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Microbial community analyses of Municipal Solids Waste landfill site soils by 16S rRNA molecular approach

(分子生物学的手法による廃棄物埋立地土壌での温暖化ガス放出に関わる微生物生態解析)

氏名 陳 安成
学位の種類 博士(工学)
学位記番号 博甲第277号
学位授与の日付 平成15年6月30日
学位論文題目 Microbial community analyses of Municipal Solids Waste landfill site soils by 16S rRNA molecular approach (分子生物学的手法による廃棄物埋立地土壌での温暖化ガス放出に関わる微生物生態解析)
論文審査委員
 主査 教授 原田 秀樹
 副査 教授 山田 良平
 副査 助教授 大橋 昌良
 副査 助教授 小松 俊哉
 副査 長岡工業高等専門学校 荒木 信夫

平成15(2003)年度博士論文題名一覧] [博士論文題名一覧]に戻る.

CONTENTS

Chapter1 INTRODUCTION p.1

Chapter2 LITERATURE REVIEW p.4

Chapter3 EXTERNAL NITRATE AND SULFATE USED AS ELECTRON ACCEPTORS IN MUNICIPAL SOLID WASTE BIODEGERADATION p.37

Chapter4 ACETATE SYNTHESIS FROM H2/CO2 INSIMYLATED AND ACTUAL LANDFLL SAMPLES p.55

Chapter5 ARCHAEAL COMMUNITY COMPOSIIONS AT DIFFERENT DEPTH (UP 30m) OF A MUNICIPAL SOLID WASTE LADFILL IN TAIWAN AS REVELED BY 16s rDNA CLONING ANALYSES p.72

Chapter6 CONCLUSIONS AND RECOMMENDATIONS p.105

Landfill is an ultimate disposal alternative and forms a significant part of MSW management. Landfill stabilization is a dynamic, microbially mediated process, which is primarily influenced by waste characteristics, availability of moisture and nutrients, and prevailing operation circumstances.
A complex series of biological and chemical reactions begins with the burial of refuse in a landfill, representing an active anaerobic ecosystem. To understand the establishment and maintenance of anaerobic communities in landfills is crucial to improve leachate quality and methane production, and thereby to reduce the impacts on environment. Development of these technologies necessitates a comprehensive understanding of the microbiology and biochemistry of refuse biodegradation.
The objectives of this research are (1) to investigate the bioprocess associated with methanogenesis and (2) to identify the specific microorganisms and their diversity involved in a landfill ecosystem by 16S rRNA molecular approach. The results of this research could be served to further study for understanding the establishment and maintenance of anaerobic communities in landfills as well as for improving leachate quality and methane production, and thereby to reduce the impacts on environment. The results and discussion of the experiment through chapter three to five are described as follows:
In Chapter 3, the simulated MSW were used to evaluate the influence of external electron acceptors, such as nitrate and sulfate in the biodegradation of MWS. We can draw the picture that the common characteristic of denitrification in solid waste biodegradation with external nitrate was that nitrous oxide produced and accumulated associated with nitrite accumulated, subsequently, as long as both of nitrous oxide and nitrite were decreased, nitrogen gas produced. This phenomenon is an adverse influence to apply the denitrification in biodegradation of solid waste. Thus, the proposed process that the leachate treated by aerobic aeration was recycled to landfill will face the serious problem of nitrous oxide. We can conclude (1) the nitrate reduction was not denitrification but it could be dissimilation nitrate reduction to ammonia (DNRA). (2) Nitrite was a key intermediate, which was increased then decreased in the denitrification process of the substrate of glucose, H2/CO2, and acetate in the buffer suspension experiment by using the nitrate as the electron acceptor. (3) Nitrous oxide was an important gas associated with nitrite concentration in the experiment. It was produced and consumed in the denitrification process. Denitrifiation could not be completed unless nitrous oxide was consumed up. In terms of environment impact, the DNRA and nitrous dioxide were adverse influence.
In Chapter 4, H2 produced and consumed in a landfill under anaerobic conditions, the traditional concept indicated that the consumed H2 would be converted to CH4. However, we found that the homoacetogenesis reaction was the significant process in the landfill for hydrogen utilization. The relationship between hydrogen utilization and acetate formation was constructed by the modified biological methane potential experiment. The consumption of supplemental H2 by landfill samples in suspension culture was concomitant with the significant acetate synthesis. The homoacetogenesis reaction was validated by that 4.01~5.43 moles of hydrogen was consumed per mole acetate synthesis in simulated as well as actual samples excavated from three full-scale landfills in the north, middle and south of Taiwan. The fact that homoacetogens outcompeted the methanogens in the landfill sample could not only be explained by the thermodynamic calculation. The metabolic versatility of homoacetogens could be the main advantage for their success in a natural anaerobic environment.
 To our best knowledge, this is the first paper reported that acetate synthesis from H2/CO2 in simulated and full-scale landfill samples.
In Chapter 5, the identification of methanogens' diversity in landfill was conducted by molecular approach, including DNA extract, polymerase chain reaction (PCR), cloning, DNA sequence, and phylogenetic tree analysis as well as terminal restriction fragment length patterns. Archaeal 16S rDNA clone libraries were constructed for samples taken at 10-, 20- and 30-m depth in a landfill, which corresponded approximately 3-, 6- and 9-years operation, respectively. Sequencing and phylogenetic analyses of representative clones showed that all of the rDNAs were closely related to typical methanogens known to date. The distributions of phylotypes in clone libraries were similar to each other. Dominant clones in all the clone libraries were closely related to thermophilic species, such as Methanothermobacter thermautotrophicus, suggesting that the temperatures at these sites were high. This was supported by the results of H2-dependent methanogenic activity tests, showing that the activities of all samples at 55°C were much higher than those at 25°C. We found relatively stable community structure from 3-9 years after burial. In addition, temperature is an important factor that improves the ability of homoacetogens to compete successfully against methanogens for hydrogen. it obvious that although homoacetogenesis was predominate at 25℃, homoacetogens cannot compete methanogens for hydrogen at 55℃ in the deep layers (> 10m-depth) of the landfill. It was proposed that temperature affected the archaea community structure in the landfill samples, demonstrated by a shift of acetotrophic to hydrogenotrophic methanogens with increasing temperature from 25℃ to 55℃ in this study. To our knowledge, this work is the first report describing the genetic diversity of Archaea in the deep layers of a MSW landfill.

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