Name: development of sulfidogenic sludge from marine sediments and trichloroethylene reduction in an upflow anaerobic sludge blanket reactor
Uploaded: 2015-10-15T14:00:00.000+00:00
Duration: 15 min 19 s
Description: Watch this Scientific Journal Video about Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor at JoVE.com

The authors are grateful for the financial support provided by Instituto Polit&#233;cnico Nacional grants 20120110, 20130399 and 20140239 SIP and also by Instituto de Ciencia y Tecnolog&#237;a del Distrito Federal Mexico (PICS 08-79, ICYT-DF, 2009-2012). Thanks also to CONACYT &#8211; Mexico for the graduate scholarship (225806) awarded to Selene Montserrat Garc&#237;a-Solares and for the financial support provided by grant 82627.

<img alt="图1" src="/files/ftp_upload/52956/52956fig1highres.jpg" width="450" /> 图1.方案协议的步骤。 <a href="https://www.jove.com/files/ftp_upload/52956/52956fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.收集海洋沉积物对污泥的形成<ol><li>确定一个平易近人海底区域或者靠近热液喷口（由于硫化物的存在下，这可能表明较高的硫酸盐还原活性），或者一个区域，其中的有机物质碎片是可检测的。 </li><li>对于这项工作的目的，需要约3或4千克沉积物和漏水关样品。是在暗塑料袋样品。无需进行制冷。 </li><li>一旦在实验室中，保持与样品袋在冰箱，如果他们不打算立即使用。对于这项工作，桑普的目的LES可在冰箱几个星期或几个月使用它们之前。 </li><li>取沉积物样品（即1或2公斤）的很大一部分，并使用适当的筛目（0.2厘米）从沉积物中的大碎片含碳材料可被发现或一些岩石可能存在消除。 注意:在这种情况下0.20厘米直径（在0.0767）的网眼被用于，但它可以根据样品中的颗粒的尺寸是不同的尺寸。 <ol><li>传递沉积物通过网后，混合选择，以促进该部分是均匀的部分。 </li><li>取分离更小的样品（即，2至3克）通过以下的标准方法11，以确定挥发性悬浮固体（VSS）的内容。 注: 参见图2步骤1.2至1.4。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52956/52956fig2.jpg" /> 图2.照片中的沉积物样品。（一）沉积物样品刚过上当受骗。（B）沉积物样品通过网格后。（C）样品采取称重之前，挥发性悬浮固体（VSS）的决心。在培养皿中不需要进行消毒。 <a href="https://www.jove.com/files/ftp_upload/52956/52956fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2.生物反应器设置<ol><li>对于这项工作的目的，使用的UASB反应器中的玻璃带3 L的总工作容积或者，使用1或2升容积玻璃反应器中。 </li><li>基于所述沉积物的VSS含量计算沉降物的数量被用作接种物，得到5克的VSS在1升</li><li>考虑到，如果沉积物计算后的量太大时，生物反应器的再约25％至30％的体积应当由沉积物代替占据。 <ol><li>记录，因为它的VSS内容将改变时，微生物群落富集的生物反应器。需要在VSS含量为硫酸盐的计算降低生物反应器中的活性。 </li></ol></li><li>确保生物反应器中的基础培养基和缓冲溶液的最终浓度是类似于所报告的格雷罗哈斯等 。 （2014）12。 <ol><li>确保沉积物，基本培养基，缓冲溶液和挥发性脂肪酸的最终体积是等于反应器中的最终工作体积。基础培养基配方12含有适当浓度的痕量金属和维生素溶液。 </li><li>制备基础培养基和缓冲液的储备溶液在适当的浓度为反应器所用的工作体积（即，2，3或4倍比报道的步骤2.4更浓缩的），以确保当它被稀释，它是在报告的格雷罗-巴拉哈斯等的浓度。 （2014年）12）。 注意:储备溶液的基础培养基是总是必要的，然而，缓冲溶液时，才需要在启动时。它没有必要在此时间后添加缓冲溶液。 </li><li>制备的挥发性脂肪酸的原液:在2.5乙酸盐，丙酸盐和丁酸盐:1:1比例的COD。考虑到为计算的乙酸钠包括在基础培养基。在反应器中的最终的COD浓度必须2.7克/升。 注意:准备这个解决方案在通风橱中。穿腈手套和护目镜此溶液的制备。考虑到硫酸与挥发性脂肪酸的反应， 示于图3的化学计量。 </li><li>制备的硫酸钠在适当浓度的储备溶液（ 用 Na 2 SO 4），可提供到反应器中的4000毫克/升的硫酸根离子（SO 4 2-）的终浓度。可替代地，包括第硫酸盐在基础培养基所需的，而不是只要由一个储液加入其作为最终硫酸盐电子量（SO 4 2-）的浓度是合适的。 </li></ol></li><li>放置在反应器中与基础培养基的一部分混合，沉积物，以确保它们到达反应器的底部。 <ol><li>添加与挥发性脂肪酸溶液和硫酸溶液混合的基础培养基和缓冲溶液的其余部分。确保的挥发性脂肪酸的溶液倒入液体。注意:进行这个步骤在通风橱中。 </li><li>设置的连接和该反应器的管道，以再循环泵。设定再循环流速为60毫升/分钟。在34°C在设定温度室的生物反应器。定期检查温度变化小（ 即 34±1.7°C） </li><li>设置连接到气体位移列。 注: 参见图4的步骤2.1至2.5。 </li> &lt;/ OL&gt; </li></ol><img alt="图3" src="/files/ftp_upload/52956/52956fig3highres.jpg" width="600" /> 图3.化学计量硫酸盐还原VFA（乙酸，丙酸和丁酸）的。 <a href="https://www.jove.com/files/ftp_upload/52956/52956fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52956/52956fig4highres.jpg" width="400" /> 图4. UASB反应器。（A）初始时间。（B）连续政权在300天的运行中。 <a href="https://www.jove.com/files/ftp_upload/52956/52956fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.操作反应器的促进Sulfidogenesis微生物和生长注意:允许用于接种物消​​耗购买Volatile脂肪酸和硫酸。为了这个目的，等待一个星期进行为硫酸盐，硫化物和COD消耗首次分析。 <ol><li>后孵育1周采取的5至7毫升液体的样品进行分析，化学需氧量，硫酸盐和硫化物含量和pH下述标准方法11,13。 <ol><li>通过以下亚甲蓝方法13分析硫化物在液体分光光度计（在670纳米的波长（λ））。 <ol><li>放置5毫升乙酸锌溶液（2％w / w的）在25毫升容量瓶中，加迅速加入200μl样品到醋酸锌溶液。 </li><li>加入2.5毫升N，N-二甲基对苯二胺草酸（DMP）溶液（0.2％w / w的20％ 的 H 2 SO 4）和125微升铁（III）硫酸铵溶液（10％重量/的瓦特在2％的H 2 SO 4）并完成与蒸馏水25毫升的容量瓶中。等待30分钟的反应发生，时间在其中蓝色颜色是稳定的。13。 注:等待至少15分钟，但不超过60分钟，以测试样品中的分光光度计。进行在分光光度计蓝色最终溶液的读数。 </li></ol></li><li>根据标准方法11分析干燥。这里，通过使用比浊法量化硫酸硫酸钡。 <ol><li>放置5毫升的调理溶液（盐酸盐酸1:1）在25毫升容量瓶中，加入1毫升先前离心样品（在11320×g）中，完成将25ml量瓶中，用蒸馏水和加1克氯化钡。 </li><li>混合在一涡旋1分钟的溶液中。等待4分钟的硫酸钡，以形成与读取在分光光度计的样品在420纳米11一个波长（λ）。 </li></ol></li><li>根据标准方法分析11的COD。或者，使用COD Determina的化套件。 <ol><li>在此之前的COD测定，彻底离心样品（在11320 XG）除去剩余的硫化物，可能会干扰测定。如果需要的话，离心两次:第一次立即服用样品和第二时间之后等待6或8小时，然后进行分析的COD。 </li><li>加2ml样品与COD测定试剂盒的反应小瓶中，密封该小瓶并均化通过温和搅拌该混合物。制备一个空白加入2毫升蒸馏水至另一个反应瓶中并均化混合物。 </li><li>放置在消化反应器中的小瓶，在150℃进行2小时。取出小瓶，让他们在黑暗中冷却下来。采取在分光光度计小瓶的读数在620纳米的波长。 </li></ol></li><li>获得从气体置换柱的气体体积。 </li></ol></li><li>最多等待另一个5到7天，直到硫酸的消耗。硫酸和COD必须在被消耗前pproximately 85％至90％，开始一个新的补料分批。 </li><li>一旦硫酸（和COD）的消耗，完全重复步骤2.4。供应新鲜培养基和营养物的新为每批次。 </li><li>重复步骤3.1和3.2。在这一点上每批次之间应该7天和10天持续。 </li><li>当3〜4批已全部完成，重复步骤2.4，但会增加COD浓度为4克/升。 </li><li>重复步骤3.1和3.2的步骤。 <ol><li>重复步骤3.3，但增加的COD浓度，以6克/升。 </li><li>重复3.6和3.6.1逐渐增加COD浓度，直到它为10克/升。 注意:请呈现硫酸浓度（mg / L）与时间（d）该曲线图。 </li></ol></li><li>当硫酸盐消耗量是80％以上，不到24小时，出现这种情况的一个多星期，开关在反应器的操作，以连续模式。对于连续模式中设置的水力停留时间（HRT），在24小时，并保持硫酸盐浓度在4克/升和COD的以10克/升。 注:随着时间的推移硫酸盐消费量应 速度更快。 </li></ol> 4.硫酸盐还原活性测试<ol><li>在此之前，测试确保连续制度下，生物反应器呈现在硫酸盐浓度剩余小于10％的变化。 </li><li>在任何给定的一天后，其中HRT周期和行为的步骤2.4停止反应器。对于步2.4.3用10克/升的COD浓度。 </li><li>一旦在生物反应器被供给，需要5到7 ml的样品的液体和化学需氧量，硫酸盐，硫化物（步骤3.1）和pH每小时执行分析。记录产生的气体量。 </li><li>计算硫酸根据文献14还原活性。 <img alt="式（1）" src="/files/ftp_upload/52956/52956eq1.jpg" /></li></ol> SRA =硫酸盐还原活性（毫克COD-H 2 S）/ gVSS量* D 米H 2 </em&gt; S =硫化物浓度单位为mg COD-H 2 S VSS =挥发性悬浮固体浓度 T =时间（D或小时） <ol start="5"><li>作出这样的显示硫酸盐消耗量与硫化物浓度的百分比随时间以mg / L对应的图形。使该显示的COD消耗随时间的百分比的曲线图。制作，显示随着时间的推移pH值变化的曲线图。 </li></ol> 5，三氯乙烯（TCE）还原试验<ol start="1"><li>在此之前，测试确保生物反应器正在连续制度下，并提出在硫酸盐浓度剩余小于10％的变化。不启动此测试如果硫酸盐还原生物反应器中低于90％。 </li><li>制备的三氯乙烯考虑到在生物反应器的液相中该化合物的最终浓度必须为300微米的储备液（TCE）。考虑partitioniNG使用Henry&#39;s法因次常数（H&#39;）的TCE在34°C的化合物的顶空。 H&#39;at 34℃，TCE为0.4722。 <img alt="公式（2）" src="/files/ftp_upload/52956/52956eq2.jpg" /> 注意:准备这个解决方案在通风柜和戴手套和护目镜。 <ol><li>例如，对于一个5000微米原液，计算如下: <img alt="公式3" src="/files/ftp_upload/52956/52956eq3.jpg" /> TCE气相浓度=（0.4722）*（5000）= 2139微米。包括这个浓度，因为这一数额TCE将是在顶空原液的制备。 然后，在原液中的液体（水），实际的TCE的浓度将是:5000 + 2139 = 7139μM。 TCE密度=1.43克/毫升。转换7139微米为mg，然后通过使用三氯乙烯的密度计算的TCE为原液的体积。 注:的TCE原液米浓度唉低于5000微米， 即 3000或1000微米，这取决于如何将该溶液的体积太大，根据其液相体积可以被传递至生物反应器。 </li></ol></li><li>制备标准曲线，在气相色谱仪的三氯乙烯， 顺 -1,2-二氯乙烯， 反式 -1,2-二氯乙烯，氯乙烯和乙烯。通过以下在5.2的三氯乙烯原液描述的相同方法制备的顺式 -1,2-二氯乙烯，反式-1,2-二氯乙烯的标准曲线从这些化合物的贮备液。通过从标准（气瓶）稀释每种气体的浓度制备氯乙烯和乙烯的标准曲线。 <ol><li>制备这些化合物的标准曲线的范围内的20至300微米。使用报告格雷罗-巴拉哈斯等人的方法 。 （2011）15，用于这些化合物在气相色谱仪的分析。 注意:这些准备站在通风柜ARD解决方案，并戴上手套和护目镜。 </li></ol></li><li>在任何给定的一天后，其中HRT周期和行为的步骤2.4停止反应器。对于步2.4.3用10克/升的COD浓度。 </li><li>一旦在生物反应器被供给，直接添加到三氯乙烯中的液体从在5.2制备的原液的生物反应器，生物反应器中的液相中的最终的TCE浓度必须为300μm。水力停留时间设定为12小时。 <ol><li>在一个HRT周期结束取液（500〜1000微升）和行为分析的COD，硫酸盐和硫化物的样品（步骤3.1.1，3.1.2和3.1.3）。取的顶部空间（100〜250微升）的样品，并进行分析，三氯乙烯，顺-1,2-二氯乙烯， 反式 -1,2-二氯乙烯，氯乙烯和乙烯在气相色谱仪。 </li></ol></li><li>重复步骤2.4。对于步2.4.3用10克/升的COD浓度。 </li><li>不要重复任何TCE还原试验，直到生物反应器中呈现超过900硫酸盐0％减少和较少的10％的变化两者，硫酸盐还原和硫酸中剩余的生物反应器。 </li><li>重复5.4，5.5和5.6两三次以上。 </li><li>取沉积物样品（0.5克）只需经过TCE减少测试完成后进行鉴定微生物。经过2或3 TCE减少测试执行此操作。 </li></ol> 6.硫酸盐还原TCE实验后还原活性测试<ol start="1"><li>完全重复步骤4。 </li></ol> 7.鉴定微生物<ol><li>取各得到大约0.5g污泥样品，并按照标准方法进行12总RNA提取。 </li><li>扩增16S rRNA基因用反转录，并进行聚合酶链反应（RT-PCR）扩增一个步骤12。 </li><li>设计引物扩增或作为初始的方法使用那些在文学11建议。按照amplif在文学12 ication程序建议。 </li><li>构建的16S rRNA库。 PCR扩增子可以通过使用克隆杰11被克隆。通常情况下，从各板（各菌落代表一个PCR产物）10个菌落可以克隆。根据在文献12提出的程序制备的质粒DNA进行测序。 </li><li>进行片段的测序。根据在文献中12建议的程序重新扩增大约1400 bp的对PCR外部产物与先前描述的方案进行PCR扩增（步骤7.4）和克隆。隔离来自大肠杆菌的质粒大肠杆菌菌落如文献12建议。不要进行部分程序进行测序应用M13通用引物12。 </li><li>进行序列分析。通过使用Clustal X对齐的核苷酸序列，并在文本编辑器中手动调整。执行NCBI databas的BLAST搜索即（http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi）12。 </li><li>得到的核苷酸序列的登录号。沉积下的对应登录号（即，JQ713915eJQ713925从扩增子序列）12确定在EMBL核苷酸序列数据库（根银行/ EMBL / DDBJ） ​​克隆的核苷酸序列。 </li></ol>

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H., Inagaki, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehalogenation+activities+and+distribution+of+reductive+dehalogenase+homologous+genes+in+marine+subsurface+sediments.">Dehalogenation activities and distribution of reductive dehalogenase homologous genes in marine subsurface sediments.</a> Applied and Environmental Microbiology. 75 (21), 6905-6909 (2009).</li><li>U.S. Environmental Protection Agency. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=List+of+priority+pollutants.">List of priority pollutants.</a> Clean Water Methods. , (2014).</li><li>Ozdemir, C., Dursun, S., Karatas, M., Sen, N., Sahinkaya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+trichloroethylene+(TCE)+in+upFlow+anaerobic+sludge+blanket+reactors+(UASB).">Removal of trichloroethylene (TCE) in upFlow anaerobic sludge blanket reactors (UASB).</a> Biotechnology and Biotechnological Equipment. 21 (1), 107-112 (2007).</li><li>Zhang, Y., Wang, X., Hu, M., Li, P. <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+hydraulic+retention+time+(HRT)+on+the+biodegradation+of+trichloroethylene+wastewater+and+anaerobic+bacterial+community+in+the+UASB+reactor.">Effect of hydraulic retention time (HRT) on the biodegradation of trichloroethylene wastewater and anaerobic bacterial community in the UASB reactor.</a> Applied Microbiology and Biotechnology. 99, 1977-1987 (2015).</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>Guerrero-Barajas, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sulfate+reduction+and+trichloroethylene+(TCE)+biodegradation+in+a+UASB+reactor+operated+with+sludge+developed+from+hydrothermal+vents+sediments:+process+and+microbial+ecology.">Enhanced sulfate reduction and trichloroethylene (TCE) biodegradation in a UASB reactor operated with sludge developed from hydrothermal vents sediments: process and microbial ecology.</a> International Biodeterioration and Biodegradation. 94, 182-191 (2014).</li><li>Tr&#252;per, H. G., Schlegel, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulphur+metabolism+in+Thiorhodaceae+I.+Quantitative+measurements+on+growing+cells+of+Chromatium+okenii.">Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells of Chromatium okenii.</a> Antoine van Leeuwenhoek. 30, 225-238 (1964).</li><li>Gallegos-Garc&#237;a, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological processes of sulfate reduction in biofilms for metals precipitation [Ph D thesis]. , (2009).</li><li>Guerrero-Barajas, C., Garibay-Orijel, C., Rosas-Rocha, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfate+reduction+and+trichloroethylene+biodegradation+by+a+marine+microbial+community+from+hydrothermal+vents+sediments.">Sulfate reduction and trichloroethylene biodegradation by a marine microbial community from hydrothermal vents sediments.</a> International Biodeterioration and Biodegradation. 65, 116-123 (2011).</li></ol>

The authors Selene Montserrat Garc&#237;a-Solares, Claudio Garibay-Orijel and Claudia Guerrero Barajas submitted (as inventors) in 2012 the application to obtain a patent entitled: &#8220;Process for the anaerobic treatment of industrial wastewater with high content of sulfate and chlorinated compounds&#8221;. This submission has been approved to the stage of the formatting. The submission has Instituto Polit&#233;cnico Nacional as the rights owner.

有sulfidogenesis的环境生物技术多个应用程序，硫酸盐还原细菌的代谢的最常用的应用程序在财团与发酵菌是在废水处理中的一个。 UASB反应器是主要的设计方法，以工业废水处理与硫酸盐高浓度之间。在这项工作中，我们提出了一个协议，以获得从海洋沉积物sulfidogenic污泥的UASB反应器。在协议中的关键步骤，以从海洋沉积物获得sulfidogenic污泥是:（1）促进沉积物样品的的同源被放置在反应器中，（...

一对环境生物技术的最重要的贡献是生物反应器，其中使用（接种物）的污泥能够硫酸盐还原条件下进行的设计。硫酸盐还原（SR）允许含有除同时除去COD，重金属和有机污染物，这一事实使SR中的污泥1的期望特性的高浓度硫酸的废水流的治疗。废水污染硫酸的一些例子来自制革，造纸，医药，化工等制造行业1。然而，大多数的文献是指sulfidogenic污泥时产甲烷颗粒污泥已适应sulfidogenesis 2。这种适应是通过操纵COD / SO 4 2-比在生物反应器和添加的化学品，以抑制在污泥2,3甲烷通常达到。除了长的时间是m唉要求sulfidogenic颗粒的形成中，甲烷和硫酸盐还原和污泥的高浓度硫化物的公差之间的竞争，是一些主要问题，如果是从适应获得在生物反应器中使用的sulfidogenic污泥可能出现主要产甲烷污泥硫酸盐还原条件。在这项工作中，我们描述的方法在上流式厌氧污泥床反应器（UASB），以获得从热液喷口沉积物（蓬美达，纳亚里特，墨西哥）主要为sulfidogenic污泥，然后我们评估它的硫酸盐还原活性随着时间的推移，进行了一个实验以评估其对还原脱氯的应用。被选择的沉积物的位置，因为它已被报道，在该网站有形成硫化物由于通过微生物群落栖息该特定地方4呈现的硫酸盐还原活性。 有SEVER人的优势获得沉积物这sulfidogenic污泥在适应产甲烷颗粒污泥sulfidogenesis。其中的一些优点是:（1）这是没有必要以形成颗粒的生物反应器进行操作，（2）将污泥容忍较高浓度硫化物的相比更为UASB与适于甲烷污泥操作，和（3）有用即使乙酸的用量为包含在培养基中以促进污泥的形成挥发性脂肪酸的混合物甲烷为基片没有竞争。 此步骤，以促进sulfidogenesis因为海洋沉积物有各种各样，如硫酸盐还原菌，发酵菌和细菌脱卤，只是仅举几例5,6微生物的天然泳池。财团从海洋沉积物开发利用这一协议的类型可能会出现在硫酸盐还原，因此，高的工作效率<html> ulfate还原活性随着时间的推移和更高的耐受性硫化物的浓度比报告为有毒的产甲烷菌和硫酸盐还原菌更高。另一方面，它很可能是该脱卤能力由以下此处协议提议的，但它可以取决于原始微生物群落也示于沉积物。这种假设是基于这样还原脱氯可以通过呼吸或共代谢发生的事实完成的，这两个条件，可能会在海洋微生物群落7得到提升。沉积物以获得污泥的培养物通过使用乙酸盐，丙酸盐和丁酸盐的混合物作为底物，因为这些挥发性脂肪酸所使用的几株硫酸盐还原细菌的进行。这些酸也经常发现在海洋沉积物碳化合物的种类，根据在文献中关于碳质材料在海沉积物5,6-若干报告。 内容"&gt;最后，一些被发现在地下水和其他水体，世界各地的最有毒化合物是氯化溶剂，如三氯乙烯（TCE）或全氯乙烯（PCE）。这些化合物是有毒的，不仅对人，而是还对微生物，特别是三氯乙烯，这仍然是在美国被认为是优先的污染物由环境保护局8。在这项工作中，我们提出，是在一个实验，其中sulfidogenic污泥减少TCE浓度的其能力进行测试系列报道的甲烷条件下，9,10含氯化合物生物降解性。值得一提的是，大多数的含氯化合物的生物降解研究已经甲烷条件9,10下进行的。我们认为，实验TCE在该协议提出的是一个的污泥的潜在应用很好的例子。该实验的目的是到e计价污泥的三氯乙烯和对硫酸盐还原活性的三氯乙烯效果的耐受性。考虑到最上氯化化合物的生物降解研究的是甲烷条件下进行的，该协议建议一个油泥的产生可以被用来同时:（1）除去硫酸，（2）除去COD和（3）除去氯化合物。进一步的步骤可以是评价对同时除去三氯乙烯和重金属的污泥（除了硫酸盐和COD），这是不能被甲烷条件下评价两个条件。

<table><tbody><tr><td>trichloroethylene&amp;nbsp;</td><td>sigma Aldrich</td><td>251402</td><td></td></tr><tr><td>&lt;em&gt;cis&lt;/em&gt;-1,2-dichlorotehylene</td><td>sigma Aldrich</td><td></td><td></td></tr><tr><td>&lt;em&gt;trans&lt;/em&gt;-1,2-dichloroethylene</td><td>sigma Aldrich</td><td>D-62209</td><td></td></tr><tr><td>vinyl chloride scotty standard</td><td>supelco</td><td></td><td>1,000 ppm v/v in nitrogen</td></tr><tr><td>ethene scotty standard</td><td>supelco</td><td></td><td>99% purity</td></tr><tr><td>pump</td><td>Masterflex</td><td></td><td>Model 7553-75</td></tr><tr><td>spectrophotometer</td><td>any</td><td></td><td></td></tr><tr><td>microcentrifuge</td><td>any</td><td></td><td></td></tr><tr><td>gas tight syringes&amp;nbsp;</td><td>any</td><td></td><td>100 and 200 microliters</td></tr><tr><td>UASB glass reactor</td><td>any</td><td></td><td>under design</td></tr><tr><td>gas chromatograph&amp;nbsp;</td><td>any</td><td></td><td>FID detector</td></tr><tr><td>capillary column SPB-624</td><td>supelco</td><td></td><td></td></tr><tr><td>pH meter</td><td>any</td><td></td><td></td></tr><tr><td>viton tubing</td><td>Masterflex</td><td></td><td></td></tr><tr><td>basal medium reagents</td><td>any</td><td></td><td></td></tr><tr><td>trace metals reagents</td><td>any</td><td></td><td></td></tr><tr><td>vitamins solution reagents</td><td>any</td><td></td><td></td></tr><tr><td>sodium sulfate</td><td>any</td><td></td><td></td></tr><tr><td>volatile fatty acids</td><td>any</td><td></td><td></td></tr><tr><td>COD determination kit</td><td>HACH</td><td></td><td>range 0-15,000 mg/L</td></tr><tr><td>TOPO-TA cloning kit pCR&amp;reg;4.0&amp;nbsp;</td><td>Invitrogen, US</td><td></td><td></td></tr><tr><td>S.N.A.P. TM Miniprep Kit&amp;nbsp;</td><td>Invitrogen, UK</td><td></td><td></td></tr><tr><td>Pure link TM Quick Plasmid Miniprep kit</td><td>Invitrogen</td><td></td><td></td></tr></tbody></table>

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.

的生物反应器中硫酸盐还原的典型行为示于图5。注意到，在硫酸操作减少第一周将是缓慢的是很重要的。然而慢，硫酸随时间的90％以上的消耗表明接种物正在开发的微生物群落能够减少干燥并因此，富含硫酸盐还原菌。图中的不同时期表明，硫酸盐还原反应增加其速度随时间。在开始时，批料用了20天的硫酸将减少（期间I）中，那么它的降低率正在增加，这需要大约10?...

Watch this Scientific Journal Video about Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor at JoVE.com

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

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 ...

development-sulfidogenic-sludge-from-marine-sediments

Research

JoVE Journal

Environment

9.6K 次观看.  Instituto Politécnico Nacional. 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.

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

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 &#176;C) and thermophilic (55 &#176;C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 &#176;C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 LCH4/kgVS&#160;(VS: volatile solids) and 121 L CH4/kgVS&#160;at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kgdry_biochar was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.

A novel Upflow Anaerobic Solid State (UASS) reactor was used for biogas production from fibrous feedstock. Digestate from UASS reactor was hydrothermally carbonized into HTC biochar in a pressurized batch reactor. The integration of the two bioenergy concepts was applied in this study to increase overall bioenergy production.

评定一体化厌氧消化和水热碳化对生物能源生产

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal ...

科研

环境科学

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.

The protocol provides a methodology to solubilize aerobic granular sludge in order to extract alginate-like extracellular polymers (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) ...

Continuously-stirred Anaerobic Digester to Convert Organic Wastes into Biogas: System Setup and Basic Operation

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier 1-3. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications 4,5. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane 6,7. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures 8, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale.
Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations 9. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs).
This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation 10.

Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.

连续搅拌厌氧消化有机废物转化为沼气系统的安装和基本操作

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier ...

生物工程

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg&#176;. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg.
Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE.
To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron OxyHydroxides, Trace Elements, and Bacteria

Fate and speciation of arsenic and mercury in aquifers are closely related to physio-chemical conditions and microbial activity. Here, we present an original experimental column setup that mimics an aquifer and enables a better understanding of trace element biogeochemistry in anoxic conditions. Two examples are presented, combining geochemical and microbiological approaches.

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

具有腐蚀性烟气的台式光生物反应器中的微藻培养和生物量定量

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

将发电厂的碳捕获与半自动开放式管道池相结合，用于微藻养殖

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

严格厌氧/缺氧条件下微生物培养的介质制备

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

生物化学

用于从猪废沼气中获取生物甲烷的微藻培养

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

作者聚焦:扩展微藻生物技术以提高生物甲烷产量 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

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

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评定一体化厌氧消化和水热碳化对生物能源生产

从好氧颗粒污泥结构胞外聚合物的提取

连续搅拌厌氧消化有机废物转化为沼气系统的安装和基本操作

用于研究铁 (氧) 氢氧化物、微量元素和细菌之间厌氧生物地球化学相互作用的实验柱装置

具有腐蚀性烟气的台式光生物反应器中的微藻培养和生物量定量

将发电厂的碳捕获与半自动开放式管道池相结合，用于微藻养殖

严格厌氧/缺氧条件下微生物培养的介质制备

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化

在半工业化高速率藻类池塘中使用微藻细菌系统进行沼气净化