Due to the toxicity and non-degradable nature, heavy metals are of great environmental concern for both developing and developed countries. They tend to pose serious health hazard to human beings and animals while accumulating in soil by adsorption. Cadmium is one of the ubiquitous and highly toxic heavy metals. Once cadmium accumulates in body, it may cause renal dysfunction ("Itai-itai disease"). In Asian countries such as China, a large area of paddy soil needs cadmium remediation. It is really a challenging and essential task to clean up the contaminated sites with heavy metals. Conventional remediation techniques (e.g. soil flushing, solidification, excavation) seem expensive and destructive in nature, since they may destroy soil structure and decrease soil productivity.

Phytoremediation (plant-based remediation) is effective for the mitigation of large area surface soil contamination with regards to extraction efficiency, duration of cleanup and economy. Usually, phytoextraction needs long time and the efficiency is not high. An important reason is that a large proportion of metals in soil are unavailable for plant uptake. In order to enhance the metal availability and sustain adequate metals in soil solution for plant uptake, various agents have been added to soil. Agents, such as EDTA, were reported to increase bioavailable lead in soil and bring greater accumulation in plants. However, the addition of chemical agents (e.g. EDTA, DTPA) provides limited benefit. Chemical agents inhibit plant uptake of essential elements, and are toxic to plant growth. Furthermore, they could pose a risk to soil and ground water environment. Thus, natural agents seem more promising than synthetic chemicals.

In this dissertation, the microbial biopolymers were extracted from activated sludge and utilized to cadmium contaminated soil. Microbial biopolymers produced from activated sludge process consist of polysaccharide, protein, RNA, and DNA. The proteinaceous biopolymers have been considered to be economical and were reported to play an important role in removing heavy metals from solution.

In the other hand, plants release root exudates (e.g. amino acid) containing biopolymers with the potential to enhance cadmium uptake, translocation and resistance. However, the concentration of the naturally excreted biopolymers is low. In this dissertation, high concentration of microbial biopolymers would be produced and used for cadmium removal by plant from contaminated soil.

There were four major objectives of this study: (1) to elucidate the heavy metal binding characteristics of microbial biopolymers obtained from activated sludge grown on different media; (2) to determine the cadmium extraction efficiency of microbial biopolymers from contaminated soil; (3) to investigate the influence of microbial biopolymers on phytoextraction of cadmium in hydroponic solutions; and (4) to evaluate the potential applications of microbial biopolymers in improving phytoextraction of cadmium as an effective method for soil remediation.

This dissertation consists of eight chapters. Background information and objectives of the present study as well as the structure of the dissertation are represented in Chapter 1. Basic information on heavy metal contamination, remediation of contaminated soils, microbial biopolymers and enhanced phytoextraction based on literatures are provided in Chapter 2. Materials and methods used in this research are described in Chapter 3. Chapter 4, 5, 6 and 7 are corresponding to the four objectives described above, respectively. Chapter 4 focuses on cadmium binding characteristics of microbial biopolymers in aqueous solution. Chapter 5 depicts the effectiveness of microbial biopolymers for cadmium extraction from soil. The influence of microbial biopolymers on phytoextraction of cadmium in hydroponic condition is documented in Chapter 6. The effectiveness of microbial biopolymers application in improving phytoextraction of cadmium from contaminated soil is shown in Chapter 7. Finally, the conclusions drawn from this study and further recommendations are outlined in Chapter 8.

Metal-binding biopolymers were reported to be induced with the existence of trace metals in growth media of activated sludge. In this research, microbial biopolymers were obtained from non-induced, copper-induced and cadmium-induced activated sludge culture, and named as ASBP, ASBPCu and ASBPCd, respectively. The adsorption of cadmium by microbial biopolymers was observed in solution. The adsorption of cadmium by microbial biopolymers was fast (10 min to reach equilibrium). The BET model fitted the best of isotherm adsorption data, with R2 values of 0.991, 0.966 and 0.982 for ASBP, ASBPCu and ASBPCd, respectively.

Microbial biopolymers, as well as EDTA, citric acid and BSA, were tested for their efficiency to extract cadmium from contaminated soil. The results demonstrated that the inclusion of extracting agents increased cadmium extraction from the soil. Microbial biopolymers extracted more cadmium than 0.005mM EDTA, however, less than 0.05mM EDTA. Cadmium extraction with ASBPCd was slightly higher than ASBP and ASBPCu.

In the later step, the influence of microbial biopolymers on phytoextraction of cadmium in hydroponic solutions was examined in this study. Biopolymers provided higher cadmium uptake by plants (Crassula lycopodioides v. pseudolycopodioides), than EDTA. The cadmium uptake in the presence of biopolymers was 5.5~6.6, 24.5~31.9 and 141.7~190.1μg/g, at the initial cadmium concentration of 0.5, 1.9 and 9.1mg/l, respectively. The cadmium uptake in the presence of EDTA was 0.4~2.7, 8.9~26.4 and 34.9~152.8μg/g, at the initial cadmium concentration of 0.5, 1.9 and 9.1mg/l, respectively.

The potential application of biopolymers in improving phytoextraction of cadmium from contaminated soil was also investigated in this research work. Microbial biopolymers, compared to other agents, were found to be more effective in improving the phytoextraction of cadmium from soil. The existence of biopolymers allowed higher cadmium content accumulated in plant biomass, than other extracting agents. In ASBP, ASBPCu and ASBPCd, the cadmium content in plants was found to be 1.52, 1.63 and 1.33μg (1.9, 2.0 and 1.6 times of the control), respectively. It was also found that in the presence of microbial biopolymers ASBP, ASBPCu and ASBPCd, 10.9%, 26.2% and 13.7% of exchangeable cadmium fraction was extracted from soil matrix to plant or liquid, higher than the control test (4.3%). Microbial biopolymers were effective in improving the available cadmium amount in the soil, thus providing a higher and subsequential potential of cadmium uptake by plants in long term phytoextraction.

The synergistic effects of the sorption of heavy metals onto plants as well as the dissolution of metals from solid phase would be the possible mechanism for the improved phytoextraction of cadmium from contaminated soil. Different mechanisms were observed between microbial biopolymers and EDTA, when they were applied to improve the phytoextraction of cadmium from contaminated soil. In this dissertation, it was found that in the existence of microbial biopolymers, the limiting step would be the extraction of cadmium from soil matrix to soil solutions. However, the transportation of EDTA-Cd complex from soil solution to plant root would be the limiting step while EDTA were used for improving the phytoextraction of cadmium from contaminated soil.

In this dissertation, microbial biopolymers, acting as a new environmentally safe extracting agent, were more effective in improving cadmium accumulation in plants than other chemical agents. Instead of current chemical agents, microbial biopolymers could significantly reduce the production costs. A further study on the characterization of heavy metal-binding biopolymers derived from protein cloning techniques would have higher potential for its application in phytoextraction of heavy metals in contaminated soils. Extensive field tests of hyperaccumulator plants with biopolymers deem vital before its large-scale implementation. Practical aspects of improved heavy metal phytoextraction by microbial biopolymers also need further research. This might include development of an economical method to prepare the microbial biopolymers in a large scale, and feasible techniques to treat plants accumulated with heavy metals after harvest, and so on.

急速な産業の発展を経験した国や現在発展している国では有害金属による汚染が起こっている場合が多い。特に多くの工業拠点を有する中国では全国的に有害金属による汚染問題を抱えていると言っても過言ではない。中国華北地域では雨量が少ないことから一度使用した水をそのまま、または、処理してから灌漑用水として利用する場合が多い。その、再利用される灌漑用水は工場や住宅地などを、開水路で導水され、田畑に供されるので、その途中で多くの汚染物質が流れ込む恐れがある。その様な状況で多くの田畑の土壌が有害な金属で汚染されている状況がある。中国の経済的な状況を考えると、その様な土地を浄化する余裕はなく、そのまま汚染された土壌で作物を作り続ける場合もある。安価で効果的な汚染土壌の浄化方法が求められている。

本研究は上記のような状況を踏まえ、稲の栽培で特に気をつける必要があるカドミウムによる汚染土壌を浄化する方法を開発すべく、新しいファイトレメディエーションの開発に取り組んだものである。ファイトレメディエーションの問題点は汚染土壌からの有害金属の除去速度が極めて遅いことである。本研究ではこの問題を解決するため、カドミウム汚染土壌に微生物から抽出したバイオポリマーを添加して、カドミウムの土壌からの溶出を促進し、同時に植物への取り込みを加速する事を狙い実験的な研究を行った。

植物はシデロフォアに代表されるタンパク質様バイオポリマーを根から分泌し、金属類の取り込みを促進することが知られている。このバイオポリマーはごく低濃度であり、また、人工的にその濃度を増加させるには植物が生産する物では限りがある。そこで、同様の作用を大量生産に向く微生物バイオポリマーで達成するという点がこの研究の要点である。微生物バイオポリマーもカドミウムを効率的かつ強固に吸着することが知られており、また、植物体への金属の取り込みを微生物が産出するバイオポリマーが促進するという報告もあり、本研究を着想するに至った。

以下、博士論文の各章毎の成果を述べる。

第1章と第2章では本研究の背景と目的を示し、さらに、関連する既往の研究に関してのレビューを行い、本研究の新規性、重要性を明らかにした。

第3章では本研究の実験方法や設定条件を詳細に解説した。

第4章では異なった環境で混合培養された活性汚泥由来の微生物からタンパク質様のバイオポリマーを抽出し、それによる金属との吸着実験を行った。培養条件は無選択培地、無選択培地に微量のカドミウムや銅を添加したもの、さらに比較のため数種のカドミウムと結合することが知られている化学物質を添加した。その結果、微生物バイオポリマーはカドミウムと短時間で吸着することが確認でき、さらに、吸着実験結果はBET等温吸着モデル型である事が確認された。

第5章では微生物バイオポリマーによる土壌からのカドミウム溶出の促進効果を検証した。その結果、高い濃度(0.05mM)のEDTAには及ばなかったが、中性pH付近でカドミウムの溶出促進効果が認められた。また、溶出効果に及ぼすpHの影響も調べたが、その結果中性pH付近で最も効果的にカドミウムを溶出することがわかった。

第6章では微生物バイオポリマーが水耕栽培状態に置かれた植物によるカドミウムの取り込みをどのように促進するか、実験的に確認した。この章は前章と同様に本研究では最も重要な章で、ひとつのバイオポリマーが土壌からのカドミウム溶出促進と、溶出されたカドミウムの植物への取り込みの促進を同時に達成可能かを確認している。その結果、第5賞の結果を併せて考えると、微生物バイオポリマーは最も優れたカドミウムの取り込み促進効果が見られた。

第7章では第4章~6章までの結論をもとに、カドミウム汚染土壌で栽培された植物のカドミウム取り込みに与えるバイオポリマーの影響を実験的に調べた。その結果、バイオポリマーを添加した系で植物への取り込みが最も高い効率で観察され、実用化への可能性を示唆できた。

本博士論文の成果で最も重要な点は土壌からの金属溶出促進と植物体への金属とり込み加速の両方を可能とする微生物由来のバイオポリマーを見出した点であり、今後、このバイオポリマーの同定、大量生産等の発展的研究が見込まれる。まだ、解明しなければならない点も多いが、将来的にはこの考えを応用した経済的で高効率なファイトレメディエーションが実用化されると思われる。

以上のように本博士論文は環境工学において、顕著な貢献がみられ、博士(工学)の学位請求論文として合格と認められる。