混合菌群共培养对偶氮染料的协同脱色及降解的研究
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摘要
印染废水是国内外公认的最难处理的废水之一,这是因为废水中所含染料具有化学性质稳定,难以被生物降解的特点。其中偶氮染料占的比例最大,占染料总量50%以上。筛选能高效脱色降解偶氮染料的微生物,将其应用到印染废水处理上已经成为研究的热点之一。单一的脱色菌对偶氮染料的脱色效率低,降解不彻底,容易形成二次污染,混合菌群则能克服这些缺点。研究混合菌群共培养协同降解偶氮染料,可为提高印染废水的生物处理效率提供技术支持和相应的理论依据。
本研究从印染废水筛选出能高效对偶氮染料直接蓝289脱色的混合菌群QM。将其分离、纯化,得到两种菌株,一株为真菌M3,一株为细菌Q1。单一的菌株进行脱色,脱色率较低,将两种菌株共培养则能显著提高脱色效率,说明两者具有协同对偶氮染料直接蓝289脱色的能力。从形态、生理生化及16SrDNA,18SrDNA基因序列分析方面对两种菌进行鉴定,确定细菌Q1为蜡状芽孢杆菌,真菌M3为白地霉。
研究了混合菌群QM对直接蓝289的脱色条件,混合菌群QM脱色较佳条件为:温度30℃、初始pH7.0、静止状态。混合菌群QM具备一定的抗盐能力。与单一的菌株相比,混合菌群的脱色对环境条件的适应范围较广。混合菌群QM对多种不同结构的偶氮染料具有脱色作用,体现较广谱的脱色能力。
研究表明混合菌群QM的对偶氮染料的脱色是在共代谢条件下进行的,葡萄糖、氯化铵为共代谢脱色较适合的碳、氮源。运用响应面设计法对共代谢脱色中的三个主要因素葡萄糖、氯化铵、染料浓度进行优化。得到三者的优化条件为:葡萄糖2.80g/L,氯化铵1.60g/L,染料0.15g/L。在染料浓度小于500mg/L下,混合菌群QM对直接蓝289脱色过程符合一级反应动力学,脱色速率与染料浓度之间的关系符合Haldane抑制方程,方程式为v = S + S2 /7122.93.202S + 94.32。
研究了单一菌株Q1、M3及混合菌群QM对直接蓝289的脱色机制。结果表明菌株Q1的脱色反应是在低氧化还原电位下进行的。菌株M3脱色过程中,初期脱色主要是由菌体吸附完成的,脱色率较低,当脱色到第4d和5d时,脱色率显著提高,脱色率与漆酶的活性成正比。使用白地霉粗酶液(漆酶的酶活为6450U/L)对直接蓝289进行脱色,1.5h脱色率达到了90.2%。在混合菌群QM对直接蓝289的脱色过程中,菌株M3在培养前期能显著降低培养液的氧化还原电位。运用紫外-可见光谱扫描、HPLC、HPLC-MS技术对直接蓝289脱色的中间产物进行分析,证实混合菌群QM能将直接蓝289降解成小分子的化合物。鉴定的中间产物有3-羟基-2,7-氨基-萘磺酸钠,邻苯二甲酸。推断其中的一条脱色降解途径为直接蓝289偶氮键断裂形成3-羟基-2,7-氨基-萘磺酸钠等芳胺化合物,芳胺化合物进一步被降解生成邻苯二甲酸。初步判断混合菌群QM协同降解染料效应的机制是脱色初期菌株M3显著降低脱色液中氧化还原电位,低氧化还原电位促进菌株Q1对直接蓝289的脱色,脱色的中间产物能被菌株M3进一步降解,其中可能与菌株M3产的漆酶相关。
使用海藻酸钙包埋菌株Q1,尼龙网载体吸附菌株M3,将两种菌株固定化。固定化菌株混合培养对直接蓝289进行脱色,结果发现固定化菌株保持较高的脱色活性。紫外-可见光谱扫描、HPLC分析证明固定化菌群能将直接蓝289降解。固定化菌群对直接蓝289的脱色符合一级反应动力学,具备连续脱色的能力。固定化菌群对实际印染废水进行处理,结果表明固定化菌群既能脱色,又能部分降低废水COD含量。
Treatment of dyeing wastewater is considered to be one of the most challenging tasks due to stable chemical structure of dyes recalcitrant to biodegradation. The majority (over 50%) of industrially synthesized dyes are azo dyes. Researchers have been focusing their attention on screening of effective microorganisms to decolorize azo dyes and its application to dyeing wastewater treatment. Individual strain has low efficiency in decolorization and cannot degrade azo dye completely, causing second pollution. Mixed consortium is capable of overcoming these shortcomings. The study of the co-culture of mixed consortium to degrade azo dye can provide technological support as well as theoretical foundation for treatment of dying wastewater.
Mixed consortium QM, with remarkable ability to decolorize the Direct Blue 289(DB289), was isolated from the dyeing wastewater. Two groups of strains were purified and obtained: fungi M3 and bacteria Q1. Decolorization rate of DB289 by individual strain was low, but the co-culture of two strains enhanced decolorization efficiency. The results showed that the two strains exhibited syngersitic ability in decolorizing the azo dyes. Morphological, biochemical characterization as well as 16SrDNA, 18SrDNA gene sequences analysis indicated that the strain Q1 was Bacillus cereus and the strain M3 was Geotrichum candidum.
The study of conditions for DB298 decolorization by mixed consortium QM was carried out. Optimal conditions was initial pH 7 and temperature at 30℃under static condition. Mixed consortium QM in this study seemed to be salt-tolerating in decolorization. Compared with individual strain, mixed consortium QM in decolorization adapt to environmental conditions on a wider scope. Besides, it was capable of decolorizing structurally different azo dyes, and exhibited a broader dye-decolorizing spectrum.
Studies have shown that decolorization of azo dyes by mixed consortium QM was carried out under conditions of co-metabolism. Glucose, ammonium chloride is more suitable as carbon and nitrogen sources for decolorization of co-metabolism. Response Surface Methodology (RSM) have been applied to optimize the three major factors for co-metabolism decolorization: glucose, ammonium chloride, dye concentration. The RSM analysis indicated that the optimized conditions for the above factors are 2.80g / L, ammonium chloride: 1.60g / L, dye: 0.15g / L respectively. At the dye concentration of less than 500mg / L, the decolorization process of mixed consoritium QM in DB 289 fits the first-class kinetics model. The relationship between the decolorization rate and dye concentration is in accordance with the Haldane Inhibition Equation. The Equation is
The study of the mechanism of DB289 decolorization by individual strain (Q1 and M3) and mixed consortium QM was investigated. The result showed that the strain Q1 was decolorized under the low oxidation-reduction potential (ORP). Initially the decolorization by the strain M3 was achieved by absorption of mycelium, and the decolorizaiton rate was low.The decolorizaiton rate increased on the 4th and 5th. The decolorization rate is in positive proportion to the activity of Laccase. Decolorization rate of DB 289 by crude Laccase (the enzyme activity is 6450U/L) produced by Geotrichum candidum reached 90.2% after 1.5h. During the decolorizaiton process of DB 289 by mixed consortium QM, the strain M3 can reduce ORP of the culture significantly. UV-Vis scan, HPLC as well as HPLC-MS was used to analyze the intermediate of DB289 decolorization. The result showed low molecular compounds produced with DB289 reduction were further degraded by mixed consortium. The intermediates were generally suggested to be aromatic amines such as 3-hydroxy-2,7-amino-naphthalene sulfonate and phthelic acid. The possible degradation pathway was therefore proposed. It is inferred that the first step of the decolorization is the cleavage in azo bonds of DB289 followed by the formation of aromatic amines, which is further degraded to phthelic acid. The mechanism of decolorization by mixed consortium QM was probably that initially ORP was reduced remarkably by the strain M3 , low ORP promoted decolorization of DB289 by the strain Q1, the intermediate of decolorization can be further degraded by the strain M3, which might involve the Laccase produced by the strain M3.
The strain Q1 was immobilized with calcium alginate, and the strain M3 with nylon net carrier. Decolorization capacity was retained during the decolorization of DB289 by immobilized mixed consortium. Scanning Electron Microscopy confirmed that after decolorization the consortium grew well in the carriers. UV-Vis scan, HPLC analysis shows that immobilized mixed consortium can degrade DB 289 efficiently. Decolorization of DB 289 by immobilized mixed consortium fits the first-class kinetics. It has a continuous decolorization capacity. Treatment of real dyeing wastewater by the immobilized mixed consortium showed that they could partially remove color and COD.
本研究从印染废水筛选出能高效对偶氮染料直接蓝289脱色的混合菌群QM。将其分离、纯化,得到两种菌株,一株为真菌M3,一株为细菌Q1。单一的菌株进行脱色,脱色率较低,将两种菌株共培养则能显著提高脱色效率,说明两者具有协同对偶氮染料直接蓝289脱色的能力。从形态、生理生化及16SrDNA,18SrDNA基因序列分析方面对两种菌进行鉴定,确定细菌Q1为蜡状芽孢杆菌,真菌M3为白地霉。
研究了混合菌群QM对直接蓝289的脱色条件,混合菌群QM脱色较佳条件为:温度30℃、初始pH7.0、静止状态。混合菌群QM具备一定的抗盐能力。与单一的菌株相比,混合菌群的脱色对环境条件的适应范围较广。混合菌群QM对多种不同结构的偶氮染料具有脱色作用,体现较广谱的脱色能力。
研究表明混合菌群QM的对偶氮染料的脱色是在共代谢条件下进行的,葡萄糖、氯化铵为共代谢脱色较适合的碳、氮源。运用响应面设计法对共代谢脱色中的三个主要因素葡萄糖、氯化铵、染料浓度进行优化。得到三者的优化条件为:葡萄糖2.80g/L,氯化铵1.60g/L,染料0.15g/L。在染料浓度小于500mg/L下,混合菌群QM对直接蓝289脱色过程符合一级反应动力学,脱色速率与染料浓度之间的关系符合Haldane抑制方程,方程式为v = S + S2 /7122.93.202S + 94.32。
研究了单一菌株Q1、M3及混合菌群QM对直接蓝289的脱色机制。结果表明菌株Q1的脱色反应是在低氧化还原电位下进行的。菌株M3脱色过程中,初期脱色主要是由菌体吸附完成的,脱色率较低,当脱色到第4d和5d时,脱色率显著提高,脱色率与漆酶的活性成正比。使用白地霉粗酶液(漆酶的酶活为6450U/L)对直接蓝289进行脱色,1.5h脱色率达到了90.2%。在混合菌群QM对直接蓝289的脱色过程中,菌株M3在培养前期能显著降低培养液的氧化还原电位。运用紫外-可见光谱扫描、HPLC、HPLC-MS技术对直接蓝289脱色的中间产物进行分析,证实混合菌群QM能将直接蓝289降解成小分子的化合物。鉴定的中间产物有3-羟基-2,7-氨基-萘磺酸钠,邻苯二甲酸。推断其中的一条脱色降解途径为直接蓝289偶氮键断裂形成3-羟基-2,7-氨基-萘磺酸钠等芳胺化合物,芳胺化合物进一步被降解生成邻苯二甲酸。初步判断混合菌群QM协同降解染料效应的机制是脱色初期菌株M3显著降低脱色液中氧化还原电位,低氧化还原电位促进菌株Q1对直接蓝289的脱色,脱色的中间产物能被菌株M3进一步降解,其中可能与菌株M3产的漆酶相关。
使用海藻酸钙包埋菌株Q1,尼龙网载体吸附菌株M3,将两种菌株固定化。固定化菌株混合培养对直接蓝289进行脱色,结果发现固定化菌株保持较高的脱色活性。紫外-可见光谱扫描、HPLC分析证明固定化菌群能将直接蓝289降解。固定化菌群对直接蓝289的脱色符合一级反应动力学,具备连续脱色的能力。固定化菌群对实际印染废水进行处理,结果表明固定化菌群既能脱色,又能部分降低废水COD含量。
Treatment of dyeing wastewater is considered to be one of the most challenging tasks due to stable chemical structure of dyes recalcitrant to biodegradation. The majority (over 50%) of industrially synthesized dyes are azo dyes. Researchers have been focusing their attention on screening of effective microorganisms to decolorize azo dyes and its application to dyeing wastewater treatment. Individual strain has low efficiency in decolorization and cannot degrade azo dye completely, causing second pollution. Mixed consortium is capable of overcoming these shortcomings. The study of the co-culture of mixed consortium to degrade azo dye can provide technological support as well as theoretical foundation for treatment of dying wastewater.
Mixed consortium QM, with remarkable ability to decolorize the Direct Blue 289(DB289), was isolated from the dyeing wastewater. Two groups of strains were purified and obtained: fungi M3 and bacteria Q1. Decolorization rate of DB289 by individual strain was low, but the co-culture of two strains enhanced decolorization efficiency. The results showed that the two strains exhibited syngersitic ability in decolorizing the azo dyes. Morphological, biochemical characterization as well as 16SrDNA, 18SrDNA gene sequences analysis indicated that the strain Q1 was Bacillus cereus and the strain M3 was Geotrichum candidum.
The study of conditions for DB298 decolorization by mixed consortium QM was carried out. Optimal conditions was initial pH 7 and temperature at 30℃under static condition. Mixed consortium QM in this study seemed to be salt-tolerating in decolorization. Compared with individual strain, mixed consortium QM in decolorization adapt to environmental conditions on a wider scope. Besides, it was capable of decolorizing structurally different azo dyes, and exhibited a broader dye-decolorizing spectrum.
Studies have shown that decolorization of azo dyes by mixed consortium QM was carried out under conditions of co-metabolism. Glucose, ammonium chloride is more suitable as carbon and nitrogen sources for decolorization of co-metabolism. Response Surface Methodology (RSM) have been applied to optimize the three major factors for co-metabolism decolorization: glucose, ammonium chloride, dye concentration. The RSM analysis indicated that the optimized conditions for the above factors are 2.80g / L, ammonium chloride: 1.60g / L, dye: 0.15g / L respectively. At the dye concentration of less than 500mg / L, the decolorization process of mixed consoritium QM in DB 289 fits the first-class kinetics model. The relationship between the decolorization rate and dye concentration is in accordance with the Haldane Inhibition Equation. The Equation is
The study of the mechanism of DB289 decolorization by individual strain (Q1 and M3) and mixed consortium QM was investigated. The result showed that the strain Q1 was decolorized under the low oxidation-reduction potential (ORP). Initially the decolorization by the strain M3 was achieved by absorption of mycelium, and the decolorizaiton rate was low.The decolorizaiton rate increased on the 4th and 5th. The decolorization rate is in positive proportion to the activity of Laccase. Decolorization rate of DB 289 by crude Laccase (the enzyme activity is 6450U/L) produced by Geotrichum candidum reached 90.2% after 1.5h. During the decolorizaiton process of DB 289 by mixed consortium QM, the strain M3 can reduce ORP of the culture significantly. UV-Vis scan, HPLC as well as HPLC-MS was used to analyze the intermediate of DB289 decolorization. The result showed low molecular compounds produced with DB289 reduction were further degraded by mixed consortium. The intermediates were generally suggested to be aromatic amines such as 3-hydroxy-2,7-amino-naphthalene sulfonate and phthelic acid. The possible degradation pathway was therefore proposed. It is inferred that the first step of the decolorization is the cleavage in azo bonds of DB289 followed by the formation of aromatic amines, which is further degraded to phthelic acid. The mechanism of decolorization by mixed consortium QM was probably that initially ORP was reduced remarkably by the strain M3 , low ORP promoted decolorization of DB289 by the strain Q1, the intermediate of decolorization can be further degraded by the strain M3, which might involve the Laccase produced by the strain M3.
The strain Q1 was immobilized with calcium alginate, and the strain M3 with nylon net carrier. Decolorization capacity was retained during the decolorization of DB289 by immobilized mixed consortium. Scanning Electron Microscopy confirmed that after decolorization the consortium grew well in the carriers. UV-Vis scan, HPLC analysis shows that immobilized mixed consortium can degrade DB 289 efficiently. Decolorization of DB 289 by immobilized mixed consortium fits the first-class kinetics. It has a continuous decolorization capacity. Treatment of real dyeing wastewater by the immobilized mixed consortium showed that they could partially remove color and COD.
引文
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