废水生物处理过程的紫外与荧光光谱解析
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摘要
废水生物处理反应器中污染物和污泥的成份复杂,而传统的化学分析方法已不能满足其实时在线监测的需要。光谱法具有非接触监测、不消耗试剂、能在线原位测定等优点,是具有应用前景的生物反应器监控方法。
本论文研究引入化学计量学解析所获得的紫外与荧光光谱,提取好氧反应器、厌氧反应器、微生物燃料电池和细菌菌液样品的光谱信息,并建立它们与反应器过程参数之间的联系,旨在构建基于紫外和荧光光谱表征生物反应器参数和运行状态的方法。主要研究内容和结果如下:
1、利用偏最小二乘法建立了硝化反应器出水的紫外光谱与硝酸盐和亚硝酸盐浓度的关系模型,探讨了偏最小二乘法解析反应器出水重叠严重光谱的可行性;以微生物产生的四种荧光物质(色氨酸、酪氨酸、维生素B6和辅酶NADH)为研究对象,证实了平行因子法解析重叠严重的荧光光谱的可行性,该方法还可以获得样品中各荧光组分的激发发射光谱和相对浓度。
2、利用三维荧光光谱分别表征了好氧反应器中的溶解性微生物产物、污泥胞内物质以及胞外聚合物,并利用平行因子分析法对荧光光谱进行了解析,获取了其反应历程中的浓度变化。胞内物质中的荧光成分主要是蛋白和辅酶NADH,其浓度与反应器状态有密切联系,胞内蛋白与VSS成线性相关,而胞内NADH与底物比增长速率相关;溶解性微生物产物中的荧光成分主要是蛋白、类富里酸和类胡敏酸物质,反应器的状态参数与溶解性微生物产物的生成可以用Leudeking-Piret方程来描述;蛋白和类胡敏酸的产生主要与底物利用相关,而类富里酸的产生则是与非生长相关的;胞外聚合物中的荧光成分主要是蛋白和类富里酸物质;好氧反应器中泥水混合液的荧光在线测定可以反映污泥浓度的高低,但是无法反映底物加入的影响,倍增管高压、光纤耦合效率和外界光源的影响都是在线测定需要考虑的因素。
3、研究了间歇厌氧产氢反应器过程的三维荧光光谱,发现蛋白和NADH是该过程产生的主要荧光物质,其强度得分之比与氢气分压之间存在着量化关系;高温厌氧产氢反应器出水的三维荧光光谱的分析结果表明,蛋白、NADH和核黄素是该反应器主要的荧光物质,并获得了光谱分析结果对反应器工艺参数(水力停留时间、底物浓度和温度)的响应关系,发现NADH的相对浓度与氢气分压具有很好的一致性;建立了定量测定了高温厌氧反应器出水中的三种荧光物质的标准加入法。
4、研究了微生物燃料电池反应过程的紫外与荧光光谱变化。伴随着微生物燃料电池的产电过程,产电微生物释放出的产物导致UV_254吸收值总体增加;这些产物中蛋白和类富里酸是主要荧光物质,蛋白峰主要源于蛋白内色氨酸残基的贡献,而类富里酸可能源于一些辅酶或富里酸物质的贡献;Fe~(2+)的投加有助于燃料电池的产电,并出现荧光物质的浓度变化。
5、利用米散射理论计算了不同折射率条件下米散射系数与无因次粒径的关系,并且分别对细菌结构所引起的消光光谱进行了计算;以模拟细菌的聚苯乙烯小球为例,用米散射结合Levenberg-Marquart最小二乘法对其紫外消光光谱进行了解析,并估计出其粒径和浓度;初步探索了巨大芽孢杆菌的多波长消光光谱。
The biological wastewater treatment reactors with various pollutants and microorganisms are complex systems. Conventional chemical analysis can not meet the requirement of real-time and on-line monitoring of bioreactors. Spectrometry method is a non-invasive and regent-free one, and can be performed on-line and in situ. Thus, it is a promising method for monitoring the bioreactor.
In this study the chemometrical method was introduced to decompose the spectra of microorganisms and the effluents from various bioreactors, including aerobic, anaerobic, and nitrifying bioreactors and a microbial fuel cell (MFC). The information was drawn and related to the bioreactor process parameters. The main objective of this work was to develop a new method of characterizing the parameter and status of bioreactors, based on UV/Vis and fluorescence spectra. Main contents and results are as follows:
In Chapter 2, the correlation model was established between the UV spectra and the concentrations of nitrate and nitriate from a mtrifying reactor. The feasibility of decomposing the overlapped spectra by partial least squares (PLS) was verified. Taking the four biogenic fluorophores as an example, the feasibility of decomposing the severely overlapped fluorescence spectra by parallel factor analysis (PARAFAC) was verified. The information about the relative concentration, the excitation and emission spectra was obtained.
In Chapter 3, the excitation-emission matrix (EEM) fluorescence spectra were used to characterize the soluble microbial products (SMP), intracellular substances and extracellular polymer substances (EPS). PARAFAC was applied to extract the pure spectra from the overlapped spectra. Results show that proteins and nicotinamide adenine dinucleotide, reduced form (NADH), were identified as the two main intracellular fluorophores in the intracellular substances. The fluorescence intensity scores of the intracellular fluorophores were closely related to the bioreactor performance. The profiles of the intracellular proteins were similar to those of volatile suspended solids (VSS), and the intracellular NADH was correlated to the specific substrate degradation rate. Three main components, i.e., proteins, fulvic- and humic-like substances, were identified from the SMP. Their fluorescence intensity scores of the three fluophores in the SMP could be connected with process parameters through the Leudeking-Piret equation. The extracellular proteins and humic-like substances were substrate-utilization-associated, and that the fulvic-like substances were non-growth-associated. Proteins and fulvic-like substances were identified as the two main fluorophores in the EPS. The on-line monitoring data of the mixture in the reactor were able to reflect the variation of the sludge concentration, but failed to reflect the effect of substrate addition. The voltage of the photomultiplier tube (PMT), the coupling efficiency of the fiber optics and the influence of the circumstance lightshould be taken into account.
In Chapter 4, the EEM of an anaerobic H_2-producing batch reactor were explored,and the main fluorophores of the reactor effluents were proteins and NADH. The ratio of the fluorescence intensity scores of the two fluorophores was related to the H_2 partial pressure, and the relationship between them could be expressed using a non-liner equation. The EEM fluorescence spectra of the effluents from a thermophilic upflow anaerobic sludge bed (UASB)H_2-producing reactor were also explored. Proteins, NADH and riboflavin were the main fluorophores. The effects of hydraulic retention time, substrate concentration and temperature on the bioreactor performance were evaluated, and the response of the three fluorophores scores was also explored. The profiles of the NADH scores were related to the variation of the H_2 partial pressure. Standard addition method was used to quantitatively determinate the concentration of these three fluorophores from the UASB effluents.
In Chapter 5, the variation of the UV and fluorescence spectra from an MFC was investigated. Accompanied with the reaction in the microbial fuel cell, the microorganisms released some microbial products, which led to an increase in UV_(254). Proteins and fulvic-like substances were identified as the main fluorophores by decomposing the EEM fluorescence spectra of the MFC with PARAFAC. The protein fluorescence was attributed to the tryptophane residues of the proteins, while the fulvic-like substances might be attributed to the coenzyme and fulvic acid. The dose of Fe~(2+) promoted the electrogenesis of the MFC, and the profiles of the fluorophores were different from those of the control.
In Chapter 6, Mie scattering theory was used to calculated the extinction coefficient as a function of dimensionless radius at different reflection indexes. The extinction spectra produced by the macrostructure and internal structure were also calculated as a function of wavelength. Taking the polystyrene pellet as an example, levenberg-marquart least squares coupled with the Mie theory were used to calculate the extinction spectrum, and the radius and concentration were estimated. The UV/Vis spectra of Bacillus megaterium was explored.
本论文研究引入化学计量学解析所获得的紫外与荧光光谱,提取好氧反应器、厌氧反应器、微生物燃料电池和细菌菌液样品的光谱信息,并建立它们与反应器过程参数之间的联系,旨在构建基于紫外和荧光光谱表征生物反应器参数和运行状态的方法。主要研究内容和结果如下:
1、利用偏最小二乘法建立了硝化反应器出水的紫外光谱与硝酸盐和亚硝酸盐浓度的关系模型,探讨了偏最小二乘法解析反应器出水重叠严重光谱的可行性;以微生物产生的四种荧光物质(色氨酸、酪氨酸、维生素B6和辅酶NADH)为研究对象,证实了平行因子法解析重叠严重的荧光光谱的可行性,该方法还可以获得样品中各荧光组分的激发发射光谱和相对浓度。
2、利用三维荧光光谱分别表征了好氧反应器中的溶解性微生物产物、污泥胞内物质以及胞外聚合物,并利用平行因子分析法对荧光光谱进行了解析,获取了其反应历程中的浓度变化。胞内物质中的荧光成分主要是蛋白和辅酶NADH,其浓度与反应器状态有密切联系,胞内蛋白与VSS成线性相关,而胞内NADH与底物比增长速率相关;溶解性微生物产物中的荧光成分主要是蛋白、类富里酸和类胡敏酸物质,反应器的状态参数与溶解性微生物产物的生成可以用Leudeking-Piret方程来描述;蛋白和类胡敏酸的产生主要与底物利用相关,而类富里酸的产生则是与非生长相关的;胞外聚合物中的荧光成分主要是蛋白和类富里酸物质;好氧反应器中泥水混合液的荧光在线测定可以反映污泥浓度的高低,但是无法反映底物加入的影响,倍增管高压、光纤耦合效率和外界光源的影响都是在线测定需要考虑的因素。
3、研究了间歇厌氧产氢反应器过程的三维荧光光谱,发现蛋白和NADH是该过程产生的主要荧光物质,其强度得分之比与氢气分压之间存在着量化关系;高温厌氧产氢反应器出水的三维荧光光谱的分析结果表明,蛋白、NADH和核黄素是该反应器主要的荧光物质,并获得了光谱分析结果对反应器工艺参数(水力停留时间、底物浓度和温度)的响应关系,发现NADH的相对浓度与氢气分压具有很好的一致性;建立了定量测定了高温厌氧反应器出水中的三种荧光物质的标准加入法。
4、研究了微生物燃料电池反应过程的紫外与荧光光谱变化。伴随着微生物燃料电池的产电过程,产电微生物释放出的产物导致UV_254吸收值总体增加;这些产物中蛋白和类富里酸是主要荧光物质,蛋白峰主要源于蛋白内色氨酸残基的贡献,而类富里酸可能源于一些辅酶或富里酸物质的贡献;Fe~(2+)的投加有助于燃料电池的产电,并出现荧光物质的浓度变化。
5、利用米散射理论计算了不同折射率条件下米散射系数与无因次粒径的关系,并且分别对细菌结构所引起的消光光谱进行了计算;以模拟细菌的聚苯乙烯小球为例,用米散射结合Levenberg-Marquart最小二乘法对其紫外消光光谱进行了解析,并估计出其粒径和浓度;初步探索了巨大芽孢杆菌的多波长消光光谱。
The biological wastewater treatment reactors with various pollutants and microorganisms are complex systems. Conventional chemical analysis can not meet the requirement of real-time and on-line monitoring of bioreactors. Spectrometry method is a non-invasive and regent-free one, and can be performed on-line and in situ. Thus, it is a promising method for monitoring the bioreactor.
In this study the chemometrical method was introduced to decompose the spectra of microorganisms and the effluents from various bioreactors, including aerobic, anaerobic, and nitrifying bioreactors and a microbial fuel cell (MFC). The information was drawn and related to the bioreactor process parameters. The main objective of this work was to develop a new method of characterizing the parameter and status of bioreactors, based on UV/Vis and fluorescence spectra. Main contents and results are as follows:
In Chapter 2, the correlation model was established between the UV spectra and the concentrations of nitrate and nitriate from a mtrifying reactor. The feasibility of decomposing the overlapped spectra by partial least squares (PLS) was verified. Taking the four biogenic fluorophores as an example, the feasibility of decomposing the severely overlapped fluorescence spectra by parallel factor analysis (PARAFAC) was verified. The information about the relative concentration, the excitation and emission spectra was obtained.
In Chapter 3, the excitation-emission matrix (EEM) fluorescence spectra were used to characterize the soluble microbial products (SMP), intracellular substances and extracellular polymer substances (EPS). PARAFAC was applied to extract the pure spectra from the overlapped spectra. Results show that proteins and nicotinamide adenine dinucleotide, reduced form (NADH), were identified as the two main intracellular fluorophores in the intracellular substances. The fluorescence intensity scores of the intracellular fluorophores were closely related to the bioreactor performance. The profiles of the intracellular proteins were similar to those of volatile suspended solids (VSS), and the intracellular NADH was correlated to the specific substrate degradation rate. Three main components, i.e., proteins, fulvic- and humic-like substances, were identified from the SMP. Their fluorescence intensity scores of the three fluophores in the SMP could be connected with process parameters through the Leudeking-Piret equation. The extracellular proteins and humic-like substances were substrate-utilization-associated, and that the fulvic-like substances were non-growth-associated. Proteins and fulvic-like substances were identified as the two main fluorophores in the EPS. The on-line monitoring data of the mixture in the reactor were able to reflect the variation of the sludge concentration, but failed to reflect the effect of substrate addition. The voltage of the photomultiplier tube (PMT), the coupling efficiency of the fiber optics and the influence of the circumstance lightshould be taken into account.
In Chapter 4, the EEM of an anaerobic H_2-producing batch reactor were explored,and the main fluorophores of the reactor effluents were proteins and NADH. The ratio of the fluorescence intensity scores of the two fluorophores was related to the H_2 partial pressure, and the relationship between them could be expressed using a non-liner equation. The EEM fluorescence spectra of the effluents from a thermophilic upflow anaerobic sludge bed (UASB)H_2-producing reactor were also explored. Proteins, NADH and riboflavin were the main fluorophores. The effects of hydraulic retention time, substrate concentration and temperature on the bioreactor performance were evaluated, and the response of the three fluorophores scores was also explored. The profiles of the NADH scores were related to the variation of the H_2 partial pressure. Standard addition method was used to quantitatively determinate the concentration of these three fluorophores from the UASB effluents.
In Chapter 5, the variation of the UV and fluorescence spectra from an MFC was investigated. Accompanied with the reaction in the microbial fuel cell, the microorganisms released some microbial products, which led to an increase in UV_(254). Proteins and fulvic-like substances were identified as the main fluorophores by decomposing the EEM fluorescence spectra of the MFC with PARAFAC. The protein fluorescence was attributed to the tryptophane residues of the proteins, while the fulvic-like substances might be attributed to the coenzyme and fulvic acid. The dose of Fe~(2+) promoted the electrogenesis of the MFC, and the profiles of the fluorophores were different from those of the control.
In Chapter 6, Mie scattering theory was used to calculated the extinction coefficient as a function of dimensionless radius at different reflection indexes. The extinction spectra produced by the macrostructure and internal structure were also calculated as a function of wavelength. Taking the polystyrene pellet as an example, levenberg-marquart least squares coupled with the Mie theory were used to calculate the extinction spectrum, and the radius and concentration were estimated. The UV/Vis spectra of Bacillus megaterium was explored.
引文
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