好氧颗粒污泥中细菌藻酸盐的研究
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摘要
以一类细菌胞外多糖物质-细菌藻酸盐为研究对象,对好氧颗粒污泥中的细菌藻酸盐进行了提取和鉴定,采用紫外-可见光谱、红外和拉曼光谱、核磁共振谱、质谱、X衍射、原子力显微镜、钌红染色-扫描电镜和透射电镜观察等先进的现代仪器分析技术,对好氧颗粒污泥细菌藻酸盐提取物的分子结构、特征基团、结晶度、超分子自组装特性等进行了全方位的剖析,证实细菌藻酸盐在好氧颗粒污泥中的含量可达到35.1%。它们化学测定式为C_6H_(10)O_6Na,是非结晶的、聚甘露糖醛酸残基和聚古洛糖醛酸残基比值为0.85、部分O-乙酰化的、分子量较小的藻酸盐寡糖的混合物,是细菌藻酸盐裂合酶作用的产物。细菌藻酸盐提取物在Ca~(2+)离子的诱导下可以发生分子自组装,聚集体由无规分布、粒径较小的球状体(平均粒径50nm)向有序的三维网状体(平均粒径>10um)转变。藻酸盐提取物与Fe3+,Ca~(2+)和Cu~(2+)等金属离子作用形成凝胶颗粒。它们具有与好氧颗粒污泥相近的比重和沉降速度。藻酸盐-金属凝胶构成了好氧颗粒污泥的骨架,在很大程度上决定着颗粒的形状、粒径、比重和沉降速度。
钌红染色-扫描电镜和透射电镜多糖原位可视化研究表明,细菌藻酸盐在好氧颗粒污泥中广泛分布,以凝胶形式存在。它们的分子自组装特性不仅使污泥表面负电荷量下降、密度增加,粒径增大,而且显著提高了污泥的沉降速率,并使颗粒污泥的含水率远低于活性污泥的含水率,改善了污泥的沉降性能,提高了泥水分离能力。在好氧颗粒污泥中丝状菌大量增殖的时期，细菌藻酸盐凝胶核心的存在对抑制丝状菌膨胀现象的出现起到了重要作用。
在综合分析细菌藻酸盐提取物的特性和对颗粒形成的作用的基础上，提出了好氧颗粒污泥以细菌藻酸盐为骨架的形成机制，即SBR颗粒污泥反应器的周期性贫营养阶段诱导细菌藻酸盐的分泌，产生的细菌藻酸盐经异构酶和裂解酶作用后，成为含有一定量聚古洛糖醛酸残基的寡糖，在反应器中Ca~(2+)等金属离子的诱导下，通过分子自组装最终形成细菌藻酸盐-金属凝胶，使好氧颗粒污泥成为以细菌藻酸盐-金属凝胶为骨架的自固定化载体。
微生物学研究方面，确立了好氧颗粒污泥中定量测定芽孢含量的方法，发现颗粒污泥干重的33.7%为芽孢。通过定性测定好氧颗粒污泥对不良环境的耐受力以及环境条件改善后活性的恢复与芽孢萌发的关系，证明了大量存在的芽孢增强了好氧颗粒污泥耐受低温、高温高压、紫外线的能力，是一种潜在的生命力。
利用微生物学技术从好氧颗粒污泥中筛选出优势菌种，培育出纯培养好氧颗粒污泥。通过种属鉴定，证明蜡样芽孢杆菌具有分泌细菌藻酸盐，并形成好氧颗粒污泥的能力。纯培养好氧颗粒污泥具有与SBR反应器中形成的好氧颗粒污泥相似的特性：规则的形状、密实的结构和良好的沉降性能。表现为SVI 34ml·g~(-1)，沉降速率18-39m·h~(-1),比重1.037，平均粒径0.1-1mm，比耗氧速率28.3mgO2·g~(-1)h~(-1)。对纯培养颗粒污泥中细菌藻酸盐的提取和分析不仅首次发现蜡样芽孢杆菌可以产生细菌藻酸盐，而且从微生物学方面证实了好氧颗粒污泥以细菌藻酸盐为骨架的机制。并将芽孢含量和细菌藻酸盐作为两个新的参数用以描述好氧颗粒污泥的特性。
最后,将好氧颗粒污泥中藻酸盐的提取、鉴定和分析方法扩展到生物膜、活性污泥和厌氧颗粒污泥,发现这些微生物聚集体与好氧颗粒污泥一样,都含有一定量O-乙酰化的藻酸盐寡糖的混合物。证实微生物聚集体中有很大一部分胞外糖类物质是分子量很小的寡糖,澄清了目前环境工程领域中认为胞外糖类聚合物都是高分子多糖的观念。
Bacterial alginates were extracted from aerobic granules, identified by FAO methods and characterized by UV/visible Spectroscopy, Infrared Spectroscopy, solid state Nuclear Magnetic Resonance Spectroscopy, Mass Spectrometry, Powder X-ray diffractometry, Atomic Force Microscopy, Scanning Electron Microscopy, Transmission Electron Microscopy and elemental composition. Alginate content in aerobic granules is determined as 351mg sodium alginate/g SS. The extracted bacterial alginates are mixtures of partially O-acetylated oligosaccharides with M/G ratio 0.85. Under the inducement of CaCl2, they demonstrate different conformations under Atomic Force Microscopy analysis, transfer from randomly distributed globules, to rod-like and flower-shaped aggregations, finally to ordered weblike networks as their concentrations increased. They are capable of forming gel beads when dropped in multiple cation metal solutions.
Ruthenium red staining was adapted in the study on extracellular polysaccharides distribution in aerobic granules. Both SEM and TEM observations manifested the existence of weblike alginate gel. The selfassembly property of bacterial alginate molecules has positive effect on increasing granules’specific gravity, particle size and settling velocity. The bacterial alginate gel matrix in the aerobic granule not only contributes to its improved setteability and enhanced solid/liquid separation capacity, but also offers bacteria the same inhabitancy and protection as the manmade alginate immobilizations do. During the period of filaments propagation, this alginate matrix aids in keeping high settling velocity and prevents sludge bulking.
A procedure for spore quantification in aerobic granules was developed. Sporecontent was determined by analyzing dipicolinic acid (dpa) extracted from aerobicgranules. Results show that dpa constitutes 33.7 mg per g SS, meaning that about337mg per g SS were spores, not the normal vegetative cells. Aerobic granules couldrecover their metabolism activity in a very short time after sterilized at 121℃15psifor 15mins; frozen at -21℃for two weeks and exposed to UV light (30w) within thedistance of 30 cm for 1 hour, for the large amount of spores exist in the aerobicgranule, which are more capable to sustain the sever conditions than the normalvegetative cells.
Pure culture aerobic granules, with outstanding low SVI, high settling rate andspecific gravity, were successfully developed by cultivating the strain selected fromthe mixed culture aerobic granules, in shaking Erlenmeyer flask by sequencing batchway. The strain was phenotypicly, biochemically and genotypicly identifiedbelonging to Bacillus cereus A. With the aerobic granule formation capability andpopularity in the environment, the genus Bacillus cereus A is required more attentionin biological wastewater treatment domain. Investigations on bacterial alginateextracted from pure culture aerobic granules prove that alginate gel is the maincomponent of aerobic granular sludge.
Bacterial alginates were also extracted from activated sludge and biofilmcultivated in SBR, and anaerobic granules. Results show that activated sludge,biofilm, and anaerobic granules all contain certain amount of bacterial alginates.Similar with aerobic granules, bacterial alginates extracted from them are alsoO-acetylated oligosaccharides blends, which demonstrates that bacterial alginatesplayed an important role in bio-aggregations.
Results of bacterial alginates characterization, spore content introduction and Bacillus cereus alginate secreting property will throw light on aerobic granules formation mechanism investigations.
钌红染色-扫描电镜和透射电镜多糖原位可视化研究表明,细菌藻酸盐在好氧颗粒污泥中广泛分布,以凝胶形式存在。它们的分子自组装特性不仅使污泥表面负电荷量下降、密度增加,粒径增大,而且显著提高了污泥的沉降速率,并使颗粒污泥的含水率远低于活性污泥的含水率,改善了污泥的沉降性能,提高了泥水分离能力。在好氧颗粒污泥中丝状菌大量增殖的时期，细菌藻酸盐凝胶核心的存在对抑制丝状菌膨胀现象的出现起到了重要作用。
在综合分析细菌藻酸盐提取物的特性和对颗粒形成的作用的基础上，提出了好氧颗粒污泥以细菌藻酸盐为骨架的形成机制，即SBR颗粒污泥反应器的周期性贫营养阶段诱导细菌藻酸盐的分泌，产生的细菌藻酸盐经异构酶和裂解酶作用后，成为含有一定量聚古洛糖醛酸残基的寡糖，在反应器中Ca~(2+)等金属离子的诱导下，通过分子自组装最终形成细菌藻酸盐-金属凝胶，使好氧颗粒污泥成为以细菌藻酸盐-金属凝胶为骨架的自固定化载体。
微生物学研究方面，确立了好氧颗粒污泥中定量测定芽孢含量的方法，发现颗粒污泥干重的33.7%为芽孢。通过定性测定好氧颗粒污泥对不良环境的耐受力以及环境条件改善后活性的恢复与芽孢萌发的关系，证明了大量存在的芽孢增强了好氧颗粒污泥耐受低温、高温高压、紫外线的能力，是一种潜在的生命力。
利用微生物学技术从好氧颗粒污泥中筛选出优势菌种，培育出纯培养好氧颗粒污泥。通过种属鉴定，证明蜡样芽孢杆菌具有分泌细菌藻酸盐，并形成好氧颗粒污泥的能力。纯培养好氧颗粒污泥具有与SBR反应器中形成的好氧颗粒污泥相似的特性：规则的形状、密实的结构和良好的沉降性能。表现为SVI 34ml·g~(-1)，沉降速率18-39m·h~(-1),比重1.037，平均粒径0.1-1mm，比耗氧速率28.3mgO2·g~(-1)h~(-1)。对纯培养颗粒污泥中细菌藻酸盐的提取和分析不仅首次发现蜡样芽孢杆菌可以产生细菌藻酸盐，而且从微生物学方面证实了好氧颗粒污泥以细菌藻酸盐为骨架的机制。并将芽孢含量和细菌藻酸盐作为两个新的参数用以描述好氧颗粒污泥的特性。
最后,将好氧颗粒污泥中藻酸盐的提取、鉴定和分析方法扩展到生物膜、活性污泥和厌氧颗粒污泥,发现这些微生物聚集体与好氧颗粒污泥一样,都含有一定量O-乙酰化的藻酸盐寡糖的混合物。证实微生物聚集体中有很大一部分胞外糖类物质是分子量很小的寡糖,澄清了目前环境工程领域中认为胞外糖类聚合物都是高分子多糖的观念。
Bacterial alginates were extracted from aerobic granules, identified by FAO methods and characterized by UV/visible Spectroscopy, Infrared Spectroscopy, solid state Nuclear Magnetic Resonance Spectroscopy, Mass Spectrometry, Powder X-ray diffractometry, Atomic Force Microscopy, Scanning Electron Microscopy, Transmission Electron Microscopy and elemental composition. Alginate content in aerobic granules is determined as 351mg sodium alginate/g SS. The extracted bacterial alginates are mixtures of partially O-acetylated oligosaccharides with M/G ratio 0.85. Under the inducement of CaCl2, they demonstrate different conformations under Atomic Force Microscopy analysis, transfer from randomly distributed globules, to rod-like and flower-shaped aggregations, finally to ordered weblike networks as their concentrations increased. They are capable of forming gel beads when dropped in multiple cation metal solutions.
Ruthenium red staining was adapted in the study on extracellular polysaccharides distribution in aerobic granules. Both SEM and TEM observations manifested the existence of weblike alginate gel. The selfassembly property of bacterial alginate molecules has positive effect on increasing granules’specific gravity, particle size and settling velocity. The bacterial alginate gel matrix in the aerobic granule not only contributes to its improved setteability and enhanced solid/liquid separation capacity, but also offers bacteria the same inhabitancy and protection as the manmade alginate immobilizations do. During the period of filaments propagation, this alginate matrix aids in keeping high settling velocity and prevents sludge bulking.
A procedure for spore quantification in aerobic granules was developed. Sporecontent was determined by analyzing dipicolinic acid (dpa) extracted from aerobicgranules. Results show that dpa constitutes 33.7 mg per g SS, meaning that about337mg per g SS were spores, not the normal vegetative cells. Aerobic granules couldrecover their metabolism activity in a very short time after sterilized at 121℃15psifor 15mins; frozen at -21℃for two weeks and exposed to UV light (30w) within thedistance of 30 cm for 1 hour, for the large amount of spores exist in the aerobicgranule, which are more capable to sustain the sever conditions than the normalvegetative cells.
Pure culture aerobic granules, with outstanding low SVI, high settling rate andspecific gravity, were successfully developed by cultivating the strain selected fromthe mixed culture aerobic granules, in shaking Erlenmeyer flask by sequencing batchway. The strain was phenotypicly, biochemically and genotypicly identifiedbelonging to Bacillus cereus A. With the aerobic granule formation capability andpopularity in the environment, the genus Bacillus cereus A is required more attentionin biological wastewater treatment domain. Investigations on bacterial alginateextracted from pure culture aerobic granules prove that alginate gel is the maincomponent of aerobic granular sludge.
Bacterial alginates were also extracted from activated sludge and biofilmcultivated in SBR, and anaerobic granules. Results show that activated sludge,biofilm, and anaerobic granules all contain certain amount of bacterial alginates.Similar with aerobic granules, bacterial alginates extracted from them are alsoO-acetylated oligosaccharides blends, which demonstrates that bacterial alginatesplayed an important role in bio-aggregations.
Results of bacterial alginates characterization, spore content introduction and Bacillus cereus alginate secreting property will throw light on aerobic granules formation mechanism investigations.
引文
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