫去除率為14 g L-1hr-1，突變株A47-1之硫化氫去除率最高可為152.5 g L-1hr-1為野生株之11倍，且效率達99%以上，此次突變所得之突變株A47-1，大幅提升生物槽鐵再生之能力，並間接提高硫化氫去除系統處理效能。

Utilization of biological-chemical H2S gas removal system can purify marsh gas efficiently. The combined system contains rapid H2S oxidation in chemical reactor and Fe3+ regeneration in biological reactor to purify methane. The microbial strain we used originally was Acidithiobacillus sp. CP1 which is a chemolithotrophic bacteria, ferric iron regeneration efficiency was limited due to slow bacteria grow rate. The maximal H2S removal capacity in the original system was only 14g L-1 hr-1. In this study, NTG(N-methyl-N’-nitro-N-nitrosoguanidine) was applied to wild type strain Acidithiobacillus sp. CP1 for random mutation. All the formative colonies were picked up and screened to obtain the highest oxidation ability and was named mutant strain Acidithiobacillus sp. A47-1. Futher studies were conducted to reveal the optimal growth condition of mutant strain Acidithiobacillus sp. A47-1(Fe2+ concentration is 20 g L-1；air flux rate is 1600 ml mim-1；carrier size is 4.75mm). The mutant strain Acidithiobacillus sp. A47-1 was examined in the biological-chemical combined system in the condition that the H2S gas and iron concentration were 2000ppm and 20g L-1, respectively. The maximal H2S removal capacity of the system was 152.4g L-1 hr-1, which was 11 folds higher than the wild type Acidithiobacillus sp. CP1. In addition, the removal efficiency was 99%. Therefore, we elevated the H2S removal capacity and efficiency via improvement of Fe3+ regeneration ability of mutant strain Acidithiobacillus sp. A47-1.

摘要...............................................................................................................i Abstract…………………………………………………………………….ii
目錄..............................................................................................................iii
圖表目錄....................................................................................................vii 第一部份: 亞鐵氧化硫桿菌之篩選

壹、前言.......................................................................................................1
1.1硫化氫來源.........................................................................................1
1.2硫化氫特性.........................................................................................1
1.3硫化氫去除法.....................................................................................2
1.3.1焚化法.........................................................................................2
1.3.2物理吸附法.................................................................................3
1.3.3水洗法.........................................................................................3
1.3.4 氧化吸收法................................................................................3
1.3.5 酸鹼吸收法................................................................................4
1.3.6 生物法........................................................................................4
1.3.7 生物化學串聯系統....................................................................4
1.4 氧化亞鐵硫桿菌簡介........................................................................6
1.5影響Acithiobacillus sp. 鐵再生效率因子.........................................6
1.5.1生物載體.....................................................................................6
1.5.2溶氧量.........................................................................................7
1.5.3二價鐵濃度..............................................................................7 1.6研究目的............................................................................................8

貳、材料與方法............................................................................................9
2.1 材料....................................................................................................9
2.1.1 微生物種類...............................................................................9
2.1.2 固、液態培養基.......................................................................9
2.1.3 儀器設備.................................................................................11
2.1.4 藥品.........................................................................................12
2.2 方法..................................................................................................12
2.2.1 突變方法.................................................................................12
2.2.2 依菌落大小篩選具高生長速度之菌株.................................13
2.2.3 依基質顏色篩選具高鐵轉換效率之菌株.............................13
2.2.4 利用連續式培養篩選具高生長速度之菌株.........................14
2.2.5 亞鐵濃度測定.........................................................................14
2.2.6 三價鐵濃度測定.....................................................................16
2.2.7 總鐵之測定.............................................................................18
2.2.8 菌種固定化.............................................................................18
2.2.9 反應器架設與操作.................................................................18 2.2.10 生物載體大小對鐵轉換速率影響.......................................22 2.2.11 通氣量對鐵轉換速率影響...................................................23 2.2.12 二價鐵濃度對鐵轉換速率影響...........................................23
2.2.13 H2S 濃度測定.......................................................................24

参、結果.....................................................................................................25
3.1 依菌落大小篩選具高生長速度之菌株..................................25
3.2 依基質顏色篩選具高鐵轉換效率之菌株..............................25
3.3 利用連續式培養篩選具高生長速度之菌株..........................26
3.4 野生株Acithiobacillus sp. CP1及突變株Acithiobacillus sp.
A47-1鐵轉換速率比較……………………….…………….27
3.5 生物載體大小對鐵轉換速率影響…………………………..28
3.6 通氣量對鐵轉換速率影響…………………………………..29
3.7 基質二價鐵濃度對鐵轉換速率影響......................................30
3.8 突變株於生物化學串聯系統中硫化氫去除效果..................31
肆、討論.....................................................................................................36
4.1 生物載體大小對鐵轉換速率影響..........................................36
4.2 通氣量對鐵轉換速率影響......................................................37
4.3 野生株Acithiobacillus sp. CP1與突變株Acithiobacillus sp.
A47-1硫化氫去除效率比較..................................................37
4.4 不同基質濃度對硫化氫去除之影響......................................38
4.5 生物化學串聯反應器操作過程中所遭遇之問題..................39

第二部份: 亞鐵氧化硫桿菌鐵氧化分子機制

壹、前言.....................................................................................................41
1.1 A. ferrooxidans亞鐵氧化分子機制………………………………41
1.2 文獻回顧.........................................................................................42
1.3 Cytochrome簡介............................................................................43
1.4 Cyc2簡介........................................................................................44
1.5 研究目的.........................................................................................46

貳、材料與方法.........................................................................................47
2.1 材料.................................................................................................47
2.1.1微生物種類.............................................................................47
2.1.2 培養基....................................................................................47
2.1.3 藥品........................................................................................47
2.1.4 設備........................................................................................49
2.2 方法.................................................................................................50
2.2.1 Acithiobacillus sp. CP1 DNA 抽取與製備...........................50
2.2.2 聚合酵素連鎖反應...............................................................51
2.2.3 DNA電泳............................................................................51
2.2.4 質體DNA抽取製備方法......................................................52
2.2.5 限制酵素處理......................................................................52
2.2.6 從電泳膠體中回收DNA片段.............................................53
2.2.7 DNA 片段的接合反應.....................................................53
2.2.8 大腸桿菌勝任細胞之製備..................................................53
2.2.9 大腸桿菌的熱休克轉型作用..............................................54
2.2.10 Cyc2 之純化......................................................................55
2.2.11 蛋白質濃度測定................................................................55
2.2.12 SDS-PAGE..........................................................................56
2.2.13 cyc2去摺疊與摺疊.............................................................57
2.2.14 cyc2活性測試.....................................................................57
2.2.15 heme stain………………………………………………...58

参、結果與討論………………………………………………………...59
3.1 cyc2 inclusion body 去折疊與再摺疊...................................59
3.2 Acithiobacillus sp. CP1 rus operon之P1 promoter於E.coli 中表
現.............................................................................................60
論文圖表...................................................................................................61
參考文獻...................................................................................................84








圖表目錄

圖一 : 生物槽反應器示意圖....................................................................61
圖二 : 生物化學串聯反應器示意圖........................................................62
圖三 : 野生株Acithiobacillus sp. CP1與突變株Acithiobacillus sp. A6、
A7、A14、A15、A35、A47、A49、A50之鐵氧化率比較….........63
圖四：野生株Acithiobacillus sp. CP1及突變株Acithiobacillus sp. A47-1
鐵氧化速率比較.............................................................................64
圖五：生物載體大小對突變株Acithiobacillus sp. A47-1亞鐵氧化率之影
響.....................................................................................................65
圖六：通氣量對突變株Acithiobacillus sp. A47-1三價鐵生成量影響.....66
圖七：基質濃度對突變株Acithiobacillus sp. A47-1三價鐵生成量之影
響.....................................................................................................67

圖八：突變株Acithiobacillus sp. A47-1對不同基質濃度之鐵轉化百分
率.....................................................................................................68
圖九：串聯系統中鐵離子變化情形，於[H2S]=2000ppm，inlet H2S
volumn=160ml min-1、[Fe]=20g L-1條件下(a)化學槽鐵離子濃度變
化 (b)生物槽鐵離子濃度變化......................................................69
圖十：串聯系統中鐵離子變化情形，於[H2S]=2000ppm、inlet H2S
volumn=360ml min-1、[Fe]=20g L-1條件下(a)化學槽鐵離子濃度
變化(b)生物槽鐵離子濃度變化....................................................70

圖十一：串聯系統中鐵離子變化情形，於[H2S]=2000ppm、inlet H2S
volumn=836ml min-1、[Fe]=20g L-1條件下(a)化學槽鐵離子濃
度變化(b)生物槽鐵離子濃度變化............................................71

圖十二：串聯系統中鐵離子變化情形，於[H2S]=4000ppm、inlet H2S
volumn=190ml min-1、[Fe]=20g L-1條件下(a)化學槽鐵離子濃
度變化(b)生物槽鐵離子濃度變化............................................72

圖十三：串聯系統中鐵離子變化情形，於[H2S]=2000ppm、inlet H2S
volumn=836ml min-1、[Fe]=50g L-1條件下(a)化學槽鐵離子濃
度變化(b)生物槽鐵離子濃度變化............................................73
圖十四：renature process 各別步驟之蛋白質變化.................................74
圖十五：CYC2活性測試 ………………………………..........................75
圖十六：Heme stain... ................................................................................76
圖十七：於E.col中CYC2 之表現..........................................................77
圖十八：RT-PCR..........................................................................................78圖十九：Proposed electron transfer pathway between ferrous iron and
oxygen in A. feerrooxidansATCC33020…...................................79

圖二十：The rus operon of A. ferrooxidans ATCC33020….......................80
表1: 不同硫化氫去除法比較...................................................................81

表2：Maximum ferric iron productivity (g hr-1) ........................................81
表3 : Refolding Process..............................................................................82
表4 : 於不同條件下野生株Acithiobacillus sp. CP1與突變株
Acithiobacillus sp. A47-1在生物化學串聯串聯系統之硫化氫去除
測試結果.........................................................................................83

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