利用豬糞作為原料探討高溫厭氧消化系統生產沼氣之微生物菌相

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張倚嘉(Yi-Chia Chang) 查詢紙本館藏

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利用豬糞作為原料探討高溫厭氧消化系統生產沼氣之微生物菌相
(Characterization of microbial communities from a thermophilic anaerobic digester running onswine manure)

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摘要(中)

厭氧消化槽為一廣泛使用於有機廢棄物之生物處理方法，如動物糞便或有機固態廢棄物皆可由此法處理並產生生質沼氣。沼氣是甲烷與二氧化碳的混合氣且為一種再生能源。厭氧消化槽產生沼氣的代謝途徑主要有三步驟:水解(hydrolysis)、產酸(acidogenesis)和產甲烷作用(methanogenesis)。本研究目的利用可培養及不可培養之方法，研究以豬糞為料源，探討在55°C高溫厭氧消化槽中微生物菌群結構及功能。根據16S rRNA 基因親緣演化樹分析結果顯示，以可培養微生物方式鑑定出細菌菌群包含95%厚壁菌門(Firmicutes) 和5%變形菌門(Proteobacteria)。以克隆庫(clone libraries)鑑定細菌菌群有54%變形菌門、38%無法培養之細菌及7%厚壁菌門；古生菌菌群有79%甲烷桿菌科(Methanobacteriaceae)、17%甲烷八疊球菌科(Methanosarcinaceae)及4%無法培養之古生菌。以denaturing gradient gel electrophoresis (DGGE)基因指紋方法鑑定13個優勢細菌帶其中含50%厚壁菌門、40%變形菌門及 10%無法培養之細菌; 7個優勢古生菌帶有14%甲烷桿菌科。其中在多樣性微生物中厚壁菌門和變形菌門負責分解豬糞中複雜的有機物質，並在水解和產酸作用中扮演重要角色。而甲烷桿菌科和甲烷八疊球菌科具有氫氣及二氧化碳轉化成甲烷的能力，另外甲烷八疊球菌科可將醋酸轉化成甲烷。本研究提供以豬糞作為唯一料源在高溫厭氧消化槽中生產沼氣之微生物菌相之訊息。

摘要(英)

Anaerobic digestion (AD) is a biological treatment using a wide range of organic wastes, such as animal manure and organic solid waste to produce biogas. Biogas, a mixture of methane and carbon dioxide, is a renewable source of energy. AD can be divided into three major stages: hydrolysis, acidogenesis and methanogenesis. This study was aimed to research structures and functions, using cultivation-dependent and cultivation-independent (clone library and DGGE) methods, of the microbial community in AD at 55°C using pig manure. Based on phylogenetic analysis of 16S rRNA genes, the isolated microbes include 95% Firmicutes, 5% Proteobacteria. The clone libraries indicated that the bacterial community was composed of 54% Proteobacteria, 38% unclassified bacteria and 7% Firmicute. The archaeal community was composed of 79% Methanobacteriaceae, 17% Methanosarcinaceae and 4% unclassified Euryarchaea. The denaturing gradient gel electrophoresis (DGGE) revealed 13 dominant bacterial bands that belonged to the bacteria phyla 50% Firmicute, 40% Proteobacteria and 10% unclassified bacteria. The 7 dominant archaeal bands were composed of 14% Methanobacteriaceae. The diverse species from the phyla Firmicutes and Proteobacteria play key roles in the hydrolysis and acidogenesis stages of degrading the complex organic matters in swine manure. Species from Methanobacteriaceae and Methanosarcinaceae are proved to be able of using H2 and CO2 to produce methane. In addition, Methanosarcinaceae species also use acetate as a substrate to produce methane. This study provides information on the microbial community in thermophilic AD using swine manure as the sole feedstock for the biosynthesis of biogas.

關鍵字(中)

★ 甲烷菌
★ 甲烷氣
★ 微生物菌相
★ 沼氣
★ 高溫厭氧消化槽
★ 豬糞

關鍵字(英)

★ methanogen
★ Biogas
★ pig manure
★ methane
★ microbial community
★ thermophilic anaerobic digestion

論文目次

中文摘要 ................................................................................................................................... I
Abstract ................................................................................................................................... II
Table of Contents ................................................................................................................... III
List of Figures ........................................................................................................................ VI
List of Tables ......................................................................................................................... VII
List of Abbreviations .......................................................................................................... VIII
Instrument .............................................................................................................................. IX
Chemicals ................................................................................................................................ X
Chapter 1: Introduction ........................................................................................................... 1
1.1 Sample Source ................................................................................................................. 1
1.2 Anaerobic Digestion ....................................................................................................... 1
1.2.1 Hydrolysis ................................................................................................................. 2
1.2.2 Acidogenesis ............................................................................................................. 2
1.2.3 Methanogenesis ........................................................................................................ 3
1.3 Microbial Community Distribution in the Environment and Anaerobic Digesters . 3
1.4 Biogas ............................................................................................................................... 5
1.4.1 Biogas Compositions ............................................................................................... 5
1.4.2 Applications and Potential of Biogas ..................................................................... 5
1.5 Applications of Microbial Techniques .......................................................................... 5
1.6 Molecular diversity analysis of environmental samples by using different primer sets ......................................................................................................................................... 7
IV
1.7 Research Aims ................................................................................................................ 9
1.8 Study Outline ................................................................................................................ 10
Chapter 2: Materials and Methods ....................................................................................... 11
2.1 Sample Source and the Operation of the Thermophilic Digester ............................ 11
2.2 Cultivation-Dependent of Microbial Isolation ........................................................... 11
2.2.1 Sample Preparation ............................................................................................... 11
2.2.2 Serial Dilutions ....................................................................................................... 12
2.2.3 Culture Medium Preparation ............................................................................... 12
2.2.3.1 Nutrient Agar (NA) Plate ............................................................................... 12
2.2.3.2 Nutrient Broth (NB) ....................................................................................... 12
2.2.4 Identification of Bacteria ...................................................................................... 12
2.2.4.1 Sequence and Phylogenetic Analysis of Culture Sample ............................ 12
2.3 Culture-Independent .................................................................................................... 13
2.3.1 DNA Extraction ..................................................................................................... 13
2.3.2 Polymerase Chain Reaction (PCR) Amplification of 16S rRNA ....................... 14
2.3.2.1 PCR Amplification of Bacteria V6-V8 16S rRNA ....................................... 14
2.3.2.2 PCR Amplification of Archaea V3-V5 16S rRNA ....................................... 14
2.3.3 PCR-DGGE analysis of Bacteria and Archaea Primer ...................................... 15
2.3.4 Cloning and Sequencing Analysis of PCR-DGGE Fragments .......................... 15
2.3.5 Cloning and Sequencing Analysis of Bacteria and Archaea of 16S rRNA ....... 16
Chapter 3: Results .................................................................................................................. 17
3.1 Performance of the Thermophilic Anaerobic Digester ............................................. 17
3.2 Analysis of the Cultivation-Dependent Bacterial Community ................................. 17
3.3 PCR-DGGE Analysis of the Dynamics of Thermophilic Anaerobic Digester using
V
Pig Manure as Feedstock Microbial Community ............................................................ 19
3.3.1 Analysis of Bacteria Community by DGGE ........................................................ 19
3.3.2 Analysis of Archaeal Community by DGGE ....................................................... 20
3.4 16S rRNA Gene Clone Library and Phylogenetic ..................................................... 21
3.4.1 16S rRNA Gene Clone Library of Bacteria Community ................................... 21
3.4.2 16S rRNA Gene Clone Library of Archaea community .................................... 22
Chapter4: Discussion .............................................................................................................. 23
4.1 Performance of the Thermophilic Anaerobic Digester ............................................. 24
4.2 The Bacteria Involved in Hydrolysis and Acidogenesis during the Thermphilic Anaerobic Digestion ........................................................................................................... 24
4.3 The Methanogenic Archaea Involved in Methanogenesis during the Thermophilic Anaerobic Digestion ................................................................................... 29
4.4 Metaproteomic Analysis of the Functional Microbial Community Present in a Thermophilic Anaerobic Digestion Running on Swine Manure .................................... 32
Chapter 5: Conclusion ............................................................................................................ 36
References: .............................................................................................................................. 38
Appendix Ⅰ. Microbial isolation using cultivation-dependent method and identification of 16S rRNA gene sequencing ........................................................................ 74
Appendix Ⅱ. Cultivation-independent method of DGGE analysis and identification of 16S rRNA gene sequencing .................................................................................................. 209
Appendix Ⅲ. Cultivation-independent method of Clone library analysis and identification of 16S rRNA gene sequencing ...................................................................... 214

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2012-8-27

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