耐冷苯胺降解菌的特性及其对苯胺废水处理的强化作用
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摘要
本课题在充分理解生物强化技术一般机理的前提下,以苯胺作为目标污染物,针对东北寒冷地区冬季水温低、特种工业废水生物处理工程效果差等问题,研究了高效耐冷苯胺降解菌JH-9的特性以及生物强化效能,并率先提出了“混合菌丝球”的概念。对发酵过程中产生的霉菌菌丝球进行了技术开发,采用混合菌种固定化技术将高效耐冷苯胺降解菌固定在霉菌Y3形成的纯生物质载体上。详细描述了混合菌丝球的形成过程及效能,并对成球机制进行了初步探讨。
在低温下对吉林化工厂污水处理厂曝气池活性污泥、低温生活污水处理系统曝气池活性污泥、实验室菌种库保存的高效菌剂,以及以上三者的混合菌群四种样品进行了变温培养、驯化,筛选到低温下降解苯胺的高效菌群—吉化污泥。该菌群属于耐冷菌群,低温下对苯胺有较好的降解效果,菌胶团形成能力较强,并且表现出很强的生物絮凝能力。从吉化污泥中分离筛选到一株低温下(10℃)能够较好降解苯胺的耐冷菌株JH-9,它能以苯胺为唯一碳源、氮源生长,通过Sherlock脂肪酸鉴定系统、16S rRNA等分析方法初步鉴定为醋酸钙不动杆菌(Acinetobacter-calcoaceticus)。从环境因子、培养基组分、接菌量、底物多样性、共代谢、反应动力学等生理生态学角度对JH-9降解特性进行了研究,得出低温下其降解苯胺的最适条件为pH 7.0,C/P 1:1~2:1,接菌量为10%(v/v)。葡萄糖和微量元素可以促进JH-9对苯胺的降解;而苯酚会抑制其对苯胺的降解。当菌体起始浓度一定时,苯胺降解率及平均降解速率主要与苯胺初始浓度有关。在较低的苯胺浓度范围内,苯胺降解率和降解速率较高,遵循Monod方程;当苯胺浓度较高时,其降解过程以基质抑制型的Haldane方程为主。
采用生物强化技术将菌株JH-9、吉化污泥及两者等量混合的菌群作为强化菌剂投加到SBR反应系统中,测定苯胺的去除能力是否提高,并考察TOC、TN及微生物活性等指标的变化,用DGGE的方法对投菌强化前后反应器中活性污泥系统菌群多样性进行分析。结果表明,在反应器内温度为12℃、摇床转速160r/min、pH 7.0、反应周期为48h等条件,三个强化系统都可以提高苯胺的降解能力,苯胺降解率在74.2%～100%,TOC的去除率83%～88.8%,而TN变化不明显。系统中异养菌的活性远远大于硝化细菌和亚硝化细菌。
考察苯胺浓度冲击负荷、pH冲击负荷、苯酚毒物负荷及贫营养条件下系统生物强化效果,发现在不同冲击负荷条件下各个强化系统体现的降解苯胺的优势不同,在pH负荷下,单菌强化效果相对较好,在苯胺浓度负荷和苯酚毒物负荷冲击下,单菌和吉化污泥的混合菌群强化效果较好,在贫营养负荷冲击下,吉化污泥菌群强化效果较好。在整个强化实验中,强化系统对TOC的去除效果要好于对照系统,而对TN的去除却没有明显的优势。
以霉菌Y3形成的菌丝球作为生物质载体分别采用同时培养法和吸附法对菌株JH-9进行固定化,并率先提出了一种新型固定化方法“同时培养法”。两种方法的对比结果表明:同时接种法在单位时间内可固定化细菌量更多、其堆积体积更大,成球总数量更多,球体直径较小,总重量和总相对密度也较小。同时培养法形成的混合菌丝球内部细菌是非常均匀的排列生长在每一根菌丝上,无论菌丝交联与否;而吸附法形成的混合菌丝球内部,细菌只存在于多条菌丝交叉形成平台的地方。
对混合菌丝球的降解效能进行考察发现,混合菌丝球对苯胺保持了良好的降解效能,同时培养法形成的混合菌丝球对苯胺的降解效能要明显好于吸附法形成的混合菌丝球,且效果稳定、持久。与固定化陶粒相比,混合菌丝球对苯胺的降解能力略显优势。菌丝球再生实验表明:经过碎片繁殖再生的混合菌丝球能很好的保持对苯胺的降解能力。为了进一步在反应器中考察混合菌丝球的生物强化效能,将混合菌丝球、高效降解苯胺混合菌群和菌株JH-9作为强化菌剂应用于SBR工艺中。结果表明:针对初始苯胺浓度为130mg/L的苯胺废水,混合菌丝球从第三个运行周期开始即实现了出水苯胺的去除率达100%、COD的去除率在60%以上,并一直稳定维持到运行的第八个周期。通过考查混合菌丝球的形成过程及机制发现:曲霉Y3和菌株JH-9同时培养形成混合菌丝球经历4个阶段,静电引力和细胞表面的粘附力是混合菌丝球形成的主要原因。
The dissertation with the premise of the full understanding of the mechanism of bioaugmentation, and choose aniline as the target pollutants. In order to solve the problems such as the low temperature in cold northeast winter and poor effect of biological treatment engineering for special industrial wastewater, study of a highly efficient cold tolerance aniline degrading bacteria JH-9, as well as the characteristics of enhanced performance, and the first to put forward a "combined pellet" concept. The technic exploiture of mycelium pellet produced during fermentation process was developed. With the immobilization technique of mixed bacteria, the high effiency cold tolerance bacteria were fixed onto biomass carrier. Formation process and performance of the combined pellet was detailed descripted, and pellet formation mechanism has been discussed.
An aniline-degrading flora, Jihua sludge, which has high efficiency at low temperature, was enriched by alternating temperature culture and screened out from four different samples. They were active sludge of aeration tank from wastewater treatment plant of Jilin chemical plant, active sludge of aeration tank from domestic wastewater treatment plant which applied at low temperature, high-efficiency bacterica preserved by laboratory and the mixture of them. As a psychrotroph flora, the flora prossessed high flocculating capability and forming zoogloea capability. Strain JH-9 which was able to use sole carbon and nitrogen source for grouth was isolated from jihua slduge at low temperature. It was classified as Acinetobacter-calcoaceticus by gas chromato-graphy of bacterial fatty acids with the MIDI Sherlock Microbial Identification System and 16S rRNA. Degradation of aniline by strain JH-9 was investigated in the physiological and ecological aspects such as environmental genes, the components of substrate, the quantity of inoculability, metabolism with two substrates mixing and the kinetics of the reaction. It was found that the optimum condition was pH 7.0, C/P 1:1~2:1, 10%(v/v) with the quantity of inoculability. Glucose and trace element could accelerate degradation of aniline while phenol was opposite. With same aniline concentration, the degradation rate depended on initial aniline concentration. With low organic compound concentration, the degradation rate was high and the degradation process was in accordance with Monod equation; with high organic compound concentration, the degradation rate was low and the degradation process was in accordance with Haldane equation.
Aniline-degrading bacterica JH-9, jihua sludge, and the flora with the same amount of JH-9 and jihua sludge mixing were casted to SBRs to look into their ability to remove aniline and see out diversification of TOC, TN, interrelated index by microorganism from a fullscale biotreatment system treating aniline. Diversity of the communities was analysed before or after casting microorganisms by DGGE. It was found that at 12℃, rotate speed of 160r/min, pH of 5 and each cycle of 48h, three bioaugmentation systems enhanced aniline removal, the rate of aniline removal was 74.2%～100% and the rate of TOC removal was 83%～88.8%. The heterophic bacteria had the dominance than nitrobacteria in SBRs.
Simultaneity we also reviewed the effect of bioaugmentation in different conditions with aniline concentration burthen, pH burthen, phenol burthen and starvling. It was found that each system emerged respective advantage. Strain JH-9 had the good effect of aniline removal in pH burthen while the flora was ascendant in aniline concentration burthen and phenol burthen. At last, jihua sludge had the finer rate of degradating aniline in hungered environment. During the experiments of bioagumentation, the effect of removal of TOC in the systems with aggrandizement was better than that in system for comparison, while there was no advantage for the removal of TN in all systems.
Immobilized strain JH-9 with simultaneity culture and adsorption respectively, with mycelium pellet forming by aspergillus Y3 as the biomass carrier. It is the first time to propose a new immobilization method-simultaneity culture method. Comparative results of the two methods showed that: simultaneity culture method has many advantages such as more immobilization bacteria, bigger cumuli volum, more pellets, and shorter pellet diameter, light gross weight and small relative density. No matter mycelium crosslinking or not, bacteria within the combined pellet foaming by simultaneity culture method are growing equably on each of the mycelium. But the bacteria within the combined pellet forming by adsorption method are existted on the cross-platform forming with number of mycelium only. Then we review the degrading performance of the combined pellet. The result shows that it maintained a good performance of strain JH-9, combined pellet forming by simultaneity culture method has better steady and permanence degrading performance than that foaming by adsorption method. Compired with immbolized ceramsite, combined pellet has a small advantage in aniline degradation performance. The result of regeneration experiment shows that combined pellet regenerated by fragment breedding can keep high aniline degradationg ability. In order to text bioaugmentation performance of combined pellet in reactor, combined pellet, strain JH-9 and Jihua sludge was casted into SBRs. The result shows that the aniline removal rate of output was 100% and the COD removal rate above 60% from the third running period, as the input aniline concentration was 130mg/L, and this result was stay the course. Through tracing the formation process and mechanism of combined pellt, we found that combined pellet forming by simultaneity culture method with aspergillus Y3 and strain JH-9 experienced four stages, and the main pellet forming reasons are the electrostatic attraction and cell surface adhesion.
在低温下对吉林化工厂污水处理厂曝气池活性污泥、低温生活污水处理系统曝气池活性污泥、实验室菌种库保存的高效菌剂,以及以上三者的混合菌群四种样品进行了变温培养、驯化,筛选到低温下降解苯胺的高效菌群—吉化污泥。该菌群属于耐冷菌群,低温下对苯胺有较好的降解效果,菌胶团形成能力较强,并且表现出很强的生物絮凝能力。从吉化污泥中分离筛选到一株低温下(10℃)能够较好降解苯胺的耐冷菌株JH-9,它能以苯胺为唯一碳源、氮源生长,通过Sherlock脂肪酸鉴定系统、16S rRNA等分析方法初步鉴定为醋酸钙不动杆菌(Acinetobacter-calcoaceticus)。从环境因子、培养基组分、接菌量、底物多样性、共代谢、反应动力学等生理生态学角度对JH-9降解特性进行了研究,得出低温下其降解苯胺的最适条件为pH 7.0,C/P 1:1~2:1,接菌量为10%(v/v)。葡萄糖和微量元素可以促进JH-9对苯胺的降解;而苯酚会抑制其对苯胺的降解。当菌体起始浓度一定时,苯胺降解率及平均降解速率主要与苯胺初始浓度有关。在较低的苯胺浓度范围内,苯胺降解率和降解速率较高,遵循Monod方程;当苯胺浓度较高时,其降解过程以基质抑制型的Haldane方程为主。
采用生物强化技术将菌株JH-9、吉化污泥及两者等量混合的菌群作为强化菌剂投加到SBR反应系统中,测定苯胺的去除能力是否提高,并考察TOC、TN及微生物活性等指标的变化,用DGGE的方法对投菌强化前后反应器中活性污泥系统菌群多样性进行分析。结果表明,在反应器内温度为12℃、摇床转速160r/min、pH 7.0、反应周期为48h等条件,三个强化系统都可以提高苯胺的降解能力,苯胺降解率在74.2%～100%,TOC的去除率83%～88.8%,而TN变化不明显。系统中异养菌的活性远远大于硝化细菌和亚硝化细菌。
考察苯胺浓度冲击负荷、pH冲击负荷、苯酚毒物负荷及贫营养条件下系统生物强化效果,发现在不同冲击负荷条件下各个强化系统体现的降解苯胺的优势不同,在pH负荷下,单菌强化效果相对较好,在苯胺浓度负荷和苯酚毒物负荷冲击下,单菌和吉化污泥的混合菌群强化效果较好,在贫营养负荷冲击下,吉化污泥菌群强化效果较好。在整个强化实验中,强化系统对TOC的去除效果要好于对照系统,而对TN的去除却没有明显的优势。
以霉菌Y3形成的菌丝球作为生物质载体分别采用同时培养法和吸附法对菌株JH-9进行固定化,并率先提出了一种新型固定化方法“同时培养法”。两种方法的对比结果表明:同时接种法在单位时间内可固定化细菌量更多、其堆积体积更大,成球总数量更多,球体直径较小,总重量和总相对密度也较小。同时培养法形成的混合菌丝球内部细菌是非常均匀的排列生长在每一根菌丝上,无论菌丝交联与否;而吸附法形成的混合菌丝球内部,细菌只存在于多条菌丝交叉形成平台的地方。
对混合菌丝球的降解效能进行考察发现,混合菌丝球对苯胺保持了良好的降解效能,同时培养法形成的混合菌丝球对苯胺的降解效能要明显好于吸附法形成的混合菌丝球,且效果稳定、持久。与固定化陶粒相比,混合菌丝球对苯胺的降解能力略显优势。菌丝球再生实验表明:经过碎片繁殖再生的混合菌丝球能很好的保持对苯胺的降解能力。为了进一步在反应器中考察混合菌丝球的生物强化效能,将混合菌丝球、高效降解苯胺混合菌群和菌株JH-9作为强化菌剂应用于SBR工艺中。结果表明:针对初始苯胺浓度为130mg/L的苯胺废水,混合菌丝球从第三个运行周期开始即实现了出水苯胺的去除率达100%、COD的去除率在60%以上,并一直稳定维持到运行的第八个周期。通过考查混合菌丝球的形成过程及机制发现:曲霉Y3和菌株JH-9同时培养形成混合菌丝球经历4个阶段,静电引力和细胞表面的粘附力是混合菌丝球形成的主要原因。
The dissertation with the premise of the full understanding of the mechanism of bioaugmentation, and choose aniline as the target pollutants. In order to solve the problems such as the low temperature in cold northeast winter and poor effect of biological treatment engineering for special industrial wastewater, study of a highly efficient cold tolerance aniline degrading bacteria JH-9, as well as the characteristics of enhanced performance, and the first to put forward a "combined pellet" concept. The technic exploiture of mycelium pellet produced during fermentation process was developed. With the immobilization technique of mixed bacteria, the high effiency cold tolerance bacteria were fixed onto biomass carrier. Formation process and performance of the combined pellet was detailed descripted, and pellet formation mechanism has been discussed.
An aniline-degrading flora, Jihua sludge, which has high efficiency at low temperature, was enriched by alternating temperature culture and screened out from four different samples. They were active sludge of aeration tank from wastewater treatment plant of Jilin chemical plant, active sludge of aeration tank from domestic wastewater treatment plant which applied at low temperature, high-efficiency bacterica preserved by laboratory and the mixture of them. As a psychrotroph flora, the flora prossessed high flocculating capability and forming zoogloea capability. Strain JH-9 which was able to use sole carbon and nitrogen source for grouth was isolated from jihua slduge at low temperature. It was classified as Acinetobacter-calcoaceticus by gas chromato-graphy of bacterial fatty acids with the MIDI Sherlock Microbial Identification System and 16S rRNA. Degradation of aniline by strain JH-9 was investigated in the physiological and ecological aspects such as environmental genes, the components of substrate, the quantity of inoculability, metabolism with two substrates mixing and the kinetics of the reaction. It was found that the optimum condition was pH 7.0, C/P 1:1~2:1, 10%(v/v) with the quantity of inoculability. Glucose and trace element could accelerate degradation of aniline while phenol was opposite. With same aniline concentration, the degradation rate depended on initial aniline concentration. With low organic compound concentration, the degradation rate was high and the degradation process was in accordance with Monod equation; with high organic compound concentration, the degradation rate was low and the degradation process was in accordance with Haldane equation.
Aniline-degrading bacterica JH-9, jihua sludge, and the flora with the same amount of JH-9 and jihua sludge mixing were casted to SBRs to look into their ability to remove aniline and see out diversification of TOC, TN, interrelated index by microorganism from a fullscale biotreatment system treating aniline. Diversity of the communities was analysed before or after casting microorganisms by DGGE. It was found that at 12℃, rotate speed of 160r/min, pH of 5 and each cycle of 48h, three bioaugmentation systems enhanced aniline removal, the rate of aniline removal was 74.2%～100% and the rate of TOC removal was 83%～88.8%. The heterophic bacteria had the dominance than nitrobacteria in SBRs.
Simultaneity we also reviewed the effect of bioaugmentation in different conditions with aniline concentration burthen, pH burthen, phenol burthen and starvling. It was found that each system emerged respective advantage. Strain JH-9 had the good effect of aniline removal in pH burthen while the flora was ascendant in aniline concentration burthen and phenol burthen. At last, jihua sludge had the finer rate of degradating aniline in hungered environment. During the experiments of bioagumentation, the effect of removal of TOC in the systems with aggrandizement was better than that in system for comparison, while there was no advantage for the removal of TN in all systems.
Immobilized strain JH-9 with simultaneity culture and adsorption respectively, with mycelium pellet forming by aspergillus Y3 as the biomass carrier. It is the first time to propose a new immobilization method-simultaneity culture method. Comparative results of the two methods showed that: simultaneity culture method has many advantages such as more immobilization bacteria, bigger cumuli volum, more pellets, and shorter pellet diameter, light gross weight and small relative density. No matter mycelium crosslinking or not, bacteria within the combined pellet foaming by simultaneity culture method are growing equably on each of the mycelium. But the bacteria within the combined pellet forming by adsorption method are existted on the cross-platform forming with number of mycelium only. Then we review the degrading performance of the combined pellet. The result shows that it maintained a good performance of strain JH-9, combined pellet forming by simultaneity culture method has better steady and permanence degrading performance than that foaming by adsorption method. Compired with immbolized ceramsite, combined pellet has a small advantage in aniline degradation performance. The result of regeneration experiment shows that combined pellet regenerated by fragment breedding can keep high aniline degradationg ability. In order to text bioaugmentation performance of combined pellet in reactor, combined pellet, strain JH-9 and Jihua sludge was casted into SBRs. The result shows that the aniline removal rate of output was 100% and the COD removal rate above 60% from the third running period, as the input aniline concentration was 130mg/L, and this result was stay the course. Through tracing the formation process and mechanism of combined pellt, we found that combined pellet forming by simultaneity culture method with aspergillus Y3 and strain JH-9 experienced four stages, and the main pellet forming reasons are the electrostatic attraction and cell surface adhesion.
引文
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