||Study on Characteristics of the Hydrogen Fermentation Utilizing Multiple Substrates Containing Nitrogen Compounds
||Department of Environmental Engineering
waste activated sludge
anaerobic hydrogen-producing processes
由複合基質產氫的研究中得知，基質組成分明顯影響生物產氫效率，最佳的產氫基質組合為60%的葡萄糖加40%水解蛋白質或80%的澱粉加20%水解蛋白質。水解蛋白質主要影響產氫生物生長與環境酸鹼值。足夠的蛋白質含量有助於生物生長，也促進生物產氫效率。而要維持穩定的中性酸鹼值環境所添加的大量磷酸緩衝液，並不利於生物產氫。含40%水解蛋白質複合基質的氫醱酵中，添加50 mM的磷酸鹽產氫量比添加10 mM產氫量少了33%。除了基質組成與鹽類濃度外，氫分壓對於生物產氫有明顯的影響。研究中發現，缺乏良好的質傳條件，液相中的溶氫可能比氣相的氫分壓高出數十倍甚至上百倍，氫分壓可能造成的影響遠比自氣相中觀察到的來的嚴重。氫分壓不但抑制產氫，更可能引發蛋白質有機物嚴重的耗氫分解。而本研究採用的振盪培養與薄膜分離技術都能明顯降低氫分壓危害。增加薄膜分離模組快速移除生物產氣，分別有效提昇生物產氫速率與產氫量達15%與10%的幅度。
本研究應用氫醱酵這項新穎的技術於廢棄活性污泥厭氧處理的預醱酵處理，將大分子的有機物降解為小分子。降解1 g 經加鹼前處理的廢棄活性污泥可產生0.54 mmole的氫氣，22 mg的乙酸及53 mg氨氮。在氫醱酵後可量測的COD及TKN部份均呈現增加的趨勢，顯示氫醱酵有效降解廢棄活性污泥微小分子型態有機物。廢棄活性污泥的氫醱酵產氫特性與水解蛋白質相似，在氫醱酵反應中除了產氫也有明顯的耗氫現象。由本研究的結果顯示，氫醱酵中的水解酵素(protease及amylase)活性與氫氣的累積動態一致，酵素活性的最大值出現於最大累積產氫時期附近。產氫量隨著amylase的添加而增加，但是卻隨著protease的添加而減少。因為在產氫反應中碳水化合物是一項良好的電子供應者，可供氫氣的生成；蛋白質可能是良好的電子接受者，故會促進氫氣的消耗。
本研究啟動並長期操作一厭氧氫醱酵生物反應器，並探討不同水力停留時間(HRT)下，氫氣生成情形、微生物生長及複合基質所扮演角色。以20,000 mg/L葡萄糖及水解蛋白質(3：2(w/w))作為基質，溫度控制於35℃，進行五試程水力停留時間分別為12、8、6、4及2小時。水力停留時間長於2小時的試程，產氫表現及微生物生長皆可維持一穩定狀態。當水力停留時間為6小時，系統表現最佳的微生物生長及最大的氫氣產率5.6 mmole H2/g glucose as COD。在不同水力停留時間下，葡萄糖主要利用於生產氫氣且可降解完全，蛋白質所扮演的角色則不盡相同，在較長的水力停留時間下，蛋白質被利用於醱酵及進行生物體合成，隨著水力停留時間縮短，其作用主要轉為生物質體合成用，或仍以有機氮的形式殘留，只有少部分被代謝至無機的氨氮形式。
This research includes to investigate the characteristic, mechanism and influential factors of hydrogen fermentation in carbohydrates, protein multiple substrates and waste activated sludge (WAS). The hydrogen production, hydrogen consumption, cell growth and products accumulation were discussed. The significant hydrogen consumption following hydrogen production was observed during the fermentation of peptone, multiple substrate and WAS. Their main fermentative byproducts were fatty acids, such as acetate and butyrate that were similar to carbohydrate’s fermentative products. The hydrogen yields of glucose, peptone, multiple substrates and WAS were 10.6、0.67、7.1 and 0.54 mmole/g-COD.
According to the investigation on hydrogen fermentation of multiple substrates, the component of multiple substrates influenced the hydrogen yield significantly. The optimal components for hydrogen production were 60% glucose plus 40% peptone or 80% starch plus 20% peptone. In the fermentation of the optimal substrates, peptone was provided as nutrient for growth and help for maintaining neutral pH. Sufficient protein made the good cell growth and more stable pH condition. However, the extra addition of phosphate buffer for neutralizing pH was not advantage for hydrogen yield. The hydrogen yield in the fermentation with 50 mM phosphate was 33% less than 10 mM phosphate. Besides substrate component and ion strength, hydrogen partial pressure influenced hydrogen production significantly. High level of hydrogen partial pressure causes a little inhibition on hydrogen production and serious hydrogen consumption. The actually dissolved hydrogen partial pressure may one hundred times higher than the apparent partial pressure observed in headspace. Dissolved hydrogen partial pressure depending on hydrogen mass transfer controls the hydrogen yield and productivity. In order to remove the dissolved hydrogen immediately, a good mass transfer system is required. In this study, membrane separation module was used to remove biogas and improved the hydrogen productivity and yield by 15% and 10%, respectively.
This work introduces the novel hydrogen fermentation process into the first stage of anaerobic digestion for treating WAS. Hydrogen fermentation was introduced to degrade large molecular organic matter and to recover hydrogen energy. One gram COD of the waste sludge hydrolysate, which was prepared by pre-treating the WAS coming from the foodstuff plane or fructose manufactory by base, could be fermented and produced approximately 0.54 mmole of H2 gas, 22 mg of acetate and 53 mg of ammonia nitrogen. After fermenting, the detectable COD and TKN increased. The fermenting characteristics are similar to those of the fermentation of peptone. Both hydrogen production and consumption were observed in the fermentation. The hydrogen production depends on the activities of amylase and protease, the starch and protein hydrolysis enzymes, because the hydrolysis is the rate-limiting step of the biodegradation. The variation in the enzymatic activity during fermentation followed the hydrogen accumulation. The peak enzymatic activities were observed near the peak of hydrogen productivity. The hydrogen production was increased by adding amylase, but decreased by adding protease, because carbohydrate is a good electron donor for producing hydrogen, whereas protein as an probable electron acceptor for consuming hydrogen.
Multiple substrates, containing glucose and peptone, were fed into an anaerobic hydrogen fermentative continuous-flow stirred tank reactor (CSTR). The CSTR was operated at various hydraulic retention times (HRT) to examine the effects of HRT on hydrogen production and cell growth, and the role played by peptone. Maximum hydrogen yield and biomass concentration were obtained at 5.6 mmole H2/g COD and 3470 mg-MLVSS/L respectively following 6 hours of HRT. Hydrogen production and cell growth were maintained stable when HRT was longer than 2 hours. The role of protein changed with HRT. Protein was fermented and used in biosynthesis when HRT was long. However, most of the protein was utilized in biosynthesis or was maintained in its organic nitrogen form, and only a slight amount of it was fermented into the ammonia when HRT was short.
To monitor the bioactivity change in hydrogen fermentors, this work presented a novel bioelectrochemical method equipped with a polyviologen modified glassy carbon as a working electrode and with ferricyanide as an electron mediator. Experimental results demonstrated that the ferricyanide can transfer electrons from hydrogen producing bacteria cells to electrode without significant inhibition on cell growth. To protect the electrode from pollution by protein and bacteria during bioassay, polyviologen film was applied to modify the working electrode, and stabilized the responding current compared to the use of bare electrode. The mediated amperometric bioassay provided the bioactivity information for indicating the statue of the fermentor and helping draw up the operation strategy when the fermentor was operated under abnormal conditions, including stop-feeding and re-feeding. The results of bioactivity monitoring suggested an interesting phenomenon that the activity of hydrogen producing bacteria transiently increased during the unsteady period and then decreased.
According to the different damage on bioactivity caused by starvation, the restoration strategy includes three set. First one is fermentor suffering from short-term starvation (< 2 hours) that could be restored by direct re-feeding with full loading. Secondly, fermentor suffering from mid-term starvation (5.5 hours) should be restored by partial loading re-feeding strategy to improve bioactivity and to avoid washout, and then the full loading applied. The last one is fermentor suffering from long-term starvation (> 7 days) that should be re-startup by batch culture, half-loading and then full-loading for inducing the spore sprouting, improving the bioactivity and then stable operating.
摘 要 I
List of Figures IX
List of Tables XI
Chapter 1 Introduction 1-1
Chapter 2 Literature Reviews 2-1
2-1. Hydrogen economy 2-1
2-2. Hydrogen production technologies 2-11
2-3. Microbiology of biohydrogen production 2-18
2-4. Biochemistry of hydrogen fermentation 2-27
2-5. Effects on hydrogen fermentation 2-33
2-6. Process technologies 2-41
2-7. Bioelectrochemical methods for monitoring
Chapter 3 Material and Methods 3-1
3-1. Biochemical hydrogen potential test (BHP
3-2 Transfer culture for acclimatizing
hydrogen producing bacteria 3-4
3-3. Hydrogen fermentation in continuos-flow
stirred tank reactor (CSTR) 3-5
3-4 Analysis of hydrolyzing enzymatic activity 3-6
Chapter 4.Hydrogen Fermentation of Protein Compound 4-1
4-1 Hydrogen producing and consuming during
the fermentation 4-2
4-2 Accumulation and composition of
fermentative products from peptone 4-4
4-3 Proposed mechanism of biohydrogen
production with degradation of peptone 4-9
4-4 Summary 4-12
Chapter 5 Hydrogen Fermentation of Multiple Substrate Composed of Carbohydrate and Protein 5-1
5-1 The characteristic of the hydrogen
fermentation of multiple substrates 5-1
5-2 Effects of protein/carbohydrate ratio on
hydrogen fermentation 5-4
5-3 Inhibiting effect on hydrogen production
caused by hydrogen partial pressure 5-18
5-4 Effects of S0/X0 on hydrogen fermentation 5-33
5-5 Effects of salts, sodium chloride
and sodium phosphate, on hydrogen
Chapter 6 Feasibility-Study of Hydrogen Fermentation Applied on Waste Sludge Treatment 6-1
6-1. Hydrogen fermentation using pretreated
was as substrate and as screened seed
6-2. Acclimating hydrogen producing bacteria
degrading was hydrolysate by transfer
6-3. Enzymatic activity in the fermentation
of sludge hydrolysate 6-13
6-4 Summary 6-17
Chapter 7 Operation of Anaerobic Hydrogen Fermentor and Monitoring Bioactivity of Hydrogen Producing Bacteria 7-1
7-1. Effects of HRT on hydrogen fermentor 7-2
7-2. Development of the bioelectrochemical
method for rapidly determining bioactivity 7-6
7-3. Stop-feeding and re-feeding: scenario
of a hydrogen fermentor 7-19
Chapter 8 Conclusions and Suggestions 8-1
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