Exiguobacterium sp.TL的性能及其处理高盐偶氮染料废水的研究
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摘要
本文利用高效耐盐偶氮染料降解微生物探索高效、稳定脱色并彻底矿化偶氮染料废水的生物处理工艺。考察了染料浓度、盐度及金属离子对野生菌群宏观处理效果及微观群落结构的影响；从野生土壤菌群中筛选耐盐偶氮还原菌株,对其进行鉴定及特性进行考察,并考察其与氧化还原介体的耦合作用强化偶氮脱色过程；针对偶氮染料脱色中间产物矿化难的问题,利用青霉菌-细菌联合体系改善了脱色过程及矿化程度。主要包括如下研究内容：
以野生土壤微生物群落为研究对象,驯化其对偶氮染料进行脱色,考察了染料浓度、盐度及金属离子对菌群脱色不同偶氮染料的影响。在一定的浓度范围内,结构相对简单的偶氮染料脱色过程受初始染料浓度影响较小,而结构复杂的染料脱色速率会随着初始浓度的提升明显下降。盐度的提升会抑制染料的生物脱色过程,但随着驯化时间的延长,活性会有所恢复,说明菌群具有一定的耐盐能力。一些金属离子会对菌群的脱色活性产生抑制作用,1 mmol／L的不同金属离子对脱色过程的抑制作用顺序为：Cu2+>Al3+和Zn2+>Mg2+、Ca2+和pb2+；随着离子浓度的提升,Al3+和Zn2+产生的抑制作用会增强,而Mg2+和Ca2+的影响几乎不变。DGGE指纹图谱一定程度上显示了群落结构在不同条件下的动态变化,序列分析结果显示,优势菌种主要属于Bacillus、Pseudomonas、Sedimentibacter、Clostridiales、Streptomyces等菌属。
从该野生土壤群落中分离得到1株耐盐偶氮染料脱色菌,命名为TL。通过形态观察、生理生化和16S rDNA序列分析,确定其归属于Exiguobacterium属,其16S rDNA全序列登录GenBank数据库,序列号为EU159578。首次验证了该菌属具有耐盐偶氮还原功能。
以活性艳红X-3B为目标物,考察了菌株TL的基本特性,确定了其生长、降解的最佳条件为：接种量6%(v／v),pH=5.4-7.0,温度为30-40℃,盐度(NaCl)≤150g／L。相比于Na+和K+的影响,Mg2+和Ca2+的加入能够促进菌株TL对偶氮染料的脱色,而Al3+、Zn2+、Fe3+和Cu2+则会不同程度上抑制菌株TL的活性。菌株TL不能很好的利用葡萄糖、蔗糖、乳糖等作为共代谢基质进行脱色,但铵盐、硝酸盐和亚硝酸盐均为较适宜的无机氮源。其脱色反应符合一级反应动力学规律,当初始浓度小等于700 mg／L时反应动力学常数为0.22-0.26 h-1,而当浓度在800-1000 mg／L时,反应动力学常数明显下降。通过UV-Vis、LC-MS等分析手段验证,菌株TL对偶氮染料的脱色过程符合典型生物还原规律。
氧化还原介体——蒽醌对菌株TL的完整细胞还原脱色活性艳红X-3B过程具有催化作用,其最佳条件为：染料浓度小等于75 mg／L,蒽醌浓度0.6 g／L,接种量6 g／L(湿重),温度30-40℃(30℃最佳),pH值为3.02-4.00(主要为吸附作用)和7.00-9.08(主要为生物还原作用)；相比于Na+,Mg2+和Ca2+可以促进脱色过程,Zn2+、Al3+、Fe3+和Cu2+则会不同程度上抑制脱色过程。利用菌TL的生物强化作用和蒽醌介体的催化作用,模拟脱色处理酸性大红GR废水,结果显示,生物强化作用能够使工艺启动时间比非强化体系缩短了约2.5 d,而蒽醌的加入能够将低负荷和高负荷进水条件下的脱色速率分别提高1000 mg／(g·d)和500 mg／(g·d),同时满足了快速启动和稳定、高效运行的双重目的,但蒽醌会对未经驯化的体系有抑制作用,需在工艺启动完毕后加入。PCR-DGGE结果显示,采用分步耦合强化的启动模式下,微生物群落结构稳定,强化菌株能够与活性污泥中的其他优势菌种共存,说明其具有较强的环境适应能力。
考察了具有偶氮脱色能力的青霉菌Penicillium sp. QQ-细菌Exiguobacterium sp. TL联合生物体系对高盐X-3B废水的脱色过程及脱色中间产物的初步解析,并利用该联合体系建立新型的“厌氧-好氧”组合工艺对高盐X-3B废水进行矿化处理。首先利用表面响应法对联合体系的最佳脱色条件进行了模拟,得出最佳条件为：青霉菌QQ接种量132.67 g／L,细菌TL接种量1.09 g／L,葡萄糖浓度2.25 g／L,酵母粉浓度2.10g／L,温度33.0℃,pH值6.50,初始X-3B浓度235.14 mg／L；ANOVA方差分析结果显示该模型及各考察因素对脱色过程均具有显著性影响,而其中青霉菌QQ的接种量与酵母粉浓度的协同作用具有显著性影响。青霉菌QQ、细菌TL联合作用条件下以及细菌TL单独作用条件下X-3B脱色产物的HPLC图谱具有明显的差别,根据图谱及宏观现象推断前者主要为小分子大极性化合物,为芳香胺化合物的进一步分解产物,而后者则很有可能为对应的芳香胺化合物及其自发氧化、聚合产物。通过比较确立了新型的“厌氧-好氧”组合生物工艺,厌氧及好氧生物单元的接种物分别为青霉菌QQ+细菌TL和活性污泥,200mg／L的活性艳红X-3B经该组合工艺处理后,可被完全矿化。
The purpose of this dissertation is to establish a high-salt azo dye treatment method which not only can effectively and stably decolorize but also mineralize dyes. The study was started from the investigation of efficient salt-tolerant and azo dye-reducing microbial community, mainly including the impact of initial dye concentration, salinity and metal ions from. An efficient salt-tolerant azo dye decolorizing bacterial strain was isolated, identified and its characteristics were investigated. Then the co-augmentation effect of the strain and anthraquinone on azo dye biodecolorization by activity sludge system for further improvement was studied. Finally, the synergism of a Penicillium and bacterial strain was used to promote the mineralization of azo dyes by only microbial treatment method. The contents of this dissertation were as follows:
The influences of initial dye concentration, salinity and metal ions on azo dye decolorization by a wild soil microbial consortium were investigated. The results showed that when the initial concentration or salinity increased, the impact to the decolorization of simpler-structure dyes was less. Some metal ions would inhibit the activity of microbial community and the order of inhibition was as follows:Cu2+> Al3+ and Zn2+> Mg2+, Ca2+and Pb2+. Besides, as the concentration of metal ions increased, the inhibition effects of Al3+and Zn2+ increased, but there was little change for Mg2+ and Ca2+. DGGE fingerprint showed the dynamic shifts of microbial community structures and the dominant species were mainly belonging to Bacillus, Pseudomonas, Sedimentibacter, Clostridiales, and Streptomyces.
A salt-tolerant bacterial strain which could decolorize azo dyes was isolated and was named as TL. It was identified as genus Exiguobacterium by morphology, physiology and biochemistry and 16S rDNA sequence analysis. The 16S rDNA sequence of strain TL had been deposited in the GenBank database under accession number EU159578. It was identified as a novel azo dye-reducing and salt-tolerant bacterium according to the previous reports.
The characteristics of strain TL were investigated and the optimal conditions for growth and decolorization were:inoculum amount 6% (v/v), pH value 5.4-7.0, temperature 30-40℃, salinity (NaCl)≤15%(w/v). Mg2+ and Ca2+ promoted decolorization, however, Al3+, Zn2+, Fe3+ and Cu2+ could inhibite the process in different extent. Strain TL could not decolorize azo dyes efficiently when using glucose, sucrose and lactose as carbon source. Whereas, it could grow and decolorize azo dyes depending on several inorganic nitrogen source. Decolorization of active brilliant red X-3B in the medium containing peptone as carbon source followed the first-order reaction kinetics. The kinetics constants were about 0.22-0.26 mg/(L-h) when the initial dye concentrations were not more than 700 mg/L, and it decreased to nearly a half when the initial dye concentrations were higher than 700 mg/L. Decolorization of azo dye by strain TL was identified as typical azo reduction pathway by UV-Vis and LC-MS.
The redox mediator anthraquinone could catalyze the reduction of azo dyes by the whole cells of strain TL and the optimal conditions were:initial dye concentration less than 75 mg/L, anthraquinone concentration 0.6 g/L, inoculum amount 6 g/L (wet weight), temperature 30-40℃(optimum of 30℃), pH value 3.0-4.0 (mainly bio-adsorption) and 7.0-9.0 (mainly bio-reduction). Compared with Na+, Mg2+ and Ca2+ could promote the decolorization; Zn2+, Al3+, Fe3+and Cu2+ inhibited it. The activated sludge system which was started-up by bioaugmentation with strain TL firstly and then enhanced by anthraquinone was determined as the most efficient mode of co-augmented system. This process realized both of fast-start-up and efficient & stable operation. Compared with non-augmented system, the start-up time was 2.5 d shorter and the color removal loadings were about 1000 mg/(g-d) (initial dye concentration was about 700 mg/L),500 mg/(g-d) (initial dye concentration was about 1000 mg/L), respectively. The results of PCR-DGGE showed that the the consortium was stable and strain TL could co-exist with the aboriginal microorganisms in the activated sludge.
Penicillium sp. strain QQ was introduced to the azo dye wastewater treatment system in order to improve the biodegradability of the effluence of anaerobic unit. The optimal decolorization conditions by the synergism of strains TL and QQ was optimized and simulated by response surface methodology (RSM), and the results were:inoculum of strains TL and QQ were 1.09 g/L and 132.67 g/L (wet weight), concentrations of glucose and yeast extract were 2.25 g/L and 2.10 g/L, temperature 33.0℃, pH value 6.50, initial concentration of active brilliant red X-3B 235.14 mg/L, respectively. The inoculum of strain QQ and yeast extract concentration showed synergistic effect on decolorization. ANOVA results demonstrated that the model and tested factors were significant to decolorization by the synergism system. HPLC results showed that some compounds of low molecular and high polarity might be produced in the synergism system. Whereas, the major decolorization products in the system only contained bacterial strain TL might be high molecular compounds, which was inferred as corresponding aromatic amines and their auto-oxidation and auto-polymerization products. The anaerobic unit inoculated with strains QQ and TL followed by the aerobic unit inoculated with activated sludge were chosen as the optimal combined biological process. The dye could be mineralized with this combined process.
以野生土壤微生物群落为研究对象,驯化其对偶氮染料进行脱色,考察了染料浓度、盐度及金属离子对菌群脱色不同偶氮染料的影响。在一定的浓度范围内,结构相对简单的偶氮染料脱色过程受初始染料浓度影响较小,而结构复杂的染料脱色速率会随着初始浓度的提升明显下降。盐度的提升会抑制染料的生物脱色过程,但随着驯化时间的延长,活性会有所恢复,说明菌群具有一定的耐盐能力。一些金属离子会对菌群的脱色活性产生抑制作用,1 mmol／L的不同金属离子对脱色过程的抑制作用顺序为：Cu2+>Al3+和Zn2+>Mg2+、Ca2+和pb2+；随着离子浓度的提升,Al3+和Zn2+产生的抑制作用会增强,而Mg2+和Ca2+的影响几乎不变。DGGE指纹图谱一定程度上显示了群落结构在不同条件下的动态变化,序列分析结果显示,优势菌种主要属于Bacillus、Pseudomonas、Sedimentibacter、Clostridiales、Streptomyces等菌属。
从该野生土壤群落中分离得到1株耐盐偶氮染料脱色菌,命名为TL。通过形态观察、生理生化和16S rDNA序列分析,确定其归属于Exiguobacterium属,其16S rDNA全序列登录GenBank数据库,序列号为EU159578。首次验证了该菌属具有耐盐偶氮还原功能。
以活性艳红X-3B为目标物,考察了菌株TL的基本特性,确定了其生长、降解的最佳条件为：接种量6%(v／v),pH=5.4-7.0,温度为30-40℃,盐度(NaCl)≤150g／L。相比于Na+和K+的影响,Mg2+和Ca2+的加入能够促进菌株TL对偶氮染料的脱色,而Al3+、Zn2+、Fe3+和Cu2+则会不同程度上抑制菌株TL的活性。菌株TL不能很好的利用葡萄糖、蔗糖、乳糖等作为共代谢基质进行脱色,但铵盐、硝酸盐和亚硝酸盐均为较适宜的无机氮源。其脱色反应符合一级反应动力学规律,当初始浓度小等于700 mg／L时反应动力学常数为0.22-0.26 h-1,而当浓度在800-1000 mg／L时,反应动力学常数明显下降。通过UV-Vis、LC-MS等分析手段验证,菌株TL对偶氮染料的脱色过程符合典型生物还原规律。
氧化还原介体——蒽醌对菌株TL的完整细胞还原脱色活性艳红X-3B过程具有催化作用,其最佳条件为：染料浓度小等于75 mg／L,蒽醌浓度0.6 g／L,接种量6 g／L(湿重),温度30-40℃(30℃最佳),pH值为3.02-4.00(主要为吸附作用)和7.00-9.08(主要为生物还原作用)；相比于Na+,Mg2+和Ca2+可以促进脱色过程,Zn2+、Al3+、Fe3+和Cu2+则会不同程度上抑制脱色过程。利用菌TL的生物强化作用和蒽醌介体的催化作用,模拟脱色处理酸性大红GR废水,结果显示,生物强化作用能够使工艺启动时间比非强化体系缩短了约2.5 d,而蒽醌的加入能够将低负荷和高负荷进水条件下的脱色速率分别提高1000 mg／(g·d)和500 mg／(g·d),同时满足了快速启动和稳定、高效运行的双重目的,但蒽醌会对未经驯化的体系有抑制作用,需在工艺启动完毕后加入。PCR-DGGE结果显示,采用分步耦合强化的启动模式下,微生物群落结构稳定,强化菌株能够与活性污泥中的其他优势菌种共存,说明其具有较强的环境适应能力。
考察了具有偶氮脱色能力的青霉菌Penicillium sp. QQ-细菌Exiguobacterium sp. TL联合生物体系对高盐X-3B废水的脱色过程及脱色中间产物的初步解析,并利用该联合体系建立新型的“厌氧-好氧”组合工艺对高盐X-3B废水进行矿化处理。首先利用表面响应法对联合体系的最佳脱色条件进行了模拟,得出最佳条件为：青霉菌QQ接种量132.67 g／L,细菌TL接种量1.09 g／L,葡萄糖浓度2.25 g／L,酵母粉浓度2.10g／L,温度33.0℃,pH值6.50,初始X-3B浓度235.14 mg／L；ANOVA方差分析结果显示该模型及各考察因素对脱色过程均具有显著性影响,而其中青霉菌QQ的接种量与酵母粉浓度的协同作用具有显著性影响。青霉菌QQ、细菌TL联合作用条件下以及细菌TL单独作用条件下X-3B脱色产物的HPLC图谱具有明显的差别,根据图谱及宏观现象推断前者主要为小分子大极性化合物,为芳香胺化合物的进一步分解产物,而后者则很有可能为对应的芳香胺化合物及其自发氧化、聚合产物。通过比较确立了新型的“厌氧-好氧”组合生物工艺,厌氧及好氧生物单元的接种物分别为青霉菌QQ+细菌TL和活性污泥,200mg／L的活性艳红X-3B经该组合工艺处理后,可被完全矿化。
The purpose of this dissertation is to establish a high-salt azo dye treatment method which not only can effectively and stably decolorize but also mineralize dyes. The study was started from the investigation of efficient salt-tolerant and azo dye-reducing microbial community, mainly including the impact of initial dye concentration, salinity and metal ions from. An efficient salt-tolerant azo dye decolorizing bacterial strain was isolated, identified and its characteristics were investigated. Then the co-augmentation effect of the strain and anthraquinone on azo dye biodecolorization by activity sludge system for further improvement was studied. Finally, the synergism of a Penicillium and bacterial strain was used to promote the mineralization of azo dyes by only microbial treatment method. The contents of this dissertation were as follows:
The influences of initial dye concentration, salinity and metal ions on azo dye decolorization by a wild soil microbial consortium were investigated. The results showed that when the initial concentration or salinity increased, the impact to the decolorization of simpler-structure dyes was less. Some metal ions would inhibit the activity of microbial community and the order of inhibition was as follows:Cu2+> Al3+ and Zn2+> Mg2+, Ca2+and Pb2+. Besides, as the concentration of metal ions increased, the inhibition effects of Al3+and Zn2+ increased, but there was little change for Mg2+ and Ca2+. DGGE fingerprint showed the dynamic shifts of microbial community structures and the dominant species were mainly belonging to Bacillus, Pseudomonas, Sedimentibacter, Clostridiales, and Streptomyces.
A salt-tolerant bacterial strain which could decolorize azo dyes was isolated and was named as TL. It was identified as genus Exiguobacterium by morphology, physiology and biochemistry and 16S rDNA sequence analysis. The 16S rDNA sequence of strain TL had been deposited in the GenBank database under accession number EU159578. It was identified as a novel azo dye-reducing and salt-tolerant bacterium according to the previous reports.
The characteristics of strain TL were investigated and the optimal conditions for growth and decolorization were:inoculum amount 6% (v/v), pH value 5.4-7.0, temperature 30-40℃, salinity (NaCl)≤15%(w/v). Mg2+ and Ca2+ promoted decolorization, however, Al3+, Zn2+, Fe3+ and Cu2+ could inhibite the process in different extent. Strain TL could not decolorize azo dyes efficiently when using glucose, sucrose and lactose as carbon source. Whereas, it could grow and decolorize azo dyes depending on several inorganic nitrogen source. Decolorization of active brilliant red X-3B in the medium containing peptone as carbon source followed the first-order reaction kinetics. The kinetics constants were about 0.22-0.26 mg/(L-h) when the initial dye concentrations were not more than 700 mg/L, and it decreased to nearly a half when the initial dye concentrations were higher than 700 mg/L. Decolorization of azo dye by strain TL was identified as typical azo reduction pathway by UV-Vis and LC-MS.
The redox mediator anthraquinone could catalyze the reduction of azo dyes by the whole cells of strain TL and the optimal conditions were:initial dye concentration less than 75 mg/L, anthraquinone concentration 0.6 g/L, inoculum amount 6 g/L (wet weight), temperature 30-40℃(optimum of 30℃), pH value 3.0-4.0 (mainly bio-adsorption) and 7.0-9.0 (mainly bio-reduction). Compared with Na+, Mg2+ and Ca2+ could promote the decolorization; Zn2+, Al3+, Fe3+and Cu2+ inhibited it. The activated sludge system which was started-up by bioaugmentation with strain TL firstly and then enhanced by anthraquinone was determined as the most efficient mode of co-augmented system. This process realized both of fast-start-up and efficient & stable operation. Compared with non-augmented system, the start-up time was 2.5 d shorter and the color removal loadings were about 1000 mg/(g-d) (initial dye concentration was about 700 mg/L),500 mg/(g-d) (initial dye concentration was about 1000 mg/L), respectively. The results of PCR-DGGE showed that the the consortium was stable and strain TL could co-exist with the aboriginal microorganisms in the activated sludge.
Penicillium sp. strain QQ was introduced to the azo dye wastewater treatment system in order to improve the biodegradability of the effluence of anaerobic unit. The optimal decolorization conditions by the synergism of strains TL and QQ was optimized and simulated by response surface methodology (RSM), and the results were:inoculum of strains TL and QQ were 1.09 g/L and 132.67 g/L (wet weight), concentrations of glucose and yeast extract were 2.25 g/L and 2.10 g/L, temperature 33.0℃, pH value 6.50, initial concentration of active brilliant red X-3B 235.14 mg/L, respectively. The inoculum of strain QQ and yeast extract concentration showed synergistic effect on decolorization. ANOVA results demonstrated that the model and tested factors were significant to decolorization by the synergism system. HPLC results showed that some compounds of low molecular and high polarity might be produced in the synergism system. Whereas, the major decolorization products in the system only contained bacterial strain TL might be high molecular compounds, which was inferred as corresponding aromatic amines and their auto-oxidation and auto-polymerization products. The anaerobic unit inoculated with strains QQ and TL followed by the aerobic unit inoculated with activated sludge were chosen as the optimal combined biological process. The dye could be mineralized with this combined process.
引文
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