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研究生：	黃楷珞 Huang, Kai-Lou
論文名稱：	結合暗醱酵產氫及微藻養殖進行永續且零CO2排放之創新生物產氫系統開發 Developing a sustainable and CO2-free biohydrogen producing system via a novel integration of dark hydrogen fermentation and microalgae cultivation
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	131
中文關鍵詞：	Chlorella vulgaris 、Clostridium butyricum 、混營培養、異營培養、光自營培養、二氧化碳固定、暗發酵產氫、系統整合
外文關鍵詞：	Chlorella vulgaris, Clostridium butyricum, mixotrophic cultivation, heterotrophic, phototrophic, CO2 fixation, dark fermentation, process integration
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隨著全球暖化和能源日益減少，尋找新的替代能源是全球熱門的議題。生質燃料，是指利用生物質經轉換所獲得能源(如生質酒精、生質柴油和生質氫氣)漸漸被受重視。近年來第三代生質料源 – 微藻逐漸受到重視，以微藻生產生質能儼然成為未來的新趨勢。本研究企圖將微藻系統串連暗發酵產氫系統，以微藻Chlorella vulgaris JSC-6吸收發酵產氫之產物二氧化碳及放流水中之揮發酸 (乙酸及丁酸) 進行零CO2排放與去除暗發酵液COD之目的，達到自給自足且零碳排放量之生質氫能系統。
首先，探討微藻在不同系統(光自營，異營以及混營)中，針對吸收CO2及有機酸和累積碳水化合物之效率進行研究，分別對環境因子如pH值，CO2曝氣速率以及有機酸的濃度進行微藻培養，使之能在後端有效的利用Clostridium butyricum CGS5在產氫過程中所排放之代謝產物。實驗結果顯示，此微藻在混營系統下，pH控制為7.5，曝氣速率為5x10-3 vvm以及乙酸與丁酸濃度分別為0.3 g/L和0.8 g/L的條件下，微藻能夠將放流水中的揮發酸(乙酸及丁酸)和氣相中之CO2完全利用，並產生能累積碳水化合物之微藻生物質，以利於後續利用。
此外，過去研究指出，水溶液中過高的HCO3-濃度會抑制Chlorella vulgaris JSC-6的生長，然而在暗醱酵液中則含有大量作為緩衝溶液的NaHCO3。為使微藻在利用暗發酵液時不受到HCO3-濃度的抑制，本研究移除在發酵產氫培養基中之NaHCO3。結果顯示，Clostridium butyricum CGS5在不添加緩衝液NaHCO3仍能夠繼續生長並進行產氫，其最大產氫速率為189 ml/h/L，產率為3.23 mol H2/mol Sucrose。另一方面，本研究亦將含高碳水化合物之微藻biomass做為發酵產氫之料源，並探討微藻biomass經不同的前處理方式進行水解並醱酵產氫之最佳條件。結果發現，Chlorella vulgaris JSC-6 以商業化酵素進行水解時，可達到100%的還原糖轉化率與80%的單糖轉化率，其累積產氫量為1407 ml/L，而氫氣產率為43.44 mmol/g alga。
最後，本研究利用連續流方式將微藻以混營培養系統串聯暗發酵產氫系統進行生物產氫。在此實驗中當發酵之HRT為16 h時，其產氫速率為350 ml/L/h，且其出流水與排放氣體皆與微藻系統結合，透過微藻系統進行混營培養，而微藻在HRT為6天時，可完全將CO2和出流液中的揮發酸100%降解，而產生的藻體其碳水化合物含量也可達65%，並可進行連續式微藻生長。因此，本研究成功地將發酵產氫與微藻系統進行串接整合且達到半連續流裝置，不但可以使CO2達到零排放之目的及去除發酵液中的COD，並能連續生產發酵所需之藻體料源，達到零碳排放能源生產及永續利用之目的。

In light of the crisis of global warming and energy deficiency, finding alternative renewable energy resources for human sustainability is one of the most important global issues at present. Biofuels (e.g., alcohols, biodiesel, or biogas) derived from biomass have caught increasing attention. In particular, using microalgae biomass as the third generation feedstock for biofuels production has become a new trend. In this study, we focused on developing a novel biohydrogen (a no-carbon fuel) technology by integrating dark hydrogen fermentation and mixotrophic microalgae growth to create a CO2-free self-sustainable biohydrogen system.
Our previous study focused on selecting a microalgae cultivation system (photoautotrophic, heterotrophic and mixotrophic) that could be used to grow microalgae to achieve a maximum efficiency of CO2 fixation, COD removal and carbohydrate accumulation. To improve the consumption rate of the metabolites coming from dark hydrogen fermentation process using Clostridium butyricum CGS5 as the bioH2 producer, the primary factors affecting the growth of the microalgae, such as pH, CO2 feeding rate and organic acid loading, were investigated. The results show that the optimal pH, CO2 feeding rate and organic acid loading were 7.5, 0.5 ml/min and 1/6X diluted fermentation effluent, respectively. The isolated microalga Chlorella vulgaris JSC-6 can grow efficiently on the liquid (acetate or butyrate) and gaseous (CO2) metabolites from dark fermentation under mixotrophic conditions and accumulates high carbohydrate content in its biomass.
Moreover, our previous study showed that C. vulgaris JSC-6 was inhibited by an excess concentration of HCO3-. However, a high concentration of NaHCO3 is present in the liquid effluent of dark H2 fermentation since it was used as the buffer in the dark fermentation medium. To avoid the inhibition of NaHCO3 to the microalgae growth, the NaHCO3 component was excluded from the fermentation medium. The results show that the Cl. butyricum CGS5 could still produce biohydrogen efficiently on the NaHCO3–free medium, obtaining a maximum H2 production of 189 ml/L/min and a yield of 3.23 mol/mol.
On the other hand, the carbohydrate-rich microalgae biomass was also used as the carbon source to produce bio-hydrogen via dark fermentation. The results show that the microalgal biomass could be effectively hydrolyzed by commercial enzyme to achieve a sugar conversion of 87%. The microalgae hydrolysate was used to produce H2 by Cl. butyricum CGS5, giving a maximum cumulative H2 production of 1407 ml/L and a yield of 43.44 mmol/g alga.
Dark fermentation and microalgae culture were integrated under the optimal conditions. The results show that H2 productivity of dark fermentation at a hydraulic retention time (HRT) of 16 h was 350 ml/L/h. C. vulgaris JSC-6 can efficiently utilize the soluble metabolites and completely remove CO2 from the gas effluent of dark fermentation for mixotrophic growth. The carbohydrate content of the yielded microalgal biomass can reach up to 65%, it suitable to serve as feedstock for dark hydrogen fermentation. This study successfully demonstrated the feasibility of integration of dark H2 fermentation and mixotrophic growth of microalgae under cyclic semi-continuous operations. This novel process was able to produce hydrogen without CO2 emissions with a significant COD reduction of the dark fermentation effluent and a production of microalgal biomass as self-sustainable feedstock.

摘要	I
Abstract	III
Acknowledgment	VI
Contents	VIII
List of Tables	XIII
List of Figures	XV
Chapter 1	Introduction	1
1	Background and motivation	1
2	Research scheme	4
Chapter 2	Literature review	7
1	Microalgae	7
1.1	Photosynthesis of microalgae	9
1.2	Microalgae cultivation	11
1.3	CO2 fixation of microalgae	12
1.4	Microalgae-based carbohydrates	13
1.5	Metabolism of microalgae	15
1.6	Environmental factors that potentially affect the production of microalgae carbohydrate	17
1.6.1	Nitrogen depletion	17
1.6.2	pH	18
1.6.3	CO2 supplement	18
2	Hydrogen	20
2.1	Hydrogen production methods	21
2.1.1	H2 production from fossil fuels by thermo-chemical method	21
2.1.2	H2 production by electrochemical method	22
2.1.3	Biologic production of H2 production	22
3	Integration of microalgae and dark fermentation systems	29
3.1	Microalgae cultivation by wastewater	29
3.2	Using the microalgae biomass as the carbon source to the biofuels	32
3.2.1	Pre-treatment methods of microalgae	32
3.2.2	Biohydrogen production from Microalgae biomass	33
Chapter 3	Materials and Methods	35
1	Chemicals and materials	35
2	Equipments	37
3	Bacterial strains and cultivation conditions	38
3.1	Hydrogen producing anaerobic bacteria and culture medium	38
3.2	Microalgae strain and culture medium	40
4	Analytical methods	41
4.1	Measurement of biomass concentration	41
4.2	Measurement of nitrate concentration	42
4.3	Determination of the total organic/inorganic carbon concentration in culturing medium	43
4.4	Determination of reducing sugar concentration by DNS method	43
4.5	Measurement of gaseous products	44
4.6	Determination of the concentration of soluble metabolites by HPLC	45
4.7	Measurement of light intensity	46
4.8	Determination of carbohydrates in the microalgal biomass	46
4.9	Analysis of transient behavior by modified Gompertz equation	47
5	Experimental methods	48
5.1	Effect of different pH controlled on biomass productivity, CO2 removal rate and carbohydrate accumulation for the microalgal cultivation	48
5.2	Effect of organic acid loading for the microalgae under the heterotrophic cultivation	49
5.3	Effect of air feeding rate for the microalgae under the photoautotrophic cultivation	49
5.4	Effect of organic acid loading and air feeding rate for the microalgae under the mixotrophic cultivation	49
5.5	Engineering strategies to enhance carbohydrate accumulation, improve the COD removal and CO2 fixation efficiency	50
5.6	Effect of NaHCO3 concentration on the biohydrogen production	50
5.7	Biohydrogen production by microalgae with acid pretreatment method and enzymatic hudrolysis	51
5.8	Microalgae growth in the supernatant of dark fermentation broth	51
5.9	Integration of dark fermentation and mixotrophic cultivation of m	52
Chapter 4	Results and discussion	54
1	Effect of different pH control under the different cultivation of the microalgae on biomass productivity and carbohydrate accumulation	54
1.1	Effect of different pH-controlled on microalgae growth under the mixotrophic cultivation of 2.5% CO2 and organic acid	54
1.2	Effect of different pH-controlled on microalgae growth under the heterotrophic cultivation of organic acid	59
1.3	Effect of different pH-controlled on microalgae growth under photoautotrophic cultivation of 40% CO2	63
1.4	Effect of different pH-controlled on microalgae grow under the mixotrophic cultivation of 40% CO2 and organic acid	68
1.5	Summary	73
2	Effect of organic acid loading on the microalgae growth under the heterotrophic cultivation	75
3	Effect of air feeding rate for the microalgae under the photoautotrophic cultivation	80
4	Effect of organic acid loading and air feeding rate on the microalgae under the mixotrophic cultivation	84
4.1	Effect of organic acid loading on microalgae growth under the mixotrophic	84
4.2	Effect of air feeding rate on microalgae growth under the mixotrophic	89
5	Engineering strategies to enhance carbohydrate accumulation and improve the COD removal and CO2 fixation efficiency	93
5.1	One-stage strategy to enhance carbohydrate accumulation, CO2 fixation and organic acid utilization of C. vulgaris JSC-6	94
5.2	Two-stage strategy to enhance carbohydrate accumulation, CO2 fixation and organic acid utilization of C. vulgaris JSC-6	96
5.3	Summary	98
6	Effect of NaHCO3 concentration on the biohydrogen production of dark fermentation	100
7	Effect of difference pretreatment ways of microalgae biomass to produce the biohydrogen	102
8	Integrated system of microalgae cultivation and dark fermentation	105
8.1	Integrated dark fermentation and microalgae systems on continuous model	106
8.2	Improving CO2 fixation and carbohydrate production by using continuous cultivation strategy	111
8.2.1	Improving CO2 fixation and carbohydrate production by increasing the volume strategy	111
8.2.2	Effects the concentration of nitrate of the microalgae growth under the continuous system of the mixotrophic	113
8.3	Summary	118
Chapter 5	Conclusions	120
Chapter 6	Future perspectives	122
References	123
 

                                    

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