氏名　Chuang Hui-Ping
学位の種類　博士（工学）
学位記番号　博甲第446号
学位授与の日付　平成19年12月31日
学位論文題目　Development of a Novel and Cost-Effective Nitrogen Removal Process by the Application of Down-Flow Hanging Sponge （DHS） Systems　（下降流懸架式スポンジリアクターによる新規低コスト型窒素除去プロセスの開発）
論文審査委員
　主査　准教授　小松　俊哉
　副査　准教授　山口　隆司
　副査　准教授　高橋　祥司
　副査　准教授　姫野　修司
　副査　准教授　湯川　高志
　副査　広島大学大学院工学研究科　教授　大橋　晶良

• 論文目次
• 論文要旨
• 論文審査の結果の要旨

［平成19（2007）年度博士論文題名一覧］ ［博士論文題名一覧］に戻る．

Chapter I　General outline and objectives
　1.1　Outline　p.I-2
　1.2　Ovjectives　p.I-3
Chapter II　Literature Review-Nitrogen removal technologies
　2.1　Nitrogen cycle　p.II-2
　2.2　Nitrification　p.II-3
　2.2.1 Autotrophic nitrification　p.II-3
　2.2.2 Heterotrophic nitrification　p.II-4
　2.2.3 Effect factors on nitrification　p.II-4
　2.3　Denitrification　p.II-9
　2.3.1 Respiratory denitrification　p.II-9
　2.3.2 Aerobic denitrification　p.II-10
　2.3.3 Lithoautotrophic denitrification　p.II-11
　2.3.4 Effect factors on denitrification　p.II-11
　2.4　Microbiology of nitrogen-related reactons　p.II-13
　2.4.1 Aerobic ammonia-oxidizing consortia　p.II-13
　2.4.2 Nitrite-oxidizing consortia　p.II-14
　2.4.3 Anaerobic ammnonia-oxidizing consortia　p.II-15
　2.4.4 Denitrifying consortia　p.II-17
　2.5　Nitrogen removal technologies　p.II-18
　2.5.1 Nitrification-denitrification process　p.II-18
　2.5.1 Nitrification-denitrification process　p.II-18
　2.5.2 Partial nitrification process　p.II-18
　2.5.3 SHARON process　p.II-19
　2.5.4 Anammox process　p.II-19
　2.5.5 Patial nitrification/SHARON-Anammox process　p.II-21
　2.5.6 CANON process　p.II-22
　2.5.7 NOx process　p.II-23
　2.5.8 OLAND process　p.II-24
　2.5.9 Deammonification process　p.II-24
　2.5.10 Comparison of nitrogen removal process　p.II-25
　2.6　Introducton of DHS reactor　p.II-27
　2.6.1 Characteristics of the DHS system　p.II-28
　2.6.2 Removal efficiencies in DHS systems　p.II-29
　2.6.3 Advantages of DHS system　p.II-30
　References　p.II-31
Chapter III　Performance and Microbial Community of Partial Nitrification to N itrite in a Down-Flow hanging Sponge （DHS） Reactor under oxygen-limited Conditio ns
　3.1　Introduction　p.III-2
　3.2　Materials and Methods　p.III-3
　3.2.1 Experimental apparatus　p.III-3
　3.2.2 Substrate and operation　p.III-3
　3.2.3 Analysis　p.III-4
　3.2.4 Constraction of bacterial, archaeal & ammonia oxidizing 16S rRNA and amoA genes clone libraries　p.III-5
　3.2.5 Clone-FISH　p.III-6
　3.2.6 Fluorescence in situ hybridization （FISH） analysis　p.III-7
　3.2.7 Nucleotide sequence accession numbers　p.III-7
　3.3　Result and discussion　p.III-8
　3.3.1 Performance of DHS reactor　p.III-8
　3.3.2 Effect of oxygen on nitrite production　p.III-15
　3.3.3 Effect of oxygen on nitrous oxide production　p.III-16
　3.3.4 Ammonium oxidation characteristics for different systems　p.III-17
　3.3.5 Microbial community catalyzes partial nitrification in a DH S　p.III-20
　3.3.6 Phylogenetic diversity of amoA genes in a DHS　p.III-25
　3.3.7 Spatial distribution of microbes in a DHS by fluorescence i n situ hybridization（FISH） analysis　p.III-26
　3.4　Conclusions　p.III-28
　References　p.III-29
Chapter IV　Performance and Microbial Community of Anoxic Ammonium Oxidation in a Dowm-Flow Hanging Sponge （DHS） Reactor
　4.1　Introduction　p.IV-2
　4.2　Material and Methods　p.IV-3
　4.2.1 Experimental apparatus　p.IV-3
　4.2.2 Substrate and operation　p.IV-4
　4.2.3 Analysis　p.IV-5
　4.2.4 Construction of bacterial, planctomycetes- and anammox-spec ific 16SrRNA clone libraries　p.IV-5
　4.2.5 Fluoresence in situ hybridization （FISH） analysis　p.IV-7
　4.3　Results and discussion　p.IV-7
　4.3.1 Performance of the DHS reactor　p.IV-7
　4.3.2 Occurrence of nitrogen removal　p.IV-12
　4.3.3 Sludge development in the DHS system　p.IV-15
　4.3.4 Nitrogen removal characteristic for different systems　p.IV-17
　4.3.5 Microbial community catalyzes anammox in a DHS　p.IV-19
　4.3.6 Spatial distribution of microbes in a DHS by fluorescence i n situ hybridization （FISH） analysis　p.IV-24
　4.4　Conclusions　p.VI-24
　References　p.VI-25
Chapter V　Combination of partial nitrification and anammox reaction （CANON） in a downflow hanging sponge （DHS） rector under oxygen limited conditions
　5.1　Introduction　p.V-2
　5.2　Material and Methods　p.V-3
　5.2.1 Experimental apparatus　p.V-3
　5.2.2 Substrate and operation　p.V-3
　5.2.3 Analysis　p.V-4
　5.3　Results and discussion　p.V-4
　5.3.1 Performance of DHS reactor　p.V-4
　5.3.2 Effect of oxygen on nitrite production adn nitrogen removal　p.V-11
　5.3.3 Effect of oxygen on nitrous oxide production　p.V-12
　5.3.4 Nitrogen removal characteristics for different systems　p.V-13
　5.4　Conclusions　p.V-14
　References　p.V-15
Chapter VI　Conclusions and recommendations for future research
　6.1　Conclusions　p.VI-2
　6.2　Recommendations for future reseach　p.VI-4

Nitrogen pollution has been the focus of more increased attention. The convent-ional process of nitrogen removal by nitrification and denitrification is expen-sive and requires considerable energy, due to needed aeration and carbon supply.Several newly biological nitrogen removal processes were developed in place of the former process. However, there were still lots of problems associated with the pervious developed reactor systems such as biomass washout or low applicablenitrogen loading. Inaddition, limitations inherent to these systems are the costof operation and maintenance due to high energy consumption. The down-flow hang-ing sponge（DHS） reactor has been successfully developed for sewage treatment incombination with an up-flow anaerobic sludge bed（UASB） reactor and recommended as a low-priced treatment system for removal of nitrogen.
Partial nitrification of ammonium to nitrite is an important primary process and it makes a suitable substrate for anammox reaction in a shortcut nitrogen removal process. Partial nitrification could be successfully achieved in a closed DHS reactor, with the maximum ammonium oxidation of 1.46kg NH4+-N･m-3・day-1. Oxygen contact in the gas-phase below 1% （0.42 mg･L-1 of DO） resulted in asatisfactory accumulation of nitrite instead of nitrate; however, it is also associated with the a N2O production. The biomass concentration of 3.84 g VSS/L showed a significantly higher nitrification activity of 0.12 kg NH4+-N･kg VS-1･d-1, which suggests that, besides the novelty of operational simplicity, the sys-tem has an adequate performance capability making it a potential system for par-tial nitrification. Furthermore, a complicated microbial community was observed in a DHS, but dominated by Nitrosomonas species for partial oxidation of ammoni-um to nitrite. Inaddition, Ammonium oxidizers and denitrifying bacteria were Bacteoridates groups were also detected by the population size of 13.0% and 10.6% of total clones, respectively. For FISH analysis, about 41% ammonia oxidi-zers and 5.4% nitrite oxidizing bacteria were detected in this system. Complica-ted microbial consortia were observed in this system supplied only ammonium wit-hout organic matter under strict low DO conditions.
Application of the down-flow hanging sponge （DHS） reactor for the anammox proc-ess is a new attempt to develop a low-priced nitrogen-removal process. Four ope-rational parameters were tested to estimate the suitability of anoxic ammonium oxidation with a DHS reactor. A significant increase in nitrogen removal was ac-hieved by raising the influent flow rate, which likely increased bacterial grow-th and effective substrate utilization. Anammox reaction coupled with nitrate reduction in a DHS could attain high nitrogen removal of up to 95%. We could co-ncluded that anoxic ammonium oxidation in a DHS reacter is useful for teating wastewater contained low nitrogen compounds. A complicated microbial community was observed in an anoxic DHS reacter and Comamonas populations by dominated in the 16S rRNA clone library for nitrate reduction. Candidatus Kuenenia sttugarti-ensis were mainly esponsible for anammox reaction under anoxic condition. Predo-minance of uncultured planctomycetes bacteria was numerous as determined in the planctomycetes-specific 16S rRNA gene-based clone library, showing some new-type anammox bacteria existed in this system. Moreover, FISH analysis showed that the phylum planctomycetes dominated in this system for anammox reaction. Based on above-mentioned, we concluded that anammox microorganisms and denitrif-ying bacteria were responsible for anoxic ammonium with nitrite as electron acc-eptor.
Finally, combination of partial nitrification and anammox in a closed DHS was investigated at different operating parameters limiting the oxygen supply. It was demonstrated that autotrophic nitrogen removal could be successfully establ-ished in a nitrifying-biofilm DHS. A co-culture of aerobic and anaerobic ammoni-um oxidizers consumed oxygen preventing the inhibition of anammox bacteria, mak-ing a suitable substrate for anammox reactor. However, lower oxygen concentrati-on is also associated with the production of noxious gas like N2O, which should be studied furter. Nitrogen removal rate finally attained was 1.53 kg N･m-3 sponge･day-1 as maximum in the DHS reactor. It was observed that nitrogen remo-val rate of the DHS reactor was higher than SBR, CSTR, UASB or RBC systems.
Based on above-mentioned, DHS system has been recommended as a useful system for removal of nitrogen in the wastewater due to its capability to provide long-er SRT for slow-growing bacteria. However, there are still some problems in app-lication of nitrogen removal process, the improvement of DHS system has an adeq-uate performance capability making it a potential system for nitrogen removal.

　本論文は、「Development of a Novel and Cost-Effective Nitrogen Removal Proces-s by the Application of Down-Flow Hanging Sponge（DHS） Systems（懸垂型スポンジろ床リアクターによる新規低コスト型窒素除去プロセスの開発）」と題し、6章より構成されている。
　第1章「緒論」では、本研究の意義と目的について述べ、論文の構成について記述している。
　第2章は、研究の背景と既往の研究について記述している。窒素サイクル、硝化、脱窒、窒素サイクルに関係する微生物、窒素除去技術、および本研究で用いたDHS技術についてまとめた。
　第3章では、酸素供給制限をした密閉型DHSを用いて、アンモニアから、嫌気的アンモニア参加で要求されるモル比で96%に相当する量の亜硝酸塩の生産に成功したことを記述している。また、16S rRNAによる遺伝子解析の結果、Nitrosomonasと共に、未培養微生物群であるOP10も高い割合で検出されたことから、これらが本反応に寄与していることを明らかにしている。
　第4章では、密閉型DHSをより低酸素の条件として、アンモニア酸化特性を評価した。低酸素DHSは、スポンジ容積当り1日に負荷する窒素量が1.85 kgという高い窒素容積負荷（1.85 kg･m-3･day-1）において窒素除去率95%を達成できることを示した。また、本システムで、亜硝酸塩の低減にComamonasが寄与しているのに対して、無酸素条件下の窒素除去反応にはKuenenia sttugartiensisが主に作用している事も明らかとした。その結果、Planctomysetesに属する未培養な細菌もクローンライブラリーで多数検出されていることから、新しいタイプの窒素除去細菌である可能性も明らかとした。
　第5章では、部分的硝化と嫌気的アンモニア酸化を、単相のDHS内で進行させることを試み、その結果、窒素容積負荷1.53 kg･m-3･day-1で窒素除去率84%を達成できたことを記述している。
　第6章では、本論文で得られた結果と考察を要約し、今後の研究への提案を行った。
　以上のように本論文は、曝気と有機物添加を必要としないDHSを用いた新規な高速窒素除去技術の開発についてまとめたものである。本論文は、水環境保全施設における窒素処理について有用な技術を提案しており、工学上および工業上貢献するところが大きく、博士（工学）の学位論文として十分な価値を有するものと認める。