臺灣博碩士論文加值系統

　　厭氧處理程序之最終產物─甲烷與二氧化碳，近年來已被認定為溫室效應之最大貢獻者之一，而於厭氧過程中會產生非溫室氣體的氫氣，但是氫氣於早期的厭氧處理程序中，大多是用於厭氧反應槽操作時的負荷指標（load indicator）或是警示指標（alarm indicator），甚少有將氫氣視為能源的觀念。另外，不像一般石化燃料，氫氣燃燒後，不會產生二氧化碳（亦為溫室氣體），僅會產生水，且氫氣亦具有高能量之特質（122 KJ/g，約石化燃料的2.75倍），因此近年來世界先進各國多有欲將氫氣取代石化燃料之研究，所以氫氣可能成為未來的重要能源。況且若能利用微生物產生大量之氫氣，除對環境無害之外亦可以節省能源，因此若能將厭氧生物處理所產生之氫氣善加研究與利用，對於未來能源問題之改善，定有相當之助益。
　　由於我國缺乏能源且各種工業均會產生大量有機廢棄物，若利用本研究之生物產氫技術，處理有機廢棄物以產生大量氫氣乾淨能源，將可同時解決能源與環保問題。因此本研究以生活污水廠之污泥處理醣類基質（glucose及sucrose），使用2座CSTR反應槽（實反應體積４升）以產生氫氣，獲致下列結果：
　　glucose反應槽以HRT 4 hr之產氫效果為佳（氫含量45.1﹪、16.4 L-H2/L/Day、516 mL-H2/g VSS/hr）；sucrose反應槽以HRT 3 hr之產氫效果為佳（氫含量47.2﹪、26.9 L-H2/L/Day、648 mL-H2/g VSS/hr）。glucose反應槽最大產氫率為1.65 mol-H2/mol-glucose；sucrose反應槽最大產氫率為4.52 mol-H2/mol-sucrose。污泥經加熱或酸鹼前處理之產氫效果確比未經前處理之污泥為佳，尤其是酸前處理。馴養pH對產氫亦有一定之影響。

This study aimed at the hydrogen production of sewage sludge that digested the carbohydrate (glucose and sucrose). To achieve this purpose, the experimental approach was initially focused on the starting-up of hydrogen-producing digesters (continuously stirred tank reactor, CSTR), then the performance of the reactors and finally the influences of enrichment of seed sludge.
The procedure for starting-up CSTR reactors for acclimating anaerobic hydrogen-producing microorganisms with sewage sludge was investigated. Initially, the feeding and mixing were in a mode of semi-continuous type; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to a low value (pH 5.18), it was adjusted to 6.7 with sodium hydroxide (1N). At the same time, the semi-continuous type operation was changed to a continuous type. Finally, the pH was continuously regulated around 6.7. The results indicate that this procedure could cultivate seed sludge for hydrogen production from sewage sludge and obtain a large hydrogen production in less than 60 days. This seed sludge had a hydrogen yield of 1.63 mol-H2/mol-glucose and specific hydrogen production rate (SHPR) of 321 mmol-H2/g VSS-d at the HRT of 13.3 h; hydrogen yield was 4.45 mol-H2/mol-sucrose and SHPR was 707 mmol-H2/g VSS-d.
The influence of substrate on anaerobic hydrogen producing reactors seeded with sewage sludge was employed with batch tests. The results indicated that a substrate inhibition phenomenon results from higher glucose concentration, but not in higher sucrose concentration. The varieties of substrate and initial concentration influence the fermentation for hydrogen production.
Using thermal pretreatment, or a low or high pH environment to obtain dominant microbes for hydrogen production from sludge is a feasible method. Based on the experimental results, hydrogen production from sludge with thermal, acid or base enrichment is higher than that of the control. The hydrogen production potential of the sludge with acid or base enrichment enhanced 200 and 333 folds than that of the control when the enrichment pH was 10 and 3, respectively. The enrichment is due to the shortening of microorganisms'' lag-time which occurs at a proper cultivation pH level.
Two CSTR reactors were started up by using sewage sludge for producing hydrogen from sucrose or glucose. The substrate was fed in a continuous mode from HRT 13.3 hrs to 10, 8, 6, 5, 4, 3, and 2 hrs. The hydrogen gas production and hydrogen percentage increased when HRT decreased at long HRT and decreased with a HRT decreased at short HRT (2 hrs). Under steady state conditions, for reactor fed with glucose and sucrose, respectively, the SHPR ranged from 60 to 516 and 82 to 886 mL-H2/g VSS-hr; the yield ranged from 0.39 to 1.65 mol-H2/mol-glucose and 1.42 to 4.52 mol-H2/mol- sucrose. Kinetic models were developed to describe and predict the experimental results from the H2-producing cultures. The major volatile fatty acid (VFA) produced was butyric acid (HBu) with acetic acid and propionic acid at less quantities. The major solvent product was ethanol, whose concentration was only 15% of that of HBu, indicating that the metabolic flow favors H2 production. The model study also suggests that product formation in the continuous hydrogen-producing cultures was essentially a linear function of biomass concentration. H2 content, SHPR and yield were HRT and substrate-dependent. For each substrate two stages of HRT-dependent relationships for these three parameters were obtained. For glucose-degradation hydrogenation, the critical HRT values of the two-stages relation were 6 hrs for these three parameters. For sucrose-degradation hydrogenation, the critical HRT values of the two-stages relation were 5, 4 and 4 hrs for H2 content, SHPR and yield, respectively.

Abstract （In Chinese） I
Abstract II
Table of Contents IV
List of Figures VII
List of Tables X
1. Introduction 1-1
1-1 Motivation and Objectives 1-1
1-2 Hydrogen Production in an Anaerobic Process 1-1
1-3 Anaerobic Microorganisms of Hydrogen Production- Clostridium spp. 1-3
1-3-1 Characteristics of the Clostridium spp. 1-3
1-3-2 Fermentation of the Clostridium spp. 1-4
1-3-3 Characteristics of the Endospores 1-8
1-3-4 Production of Hydrogen Gas 1-9
1-4 Literature Reviews about operational parameters 1-11
1-5 Outline of this Thesis 1-15
1-6 References 1-15
2. Start-up of CSTR Digesters for Hydrogen Production
2-1 Introduction 2-1
2-2 Materials and Methods 2-2
2-2-1 Operation of the Reactors at the Start-up Period 2-2
Seed Sludge 2-2
Substrate 2-2
CSTR Reactor 2-2
Assessing Protocol 2-3
Analysis 2-4
2-2-2 Batch Experiments- Influence of Substrate 2-4
Biomass 2-4
Materials 2-4
Experimental Protocol 2-5
Analysis and Calculation 2-5
2-3 Results and Discussion 2-7
2-3-1 Operation of the Reactors at the Start-up Period (HRT 20 to 2.5 days)
Glucose Reactor 2-7
Sucrose Reactor 2-9
VFA Concentrations 2-9
2-3-2 Influence of Substrate 2-13
Hydrogen Production 2-17
Kinetic Analysis and Specific Hydrogen Production Rate 2-17
Variation of VFA Concentrations 2-20
Production Yields of Hydrogen, VFA and SMP 2-29
2-4 Conclusions 2-30
2-5 References 2-30
3. Performance of the Digesters
3-1 Introduction 3-1
3-2 Materials and Methods 3-2
3-2-1 Seed Sludge 3-2
3-2-2 Substrate 3-2
3-2-3 CSTR Reactor Operation 3-2
Start-up Period (HRT 20 to 2.5 days) 3-2
Continuous Feeding Period (HRT 13.3 to 2 hours) 3-2
3-2-4 Analysis 3-3
3-2-5 Development of Kinetic Models 3-3
Model System 3-3
Material Balances 3-4
3-3 Results and Discussion 3-8
3-3-1 Reactor Operation from Day 1 to Day 200 (HRT 13.3 to 2 hours) 3-8
Intermediate Liquid Products 3-8
Gas Production 3-8
3-3-2 Reactor Operation under Steady State Conditions (HRT 2-13.3 hrs) 3-15
pH and Alkalinity 3-15
Biomass 3-15
Intermediate Liquid Product Concentrations 3-21
Hydrogen Gas Production 3-23
3-3-3 Kinetics 3-26
Kinetics of EHP 3-26
Kinetics of IHP 3-39
3-3-4 Two-Stages Model 3-43
3-4 Conclusions 3-48
3-5 References 3-49
4. Influences of Enrichment of Seed Sludge
4-1 Introduction 4-1
4-2 Materials and Methods 4-3
4-2-1 Experimental Protocol 4-3
Thermal Enrichment Enhancement Test 4-3
Influences of Thermal Enrichment and Incubation Environment 4-4
Acid/Base Enrichment Enhancement Test 4-5
4-2-2 Analysis and Calculation 4-7
4-3 Results and Discussion 4-9
4-3-1 Thermal Enrichment Enhancement Test 4-9
Hydrogen Production 4-9
Kinetic Analysis 4-13
Specific Hydrogen Production Rate and Production Yields of Hydrogen 4-14
Variation of VFA Concentrations during the Enrichments 4-14
4-3-2 Influences of Thermal Enrichment and Incubation Environment 4-16
Hydrogen Production 4-16
Kinetic Analysis 4-18
Specific Hydrogen Production Rate and Production Yields of Hydrogen 4-18
Main Factor Effect and Best Factor Level 4-20
4-3-3 Acid/Base Enrichment Enhancement Test 4-22
Hydrogen Production 4-22
Specific Hydrogen Production Rate 4-24
Variation of VFA Concentrations during the Enrichments 4-25
Production Yields of Hydrogen and VFA 4-28
Kinetic Analysis 4-34
4-4 Conclusions 4-37
4-5 References 4-38
5. Conclusions and Suggestions
5-1 Conclusions 5-1
5-2 Topics Suggested for the Future Research 5-2
Appendix
Notations i
Curriculum Vitae iii
Acknowledgments vii

Akano T., Miura Y., Fukatsu K., Miyasaka H., Ikuta Y., Matsumoto H., Hamasaki A., Shioji N., Mizoguchi T., Yagi K. and Maeda I. (1996). Hydrogen production by photosynthetic microorganisms. Appl. Biochem. Biotechnol., Vol. 57, pp. 677-688.
Akashah M., Yoshida M., Watanabe M., Nakamura M. and Matsumoto J. (1997). Hydrogen gas production from glucose and its microbial kinetics in anaerobic systems. Proc. 8th International Conf. On Anaerobic Digestion, Vol. 1, pp.261-268.
Annous B. A., Shieh J. S., Shen G. J., Jain M. K. and Zeikus J. G. (1996). Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH. Appl. Microbiol. Biot., Vol. 45, No. 6, pp. 804-810.
Aoyama K., Uemura I., Miyake J. and Asada Y. (1997). Fermentative metabolism to produce hydrogen gas and organic compounds in a Cyanobacterium, spirulina platensis. J. Ferment. Bioeng., Vol. 83, pp. 17-20.
APHA. (1995). Standard Methods for the Examination of Water and Wastewater, 19th edition, American Public Health Association, New York, USA.
Atlas R. M. (1988). Microbiology: Fundamentals and Applications, MacMillan Publishing Company, New York.
Bailey J. E. and Ollis D. F. (1986). Biochemical engineering fundamentals, 2nd edition, McGraw-Hill Book Co., N. Y.
Bhattacharya S. K., Jr Sluder J. L. and Uberoi V. (1995). Effects of 4-nitrophenol on H2 and CO levels in anaerobic propionate systems. Wat. Res., Vol. 29, No. 5, pp. 1249-1258.
Brock T. D., Madigan M. T., Martinko J. M. and Parker J. (1994). Biology of Microorganisms. Pretice-Hall , Inc.,USA.
Brosseau J. D. and Zajic J. E. (1982). Hydrogen-gas production with Citrobacter intermedius and Clostridium pasteurianum. J. Chem. Tech. Biotechnol., Vol. 32, pp. 496-502.
Cha G. C. and Noike T. (1997). Effect of rapid temperature change and HRT on anaerobic acidogenesis. Wat. Sci. Tech., Vol. 36, pp. 247-253.
Chen K. C., Chen S. J. and Houng J. Y. (1996). Improvement of gas permeability of denitrifying PVA gel beads. Enzyme Microb. Technol., Vol. 18, pp. 502-506.
Christopher F. (1996). Sustainable energy for tomorrow’s World: The case for an optimistic future. In: The Energy Crisis, Feldman, D.L. (ed.), The Johns Hopkins University Press. Part 4, p.230.
Dinopoulou G., Rudd T. and Lester J. N. (1988). Anaerobic acidogenisis of complex wastewater: I. The influence of operational parameters on reactor performance. Biotechnol. Bioeng., Vol. 31, pp. 958-968.
Edwards V. H. (1970). The influence of high concentrations on microbial kinetics. Biotechnol. Bioeng., Vol. 12, pp. 679-712.
Endo G., Noike T., and Matsumoto J. (1982). Characteristics of cellulose and glucose decomposition in acidogenic phase of anaerobic digestion. Proc. Soc. Civ. Engrs, No. 325, pp. 61-68 (In Japanese).
Girbal L., Croux C., Vasconcelos I. and Soucaille P. (1995). Regulation of metabolic shifts in Clostridium acetobutylicum ATCC-824. FEMS Microbiol. Rev., Vol. 17, No. 3, pp. 287-297.
Guiot S. R., Pauss A. and Costerton J. W. (1992) A structured model of the anaerobic granule consortium. Wat. Sci. Tech. 25, 1-10.
Guwy A. J., Hawkes F. R., Hawkes D. L. and Rozzi A. G. (1997). Hydrogen production in a high rate fluidised bed anaerobic digester. Wat. Res., Vol. 31, pp. 1291-1298.
Haandel A. C. and Lettinga G. (1994). Anaerobic Sewage Treatment- a practical guide for regions with a hot climate. John Wiley & Sons Ltd., England.
Hallenbeck P. C. (1983). Immobilized microorganisms for hydrogen and ammonia production. Enzyme Microb. Technol., Vol. 5, pp. 171-180.
Huang C. L. (2001). Diversity studies on hydrogen-producing anaerobic microorganisms by molecular approaches. M.S. Thesis, National Central University, Chung-li, Tao-Yuan, Taiwan, R.O.C. (In Chinese)
Hwang J. L., Chen C. C., Lin C. Y. and Liu W. C. (2000). Diversity studies on hydrogen-producing anaerobic microorganisms by molecular approaches. Proceedings 25th Wastewater Treatment Technology Conference, Yun-Lin, Taiwan, Dec. 1-2, 2000, pp. 321-326 (In Chinese).
Kashket E. R. and Cao Z. Y. (1995). Clostridial Strain Degeneration. FEMS Microbiol. Reviews, Vol. 17, pp. 307-315.
Kataoka N., Miya A. and Kiriyama K. (1997). Studies on hydrogen production by continuous culture system of hydrogen producing anaerobic bacteria. Wat. Sci. Tech., Vol. 36, pp. 41-47.
Kumar N., Monga P. S., Biswas A. K. and Das D. (2000). Modeling and simulation of clean fuel production by Enterobacter cloacae IIT-BT 08. Int. J. Hydrogen Energy, Vol. 25, pp. 945-952.
Lay J. J. (2000). Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol. Bioeng., Vol. 68, pp. 269-278.
Lay J. J., Lee Y. J. and Noike T. (1999). Feasibility of biological hydrogen production from organic fraction of municipal solid waste. Wat. Res., Vol. 33, pp. 2579-2586.
Lay J. J., Li Y. Y. and Noike T. (1998). The influences of pH and ammonia concentration on the methane production in high-solids digestion. Wat. Environ. Res., Vol. 70, No. 5, pp. 1075-1082.
Lee Y. J., Miyahara T. and Noike T. (1999). Effect of pH on the microbial hydrogen fermentation. Asian Waterqual '99, 7th IAWQ Asia-Pacific Regional Conference, Vol. 1, pp. 215-220.
Lin C. Y. and Chang R. C. (1999). Hydrogen Production During the Anaerobic Acidogenic Conversion of Glucose. J. Chem. Technol. Biotechnol., Vol. 74, pp. 498-500.
Lin C. Y., Chen C. C. and Lin M. C. (2000a) Hydrogen production in anaerobic acidogenesis process－Influences of thermal isolation and acclimation environment. J. Chinese Institute of Environ. Eng., Vol. 10, pp. 163-168.
Lin C. Y., Chen C. C. and Lin M. C. (2000b). Enhancements of acid-base isolation on hydrogen production in anaerobic process. Biotechnology 2000 the World Congress on Biotechnology, Berlin, Germany, Sept. 3-8, 2000, Vol.3, pp.184-185.
Majizat A., Mitsunori Y., Mitsunori W., Michimasa N. and Junichiro M. (1997). Hydrogen gas production from glucose and its microbial kinetics in anaerobic systems. Wat. Sci. Tech., Vol. 36, No. 6-7, pp. 279-286.
Mizuno O., Dinsdale R., Hawkes F. R., Hawkes D. L. and Noike T. (2000). Enhancement of hydrogen production from glucose by nitrogen gas sparging. Bioresource Technol., Vol. 73, pp. 59-65.
Mosey F. E. and Fernandes X. A. (1989). Patterns of hydrogen in biogas from the anaerobic digestion of milk-sugars. Wat. Sci. Tech., Vol. 21, pp. 187-196.
Mosolova T. P., Kalyuzhnyi S. V., Belogurova N. G. and Varfolomeev S. D. (1991). Effects of antibiotics, temperature, and pH on the growth and metabolism of Clostridium Thermosaccharolyticum. Microbiology, Vol. 60, No. 3, pp. 340-345.
Nakamura M., Kanbe H. and Matsumoto J. (1993). Fundamental studies on hydrogen production in the acid-forming phase and its bacteria in anaerobic treatment processes－The effects of solids retention time. Wat. Sci. Tech., Vol. 28, pp. 81-88.
Nandi R. and Sengupta S. (1998). Microbial-Production of Hydrogen - An Overview, Critical Reviews in Microbiol., Vol. 24, No. 1, pp. 61-84.
Onodera H., Miyahara T. and Noike T. (1999). Influence of ammonia concentration on hydrogen transformation of sucrose. Asian Waterqual '99, 7th IAWQ Asia-Pacific Regional Conference, Vol. 2, pp. 1139-1144.
Podesta J. J., Gutierrez-Navarro A. M., Estrella C. N. and Esteso M. A. (1997). Electrochemical measurement of trace concentrations of biological hydrogen produced by Enterobacteriaceae. Res. Microbiol., Vol. 148, pp. 87-93.
Rachman M. A., Nakashimada Y., Kakizono T. and Nishio N. (1998). Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor. Appl. Microbiol. Biotechnol., Vol. 49, pp. 450-454.
Reimann A., Biebl H. and Deckwer W. D. (1996). Influence of iron, phosphate and methyl viologen on glycerol fermentation of Clostridium-butyricum. Appl. Microbiol. Biot., Vol. 45, No. 1-2, pp. 47-50.
Ren N., Wang B. and Huang J. C. (1997). Ethanol-type fermentation from carbohydrate in high rate acidogenic reactor. Biotech. Bioeng., Vol. 54, pp. 428-433.
Shuler M. L. and Kargi F. (1992) Bioprocess engineering, Prentice-Hall International, Inc, Englewood Cliffs, New Jersey.
Sparling R., Risbey D. and Poggi-Varalldo H. M. (1997). Hydrogen production from inhibited anaerobic composters. Int. J. Hydrogen Energy, Vol. 22, No. 6, pp. 563-566.
Stronach S. M., Lester J. N. and Rudd T. (1986). The influence of variable organic loading on the start-up of an anaerobic fluidized-bed reactor. Biotechnol. Lett., Vol. 8, No. 7, pp. 521-524.
Stronach S. M., Rudd T. and Lester J. N. (1986). Anaerobic Digestion Processes in Industrial Wastewater Treatment, Springer-Verlag, Berlin, Germany.
Taguchi F., Chang J. D., Takiguchi S. and Morimoto M. (1992). Efficient hydrogen production from starch by a bacterium isolated from termites. J. Ferment. Bioeng., Vol. 73, pp. 244-245.
Taguchi F., Mizukami N., Hasegawa K. and Saitotaki T. (1994). Microbial Conversion of Arabinose and Xylose to Hydrogen by a Newly Isolated Clostridium Sp No-2. Can. J. Microbiol., Vol. 40, No. 3, pp. 228-233.
Taguchi F., Mizukami N., Taki T. S. and Hasegawa K. (1995a). Hydrogen Production from Continuous Fermentation of Xylose During Growth of Clostridium Sp. Strain No-2. Can. J. Microbiol., Vol. 41, No. 6, pp. 536-540.
Taguchi F., Mizukami N., Yamada K., Hasegawa K. and Taki-saito T. (1995b). Direct conversion of cellulosic materials to hydrogen by Clostridium sp. Strain No. 2. Enzyme Microb. Tech., Vol. 17, pp. 147-150.
Taguchi F., Yamada K., Hasegawa K., Taki-saito T. and Hara K. (1996). Continuous hydrogen production by Clostridium sp. Strain No. 2 from cellulose hydrolysate in an aqueous two-phase system. J. Ferment. Bioeng., Vol. 82, No. 1, pp. 80-83.
Ueno Y., Kawai T., Sato S., Otsuka S. and Morimoto M. (1995). Biological production of hydrogen from cellulose by natural anaerobic microflora. J. Ferment. Bioeng., Vol. 79, No. 4, pp. 395-397.
Ueno Y., Otsuka S. and Morimoto M. (1996). Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture. J. Ferment. Bioeng., Vol. 82, No. 2, pp. 194-197.
Vieth W. R. (1994) Bioprocess Engineering, John Wiley & Sons, Inc., pp. 127-129.
White D. (1995). The Physiology and Biochemistry of Prokaryotes. Oxford University Press, Inc., New York.
Wiseman A. (1987). Handbook of Enzyme Biotechnology, Halsted Press, New York.
Yan R. T., Zhu C. X., Golemboski C. and Chen J. S. (1988). Expression of solvent-forming enzymes and onset of solvent production in batch culture of Clostridium butyricum. Appl. Environ. Microbiol., Vol. 54, pp. 642-648.
Yokoi H., Maeda Y., Hirose J., Hayashi S. and Takasaki Y. (1997a). H2 production by immobilized cells of Clostridium butyricum on porous glass beads. Biotechnol. Tech., Vol. 11, No. 6, pp. 431-433.
Yokoi H., Tokushige T., Hirose J., Hayashi S. and Takasaki Y. (1997b). Hydrogen production by immobilized cells of aciduric Enterobacter aerogenes strain HO-39. J. Ferment. Bioeng., Vol. 83, No. 5, pp. 481-484.
Zoetemeyer R. J., Van Den Heuvel J. C. and Cohen A. (1982). pH influence on the anaerobic dissimilation of glucose in an anaerobic digestor. Wat. Res., Vol. 16, pp. 303-311.