The textile wastewater, which is rated as the most polluting among all industrial sectors considering both volume and composition of effluent, is a complex and highly variable mixture of many polluting substances ranging from inorganic and low molecular weight organic compounds to polymers. It induces persistent color coupled with organic load leading to disruption of the total ecological/symbiotic balance of the receiving water stream. Dyes with striking visibility in recipients may significantly affect photosynthetic activity in aquatic environment due to reduced light penetration and may also be toxic to some aquatic lives. It is difficult to remove dyes from effluents since dyes are stable to light, heat and oxidizing agents and are hardly biodegradable.

Several physico-chemical decolorization techniques have been reported (e.g. coagulation, adsorption, membrane separation, advanced oxidation processes), few, however, have been accepted by the textile industries due to high cost, low efficiency and inapplicability to a wide variety of dyes. Biodegradation is an environmentally friendly and cost competitive alternative but the conventional aerobic treatments have been proved ineffective while highly toxic aromatic amines can be formed by reductive fission under anaerobic conditions. However, wood rotting 'White-rot fungi' are able to degrade aerobically a wide variety of recalcitrant organic pollutants, including various types of dyes through extracellular secretion of non-specific oxidative enzymes.

This study proposed a hybrid membrane-coupled fungi reactor for treatment of textile dye wastewater. The application of white-rot fungi in large-scale waste treatment has so far been impeded by the lack of bioreactor systems that can sustain long-term steady production of high levels of extracellular enzymes under non-sterile environment together with a controlled growth of fungi. On the other hand, although membrane bioreactor (MBR) has become a reliable alternative to conventional activated sludge processes and an option of choice for many domestic and industrial applications, membrane fouling and its consequences in terms of plant maintenance and operating costs limit the widespread application of this technology.Hence, the ultimate aim of this study was to enhance microbial degradation by adopting appropriate process designs and to mitigate membrane fouling by developing fouling-resistant membrane module.

A fouling-resistant compact hollow-fiber module was developed for utilization in the proposed membrane-coupled fungi reactor to treat high strength textile wastewater. Under similar conditions, while the usual hollow-fiber bundles exhibited fatal cake-layer fouling within a day or so, the modules with spacer sustained stable performance for a month.Among the explored modules, a hybrid module (fiber packing density =61.5 %, surfacearea=1.07 m2) obtained by winding a rigid spacer (thickness=1 mm, opening=7 mm x 7 mm) on the surface of a module originally containing a thin spacer (opening=1 mm x 1 mm) exhibited the optimum compactness so as to minimize intrusion of sludge while simultaneously allowing wash-out of the small amount of sludge trapped within it. Periodic low-dose cleaning from the initiation of operation was found to be more effective than application of high-dose cleaning after occurrence of severe fouling. Periodic in situ chemical backwashing with a low dose (500 mg C1 /L, 100 ml/m2, twice/week) and intermittent surface-cleaning with a specially designed aeration device (1 L air/ min, 1 min per 30 min)enabled stable operation for a prolonged period under the selected average flux (1.27x10(-7) m3 /m2. s) and MLSS concentrations (up to 25 g/ L). Under the similar conditions, four-foldreduction in total consumptions of both chemical and air was possible when the developed module was placed within a coarse-pore (50-200um) pre-filtration cage. However, a three-fold reduction in compactness (membrane area per unit volume of composite module) was inevitable in this case.

On the other hand, an in-depth assessment of the enzymatic activity and efficiency of &coloration of wide varieties of dyes by pure fungi culture was conducted in preliminary batch tests. Almost complete color and reasonable TOC removal performances (66%) concurrent with detectable level of expression of extracellular enzyme were observed. A significant role of biosorption along with biodegradation in decoloration, especially at the initial stage, was noticed. Batch tests also confirmed significant dye decoloration (84.2%) in presence of polyvinyl alcohol-another hardly biodegradable common pollutant in textile wastewater.

The critical factors which influence the dye degradation performance of fungi, namely,morphology, hydrodynamic conditions, bacterial contamination etc., were also thoroughly investigated in batch tests. Fungi sludge contaminated with bacteria exhibited moderate decoloration, no enzymatic activity and much faster rate of TOC consumption. Under sequential replenishment of the liquid media the sludge progressively agglomerated and demonstrated much improved decoloration; however, enzyme in the media was not within detection limit. Investigation under sterile environment with crude enzyme solution employing different modes (reciprocal/rotary) and intensities (shaking speed 80-150 rpm) of agitation revealed negligible mechanical inactivation of enzyme. Disintegrated pure culture inoculated aseptically under strong agitation (150 rpm) exhibited an enzymatic activity approximately four times higher than that in agglomerated culture under lower agitation (80 rpm). Conversely, disintegrated pellets of fungi were found to be more prone to bacterial contamination. Monitoring fate of crude enzyme solution contaminated with bacteria also confirmed bacterial disintegration of fungal enzyme.

A frequently reported problem associated with the long-term operation of reactor containing white-rot fungi is the intensive sludge-growth due to the high dose of carbon source as required for maintaining the viability of the fungi. On the other hand, it has been reported that,depending on the type of dye and level of enzymatic activity, sorption on fungal mass may play a significant role in total decoloration. In this study, the extent of removal of dyes possessing different biosorption properties was examined in an MBR bearing sludge bed to promote sorption of dyes onto settled biomass. The reactor design with sludge bed and a split-mode feeding strategy proved to be an efficient means to control the MLSS concentration (MLSSaerobic) in direct contact with the membrane. The average stable MLSSaerobic concentrations in case of feeding from top, bottom or simultaneously from top (60%)-bottom (40%) were 25, 4 and 11 g/L, respectively. Feeding mode and MLSS concentration played a significant role in color and TOC removal. The respective average color and TOC removals in case of feeding from top, bottom or simultaneously from top (60%)-bottom (40%) were as follows: Color (93.2%, 57.5%, 91.3%), TOC (97%, 94%, 97%).In case of feeding from bottom, marked dependence of decoloration on pH and hindered TOC removal in presence of chemical cleaning of membrane were observed. Depending on the type of dye, the biomass at the settling zone offered considerable sorption while the wastewater passed through this zone, thereby aiding in overall decoloration. The MBR in this case achieved an excellent 99.7% decolol'ation. The removal by the MBR of a dye showing negligible sorption on biomasS was, however, incomplete and necessitated GAC post treatment.

Prevention of continuous loss of extracellular enzyme along with treated effluent may be critical in maintenance of stable biological degradation of dye in an MBR. Such retention of enzyme may be achieved by application of simultaneous adsorption within MBR. The effect of reactor-operation mode (continuous versus sequencing batch) and simultaneous adsorption on the performance of the membrane-coupled fungi reactor was investigated. Co-adsorption of dye and enzyme onto activated carbon and subsequent enzymatic dye degradation was observed to occur. The dye degradation potential on activated carbon per enzymatic activity was estimated as 1.33 mg dye/ (uM substrate/min). The performance of the MBR under sequencing batch mode without any GAC-coating on the membrane was much worse than that when GAC-coated mesh was wrapped on the membrane (color absorbance of 2 and 0.6, respectively, within 5 days). In the sequencing batch MBR, the dye concentration in the feed apparently had a little influence on the permeate quality under the same HRT and withdrawal rate. On the other hand, HRT (along with withdrawal rate) appeared to impose significant influence on permeate quality. The fact that the permeate quality deteriorated in case of the shorter HRT (color absorbance 0.6 under HRT of 1 day as compared to 0.01 under HRT of 3 days) even though the dye loading in the feed was kept the same suggested that the utilized amount of GAC was not enough to completely prevent the leakage of enzyme from the reactor under the applied withdrawal rate. The performance of the continuous flow MBR, in case of which a fixed lower withdrawal rate was applied, was comparatively less affected by HRT and amount of GAC on the membrane. Under similar conditions (except the withdrawal rate), the performance of the continuous-flow MBR was always better than that of the sequencing batch MBR (color absorbance varying from 0.01 to 0.225 and from 0.01 to 0.9 during different trials, respectively). The difference was particularly significant under shorter HRT and/or when a higher flow rate of withdrawal from the sequencing batch MBR was applied. The main reason of difference of performance between the two types of MBRs were more related to the inevitable differences in withdrawal rates that can be applied in association with those reactor types and the consequent differences in the required amount of GAC on the membrane to effectively prevent leakage of enzyme from the reactor.

The importance of agglomerated fungal morphology on the performance of the proposed reactor was assessed. Mild stirring resulted in granular morphology and concomitantly triggered high enzymatic activity of fungi (around 40 uM substrate/min) which allowed excellent decoloration. However, in the course of operation under non-sterile environment in sequencing batch mode, bacterial contamination eventually occurred. Following this, the fungi granules suffered irrecoverable damage which eventually led to disintegrated morphology with limited enzymatic activity. Promotion of agglomerated growth along with granular morphology was found to ensure resistance against bacterial disruption of fungal body as well as high enzymatic activity (around 30 uM substrate/min). In the course of operation, the granular pellets were observed to extend branches, gradually bond to each other and form networked masses which eventually settled on the reactor-floor. Despite this change, the excellent enzymatic activity and decoloration performance continued. During continuous operation of an MBR containing attached growth under a shortened HRT (1 day as compared to 4.5 days as applied in case of SBR), the level of extracellular enzyme within the reactor plummeted significantly (79%).Nevertheless, the decoloration efficiency sustained. The agglomerated growth, however, was found to be very prone to mechanical shear imposed by accidental increase in stirring speed. Conversely, despite excellent decoloration, the TOC removal ranged from 31 to 70%.

Based on the trials with different reactor arrangements, a two-step removal process-decoloration in a fungi-dominated reactor followed by TOC removal in an MBR containing mixed microbial community-was proposed. By preventing over-flow of fungi from the first reactor to the subsequent MBR by placing a coarse-pore screen at the over-flow outlet, the expected fungal dominance in the stirred reactor and the congenial atmosphere for bacterial TOC removal in the MBR was achieved. The two-step process could attain excellent decoloration of wide varieties of dyes as well as impressive TOC removal (99%). Marked adsorption of dyes onto biomass, especially in the stirred reactor, was observed. Considerable number of white-patches amidst the colored biomass indicated local zones of high enzymatic activity. The fact that the permeate quality did not deteriorate during prolonged period of operation confirmed that the biosorbed dye was subsequently degraded.

The proposed system shows great potential in terms of devising an efficient membrane-based biological treatment system for textile wastewater.

本論文は「Enhancement of dye degradation and mitigation of membrane fouling in a membrane-coupled fungi reactor treating textile wastewater (染色工場排水処理のための膜分離菌類リアクターにおける染料分解促進と膜ファウリング制御」と題し、新たな膜分離菌類リアクターを提案し、これまで染料含有排水処理に適用が試みられながら安定した処理が達成できず実用化されてこなかった白色腐朽菌を用いた連続処理に成功したものであり、また染料分解促進の方法論を提示し、さらに中空糸膜モジュールにスペーサを導入した画期的なコンパクトモジュールの開発に成功した独創的研究である。

第1章は「序論」である。研究の背景、染色工場排水処理の抱える課題、それを受けた研究の必要性、目的と位置づけ、及び論文構成等を述べている。

第2章は「文献レビュー」である。染色工場排水の特徴、含有難分解性物質、毒性、従来の処理法、菌類が産生する分解酵素、その他の染料含有排水処理に関する既往の知見をまとめている。

第3章は「実験材料及び方法」である。用いた菌類や染料の種類、分析方法、実験装置および方法についてまとめている。

第4章は「ファウリング抑制型スペーサ付コンパクト中空糸モジュールの開発」である。根元を織り込んだ中空糸レイヤーとスペーサを重ねて巻き込みモジュール化し、その外周部をプラスチックメッシュで固定した膜モジュールを開発し、染色含有排水の処理に適用したところ、スペーサが膜糸の動きを適度に拘束し膜モジュールの変形を抑制することによりモジュール内への汚泥の進入を防止することが可能となった。また、膜モジュール内に浸入する溶解性ファウリング物質については定期的なインライン化学洗浄を用いて除染するが、その場合にスペーサが汚染物質の排水路となり効果的な化学洗浄が実現した。また、洗浄のためのエアレーションはモジュール外周部だけに、かつ間歇的に与えるだけでよく、洗浄用エアレーション必要量を従来型モジュールより大きく削減することに成功した。ここで開発したモジュールは汎用性が高いと考えられ、今後の浸漬型メンブレンバイオリアクター(MBR)に適用するモジュールの革新への一歩を踏み出すものであると評価できる。

第5章は「菌類の産生する酵素活性及び染料分解効率に及ぼす諸因子」である。本研究で用いた白色腐朽菌Coriolus versicolor, NBRC 9791について、アゾ染料の一種であるPoly S119、Acid Orange II、及び Poly R-478を用いて染料分解性能に関するバッチ試験を行ったものである。染料分解能力に及ぼす主要な因子として菌の形態、攪拌条件等が抽出されたが、それに加え、特に実処理を想定するときに避けられない細菌との競合関係が分解性能に大きく影響を及ぼすことがわかった。その場合、菌類の形態が分散的であると産生した酵素の細菌による分解の影響を大きくうけるため、集塊状態を保持することの重要性が指摘された。

第6章は「汚泥床付き菌類MBRにおける汚泥吸着能の異なる染料の脱色」である。MBR下部に汚泥床を設け、原水を槽上部60%、下部から40%に分配して流入させる方法により、全体の処理性能(脱色率及び全有機炭素除去率(TOC))を向上させることができた。汚泥床は、槽内の汚泥保持量の増大を達成しつつ膜モジュールへの汚泥負荷を減少させる効果があり、また染料を吸着保持する効果が認められた。特に汚泥吸着能の高い染料の場合は、染料が汚泥に吸着された後、徐々に分解させるため、処理効率は非常に高くなる。しかし、汚泥吸着能の低い染料の場合は、なお処理水に着色成分が若干流出することが認められ、その場合は後処理として活性炭カラムに通水することで解決できることを実証した。

第7章は「MBR性能に及ぼす運転モード及び同時吸着デバイスの影響」である。前章までに開発した膜モジュールに、さらに染料及び菌類産生染料分解酵素の吸着能を持たせるため、膜モジュール外周部に活性炭層をコートさせたハイブリッドモジュールを開発した。別に活性炭層が染料及び菌類産生染料分解酵素を吸着し活性炭層で染料の分解が生じていることを確認し、また連続処理実験によりハイブリッドモジュールにより安定処理ができることを実証した。また、コートする活性炭層の量が限られるので、処理性能には通水速度が大きく影響し、その点でろ過時の通水量が大きくならざるを得ないSBR(反復回分槽)運転法よりも連続流運転法の方が優れていることを示した。

第8章は「MBRの処理性能に及ぼす菌類形態の影響」である。MBRを用いた染料含有排水の連続処理試験において、5章で示した集塊を保持する運転条件が重要であることが確認された。また、積極的に集塊を形成するために支持体の存在が効果的であることが示唆された。

第9章は「菌類付着反応槽-MBRシステムの処理性能」である。前章で示唆された結果を受け、菌類を積極的に保持させるための付着型反応槽をMBRに前置する2段処理システムを開発し、その処理性能を調べた。菌類付着反応槽とMBRの間に粗目スクリーンを挿入することにより菌類の流出を出来るだけ防いだ結果、期待通りの処理性能を達成することができた。排水処理として混合培養系にならざる得ない状況において、いかに菌類の有する機能を保持することができるかが実用化の鍵を握り、その意味で菌類を優占させる方法論を提示できた意義は大きい。

第10章は「結論」である。

以上要するに、本論文は、ユニークな発想により、これまでにない画期的なコンパクト膜モジュールの開発に成功し、それを用いた新たな膜分離菌類リアクターを提案し、菌類を用いた染色工場排水処理法の実用化への道を切り開いたものであり、同時に膜分離菌類リアクターにおける基礎的、学術的情報を与える独創の極めて高い研究であると評価できる。また、本研究で得られた知見は、都市環境工学の学術の発展に大きく貢献するものである。

よって本論文は博士(工学)の学位請求論文として合格と認められる。