氏名　邱　艶玲
学位の種類　博士（工学）
学位記番号　博甲第310号
学位授与の日付　平成16年3月25日
学位論文題目　Cultivation and Dectection of Anaerobic Syntrophic Phthalate Isomers-and Benzoate-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephalate Manufacturing　（テレフタル酸含有廃水嫌気性処理プロセスに存在するフタル酸類・安息香酸を分解する微生物の培養と検出）
論文審査委員
　主査　教授　丸山　暉彦
　副査　助教授　宮木　康幸
　副査　助教授　高橋　修
　副査　助教授　佐野　可寸志
　副査　東北大学大学院情報　科学研究科　助教授　武山　泰

• 論文目次
• 論文要旨

［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

Abstract　p.i

Chapter1 General Introduction　p.1-1
1.1 Background　p.1-1
1.2 Aims of the study　p.1-2
1.3 Literature review　p.1-3
1.4 Outline of the thesis　p.1-12
Reference　p.1-13

Chapter2 Identification and isolation of asaerobic,syntrophic phthalate isomer-degrading microbes　p.2-1
2.1 Introduction　p.2-2
2.2 Materials and methods　p.2-3
2.3 Results　p.2-8
2.4 Discussion　p.2-19
2.5 Conclusion　p.2-24
Reference　p.2-25

chapter3 Isolation and characterization of two anaerobic,syntrophic phthalate isomer-oxidizers within the genus of Pelotomaculum　p.3-1
3.1 Introduction　p.3-2
3.2 Materials and methods　p.3-3
3.3 Results and discussion　p.3-6
Reference　p.3-17

Chaoter4 Isolation of anaerobic,syntrophic Phthalate isomer-oxidizer in group TA of delta-Proteobacteria　p.4-1
4.1 Introduction　p.4-1
4.2 Materials and methods　p.4-3
4.3 Results and discussion　p.4-5
Reference　p.4-8

Chapter5 Insolation of a novel lineage of anaerobic,syntrophic benzoate-degrading bacterium　p.5-1
5.1 Interoduction　p.5-1
5.2 Materials and methods　p.5-2
5.3 Results　p.5-5
5.4 discussion　p.5-10
Reference　p.5-13

Chapter6 In situ detection of phthate isomer and benzoate-degrading microbes　p.6-1
6.1 Introduction　p.6-1
6.2 Materials and methods　p.6-2
6.3 Results and discussion　p.6-4
Reference　p.6-6

Chapter7 Summary　p.7-1
Publication list　p.ii
Acknoeledgement　p.iii

Phthalate isomers are primarily anthropogenic compounds, have been produced in massive amounts for use in manufacturing plastic bottles, polyester fibers, and other petroleum-based products in the world, and are consequently eluted in the wastewater generated in the corresponding industries. From economical and energetic aspects, anaerobic （methanogenic） treatment technologies are increasingly applied to treat those complex wastewaters. However, all laboratory-, pilot-, and full-scale engineering studies have demonstrated that the processes require a long start-up time （lag phase） for the removal of phthalate isomers, and that the removal is often stagnated during the treatment; these have led to the difficulties to stably operate such processes. Since these phenomena are highly attributed to the microbial populations responsible for the degradation of phthalate isomers under methanogenic conditions, much attention has to be paid to the phthalate isomers degrading populations in those processes. However, so far no methanogenic phthalate isomers degrading microbes have been isolated. Subsequently their substrate specificities, biochemical pathways, nutritional requirements and functional roles are unknown. The thesis aimed for accumulating basic knowledge on phthalate isomers and its intermediate （i.e., benzoate） degrading microbes under methanogenic conditions.
The microorganisms associated with the degradation of anaerobic phthalate isomers and benzoate was investigated by using conventional cultivation techniques combined with molecular approaches. To isolate benzoate and phthalate isomer-degrading microbes, primary enrichments were made with ortho-phthalate, isophthalate, terephthalate and benzoate as the sole energy source at 37°C anaerobically with two different anaerobic sludges as inocula; both of the sludges had been treating real wastewaters containing high strengths of phthalate　isomers and benzoate. In total, six methanogenic phthalate isomer-degrading enrichment cultures were obtained, which degraded not only the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with the concomitant production of methane, presumably involving strictly syntrophic substrate-degradation. 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization （FISH） revealed that the predominant bacteria in the phthalate isomers enrichment cultures affiliated with a recently recognized non-sulfate-reducing subcluster in the group 'Desulfotomaculum lineage I', or a clone cluster （group TA） in the class delta-Proteobacteria. After several attempts three anaerobic, syntrophic phthalate isomer-degrading bacteria were isolated, including two species within genus Pelotomaculum of the group Desulfotomaculum lineage I and one in Group TA of the delta-Proteobacteria.
In the three benzoate-degrading cultures, the predominant population was found related to Syntrophus gentianae or Sporotomaculum hydroxybenzoicum. Since the genus Syntrophus as the benzoate-degrading syntrophs has been well studied, this study focus only on the isolation of the bacterium belonging to the genus Sporotomaculum. After several attempts the benzoate-degrader, designated strain FB, was obtained in pure culture with crotonate. In syntrophic co-culture with hydrogenotrophic methanogens, strain FB can metabolize benzoate. Phylogenetically, the strain belongs to the genus Sporotomaculum in the phylum Firmicutes, indicating that the isolate represents a second bacterial lineage of benzoate-degrading syntrophic bacteria.
In order to elucidate the abundance and spatial distribution of those cultured microbes in their original sludge granules, FISH technique combined with confocal laser scanning microscopy was applied to thin sections of the granules. Firstly several rRNA-targeted oligonucleotide probes specific for detection of benzoate and phthalate isomer-degrading microbes were designed. FISH analysis revealed that those microbes were abundantly present in the original sludge, suggesting that these microbes actually play significant roles in the removal of pollutants in terephthalate wastewater.
This thesis provides the first information on the microbes associated with mineralization of phthalate isomers under methanogenic conditions. In the future, the study of the isolated bacterial cultures might give important information about the metabolic capabilities of microorganisms and their potential function in their natural habitat.In addition, this research builds a convenient monitoring technology for quickly detect and quantify these specific phthalate and benzoate-degrading microbes in their wastewater treating process or polluted area.