Department of Environmental Engineering, Technical University of Denmark, Miljøvej Building 113, 2800 Kongens Lyngby, Denmark.
Water Res. 2013 Oct 1;47(15):5564-74. doi: 10.1016/j.watres.2013.06.026. Epub 2013 Jun 26.
Extracellular polymeric substances (EPS) have a presumed determinant role in the structure, architecture, strength, filterability, and settling behaviour of microbial solids in biological wastewater treatment processes. Consequently, numerous EPS extraction protocols have recently been published that aim to optimize the trade off between high EPS recovery and low cell lysis. Despite extensive efforts, the obtained results are often contradictory, even when analysing similar biomass samples and using similar experimental conditions, which greatly complicates the selection of an extraction protocol. This study presents a rigorous and critical assessment of existing physical and chemical EPS extraction methods applied to mixed-culture biomass samples (nitrifying, nitritation-anammox, and activated sludge biomass). A novel fluorescence-based method was developed and calibrated to quantify the lysis potential of different EPS extraction protocols. We concluded that commonly used methods to assess cell lysis (DNA concentrations or G6PDH activities in EPS extracts) do not correlate with cell viability. Furthermore, we discovered that the presence of certain chemicals in EPS extracts results in severe underestimation of protein and carbohydrate concentrations by using standard analytical methods. Keeping both maximum EPS extraction yields and minimal biomass lysis as criteria, it was identified a sonication-based extraction method as the best to determine and compare tightly-bound EPS fractions in different biomass samples. Protein was consistently the main EPS component in all analysed samples. However, EPS from nitrifying enrichments was richer in DNA, the activated sludge EPS had a higher content in humic acids and carbohydrates, and the nitritation-anammox EPS, while similar in composition to the nitrifier EPS, had a lower fraction of hydrophobic biopolymers. In general, the easily-extractable EPS fraction was more abundant in carbohydrates and humic substances, while DNA could only be found in tightly bound EPS fractions. In conclusion, the methodology presented herein supports the rational selection of analytical tools and EPS extraction protocols in further EPS characterization studies.
细胞外聚合物（EPS）在生物废水处理过程中微生物固体的结构、架构、强度、过滤性和沉降行为中具有假定的决定作用。因此，最近已经发布了许多 EPS 提取方案，旨在优化高 EPS 回收率和低细胞裂解之间的权衡。尽管进行了广泛的努力，但即使在分析类似的生物质样本和使用类似的实验条件时，得到的结果也常常相互矛盾，这极大地增加了提取方案的选择难度。本研究对应用于混合培养生物质样本（硝化、亚硝化-厌氧氨氧化和活性污泥生物质）的现有物理和化学 EPS 提取方法进行了严格和批判性评估。开发并校准了一种新的基于荧光的方法来量化不同 EPS 提取方案的裂解潜力。我们得出的结论是，通常用于评估细胞裂解的方法（EPS 提取物中的 DNA 浓度或 G6PDH 活性）与细胞活力不相关。此外，我们发现 EPS 提取物中某些化学物质的存在会导致使用标准分析方法严重低估蛋白质和碳水化合物的浓度。保持最大 EPS 提取产率和最小生物质裂解作为标准，确定和比较不同生物质样本中紧密结合的 EPS 分数的最佳方法是基于超声的提取方法。在所有分析样本中，蛋白质都是 EPS 的主要成分。然而，硝化富集物的 EPS 富含 DNA，活性污泥 EPS 中的腐殖酸和碳水化合物含量较高，而亚硝化-厌氧氨氧化 EPS 与硝化器 EPS 的组成相似，但疏水性生物聚合物的比例较低。一般来说，易提取的 EPS 部分在碳水化合物和腐殖质中更为丰富，而 DNA 只能在紧密结合的 EPS 部分中找到。总之，本文提出的方法学支持在进一步的 EPS 特性研究中合理选择分析工具和 EPS 提取方案。
