This research was financially supported by the SIAM Gravitation Grant 024.002.002, the Netherlands Organization for Scientific Research and by the Dutch Technology Foundation (STW - Simon Stevin Meester 2013). The authors want to thank Mario Pronk for providing the granular sludge samples.

注:AGS是从污水处理厂荷兰乌得勒支的Nereda飞行员反应器收集。反应器用市政污水送入。颗粒污泥有59.5毫升/克VSS污泥容积指数（SVI 5分钟 ）。污泥是在4月在需氧周期结束采样。采样后，将污泥立即运送到实验室，过筛并储存在-20℃直至使用。 1. EPS提取注意:在4000×g下4℃20分钟离心的颗粒污泥，倾倒出上清液。收集颗粒在颗粒的提取。颗粒的总固体（TS）和挥发性固体（VS）是由标准方法23测定。颗粒湿重之间的转换因子 - 直接从粒料取颗粒的重量 - 和TS之前的提取来确定。所有提取均一式三份进行。 注:3G湿克ranules用于每个提取方法。其TS和VS值（0.39克TS和0.34 g与），一式三份测定，用于计算提取率。 <ol><li>离心提取11 <ol><li>颗粒传输3克（湿重）到离心管中，离心管填充至50ml用软化水。 </li><li>微微抖动的手离心管。 </li><li>离心机含有4000×g下4℃20分钟将混合物离心管。 </li><li>收集上清液在玻璃烧杯中，弃沉淀，并与上清液继续如在第1.7节所述。 </li></ol></li><li>超声提取10 <ol><li>颗粒传输3克（湿重）到离心管中，离心管填充至50ml用软化水。 </li><li>在40应用在冰上脉冲超声处理2.5分钟W至该混合物中。 </li><li>离心机离心含有4000×g下4℃20分钟将混合物管。 </li><li>收集上清液在玻璃烧杯中，弃沉淀，并与上清液继续如在第1.7节所述。 </li></ol></li><li>乙二胺四乙酸（EDTA）萃取11 <ol><li>颗粒传输3克（湿重）的100毫升的玻璃瓶和瓶填充至50ml，用2％（重量/体积）的EDTA溶液。 </li><li>稍微摇动瓶子的手，并在4℃下存放于冰箱中3小时。 </li><li>将混合物转移到50毫升离心管中。 </li><li>离心机含有4000×g下4℃20分钟将混合物离心管。 </li><li>收集上清液在玻璃烧杯中，弃沉淀，并与上清液继续如在第1.7节所述。 </li></ol></li><li>甲酰胺-氢氧化钠萃取氢氧化钠（NaOH）13 <ol><li>颗粒传输3克（湿重）的100ml玻璃瓶和瓶填充至50ml用软化水。 </li><li>加0.3ml 99％的甲酰胺。 </li><li>稍微摇动瓶子的手，并在4℃下存放于冰箱中1小时。 </li><li>加入20毫升的1摩尔的NaOH到颗粒悬浮液中。 </li><li>稍微摇动瓶子的手，并在4℃下存放于冰箱中3小时。 </li><li>混合物均匀地转移到50mL的离心管中。 </li><li>离心机含有4000×g下4℃20分钟将混合物离心分离管中。 </li><li>收集上清液在玻璃烧杯中，弃沉淀，并与上清液继续如在第1.7节所述。 </li></ol></li><li>甲醛-氢氧化钠提取11 <ol><li>颗粒传输3克（湿重）的100毫升的玻璃瓶和瓶填充至50ml用软化水。 </li><li>加0.3ml 37％甲醛。 </li><li>稍微摇动瓶子由手工和存储它在4℃的冰箱中1小时。 </li><li>加入20毫升的1摩尔的NaOH到颗粒悬浮液中。 </li><li>稍微摇动瓶子的手，并在4℃下存放于冰箱中3小时。 </li><li>混合物均匀地转移到50mL的离心管中。 </li><li>离心机含有4000×g下4℃20分钟将混合物离心分离管中。 </li><li>收集上清液在玻璃烧杯中，弃沉淀，并与上清液继续如在第1.7节所述。 </li></ol></li><li>高温-碳酸钠萃取（ 的 Na 2 CO 3）9,22,24 <ol><li>预热150毫升自来水中的1000ml的玻璃烧杯中在磁力搅拌器上至80℃。 </li><li>传输3克（湿重）颗粒在250毫升带挡板烧瓶中并将烧瓶填充至50ml用软化水。 </li><li>加入0.25g克Na 2 CO 3无水0.67克Na 2 CO 3•10H <sUB&gt; 2 O到烧瓶中，得到0.5％（重量/体积） 的 Na 2 CO 3浓度。 </li><li>把含有该混合物放入水浴的烧瓶中。用铝箔分别覆盖烧瓶并将烧杯玻璃以防止蒸发。 </li><li>搅拌该混合物在400rpm 35分钟和80℃。 </li><li>将混合物转移到50毫升离心管中。 </li><li>离心机含有4000×g下4℃20分钟将混合物离心管。 </li><li>收集上清液并弃沉淀。 </li></ol></li><li> TS和VS根据标准方法23所有的提取物的测定。 <ol><li>取上清并透析它用于对抗千毫升超纯水24小时（透析袋3500大分子量截断（MWCO））11,12。改变透析水后12小时，以提高透析的效果。 </li><li>传送一个合理部分（周围1/3）的渗析上清液至铝皿对TS和VS测量23。 注意:过夜，在105℃下干燥的样品。空铝盘和含有干燥的样品的铝盘的重量差是TS的内容。然后刻录包含在550℃的样品2小时，以同一铝盘。空铝盘和含烧成样品的铝盘之间的重量差是灰分含量。 TS和灰分含量之间的差异是在VS内容。 </li><li>对于各提取物，透析上清液的残余馏分转移至10毫升玻璃烧杯。在60℃下增稠2天的上清液以1-2毫升的最终体积，以增加在上清液中的聚合物浓度。 </li></ol></li></ol> 2.海藻类聚合物外（ALE）提取<ol><li>透析根据步骤1.7.1在步骤1.6.8中获得的提取物。 </li><li>转移透析提取到250ml玻璃烧杯中。慢LY搅拌转速100rpm和室温的提取。不断地监测用pH电极的pH变化，同时加入1M盐酸（HCl），以2.2±0.05的最终pH在酸性形式得到ALE。 </li><li>调节pH至2.2后，转移提取到50ml离心管中并离心以4,000×g下4℃20分钟。 </li><li>弃去上清液，并收集凝胶状颗粒。胶状沉淀是在酸性形式ALE。 </li><li>以获得钠（或钾）ALE的形式，缓慢加入0.5M的氢氧化钠（或0.5M的氢氧化钾）以在步骤2.4中得到的凝胶，边用玻璃棒用手慢慢混合该凝胶达到pH值8.5，直到。 </li></ol> 3.离子水凝胶地层测试注:为了检查是否提取了EPS离子水凝胶地层特性，与钙离子的珠形成实验25。 <ol><li>在步骤1.7.3提取物的增稠到后的1-2毫升的体积，慢慢地搅拌用玻璃棒的混合物中，并调节其pH至8.5用0.5M NaOH中。 </li><li>取步骤3.1或步骤2.5的钠ALE的提取物和慢慢滴用巴氏吸管将萃取到2.5％（重量/体积）氯化钙（ 氯化钙 ）的解决方案。 注意:如果提取的EPS具有离子型水凝胶凝胶成形加工性能，水滴形（球形）珠将形成。如果所提取的车不具有离子性水凝胶的凝胶形成性质，萃取将在氯化钙溶液分散。 </li></ol> 4.离子水凝胶的稳定性试验注意:为了进一步了解在AGS结构形成的离子的EPS水凝胶的作用，稳定性试验对的 Na 2 CO 3萃取的离子的水凝胶珠，在步骤3.2收集进行。 <ol><li>保持水凝胶珠中的CaCl 2溶液30分钟。 </li><li>用勺子从氯化钙取出水凝胶珠</suB&gt;溶液和在四个相等的部分分割珠。 </li><li>在10ml商店馏分1为在4℃下4小时软化水。 1.5 - 如在提取方法1.3中描述的以下稳定性试验中相同的方式进行的。 </li><li>商店级分2在10毫升的2％（重量/体积）乙二胺四乙酸3小时，在4℃下溶液。 </li><li>在7.15毫升商店馏分3软化水用60微升99％的甲酰胺，在4℃下1小时。然后于4℃下添加2.85毫升的1M NaOH和存储级分3 3小时。 </li><li>在7.15毫升商店馏分4软化水用60微升37％甲醛在4℃下1小时。然后于4℃下添加2.85毫升的1M NaOH和存储分数4 3小时。 </li><li>监控，如果在4.3中描述的条件下的储存过程中的小珠可见崩解 - 4.6如果珠承受提取条件来评价。 </li></ol>

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J., Elimelech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+polysaccharide+gel+layers+in+the+presenceof+Ca2++and+K++ions:+Measurements+and+mechanisms.">Formation of polysaccharide gel layers in the presenceof Ca2+ and K+ ions: Measurements and mechanisms.</a> Biomacromolecules. 8 (1), 113-121 (2007).</li><li>Jiang, H. L., Tay, J. H., Liu, Y., Tay, S. T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++augmentation+for+enhancement+of+aerobically+grown+microbial+granules+in+sludge+blanket+reactors.">Ca2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors.</a> Biotechnol. Lett. 25 (2), 95-99 (2003).</li><li>Liang, Z., Li, W., Yang, S., Du, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+structural+characteristics+of+extracellular+polymeric+substances+(EPS),+pellets+in+autotrophic+nitrifying+biofilm+and+activated+sludge.">Extraction and structural characteristics of extracellular polymeric substances (EPS), pellets in autotrophic nitrifying biofilm and activated sludge.</a> Chemosphere. 81 (5), 626-632 (2010).</li><li>Guo, X., Liu, J., Xiao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+damage+of+cell+wall+and+cell+membrane+for+various+extracellular+polymeric+substance+extractions+of+activated+sludge.">Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.</a> J. Biotechnol. 188, 130-135 (2014).</li></ol>

The authors have nothing to disclose.

备注的协议部分 车/ ALE的提取为50ml体积和3克的颗粒进行说明。这些值仅作为指导原则。具有较高颗粒浓度的提取可降低所提取的EPS的产率。 ALE的提取过程中的温度应在80℃下进行30分钟，保持恒定。升温所需的混合物中的时间（约5分钟）被包括在该协议。此外，提取功效是通过使用大小相同烧瓶底部的直径的磁性搅拌棒增强。这将导致良好的混合性能和研磨效果，促进EPS的提取。 后来在协议部分，所有提取的TS和VS产量（上清步骤1.1-1.6收集）被确定。透析需要之前的TS和VS测量被执行，以减少由于用于提取的化学物质的存在可能的错误。一个3500大的MWCO建议删除这些化学物质，同时保留透析袋内的EPS大分子。透析袋应具有比提取物的体积更大的体积。这是必要的，因为该提取物的体积将在透析期间增加（ 例如，对于EDTA提取多达40％的体积增加）。化学去除透析的程度可通过事先和透析后测量样品中的pH值来确定。可替代地，透析用水的电导率的测量显示出离子去除的程度。 从总提取的EPS得到ALE（步骤1.6和2）的透析步骤是可选的。然而，透析有三个优点:它减少所需的沉淀的盐酸量，它改善了在提取物中的酸的传质，并降低了所得ALE的灰分含量。为ALE的析出，建议使用玻璃烧杯用比为Extrac体积大得多吨。 的 Na 2 CO 3在提取通常过量。在加入HCl将首先与的 Na 2 CO 3反应，残留在提取物，产生二氧化碳的形成，如果该样品没有之前透析，在发泡。加入HCl的过程中，提取应该慢慢地与同样大小的磁力搅拌棒的烧杯底部搅拌。这种尺寸和缓慢搅拌的搅拌棒，将导致不破坏沉淀物的结构，甚至混合。如果在提取物中形成的酸性凝胶团块后，将烧杯应该稍微用手回荡。沉淀用1M的酸浓度进行，以避免在提取的大量增加，而仍然获得的酸的样品中的均匀分布。更高的酸浓度可能导致区域的pH降低和酸性凝胶团块的形成。低于2.0 pH值降低，可以恢复的ALE量，可能是由于结构性变化在较低pH值的聚合物。它保持最终pH为2.20±0.05是很重要的。 限制 在ALE萃取法旨在提取AGS或生物膜在EPS中一般的结构外的聚合物，但不适合于提取所有本车。提取所有车，一个以上的提取方法的组合是必要的。此外，如示出通过施加两倍和四倍提取的增加对VS 的EPS产量的，一个单一的萃取将不能提取所有结构的EPS。 ALE提取是一个严酷的EPS提取方法，不断搅拌热和碱性条件下的完美组合。由于这个原因，可能是一些胞内物质与所述EPS萃取在一起。虽然细胞裂解可通过物理和化学的提取技术（超声处理31,32，氢氧化钠31,32，EDTA 11,32，CER 32，耐热32和高剪切速率由μm所引起ixing 19），细胞内物质的回收EPS的存在还有待验证。所提取的EPS的离子凝胶形成性是本研究的主要焦点，回收的车是否包含没有分析胞内的材料。未来的研究将集中在提取的EPS识别细胞内物质。 增溶的AGS的水凝胶基质是至关重要的提取结构车 车形式AGS致密和紧凑的水凝胶基质中。虽然车包含各种类的有机大分子，如多糖，蛋白质，核酸，（磷酸）脂质，腐殖物质和一些细胞间的聚合物7,5,8，不是所有的人形成凝胶。只有那些凝胶形成的聚合物在这里视为EPS中的结构的聚合物。 车提取的目标是首先溶解的EPS然后收集溶解的EPS。如果结构EPS（ 即 T他EPS形成水凝胶）是萃取的目标，AGS的凝胶基质，必须首先溶解。只有能够溶解凝胶基质方法能够提取结构的EPS。在这项研究中，一些常用的EPS提取方法，如离心10 - 15，超声10,14,15，EDTA 10 - 12,14,15，甲醛+氢氧化钠10 - 15和甲酰胺+氢氧化钠13不能有效分离的结构EPS。这是由于这样的事实，即好氧颗粒的水凝胶基质不被这些方法溶解。出于这个原因，在第4稳定性试验仅与存在于EDTA，甲酰胺+氢氧化钠甲醛的NaOH提取条件和+进行。这三次采油是不能够分离结构EPS，但仍获得除的 Na 2 CO 3萃取最高VS EPS产量。 Ø条件F中的的 Na 2 CO 3萃取并没有采用，因为这萃取法明确溶解的AGS矩阵。因此稳定性测试过程中应用的条件被认为是代表。 用阳离子交换树脂（CER），另一常用的EPS提取方法，提取不被认为对于这种比较，作为与CER车提取以前的研究没有屈服比这里所使用的化学提取更好的结果。 凝胶形成EPS在AGS 凝胶形成车被视为在AGS的水凝胶基质的结构的EPS。值得指出的是，有各种水凝胶如离子凝胶，温度引起的凝胶和pH诱导的凝胶。这项研究仅仅关注每股盈利形成离子凝胶。关于提取的结构的凝胶材料的大分数，这很可能是占主导地位的结构的EPS。当然，还有可能是其他种类的EPS这种形式的不同类型的水凝胶（ 例如 ，pH值诱发的凝胶28）中的相同或其它类型的好氧颗粒的存在。然而，不管是什么样的水凝胶是有针对性的，增溶EPS凝胶基质是提取凝胶形成EPS最重要的一步。 目前，一些研究已经在颗粒污泥结构EPS完成。在这个协议中所描述的，ALE提取是能够从AGS中提取凝胶形成的EPS的，将在未来的研究用于表征结构的EPS。更多的研究需要在AGS，EPS结构和非结构EPS做到更好地了解造粒和EPS的过程和功能。尤其是以下三点需要调查:为什么微生物产生如此大量的EPS的，什么是EPS的确切组成和改性是如何取决于环境变化的EPS的组成。检测和分析所有相关化合物及其interacti组件将有助于了解生物膜和如何使用它们来我们的优势。

近年来好氧颗粒污泥（AGS）过程已成为一种流行的污水生物处理过程中，在几个全规模的污水处理厂1成功应用。在与传统的活性污泥法，在AGS处理的微生物形成颗粒代替絮凝物2。这些颗粒具有更好的沉降，可以承受更高的有机负荷率，并有较高的耐受性的毒性比活性污泥絮凝物3。 与生物膜，AGS自发没有任何载体材料4的参与而形成的。在AGS，像在生物膜，微生物产生（EPS）5的高度水合胞外聚合物物质显著量形成水凝胶基质中，它们是自固定4 - 6。车是一种复杂的混合物，包括多糖，蛋白质，核酸，（phosp浩）脂类，腐殖质和一些间聚合物5,7,8。这些聚合物质通过静电力，氢键，有吸引力的离子力和/或生化反应等等 5彼此相互作用，形成了致密的三级网络结构。在EPS中，其能够形成水凝胶4,9和向第三网络结构的形成的聚合物是在这方面，认为是结构的EPS，总的EPS的子集。 车是负责的化学结构和颗粒5的物理性质。因此，关键是要了解每种EPS复合的功能。各种方法应用于EPS中提取10 - 15。然而，由于他们的极端复杂性，这几乎是不可能由一个单一的方法提取所有的EPS部件。到目前为止，不存在"一刀切"的EPS提取方法。提取方法的选择不仅影响的总量，而且回收的聚合物13,16的组合物- 20。取决于污泥的种类和感兴趣的不同方法在EPS是必需的。 提取凝胶形成的聚合物，表征它们的性质和调查其彼此和与非凝胶形成车交互将有助于揭示的EPS中颗粒污泥的形成中的作用。此外，凝胶形成的聚合物，可以在工业应用中有用的生物聚合物。一个可能的应用程序是已经由使用凝胶-成型聚合物从AGS作为涂层材料以增加的纸张21的耐水性示出。 因此，需要提取方法，具体为凝胶形成的EPS。这项研究的目的是开发一种方法，从AGS提取凝胶形成EPS。六提取方法10 - 15,22，这是经常在文献中，选择从AGS提取EPS。总量和所提取的EPS的凝胶形成特性每种方法进行了比较。

<table border="0" cellpadding="0" cellspacing="0" width="576">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">250 ml baffled flask</td>
			<td style="width:71px;">Kimble</td>
			<td style="width:119px;">25630-250</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">1000 ml glass beaker</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">213-1128</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:219px;">RCT basic, magnetic stirrer with thermometer</td>
			<td style="width:71px;">IKA</td>
			<td style="width:119px;">3810000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">sodium carbonate decahydrate</td>
			<td style="width:71px;">Merck KGaA</td>
			<td>1063911000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">50 ml centrifugation tubes</td>
			<td style="width:71px;">greiner bio-one</td>
			<td align="right" style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:219px;">Multifuge 1 S-R, centrifuge</td>
			<td style="width:71px;">Heraeus/Thermo Scientific</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">hydrochloric acid, 37 %</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">30721-1L-GL-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">250 ml glass beaker</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">213-1124</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">calcium chloride dihydrate</td>
			<td style="width:71px;">Merck KGaA</td>
			<td align="right" style="width:119px;">1023821000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">1 ml Pasteur Pipette</td>
			<td style="width:71px;">Copan</td>
			<td style="width:119px;">201C</td>
			<td></td>
		</tr>
	</tbody>
</table>

extraction of structural extracellular polymeric substances from aerobic granular sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na2CO3) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca2+). From the six extraction methods used, only the Na2CO3 extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca2+. The Ca2+-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.

EPS提取 颗粒施加不同的EPS萃取程序后的外观示于图1。颗粒的形状和凝胶结构完好离心（ 图1a）和EDTA提取（ 图1c）之后。颗粒破碎成不同尺寸通过超声处理的片段。在液相中的浊度可能是由于悬浮液的小片段（ 图1b）的作为浊度离心后高度降低。甲酰胺和甲醛单独没有对改变颗粒的形状和其凝胶结构的任何影响（数据未显示）。添加NaOH后，将液相变成淡黄色。某些蓬松材料从颗粒表面脱离，形成在结算颗粒（ 图1D和1E）的顶部的层。仍然，的形状颗粒没有改变。加入NaOH明显改善车溶解，但不能破坏凝胶基质结构。相比较而言，颗粒剂的 Na 2 CO 3萃取（ 图1F）后完全消失。代替溶胶状液体和细小果冻状颗粒的混合物中形成的，显示出确实溶解颗粒的凝胶基质。 <img alt="图1" src="/files/ftp_upload/54534/54534fig1.jpg" /> 图1.好氧颗粒污泥EPS提取。对于每提取方法对颗粒的影响，更好的视觉效果，实验在25毫升玻璃瓶进行。在提取过程之后，萃取物保持1小时，在室温下，以允许悬浮物沉降。 （ 一 ）离心提取，（ 二 ）超声提取，（ 三 ）的EDTA提取，（D）甲酰胺-氢氧化钠额外ction，（E）甲醛-氢氧化钠提取，（F）高温- 的 Na 2 CO 3萃取<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54534/54534fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 车的产量相对于VS分数为每个方法在图2中示出。产率以mg VS 车每克初始VS 颗粒呈现。由甲醛+氢氧化钠所得EPS的量，甲酰胺+ NaOH和的 Na 2 CO 3 +热量+混合比离心，超声处理和EDTA提取的更高。 13,15表示在碱性条件提高车溶解度26,27 -对于这些提取技术类似的结果也由先前的研究11所示。车的通过的 Na 2 CO 3中回收的量<html>最高的，只有通过离心分离得到的20倍以上。此外， 的 Na 2 CO 3萃取的总的EPS产率可进一步由多个提取增强。使用第一次提取的步骤1.6.8（协议部分）废旧沉淀第二提取28％增加总产，四倍提取甚至46％提高总产量。 <img alt="图2" src="/files/ftp_upload/54534/54534fig2.jpg" /> 图2.所有的提取方法相对于VS产量和灰分含量的结果。对于每次提取第一条表示毫克VS产量VS EPS每克初始VS 颗粒 。第二条代表灰中所提取的TS中的重量百分比。误差条示出了用于每个提取技术进行三次萃取的标准偏差。"_blank"&gt;请点击此处查看该图的放大版本。 海藻般的胞外多聚物（ALE）提取 由的 Na 2 CO 3萃取萃取EPS的pH调节至2.2后，总的VS的63％沉淀。沉淀物是酸性的ALE 25。该残余馏分是可能的EPS可在萃取条件下被溶解，但不能形成在pH 2.2沉淀。 离子水凝胶形成试验 好氧颗粒已被描述为类似于水凝胶。造粒过程已被视为与糖苷作为胶凝剂4,9,25,28形成凝胶的现象。正常情况下，钙离子是废水中的最常见的阳离子之一。此外，它很容易与酸性多糖结合（ 例如，藻酸盐和聚半乳糖醛酸），大概是作为反离子调解凝胶29。由此导致的离子交联的水凝胶。据观察，加入钙离子能加速好氧污泥颗粒30。因此，钙离子 -EPS（离子凝胶）可以发挥在颗粒污泥建立凝胶基质结构中起重要作用。在这方面，无论所提取的EPS形成具有钙离子的离子的水凝胶可以被用来作为测试，以检查是否所提取的EPS是有助于在颗粒污泥9形成凝胶基质的结构的聚合物。 在这项研究中，通过各种方法从AGS（ 图3a）萃取EPS，只有通过的 Na 2 CO 3萃取的EPS中的2.5％（重量/体积）的CaCl 2溶液保持的液滴的形状，形成稳定的离子的水凝胶珠粒。此外，由此得到的钠ALE EPS由额外的步骤（ALE提取聚合物， 图3b）显示相同的属性也是如此。的钙离子 -ALE凝胶珠（ 图3c）的颜色和形态相似颗粒污泥（ 图3a）。显然，通过的 Na 2 CO 3的方法提取的EPS有助于在颗粒污泥凝胶基质的形成。 ALE，这是本车的主要成分是结构的聚合物，能够形成离子的水凝胶。 离子水凝胶的稳定性试验 据观察，车提取过程中，好氧颗粒保持其球形形状在EDTA中，甲醛+ NaOH和甲酰胺+氢氧化钠（ 图1）。以理解，如果所提取的结构的聚合物起到颗粒的稳定性的作用， 钙 -ALE珠恰好相同的方式处理作为提取过程中有氧颗粒。有趣的是，钙离子 -ALE珠粒显示类似稳定性作为AGS的（ 图3d - 3F）， 即 ， 钙 -ALE珠在EDTA中极稳定的。有从Ca的表面分离的ALE的少量2+ -ALE珠粒（在图3e微小褐色絮和3f）中，当钙离子 -ALE珠粒浸泡过甲醛+ NaOH和甲酰胺+氢氧化钠三小时， 分别。这种相似性在钙 -ALE珠子和好氧颗粒之间稳定性方面表明，ALE是形成AGS凝胶基质中的重要的结构的聚合物的一部分。 <img alt="图3" src="/files/ftp_upload/54534/54534fig3.jpg" /> 图3.有氧颗粒和萃取ALE（a）中在软化水公关颗粒剂IOR提取。 （ 二 ）酸性ALE在4000×g下离心4℃20分钟后（根据段落1.6和2萃取）。离子水凝胶的稳定性试验的结果。存储在软化水（ 三 ） 钙 -ALE珠在4℃下4小时。存储在2％的EDTA（ 四 ） 的 Ca 2+ -ALE珠，在4℃下3小时。 （ 五 ）储存在甲酰胺+ NaOH溶液在4℃下4小时的 Ca 2+ -ALE珠。 （F）钙离子在4℃下保存在甲醛+ NaOH溶液4小时-ALE珠。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54534/54534fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge at JoVE.com

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

该协议提供了一种方法，以溶解好氧颗粒污泥以提取藻样胞外聚合物（ALE）。

从好氧颗粒污泥结构胞外聚合物的提取

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) ...

extraction-structural-extracellular-polymeric-substances-from-aerobic

Nanyang Technological University

Research

JoVE Journal

Environment

22.4K 次观看.  Delft University of Technology. 该协议提供了一种方法，以溶解好氧颗粒污泥以提取藻样胞外聚合物（ALE）。 

视频: Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. 
This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.

Microbial sulfate reduction is a process of great importance in environmental biotechnology. The success of the sulfidogenic reactors depends among other factors on the microbial composition of the sludge. Here, we present a protocol to develop sulfidogenic sludge from hydrothermal vents sediments in a UASB reactor for reductive dechlorination purposes.

Sulfidogenic污泥海洋沉积物和三氯乙烯减少在上流式厌氧污泥床反应器开发

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

科研

环境科学

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

提取和表面活性剂的特性，从大气气溶胶

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Fabrication of Anisotropic Polymeric Artificial Antigen Presenting Cells for CD8+ T Cell Activation

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular substrates offer key advantages over cell-based aAPC, including precise control of signal presentation parameters and physical properties of the aAPC surface to modulate its interactions with T cells. aAPC constructed from anisotropic particles, particularly ellipsoidal particles, have been shown to be more effective than their spherical counterparts at stimulating T cells due to increased binding and larger surface area available for T cell contact, as well as reduced nonspecific uptake and enhanced pharmacokinetic properties. Despite increased interest in anisotropic particles, even widely accepted methods of generating anisotropic particles such as thin-film stretching can be challenging to implement and use reproducibly.
To this end, we describe a protocol for the rapid, standardized fabrication of biodegradable anisotropic particle-based aAPC with tunable size, shape, and signal presentation for T cell expansion ex vivo or in vivo, along with methods to characterize their size, morphology, and surface protein content, and to assess their functionality. This approach to fabricating anisotropic aAPC is scalable and reproducible, making it ideal for generating aAPC for &#34;off-the-shelf&#34; immunotherapies.

Here, we present a protocol to quickly and reproducibly generate biologically inspired, biodegradable articifical antigen presenting cells (aAPC) with tunable size, shape, and surface protein presentation for T cell expansion ex vivo or in vivo.

CD8+ T 细胞活化的各向异性聚合物人工抗原呈现细胞的制备

Artificial antigen presenting cells (aAPC) are a promising platform for immune modulation due to their potent ability to stimulate T cells. Acellular ...

Measuring the Shape and Size of Activated Sludge Particles Immobilized in Agar with an Open Source Software Pipeline

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and shape of activated sludge flocs can indicate the conditions at the microscale, and also directly affect how well the sludge settles in a clarifier.
Particle size and shape are both misleadingly &#39;simple&#39; measurements. Many subtle issues, often unaddressed in informal protocols, can arise when sampling, imaging, and analyzing particles. Sampling methods may be biased or not provide enough statistical power. The samples themselves may be poorly preserved or undergo alteration during immobilization. Images may not be of sufficient quality; overlapping particles, depth of field, magnification level, and various noise can all produce poor results. Poorly specified analysis can introduce bias, such as that produced by manual image thresholding and segmentation.
Affordability and throughput are desirable alongside reproducibility. An affordable, high throughput method can enable more frequent particle measurement, producing many images containing thousands of particles. A method that uses inexpensive reagents, a common dissecting microscope, and freely-available open source analysis software allows repeatable, accessible, reproducible, and partially-automated experimental results. Further, the product of such a method can be well-formatted, well-defined, and easily understood by data analysis software, easing both within-lab analyses and data sharing between labs.
We present a protocol that details the steps needed to produce such a product, including: sampling, sample preparation and immobilization in agar, digital image acquisition, digital image analysis, and examples of experiment-specific figure generation from the analysis results. We have also included an open-source data analysis pipeline to support this protocol.

The size and shape of particles in activated sludge are important parameters that are measured using varying methods. Inaccuracies arise from non-representative sampling, suboptimal images, and subjective analysis parameters. To minimize these errors and ease measurement, we present a protocol specifying every step, including an open source software pipeline.

利用开源软件管道测量琼脂中固定活性污泥颗粒的形状和大小

Experimental bioreactors, such as those treating wastewater, contain particles whose size and shape are important parameters. For example, the size and ...

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and increasing the recovery of coalbed methane. A visualized and constant-volume gas-solid coupling system is introduced here to investigate the influence of CO2 sorption on the physical and mechanical properties of coal. Being able to keep a constant volume and monitor the sample using a camera, this system offers the potential to improve instrument accuracy and analyze fracture evolution with a fractal geometry method. This paper provides all steps to perform a uniaxial compression experiment with a briquette sample in different CO2 pressures with the gas-solid coupling test system. A briquette, cold-pressed by raw coal and sodium humate cement, is loaded in high-pressure CO2, and its surface is monitored in real-time using a camera. However, the similarity between the briquette and the raw coal still needs improvement, and a flammable gas such as methane (CH4) cannot be injected for the test. The results show that CO2 sorption leads to peak strength and elastic modulus reduction of the briquette, and the fracture evolution of the briquette in a failure state indicates fractal characteristics. The strength, elastic modulus, and fractal dimension are all correlated with CO2 pressure but not with a linear correlation. The visualized and constant-volume gas-solid coupling test system can serve as a platform for experimental research about rock mechanics considering the multifield coupling effect.

A Uniaxial Compression Experiment with CO2-Bearing Coal Using a Visualized and Constant-Volume Gas-Solid Coupling Test System

This protocol demonstrates how to prepare a briquette sample and conduct a uniaxial compression experiment with a briquette in different CO2 pressures using a visualized and constant-volume gas-solid coupling test system. It also aims to investigate changes in terms of coal&#8217;s physical and mechanical properties induced by CO2 adsorption.

使用可视化和恒定体积气体-固体耦合测试系统使用 CO2轴承煤的单轴压缩实验

Injecting carbon dioxide (CO2) into a deep coal seam is of great significance for reducing the concentration of greenhouse gases in the atmosphere and ...

Cortisol Extraction from Sturgeon Fin and Jawbone Matrices

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) and quantify any differences in fin cortisol levels among three main sturgeon species. Fins were harvested from 19 sacrificed sturgeons including seven beluga (Huso huso), seven Siberian (Acipenser baerii), and five sevruga (A. stellatus). The sturgeons were raised in Iranian farms for 2 years (2017-2018), and cortisol extraction analysis was conducted in South Korea (January-February 2019). Jawbones from five H. huso were also used for cortisol extraction. Data were analyzed using the general linear model (GLM) procedure in the SAS environment. The intra- and inter-assay coefficients of variation were 14.15 and 7.70, respectively. Briefly, the cortisol extraction technique involved washing the samples (300 &#177; 10 mg) with 3 mL of solvent (ultrapure water and isopropanol) twice, rotation at 80&#160;rpm for 2.5 min, air-drying the washed samples at room temperature (22-28 &#176;C) for 7 days, further drying the samples using a bead beater at 50 Hz for 32 min and grinding them into powder, applying 1.5 mL methanol to the dried powder (75 &#177; 5 mg), and slow rotation (40 rpm) for 18 h at room temperature with continuous mixing. Following extraction, samples were centrifuged (9,500 x g for 10 min), and 1 mL supernatant was transferred into a new microcentrifuge tube (1.5 mL), incubated at 38 &#176;C to evaporate the methanol, and analyzed via enzyme-linked immunosorbent assay (ELISA). No differences were observed in fin cortisol levels among species or in fin and jawbone cortisol levels between washing solvents. The results of this study demonstrate that the sturgeon jawbone matrix is a promising alternative stress indicator to solid matrices.

In this study, we present a protocol for cortisol extraction from the fin and jawbone of sturgeon species. Fin and jawbone cortisol levels were further examined by comparing two washing solvents followed by ELISA assays. This study piloted the feasibility of jawbone cortisol as a novel stress indicator.

皮质醇从鱼翅和爪骨基质提取

The aims of this study were to develop a technique for the extraction of cortisol from sturgeon fins using two washing solvents (water and isopropanol) ...

Extracellular Multi-Unit Recording from the Olfactory Nerve of Teleosts

Recent studies have shown that ocean acidification affects olfactory-driven behavior in fish. This may be due in part to a reduction in olfactory sensitivity in high PCO2/low pH water. To assess the effects of ocean acidification, or olfactory sensitivity in marine fish in general, we propose that extracellular multi-unit recording from the olfactory nerve is the method of choice. Although invasive, it is sensitive, robust, reproducible and independent of external salinity (unlike the electro-olfactogram [EOG], for example). Furthermore, it records a primary sensory input into the CNS, prior to any central processing. We show that this method can show a reduction in olfactory sensitivity that is both temporary and odorant-dependent, using a range of amino acids to construct concentration-response curves and calculate the thresholds of detection.

Extracellular multi-unit recording from the olfactory nerve is a sensitive, robust and reproducible method for assessing olfactory sensitivity in marine fish. It records primary sensory input and is independent of external salinity.