臺灣博碩士論文加值系統

人類過度使用化石燃料，不僅造成溫室效應等嚴重環境汙染，也使得全球蘊藏量日漸枯竭，因此積極尋找新且潔淨的替代能源就成了當務之急，而氫氣能源的開發即為其中之一。氫氣可經由微生物或酵素分解木質纖維素(lignocellulose)再進一步發酵生成，特別是以木質纖維素作為原料時，具有在自然界中存在量極為豐富、成本低廉，及其生產對環境友善等優點。本研究的目的為應用水解酵素進行木質纖維素之「分開酵素水解與發酵製程」(SHF)及「同步糖化與發酵製程」(SSF)兩種不同產氫程序之差異評估。主要探討二株具有纖維素分解能力的真菌Trichoderma longibrachiatum、Aspergillus niger，及本實驗室分離得到的一株具有產生高活性半纖維素分解酵素、氫氣與乙醇等能力的厭氧細菌Clostridium xylanolyticum Ter3，分析它們所產生的胞外carboxymethyl cellulase (CMCase)、Avicelase及xylanase之特性與活性表現。在最適作用條件的測試中，各酵素的最適作用溫度皆介於40℃~60℃之間，最適作用pH值則大多為pH 6。經定量分析比較本研究使用的三株微生物所產生的各類型木質纖維素分解酵素粗萃取液的活性後得知，T. longibrachiatum以MR medium培養12天後可產生最高活性的CMCase (38.13 U/L)；T. longibrachiatum以MA medium培養3天後可產生最高活性的Avicelase (9.3 U/L)，而A. niger則以MX medium培養4天後可產生最高活性的xylanase (114.97 U/L)。此外，活性定量結果也顯示各菌株產生的xylanase活性皆比CMCase及Avicelase高許多。經SDS-PAGE與zymogram分析各菌株之xylanase後發現，T. longibrachiatum產生的酵素蛋白有兩種，分子量分別約21、17 kDa；A. niger也是產生兩種，分別約20、30 kDa；C. xylanolyticum Ter3則只產生一種，其分子量為37 kDa。為了進一步瞭解利用天然木質纖維素來產氫的可行性，因此將各菌株所產生的三種水解酵素以不同的搭配組合進行碎稻桿之水解測試。結果顯示，將T. longibrachiatum CMCase (2 units)、T. longibrachiatum Avicelase (0.1 unit)及A. niger xylanase (2 units)一起添加到總體積150 ml內含0.5% 碎稻桿的緩衝溶液中，進行分解反應後可得到最高的還原糖生成量(1077.92 mg/L)。當此酵素組合再搭配一株好的產氫菌株，Clostridium acetobutylicum ATCC 824，一起添加到總體積10 ml內含0.5% 碎稻桿以取代葡萄糖與可溶性澱粉作為單一碳源的液態RCM培養基中時，以SSF製程培養240小時後的氫氣產量(2.68 ml)多過於使用SHF製程所獲得的氫氣量(0.42 ml)。

Excessive use of fossil fuels by human beings not only caused greenhouse effect and serious environmental pollution but also graduately exhaust its deposits in the world. So, it has become a top priority to search for new and clean alternative energy sources, the development of hydrogen energy is one of them. Hydrogen could be produced from lignocellulosic biomass through microbial or enzymatic decomposition and further fermentation, in particular, lignocellulose has the advantage of its large-scale availability, low cost and environmentally friendly production. The purpose of this study was to evaluate the application of hydrolytic enzymes in two different processes of hydrogen production from lignocellulosic biomass, separate hydrolysis and fermentation (SHF); and simultaneous saccharification and fermentation (SSF). Carboxymethyl cellulase (CMCase), Avicelase, and xylanase used were produced by two fungi, Trichoderma longibrachiatum and Aspergillus niger, and a strict anaerobic bacterium, Clostridium xylanolyticum Ter3 which can also produced some hydrogen and ethanol. When the crude preparation of these enzymes were used to examined activity, their optimal temperature were between 40℃ and 60℃, and their optimal pH were mostly at 6.0. When compared the activities of each kind of lignocellulolytic enzymes among the three microorganisms used in this study, T. longibrachiatum expressed the highest CMCase activity (38.13 U/L) and Avicelase activity (9.3 U/L) when it was cultivated in MR medium for 12 days and in MA medium for 3 days, respectively, and A. niger expressed the highest xylanase activity (114.97 U/L) when it was cultivated in MX medium for 4 days. In addition, quantitative results also showed that all the three microorganisms expressed higher xylanase activity than CMCase and Avicelase activities. SDS-PAGE and zymogram analyses revealed that the two proteins with obvious xylanolytic activity produced by T. longibrachiatum had molecular weight about 21 kDa and 17 kDa, respectively; A. niger also produced two proteins with xylanase activity, their molecular weight were about 20 and 30 kDa, respectively; C. xylanolyticum Ter3 produced single xylanase protein with molecular weight about 37 kDa. In order to realize the possibility of utilizing natural lignocellulosic biomass to produce hydrogen, various combinations of the three kinds of hydrolytic enzymes from the three microorganisms were used to test the decomposition of rice straw. It was found that the combination of 2 units of T. longibrachiatum CMCase, 0.1 unit of T. longibrachiatum Avicelase, and 2 units of A. niger xylanase produced the highest amount of reducing sugars (1077.92 mg/L) from 0.5% rice straw in 150 ml of reaction buffer. When this enzyme combination and a good hydrogen producer, C. acetobutylicum ATCC 824, were added to 10 ml of RCM medium in which 0.5% rice straw substituted for glucose and soluble starch as sole carbon source, then the cultivation in SSF mode produce more H2 (2.68 ml) than the SHF mode produced (0.42 ml) in 240 hours.

中文摘要…………………………………………………………………………………i
英文摘要………………………………………………………………………………iii
目錄……………………………………………………………………………………v
表目錄…………………………………………………………………………………ix
圖目錄…………………………………………………………………………………x

第一章　緒論…………………………………………………………………………1
1.　前言……………………………………………………………………………1
1.1　全球能源危機與環境議題-化石燃料……………………………………1
1.2　再生能源的開發-生質能…………………………………………………1
1.3　潔淨新能源-生質氫能……………………………………………………2
2.　文獻回顧………………………………………………………………………3
2.1　木質纖維素的結構組成……………………………………………………3
2.2　木質纖維素的前處理(pretreatment)………………………………………4
2.3　木質纖維素分解系統………………………………………………………4
2.3.1　木質纖維素分解酵素的組成及來源…………………………………4
2.3.2　纖維素分解酵素(cellulase)……………………………………………5
2.3.2.1　內切型纖維素分解酵素(endo-β-1,4-D-glucanase)………………5
2.3.2.2　外切型纖維素分解酵素(exo-β-1,4-D-glucanase)………………5
2.3.2.3　葡萄糖苷酵素(β-glucosidase)……………………………………6
2.3.3　半纖維素分解酵素(hemicellulase)……………………………………7
2.3.4　木質素分解酵素(ligninase)……………………………………………8
2.3.5　木質纖維素分解酵素的應用…………………………………………8
2.4　微生物產氫系統……………………………………………………………9
2.4.1　產氫微生物的種類……………………………………………………9
2.4.2　Clostridium屬細菌產氫代謝…………………………………………10
2.4.3　氫酶(Hydrogenase)……………………………………………………11
3.　研究動機與目的………………………………………………………………12
第二章　材料與方法…………………………………………………………………13
1.　實驗材料………………………………………………………………………13
1.1　菌種來源…………………………………………………………………13
1.1.1　糖化菌株……………………………………………………………13
1.1.2　發酵產氫菌株………………………………………………………13
1.2　藥品………………………………………………………………………13
1.3　培養基……………………………………………………………………13
1.4　誘導生成木質纖維素分解酵素之培養基………………………………15
1.5　試劑………………………………………………………………………16
1.5.1　剛果紅染色…………………………………………………………16
1.5.2　DNS試劑…………………………………………………………16
1.6　蛋白質電泳用試劑………………………………………………………16
1.6.1　設備…………………………………………………………………16
1.6.2　SDS-PAGE…………………………………………………………16
1.6.3　Zymogram…………………………………………………………17
1.6.4　膠體染色……………………………………………………………17
1.7　氫氣量測定………………………………………………………………17
2.　實驗方法………………………………………………………………………17
2.1　菌種保存…………………………………………………………………17
2.2　固態厭氧培養……………………………………………………………17
2.3　剛果紅測試法……………………………………………………………18
2.4　木質纖維素分解酵素活性測試…………………………………………18
2.4.1　DNS還原糖測試法………………………………………………18
2.4.2　胞外粗酵素液的萃取………………………………………………18
2.4.3　胞外粗酵素液的濃縮製備…………………………………………19
2.4.4　最適作用溫度測定…………………………………………………19
2.4.5　最適作用pH值測定………………………………………………19
2.5　蛋白質電泳分析…………………………………………………………20
2.5.1　SDS-PAGE……………………………………………………………20
2.5.2　Zymogram……………………………………………………………21
2.6　分開酵素水解與發酵製程(separate hydrolysis and fermentation,
　　 SHF)………………………………………………………………………21
2.6.1　碎稻桿之酵素水解…………………………………………………21
2.6.2　厭氧產氫菌之碎稻桿水解液發酵…………………………………21
2.7　同步糖化與發酵製程(simultaneous saccharification and fermentation,
SSF)………………………………………………………………………22
第三章　實驗結果……………………………………………………………………23
1.　T. longibrachiatum、A. niger及C. xylanolyticum Ter3之木質纖維素分解酵
素活性分析……………………………………………………………………23
1.1 T. longibrachiatum、A. niger及C. xylanolyticum Ter3之纖維素及半纖
維素分解酵素的最適作用條件…………………………………………23
1.1.1　T. longibrachiatum之纖維素及半纖維素分解酵素的最適作用條
件……………………………………………………………………23
1.1.2　A. niger之纖維素及半纖維素分解酵素的最適作用條件…………24
1.1.3　C. xylanolyticum Ter3之纖維素及半纖維素分解酵素的最適作用條
件……………………………………………………………………25
1.2 T. longibrachiatum、A. niger及C. xylanolyticum Ter3之木質纖維素分
解酵素於不同培養時間之活性表現……………………………………26
1.2.1　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經CMC誘導
生成之CMCase酵素活性分析………………………………………26
1.2.2　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經碎稻桿誘導
生成之CMCase酵素活性分析………………………………………27
1.2.3　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經Avicel誘導
生成之Avicelase酵素活性分析……………………………………27
1.2.4　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經碎稻桿誘導
生成之Avicelase酵素活性分析……………………………………28
1.2.5　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經xylan誘導
生成之xylanase酵素活性分析………………………………………29
1.2.6　T. longibrachiatum、A. niger及C. xylanolyticum Ter3經碎稻桿誘導
生成之xylanase酵素活性分析………………………………………29
1.3　T. longibrachiatum、A. niger及C. xylanolyticum Ter3之半纖維素分解
酵素的SDS-PAGE及zymogram分析……………………………………30
2.　以T. longibrachiatum、A. niger及C. xylanolyticum Ter3產生的木質纖維素
　　　分解酵素進行天然基質的水解測試…………………………………………30
2.1　以T. longibrachiatum之三種水解酵素的不同搭配組合進行碎稻桿之糖
　　化測試……………………………………………………………………31
2.2　以A. niger之三種水解酵素的不同搭配組合進行碎稻桿之糖化測試…31
2.3　以C. xylanolyticum Ter3之三種水解酵素的不同搭配組合進行碎稻桿之
　　糖化測試…………………………………………………………………31
2.4　以T. longibrachiatum CMCase、A. niger xylanase及T. longibrachiatum
Avicelase之混合酵素系統進行碎稻桿之糖化測試……………………32
3.　C. acetobutylicum之碎稻桿水解液發酵產氫………………………………32
第四章　討論…………………………………………………………………………34
第五章　結論…………………………………………………………………………38
參考文獻………………………………………………………………………………39
表與圖…………………………………………………………………………………46

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