Extracellular polymeric substances (EPS) play significant roles in the formation, function, and interactions of microalgal-bacteria consortia. Understanding the key roles of EPS depends on reliable extraction and quantification methods, but differentiating of EPS from microalgae versus bacteria is challenging. In this work, cation exchange resin (CER) and thermal treatments were applied for total EPS extraction from microalgal-bacteria mixed culture (MBMC), flow cytometry combined with SYTOX Green staining was applied to evaluate cell disruption during EPS extraction, and auto-fluorescence-based cell sorting (AFCS) was used to separate microalgae and bacteria in the MBMC. Thermal extraction achieved much higher EPS yield than CER, but higher temperature and longer time reduced cell activity and disrupted the cells. The highest EPS yield with minimal loss of cell activity and cell disruption was achieved using thermal extraction at 55℃ for 30 min, and this protocol gave good results for MBMC with different microalgae:bacteria (M:B) mass ratios. AFCS combined with thermal treatment achieved the most-efficient biomass differentiation and low EPS loss (<4.5 %) for the entire range of M:B ratios. EPS concentrations in bacteria were larger than in microalgae: 42.8 ± 0.4 mg COD/g TSS versus 9.19 ± 0.38 mg COD/g TSS. These findings document sensitive and accurate methods to extract and quantify EPS from microalgal-bacteria aggregates.

中文翻译：

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混合培养物中微藻和细菌胞外聚合物的分化和定量

胞外聚合物（EPS）在微藻-细菌群落的形成、功能和相互作用中发挥着重要作用。了解 EPS 的关键作用取决于可靠的提取和定量方法，但将 EPS 与微藻和细菌区分开来具有挑战性。在这项工作中，应用阳离子交换树脂（CER）和热处理从微藻-细菌混合培养物（MBMC）中提取总EPS，应用流式细胞术结合SYTOX Green染色来评估EPS提取过程中的细胞破坏，以及自发荧光基于细胞分选（AFCS）用于分离 MBMC 中的微藻和细菌。热提取获得的 EPS 产量比 CER 高得多，但较高的温度和较长的时间会降低细胞活性并破坏细胞。使用 55℃ 热提取 30 分钟实现了最高的 EPS 产量，同时细胞活性损失和细胞破碎最小，并且该方案对于不同微藻：细菌 (M:B) 质量比的 MBMC 给出了良好的结果。 AFCS 与热处理相结合，在整个 M:B 比例范围内实现了最有效的生物质分化和较低的 EPS 损失 (<4.5%)。细菌中的 EPS 浓度高于微藻中的浓度：42.8 ± 0.4 mg COD/g TSS 对比 9.19 ± 0.38 mg COD/g TSS。这些发现记录了从微藻-细菌聚集体中提取和量化 EPS 的灵敏而准确的方法。