资助这项工作的各个方面是由各种来源，包括农业部美国农业部的具体合作协议58-6408-1-595和美国国家科学基金会（奖1049911）提供。

胞外酶活性土壤和沉积物中的比色分析 1。对酶活性的比色分析底物的制备及缓冲溶液<ol><li>通过混合50毫升0.1M乙酸（2.87毫升冰醋酸在500毫升水中）制备50mM醋酸缓冲液（pH 5.0-5.5），加入150ml的0.1M醋酸钠，和200毫升蒸馏水的H 2 O。如有必要，调整pH值至5.0-5.5用0.1M乙酸。 </li><li>在蒸馏H 2 O制备的1M氢氧化钠（NaOH）的溶液</li><li>在50mM醋酸缓冲液制备p NP-连接的底物溶液。为了测定磷酸酶制备5mM对NP-磷酸盐，在50mM乙酸盐缓冲液;以测定β-葡糖苷酶制备5mM对NP-β-吡喃葡糖苷;以测定NAG酶制备2毫米的pNP-β-N-acetylglucosaminide。准备在无菌的15毫升或50ml离心管中所有衬底的解决方案。解决方案可以被存储在4℃下2-3周。 </li></ol> 2。测定标准的转换吸光度为 p的NP浓度<ol><li>在50mM醋酸缓冲液制备的对硝基苯酚的标准溶液。浓度范围应为0.025-1毫米。 </li><li>转移100μl的各浓度重复三次，以清晰的96孔微板。加10μl1 M氢氧化钠和190微升蒸馏水H 2 O至每口井。 </li><li>在410nm下用酶标仪记录吸光度。 </li><li>乘法浓度在标准曲线0.3得到每300微升反应体积微摩尔p NP。绘制吸光度曲线与p NP的微摩尔。该曲线的斜率作为转换系数（C），这将涉及到的吸光度在各酶反应微摩尔p NP的。 </li></ol> 3。进行酶分析<ol><li>对于土壤，制备各样品的浆液在concentr待测定在无菌15ml离心管振动性为1g /毫升-1用50mM醋酸缓冲液约的。对于沉积物，补充足够的醋酸盐缓冲液，使料浆容易pipettable。所需浆料的​​确切量将根据酶测定的数目而变化，但至少为5毫升的建议。每个涡浆，直到土壤或沉积物的所有团块纷纷散去，并注意最终体积。 </li><li>再涡流各浆料并立即吸取150微升到每个6孔96孔深孔块（ 图1）上。重要的是要彻底涡流和频繁，以保持土壤的颗粒在悬浮液中。离开每块至少有两个井空作为底物的控制。注:剪辑的移液器吸头与之前的移液剪刀年底的土壤泥浆容易堵塞的提示。准备一台96井区的每种酶进行检测。 </li><li>倒入大约5毫升的醋酸缓冲液入吸管水库和使用8道移液器到150微升的缓冲液加入至升AST各样品2孔（这将样品缓冲液对照组）和2基板对照孔（ 图1） </li><li>倾大约10毫升正常 P NP-底物溶液成吸管水库和使用8道移液器至150微升底物溶液加至前四个孔中每一个样品和两个基板对照孔（ 图1）。注意时间作为反应只要加入底物溶液开始。 </li><li>孵育板在室温（22℃）为0.5-4小时。确切的温育时间将取决于样品中的活性水平和待测定的酶而有所不同。对于大多数土壤和沉积物，磷酸酶和β-葡萄糖苷酶需要0.5-1.5小时的孵育时间，而NAG酶和其他酶需要&gt; 2小时孵育时间。 </li><li>虽然试验是孵化制订明确的96孔微孔板读吸光度。准备一台微孔板的每个深孔块（ 即每封nzyme测定）。移液管加入10μl的1M NaOH和190微升蒸馏水到微孔板的每个孔中。注意:氢氧化钠减慢酶促反应，并且提高，从而提高了反应过程中释放的P NP的颜色的pH值。 </li><li>培养后，离心分离96孔块在2,000-5,000×g离心5分钟以沉淀土壤颗粒。 </li><li>使用多道移液器从每孔撤出100微升，请小心操作以免颗粒，并将其传输到编制明确的96孔板对应得很好。 </li><li>打开酶标仪，并设立任何必要的软件。在410nm处记录吸收值。如果一个特定的孔的吸光度高于酶标仪的线性检测限，淡化了良好1:1的水和重新测量。如果吸光率仍然太高，该测​​定应反复用更短的孵育时间。 </li></ol> 4。样品的干质量的测定<ol><li>移液器各1ml SAMPL的<html> Ë浆到一个预先称重的铝盘中。 </li><li>干在75℃烘箱中干燥48小时后称量。从这个值减去锅的重量，得到土壤或沉积物的干质量的1毫升的浆料。通过0.15的因子相乘，以确定在150微升的酶测定加入到每孔样品的干质量。 </li></ol> 5。每个土壤干质量或沉积物酶活性的计算<ol><li>减去从样品测定吸光度采样控制吸光度计算出每个样品的最终吸光度。如果基板的控制有较高的吸收率（约&gt; 0.060），然后减去者也。 </li><li>计算酶的活性在微摩尔小时-1 g干质量-1从以下公式计算: </li></ol>酶的活性=最终吸光度/（C X孵育时间×样品干重） 胞外酶活性的天然水域的荧光分析对酶活性的荧光分析幔"&gt; 1。底物的制备，标准和缓冲溶液<ol><li>通过在无菌溶解适当的衬底制备200μM的溶液MUB联的底物（ 如 4 - MUB-β-吡喃葡糖苷，4 - MUB-磷酸盐，4 - MUB-N-乙酰基-β-D-氨基葡糖苷）的（高压灭菌）蒸馏水H 2 O的在无菌的15毫升或50ml离心管中。包装在铝箔管，以排除光线并存储在使用之前冰箱。基材应至少1周是稳定的，如果以这种方式存储。 </li><li>通过使100微米4 -甲基伞形酮在无菌蒸馏水H 2 O的原液准备MUB标准冷藏储存在琥珀色或箔纸包裹的瓶子。在使用前立即由1/10稀释为100μM原液到无菌H 2 O的使10μM的用于酶测定的工作溶液。 </li><li>通过溶解8.4克碳酸氢钠&lt;作好准备的100毫米碳酸氢盐缓冲液的原液子&gt; 3到1 LH 2 O和高压灭菌。根据需要使5毫米的酶的测定工作液稀释此原液的1/20到无菌H 2 O </li></ol> 2。在96孔黑色微孔板组织水样<ol><li>组织微板的每个酶按照图2所示的例子。请注意，以允许足够的复制，标准和控制，这个程序可以测定单个酶的活性达九水样在一个96孔微孔板黑色。 </li><li>倒入大约5毫升第一样品放入吸管水库和使用8道移液器以微升pipette200到所有的水井在微孔板（S）的第1列的。丢弃用过的枪头，并根据需要对每个水样，以填补列1-9重复。 </li></ol> 3。设置样品，标准，淬火和控制基板<ol><li>设置控件占样品，标准秒，基板和淬火在相同的黑色微孔板的样品（ 图2）。 </li><li>样品对照样品含有水和碳酸氢盐缓冲液中，并没有在活性计算中使用，但将演示读取整个实验过程中保持一致。淬火控制包括样品的水和荧光标记的标准量的和用于测量荧光的样品水的衍射。基板和标准控件要么基板联或标准的荧光标记，分别与碳酸氢盐缓冲液的组成。 </li><li>倒入大约5毫升5 mM的碳酸氢盐缓冲液到一个干净的移液管水库。吸取50μL缓冲到微孔板1通过9排D和E形成两个复制的每个样品采样控制井。变化的枪头然后行转移200微升碳酸氢盐缓冲液中，以井10到12 A和H。 </li><li>通过调光或关闭李降低环境照明亮灯作为荧光标准对光敏感。 </li><li>倒入大约5毫升10微米4 - 甲基伞形酮到一个干净的移液管水库。吸取50μL进微孔板1通过行H 12，和成井1到9成排G和F，形成每个样品淬火控制和整体水平控制三个重复。无论是将微孔板在黑暗中或覆盖用不透明的盖子，以减少MUB的光降解。 </li><li>打开的荧光计和设置任何必要的软件以准备加入底物之前阅读。注意:某些荧光灯泡可能需要3分钟以上的预热时间。 </li><li>倒入大约5毫升适当MUB联基板（ 如 4-MUB -磷酸）放入干净的移液管水库。使用12通道移液器吸取到50微升到微孔板1通过A排12，进水井1至9中的行B和C形成三个重复测定每个样品和三个基板的控制。立即出现Ÿ继续执行步骤4.1，记录荧光。 </li></ol> 4。记录荧光<ol><li>基板除了微孔板后立即读出初始荧光。阅读荧光后，孵育微孔板在室温（22℃），无论是在黑暗中或覆盖着不透明的盖子，以减少MUB的光降解。 </li><li>以测量水样品中的最大的潜在酶活性所需的温育时间将依赖于酶浓度的样品内。由于这是未知的测定完成之前，该微板将要读出的多个时刻的步骤。通常情况下，在10〜15分钟的间隔读了1个小时的过程中是可以接受的许多酶，虽然与某些酶的活性非常高的样品可以在10分钟之前见顶。 </li><li>继续阅读荧光微孔板在你指定的时间间隔至少1小时。一定要保持微孔板盖或读数之间的暗秒。 </li></ol> 5。每个供水量酶活性的计算<ol><li>在每个时间间隔中的每个样本计算:平均初始样品的荧光（孔D和E），平均最终样品的荧光（井AC），平均标准荧光（孔H10-11），且平均急冷控制荧光（井FG）。 </li><li>对于每个时间间隔，计算在纳摩尔小时酶活-1毫升-1由以下公式计算: </li></ol>酶活性=（平均样品荧光 - 平均初始样品荧光）/（（平均标准的荧光/ 0.5摩尔）×（平均淬火控制荧光/均值标准的荧光）×（0.3毫升）×（时间以小时为单位）） <ol start="3"><li>检查计算每个时间步骤中的活性值。从最高的活动时间步长决定最终的潜在活性。如果活性值继续增加再后来的时间步长，可能需要，如果活性值下降throug豪特的运行过程中，然后用更短的时间的步骤再次运行。最终活性是在衬底消耗小时-1毫升-1的纳摩尔但可以按比例增加，表示为微摩尔小时-1·L-1。 </li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+scale+patterns+in+microbial+extracellular+enzyme+activity+during+leaf+litter+decomposition+in+a+stream+and+its+floodplain.">Fine scale patterns in microbial extracellular enzyme activity during leaf litter decomposition in a stream and its floodplain.</a> Microbial ecology. 58, 591-598 (2009).</li></ol>

作者宣称，他们有没有竞争的财务权益。

确定各种土壤和沉积物中微生物胞外酶的活性可以有效地洞察养分的矿化与有机物处理17的速率。然而，土壤可以在其水分含量而有所不同，所以标准化活动，以土壤干重是很重要的。这就要求（通常两天）超越了简单的测量酶活性的附加干燥步骤。因此，相对于酶活性的水样，接近瞬时的结果提供了在测定中，在土壤和沉积物酶活性的可靠的检测需要几天。对于一些土壤，它甚至可能是更合适的，表达为每克活性有机物或无灰干重，需要一个额外的灰化步骤（通常为2小时，在500℃）以外的烘干程序。无论如何，在土壤或沉积物酶活性的比色测定法中最关键的步骤是将上清液从深阱方框f撤跟据一个离心培养期的结束。因为这个过程依赖于测量吸光度，几个流浪土壤颗粒甚至在最后的微孔板的存在可能会导致虚假的结果。较高的离心速度（&gt; 5,000 XG）可能有助于缓解这个问题，但在我们的经验离心机转子接受微孔板通常有低于这个阈旋转极限，而块本身可能不会容忍更高的速度。本质上，这个特定的移液步骤需要小心和速度的组合，以使用多通道移液器以快速地从深阱块传送所需的上清液的量为微量。 胞外酶活性的水样中的测定既没有必要干燥时间，也不是离心步骤，后者因为无论是酶 - 底物反应和最终产品的荧光的测量发生在相同的微孔板。因此，这些一ssays通常更快，最初可能会出现更容易运行。但是，他们有自己的局限性在于，只要有可能，在MUB标准和MUB联基材不应该暴露在光线下。在我们的经验，调暗灯光尽可能多的可能的移液过程中和温育微孔板在黑暗中（或覆盖有一个不透明的盖子）是必要的。因为这些测定也需要在板读取在多个时间点，这同时测定多种酶时常常会导致板之间需要非常快速的切换。例如，为了测定9水样本进行的同时（ 即用六种不同的微板）6的酶的活性，有必要读出的板，每2分钟1小时，以确保每个单独的板读取每10 -15分钟（在这种情况下，每12分钟）。必须小心，以保持特定的板被读的轨道，以及以确保没有液体溢出的往复米的板，因为它被转移进和流出的微孔板荧光计。当测定多种酶一次，同样重要的是要记住，它需要的酶标仪实际读板，并相应地错开读间隔时间。与样品是浑浊工作时与水样中胞外酶活性的荧光检测的最终关注的是。含有悬浮颗粒的水样前应首先涌入吸管水库动摇和退出和退出与被加载到微孔板前移液器再次混合。较高的数字样本中的颗粒也通常会导致荧光信号的较大的淬火，以及淬火控制每个样品的重要性，不能充分强调。 虽然MUB- 和 p NP联基材通常显示为环保酵素，对K M值类似V 最大价值可以不同，和酶，如β-葡糖苷酶和磷酸酶可能对MUB联衬底更高的亲和力比它们的 p NP-连接的类似物14。因此，MUB联的底物是可能比那些链接到P NP，这表明它们将是一个较好的选择测量土壤和沉积物中的酶活性时，使用更敏感。然而，它是在MUB实验测得的荧光最终产品，须在某些土壤提取物潜在淬火和荧光读数可能不稳定随着时间的推移12。土壤和沉积物中也往往有比天然水域较高外酶的活性，使荧光MUB联检测的敏感性增加可能会提供一点优势分析某些土壤时给出淬火的潜在问题的色度​​p NP方法。作为一个方面说明，在P NP联基板和p NP标准本身一般都比较afforda竹叶提取比他们MUB同行;一个额外的理由为他们的使用，如果预算限制适用。 也许能够测定微生物胞外酶的活性在水生和陆生环境中最重要的方面是，虽然这些技术测量微生物的生理过程，这些过程对碳和养分的生态系统层面转变有直接的影响。有机物的分解率已经直接关系到沉积物中18,19胞外纤维素酶的活性，使酶的活性快速的测量可能有助于瞬间原位分解速率环境样品中的测定。使用高通量微孔板方法允许在更大的样本数酶活性的同时测量超过典型的单管的方法，所以在酶活性的变化（通过扩展的生态系统级进程）在RESPONSE等因素深度20或环境扰动9,21可以进行检查。同样，参与养分循环的酶，如磷酸酶和NAG酶的检测，可以土壤发育8时提供深入了解营养限制在特定的环境中，例如，有机磷的相对重要性，以有机氮。 由于酶活性进行了测量环境样品中超过三十年，采用先进的荟萃分析研究之间的比较正在成为可能。这些研究表明，在有机物质的分解和养分矿化的全球范围内，酶活性，并因此率，被栓至pH，底物的可用性，营养物质和化学计量17。同时，利用一个微板为基础的协议的开始，以允许使用地质统计制图技术22的酶活性细尺度模式的分析</suP&gt;。这种技术可以检查空间变异模式和未来发展可​​能包括采用384孔微孔板为基础，以允许更多的样品和空间格局的更精细的分辨率的瞬时分析。

微生物如细菌和真菌通过生产胞外酶的取得从复杂的有机化合物的营养物质和碳。这些酶通常水解的聚合物为可以被取入细胞小亚基。因此，在生态层面上，这些微生物胞外酶是负责大部分的养分矿化和发生在自然的环境中有机物的分解。酶，例如纤维二糖水解（CBH）和β-葡糖苷酶是纤维素降解和工作重要一致来催化纤维素水解成葡萄糖1,2，它提供了对微生物的吸收和同化一个可利用的碳底物。酶磷酸酶释放的有机磷易溶于无机磷酸基团，基本上矿化磷酸盐和使其可用于大多数生物3使用。其他酶，如N-乙酰氨基（NAG酶），是的importan吨中几丁质的降解，可以使碳和氮供微生物采集4。 一项所述的程序，用于在自然环境中的微生物胞外酶活性的测定是使用人工对硝基苯基（ 对 NP）相连的基板，该最初被开发，以检测土壤磷酸酶活5的方法。此方法依赖于有色终产物， 对硝基苯酚，当人工基板通过适当的酶水解时释放的检测。 对硝基苯酚的随后可以通过测量其吸光度约为400-410 nm的比色定量。这种方法已经被应用到检测其它酶如NAG酶6，并已应用于各种研究着眼于在土壤和沉积物7-9微生物胞外酶活性。 这是originall一种替代方法Ý用于评估细胞外糖苷酶的活性在水生环境10,11是利用4 -甲基伞形酮（MUB）相连的底物。释放（4 - 甲基伞形酮）的最终产物是高荧光的，并且可以使用与周围四百六十分之三百六纳米的激发/发射设置一个荧光计来检测。各种MUB联的人工基质的情况下，允许至少为许多酶（ 如 β-葡糖苷酶，纤维二糖水解，NAG酶，磷酸酶）的活性的荧光测定作为可使用p NP-衬底比色步骤进行测定。其它微生物的胞外酶，如蛋白降解的亮氨酸氨肽酶，可以使用7 - 氨基-4 - 甲基香豆素（COU）键合底物荧光计进行测定。既MUB-和COU联基板已被用来确定各种陆地和水中的样品12,13中酶的活性。 虽然以前的研究单位描述离子束增强沉积荧光或比色微孔板方法来确定胞外酶的活性14，有必要建立一个清晰的介绍了如何进行这样的试验。在这里，我们展示了进行高通量微孔板技术的使用比色p NP联基板的做法土壤和沉积物，并在使用荧光MUB联基板技术自然水域胞外酶的活性分析程序。我们专注于β-葡糖苷酶，NAG酶和磷酸酶的活性的测量，因为这些酶可以分别连接到碳，氮，磷和骑自行车。然而，此处描述的步骤可以应用到使用不同的人工基质的其他胞外酶的测定。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS AND MATERIALS</td>
		 </tr>
		 <tr>
			<td>Glacial acetic acid</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sodium acetate</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sodium hydroxide</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>p-Nitrophenol</td>
			<td>Fisher</td>
			<td>BP612-1</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>p-Nitrophenyl (pNP)-phosphate</td>
			<td>Sigma</td>
			<td>N3234</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>pNP-&beta;-glucopyranoside</td>
			<td>Sigma</td>
			<td>N7006</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>pNP-&beta;-N-acetylglucosaminide</td>
			<td>Sigma</td>
			<td>N9376</td>
			<td>pNP-substrate</td>
		 </tr>
		 <tr>
			<td>Clear 96-well microplates</td>
			<td>Fisher</td>
			<td>12-563-301</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>96-well deep well blocks</td>
			<td>Costar</td>
			<td>3958</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>Aluminum weigh pans</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sterile 15 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Sterile 50 ml centrifuge tubes</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>4-Methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td></td>
		 </tr>
		 <tr>
			<td>4-Methylumbelliferyl (MUB)-phosphate</td>
			<td>Sigma</td>
			<td>M8883</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>4-MUB-glucopyranoside</td>
			<td>Sigma</td>
			<td>M3633</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>4-MUB-N-acetylglucosaminide</td>
			<td>Sigma</td>
			<td>M2133</td>
			<td>MUB-substrate</td>
		 </tr>
		 <tr>
			<td>Sodium bicarbonate</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td>Black 96-well microplate</td>
			<td>Costar</td>
			<td>3792</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipette reservoir</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		 </tr>
		 <tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge rotor</td>
			<td>Eppendorf</td>
			<td>A-4-81</td>
			<td>For microplates/deep-well blocks</td>
		 </tr>
		 <tr>
			<td>Microplate reader</td>
			<td>BioTek</td>
			<td>Synergy HT</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>Microplate fluorometer</td>
			<td>BioTek</td>
			<td>FLx 800</td>
			<td>Alternates available</td>
		 </tr>
		 <tr>
			<td>8-channel pipettor</td>
			<td></td>
			<td></td>
			<td>Various suppliers</td>
		 </tr>
		</tbody>
 </table>

determination of microbial extracellular enzyme activity in waters, soils, and sediments using high throughput microplate assays

大部分的养分循环和碳处理的自然环境是通过微生物释放胞外酶的活性。因此，这些胞外酶的活性的测定可以给见解生态系统水平的过程，如有机物分解或氮和磷的矿化速率。环境样品中的胞外酶活性的测定通常涉及暴露样品到人工比色或荧光底物和跟踪底物水解的速率。在这里，我们描述了基于微板的方法，这些方法允许对大量样品的一个短的时间内进行分析。样品被允许与人工基质内的96孔微孔板或深孔微孔板块进行反应，并且酶的活性，随后通过吸收所产生的终产物的荧光或使用典型的微孔板里德确定r或荧光。这种高通量的程序不仅有利于空间上不同的网站或生态系统之间的比较，而且还通过减少每个样品所需的试剂整体体积大大减少这种试验的费用。

土壤和水体沉积物通常有胞外酶的活性明显的水平附着的微生物群落（生物膜），颗粒的表面上生长的结果。 图3显示了本次活动依赖于从第三表层沉积物得到颗粒的大小如何变化在密西西比州北部，美国订单源源不断。先前的研究已经表明，在此流泥沙颗粒的细菌群落可以分为基于他们的社会结构的分子分析三个不同的群体:那些在0.063毫米颗粒，那些在0.125，0.25，和0.5毫米的颗粒，而这些1毫米的颗粒15。支持这一结论的胞外酶活性的模式数据;与磷酸酶（ 图3A），即相似于0.125，0.25和0.5毫米的颗粒，但在1和0.063毫米馏分当使用p NP-连接基板技术测定高得多。 Ø热敏酶，如β-葡糖苷（ 图3B），和NAG酶（ 图3C）表示的最细的颗粒相似的峰，但升高1毫米的颗粒，突出的事实，不同的酶可表现出不同的环境分布和这些可阐明使用该法。相对较低的误差线显示，比较不同环境样品时，酶的活性在沉积物比色法测定重复性好，因此适合进行统计分析。 天然水域往往有每毫升低于胞外酶的活性比土壤或沉积物每克做。因此，它们应该被荧光计使用MUB联的基质进行测定。 图4显示了酶的磷酸酶（ 图4A）的活性，β-葡糖苷（ 图4B），和NAG酶（ 图4C）随深度而变化的浅湖在密西西比州北部，美国答湖（皮绳湖）被称为是营养差16，也是由磷酸酶（ 图4A）的活性比较高建议，一种酶，微生物，以便从有机化合物收购磷矿生产。对于所有三个测定的酶，在水的表面（0厘米）和50厘米深度采集的样品表现出相似的活性，而活性升高100厘米的样品中。该样品基本上取自水 - 沉积物界面和沉淀颗粒的样品中是否存在可能占了较高的活性看出本示例中，特别是对β-葡糖苷酶和NAG酶。作为与p NP基板，低误差棒表明，即使只有三个重复读数，使用MUB基板上的荧光测定法的重现性高。 注意，单位为图4是基板的纳摩尔消耗而那些用于图3是在衬底消耗的微摩尔，即使每单位面积可比（无论是每毫升或每克）。这突出了一个事实，即土壤和沉积物倾向于具有比天然水域高得多的胞外活性（ 图3中每一个特定的酶的活性是约大于图4中的等效酶的活性10 - 100倍以上）。它也显示了MUB联基板技术，使用这种技术用于测定胞外酶的活性水样中的必要性的敏感性增加。 <img alt="图1" fo:content-width="3.5in" fo:src="/files/ftp_upload/50399/50399fig1highres.jpg" src="/files/ftp_upload/50399/50399fig1.jpg" /> 图1。 96孔深孔板中使用比色p NP联s的土壤或沉积物中胞外酶活性的测定建议布局ubstrate技术。各孔应接受总共300微升的，由样品浆料中，底物溶液，或醋酸盐缓冲液的取决于它是否是一个样本来说，样本控制，或控制衬底。 <img alt="图2" fo:content-width="5.5in" fo:src="/files/ftp_upload/50399/50399fig2highres.jpg" src="/files/ftp_upload/50399/50399fig2.jpg" /> 图2。 96孔黑色建议布局微孔板的胞外酶的活性用荧光MUB联基板技术水样中检测。九个样品可垂直于板（一）各占据八口井布置。这八个孔用于样品运行，采样控制，和淬火的控制，同时保持井用于基板的控制和MUB标准（B）。 <img alt="图3" fo:content宽度="3.5英寸"FO:SRC ="/ files/ftp_upload/50399/50399fig3highres.jpg"SRC ="/ files/ftp_upload/50399/50399fig3.jpg"/&gt; 图3。对不同规模从一条小溪在北密西西比，美国收集表层沉积物颗粒的胞外酶的活性。每个粒度组分测定磷酸酶（A），β-葡萄糖苷酶（B）的活性，NAG酶（C）以下的p NP-底物显色过程。活性被报道在微摩尔底物消耗小时-1克沉淀物-1的干质量，并为每个粒度级分三组重复读数的平均值（+ SE）。 <img alt="图4" fo:content-width="3.25in" fo:src="/files/ftp_upload/50399/50399fig4highres.jpg" src="/files/ftp_upload/50399/50399fig4.jpg" /> 图4。水在三个DIF采取外酶活性ferent深度（0，50，和100公分）从一个浅湖在北美国密西西比州。水测定磷酸酶（A），β-葡萄糖苷酶（B）的活性，NAG酶（C）以下的MUB基板荧光程序。活性被报道在纳摩尔衬底消耗h -1的毫升水和-1是每样本3重复读数的平均值（+ SE）。

Watch this Scientific Journal Video about Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays at JoVE.com

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

基于微板程序描述了用于胞外酶活性的比色或荧光分析。这些程序允许在大量环境样品等活动的管理的时间范围内的快速检测。

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

determination-microbial-extracellular-enzyme-activity-waters-soils

Nanyang Technological University

Research

JoVE Journal

Environment

38.7K 次观看.  The University of Mississippi. 基于微板程序描述了用于胞外酶活性的比色或荧光分析。这些程序允许在大量环境样品等活动的管理的时间范围内的快速检测。 

视频: Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer&#39;s particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e. C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e. colorimetric) assays,&#160;but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ soil type and temperature can influence enzyme kinetics.

To measure potential rates of soil extracellular enzyme activities, synthetic substrates that are bound to a fluorescent dye are added to soil samples. Enzyme activity is measured as the fluorescent dye is released from the substrate by an enzyme-catalyzed reaction, where higher fluorescence indicates more substrate degradation.

潜在土壤胞外酶活性的高通量荧光测量

Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be ...

科研

环境科学

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

从农业土壤使用静态钱伯斯温室气体流量测量

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

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 ...

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

沉积物核心切片和孔隙水萃取缺氧条件下

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

清洁取样和痕量金属研究河和河口水域分析

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

A Bending Test for Determining the Atterberg Plastic Limit in Soils

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a considerable subjective judgment from the operator that carries out the test is involved during its performance, which may affect the final result significantly. Different alternative methods have been put forward, but they cannot compete with the standard rolling test in speed, simplicity and cost.
In an earlier study by the authors, a simple method with a simple device to determine the PL was presented (the &#34;thread bending test&#34; or simply &#34;bending test&#34;); this method allowed the PL to be obtained with minimal operator interference. In the present paper a version of the original bending test is shown. The experimental basis is the same as the original bending test: soil threads which are 3 mm in diameter and 52 mm long are bent until they start to crack, so that both the bending produced and its related moisture content are determined. However, this new version enables the calculation of PL from an equation, so it is not necessary to plot any curve or straight line to obtain this parameter and, in fact, the PL can be achieved with only one experimental point (but two experimental points are recommended).
The PL results obtained with this new version are very similar to those obtained through the original bending test and the standard rolling test by a highly experienced operator. Only in particular cases of high plasticity cohesive soils, there is a greater difference in the result. Despite this, the bending test works very well for all types of soil, both cohesive and very low plasticity soils, where the latter are the most difficult to test via the standard thread rolling method.

The traditional standardized test for determining the plastic limit in soils is performed by hand, and the result varies depending on the operator. An alternative method based on bending measurements is presented in this study. This allows the plastic limit to be obtained with a clear and objective criterion.

弯曲试验在土壤确定阿太堡限塑

The thread rolling test is the most commonly used method to determine the plastic limit (PL) in soils. It has been widely criticized, because a ...

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

提取及土壤微生物磷脂脂肪酸分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and organiclastic sulfate reduction (OSR) in marine sediments. However, unravelling the complex pyritization sequence is a challenge because of the coexistence of different sequentially formed pyrite phases. This manuscript describes a sample preparation procedure that enables the use of secondary ion mass spectroscopy (SIMS) to obtain in situ &#948;34S values of various pyrite generations. This allows researchers to constrain how SO4-AOM affects pyritization in methane-bearing sediments. SIMS analysis revealed an extreme range in &#948;34S values, spanning from -41.6 to +114.8&#8240;, which is much wider than the range of &#948;34S values obtained by the traditional bulk sulfur isotope analysis of the same samples. Pyrite in the shallow sediment mainly consists of 34S-depleted framboids, suggesting early diagenetic formation by OSR. Deeper in the sediment, more pyrite occurs as overgrowths and euhedral crystals, which display much higher SIMS &#948;34S values than the framboids. Such 34S-enriched pyrite is related to enhanced SO4-AOM at the sulfate-methane transition zone, postdating OSR. High-resolution in situ SIMS sulfur isotope analyses allow for the reconstruction of the pyritization processes, which cannot be resolved by bulk sulfur isotope analysis.

Preparation of Authigenic Pyrite from Methane-bearing Sediments for In Situ Sulfur Isotope Analysis Using SIMS

Analyses of the sulfur isotopic composition (&#948;34S) of pyrite from methane-bearing sediments have typically focused on bulk samples. Here, we applied secondary ion mass spectroscopy to analyze the &#948;34S values of various pyrite generations to understand the diagenetic history of pyritization.

原位含甲烷沉积物中自黄铁矿的制备及应用模拟器硫同位素分析

Different sulfur isotope compositions of authigenic pyrite typically result from the sulfate-driven anaerobic oxidation of methane (SO4-AOM) and ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Sampling Soils in a Heterogeneous Research Plot

Soils are highly heterogeneous. In general, the number of soil samples required for soil research has always been determined arbitrarily and the associated accuracy is unknown. Here, we present a detailed protocol for efficient and clustered soil sampling in a research plot and, relying on a pilot sampling using this design, for demonstrating soil spatial heterogeneity and informing reasonable sample sizes and associated accuracy for future study. The protocol mainly comprises four steps: sampling design, field collection, soil analysis, and geostatistical analysis. The step-by-step procedure is modified according to former publications. Two examples will be presented to demonstrate contrasting spatial distributions of soil organic carbon (SOC) and soil microbial biomass carbon (MBC) under different management practices. In addition, we present a strategy to determine the sample size requirement (SSR) given a certain level of accuracy based on the plot-level coefficient of variation (CV). The field sampling protocol and the quantitative determination of the sample size will assist researchers in seeking feasible sampling strategies to meet research needs and resources' availability.

The traditional soil-sampling procedure determines the number of soil samples arbitrarily. Here, we provide a simple yet efficient clustered soil-sampling design to demonstrate soil spatial heterogeneity and quantitatively determine the number of soil samples required and the associated sampling accuracy.