影响好氧颗粒污泥形成与结构稳定的胞外多聚物关键组分研究
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摘要
好氧颗粒污泥是一类物理结构致密、沆降性能优异的生物聚集体,胞外多聚物(Extracellular Polymeric Substances, EPS)对其形成与结构稳定化具有重要作用。然而,目前污泥EPS提取与分析方法存在局限性,影响颗粒污泥主要成分EPS的组成含量分析,限制颗粒污泥形成机理的探析。论文比较研究了不同提取方法对生物聚集体EPS主要组分提取效率的影响；在此基础上,应用三维荧光光谱(3D-EEM)、傅里叶红外光谱(FTIR)等先进技术研究好氧污泥颗粒化过程EPS组分变化,并结合不同形态结构的颗粒污泥EPS组分分析,初步揭示影响好氧颗粒污泥形成及结构稳定化的EPS关键组分。主要获得以下研究结论：
1、鉴于目前污泥EPS提取方法存在不同组分提取效率差异大等问题,以普通絮体污泥、好氧颗粒污泥以及结构失稳的好氧颗粒污泥为研究对象,应用3D-EEM技术分析阳离子交换树脂法(CER)、硫化物法(Sulfide)、CER+Sulfide法、甲酰胺+NaOH法、加热法(Heat)五种常见提取方法对污泥EPS提取效率的影响。结果表明,实验选取的五种污泥EPS提取方法的破细胞程度均较小,其中甲酰胺+NaOH法、Heat法处理后的污泥EPS提取量远高于CER法、Sulfide法及CER+Sulfide法,表明甲酰胺+NaOH法、Heat法具有较高的EPS提取效率。3D-EEM分析进一步揭示,CER法、Sulfide法及CER+Sulfide法提取的污泥EPS组分较单一、不全面；甲酰胺+NaOH法虽具有最高的EPS提取效率,但不能有效提取污泥EPS中的芳香族类蛋白,不适于污泥EPS后续功能组分分析。相比而言,Heat法可全面提取污泥EPS中的三大类具有荧光特性的芳香族类蛋白、类富里酸、类胡敏酸,可作为较优的污泥EPS提取方法。
2、应用3D-EEM技术研究处理模拟城镇污水好氧颗粒污泥反应器启动过程污泥EPS组分变化发现,颗粒化过程代表芳香族类蛋白的荧光特征峰PeakB、D和F (Ex/Em=220-230/358.5-364、280-290/360-368、230/308.5)的响应值逐渐增强,其中酪氨酸类蛋白(Peak F)在颗粒污泥中的高度富集揭示其在颗粒结构维持中的重要作用；代表类胡敏酸的荧光特征峰Peak C (Ex/Em=350-360/445.5-452.5)的响应值在污泥颗粒化过程中呈先增后减再稳定的趋势,结合有关文献分析其在污泥颗粒化过程中利于微生物聚集。结合FTIR分析发现,p-折叠蛋白(1650-1640cm-1)、天冬氨酸(1400cm-1)在污泥颗粒化过程大量存在,且其响应值变化趋势与PN含量变化基本一致；属芳香族类蛋白的酪氨酸(1517cm-1)亦在成熟颗粒内富集,印证了3D-EEM的分析结果；此外,吸收波段为1200-1000cm-1的多糖组分在污泥颗粒化过程发生显著的结构转变,结合有关文献认为不同结构多糖物质的转化影响污泥颗粒化,有关机理待进一步探析。
3、研究不同形态结构的好氧颗粒污泥发现,相比稳态好氧颗粒,失稳颗粒污泥的PN, PS含量大幅增加,但芳香族类蛋白(Peak B、D)明显减少,证实芳香族类蛋白在颗粒污泥结构中具有重要作用；类胡敏酸(Peak C)响应值随颗粒污泥的失稳而增加,表明该组分的过量富集影响颗粒污泥稳定性。FTIR分析亦证实酪氨酸类蛋白(1517cm-1)的存在以及颗粒化过程多糖物质(1200-1000cm’)结构的转变对颗粒污泥形成与稳定化影响较大,揭示这两类物质是颗粒污泥EPS的关键组分。
The aerobic granular sludge is a class of microbial aggregates with compact physical structure and excellent settleability. Extracellular polymeric substances(EPS), as major compents of aerobic granular sludge, plays an important role in the formation and stability of aerobic granule. As the limitation of EPS extract and analytical methods, it's difficult to analysis the main EPS components and reveal the mechanism of aerobic sludge granulation. Based on the comparison of different EPS extraction methods for microbial aggregates, three-dimensional fluorescence spectroscopy (3D-EEM) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the key components of EPS during the formation and stablility of aerobic granular sludge. Main conclusions were as follows:
1、In view of the different effectiveness of various methods for EPS extraction from flocs sludge, unstable and stable aerobic granular sludge,3D-EEM was applied to investigate EPS extract efficiencies of cation exchange resin (CER), Sulfide, CER combined Sulfide, Formamide combined NaOH and Heat methods. It was found that the cells was negligibly broken to all of the extract methods. EPS concents extracted by Formamide+NaOH and Heat methods were higher than those by CER, Sulfide and CER+Sulfide methods, which suggested that Formamide+NaOH and Heat method had a higher EPS extraction efficiency. Analysis of 3D-EEM revealed that the EPS components extracted by CER, Sulfide and CER+Sulfide methods were incomplete, and Formamide+NaOH method, which could not effectively extract aromatic protein-like, was not suitable for the following analysis of sludge EPS composition. In contrast, the Heat method, which could fully extract EPS components including aromatic protein-like, fulvic acid-like and visible humic acid-like, was the best EPS extraction method for microbial aggregates.
2, The results of sludge EPS analysis during aerobic sludge granulation using 3D-EEM showed that the response values of fluorescence peak B, D and F (Ex/Em=220-230/358.5-364,280-290/360-368,230/308.5) which represent aromatic protein-like gradually increased along with sludge granulation, and it's found that tyrosine protein-like(peak F, one of the aromatic protein-like) was enhanced granular sludge. The response value of fluorescence peak C (Ex/Em=350-360/445.5-452.5) which represent humic acid-like firstly increased and then decreased to a stable value, it's speculated that humic acid-like also favor microbial aggregates. Further analysis with FTIR indicated thatβ-sheet protein (1650-1640cm-1) and aspartic acid (1400cm-1) existed in the whole sludge granulation, and their variation tendency was consistent with PN content. The enrichment of tyrosine protein-like (1517cm-1) was also found in mature granule, and it's further confirmed its importance in the granular stability. In addition, the variation of absorption band between 1200-1000cm"1 which represent polysaccharide demonstrated that the structural shift of polysaccharide favors the aerobic sludge granulation and the mechanism about it need further research.
3. The analysis of stable-and unstable aerobic granule EPS showed that the PN and PS contents of unstable aerobic granule significantly increased compared to the stable granule, but it's worth noting that aromatic protein-like (Ex/Em=220-230/342-368.5,230/308.5) was greatly decreased, which comfirmed the importance of aromatic protein-like in maintaining structural stability of aerobic granular sludge. Futhermore excessive humic acid-like (Ex/Em=360/447.5-452) would result in the loss of granular stability. FTIR results demonstrated that the existence of tyrosine protein-like(1517cm-1) and the structural shift of polysaccharide(1200-1000cm-1) played a critical role in the formation and stability of aerobic granule, and they are the key EPS components of aerobic granular sludge.
1、鉴于目前污泥EPS提取方法存在不同组分提取效率差异大等问题,以普通絮体污泥、好氧颗粒污泥以及结构失稳的好氧颗粒污泥为研究对象,应用3D-EEM技术分析阳离子交换树脂法(CER)、硫化物法(Sulfide)、CER+Sulfide法、甲酰胺+NaOH法、加热法(Heat)五种常见提取方法对污泥EPS提取效率的影响。结果表明,实验选取的五种污泥EPS提取方法的破细胞程度均较小,其中甲酰胺+NaOH法、Heat法处理后的污泥EPS提取量远高于CER法、Sulfide法及CER+Sulfide法,表明甲酰胺+NaOH法、Heat法具有较高的EPS提取效率。3D-EEM分析进一步揭示,CER法、Sulfide法及CER+Sulfide法提取的污泥EPS组分较单一、不全面；甲酰胺+NaOH法虽具有最高的EPS提取效率,但不能有效提取污泥EPS中的芳香族类蛋白,不适于污泥EPS后续功能组分分析。相比而言,Heat法可全面提取污泥EPS中的三大类具有荧光特性的芳香族类蛋白、类富里酸、类胡敏酸,可作为较优的污泥EPS提取方法。
2、应用3D-EEM技术研究处理模拟城镇污水好氧颗粒污泥反应器启动过程污泥EPS组分变化发现,颗粒化过程代表芳香族类蛋白的荧光特征峰PeakB、D和F (Ex/Em=220-230/358.5-364、280-290/360-368、230/308.5)的响应值逐渐增强,其中酪氨酸类蛋白(Peak F)在颗粒污泥中的高度富集揭示其在颗粒结构维持中的重要作用；代表类胡敏酸的荧光特征峰Peak C (Ex/Em=350-360/445.5-452.5)的响应值在污泥颗粒化过程中呈先增后减再稳定的趋势,结合有关文献分析其在污泥颗粒化过程中利于微生物聚集。结合FTIR分析发现,p-折叠蛋白(1650-1640cm-1)、天冬氨酸(1400cm-1)在污泥颗粒化过程大量存在,且其响应值变化趋势与PN含量变化基本一致；属芳香族类蛋白的酪氨酸(1517cm-1)亦在成熟颗粒内富集,印证了3D-EEM的分析结果；此外,吸收波段为1200-1000cm-1的多糖组分在污泥颗粒化过程发生显著的结构转变,结合有关文献认为不同结构多糖物质的转化影响污泥颗粒化,有关机理待进一步探析。
3、研究不同形态结构的好氧颗粒污泥发现,相比稳态好氧颗粒,失稳颗粒污泥的PN, PS含量大幅增加,但芳香族类蛋白(Peak B、D)明显减少,证实芳香族类蛋白在颗粒污泥结构中具有重要作用；类胡敏酸(Peak C)响应值随颗粒污泥的失稳而增加,表明该组分的过量富集影响颗粒污泥稳定性。FTIR分析亦证实酪氨酸类蛋白(1517cm-1)的存在以及颗粒化过程多糖物质(1200-1000cm’)结构的转变对颗粒污泥形成与稳定化影响较大,揭示这两类物质是颗粒污泥EPS的关键组分。
The aerobic granular sludge is a class of microbial aggregates with compact physical structure and excellent settleability. Extracellular polymeric substances(EPS), as major compents of aerobic granular sludge, plays an important role in the formation and stability of aerobic granule. As the limitation of EPS extract and analytical methods, it's difficult to analysis the main EPS components and reveal the mechanism of aerobic sludge granulation. Based on the comparison of different EPS extraction methods for microbial aggregates, three-dimensional fluorescence spectroscopy (3D-EEM) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the key components of EPS during the formation and stablility of aerobic granular sludge. Main conclusions were as follows:
1、In view of the different effectiveness of various methods for EPS extraction from flocs sludge, unstable and stable aerobic granular sludge,3D-EEM was applied to investigate EPS extract efficiencies of cation exchange resin (CER), Sulfide, CER combined Sulfide, Formamide combined NaOH and Heat methods. It was found that the cells was negligibly broken to all of the extract methods. EPS concents extracted by Formamide+NaOH and Heat methods were higher than those by CER, Sulfide and CER+Sulfide methods, which suggested that Formamide+NaOH and Heat method had a higher EPS extraction efficiency. Analysis of 3D-EEM revealed that the EPS components extracted by CER, Sulfide and CER+Sulfide methods were incomplete, and Formamide+NaOH method, which could not effectively extract aromatic protein-like, was not suitable for the following analysis of sludge EPS composition. In contrast, the Heat method, which could fully extract EPS components including aromatic protein-like, fulvic acid-like and visible humic acid-like, was the best EPS extraction method for microbial aggregates.
2, The results of sludge EPS analysis during aerobic sludge granulation using 3D-EEM showed that the response values of fluorescence peak B, D and F (Ex/Em=220-230/358.5-364,280-290/360-368,230/308.5) which represent aromatic protein-like gradually increased along with sludge granulation, and it's found that tyrosine protein-like(peak F, one of the aromatic protein-like) was enhanced granular sludge. The response value of fluorescence peak C (Ex/Em=350-360/445.5-452.5) which represent humic acid-like firstly increased and then decreased to a stable value, it's speculated that humic acid-like also favor microbial aggregates. Further analysis with FTIR indicated thatβ-sheet protein (1650-1640cm-1) and aspartic acid (1400cm-1) existed in the whole sludge granulation, and their variation tendency was consistent with PN content. The enrichment of tyrosine protein-like (1517cm-1) was also found in mature granule, and it's further confirmed its importance in the granular stability. In addition, the variation of absorption band between 1200-1000cm"1 which represent polysaccharide demonstrated that the structural shift of polysaccharide favors the aerobic sludge granulation and the mechanism about it need further research.
3. The analysis of stable-and unstable aerobic granule EPS showed that the PN and PS contents of unstable aerobic granule significantly increased compared to the stable granule, but it's worth noting that aromatic protein-like (Ex/Em=220-230/342-368.5,230/308.5) was greatly decreased, which comfirmed the importance of aromatic protein-like in maintaining structural stability of aerobic granular sludge. Futhermore excessive humic acid-like (Ex/Em=360/447.5-452) would result in the loss of granular stability. FTIR results demonstrated that the existence of tyrosine protein-like(1517cm-1) and the structural shift of polysaccharide(1200-1000cm-1) played a critical role in the formation and stability of aerobic granule, and they are the key EPS components of aerobic granular sludge.
引文
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