脱色偶氮染料细菌筛选及固定化强化SBR生物脱色的研究
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摘要
偶氮类活性染料是纺织印染工业中应用最为广泛的一类染料。偶氮染料和其代谢物具有的致癌、致畸、致突变的作用,对人类及其所生存的环境造成了极大的危害。细菌极强的繁殖力和环境适应性,为其在印染废水处理的应用中奠定了基础。
本研究采用梯度平板分离法,从活性污泥中筛选分离得到了一株高效脱色偶氮染料活性黑5的细菌RB5-M1。经细菌形态学观察、生理生化试验及16S rRNA基因序列分析,鉴定菌株RB5-M1为嗜水气单胞菌,命名为Aeromonas hydrophila strain RB5-M1。取10 mL菌液加入90 mL的染料培养基中,温度为35℃,pH 8.0,厌氧条件(氮气85%、二氧化碳6%)培养。24 h测定的平均脱色率可达94.1%,最高脱色率为99.8%。菌株RB5-M1在温度20-40℃、pH 5.0-9.0时,均可对染料脱色,其中脱色最适温度为35℃、最佳pH为8.0。且该菌可适当调节环境酸碱度,有降低染料废水碱污染的能力。
菌株RB5-M1在厌氧条件(氮气85%、二氧化碳6%)下培养时,在培养初期0-24 h内,脱色速率上升很快,脱色效果明显高于静置培养和好氧培养方式；而进入中后期24-72 h,静置培养方式的脱色速率显著上升；在培养72 h后静置培养与厌氧培养方式最终的脱色效率持平。从经济成本考虑,若实际生产中设计适当的反应器,采用静置培养方式来对染料废水进行脱色处理,相比于厌氧或好氧方式或可在达到预期效果的同时,大大节约处理成本。
当活性黑5初始浓度C0为50 mg/L时,菌体延滞期低于5 h,6h测得OD600为0.313；当C0为200 mg/L时,菌体的生长延滞期为12 h,当c0为300 mg/L时,菌体的生长延滞期接近60 h。初始浓度(C0)为50 mg/L～250 mg/L时,用Monod零级反应方程拟合具有较高的相关性,说明零级反应方程能够较好地描述脱色过程,即菌株RB5-M1能以较为恒定的速率脱色活性黑5。而初始浓度(C0)为300 mg/L时,零级反应方程、一级反应方程、二级反应方程的相关系数R2较为接近,考虑到数据点选取的不同可能造成的误差,因此初始浓度大于300 mg/L的染料溶液脱色规律或更符合一级以上动力学方程模型。
当冷却温度为30℃、PVA与海藻酸钠浓度比为10：1、氯化钙浓度为2%、明胶浓度为0.2%时,获得的固定化颗粒I1机械强度较好、包埋量较多,在SBR反应器中初期对模拟染料废水的脱色率较高,后期脱色率明显下降,运行10个周期依然具有脱色效果,颗粒物理形态良好。当氯化钙浓度2%、明胶浓度0.3%、PVA与海藻酸钠浓度9：2、冷却温度20℃,获得的固定化颗粒I2的渗漏量较低,在SBR反应器中运行7个周期内脱色率均可保持在66%以上。
固定化颗粒连续脱色10次以内,脱色率高于61%,渗漏量低于24.53×107个/mL。固定化颗粒在储存前30天中活性良好。脱色率稳定在80%以上。低温条件下,储存时间超过30天的固定化颗粒,直接进行脱色试验,脱色率约为65%。单独含有固定化颗粒或驯化污泥的反应器C1、C2,其在COD去除率、TOC去除率、脱色率方面的表现均不如同时含有两者的反应器C3。由此可见固定化优势脱色菌强化SBR法处理模拟染料废水试验有良好的应用前景。
Azo dye is the most widely used dye in textile and dyeing industry. Azo dyes and its metabolins usually are carcinogenic, teratogenic and mutagenic, which has made a great danger to nature and human survival environment. Bacterial, which was considered to have an exuberant fertility and strong adaptability, shows bright prospect in application of textile and dyeing wastewater treatment.
Through isolating by the gradient plate, we got a strain named RB5-M1 which has good decolorization ability. According to its morphological, physiological characteristics and the analysis of 16S rRNA gene analyses for strain identification,>99.86% of gene sequences in isolated strain RB5-M1 were similar to Aeromonas hydrophila compared to available gene sequences in the NCBI BLAST gene bank. Added 10 mL bacterial culture solution into 90 mL Dye-BPA medium, and cultivated in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%),35℃, pH 8.0. The measurement of decolorization rate showed that decolorization rate performance is 94.1% for average value,99.8% for maximum value. The strain RB5-M1 can decolor reactive black 5 at 20-40℃, pH 5.0-9.0. The optimum conditions are 35℃, pH 8.0. What's more, the strain can also adjust pH value of environment and reduce pollution.
When we cultivated RB5-M1 in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%), for the first 24 hours, decolorization rate increased fast, and it was much better than in static culture and aerobic culture. For the second and third 24 hours, decolorization rate in static culture increased rapidly; after 72 hours, decolorization rate in static culture approximated the decolorization rate in anaerobic culture. From an economic cost consideration, if we can design the appropriate reactor, using this strain by static fermentation, it probably achieves the desired results as anaerobic fermentation, with the significant cost savings.
When initial concentration of reactive black 5 is 50 mg/L, the growth of RB5-M1 hardly delay, the OD600 at 6h is 0.313; if initial concentration of reactive black is 200 mg/L, the lag phase will be 12h; if initial concentration of reactive black is 300 mg/L, the lag phase will need nearly 60 h. When initial concentration of reactive black 5 is 50,100,150,200,250 mg/L, kinetic processing of decoloring highly corresponded with the Monod zero level reaction model, the relative coefficient R2 is 0.9878,0.9725,0.9465,0.964 and 0.902, respectively. When initial concentration of reactive black 5 is 300 mg/L, the relative coefficient R2 of Monod zero level reaction model is close to R2 of Monod 1st level and 2nd reaction model, kinetic processing of decoloring may highly corresponded with the Monod 1st level reaction model.
When stain RB5-M1 was immobilized at the condition of 30℃cooling temperature, PVA: Sodium alginate equals to 10:1,2% concentration of CaCl2,0.2% concentration of gelatin, it could form immobilized globules with higher mechanical strength and embedding biomass. When stain RB5-M1 was immobilized at the condition of 2% concentration of CaCl2,0.3% concentration of gelatin, PVA:Sodium alginate equals to 9:2,20℃cooling temperature, it could form immobilized globules with lower leakage biomass. Meanwhile, result of experiment in SBR (Sequencing Batch Reactor) confirmed the conclusion about immobilization conditions.
The immobilized globules can reapply 10 times while the decolorization rate is higher than 61%, leakage biomass is less than 24.53×107 cells/mL. Immobilized globules are active in the first 30 days; decolorization rate is more than 80%. When the immobilized globules were stored more than 30 days at a low temperature (4℃), do the decoloring test immediately, the decolorization rate will be about 65%. The removal of COD, TOC and decolorization rate of reactor C1 and C2 are worse than C3. Thus it can be seen immobilized advantage decoloring bacteria to enhance the SBR process has a bright prospect in application of textile and dyeing wastewater treatment.
本研究采用梯度平板分离法,从活性污泥中筛选分离得到了一株高效脱色偶氮染料活性黑5的细菌RB5-M1。经细菌形态学观察、生理生化试验及16S rRNA基因序列分析,鉴定菌株RB5-M1为嗜水气单胞菌,命名为Aeromonas hydrophila strain RB5-M1。取10 mL菌液加入90 mL的染料培养基中,温度为35℃,pH 8.0,厌氧条件(氮气85%、二氧化碳6%)培养。24 h测定的平均脱色率可达94.1%,最高脱色率为99.8%。菌株RB5-M1在温度20-40℃、pH 5.0-9.0时,均可对染料脱色,其中脱色最适温度为35℃、最佳pH为8.0。且该菌可适当调节环境酸碱度,有降低染料废水碱污染的能力。
菌株RB5-M1在厌氧条件(氮气85%、二氧化碳6%)下培养时,在培养初期0-24 h内,脱色速率上升很快,脱色效果明显高于静置培养和好氧培养方式；而进入中后期24-72 h,静置培养方式的脱色速率显著上升；在培养72 h后静置培养与厌氧培养方式最终的脱色效率持平。从经济成本考虑,若实际生产中设计适当的反应器,采用静置培养方式来对染料废水进行脱色处理,相比于厌氧或好氧方式或可在达到预期效果的同时,大大节约处理成本。
当活性黑5初始浓度C0为50 mg/L时,菌体延滞期低于5 h,6h测得OD600为0.313；当C0为200 mg/L时,菌体的生长延滞期为12 h,当c0为300 mg/L时,菌体的生长延滞期接近60 h。初始浓度(C0)为50 mg/L～250 mg/L时,用Monod零级反应方程拟合具有较高的相关性,说明零级反应方程能够较好地描述脱色过程,即菌株RB5-M1能以较为恒定的速率脱色活性黑5。而初始浓度(C0)为300 mg/L时,零级反应方程、一级反应方程、二级反应方程的相关系数R2较为接近,考虑到数据点选取的不同可能造成的误差,因此初始浓度大于300 mg/L的染料溶液脱色规律或更符合一级以上动力学方程模型。
当冷却温度为30℃、PVA与海藻酸钠浓度比为10：1、氯化钙浓度为2%、明胶浓度为0.2%时,获得的固定化颗粒I1机械强度较好、包埋量较多,在SBR反应器中初期对模拟染料废水的脱色率较高,后期脱色率明显下降,运行10个周期依然具有脱色效果,颗粒物理形态良好。当氯化钙浓度2%、明胶浓度0.3%、PVA与海藻酸钠浓度9：2、冷却温度20℃,获得的固定化颗粒I2的渗漏量较低,在SBR反应器中运行7个周期内脱色率均可保持在66%以上。
固定化颗粒连续脱色10次以内,脱色率高于61%,渗漏量低于24.53×107个/mL。固定化颗粒在储存前30天中活性良好。脱色率稳定在80%以上。低温条件下,储存时间超过30天的固定化颗粒,直接进行脱色试验,脱色率约为65%。单独含有固定化颗粒或驯化污泥的反应器C1、C2,其在COD去除率、TOC去除率、脱色率方面的表现均不如同时含有两者的反应器C3。由此可见固定化优势脱色菌强化SBR法处理模拟染料废水试验有良好的应用前景。
Azo dye is the most widely used dye in textile and dyeing industry. Azo dyes and its metabolins usually are carcinogenic, teratogenic and mutagenic, which has made a great danger to nature and human survival environment. Bacterial, which was considered to have an exuberant fertility and strong adaptability, shows bright prospect in application of textile and dyeing wastewater treatment.
Through isolating by the gradient plate, we got a strain named RB5-M1 which has good decolorization ability. According to its morphological, physiological characteristics and the analysis of 16S rRNA gene analyses for strain identification,>99.86% of gene sequences in isolated strain RB5-M1 were similar to Aeromonas hydrophila compared to available gene sequences in the NCBI BLAST gene bank. Added 10 mL bacterial culture solution into 90 mL Dye-BPA medium, and cultivated in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%),35℃, pH 8.0. The measurement of decolorization rate showed that decolorization rate performance is 94.1% for average value,99.8% for maximum value. The strain RB5-M1 can decolor reactive black 5 at 20-40℃, pH 5.0-9.0. The optimum conditions are 35℃, pH 8.0. What's more, the strain can also adjust pH value of environment and reduce pollution.
When we cultivated RB5-M1 in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%), for the first 24 hours, decolorization rate increased fast, and it was much better than in static culture and aerobic culture. For the second and third 24 hours, decolorization rate in static culture increased rapidly; after 72 hours, decolorization rate in static culture approximated the decolorization rate in anaerobic culture. From an economic cost consideration, if we can design the appropriate reactor, using this strain by static fermentation, it probably achieves the desired results as anaerobic fermentation, with the significant cost savings.
When initial concentration of reactive black 5 is 50 mg/L, the growth of RB5-M1 hardly delay, the OD600 at 6h is 0.313; if initial concentration of reactive black is 200 mg/L, the lag phase will be 12h; if initial concentration of reactive black is 300 mg/L, the lag phase will need nearly 60 h. When initial concentration of reactive black 5 is 50,100,150,200,250 mg/L, kinetic processing of decoloring highly corresponded with the Monod zero level reaction model, the relative coefficient R2 is 0.9878,0.9725,0.9465,0.964 and 0.902, respectively. When initial concentration of reactive black 5 is 300 mg/L, the relative coefficient R2 of Monod zero level reaction model is close to R2 of Monod 1st level and 2nd reaction model, kinetic processing of decoloring may highly corresponded with the Monod 1st level reaction model.
When stain RB5-M1 was immobilized at the condition of 30℃cooling temperature, PVA: Sodium alginate equals to 10:1,2% concentration of CaCl2,0.2% concentration of gelatin, it could form immobilized globules with higher mechanical strength and embedding biomass. When stain RB5-M1 was immobilized at the condition of 2% concentration of CaCl2,0.3% concentration of gelatin, PVA:Sodium alginate equals to 9:2,20℃cooling temperature, it could form immobilized globules with lower leakage biomass. Meanwhile, result of experiment in SBR (Sequencing Batch Reactor) confirmed the conclusion about immobilization conditions.
The immobilized globules can reapply 10 times while the decolorization rate is higher than 61%, leakage biomass is less than 24.53×107 cells/mL. Immobilized globules are active in the first 30 days; decolorization rate is more than 80%. When the immobilized globules were stored more than 30 days at a low temperature (4℃), do the decoloring test immediately, the decolorization rate will be about 65%. The removal of COD, TOC and decolorization rate of reactor C1 and C2 are worse than C3. Thus it can be seen immobilized advantage decoloring bacteria to enhance the SBR process has a bright prospect in application of textile and dyeing wastewater treatment.
引文
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