【作者】 司静；

【导师】 戴玉成； 崔宝凯；

【作者基本信息】 北京林业大学 ， 森林保护学， 2014， 博士

【摘要】 近年来,合成染料的广泛应用造成大量印染废水,这些废水不仅会影响水体的美观性,还会使水体的透光性和气体溶解度减小,从而阻碍光照的投射,造成自然水体的污染、水生动植物的死亡、生态系统的失衡,直接或间接对人体产生危害。因此,对染料的脱色和降解己成为处理废水的关键问题,而寻找高效稳定且能够对多种染料均有脱色作用的白腐真菌菌株就显得十分必要。本研究的主要目的是从42株白腐真菌菌株中筛选获得相对高效的脱色菌株,分析脱色过程中生物降解及吸附作用两个方面,并对在降解过程中起重要作用的漆酶进行了纯化。研究结果表明,通过固体及液体培养,从42株白腐真菌菌株中筛选得到菌株绒毛栓孔菌(Trametes pubescens (Schumach.) Pilat) Cui7571对偶氮染料刚果红的脱色能力最强。在此过程中,其菌丝体可被连续使用3次,且循环次数与染料脱色率之间存在着负相关线性关系。红外光谱(Fourier transform-infrared spectroscopy, FT-IR)分析和气相色谱-质谱联用(Gas chromatography-mass spectroscopy, GC-MS)分析确定了经绒毛栓孔菌降解的染料刚果红代谢产物为萘胺、联苯胺、联苯和叠氮萘。植物毒性试验证明绒毛栓孔菌对偶氮染料具有一定的脱毒作用。液体培养过程中,绒毛栓孔菌的产漆酶能力与丙二醛(Malondialdehyde, MDA)含量(P<0.01)、超氧化物歧化酶(Superoxide dismutase, SOD)活性(P<0.05)、总抗氧化能力(Total antioxidant capacity, T-AOC)(P<0.01)、过氧化氢(Hydrogen peroxide, H2O2)水平(P<0.05)和抗坏血酸(Ascorbic acid, AA)含量(P<0.05)呈正相关,与过氧化氢酶(Catalase, CAT)活性(P<0.01)和抑制羟自由基能力(Restraining ability to hydroxyl free radical, RAHFR)(P<0.05)呈负相关,这说明白腐真菌绒毛栓孔菌合成漆酶的能力与其自身的抗氧化能力密切相关,而这一过程可通过活性氧进行调控。绒毛栓孔菌在对偶氮染料刚果红的脱色过程中,漆酶Tplac的活性最高,经三步纯化后,比活力可达18.543U/mg,是粗酶液的16.016倍。经纯化的酶是一类单体蛋白,分子量为68.0kDa。最适底物为2,2’-连氮-双(3-乙基苯并噻唑-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS),最适pH为5.0,最适温度为50℃,此时催化速率(kcat/Km)为8.34s1-μM-1。此外,Tplac对碱性条件(pH7.0-10.0)具有较高的活性、稳定性和耐受性。添加终浓度为250mmol/L的金属离子后酶活力仍可维持起始活力的88%,此过程中酶对底物的亲和力也较高,说明Tplac对金属离子的耐受力较强。低浓度的叠氮化钠(NaN3)、二硫苏糖醇(DDT)和L-半胱氨酸可导致Tplac活力下降,而金属螫合剂乙二胺四乙酸(EDTA)对Tplac活力没有明显影响。由于漆酶Tplac具有与众不同的性质,因此可被应用于较多工业领域,如染料废水处理等。为提高漆酶Tplac的稳定性和回收率,通过采用壳聚糖作为载体、戊二醛作为交联剂对其进行了固定化处理。在此过程中,交联剂戊二醛的最适浓度为0.8%(v/v)最适交联时间为3h,最适给酶量为2.0mL(约为43.672U/mL),最适固定时间为4h。与游离漆酶相比,固定化漆酶Tplac的pH适应能力和抗热变性能力均有所增强,具有良好的使用稳定性。此外,利用热处理(121℃、20min)方法将绒毛栓孔菌菌丝体制备成的生物吸附剂对偶氮染料刚果红同样具有较好的脱色效果。Box-Behnken Design全因子设计得到脱色过程中最适盐度、吐温80添加浓度、温度、pH和染料浓度分别为1.1%(w/v)、3.5%(v/v)、41℃、6.2和114.3mg/L。通过红外光谱分析、化学改性和扫描电镜(Scanning electron microscope, SEM)观察得到,生物吸附剂对染料的吸附作用主要是由它们结构中所含基团之间的静电作用力所致。试验发现利用海藻酸钠或大孔树脂D201固定化处理也可显著提高绒毛栓孔菌菌丝体生物吸附剂对偶氮染料刚果红的吸附能力。在此过程中最适pH为2.0,最适初始染料浓度为100mg/L,并且该吸附作用受离子强度的影响较小。此外,绒毛栓孔菌菌丝体生物吸附剂对刚果红的吸附行为符合Freundlich等温线模型和假二级动力学模型。通过扫描电镜、红外光谱分析和X-射线衍射(X-ray diffraction, XRD)分析发现,生物吸附剂吸附染料前后的显微结构、表面官能团及晶体性质等发生了明显变化。解吸附试验证明该生物吸附剂可被连续使用。无营养条件并未明显减弱绒毛栓孔菌菌丝体对偶氮染料刚果红的脱色能力,表明该种方法可有效地减少成本,提高染料处理的实用性。在此过程中,菌丝体可被连续使用2次,且其所分泌的酶系可降解染料；最适初始pH、温度、染料浓度和盐度分别为2.0、30。C、80mg/L和2.5%(w/v),此时染料脱色率达80.52%。气相色谱-质谱联用分析得到其降解产物为萘胺、联苯胺和叠氮萘。植物毒性试验显示无营养条件下经绒毛栓孔菌菌丝体降解后的染料代谢产物对植物的毒性大大降低。上述通过对白腐真菌绒毛栓孔菌催化偶氮染料刚果红脱色过程中涉及的生物降解及吸附作用两个方面进行系统性研究与分析,阐明了绒毛栓孔菌对染料刚果红的脱色途径,意在为染料的无污染化处理奠定基础,同时也为白腐真菌的工业化应用提供相应的理论依据和技术手段。更多还原

【Abstract】 Recently, synthetic dyes extensively used in many industries can be found in wastewaters. Such effluents lead to a reduction in sunlight penetration, which in turn decrease photosynthetic activity, dissolved oxygen concentration, and water quality, and have acute toxic effects in aquatic flora and fauna, causing severe environmental problems worldwide. Cleaning up of the environment by removal of hazardous contaminants from textile effluents is a crucial and challenging problem needing numerous approaches to reach long-lasting suitable solutions. Accordingly, main objectives of the present study were:(i) to screen the white rot fungal strain with higher dye decolorization capability,(ii) to study the biodegradation and adsorption during decolorization process and (iii) to purify the extracellular laccase from the target strain by a three-step method.The experimental results displayed that, among the42white rot fungal strains, Trametes pubescens Cui7571was the target strain with higher decolorization capacity for azo dye Congo Red. During the decolorization process, the strain’s biomass could be continuously utilized for three times with declining decolorization rates. The biodegraded metabolites of azo dye Congo Red were identified as naphthalene amine, biphenyl amine, biphenyl and naphthalene diazonium by fourier transform-infrared spectroscopy (FT-IR) and gas chromatography-mass spectroscopy (GC-MS). Phytotoxicity studies revealed that the dye biodegradation by T. pubescens resulted in its detoxification.During the liquid cultivation of T. pubescens, increases in the contents of malondialdehyde (MDA)(P<0.01), hydrogen peroxide (H2O2)(P<0.05) and ascorbic acid (AA)(P<0.05) were observed. The higher activities of superoxide dismutase (SOD)(P<0.05) and total antioxidative capacity (T-AOC)(P <0.01) still could be detected during this period. However, higher restraining ability to hydroxyl free radical (RAHFR)(P<0.05) and catalase activity (CAT)(P<0.01) had negative influence on laccase activity. It was concluded that a relationship between laccase synthesis and antioxidative capability exists in fungal cells, which can be regulated by reactive oxygen.Extracellular laccase (Tplac) from T. pubescens was purified to homogeneity by a three-step method, which resulted in a high specific activity of18.543U/mg,16.016-fold greater than that of crude enzyme at the same level. Tplac is a monomeric protein that has a molecular mass of68.0kDa. The enzyme demonstrated high activity toward1.0mmol/L2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) at an optimum pH of5.0and temperature of50℃, and under these conditions, the catalytic efficiency(kcat/Km) is8.34s-1μM-1. Tplac is highly stable and resistant under alkaline conditions, with pH ranging from7.0to10.0. Interestingly, above88%of initial enzyme activity was maintained in the presence of metal ions at25.0mmol/L, leading to an increase in substrate affinity, which indicated that the laccase is highly metal-tolerant. These unusual properties demonstrated that the new fungal laccase Tplac has potentials for the specific industrial or environmental applications.In order to improve the stability and recovery, chitosan as carrier and glutaraldehyde as cross-linking agent were employed for immobilization of T. pubescens purified laccase Tplac. During the immobilization process, optimal concentration of cross-linking agent glutaraldehyde, cross-linking time, enzyme solution and immobilized time were0.8%(v/v),3h,2.0mL (about43.672U/mL) and4h respectively. Additionally, pH adaptability and thermal stability of immobilized Tplac were much higher than those of free laccase. These findings promoted the development of immobilized laccase Tplac for environmental and other fields.Meanwhile, a novel design of dye decolorization was conducted with the sorbents obtained from T. pubescens biomass in submerged cultivation. The results demonstrated that the sorbents prepared by heat treatment (121℃,20min) exhibited preferable decolorization capacity for azo dye Congo Red. Moreover, the decolorization rate-dependent influencing factors, i.e. salinity, Tween80, temperature, pH and dye concentration, were optimized by Box-Behnken full factorial design. When they were1.1%(w/v),3.5%(v/v),41℃,6.2and114.3mg/L respectively, the decolorization rate was up to99.23%after a7-day incubation period. Adsorptive decolorization was caused by the electrostatic forces between dye molecules and sorbents obtained from T. pubescens biomass, as evidenced by FT-IR, chemical modifications and scanning electron microscope (SEM) data.The T. pubescens biomass immobilized by sodium alginate or macroporous resin D201enhanced the sorption capacity for azo dye Congo Red approximately4-fold of the free biomass. Dye uptake was favored by acidic conditions at pH2.0with increasing initial dye concentration up to100mg/L using less biomass at room temperature and agitation speed. Adsorption of dyes onto the biomass was weakly dependent on ionic strength. Additionally, the adsorption process followed the pseudo-second-order kinetic and Freundlich isotherm models. During this process, the morphological changes on the biomass surface occurred, the amine functional groups present on the cell surface were mainly responsible for this process, and reduction in crystallinity of the biomass was observed, as confirmed by SEM, FT-IR and XRD respectively. Desorption experiments were performed to regenerate the sorbent, making the process more economic and environment friendly.Compared to the general treatment with fresh biomass, no dramatic variations were observed from the dye removal efficiencies by T. pubescens biomass without addition of nutrition. Thus, the results indicated that the treatment by fungal biomass without addition of nutrition performed well at lowering the capital and running cost, and enhancing convenient availability for dye decolorization. During the decolorization process, the biomass presented good persistence in repetitive decolorization operations, as well as high potentials towards the biodegradation of dyes which could be attributed to the presence of the biotransformation enzymes. After a7-day incubation period, a higher dye removal efficiency of80.52%was obtained with the optimal parameters initial pH at2.0, temperature at30℃, dye concentration at80mg/L and salinity at2.5%(w/v) respectively. In addition, the biodegraded metabolites were identified as naphthalene amine, biphenyl amine and naphthalene diazonium by GC-MS. Phytotoxicity studies displayed the less toxic nature of the biodegraded metabolites with respect to plant seeds.In this study, white rot fungus T. pubescens was observed to decolorize azo dye Congo Red more efficiently. During the decolorization process, the two simultaneous steps, adsorption between the biomass and the macromolecular dyes and enzymatic biodegradation, were elucidated systematically. The results may facilitate a better application of pollution-free treatment of various dyes and support the utilization of fungi in many industrial fields.更多还原