摘要
我国染料业、纺织印染业发达,染料废水对环境的污染更为严重。80%以上的染料为含偶氮键、多聚芳香环的复杂有机化合物,为有毒难降解有机物,化学稳定强,具有致癌、致畸、致突变的“三致”作用。因此寻找降解和吸附染料的微生物并研究其机理,成为当今环境微生物领域的热点。本文详细的研究了Shewanella oneidensis MR-1菌株降解偶氮染料的性质并克隆表达了偶氮还原酶,筛选并研究了真菌吸附剂对染料的吸附行为。论文的主要研究结果如下：
(1) Shewanella oneidensis MR-1菌株降解特征及偶氮还原酶性质
Shewanella oneidensis MR-1在静止的条件下对甲基橙和酸性黄199染料的脱色率可达99.36%和78.25%。该菌对甲基橙和酸性黄199最适的pH范围分别为4.0-7.0和6.0-8.0。外源添加Mg2+能够轻微的促进偶氮染料的降解,而Pb2+,Cd2+,Cu2+,Fe3+和Fe2+则显著抑制染料的脱色降解过程。酶学分析发现NADH-DCIP和偶氮还原酶活性在降解过程中显著升高。经过PCR获得偶氮还原酶序列,克隆表达之后具有偶氮还原酶活性,最大酶催化速率可达220.59 U/mg,在pH6.5的时候,达到最大比活为153.16 U/mg,对NADH具有偏好性。25 mM Mg2+能够增强偶氮还原酶的活性。生物信息学分析发现该偶氮还原酶属于FMN依赖型的NADH偶氮还原酶家族。
(2)真菌吸附染料的机理研究
青霉YW01吸附剂对酸性黑172和刚果红在pH 3.0、40℃和初始染料浓度为800 mg L-1的条件下的最大吸附量分别为225.38和411.53 mg g-1。吸附过程能够被Langmuir等温吸附方程拟合,说明单层吸附现象发生。动力学方程的分析发现,内部扩散为吸附限制因素。经遗传算法改进的人工神经网络分析得知,最适染料浓度和温度分别为吸附酸性黑172和刚果红的最大影响因素。CPC改性的青霉YW01吸附剂能够提高吸附量。磷酸盐缓冲液体系虽能够提高吸附速率,但产生更大的边界层效应。
在单一吸附体系中,CDAB改性的米曲霉吸附剂对酸性蓝25和酸性红337的吸附量分别为160.36和280.39 mg g-1,是未改性吸附剂的1.52和1.66倍。在双相吸附体系中,未改性的和CDAB改性的吸附剂与单一吸附体系相比对两种染料的吸附能力显著降低,说明存在竞争吸附现象。相对竞争性分析发现,存在浓度关键节点决定吸附剂对两类染料的选择性。[AB 25]/[AR 337]浓度之比大于0.63时,酸性蓝25的生物吸附占优势地位。通过神经网络研究9种真菌吸附剂表征和操作条件对吸附活性黑5染料的过程影响时发现,pH为影响吸附过程最重要的因素,其次依次为氮元素含量,初始染料浓度、碳元素含量。而且真菌对染料的吸附能力并不与比表面积成正比。
The dye and textile industries are booming and important for economic growth in China, but the pollutants cased by these industries are crucial problems we should not ignore for sustainable development. More than 80% dyes having azo bond and aromatic structure are of chemical stability, carcinogenicity, teratogenicity and mutagenesis. Therefore, to find effective microorganisms having biosorption and/or biodegradation capabilities for dyes are received more and more attention from environmental scientists. In this study, the decolorization characteristics of Shewanella oneidensis MR-1 and adsorption behavior of fungal biosorbents for dyes were investigated. The main results are as follows:
(1) Decolorization and azoreductase characteristics of Shewanella oneidensis MR-1
Shewanella oneidensis MR-1 was found to reach 99.36% and 78.25% decolorization for Methyl Orange and Acid Yellow 199 in solutions, respectively. The suitable pH range for decolorization of Methyl Orange and Acid Yellow 199 by S. oneidensis MR-1 was 4.0-7.0 and 6.0-8.0, respectively The azo dyes removal by S. oneidensis MR-1 was slightly enhanced by addition of Mg2+, but inhibited by Pb2+, Cd2+, Cu2+, Fe3+and Fe2+. The enzyme activities of NADH-DCIP reductase and azoreductase were 2.67 and 3.0 times higher, and 1.92 and 2.48 times higher, respectively in the Methyl Orange treatment and in the Acid Yellow 199 treatment as compared to the control treatment. These findings indicated that the azo dyes decolorization by S. oneidensis MR-1 was via reduction mechanism. The azoreductase was found to reach maximum enzyme velocity 220.59 U/mg, while no enzyme activities were found for the putative azoreductase toward Methyl Red. Azoreductase had highest specific activity (153.16 U/mg) at pH 6.5, which also showed a preference for NADH compared to NADPH as electron donor.
(2) Biosorption mechanism of fungi for dyes
Maximum biosorption capacities of 225.38 and 411.53 mg g"1 under initial dye concentration of 800 mg L-1, pH 3.0 and 40℃conditions were observed for Acid Black 5 (AB) and Congo Red (CR) for Penicillium YW 01, respectively. The Weber-Morris model analysis indicated that intraparticle diffusion was the limiting step for biosorption of AB and CR onto biosorbent. Analysis based on the artificial neural network and genetic algorithms hybrid model indicated that initial dye concentration and temperature appeared to be the most influential parameters for biosorption process of AB and CR onto biosorbent, respectively. The values of initial biosorption rate of biosorbent in phosphoric-phosphate buffer were found to be higher than that of corresponding values in aqueous solution, indicating phosphoric-phosphate buffer enhanced the initial biosorption rate of biosorption process. Weber-Morris model analysis indicated that the boundary layer effect had more influence on the biosorption process in phosphoric-phosphate buffer.
In single system, the biosorption capacities of CDAB-modified biosorbent reached 160.36 and 280.39 mg g-1 for Acid Blue 25 (AB 25) and Acid Red 337 (AR) 337, respectively, which were 1.52 and 1.66 times higher than that of unmodified biosorbent. In binary system, the biosorption capacities of unmodified and CDAB-modified biosorbents for both dyes decreased significantly compared to that in single system. Relative competitiveness analysis demonstrated that there existed critical initial concentration ratio which determined the predominance of dyes during biosorption process. The biosorption of AB 25 was found to be in dominant position at initial concentration ratio of [AB 25]/[AR 337] above 0.63. Sensitivity analysis of the factors affecting biosorption examined by an artificial neural network model showed that pH was the most important parameter, explaining 22%, followed by nitrogen content (16%), initial dye concentration (15%) and carbon content (10%). The biosorption capacities were not proportional to surface areas of the sorbents, but were instead influenced by their surface chemical characteristics. The data further suggest that differences in carbon and nitrogen contents may be used as a selection index for identifying effective biosorbents.
引文
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