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>

<ol>
	<li>Pronk, M., de Kreuk, M. K., de Bruin, B., Kamminga, P., Kleerebezem, R., van Loosdrecht, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+scale+performance+of+the+aerobic+granular+sludge+process+for+sewage+treatment.">Full scale performance of the aerobic granular sludge process for sewage treatment.</a> Water Res. 84, 207-217 (2015).</li><li>Kreuk, M. K., Kishida, N., van Loosdrecht, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+granular+sludge+-+state+of+the+art.">Aerobic granular sludge - state of the art.</a> Water Sci. Technol. 55 (8-9), 75 (2007).</li><li>Adav, S. S., Lee, D. J., Show, K. Y., Tay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+granular+sludge:+Recent+advances.">Aerobic granular sludge: Recent advances.</a> Biotechnol. Adv. 26, 411-423 (2008).</li><li>Seviour, T., Pijuan, M., Nicholson, T., Keller, J., Yuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+properties+of+aerobic+sludge+granules+as+hydrogels.">Understanding the properties of aerobic sludge granules as hydrogels.</a> Biotechnol. Bioeng. 102 (5), 1483-1493 (2009).</li><li>Flemming, H. -. C., Wingender, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm+matrix.">The biofilm matrix.</a> Nat. Rev. Microbiol. 8 (9), 623-633 (2010).</li><li>Seviour, T., Yuan, Z., van Loosdrecht, M. C. M., Lin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerobic+sludge+granulation:+A+tale+of+two+polysaccharides?.">Aerobic sludge granulation: A tale of two polysaccharides?.</a> Water Res. 46 (15), 4803-4813 (2012).</li><li>Wingender, J., Neu, T. R., Flemming, H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+are+Bacterial+Extracellular+Polymeric+Substances.">What are Bacterial Extracellular Polymeric Substances.</a> Microb. Extracell. Polym. Subst. Charact. Struct. Funct. , 27-53 (1999).</li><li>Flemming, H. -. C., Neu, T. R., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+EPS+Matrix:+The+&#34;House+of+Biofilm+Cells.&#34;.">The EPS Matrix: The &#34;House of Biofilm Cells.&#34;.</a> J. Bacteriol. 189 (22), 7945-7947 (2007).</li><li>Lin, Y. M., Sharma, P. K., van Loosdrecht, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+and+mechanical+differences+between+alginate-like+exopolysaccharides+isolated+from+aerobic+flocculent+sludge+and+aerobic+granular+sludge.">The chemical and mechanical differences between alginate-like exopolysaccharides isolated from aerobic flocculent sludge and aerobic granular sludge.</a> Water Res. 47 (1), 57-65 (2013).</li><li>Fang, H. H. P., Jia, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+extracellular+polymer+from+anaerobic+sludges.">Extraction of extracellular polymer from anaerobic sludges.</a> Biotechnol. Tech. 10 (11), 803-808 (1996).</li><li>Liu, H., Fang, H. H. 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+of+extracellular+polymeric+substances+(EPS)+of+sludges.">Extraction of extracellular polymeric substances (EPS) of sludges.</a> J. Biotechnol. 95, 249-256 (2002).</li><li>Comte, S., Guibaud, G., Baudu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+extraction+method+on+EPS+from+activated+sludge:+An+HPSEC+investigation.">Effect of extraction method on EPS from activated sludge: An HPSEC investigation.</a> J. Hazard. Mater. 140 (1-2), 129-137 (2007).</li><li>Adav, S. S., Lee, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+extracellular+polymeric+substances+from+aerobic+granule+with+compact+interior+structure.">Extraction of extracellular polymeric substances from aerobic granule with compact interior structure.</a> J. Hazard. Mater. 154, 1120-1126 (2008).</li><li>Pan, X., Liu, J., Zhang, D., Chen, X. I., Li, L., Song, W., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+five+extraction+methods+for+extracellular+polymeric+substances+(EPS)+from+biofilm+by+using+three-dimensional+excitation-emission+matrix+(3DEEM)+fluorescence+spectroscopy.">A comparison of five extraction methods for extracellular polymeric substances (EPS) from biofilm by using three-dimensional excitation-emission matrix (3DEEM) fluorescence spectroscopy.</a> Water SA. 36 (1), 111-116 (2010).</li><li>D'Abzac, P., Bordas, F., Van Hullebusch, E., Lens, P. N. L., Guibaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+extracellular+polymeric+substances+(EPS)+from+anaerobic+granular+sludges:+Comparison+of+chemical+and+physical+extraction+protocols.">Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: Comparison of chemical and physical extraction protocols.</a> Appl. Microbiol. Biotechnol. 85 (5), 1589-1599 (2010).</li><li>Comte, S., Guibaud, G., Baudu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relations+between+extraction+protocols+for+activated+sludge+extracellular+polymeric+substances+(EPS)+and+EPS+complexation+properties:+Part+I.+Comparison+of+the+efficiency+of+eight+EPS+extraction+methods.">Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods.</a> Enzyme Microb. Technol. 38 (1-2), 237-245 (2006).</li><li>Adav, S. S., Lee, D. J., Tay, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+polymeric+substances+and+structural+stability+of+aerobic+granule.">Extracellular polymeric substances and structural stability of aerobic granule.</a> Water Res. 42, 1644-1650 (2008).</li><li>Caudan, C., Filali, A., Lefebvre, D., Sp&#233;randio, M., Girbal-Neuhauser, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+polymeric+substances+(EPS)+from+aerobic+granular+sludges:+Extraction,+fractionation,+and+anionic+properties.">Extracellular polymeric substances (EPS) from aerobic granular sludges: Extraction, fractionation, and anionic properties.</a> Appl. Biochem. Biotechnol. 166 (7), 1685-1702 (2012).</li><li>Fr&#248;lund, B., Palmgren, R., Keiding, K., Nielsen, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+extracellular+polymers+from+activated+sludge+using+a+cation+exchange+resin.">Extraction of extracellular polymers from activated sludge using a cation exchange resin.</a> Water Res. 30 (8), 1749-1758 (1996).</li><li>Nielsen, P. H., Jahn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+EPS.">Extraction of EPS.</a> Microb. Extracell. Polym. Subst. Charact. Struct. Funct. , 49-72 (1999).</li><li>Lin, Y. M., Nierop, K. G. J., Girbal-Neuhauser, E., Adriaanse, M., van Loosdrecht, M. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+polysaccharide-based+biomaterial+recovered+from+waste+aerobic+granular+sludge+as+a+surface+coating+material.">Sustainable polysaccharide-based biomaterial recovered from waste aerobic granular sludge as a surface coating material.</a> Sustain. Mater. Technol. 4, 24-29 (2015).</li><li>Lin, Y. M., Wang, L., Chi, Z. M., Liu, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Alginate+Role+in+Aerobic+Granular+Bio-particles+Formation+and+Settleability+Improvement.">Bacterial Alginate Role in Aerobic Granular Bio-particles Formation and Settleability Improvement.</a> Sep. Sci. Technol. 43 (7), 1642-1652 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Standard Methods for the Examination of Water and Wastewater. , (1998).</li><li>Mchugh, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A guide to the seaweed industry. , (2003).</li><li>Lin, Y., de Kreuk, M., van Loosdrecht, M. C. M., Adin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+alginate-like+exopolysaccharides+isolated+from+aerobic+granular+sludge+in+pilot-plant.">Characterization of alginate-like exopolysaccharides isolated from aerobic granular sludge in pilot-plant.</a> Water Res. 44 (11), 3355-3364 (2010).</li><li>Zorel, J. A., Aquino, S. F., Sanson, a. L., Castro-Borges, W., Silva, S. Q. <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+EPS+extraction+protocols+from+anaerobic+sludge+for+gel-based+proteomic+studies.">Evaluation of EPS extraction protocols from anaerobic sludge for gel-based proteomic studies.</a> Water Sci. Technol. 72 (4), 535 (2015).</li><li>Ruiz-Hernando, M., Cabanillas, E., Labanda, J., Llorens, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound,+thermal+and+alkali+treatments+affect+extracellular+polymeric+substances+(EPSs)+and+improve+waste+activated+sludge+dewatering.">Ultrasound, thermal and alkali treatments affect extracellular polymeric substances (EPSs) and improve waste activated sludge dewatering.</a> Process Biochem. 50 (3), 438-446 (2015).</li><li>Seviour, T., Pijuan, M., Nicholson, T., Keller, J., Yuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-forming+exopolysaccharides+explain+basic+differences+between+structures+of+aerobic+sludge+granules+and+floccular+sludges.">Gel-forming exopolysaccharides explain basic differences between structures of aerobic sludge granules and floccular sludges.</a> Water Res. 43, 4469-4478 (2009).</li><li>de Kerchove, A. 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><tbody><tr><td>250 ml baffled flask</td><td>Kimble</td><td>25630-250</td><td></td></tr><tr><td>1,000 ml glass beaker</td><td>VWR</td><td>213-1128</td><td></td></tr><tr><td>RCT basic, magnetic stirrer with thermometer</td><td>IKA</td><td>3810000</td><td></td></tr><tr><td>sodium carbonate decahydrate</td><td>Merck KGaA</td><td>1063911000</td><td></td></tr><tr><td>50 ml centrifugation tubes</td><td>greiner bio-one</td><td>227261</td><td></td></tr><tr><td>Multifuge 1 S-R, centrifuge</td><td>Heraeus/Thermo Scientific</td><td>-</td><td></td></tr><tr><td>hydrochloric acid, 37%</td><td>Sigma-Aldrich</td><td>30721-1L-GL-D</td><td></td></tr><tr><td>250 ml glass beaker</td><td>VWR</td><td>213-1124</td><td></td></tr><tr><td>calcium chloride dihydrate</td><td>Merck KGaA</td><td>1023821000</td><td></td></tr><tr><td>1 ml Pasteur Pipette</td><td>Copan</td><td>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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54534/54534fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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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

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

22.5K 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

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

科研

环境科学

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 ...

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These biomolecules arrange in paracrystalline two-dimensional arrays on surfaces, bind metals, and are thus interesting for several biotechnical applications, such as biosorptive materials for the removal or recovery of different elements from the environment and industrial processes. The deposition of Au(0) nanoparticles on S-layers, either by S-layer directed synthesis 1 or adsorption of nanoparticles, opens new possibilities for diverse sensory applications. Although numerous studies have described the biosorptive properties of S-layers 2-5, a deeper understanding of protein-protein and protein-metal interaction still remains challenging. In the following study, inductively coupled mass spectrometry (ICP-MS) was used for the detection of metal sorption by suspended S-layers. This was correlated to measurements of quartz crystal microbalance with dissipation monitoring (QCM-D), which allows the online detection of proteinaceous monolayer formation and metal deposition, and thus, a more detailed understanding on metal binding.
The ICP-MS results indicated that the binding of Au(III) to the suspended S-layer polymers is pH dependent. The maximum binding of Au(III) was obtained at pH 4.0. The QCM-D investigations enabled the detection of Au(III) sorption as well as the deposition of Au(0)-NPs in real-time during the in situ experiments. Further, this method allowed studying the influence of metal binding on the protein lattice stability of Slp1. Structural properties and protein layer stability could be visualized directly after QCM-D experiment using atomic force microscopy (AFM). In conclusion, the combination of these different methods provides a deeper understanding of metal binding by bacterial S-layer proteins in suspension or as monolayers on either bacterial cells or recrystallized surfaces.

Au-Interaction of Slp1 Polymers and Monolayer from Lysinibacillus sphaericus JG-B53 - QCM-D, ICP-MS and AFM as Tools for Biomolecule-metal Studies

To obtain basic information on the sorption and recycling of gold from aqueous systems the interaction of Au(III) and Au(0) nanoparticles on S-layer proteins were investigated. The sorption of protein polymers was investigated by ICP-MS and that of proteinaceous monolayers by QCM-D. Subsequent AFM enables the imaging of the nanostructures.

SLP1聚合物和单层从金交互<em&gt; Lysinibacillus球形</em&gt; JG-B53 - QCM-D，ICP-MS和原子力显微镜的工具生物分子金属研究

In this publication the gold sorption behavior of surface layer (S-layer) proteins (Slp1) of Lysinibacillus sphaericus JG-B53 is described. These ...

化学

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

生物化学

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

生物工程

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

一种新型生物反应器为不同的微生物群落的高密度栽培

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

<meta charset="utf8"></head><body>模拟甲烷菌生态位 – 梯度注射器模型生态系统

Agarose-Based Model Ecosystem for Cultivating Methanotrophs in a Methane-Oxygen Counter Gradient

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental niche within methane-oxygen counter gradients found in soils and sediments, which influences their behavior on an individual and community level. However, conventional methods to study the physiology of these greenhouse gas-mitigating microorganisms often use homogeneous planktonic cultures, which do not accurately represent the spatial and chemical gradients found in the environment. This hinders scientists' understanding of how these bacteria behave in situ. Here, a simple, inexpensive model ecosystem called the gradient syringe is described, which uses semi-solid agarose to recreate the steep methane-oxygen counter gradients characteristic of methanotrophs' natural habitats. The gradient syringe allows for the cultivation of methanotrophic strains and the enrichment of mixed methane-oxidizing consortia from environmental samples, revealing phenotypes only visible in this spatially resolved context. This protocol also reports various biochemical assays that have been modified to be compatible with the semi-solid agarose matrix, which may be valuable to researchers culturing microorganisms within other agarose-based systems.

<meta charset="utf8"></head><body>作者聚焦:设计简单且廉价的技术在实验室中培养甲烷氧化细菌

A protocol is described for preparing a simple model ecosystem that recreates the methane-oxygen counter gradient found in the natural habitat of aerobic methane-oxidizing bacteria, enabling the study of their physiology in a spatially resolved context. Modifications to common biochemical assays for use with the agarose-based model ecosystem are also described.

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

Aerobic methane-oxidizing bacteria, known as methanotrophs, serve important roles in biogeochemical cycling. Methanotrophs occupy a specific environmental ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

胞外多糖的OLIGO质谱分析（OLIMP）

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

从好氧颗粒污泥结构胞外聚合物的提取

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此视频中的章节

方法对纸浆厂污水流和培养微生物的生物降解能力的表征促进微生物生长

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

SLP1聚合物和单层从金交互

向外观察:分离氰化物释放的碳水化合物聚合物和蛋白质

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

一种新型生物反应器为不同的微生物群落的高密度栽培

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

基于琼脂糖的模型生态系统，用于在甲烷-氧反梯度中培养嗜甲烷菌

胞外多糖的OLIGO质谱分析（OLIMP）