胞外多聚物对好氧颗粒污泥形成与结构稳定化的影响研究
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摘要
好氧颗粒污泥是一种特殊的生物聚集体,因具有沉降性能优异、多功能微生物分区定殖、抗污染负荷能力强等特点,在高浓度有机废水处理,脱氮除磷及有毒物质降解等方面具有极大的应用潜力。然而,目前好氧颗粒污泥的形成机制尚不清楚,且在长期运行过程中结构易失稳,一定程度上限制了该技术的工程化应用。本论文鉴于胞外多聚物(EPS)在生物聚集体的形成与结构维持中的重要作用,重点研究好氧颗粒污泥形成过程EPS的变化规律,并在此基础上解析不同水力剪切条件下EPS对好氧颗粒污泥结构稳定性的影响,主要结果如下:
1、通过综合比较6种不同的物理、化学提取方法的EPS提取效率及对细胞的破坏程度发现,甲酰胺、甲醛等化学提取方法对EPS组分及TOC含量测定干扰较大;热碱提取法可获得较高的EPS提取量,但其对细胞的破坏程度最大,相应EPS定量及定性分析误差大;相比60℃水浴30min,80℃水浴60min可提取较多的EPS,然而其造成的细胞破坏程度显著增加。为此,最终优选60℃水浴30min污泥EPS提取方法。此外,比较不同提取方法下获得的两种污泥样品EPS主要成分胞外蛋白(PN)和胞外多糖(PS)发现,好氧颗粒污泥的EPS含量高于活性污泥,且PN是其EPS中的优势组分。
2、低水力剪切条件下启动序批式气提生物反应器(SABR),有机污染物得到完全去除,但SABR运行半个月后发生污泥膨胀;通过有机负荷及水力剪切条件的联合调控,污泥膨胀得以有效控制,最终反应器实现完全颗粒化,污泥浓度明显增加、沉降性能有效改善。
在好氧颗粒污泥增殖阶段(阶段Ⅲ),EPS各组分含量均显著增加,尤其是PN由61.8 mg·gVSS~(-1)增至147.7 mg·gVSS~(-1),同时SDS-PAGE结果表明污泥EPS的PN条带明显增多;在之后的阶段Ⅳ,EPS中PS含量相对增加,PN/PS比值最终稳定在1.0～1.4。推测污泥EPS中PN含量与种类的增加可能对好氧颗粒污泥初期形成具有重要作用,而合适的PN/PS比通过影响微生物表面电荷与疏水性,调控微生物的聚集状态,促进好氧污泥颗粒化进程与维持其聚集性结构。
3、水力剪切力对好氧颗粒污泥结构稳定性与EPS产生的影响研究表明,低水力剪切条件(表面气速为0.5 cm·s~(-1))下启动的R1反应器在运行期间污泥PS含量下降,而PN含量与PN/PS(最高达3.01)比明显增加,随之颗粒污泥迅速瓦解;而在较高水力剪切条件(1.5 cm·s~(-1)和3.5 cm·s~(-1))下启动的R2、R3反应器,运行过程污泥PN、PS均有所增加,但PN/PS比相对稳定在1.39～1.81和1.15～1.39,其颗粒污泥的结构也较为稳定;相比而言,更高水力剪切条件下获得的低PN/PS比值更有利于好氧颗粒污泥的结构稳定性。周期内EPS监测结果表明,高水力剪切条件下基质消耗较快,产生的PS在相对较长的饥饿期内被微生物代谢利用,最终获得的适当PN/PS比有利于维持颗粒污泥的结构稳定性。颗粒污泥原位荧光染色结果进一步揭示,PS主要分布在颗粒污泥的菌胶团之间,而PN在整个颗粒污泥中均有分布,推测污泥EPS中PN、PS共同构建了好氧颗粒污泥框架结构,控制合适的PN/PS比有利于颗粒污泥结构的维持。
对3组反应器内污泥进行PCR-DGGE分析发现,不同水力剪切运行条件下微生物群落演替显著,微生物分属Betaproteobacteria,Alphaproteobacteria、Flavobacteriales等类群,其中低水力剪切条件与高水力剪切条件下污泥优势菌分别与Chryseobacteriumsp.和Balneimonas sp.具有较高的同源性,分析认为不同菌株直接影响EPS组分含量,进而影响污泥的絮凝性能与好氧颗粒污泥的结构特性。
Aerobic granulation, a novel environmental biotechnological process, was increasingly drawing interest in the area of biological wastewater treatment, it was exciting research work that explored beyond the limits of aerobic wastewater treatment such as treatment of high strength organic wastewaters, removal of nitrogen, phosphate, and bioremediation of toxic aromatic pollutants. But the formation mechanism of aerobic granule is unclear, and accordant viewpoint is that the formation of granule is related to extracellular polymeric substances (EPS). The effect of EPS on formation and stabilization of aerobic granule were discussed in the paper. Results are as follows:
1. Six different physical and chemical methods were chosen for extracting EPS from activated sludge and granular sludge. Through comprehensive comparing extraction efficiency and destruction of cells about different extraction methods, 60℃water-bathing was finally chosen for quantitative analysis of EPS. Analysis for EPS extraction by different methods showed that quantity of EPS in aerobic granules was higher than that of activated sludge, and proteins were more dominant than polysaccharides in all sludge samples.
2. The SABR reactor was started under low hydraulic shear stress (StageⅠ). Under the combined regulation of hydraulic and loading selection pressures, the control of Filamentous Sludge Bulking (StageⅡ) and multiplication of aerobic granules (StageⅢ) were effectively realized. Eventually, the tiny and homogeneous aerobic granules were formed when the C/N ratio was adjusted from 16 to 28 (StageⅣ). Aerobic granulation process was accompanied by the improvement of settling ability, increases of biomass concentration, and decrease of sludge loading rate.
More aerobic granules were found in stageⅢ, and the EPS content increased significantly, especially PN content which was raised from 61.8 mg·gVSS~(-1) to 147.7 mg·gVSS~(-1). The process was accompanied with the increasing lanes in the SDS-PAGE. In stageⅣ, the relative content of PS was increased, and complete granulation was obtained in the reactor, with PN/PS decreased. The granular reactor operated very well with the PN/PS ration controlled between 1.0 and 1.4. Thus, it was expected that the increase of PN content and proteins varieties played an important role in the initial formation of the granule, the PN/PS ratio had an great influence on the surface charge and hydrophobicity of aerobic granule, and could effectively regulate aggregation morphology of the microorganism, appropriate PN/PS could enhance structural performance of the granules.
3. The hydraulic shear stress had a great influence on the structural stability of aerobic granules and production of EPS. When R1 was operated under lower hydraulic shear stress (surficial gas velocity was 0.5cm/s), the PS content was decreased, however, the PN content was increased, with the ratio of PN/PS significantly improved reaching as high as 3.01, which was accompanied by rapid disintegration of the granules. When R2 and R3 were operated under higher hydraulic shear stress (surficial gas velocity in R2 and R3 were 1.5cm/s and 3.5cm/s, respectively), PN and PS were both increased to some degree, a relatively stable ratio of PN/PS was obtained in the two reactors, achieved 1.39～1.81 and 1.15～1.39 respectively, at the same time the granule structure was maintained in the two reactors. Moreover, compared R2 with R3, it was found that relative higher content of PS , then lower PN/PS ratio in EPS could improve the strength of aerobic granules to ensure that more granules were obtained. Thus, appropriate ratio of PN/PS was important for the maintenance of granule structure, while lower PN/PS ratio was beneficial to the stability and reinforcement of aerobic granules. In a single operation period, PS was significantly consumed under higher hydraulic shear stress. When there was a longer starvation time, the production and consumption of PS might be resulted by microbes' resistance to hostile environmental through controlling their energy metabolism. The production and consumption of PS kept the PN/PS ratio within a particular range during the operation period to realize the stability of granule structure. The in-situ fluorescence staining results showed that PN was distributed in the whole zoogloea, and PS was mainly distributed among the zoogloea. So PN and PS were expected to build the granule structure by jointing together, and formed the backbone of the whole granule with embedded cells to support the mechanical stability of granules.
PCR-DGGE analysis indicated that microbial population differed obviously in all three reactors, which operated under different hydraulic shear stress. Phylogenetic analysis indicated that Betaproteobacteria, Alphaproteobacteria, Flavobacteriales were the dominant classes in the three reactors, and the predominant bacterias in low and high hydraulic shear stress conditions were closely related to Chryseobacterium sp. and Balneimonas sp. respectively. It was expected that flocculability of the sludge was closely related to the EPS, which finally determined the maintenance of the granule structure.
1、通过综合比较6种不同的物理、化学提取方法的EPS提取效率及对细胞的破坏程度发现,甲酰胺、甲醛等化学提取方法对EPS组分及TOC含量测定干扰较大;热碱提取法可获得较高的EPS提取量,但其对细胞的破坏程度最大,相应EPS定量及定性分析误差大;相比60℃水浴30min,80℃水浴60min可提取较多的EPS,然而其造成的细胞破坏程度显著增加。为此,最终优选60℃水浴30min污泥EPS提取方法。此外,比较不同提取方法下获得的两种污泥样品EPS主要成分胞外蛋白(PN)和胞外多糖(PS)发现,好氧颗粒污泥的EPS含量高于活性污泥,且PN是其EPS中的优势组分。
2、低水力剪切条件下启动序批式气提生物反应器(SABR),有机污染物得到完全去除,但SABR运行半个月后发生污泥膨胀;通过有机负荷及水力剪切条件的联合调控,污泥膨胀得以有效控制,最终反应器实现完全颗粒化,污泥浓度明显增加、沉降性能有效改善。
在好氧颗粒污泥增殖阶段(阶段Ⅲ),EPS各组分含量均显著增加,尤其是PN由61.8 mg·gVSS~(-1)增至147.7 mg·gVSS~(-1),同时SDS-PAGE结果表明污泥EPS的PN条带明显增多;在之后的阶段Ⅳ,EPS中PS含量相对增加,PN/PS比值最终稳定在1.0～1.4。推测污泥EPS中PN含量与种类的增加可能对好氧颗粒污泥初期形成具有重要作用,而合适的PN/PS比通过影响微生物表面电荷与疏水性,调控微生物的聚集状态,促进好氧污泥颗粒化进程与维持其聚集性结构。
3、水力剪切力对好氧颗粒污泥结构稳定性与EPS产生的影响研究表明,低水力剪切条件(表面气速为0.5 cm·s~(-1))下启动的R1反应器在运行期间污泥PS含量下降,而PN含量与PN/PS(最高达3.01)比明显增加,随之颗粒污泥迅速瓦解;而在较高水力剪切条件(1.5 cm·s~(-1)和3.5 cm·s~(-1))下启动的R2、R3反应器,运行过程污泥PN、PS均有所增加,但PN/PS比相对稳定在1.39～1.81和1.15～1.39,其颗粒污泥的结构也较为稳定;相比而言,更高水力剪切条件下获得的低PN/PS比值更有利于好氧颗粒污泥的结构稳定性。周期内EPS监测结果表明,高水力剪切条件下基质消耗较快,产生的PS在相对较长的饥饿期内被微生物代谢利用,最终获得的适当PN/PS比有利于维持颗粒污泥的结构稳定性。颗粒污泥原位荧光染色结果进一步揭示,PS主要分布在颗粒污泥的菌胶团之间,而PN在整个颗粒污泥中均有分布,推测污泥EPS中PN、PS共同构建了好氧颗粒污泥框架结构,控制合适的PN/PS比有利于颗粒污泥结构的维持。
对3组反应器内污泥进行PCR-DGGE分析发现,不同水力剪切运行条件下微生物群落演替显著,微生物分属Betaproteobacteria,Alphaproteobacteria、Flavobacteriales等类群,其中低水力剪切条件与高水力剪切条件下污泥优势菌分别与Chryseobacteriumsp.和Balneimonas sp.具有较高的同源性,分析认为不同菌株直接影响EPS组分含量,进而影响污泥的絮凝性能与好氧颗粒污泥的结构特性。
Aerobic granulation, a novel environmental biotechnological process, was increasingly drawing interest in the area of biological wastewater treatment, it was exciting research work that explored beyond the limits of aerobic wastewater treatment such as treatment of high strength organic wastewaters, removal of nitrogen, phosphate, and bioremediation of toxic aromatic pollutants. But the formation mechanism of aerobic granule is unclear, and accordant viewpoint is that the formation of granule is related to extracellular polymeric substances (EPS). The effect of EPS on formation and stabilization of aerobic granule were discussed in the paper. Results are as follows:
1. Six different physical and chemical methods were chosen for extracting EPS from activated sludge and granular sludge. Through comprehensive comparing extraction efficiency and destruction of cells about different extraction methods, 60℃water-bathing was finally chosen for quantitative analysis of EPS. Analysis for EPS extraction by different methods showed that quantity of EPS in aerobic granules was higher than that of activated sludge, and proteins were more dominant than polysaccharides in all sludge samples.
2. The SABR reactor was started under low hydraulic shear stress (StageⅠ). Under the combined regulation of hydraulic and loading selection pressures, the control of Filamentous Sludge Bulking (StageⅡ) and multiplication of aerobic granules (StageⅢ) were effectively realized. Eventually, the tiny and homogeneous aerobic granules were formed when the C/N ratio was adjusted from 16 to 28 (StageⅣ). Aerobic granulation process was accompanied by the improvement of settling ability, increases of biomass concentration, and decrease of sludge loading rate.
More aerobic granules were found in stageⅢ, and the EPS content increased significantly, especially PN content which was raised from 61.8 mg·gVSS~(-1) to 147.7 mg·gVSS~(-1). The process was accompanied with the increasing lanes in the SDS-PAGE. In stageⅣ, the relative content of PS was increased, and complete granulation was obtained in the reactor, with PN/PS decreased. The granular reactor operated very well with the PN/PS ration controlled between 1.0 and 1.4. Thus, it was expected that the increase of PN content and proteins varieties played an important role in the initial formation of the granule, the PN/PS ratio had an great influence on the surface charge and hydrophobicity of aerobic granule, and could effectively regulate aggregation morphology of the microorganism, appropriate PN/PS could enhance structural performance of the granules.
3. The hydraulic shear stress had a great influence on the structural stability of aerobic granules and production of EPS. When R1 was operated under lower hydraulic shear stress (surficial gas velocity was 0.5cm/s), the PS content was decreased, however, the PN content was increased, with the ratio of PN/PS significantly improved reaching as high as 3.01, which was accompanied by rapid disintegration of the granules. When R2 and R3 were operated under higher hydraulic shear stress (surficial gas velocity in R2 and R3 were 1.5cm/s and 3.5cm/s, respectively), PN and PS were both increased to some degree, a relatively stable ratio of PN/PS was obtained in the two reactors, achieved 1.39～1.81 and 1.15～1.39 respectively, at the same time the granule structure was maintained in the two reactors. Moreover, compared R2 with R3, it was found that relative higher content of PS , then lower PN/PS ratio in EPS could improve the strength of aerobic granules to ensure that more granules were obtained. Thus, appropriate ratio of PN/PS was important for the maintenance of granule structure, while lower PN/PS ratio was beneficial to the stability and reinforcement of aerobic granules. In a single operation period, PS was significantly consumed under higher hydraulic shear stress. When there was a longer starvation time, the production and consumption of PS might be resulted by microbes' resistance to hostile environmental through controlling their energy metabolism. The production and consumption of PS kept the PN/PS ratio within a particular range during the operation period to realize the stability of granule structure. The in-situ fluorescence staining results showed that PN was distributed in the whole zoogloea, and PS was mainly distributed among the zoogloea. So PN and PS were expected to build the granule structure by jointing together, and formed the backbone of the whole granule with embedded cells to support the mechanical stability of granules.
PCR-DGGE analysis indicated that microbial population differed obviously in all three reactors, which operated under different hydraulic shear stress. Phylogenetic analysis indicated that Betaproteobacteria, Alphaproteobacteria, Flavobacteriales were the dominant classes in the three reactors, and the predominant bacterias in low and high hydraulic shear stress conditions were closely related to Chryseobacterium sp. and Balneimonas sp. respectively. It was expected that flocculability of the sludge was closely related to the EPS, which finally determined the maintenance of the granule structure.
引文
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