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研究生:張晉諺
研究生(外文):Chin-YenChang
論文名稱:結合暗發酵與微藻混營培養之創新生物產氫整合系統
論文名稱(外文):An innovative biohydrogen production system integrating dark fermentation process and mixotrophic growth of microalgae
指導教授:張嘉修張嘉修引用關係
指導教授(外文):Jo-Shu Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:102
中文關鍵詞:小球藻梭狀芽孢桿菌暗醱酵產氫二氧化碳固定還原糖揮發酸食微比光強度混營作用光異營
外文關鍵詞:Chlorella vulgarisClostridium butyricumdark fermentationCO2 fixationreducing sugarsvolatile fatty acidsfood to microorganism ratiolight intensitymixotrophic growthheterotrophic growth
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由於氣候變遷和環境的改變,減少碳排放已經是全球熱門的議題。微藻能有效的吸收二氧化碳並且將其轉換成生物質(biomass)(約1.6-2.0克的二氧化碳會產生1克的微藻biomass);而許多綠藻具有相當高的碳水化合物含量,因此非常適合作為醱酵生產生質能源(如生質氫氣)的料源。本研究即利用自行篩選之高碳水化合物含量微藻菌株Chlorella vulgaris ESP-6當作Clostridium butyricum CGS5進行暗發酵產氫的料源,並探討微藻藻體水解與醱酵產氫之最佳條件。結果發現C. vulgaris ESP-6以1.5% HCl水解時,可達99 %的碳水化合物轉化率;而以此微藻水解液進行醱酵產氫之最佳操作條件如下:溫度為37oC,初始pH值為 7.0,微藻濃度為20 g/L (相當於10 g還原糖(RS)/L),控制pH為5.5。於此條件下,其氫氣產量為1475 ml/L,最大產氫速率為246 ml/L/h,而氫氣產率為1.09 mol H2/mol reducing sugar。
由於暗醱酵產氫時會產生揮發酸為最終代謝產物,因此暗醱酵之放流水需經過後續處理去除大部分的COD後,才能進行排放。因此,本研究企圖以微藻菌株C. vulgaris ESP-6藉由光異營生長的方式,吸收利用從暗發酵中產生的揮發酸(主要為丁酸、乙酸和乳酸),除了可降低其COD外,亦能生產微藻藻體,以利於後續之利用。結果發現,該藻株能有效地利用稀釋成1/4倍的暗醱酵上清液進行生長,但是過高的揮發酸濃度卻會抑制該藻株的生長,因此揮發酸必須控制在適當的濃度下才有利於該微藻之生長。此外,為了增進微藻該藻株對揮發酸的利用,本研究針對不同的光強度以及食微比進行探討,結果發現在光強度125 mol m-2s-1下和食微比為4.5的時候有最適當的微藻生長速率以及揮發酸利用速率。因此,C. vulgaris ESP-6可利用光異營的方式有效地分解與利用暗發酵代謝產物中的揮發酸以進行生長,並去除暗醱酵廢水中之COD。
最後,本研究將微藻系統串聯暗醱酵產氫系統並以混營培養的方式同時吸收暗醱酵產氫槽所產生的二氧化碳以及放流水中之揮發酸,以同時進行CO2減量、移除COD並生產高碳水化合物含量之藻體。於此實驗中,產氫系統進行了9天的穩定連續式操作,其產氫速率為212.8±9.2 ml/h/L;且其放流水與氣體出流皆與微藻系統連結,透過微藻系統進行混營培養,可去除95%的二氧化碳以及97%的揮發酸。因此,本研究成功地將醱酵產氫與微藻系統進行創新整合,不但可達到零CO2排放與去除醱酵酵液中揮發酸之目的,並能生產可作為暗醱酵料源的微藻藻體,進行永續回收再利用,實可謂一舉數得。

Mitigation of CO2 emissions has become a globally hot issue due to its adverse effects on our environment and climate pattern. Microalga is an effective natural CO2 sink, converting CO2 to its biomass with a ratio of 1.6-2.0 (g CO2/g biomass). Some microalgae contain a large amount of carbohydrates (over 50% per dry weight of biomass), which can serve as carbon source for the fermentative production of biofuels, such as bioH2. In this study, the biomass of a green microalga, Chlorella vulgaris ESP-6, was used as the carbon source to produce hydrogen by dark fermentation with an isolated H2-producing strain Clostridium butyricum CGS5. The results show that C. vulgaris ESP-6 biomass was effectively hydrolyzed by 1.5% HCl (121oC, 20 min) to achieve sugar conversion of 99%. The microalgal hydrolysate was used to produce H2 by Cl. butyricum CGS5 under an optimal condition of 37oC, pH 7.0, and a microalgal biomass of 20 g/L (equivalent to 10 g/L of reducing sugar) with pH control at 5.5, giving a cumulative H2 production of 1475 ml/L, a maximum H2 production rate of 246 ml/L/h, and a H2 yield of 1.09 mol H2/mol reducing sugar, respectively.
Moreover, C. vulgaris ESP-6 was also used to assimilate the volatile fatty acids (mainly acetate, butyrate, and lactate) from dark hydrogen fermentation processes. The results show that the microalgal strain could grow on the 1/4x diluted supernatant of the dark fermentation broth. It was found that the microalgal growth was inhibited by lactate, butyrate, or HCO3- when their concentration was higher than 0.5, 0.1, or 2.72 g/L, respectively. To improve the consumption rate of those volatile fatty acids, the primary factors (e.g., light intensity and food to microorganism (F/M) ratio) affecting photoheterotrophic growth of the microalgal strain were investigated. The optimal condition was light intensity at 125 mol m-2s-1 and F/M ratio at 4.8. The results demonstrated that C. vulgaris ESP-6 can efficiently utilize the soluble metabolites of dark H2 fermentation for photoheterotrophic growth.
In addition, this work was also undertaken to evaluate the feasibility of using the microalgal strain to simultaneously assimilate the volatile fatty acids (soluble metabolites of dark fermentation) and fix the CO2 produced from dark fermentation under mixotrophic cultivation conditions. In these experiments, H2 was produced from continuous dark fermentation with a stable H2 production rate of 212.8±9.2 ml/h/L and a cell concentration of 1.86±0.1 g/L for 9 days. Then, the liquid the gas effluent of dark fermentation was connected with microalgae mixotrophic growth system, which was able to remove over 95% of CO2 and 97% of volatile fatty acids. The microalgal biomass produced from mixotrophic growth system was also used as feedstock to produce more H2 via dark fermentation and then the soluble metabolites and CO2 produced during dark fermentation were again assimilated by microalgae, resulting in a recycle system to produce hydrogen without CO2 emission and with a significant COD reduction of the dark fermentation effluent.

摘要……………………………………………………….I
Abstract………………………………………………III
Acknowledgment……………………………………………V
Contents………………………………………VII
List of tables…………………………………………X
List of figures…………………………XII


Chapter 1 Introduction……………………1
1.1 Background and motivation……………………1
1.2 Research scheme……………………5


Chapter 2 Literature review…………………………7
2.1 Microalgae…………………………7
2.1.1 Composition of green algae…………………………9
2.1.2 Microalgal cultivation…………………………11
2.1.3 Metabolism of green algae…………………………12
2.1.4 Microalgae as feedstocks for biofuels…………………………14
2.2 Hydrogen production…………………………16
2.2.1 Conventional methods for H2 production…………………………17
2.2.2 Biological production of hydrogen…………………………19
2.2.2.1 H2 production from algae by photosynthesis…………………23
2.2.2.2 H2 production by dark fermentation…………………………25
2.3 Integration of microalgae and dark fermentation systems…………………………29
2.3.1 Using discharged gas or liquid effluent of dark fermentation to cultivate microalgae…………………………29
2.3.2 Conversion of microalgal biomass to biohydrogen…………………………30

Chapter 3 Materials and Methods…………………………34
3.1 Chemicals and materials…………………………34
3.2 Equipment…………………………36
3.3 Microbial strains and cultivation conditions…………………………38
3.3.1 Hydrogen-producing anaerobic bacteria and culture condition…………………………38
3.3.2 Microalgal strain and culture medium…………………………40
3.4 Analytical methods…………………………42
3.4.1 Determination of reducing sugar concentration by DNS method…………………………42
3.4.2 Measurement of gaseous products…………………………43
3.4.3 Determination of the concentration of soluble metabolites by HPLC…………………………44
3.4.4 Measurement of light intensity…………………………45
3.4.5 Determination of carbohydrates in the microalgal biomass…………………………45
3.4.6 Analysis of transient behavior by modified Gompertz equation…………………………46
3.5 Experimental methods…………………………48
3.5.1 BioH2 production by microalgal biomass with alkaline pretreatment method and enzymatic hydrolysis…………………………48
3.5.2 BioH2 production by acidic hydrolysate of the microalgal biomass…………………………48
3.5.3 Optimal condition for bioH2 production…………………………49
3.5.4 Improvement of H2 production by pH-control…………………………49
3.5.5 Microalgal growth on the supernatant of dark fermentation broth…………………………49
3.5.6 Effect of sole organic acid and growth kinetics analysis…………………………50
3.5.7 Integration of dark fermentation and mixotrophic cultivation of microalgae…………………………50

Chapter 4 Results and discussion…………………………52
4.1 Pretreatment of microalgal biomass………………………52
4.2 Optimization of biohydrogen production using acidic hydrolysate of Chlorella vulgaris ESP-6………………………56
4.3 Enhancement of hydrogen production by pH-control ………………………60
4.4 Microalgal growth on the soluble metabolites in dark fermentation broth with Clostridium butyricum CGS5…………………64
4.5 Effect of pH control on microalgal cultivation using diluted dark fermentation broth………………………66
4.6 Substrate inhibition on microalgal growth………………………69
4.6.1 Effect of HCO3- on microalgal cultivation………………………69
4.7 Utilization efficiency of dark fermentation metabolites under different food to microorganism (F/M) ratio………………………77
4.8 Effect of light intensity on the consumption of metabolites from dark fermentation………………………79
4.9 Integrated system of microalgal cultivation and dark fermentation………………………81

Chapter 5 Conclusions and Prospects………………………92
Reference ………………………………………………………………95

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