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Adhesion and aggreation mechanisms of mathanogenic sludge consorrtia developedin UASB reactors

(UASBリアクターで形成されたメタン生成混合微生物集団の自己固定化機構の解明)

氏名 曽 怡禎
学位の種類 博士(工学)
学位記番号 博甲第112号
学位授与の日付 平成7年3月24日
学位論文の題目 Adhesion and aggregation mechanisms of methanogenic sludge consortia developed in UASB reactors(UASBリアクターで形成されたメタン生成混合微生物集団の自己固定化機構の解明)
論文審査委員
 主査 助教授 原田 秀樹
 副査 教授 桃井 清至
 副査 教授 森川 康
 副査 東北大学 教授 野池 達也
 副査 東京大学 助教授 滝沢 智

平成6(1994)年度博士論文題名一覧] [博士論文題名一覧]に戻る.

TABLE OF CONTENTS
Chapter
Title Page p.i
Abstract p.ii
Acknowedgements p.iii
Table of contents p.iv
List of Tables p.viii
List of Figures p.ix
Key to Abbreviation p.xii
1.INTRODUCTION p.1
2.GENERAL LITERATUREREVIEW p.2
2.1 Overview of anaerobic degradation process p.2
2.2 The methanogenic bacteria p.2
2.3 Obligate proton-reducing acetogens p.3
2.4 Sulfate-reducing bacteria p.4
2.5 Competition between sulfate reducing bacteria and methanogens p.4
2.6 Concept of granulation p.5
3.BEHAVIOR OF EXTRA CELLULAR BIOPOLYMER AND METHANOGENIC ACTIVITY OF GRANULER SLUDGE GROWN IN AN UASB REACTOR p.13
3.1 INTRODUCTION p.13
3.2 MATERIALS AND METHODS p.15
3.2.1 Experimental apparatus p.15
3.2.2 Extraction of extracelluar polymer p.16
3.2.3 Methanogenic activity measurement p.16
3.2.4 Element composition of granular sludge p.17
3.2.5 Preparation of scanning electron microscopy p.17
3.2.6 Bacterial enumeration p.17
3.3 RESULTS p.19
3.3.1 Performance of the reactor p.19
3.3.2 Distribution of granular size p.21
3.3.3 The yield of extracted extracelluar polymer (ECP) p.21
3.3.4 Methanogenic specific activity p.23
3.3.5 Microbial composition of granules p.26
3.3.6 The mineral composition of the granules p.26
3.3.7 Observation of granules p.26
3.4 DISCUSSION p.31
4.MICROBIAL COMPOSITTION AND CHARACTERIZATION OF PREVALENT SPECIES IN GRANULAR SLUDGE p.33
4A: Characterization of MethanogenicBacteria p.33
4A.1 INTRODUCTION p.33
4A.2 MATERIALS AND METHODS p.36
4A.2.1 Sample collection and handling p.36
4A.2.2 Culture and growth condition p.36
4A.2.3 Isolation and cultivation ofMethanosarcina strain p.36
4A.2.4 Isolation and cultivation ofMethanothrix strain p.37
4A.2.5 Isolation and cultivation ofhydrogen-utilizing methanogens p.37
4A.2.6 Purification check p.38
4A.2.7 Disaggregation of strain MiAA under low and high calcium condition p.38
4A.2.8 Product and substrate determination p.38
4A.3 RESULTS p.39
4A3.1 Isolation of methanogenic bacteria from UASB reactor p.39
4A3.2 Isolation and characterizationof Methanosarcina mazei strain MiAA p.40
4A.3.3 Disaggregation characteristics of strain MiAA p.40
4A.3.4 Effect of calcium and acetate concentration on disaggregation p.46
4A3.5 Characteristics of Methanothrix strain MiAT p.46
4A.3.6 Characteristics of ethanobrevibacter strain MiHM p.48
4A.4 DISCUSSIONS p.49
4B: Characterization of Acetate and Propionate Degradation Sulfate-Reducing Bacteria p.51
4B.1 INTRODUCTION p.51
4B.1.1 Role of sulfate-reducing bacteria p.51
4B.1.2 Metabolism characteristics ofDesulfobulbus p.51
4B.1.3 Metabolism characteristics of Desulfobibrio p.53
4B.1.4 Metabolism characteristics of Desulfotomaculum p.54
4B.2 MATERIALS AND METHODS p.55
4B.2.1 Culture medium and conditions of cultivation p.55
4B.2.2 Isolation of acetate and propionate-degradation SRB p.57
4B.2.3 Growth of sulfate-reducing bacteria p.56
4B.2.4 Chemical and other determinations p.56
4B.2.5 Determanation of biomass p.56
4B.3 RESULTS p.57
4B.3.1 Isolation and characterization of sulfate reducing bacteria p.57
4B.3.2 Isolation of propionate-degradation SRB p.57
4B.3.3 Characteristics of substrates utilization p.60
4B.3.4 Growth condition of propionate-degradation SRB p.60
4B.3.5 Degradation of propionate inthe presence of sulfate p.61
4B.3.6 Degradation of 1-propanol inthe presence of sulfate p.67
4B.3.7 Degradation of ethanol in the presence of sulfate p.67
4B.3.8 Degradation of ethanol in the absence of sulfate p.67
4B.3.9 Isolation of acetate-degradation SRB p.67
4B.3.10 Growth condition of acetatedegradation SRB p.70
4B.4 DISCUSSION p.72
5.HYDROPHOBICITIES OF METHANOGENS AND SULFATE-REDUCING BACTERIA ISOLATED FROM UASB REACTOR p.74
5.1 INTRODUCTION p.74
5.2 MATERIALS AND METHODS p.76
5.2.1 Organisms and culture conditions p.76
5.2.2 Assay procedure of bacteria adhesion to hydrocarbon (BATH) p.77
5.2.3 Assay procedure of hydrophobic interaction chromatography (HIC) p.77
5.2.4 Assessment of number of attached cells on glass p.77
5.2.5 The attachment of Methanosarcina strain MiAA onto p.77
Methanothrix strain MiAT p.78
5.3 RESULTS p.78
5.3.1 Bacteria adhesion to hydrocarbon (BATH) p.78
5.3.2 Hydrophobic interaction chromatography (HIC) p.78
5.3.3 The effect of growth cycle onthe attachment p.82
5.3.4 The attachment of Methanosarcina strain MiAA onto Methanothrix strain MiAT p.84
5.3.5 Coculture of Methanothrix strain MiAT, Methanosarcina strain MiAA and Methanobrevibacter strain MiHM p.88
5.4 DISCUSSIONS p.94
6.DEVELOPMENT OF ANAEROBIC GRANULARSLUDGE CONSORTIA p.96
6.1 INTRODUCTION p.96
6.2 MATERIALS AND METHODS p.98
6.2.1 Apparatus and sampling p.98
6.2.2 Enumeration of methanogens and sulfate reducing bacteria p.100
6.2.3 Extracellular polymer (ECP) analysis p.101
6.2.4 Microscopic examination p.101
6.3 RESULTS p.102
6.3.1 Morphological observation of biofilm development p.102
6.3.2 Morphological observation of sludge retained in UASB p.104
6.3.3 Variation in biomass and ECP contents p.109
6.3.4 Behavior of the viable cell number p.112
6.3.4-A Methanogens (MPB) and propionate-degrading acetogen (PDA) p.112
6.3.4-B Sulfate-reducing bacteria (SRB) p.112
6.4 DISCUSSION p.115
7.POSSIBLE MECHANISM of GRANULATIONGOVERNED BY CTERIAL ATTACHMENT p.117
7.1 INTRODUCTION p.117
7.2 MATERIALS AND METHODS p.119
7.3 RESULTS p.120
7.3.1 Surface of the granule p.120
7.3.2 Exterior layer of the granule p.124
7.3.3 Middle layer of granule p.124
7.3.4 Central layer of granule p.126
7.3.5 The construction of granule p.128
7.4 DISCUSSIONS p.130
8.SUMMARY AND CONCLUSIONS p.133
LIST OF REFERENCES p.136
LIST OF TABLES
Table Page
2.1 Methanogenic bacteria in anaerobic ecosystems p.7
2.2 Syntrophic hydrogen producing acetogenic bacteria p.8
2.3 Redox half-reactions responsible for anaerobic microbial conversionof selected substrates p.9
2.4 Compounds that can be used as substrates for dissimilatory sulfate reduction p.10
2.5 Early taxonmic organization of sulfate-reducing bacteria according to the scheme of postgate and Campbell p.11
2.6 Characteristics of some contemporary sulfate-reducing bacterial genera p.11
2.7 Fermentative processes carried out by sulfate-reducing bacteria p.12
2.8 H2 metabolism on lactate-sulfate medium by Desulfovibrio species p.12
3.1 Performance of laboratory UASB reactor p.19
3.2 The bacterial counts of the grnular sludgrs p.29
3.3 The major elements composition of the granular sludges p.29
3.4 Comparsion of polymer yields ofgranular sludge extracted with thermal extraction method p.31
4A.1 Methanogens isolated from UASBreactor p.39
4A.2 General properties of Methanosarcina strain p.39
4A.3 General properties of Methanothrix strain p.40
4A.4 General properties of hydrogentrophic methanogens p.40
4A.5 Comparsion of Methanobrevibacter species p.48
4B.1 Isolation of acetate and propionate degradation sulfate reducing bacteria (A-SRB and P-SRB) from UASB reactor p.57
4B.2 Morphological and physiological characteristics of propionate oxidation sulfate resuction isolated andenrichment from UASB reactor p.59
4B.3 Compounds test as electron dornors and carbon source with 20 mM sulfate p.59
4B.4 Compoundsutilization by Desulfobulbus strainsin the absence of sulfate p.59
4B.5 Comparative characteristics ofthe genus Desulfobulbus, strain MiPS, SSPS, and SSPS. p.61
4B.6 Comparative characteristics ofspecies of the genus Desulfoto-maculum, strain suAS and SSAS p.70
5.1 Relative hydrophobicity of bacteria determined by adherence to hydrocarbon test p.79
5.2 Relative hydrophobicity of bacteria determined by bydrophobic interaction chromatography p.81
5.3 The hydrophobicity of isolates from UASB reactor p.94
7.1 Comparison of granular structure models p.130
LIST OF FIGURES
Figure Page
2.1 Degradation pathways in anaerobic digestion process p.6
3.1 Profiles of COD along reactor height at different loading rates p.20
3.2 Profiles of MLSS along reactor height at different loading rates p.20
3.3 Distribution of granular size at different loading rares p.22
3.4 The changes in the yield of extracellular polymer along reaJ喇 0 ASCII data connection for K101-28A.sjis (133.44.10.4,1874).ITED 1993 ctor height p.25
3.8 Dependence of methanogenic activity upon granular size, A and B p.27
3.9 The changes of Methanothrix like-filaments with the increasing of loading rates p.30

4A.1 Fluirescent photomicrograph.Various stages in the life cycle of Methanosarcina strain MiAA A, B, C and D p.41
4A.2 Production of methane and uronic acid during growth of strain MiAAon HAHCa medium inoculated with aggregate cells p.44
4A.3 Production of methane and uronic acid during growth of strain MiAAon HALCa medium inoculated with aggregate cells p.44
4A.4 Production of methane during growth of strain MiAA on HAHCa and HALCa medium inoculated with aggregateand coccoid cells p.45
4A.5 Production of methane during growth of strain MiAA on MAHCa and MALCa medium inoculated with aggregateand coccoid cells p.45
4B.1 Microphotograph of propionate-oxidzing sulfate-reduction bacteria p.58
4B.2 Degradation of propionate by strain SuPS with 25 mM sulfate p.62
4B.3 Degradation of propionate by strain SSPS with 25 mM sulfate p.62
4B.4 Degradation of propionate by strain MiPS with 25 mM sulfate p.62
4B.5 Degradation of propionate by strain SuPS, SSPS, and MiPS with 25 mM sulfate p.63
4B.6 Growth of strain SuPS, SSPS, andMiPS during oxidation of propionatewith 25 mM sulfate p.63
4B.7 Degradation of I-propanol by strain SuPS, SSPS, and MiPS with 25 mM sulfate p.64
4B.8 Degradation of ethanol by strain SuPS with 25 mM sulfate p.65
4B.9 Degradation of ethanol by strain SSPS with 25 mM sulfate p.65
4B.10 Degradation of ethanol by strain MiPS with 25 mM sulfate p.65
4B.11 Degradation of ethanol by strain SuPS, SSPS, and MiPS with 25 mM sulfate p.66
4B.12 Degradation of ethanol by strain SuPS in the absence of sulfate p.68
4B.13 Degradation of ethanol by strain SSPS in the absence of sulfate p.68
4B.14 Degradation of ethanol by strain MiPs in the absence of sulfate p.68
4B.15 The changes of ratio of propionate to acetate during fermenta-tion of ethanol by strain SuPS, SSPS, and MiPPPS p.69
4B.16 Microphotograph of acetate degradation sulfate reducing bacteria p.70
4B.17 Degradation of acetate by strain SuAS and SSAS with 25 mM sulfate p.71
4B.18 Degradation of acetate by strain SSAS and SSAS with 25 mM sulfate p.71
5.1A Microphotograph showing strainMiAT cells adhering to hexadceane following mixing p.80
5.1B Microphotograph showing strainMiHM cells adhering to hexadceane following mixing p.80
5.2 The relationship between growthphase of strain MiAT and the numberof cells which attached on the glass after 4h attachment period p.83
5.3 The relationship between growthphase of strain MiHM and the number of cells which attached on the glass after 4h attachment period p.83
5.4 The relationship between growthphase of strain MiAA and the number of cells which attached on the glass after 4h attachment period p.84
5.5 The relationship between growthphase of strain MiPS and the number of cells which attached on the glass after 4h attachment period p.85
5.6 The relationship between growthphase of strain SSAS and the number of cells which attached on the glass after 4h p.85
5.7 Degradation of acetate by strain MiAT, MiAA, and coculture of MiAT and MiAA on acetate medium with 2.5 mMcalcium p.87
5.8A The appearance of fluoresence on the strain MiAT filament attachedby the coccoid cells p.89
5.8B Microphotograph showing the attachment of strain MiAA coccoid cells onto filaments of strain MiAT p.89
5.9A The conjuction of strain MiAA with calcium-phosphate complex p.90
5.9B Microphotograph showing the conjuction of calcium-phosphate complex with strain MiAA cells p.90
5.10A Florescenct photomicrograph showing only strain MiAA fluoresence was observed p.91
5.10B Microphotograph showing the conjuction of strain MiAT and MiAA combined together with the calcium-phosphate complex p.91
5.11A Fluorescent photomicrograph showing the appearance of storng fluoresence from strain MiHM surrounded the conjuction of strain MiAA and calcium-phosphate complex p.92
5.11B Microphotograph showing the conjuction of strain MiAT and MiHM with calcium-phosphate complex p.92
5.12A Floresence microphotograph showing the tri-culture of strain MiAA, MiAT, and MiHM p.93
5.12B The aggragates formed by 3 strains were combined together with calcium-phosphate complex p.93
5.12C The appearance of strain MiATfilaments after pressing the aggregates p.93
6.1 Experiment set-up. p.99
6.2 SEM observation of the progression of biofilm formation A, B, C and D p.103
6.3 Characteristics structure of each stage during biofilm development A, B, C, and D p.105
6.4 Optical micrographs of aggregate struture developed on 90 th day A and B p.106
6.5 Photograph of biofilm taken outfrom the reactor on 120th day A andB p.107
6.6 SEM observations of sludge retained in the UASB reactor A, B, C, and D p.108
6.7 The increment of biomass duringbiofim development p.110
6.8 The variation of the protein and carbohydrate contents in the polymer and the alternation of the ratio of carbohydrate and protein during biofilm development p.111
6.9 The adherence of methanogens and propionate degradation bacteria during biofilm development p.113
6.10 Changes in the lactate, propionate and acetate degradation sulfate reducing bacteria during biofilm development p.114
7.1 The filamentous orgaisms were the predominant population on the surface of granule p.121
7.2 The funne-like opening structure on the surface of granule p.122
7.3 The SEM photograph of granule with funnel-like pore p.122
7.4 The structure of the cross section of a granule with 3 distin-guished area : the outer lamella-like layer, the middle plain layer,and the central cavity layer p.122
7.5 The outer layer of the granule cross section A, B and C p.123
7.6 The cross section of the middlelayer of the granule A, B, and C p.125
7.7 The centrallayer of the cross section of the granule A, B and C p.127
7.8 Microphotograph of the cross section of granule revealed layered-structure, A-F p.129
7.9 A poaaible model of granulation p.131

 近年、UASB(Upflow Anaerobic Sludge Blanket)や固定床、流動床法等の反応器形式による高速メタン発酵型の嫌気性排水処理プロセスが開発され、中、高濃度の有機性産業廃水への適用が活発に進展している。これらの反応器形式はいずれも嫌気性微生物群の自己固定化(Self-immbilization)機能を利用し、生物膜やグラニュール状の微生物集塊(Microbial-aggregates)増殖体を形成させ、反応器内に高濃度の生物量を保持させて、その結果従来の浮遊増殖型反応器よりも飛躍的に高負荷・高速の処理性能を可能にしている。しかしながら、嫌気性自己固定型反応器のこれまでの研究は、多分にブラックボックス的取り扱いで処理機能のみが論じられ、その技術開発も経験工学的ステップを踏んできたと言える。嫌気性生物膜法の本質的機能を適切に把握し、さらに新たな技術革新を進め、より信頼性、安定性、融通性のある処理技術に発展させるためには、生物膜・造粒化(グラニューレーション)形成=細胞アグリゲーションメカニズムを微生物学的視点から解明する必要がある。
 本研究は(1)グラニュール状微生物コーンソーシアの微細構造の形態学的特性把握、(2)グラニュール状微生物コーンソーシアを構成する菌学的組成の把握とそれらの生態学的相互作用の解明、(3)グラニュールからの主要菌種の単離とそれらの細胞表層特性と付着能の関係の把握、(4)グラニュレーション過程での主要菌種の初期付着現象とアグリゲート形成現象の主な菌種の役割の把握等の課題について、以下の第3章から第7章まで構成される実験検討を行ったものである。
 第3章では、UASB反応器の長期連続実験によって、反応器の異なる高さから採取したグラニユール生物体をサイズにより分級し、細胞外ポリマー(ECP)成分とメタン生成活性に及ぼす有機物負荷(流入有機物強度)の影響を検討した。この結果、ECPの(タンパク質/炭水化物)比は、ベッド上部にいくに従い増加傾向を示し、グラニュール径の増大に伴い減少する傾向を示し、グラニュレーションにはECP中の炭水化物画分がより緊密に関与していることが明らかになった。また、グラニュール体の水素利用メタン生成活性は、グラニュール径が増大するに従い低下し、基質の拡散移動抵抗が存在した。酢酸利用メタン生成活性及びプロピオン酸投与メタン生成活性は、いずれの負荷条件でもグラニュール径が中位(1~2.5mm)のものが最も大きな活性を示したことから、グラニュール径の過度の肥大化は基質拡散抵抗が増大する結果不利になり、最適径が存在することが判明した。
 第4章では、いくつかのUASB反応器で形成されたグラニュール・コンソーシアを構成する主要な酢酸資化性メタン生成菌として、3株のMethanothrixと5株のMethanosarcina、水素資化性メタン生成菌として4株のMethanobrevibacter、及び硫酸還元菌として3株のDesulfobulbus(プロピオン酸分解硫酸還元菌)、2株のDesulfotomaculum(酢酸分解硫酸還元菌)の単離に成功し、それらの生理学的キャラクタリゼーションを行った。特に、Methanosarcina属のメタン生成菌は特異的なライフ・サイクルを呈し、シストからCocoidへのDisaggregation過程は培地のCaイオン濃度と酢酸濃度に依存し、ウロン酸の放出を伴うことが明らかになった。
 第5章では、前章で単離したメタン生成菌と硫酸還元菌に関して、異なる増殖フェイズにおける細胞表層構造と付着能の関係を、BATHテスト(Bacteria Adhesion toHydrocarbon)、HICテスト(Hydrophobic Interaction Chromatography)によって検討した。この結果、Methanothrix sp.とMethanobrevibacter sp.の細胞表層はいずれの増殖フェイズでも高い疏水性を示した。一方Methanosarcina sp.はいずれの増殖相でも高い親水性を示したが、その付着能は定常増殖相においてのみ培地中のCaイオン濃度にしたがって増加した。Desulfobulbus sp.は初期対数増殖相から定常増殖相へ移行するにつれて細胞表層の疏水性が増加し、その傾向は硫酸塩が存在しない発酵型式の方がより顕著であった。さらに、各単離菌株の増殖過程でのガラス担体への付着菌数測定によって、細胞表層の疏水性が高いほど付着能が高いことが判明した。
 第6章では、UASB反応器中に装着した付着担体への嫌気性微生物群の付着・生物膜形成過程における形態学的観察、細胞外ポリマーの挙動及び各栄養菌群の付着細胞菌数の推移を追跡した。その結果、嫌気性微生物の付着・凝集過程は4段階の継起プロセス(Adhesion、Clump形成、Conglomerate形成、およびAggregate形成過程)によって特徴付けられることが判明し、嫌気性微生物の生物膜形成とグラニュレーション機構の普遍的なアナロジーを明らかにした。
 第7章では、UASB反応器から得られた異なる増殖ステージにあるグラニュール・コンソシアの微細構造を比較検討し、グラニュールの3層構造の形態学的特性と微生物生態学的構造を明らかにした。さらに、各章で得られた知見を総合して嫌気性微生物の付着・凝集・集塊化増殖過程を説明するモデルを提示し、グラニュレーション=細胞自己固定化の生物学的機構とその意義を解明した。

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