Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5889
標題: 纖維素水解菌Cellulomonas sp.和酵母菌於SSF程序以藻粉產生生質酒精之研究
Hydrolysis of microalgal biomass for bioethanol production by simultaneous saccharification and fermentation(SSF)with Cellulomonas sp.
作者: 高婉婷
Kao, Wan-Ting
關鍵字: 微藻;microalgae;纖維素分解酵素;生質酒精;SSF;cellulase;bioethanol;SSF
出版社: 環境工程學系所
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摘要: 
使用微藻所含纖維素作為生產生質酒精之原料可改善能源與糧食供給之衝突,同時又能解決稻稈等農業作物生長緩慢的缺點。另一方面,由於微藻所含木質素少於農業廢棄物,可減少前處理的困難性。利用藻類生產生質酒精需先將纖維素水解產生醣類後,再進一步使用酵母菌進行醱酵產酒精,而相較於分步水解與醱酵程序(SHF),同步水解與醱酵程序(SSF)具有降低產物抑制纖維素分解酵素活性,以提升酒精產量之優點,因此本實驗目的為利用纖維素水解菌Cellulomonas sp.與酵母菌Saccharomyces cerevisiae以SSF程序對藻株生產生質酒精進行研究。
本研究利用Chlorella sp.藻粉分別探討前處理與水解步驟中HCl 或NaOH 濃度、無機鹽組成、藻粉濃度與水解溫度對於還原醣產量之影響,並探討酵母菌之最適醱酵溫度、pH與初始酵母菌添加量,並於最佳條件下比較不同纖維素分解酵素與不同藻粉種類對於SHF與SSF程序中酒精產量與最大酒精生產速率之影響。另外,由於考慮到纖維素水解菌Cellulomonas sp.會利用纖維素水解所產生的醣類,因此以去除菌體保留胞外酵素之粗酵素液以了解各實驗參數對還原醣產量之影響以及產物累積對於酵素活性之抑制情形。
研究發現藻株Graesiella sp.於5 L反應槽培養有最高之纖維素含量(55.18%)與200 mL三角瓶培養方式相比,纖維素含量提升9.6倍。於水解條件試驗中,以粗酵素液水解Chlorella sp.之最適產醣條件為水解溫度30℃於20 g/L藻粉濃度之Mandels Reese無機鹽液中進行水解,而於前處理條件試驗中發現以2%NaOH前處理藻粉並添加前處理上層液之方式有助於粗酵素液縮短水解產醣時間並提升88%水解產醣。此外,酵母菌之最適產酒精條件為30℃,pH6.5,初始酵母菌添加量為0.07 g/L時,以10 g/L葡萄糖為碳源有最大酒精產量43.0 g/L。
結合前處理、水解與醱酵之最佳條件於SHF與SSF程序比較粗酵素液與商業酵素,可發現於SHF程序中對未前處理之藻株Chlorella sp.水解時,使用商業酵素可得到較多還原醣產量(0.6 g/L)與酒精產量(4.09 g/L),然而其內切型酵素活性受抑制現象也較為明顯(57%),而於SSF中則為使用粗酵素液可得到較好之酒精產量(6.59 g/L)。而改用2%NaOH前處理之藻粉Chlorella sp.為原料且其他條件皆相同下,相較於SHF程序,SSF程序有助於提升3.03倍之乙醇產量。另外,不論以有無前處理之Graesiella sp.藻粉為原料時,其在SHF或SSF程序中皆相較於Chlorella sp.藻粉有較高之酒精產量與產酒精速率。

Microalgae biomass is a potential substrate for bioethanol production because (1) microalgae can exhibit a higher productivity than lignocellulosic crops; (2) cultivation of microalgae can avoid the competition between energy production and food supply when using energy crops; (3) microalgae, compared with terrestrial plants, have a low lignin content to facilitate the lignin removal. In order to generate bioethanol from microalgae biomass, cellulose in the microalgae biomass should be hydrolyzed by a variety of cellulases and the hydrolysates (reducing sugars) are subsequently fermented by yeast to produce ethanol. However, this traditional process, named separate hydrolysis and fermentation (SHF), revealed a great inhibition in the cellulase activity that is reduced by the accumulated reducing sugars. Nevertheless, it has been indicated that a simultaneous saccharification and fermentation (SSF) process can achieve a high ethanol yield than SHF process due to its maintenance of cellulase activity. Therefore, the aim of this study was to investigate the feasibility of bioethanol production from microalgae biomass by Cellulomonas sp. and Saccharomyces cerevisiae in a batch-mode SSF system.
In this study, a commercial powder of Chlorella sp. biomass and a self-cultivated Graesiella sp. biomass, were used to estimate their bioethanol production. The effect of HCl or NaOH concentrations for hydrolysis pretreatment, composition of culture medium, microalgae biomass concentration, and the temperature for cellulose hydrolysis on the production of reducing sugars were all carried out, using the commercial powder of Chlorella sp. biomass as experimental substrate. On the other hand, the optimal conditions of temperature, pH value, and the initial amount of Saccharomyces cerevisiae inoculating biomass in the fermentation of glucose were examined. Considering the cellulase-producing bacterium, Cellulomonas sp. would consume the reducing sugars after the hydrolysis, the spent medium that contained the extracellular cellulase (crude cellulase) was used to understand the effect of above experimental parameters on the reducing sugar production and the inhibition of enzyme activity by hydrolysates. Finally, the efficiency of producing reducing sugar by the crude cellulase would be compared with that by a commercial cellulase.
The results showed that the cellulose content of 55.18% in Graesiella sp. was observed in a 5-liter-photobioreactor cultivation, which can improve the cellulose content in 9.6 fold than a 200-milliliter-flask cultivation. For the hydrolysis of Chlorella sp. biomass by the crude cellulase, the maximum amount of reducing sugars was achieved in the Mandels Reese culture medium with 20 g/L inoculum at 30ºC. With respect to the pretreatment procedure, treating the Chlorella sp. biomass with 2% NaOH and further mixing the treated biomass with neutralized supernatant from earlier base pretreatment can shorten the hydrolysis period and increase the reducing sugars production in 88%. In addition, the highest ethanol content of 43.0 g/L was obtained when the fermentation process was performed under 10 g/L of glucose with a inoculum of 0.07 g/L at 40ºC and pH 6.5.
Integrating those optimal parameters and utilizing the crude and commercial cellulase to hydrolyze the Graesiella sp. or Chlorella sp. biomass in both SHF and SSF processes, the results indicated that the commercial cellulase can yield reducing sugars of 0.6 g/L and ethanol of 4.09 g/L in the SHF process with unpretreated Chlorella sp. biomass. However, an obvious inhibition to endo-β-1, 4-gulcanase was observed and its enzyme activity decreased in 57% within this SHF experiment. On the contrary, using the crude cellulase in the SSF process showed a high ethanol production (6.59 g/L). Using the pretreated Chlorella sp. biomass as the substrate, the SSF process can improve the ethanol production in 3.03 fold than SHF process. Furthermore, hydrolysis of Graesiella sp. biomass produced more ethanol and exhibited a higher ethanol-producing rate than those of Chlorella sp. biomass no matter what kind of cellulase and process were performed.
URI: http://hdl.handle.net/11455/5889
其他識別: U0005-3107201209255700
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