学位論文要旨



No 128735
著者(漢字) 李,寧
著者(英字)
著者(カナ) リ,ネイ
標題(和) 活性汚泥の脱水性と細菌群集構造の関係に関する研究
標題(洋) Study on Relationship between Dewaterability and Bacterial Population in Activated Sludge
報告番号 128735
報告番号 甲28735
学位授与日 2012.09.27
学位種別 課程博士
学位種類 博士(環境学)
学位記番号 博創域第838号
研究科 新領域創成科学研究科
専攻 社会文化環境学専攻
論文審査委員 主査: 東京大学 准教授 佐藤,弘泰
 東京大学 教授 味埜,俊
 東京大学 教授 服部,正平
 東京大学 教授 古米,弘明
 東京大学 准教授 鯉渕,幸生
内容要旨 要旨を表示する

In this study, relationship between dewaterability and bacterial population in activated sludge was of interest. With the developed dewaterability testing method, water content of dewatered sludge (WCDS) testing method, and combination of two molecular biology methods-T-RFLP and pyrosequencing, dynamics of dewaterability and microbial population were monitored and their correlations were analyzed by multiple regression analysis. The detailed compositions of this thesis are introduced as follows:

Chapter 1 and 2 introduced the background of this study and relevant research results. Additionally, the significance of this study was mentioned as well.

Dewatering is a physical unit process to reduce the volume of sludge by removing water from wet sludge. Many factors, such as extracellular polymer substances (EPS) concentration, particle size distribution, specific surface area, density, particle charge, bound water content, pH, and organic concentration, have been reported to affect dewaterability of sludge.

In the case of dewaterability of excess activated sludge, bacterial population in the sludge would be one of possible factors that affect dewaterability. Yet, so far, very limited studies have been done to clarify the effects of bacterial population on dewaterability. And those limited studies often focused on the morphological characteristics of bacteria. However, bacterial characteristics other than morphology might also affect dewaterability. Thus, it is worth to study the relationships between whole bacterial population in activated sludge and its dewaterability.

Chapter 3 demonstrated the development of WCDS testing method and the details of selected molecular biological methods.

Dewaterability testing methods, such as capillary suction time (CST), specific filtration resistance (SRF) are popularly applied by other researchers on dewaterability comparison and improvement aspects. However, CST and SRF come with quite high deviation (up to 10% of CST and 24% of SRF) which may not reflect the dynamics of dewaterability during operation. Therefore, there had not been a convenient method to determine dewaterability of excess sludge with small amount from laboratory scale activated sludge reactors.

In present study, firstly, 4×25 ml mixed liquor was centrifuged at 2000g (5 min) for thickening at the same time, supernatant was decanted, and then around 300mg-wet thickened sludge pellets each were loaded on piece membrane filters (for 4 samples) for dewatering. The filters were placed in a Swinnex filter holder (25mm, Millipore) individually, and the filter holder was set in a bucket for 50 mL conical tubes (No. 053-5010, Kubota). Then, further the loaded sludge pellets were again centrifuged at 2000g for 5 min to exert dewatering process. At last, the dewatered sludge cake (about 100 mg) was moved onto a piece of prepared aluminum foil (for which dry weight (A) had been predetermined), and the dewatered sludge weight including aluminum foil (B) was measured. In measuring the dewatered sludge weight, the foil was folded to cover the sludge to avoid drying during weight measurement. The sample was unwrapped, dried at 105℃ for 60 min, cooled to room temperature in a desiccators for 30 min, and the weight after drying was measured. Water content of dewatered sludge (WCDS) was calculated to assess dewaterability by (B-C)/(B-A). Measurement of weight was done with an analytical balance XS105 (Mettler Toledo, USA) with a resolution of 0.01 mg. Analyzes were done in quadruplicate.

Meanwhile, there are different methods available to analyze whole bacterial community in activated sludge, such as polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE) and PCR/terminal-fragment length polymorphism (T-RFLP). Recently, pyrosequencing technology has been developed, the resolution of bacterial community analysis has also rapidly improved with this technique. DGGE, with its drawbacks on difficult comparison with others implementation, was not adopted in this study. PCR/T-RFLP was reported as a semi-quantification method which had been applied in this study. Meanwhile, pyrosequencing offered the possible way on identification of the T-RFLP profilings and was utilized in this study as well.

Based on the analysis of relationship above, the responsible bacteria were further identified on monitored dewaterability dynamics of activated sludge with the pyrosequencing pipeline and QIIME basing software-OTUMAMI.

Chapter 4 discussed the results obtained by methods introduced in Chapter 3.

Totally, three reactors were monitored with dynamics of dewaterability of activated sludge. As the main results, monitored dewaterabiity of activated sludge in reactor III was shown in Figure 2.

Apparently, from day 50th to Day 120th, WCDS value increased at a relatively rapid speed. (Figure 2), while, from day 130th to the end, the fluctuation of WCDS value fluctuated within more narrowed range. With about initial 60 days of relatively lower value, WCDS increased to more than 90% which meant the general deterioration of dewaterability during the operation. Those changes may come from the shift of bacteria consortia and the competition among them also resulted in the fluctuation during the monitoring.

Then, the microbial population was analyzes by the PCR/T-RFLP method and further identified with the corresponding T-RFs by virtual digestion of pyrosequencing.

For instance, relationship was revealed by the following equation which showed the corresponding T-RFs and their correlationship with dewaterability.

Reactor III:

In particular, the estimated WCDS value well matched the observed WCDS value with R2 of 0.84.

Regarding corresponding bacteria, virtual digested corresponding T-RFs were selected and by classification with QIIME, the T-RFs were successfully classified though some could not be classified at genus level.

EPS was also analyzed with the correlation to dewaterability. No obvious correlation was found.Besides the 3 reactors, samples from 2 WWTPs were also collected to see the correlation between bacterial population and dewaterability.

Chapter 5 showed the results from 2 trial experiments to clarify the impacts of two factors -overloading and storage time. Overloading may to some extent improve dewaterability of activated sludge (4% in terms of WCDS value). Storage time certainly influences dewaterability of activated sludge because of the changed nature. However, different sludge showed different trend which on the other hand confirmed the behavior of dominant bacteria has different impact on dewaterability.

Chapter 6 summarized the present study and gave some suggestions on the future study.

In summary, this study demonstrated that the specific relationship between dewaterability and bacterial population was worth investigating. The approach established in this study to some extent obtained the preliminary results on the correlation toward some specific bacteria.

Figure 1 Flowchart of dewaterability testing. (a) Whole procedure, and (b) Schematic of dewatering step.

Figure 2.WCDS dynamics of activated sludge from operated reactor III (n=4) Average values are connected by solid line

Figure 3 Correlation between estimated WCDS and measured WCDS of reactor III

審査要旨 要旨を表示する

下廃水処理の結果発生する汚泥は、焼却したり乾燥させて減容するが、その際のエネルギー収支に汚泥の含水率や脱水性が大きな影響を与える。活性汚泥の脱水性については、これまで細胞外ポリマーの量や化学組成、あるいはフロックの形態との関連について多くの研究が行われてきた。活性汚泥中の細菌群集構造も汚泥の脱水性に大きな影響を与えるものと考えられるが、そうした観点からの研究はこれまでほとんどなかった。

本研究は活性汚泥の脱水性と活性汚泥中の細菌群集構造との関連を明らかにすることを目的として行われたものである。本論文は全七章で構成されている。第一章(序章)、第二章(文献レビュー)に続き、第三章では既存の手法に大幅な改良を加え、実験室活性汚泥の脱水性を評価する手法を開発した。第四章は第五章と第六章で共通して用いられる手法について説明している。第五章は実験室活性汚泥プロセスの活性汚泥を対象とし、また、第六勝は実下水処理場の活性汚泥を対象とし、いずれも一定期間脱水性と細菌群集構造をモニタリングし、活性汚泥の脱水性に影響を与える細菌種を特定することを試みた。第七章では論文全体の総括と、今後の展望を述べている。

第三章では、実験室活性汚泥の脱水性を評価する手法の開発について述べている。既存の手法は実下水処理場で実施されることを念頭に、数グラム程度の濃縮汚泥や脱水汚泥を必要としていた。しかし、実験室活性汚泥リアクターでは一日あたり1グラム程度の余剰汚泥が発生するにすぎず、もっと少量で脱水性を評価することができる手法を開発する必要があった。メンブレンフィルターに少量の遠心濃縮汚泥を載せ、フィルターフォルダーに装着し、そのまま遠心分離するよう、既存の手法を改良した。乾燥重量として数十mg程度の汚泥量で脱水性を評価することが可能となった。本論文ではここで確立された手法をWCDS法と称している。

第四章では実験室活性汚泥リアクターの運転法や実施設汚泥の採取スケジュール、細菌群集構造の変化の分析法、および、脱水性と関連する細菌群の絞り込みの方法について述べている。細菌群集構造の分析には、主としてT-RFLP法(末端標識制限酵素切断断片多型法)を用い、また、高速シークエンシング法を緩用した。脱水性と関連する細菌種を特定するために、本研究ではまずT-RFLPで検出された各標識断片(T-RF)と脱水性との関連を調べ、関連のありそうなT-RFを特定した。一方、高速シークエンシングの結果から、当該T-RFを生成すると考えられる細菌種(OUT、便宜上の種単位)を特定し、そのOTUの挙動と脱水性の変動との関連を比較した。

第五章では、3つの実験室活性汚泥リアクターについて数日おきに脱水性と細菌群集構造をモニタリングした結果について報告している。WCDSの値は84%~94%の間にあり、運転に伴って徐々に変動する様子を捉えることができた。4つ~7つのT-RFの存在比率を用いてWCDSを重回帰分析したところ、決定係数は0.71~0.77であり、良好な一致が見られた。重回帰式にあらわれた各T-RFと関連するOTUを高速シークエンシング法による分析結果と比較した。各T-RFには複数のOTUが対応していたが、中には一つまたは二つのOTUが過半を占める場合もあり、そうした場合について、それらOTUと関連するT-RF、および、脱水性との関連を比較した。その結果、Meganema属、Haliscomenobacter属、Thiothrix属の各OTUについて脱水性との関連が強く示唆された。ただし、Haliscomenobacter属とThiothrix属は脱水性がよいときに多かったが、Meganemaについては脱水性のよいときに多いケースと脱水性の悪いときに多いケースの双方があった。

第六章では二つの下水処理施設について、年間を通じて毎月1回試料採取を行った月毎調査と、冬・春・夏・秋の4回、一週間月曜日から金曜日まで5日連続で採取した季節毎調査の結果を報告した。脱水性との関連が示唆されたT-RFは、Zoogloea属のOTUが主体となっており、どちらかというと、その量が多いと脱水性が悪くなった。

第七章では以上の結果を総括し、また、今後の展開の方向について述べている。いくつか脱水性との関連が示唆される細菌群を抽出することができた。しかし、明確な因果関係を明らかにするためには今後の研究を待たなければならない。

なお、本論文第三章および第五章は共著論文として公表されているが、論文提出者が主体となって行なったものであり、論文提出者の寄与が十分であると判断する。

以上より、博士(環境学)の学位を授与できると認める。

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