摘要
印染工业废水是当前水体主要污染源之一。由于传统理化处理技术成本高、二次污染大,亟待研制成本低、高效、环保的生物处理技术。某些真菌具有底物降解的广谱性、对高pH及底物毒性耐受能力高等特性,在印染工业废水处理中具有特殊的应用价值。探究这类真菌对染料脱色的机理及相关酶学研究,可为利用真菌处理染料废水尤其是碱性染料废水提供理论依据。
以蒽醌染料RBBR为降解底物,在不同pH(5-10)下比较了实验室10株具有潜在降解特性的真菌对染料脱色的效果,结果发现菌株IMER1在碱性条件下对RBBR有很好的脱色属性。采用分子鉴定结合形态分类结果显示:其ITS序列与漆斑菌属(Myrothecium sp.)同源性达到90%以上;菌落形态、菌丝、分生孢子以及孢子梗的观察结果与漆斑菌属形态特征完全相似,因此确定此菌为Myrothecium sp,命名为Myrothecium sp.IMER1。
比较了IMER1对5种不同染料的脱色结果,同时系统地研究了其对蒽醌染料RBBR的脱色机理。结果表明菌株IMER1对不同染料的脱色效果不同,依次为酸性靛蓝>RBBR>刚果红>孔雀绿>溴酚蓝,对酸性靛蓝和RBBR染料脱色效果较好。对RBBR的脱色过程研究表明,胞外胆红素氧化酶(bilirubin oxidase,BOX)活性与RBBR脱色率有良好的相关性。脱色过程的前期,以菌体的生物吸附为主,吸附率约占脱色率的71.3%,而降解率只占29%左右;中后期,以BOX降解作用为主,最终的降解率约占脱色率的90%。对菌株IMER1降解RBBR动力学研究表明,降解过程符合一级动力学方程,其反应动力学常数随染料浓度的增加而减小。
将菌株IMER1发酵后的滤液经五步分离纯化,SDS和活性电泳检测纯化的蛋白为BOX酶蛋白,分子量约为64kD,纯化倍数为152.54。BOX酶学性质研究结果表明:氧化ABTS的最适温度为55°C左右,pH值在4左右;氧化胆红素的最适pH值约为7.5;Km和Vmax值表明其最适底物为胆红素。不同金属离子和有机小分子的存在影响BOX活性,Fe2+能完全抑制BOX酶活;而EDTA、乙醇、冰乙酸和甘氨酸对BOX酶活抑制作用较小。纯化的BOX酶能使5种染料发生不同程度的脱色。
研究了BOX及其介体系统对RBBR的作用机理及降解途径。与对照BOX酶相比,BOX-ABTS介体体系对RBBR的脱色速率可提高11倍,最适脱色pH从5上升到8。紫外-可见光全波长扫描RBBR降解液的图谱分析结果表明,RBBR的发色基团(蒽醌环上的羰基)和助色基团(-NH3)被改变;进一步采用GC-MS和LC-MS分析结果表明RBBR降解的主要产物为邻苯二甲酸及其衍生物。BOX-ABTS介体系统对RBBR的降解动力学研究表明,其反应动力学符合假一级反应动力学。
对菌株IMER1在液体和固体培养体系分泌BOX酶进行了研究。液体培养体系条件优化结果表明:当pH、温度和葡萄糖分别为5.7、28°C、8g/L时,BOX酶产量在PDB中可达到最优值0.643U/mL。且在PDB中BOX酶的生物合成模式为中期合成型。固体培养体系条件优化结果表明:当含水量、pH和温度分别为83.5%,4.6,23°C时,BOX酶产量可达到优化值13.45U/g。
研究了菌株IMER1及BOX对工业碱性染料废水脱色以及工艺。结果显示菌株IMER1及BOX在中性到碱性范围内对不同浓度碱性染料废水均有较好的脱色作用。菌株的脱色机理主要是吸附和降解,高浓度的废水对菌体生长有强烈的抑制作用。ABTS介体能够缩短BOX处理染料废液的时间并提高脱色效果。在鼓泡式反应器中,菌株IMER1的麸皮固体发酵菌剂对染料酸性靛蓝、RBBR脱色效果明显,但对工业碱染料废水的脱色效果一般,有待进一步研究。
Dye wastewaters have been one of the main sources of severe pollution problems worldwide. Conventional chemical and physical techniques possess inherent limitations such as high cost, formation of hazardous by-products, and intensive energy requirements. As a feasible alternative, dye decolorization using microorganisms, has recently received much attention owing to its ease of application, low cost, and environmental benignity. Some fungi have been applied to the treatment of dye effluent, owing their characterizations of broad substrates and tolerance to high concentration of toxic xenobiotics and alkali. To understand the decolorization mechanism of these fungi will provide the theoretical foundation to expand their application to decolorize dye waste, especially basic dye waste.
A comparative study on decolorization of RBBR dye by 10 strains in pH5-10 was performed and the results showed that IMER1 has high-efficient decolorization of RBBR, especially including basic pHs. To identify the species of this strain, its ITS sequence is cloned and its colony, spore, conidiophores, and mycelium morphologies are observed under microscope. These results showed that this strain is Myrothecium sp, and named as Myrothecium sp.IMER1..
The mechanism of decolorization of RBBR by strain IMER1 was investigated comprehensively. Decolorizing efficiency is indigo carmine>RBBR>congo red>malachite green> bromophenol blue, and the ability of triphenylmethane dyes decolorized by strain IMER1 is poor. It was proved that in the course of decolorization of RBBR the extracellular enzyme, bilirubin oxidase (BOX), has a good correlation with decolorization and playes a key role. The adsorption of the dye by cells is observed at the beginning of the decolorization, then the color becomes faint and finally disappeares when BOX is released by the strain. Additionally, the visual observation and UV spectral analysis demonstrated that decolorization involved biosorption and biodegradation. The kinetics of RBBR decolorization can be described by first-order reaction rate equation, and the K value decreases with the RBBR concentration.
Bilirubin oxidase is purified by the five steps, which is demonstrated to be bilirubin oxidase by SDS and native PAGE, and its molecular weight is estimated to be about 64kD.The optimum temperature for enzyme activity is 55°C and the optimum pH for bilirubin and ABTS are 7.5 and 4, respectively. Fe2+ has completely inhibition action, while EDTA、ethanol、acetic acid、glycine have little. The purified BOX can decolorize the 5 dyes.
The mechanisms of decolorization of RBBR by BOX and BOX-ABTS were investigated comprehensively. The results of the visual observation and UV spectral analysis of RBBR show that the chromophores of RBBR are decomposed resulting in degradation of RBBR at last. The analysis results of GC-MS and LC-MS indicate that the main intermediate products are phthalic acid and derivatives. The kinetics of RBBR degradation could be described by first-order reaction rate equation.
The optimum conditions for the production of BOX show that pH, temperature, glucose are 5.7, 28°C, 8g/L, respectively, the largest production of BOX is 0.643U/mL. The biosynthesis of BOX in PDB is middle phase model. On the other hand, the optimized water content, pH, temperature in solid culture are 83.5%,4.6,23°C, respectively, the largest production of BOX is13.45U/g .
Decolorization of basic dye effluent by strain IMER1 and BOX were investigated, including the feasibility of technology in reactor. Both IMER1 and BOX are able to decolorize basic dye effluent, while high concentration dye effluent will inhibit the growth of IMER1. Decolorization rate by ABTS is improved. The mixture of IMER1 and wheat bran is better for decolorization of single dye such as indigo carmine and RBBR than basic-dye-effluent in air lift bioreactor, so it is necessary to redesign the structure of reactor and optimize the program and conditions in order to improve the treatment effect for dye effluent.
引文
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