The present study focused to grasp bacterial population in treated water of the activated sludge processes. The activated sludge processes are widely used as a method of biological wastewater treatment. In these processes, treated water and activated sludge are usually separated by gravimetric settling, but the separation is not perfect and small amount of bacteria are left in treated water.

Bacteria in treated water affect the quality of treated water in three points of view. First is from the view of controlling pathogenic bacteria. Because this aspect is strongly related to human health, tremendous studies have been and are being done. Secondly, bacteria in treated water might affect ecosystems in receiving water bodies. And thirdly, these bacteria in treated water can interfere with advanced treatment processes for its reuse. Especially, considering the effects of these bacteria on receiving water bodies and water reuse, knowledge on whole bacterial population in treated water is essential. The amount of suspended solids (SS) and heterotrophic counts are indicators for the amount of whole bacterial population in treated water. Yet, these methods do not give the content of the bacterial population in treated water. Here is the need to understand the whole bacterial population in treated water.

Bacterial population in treated water is thought to reflect that in activated sludge. It is because treated water most probably contains small flocs of activated sludge. But there may also be differences. It is because some bacteria may prefer to live freely outside flocs and others may prefer to live in flocs. Therefore, it is expected that there are similarities and differences in bacterial populations in treated water and in activated sludge. Thus, the first objective of the study is to clarify the relationships of bacterial populations in treated water and in activated sludge.

In order to analyze bacterial populations in treated water and in activated sludge, the author employed one of molecular methods for bacterial population analysis in combination with a new method to prepare PCR compatible DNA extract from samples. The molecular method employed was the polymerase chain reaction (PCR) followed by terminal restriction fragment length polymorphisms (T-RFLP) method. On the other hand, the new method to prepare PCR compatible DNA extract from samples is based on the destruction of cells by sonication followed, if necessary, by dilution to reduce inhibitory effects of substances in samples. Development of the new method is the second objective of this study. By the developed method, treated water samples are sonicated, then directly applied to PCR/T-RFLP analysis.

While analyzing the bacterial populations in treated water, the author found that filtrate of treated water through 0.2μm membrane filter contained small amount of DNA fragments, which could be amplified by polymerase chain reaction (PCR) even without extraction of DNA. These DNA fragments (referred to as free DNA hereafter) might affect analysis of bacterial population in treated water from analytical point of view, but they may give any reflection of the dynamics of bacterial population. Thus, the third objective of the study was to investigate the behavior of free DNA in treated water.

With the above three objectives as the goal, this thesis contains seven Chapters. Chapter 1 is the introduction, and Chapter 2 is literature review. In Chapter 3, development of the methodology to prepare DNA template from treated water and activated sludge samples by the sonication based method was investigated. In Chapter 4, the developed method was applied to analyze the bacterial communities in treated water and activated sludge from laboratory scale activated sludge reactors. In Chapter 5, the bacterial communities in treated water and activated sludge in two full-scale wastewater treatment plants (WWTPs) were analyzed. In Chapter 6, composition of free DNA and its dynamics was studied. The source of the samples was filtrate of treated water analyzed in Chapters 4 and 5.

In Chapter 3, the experimental design and outcomes on the methodology development on the preparation of PCR-compatible DNA extracts from activated sludge and treated water were discussed. Treated water and activated sludge samples from full scale WWTP were used as the samples. They were sonified, and were diluted to different template concentrations. Then, using the 27f/519r primer set targeted at partial 16SrRNA gene, PCR was performed with different numbers of thermal cycles. The best results were obtained with the template DNA concentrations around 1-10pg/μL with 30 thermal cycles. The reproducibility was examined by comparing the composition of the PCR products by T-RFLP for triplicate analyses of both activated sludge and treated water samples; outcomes showed that the reproducibility of the developed method is satisfactory. The method of PCR template preparation developed here is easy and rapid to implement without the needs for chemicals and takes only a couple of minutes per sample.

In Chapters 4 and 5, the method developed in Chapter 3 was applied to investigate the bacterial population in treated water and in activated sludge. In Chapter 4, samples were obtained from laboratory scale reactors, while in Chapter 5, they were obtained from full scale treatment plants. In Chapter 4, samples were obtained two laboratory scale reactors operated with synthetic wastewater as the feed. For one of the reactors, samples were daily collected for 11 days, while for another; samples were collected weekly for 4 weeks. The bacterial community structures in the activated sludge and in treated water were successfully profiled by the template DNA, which were prepared by the developed method. The results showed that the bacterial community structures in treated water had significant differences from those in activated sludge. Some of peaks in the T-RFLP profiles were found more intense in treated water while other peaks were found more intense in activated sludge. The principal component analysis (PCA) on T-RFLP data showed that there was a similar trend of bacterial population changes in activated sludge and in treated water samples.

In Chapter 5, bacterial population in activated sludge and in treated water samples collected from full scale WWTPs were analyzed. Monthly samples were collected for a year from February 2010 to January 2011 to observe seasonal fluctuations, while daily samples for 5 successive days were collected in February, May, August, and November 2010 to observe daily fluctuations. The suspended solids (SS) in treated water had an average of 6mg/L and 7mg/L in plant A and B respectively, and total DNA concentrations in sonicated treated water was in the order of 100μg/L. The outcomes clearly suggested that the bacterial community structures in treated water had clear differences from those in activated sludge, as same as observed in laboratory reactors. Yet, the outcomes of PCA analysis on the T-RFLP data for monthly collected samples clearly showed a similar trend of bacterial populations changes in activated sludge and in treated water from both WWTPs. Year-round trend was confirmed by the PCA analysis, as the plots had a tendency to draw year-round circles. An additional experiment in Chapter 5 suggested that abundance of some of bacterial species was increased in treated water after the sludge had been stored for a couple of hours. The outcomes of experiments in Chapters 4 and 5 can be interpreted as follows. Peaks or bacterial species corresponding to those peaks, found more intense in treated water are associated with pin flocs or freely living in the bulk liquid. Those found more intense in activated sludge are associated with more stable flocs.

In Chapter 6, the behavior of free DNA in treated water of the activated sludge processes was investigated with basically the same samples as used in Chapters 4 and 5. As additional samples, influent wastewater to the full scale WWTPs were collected on the sampling occasions of October and December 2010. The average concentrations of free DNA in treated water were around 10pg/μL, while those of activated sludge mixed liquor and treated water samples were around 10.000pg/μL and 100pg/μL respectively. The concentration of free DNA was only about one tenth of that of total DNA in treated water. And major T-RFLP peaks in free DNA were not observed in treated water. Thus, it was concluded that free DNA does not affect the analysis of bacterial population in treated water by the method developed here.

The T-RFLP patterns of free DNA were very different from those of activated sludge. Yet, by PCA analysis, a similar trend in bacterial population change in activated sludge and free DNA were found. This outcome suggests that one of the sources of free DNA is bacteria in activated sludge. On the other hand, there were peaks that found in free DNA and in fluent samples but not in activated sludge samples. This suggests that another source of the free DNA is bacteria in influent. Yet, many of the peaks that were found intense in free DNA were weak or not detected in influent nor in activated sludge.

In conclusion, the outcomes showed that, it is enabled to use the developed methodology to analyses bacterial populations in treated water and in activated sludge. The bacterial populations found in treated water had significant differences from those in activated sludge from both laboratory reactors and WWTPs. However, similar seasonal trend of the changes of bacterial populations in treated water and in activated sludge was found. The free DNA concentration in treated water was only about one tenth of that of total DNA in treated water and free DNA was not affected the analysis of bacterial population in treated water by the method developed here. The outcomes of this study are important in both from the practical point and scientific points of wives. From the practical point of view, studies on the investigation of the possibility to suppress leaking of dominant bacteria species in treated water are very important. From the scientific point of view; the outcomes of free DNA in treated water are important as it may reflect the microbial ecology in activated sludge.

本研究は下水処理水中に出現する細菌群集とその経時的な変動について明らかにしようとしたものである。分子生物学的手法の発展によって、下水処理プロセス中で水質浄化に寄与する微生物群についての研究は大きく前進しつつあるが、それに比べると、下水処理水中の微生物群集に関する研究は非常に限られている。しかし、処理水の水質を向上させるために、また、下水処理水の再利用を行いやすくするために、処理水中の微生物群集についての理解を深める事は重要である。

また、本研究において著者は下水処理水を0.2μmのメンブレンフィルターによりろ過したろ液をそのまま鋳型とし、16SrRNA遺伝子を対象としてPCR反応を行うと、増幅産物が得られる事を見いだした。著者はろ液に含まれるDNAを遊離DNA(free DNA)とよび、その挙動についても検討した。

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、第三章では活性汚泥や処理水中からのDNAの抽出方法について検討した。第三章で導入した手法を用いて、第四章では実験室活性汚泥リアクターについて、また、第五章では二つの実下水処理場について、活性汚泥および処理水中の細菌群集構造とその変動を解析した。第六章では第四章、第五章で分析したのと同じ試料について、遊離DNAの挙動を解析した。第七章では論文全体の総括と、今後の展望が述べられている。

第三章では、簡易かつ再現性良くDNAを抽出することができる技術として、近年開発された超音波破砕・希釈法の適用可能性について、最適な試料の希釈倍率やPCR反応のサイクル数、再現性について検討している。PCR反応の対象部位は16SrRNA遺伝子の部分塩基配列である。試料を超音波破砕した後、適宜希釈してPCR反応液中における鋳型DNAの濃度を1~10μg/L程度前後とし30サイクルPCR反応を行う事で、再現性良く良好なPCR反応産物を得ることができたとしている。

第四章では実験室活性汚泥リアクターを運転し、活性汚泥と処理水中の細菌群集構造を16SrRNA部分塩基配列を対象とするPCR/T-RFLP法(PCR反応の後に末端標識制限酵素切断断片多型解析を行う方法)で検討した。両者に共通したピークが見られたものの、その強度は大きく異なっていた。処理水に出現しやすい傾向のある細菌群、活性汚泥から抜け出てくる傾向の小さい細菌群が存在する事が明らかになった。

第五章では、実下水処理場について活性汚泥と処理水中の細菌群集の変動を一年間にわたって調査した。また、一部の月では平日5日連続での試料採取・分析を行った。実験室リアクターの場合と同様、処理水に出てきやすい細菌群、あるいはその逆の細菌群が存在する事がわかった。また、通年で周期的に変動するような傾向がある事、特に処理水については数日間のうちにも大きく変動する場合がある事が明らかになった。また、汚泥を数時間放置し再混合すると、上清中の浮遊物質が増えるだけでなく、一部の微生物群が上清中に増加することがわかった。

第六章では、処理水ろ液中の遊離DNAの挙動を第四章・第五章で検討したと同じ試料について検討した。処理水を0.2μmのメンブレンフィルターでろ過し、そのろ液を直接PCR反応の鋳型としたところ、PCR反応産物を得ることができた。その構成や挙動をPCR/T-RFLP法で分析したところ、処理水や活性汚泥と共通する細菌群の遺伝子も検出されたが、その組成はいずれとも大きく異なっていた。遊離DNAがどのように生成しているのかはわからないが、微生物群集構造の変動に関連する可能性もある。

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。明らかにすることができた主な事柄は、処理水中の細菌群集構造は活性汚泥のそれとは異なる事、経年的な変動をすること、汚泥をしばらく静置し再懸濁したのちに上清を得ると、上清中の細菌群集構造が変化する事、処理水のろ液にはPCR反応の鋳型となる16SrRNA遺伝子またはその断片が存在する事、またその構成は活性汚泥や処理水のそれとも異なる事、の6点である。処理水の水質を向上させるために、処理水に出てきやすい細菌群の特性を調査する必要がある事を述べるとともに、遊離DNAの起源を明らかにする事で、活性汚泥の微生物生態系について新たな知見が得られる可能性があるとしている。

本研究から、今まであまり検討された事のなかった下水処理に関連する微生物世界について、新たな側面が浮かび上がってきたといえる。

なお、本論文3章および4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。