Name: microbiologically induced calcite precipitation mediated by sporosarcina pasteurii
Uploaded: 2016-04-16T14:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

We wish to acknowledge the partners in the Helmholtz-Alberta Initiative, the Helmholtz Association and the University of Alberta, for the support resulting from participation in this collaboration. Research funding is provided by the Helmholtz Association's Initiative and Networking Fund, the participating Helmholtz Centers and by the Government of Alberta through Alberta Environment's ecoTrust program.
Dr. Tanushree Ghosh is gratefully acknowledged for her critical inputs at a number of crucial stages.

注意:在下面描述的顺序执行的实验方案。细菌培养协议在第一节中讨论（也如图1所示 ）。第2节描述欲得使用外部添加剂的培养基中的协议。部分3描述了一种用于多模式显微镜的协议。所有的单个组分的权重可以使用分析天平进行测定。各溶液的体积可以使用量筒来测定。 注意:正确的生物安全协议必须遵守的第1 - 2咨询机构安全办公室了解详情。 1.细菌培养<ol><li> 文化细菌-琼脂平板培养基的制备 <ol><li>组装设备和成分如陪替氏培养皿，烧瓶，Tris碱，盐酸，琼脂，Millipore公司水，pH计etc.Sterilize使用前在121℃的所有容器高压灭菌。 </li><li>制备1升的0.1通过混合15.75克Tris-碱用1L微孔水的Tris-缓冲液的3M水溶液。为了降低原溶液（pH 10.4）的pH值增加2800微升盐酸（50％浓度）。连续查用pH计来设置的pH = 9。 </li><li>除以1升缓冲溶液分成两部分，如下所示: <ol><li>取800毫升此溶液。同样将其划分成各为400毫升双部分。溶解8克（NH 4）2 SO 4的一个溶液中，16克酵母提取物的其他解决方案。 </li><li>取剩余（200毫升）溶液，并再次将其分成的每次100ml两部分。混合2克（NH 4）2 SO 4为一个。添加4个克酵母提取物和4g琼脂到另一个。 </li></ol></li><li>在铝箔包装各自的瓶贴及胶带高压锅分开后的高压灭菌4解决方案。 注意:如果一个台式高压釜单元被使用时，体积应设定为500毫升（温度和压力下自动贩卖机ically指定为体积的函数）。 </li><li>从高压釜中取出来后，静置步1.3.1（下）设置两个400毫升解决方案。混合两个100ml的解决方案，有200毫升的溶液。将混合物倒入10 - 12培养皿中。 </li></ol></li><li> 文化细菌-琼脂平板样品制备 <ol><li>从冰箱（-80°C）中取出细菌股票，并允许它解冻。正确解冻后，将细菌股票和生物安全罩内的琼脂平板上。 </li><li>选择最小可用尺寸的微量（0.5 - 10微升是一个不错的选择）侵扰与Sporosarcina巴斯德股票一角。条纹的琼脂平板用枪头。将非振荡培养箱里面的条纹琼脂平板在31℃48小时。 </li><li> 48小时后，从孵化器中取出板，并目视检查单个菌落的存在。如果没有单个菌落，然后将其放置在叔他孵化另外24小时。 </li><li>直到检测单菌落重复上述过程。不要超过7天试用。 注:如果甚至一个星期后，然后可以得出结论的步骤没有正确地遵循和全过程必须从步骤1重复不会出现单菌落。 </li></ol></li><li> 文化细菌-最终样品制备 <ol><li>混合两种400毫升溶液（Tris缓冲液+（NH 4）2 SO 4和Tris缓冲液+酵母提取物（1.5.1）在一起，得到一个800 ml溶液。转移125毫升此溶液加入到烧瓶中。 </li><li>执行琼脂平板的表面上的可视检查，以确定与单个菌落的高浓度区。轻轻轻推，打破了枪头的殖民地之一。 </li><li>浸在同一枪头到125毫升烧瓶中，充分搅拌，以确保有足够数量的细胞的稳健乘法得到转移。放置烧瓶在振荡培养箱中于150rpm下，30℃下2 - 3天。经过2 - 3天，取出从孵化器的烧瓶中。 </li></ol></li><li> 文化细菌-最终的细胞计数 <ol><li>执行使用PBS以获得至少十元（10 -7）的稀释液，以确保可数单个菌落出现的非稀释的培养液连续稀释。借鉴琼脂平板一七平行等距线。 <ol><li>在培养皿中，足以突出的是由上可见的底面上画上粗线做到这一点。降3个非稀溶液的小滴成一个段。 1毫升未稀释的溶液添加到9毫升磷酸盐缓冲盐水（PBS）中以获得1:10稀释。 </li></ol></li><li>采取用吸管这个新稀释的溶液中的一小等分试样（〜0.1ml）中，并滴在下一段3更小滴。新近稀释溶液转移到一个新的烧瓶中，并进一步稀释至10倍（10倍）加入PBS。这使向下稀释至10 -2或1:100。 <ol><li>使用此1:在接下来的段100的解决方案。 1000一路:通过连续将其转让给新瓶和串联不断稀释十倍（10X）用PBS从10 -3或1获得越来越稀的样品重复这一点，他与新鲜稀释溶液的小体积处理下降到10 -7或1:10万成最后一段。 </li></ol></li><li>执行集落形成单位（CFU）平板计数孵育该板1之后，计数存在于琼脂平板上的细胞数量 - 在31℃2天。这使未稀释样本中的细菌计数的定量量度。 注:CFU值是基于系统对产生的菌落的营养培养基，温度和时间的假定每菌落是单独的，并通过一个单一的存活的微生物细胞创办在特定条件下的能力进行测定。 </li><li>密封培养皿具有自密封膜和储存以备将来使用冰箱剩余项目。 </li></ol></li></ol> 2.协议养分富集加速降水<ol><li>传输9传毫升制备的培养液（细胞+培养基）的成若干灭菌离心管，每次10毫升体积。 </li><li>准备在新鲜培养基下列浓度包含四个组件的外部富集的100毫升原液:尿素:2克/升，氯化铵1克/升，碳酸氢钠:212毫克/升，氯化钙:280毫克/升。使用分析的平衡仔细测量所有的成分，并与在烧杯中的高压釜新鲜培养基和地点（121℃，15psi的，15分钟）混合所有但尿素。 <ol><li>以下高压釜中，混合脲（2毫克）用1ml新鲜培养基的所需量和穿过装配有0.22微米的注射器过滤器，以完成浓缩过程的注射器。 </li><li>加1毫升添加剂，由9毫升准备好的培养液（细胞+培养基）的无菌离心管这种增菌培养基中（见步骤2.1）。 注:所有的这些组分中，尿素是在高温下可降解的，不能在高压灭菌器灭菌。因此，其它成分被高压灭菌后（121℃，15磅，15分钟），尿素最后通过0.22μm注射器式滤器加入。 </li></ol></li><li>涡流每个管彻底使用机械涡旋。放置富集液体（细胞+培养基+添加剂）在非振荡培养箱中于30℃。定期监测所有的单位降水开始。使用光学显微镜，一旦沉淀发作肉眼检测开始显微镜观察。实验开始后的36小时 - 这通常30之间。 </li></ol> 3.协议的多模式显微镜<ol><li>转移的流体的小体积（〜1ml）中，以高尊离子显微镜 - 腔玻璃罩系统执行与亮场模式初步意见。首先，先从最低放大倍率（4X）所有观测。其提供覆盖和关于析出物的分布，因此全局信息的广泛区域。 </li><li>选择特定区域与析出物显著卷和通过逐渐增大放大倍数放大（20X，40X，60X）。 注:由于这是不可能妥善解决从胞外聚合物物质（EPS）在明场细胞（由于非常接近的折射率），开启相衬模式必要时。在这种模式下，小区膜（作为一个不同的相位比体细胞）的仅有的轮廓是可见的，从而使视觉分化。 </li><li>最后，一​​些染色与商会选择性染带电部件的绿色，而着色红色死者组件活/死染色。参阅标准协议28 &lt;/ SUP&gt;。 </li><li>交换机上的荧光模式，以适当的红色和绿色区域之间区分。红（红色滤光器立方体540激发波长 - 580纳米，600 595 nm和屏障过滤器的分色镜 - 660 nm），而绿色（GFP长通绿色滤波器立方体440激发波长 - 480纳米，分色镜的510纳米）过滤器立方体505纳米和屏障过滤器是用来促进荧光显微镜照片。 </li><li>一旦所有的多模式数据已经被收集，选择最佳的图像和手动覆盖（个体明，相衬和荧光图像），以获得最佳的对比度和清晰度。 </li><li>在干涸的固体样品扫描电镜进行了解的晶体结构。这是一个行之有效的标准和程序。22执行XPS找出官能团（碳酸酯）的化学签名。这是一个行之有效的标准和程序以及23 </li></ol>

<ol>
	<li>Gibson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+Bacillus+pasteurii+group.">An investigation of the Bacillus pasteurii group.</a> Journal of Bacteriology. 28, 491-502 (1934).</li><li>Greenfield, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+concentration+of+calcium+and+magnesium+and+precipitation+of+calcium+carbonate+by+a+marine+bacterium.">Metabolism and concentration of calcium and magnesium and precipitation of calcium carbonate by a marine bacterium.</a> Annals of the New York Academy of Sciences. 109, 23-45 (1963).</li><li>Phillips, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+applications+of+ureolytic+biomineralization:+a+review.">Engineered applications of ureolytic biomineralization: a review.</a> Biofouling. 29, 715-733 (2013).</li><li>Dhami, N. K., 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=Biomineralization+of+calcium+carbonates+and+their+engineered+applications:+a+review.">Biomineralization of calcium carbonates and their engineered applications: a review.</a> Frontiers in microbiology. 4, 314 (2013).</li><li>Cuthbert, M. O., 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=Controls+on+the+rate+of+ureolysis+and+the+morphology+of+carbonate+precipitated+by+S.+Pasteurii+biofilms+and+limits+due+to+bacterial+encapsulation.">Controls on the rate of ureolysis and the morphology of carbonate precipitated by S. Pasteurii biofilms and limits due to bacterial encapsulation.</a> Ecological Engineering. 41, 32-40 (2012).</li><li>Okwadha, G. D., 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=Optimum+conditions+for+microbial+carbonate+precipitation.">Optimum conditions for microbial carbonate precipitation.</a> Chemosphere. 81, 1143-1148 (2010).</li><li>Stocks-Fischer, S., 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=Microbiological+precipitation+of+CaCO3.">Microbiological precipitation of CaCO3.</a> Soil Biology and Biochemistry. 31, 1563-1571 (1999).</li><li>Lauchnor, E. G., 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=Bacterially+induced+calcium+carbonate+precipitation+and+strontium+coprecipitation+in+a+porous+media+flow+system.">Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.</a> Environmental science &amp; technology. 47, 1557-1564 (2013).</li><li>Al Qabany, A., 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=Factors+Affecting+Efficiency+of+Microbially+Induced+Calcite+Precipitation.">Factors Affecting Efficiency of Microbially Induced Calcite Precipitation.</a> Journal of Geotechnical and Geoenvironmental Engineering. 138, 992-1001 (2012).</li><li>Morita, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcite+precipitation+by+marine+bacteria.">Calcite precipitation by marine bacteria.</a> Geomicrobiology Journal. 2, 63-82 (2009).</li><li>Chafetz, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+peloids:+A+product+of+bacterially+induced+carbonate+precipitation.">Marine peloids: A product of bacterially induced carbonate precipitation.</a> Journal of Sedimentary Petrology. 56, 812-817 (1986).</li><li>Whiffin, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbial CaCO3 precipitation for the production of biocement. , (2004).</li><li>Paassen, L. A., 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=Scale+up+of+BioGrout:+a+biological+ground+reinforcement+method.">Scale up of BioGrout: a biological ground reinforcement method.</a> Proceedings of the 17th International Conference on Soil Mechanics and Geotechnical Engineering. , 2328-2333 (2009).</li><li>Cunningham, A. B., 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=Microbially+enhanced+geologic+containment+of+sequestered+supercritical+CO2.">Microbially enhanced geologic containment of sequestered supercritical CO2.</a> Energy Procedia. 1, 3245-3252 (2009).</li><li>Mitchell, A. 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=Biofilm+enhanced+geologic+sequestration+of+supercritical+CO2.">Biofilm enhanced geologic sequestration of supercritical CO2.</a> International Journal of Greenhouse Gas Control. 3, 90-99 (2009).</li><li>Cunningham, A. B., 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=Reducing+the+risk+of+well+bore+leakage+of+CO2+using+engineered+biomineralization+barriers.">Reducing the risk of well bore leakage of CO2 using engineered biomineralization barriers.</a> Energy Procedia. 4, 5178-5185 (2011).</li><li>Jonkers, H. M., 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=Application+of+bacteria+as+self-healing+agent+for+the+development+of+sustainable+concrete.">Application of bacteria as self-healing agent for the development of sustainable concrete.</a> Ecological Engineering. 36, 230-235 (2010).</li><li>Tobler, D. J., 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=Transport+of+Sporosarcina+pasteurii+in+sandstone+and+its+significance+for+subsurface+engineering+technologies.">Transport of Sporosarcina pasteurii in sandstone and its significance for subsurface engineering technologies.</a> Applied Geochemistry. 42, 38-44 (2014).</li><li>Mortensen, B. M., 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=Effects+of+environmental+factors+on+microbial+induced+calcium+carbonate+precipitation.">Effects of environmental factors on microbial induced calcium carbonate precipitation.</a> Journal of applied microbiology. 111, 338-349 (2011).</li><li>Phillips, A. J., 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=Potential+CO2+leakage+reduction+through+biofilm-induced+calcium+carbonate+precipitation.">Potential CO2 leakage reduction through biofilm-induced calcium carbonate precipitation.</a> Environmental science &amp; technology. 47, 142-149 (2013).</li><li>vander Heide, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-ray Photoelectron Spectroscopy: An introduction to Principles and Practices. , (2011).</li><li>Wiley, W. R., 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=Requirement+of+an+alkaline+pH+and+ammonia+for+substrate+oxidation+by+Bacillus+pasteurii.">Requirement of an alkaline pH and ammonia for substrate oxidation by Bacillus pasteurii.</a> Journal of Bacteriology. 84, (1962).</li><li>Tagliaferri, F., 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=Observing+strain+localisation+processes+in+bio-cemented+sand+using+x-ray+imaging.">Observing strain localisation processes in bio-cemented sand using x-ray imaging.</a> Granular Matter. 13, 247-250 (2011).</li><li>Kumar, A., 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=Microscale+confinement+features+can+affect+biofilm+formation.">Microscale confinement features can affect biofilm formation.</a> Microfluidics and Nanofluidics. 14, 895-902 (2012).</li><li>Valiei, A., 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=A+web+of+streamers:+biofilm+formation+in+a+porous+microfluidic+device.">A web of streamers: biofilm formation in a porous microfluidic device.</a> Lab on a chip. 12, 5133-5137 (2012).</li><li>. LIVE/DEAD Bacterial Viability kit, Two-color bacterial viability assay Available from: <a target="_blank" href="https://tools.lifetechnologies.com/content/sfs/manuals/mp07007.pdf">https://tools.lifetechnologies.com/content/sfs/manuals/mp07007.pdf</a> (2004)</li></ol>

The authors have nothing to disclose.

关键步骤:该手稿详细介绍了协议的培养S的一个可行的样本巴斯德 。一旦培养已准备好，必须适当地富集。这是在实验的成功是至关重要的关键步骤，因为未提供适当的化学环境导致要么沉淀或其完全缺乏的很长的时间尺度。S.巴氏是几个外部机构相当敏感，并且必须以高度小心和精确的进行培养，以确保生化鲁棒性和可重复性。浓缩的程度和剂量现在已知口授?...

Sporosarcina巴氏是革兰氏阳性细菌能够在高碱性环境（pH约10）1生存并且是细菌种类，可以成为一个所谓微生物学诱导方解石沉淀（MICP）2-4现象病原体之一。 MICP是一种方法，其中碳酸钙的沉淀是通过合适的环境的条件下某些微生物引起的。S.巴氏已假定近年来重要性由于其识别为在特定条件下诱导MICP的显著体积的可能剂。这种可能性来源于这样的事实即S.巴氏具有分泌丰富量的脲酶的独特能力。这种酶作为催化剂，促进尿素水分子的存在加速溶解（具有广泛和供应充足的天然存在的生化化合物）。通过反应的级联，该方法的最终LY导致带负电的碳酸根离子的生成。这些离子，又与正金属离子如钙，最终形成碳酸钙（方解石）的沉淀反应;因此标签MICP 5-9。 MICP的方法已经知道并研究了数十年10,11。在过去的几年中，MICP已经研究了一系列工程和环境应用，包括自下而上的绿化建设12，加强大型结构13,14和碳吸收和储存15,16的。 例如，Cunnigham 17等。人设计包含贝雷砂岩核心的高压中温流反应器。将反应器与细菌S.接种fridgidimarina和高压的超临界二氧化碳注入，生物量的孔隙体积内的大量积累的条件下超微电极观察到，这导致在透过性降低95％以上。 Jonkers和施兰根18研究在混凝土的自愈过程细菌的某些特殊菌株的效果。运入通过表面气孔进入孔隙网络外部的水有望激活休眠的细菌从而有助于结构强度通过MICP。托布勒19等。比较了S的尿素分解活性有利于大型MCIP条件下的地下水本土尿素分解缩影巴斯德发现S.巴斯德具有一致的能力，以提高方解石沉淀即使在土著社区缺乏事先脲酶活性。莫滕森20 et.al已经研究的外部因素，如土壤类型，氯化铵，盐度，氧浓度和MICP裂解细胞浓度的影响。他们论证了生物处理工艺非常稳健与RESP等在参数空间中的巨大差异证实了这一过程提供了一个合适的浓缩工艺，以加强细菌进行各种大型整治应用的健康。菲利普斯21等。人的实验设计与S.被注射后，研究在砂柱和砂岩核心的渗透性和强度的变化巴氏文化。他们发现，尽管渗透性下降了2 - 4倍，而断裂强度提高三倍。 S.巴氏及其在MICP的作用是积极的研究和有关化学沉淀机制若干问题的主题仍然没有完全理解。鉴于此，它拥有一套一致的标准化协议，以准确地文化S的适当丰富的股票是非常重要的巴斯德实现MICP。在这里，我们勾勒出一个严格的协议，以确保重复性和再现性。此马nuscript描述了详细的协议，培养S.巴斯德和适当丰富培养基，以诱使降雨。该过程通过各种显微镜技术如光学和扫描电子显微镜（SEM）和X射线光电子能谱（XPS）研究。手稿的重点是MICP的过程。程序，如:SEM和SIMS，是行之有效的标准协议，不单独介绍。

<table><tbody><tr><td>Petridish</td><td>Fisher Scientific</td><td>FB0875712</td><td>Petridishes being used as Agar plate</td></tr><tr><td>Pyrex Flasks</td><td>Fisher Scientific</td><td>S63268</td><td>Corning Erlenmeyer</td></tr><tr><td>Tris-Base</td><td>Promega</td><td>H5133</td><td>being used to make Tris-Buffer</td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>H9892</td><td>1.0 N, Bioreagent, suitable for cell culture</td></tr><tr><td>Agar Powder</td><td>Sigma-Aldrich</td><td>A1296</td><td>microbiology tested, plant cell culture tested, cell culture tested, powder</td></tr><tr><td>Ammonium Sulphate</td><td>Sigma-Aldrich</td><td>A4418</td><td>for Molecular Biology</td></tr><tr><td>Yeast extract powder</td><td>Sigma-Aldrich</td><td>51475</td><td></td></tr><tr><td>Measuring Cylinder</td><td>Cole-Parmer</td><td>CP08559GC</td><td>Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC; 1,000 ml,1/Pk</td></tr><tr><td>Analytical Balance</td><td>OHAUS</td><td>AX124E</td><td>being used to measure weight of reagents</td></tr><tr><td>Autoclave</td><td>Brinkmann</td><td>58619000</td><td></td></tr><tr><td>Autoclave Tape</td><td>VWR</td><td>52428864</td><td></td></tr><tr><td>Aluminum Foil</td><td>Sigma-Aldrich</td><td>Z185140</td><td>being used to seal the flask before placing it in Autoclave</td></tr><tr><td>Bacterial Stock</td><td>Cedarlane</td><td>11859</td><td>-80 &amp;deg;C stock of &lt;em&gt;S. pasteurii&lt;/em&gt;, ATCC No. is mentioned against Cat. No.</td></tr><tr><td>Mline Single-Channel Mechanical Pipettors, Variable Volume</td><td>Biohit</td><td>725010</td><td>Marketed by VWR under catalog number 14005976</td></tr><tr><td>Micropipette Tip</td><td>Fisher Scientific</td><td>212772B</td><td>Used for scratching Agar plates</td></tr><tr><td>Incubator</td><td>Binder</td><td>80079098</td><td>Microbiology Incubator,BF Series</td></tr><tr><td>Shaking Incubator</td><td>VWR</td><td>14004300</td><td>VWR Signature Benchtop Shaking Incubators</td></tr><tr><td>Phosphate Buffer Saline (PBS)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7059</td><td></td></tr><tr><td>BD Falcon Express Pipet-Aid Pipetting Device</td><td>BD Biosciences</td><td>357590</td><td>Marketed by VWR under catalog number 53106220</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P7793</td><td>Being used to seal Agar plates</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>U1250</td><td>Enrichment for nutrient medium</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S8875</td><td>Enrichment for nutrient medium</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td>Enrichment for nutrient medium</td></tr></tbody></table>

microbiologically induced calcite precipitation mediated by sporosarcina pasteurii

被调查的特定的细菌在这里（S.巴氏 ）是其独特的能力，在合适的条件下，促使尿素（尿素水解）的水解通过脲酶分泌的自然发生的环境。尿素水解的过程中，通过化学反应的链，导致碳酸钙沉淀的形成。这被称为微生物学诱导方解石降水（MICP）。对于MICP适宜的培养协议这里详述。最后，在显微镜下的不同模式的可视化实验以了解沉淀过程的各个方面。像光学显微技术，扫描电子显微镜（SEM）和X射线光电子能谱（XPS）进行了用于化学表征终产物。此外，这些沉淀物会堵塞一个天然的多孔介质毛孔里面的能力，通过定性实验，其中海绵证明棒用来模仿细孔网络与一系列长度尺度。在含有细菌细胞培养液中浸渍的海绵棒变硬由于其孔从化学沉淀的连续方法得到的的堵塞。相比其中被压缩和施加的外部负载的作用下挤压的控制海绵条时，而硬化杆能够支持相同重量很少变形此硬化海绵棒表现出优异的强度。

S.巴斯德作为一个alkaliphile 24能够生存条件比较苛刻。当上述培养协议之后，和S.巴氏生长的腔室的内部，该细菌会导致碳酸钙的随时间（ 图2A）的沉淀。 图2（b）表示在培养基中的细菌细胞群体的相差光学显微镜图像。单个细胞能够清楚地区分，与芽孢杆菌类的细菌很明显的特征棒状的形状。小棕色箭头已用于特异性突出两...

Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

微生物诱导方解石沉淀通过介导<em&gt; Sporosarcina巴斯德</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

microbiologically-induced-calcite-precipitation-mediated-sporosarcina

Research

JoVE Journal

Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

21.8K 次观看.  University of Alberta. Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

视频: Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a morpho-physiological resting state that enhances survival under adverse environmental conditions. Even though endospore-forming Firmicutes are one of the most frequently enriched and isolated bacterial groups in culturing studies, they are often absent from diversity studies based on molecular methods. The resistance of the spore core is considered one of the factors limiting the recovery of DNA from endospores. We developed a method that takes advantage of the higher resistance of endospores to separate them from other cells in a complex microbial community using physical, enzymatic and chemical lysis methods. The endospore-only preparation thus obtained can be used for re-culturing or to perform downstream analysis such as tailored DNA extraction optimized for endospores and subsequent DNA sequencing. This method, applied to sediment samples, has allowed the enrichment of endospores and after sequencing, has revealed a large diversity of endospore-formers in freshwater lake sediments. We expect that the application of this method to other samples will yield a similar outcome.

A method to single out bacterial endospores from complex microbial communities was developed to perform tailored culture or molecular studies of this group of bacteria.

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

Endospore formation is a survival strategy found among some bacteria from the phylum Firmicutes. During endospore formation, these bacteria enter a ...

科研

环境科学

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

工程学

Two-way Valorization of Blast Furnace Slag: Synthesis of Precipitated Calcium Carbonate and Zeolitic Heavy Metal Adsorbent

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with sufficient heavy metal adsorption capacity. To this end, blast furnace slag, a residue from iron-making, is utilized as the starting material. Calcium is selectively extracted from the slag by leaching with acetic acid (2 M CH3COOH) as the extraction agent. The filtered leachate is subsequently physico-chemically purified and then carbonated to form precipitated calcium carbonate (PCC) of high purity (&lt;2 wt% non-calcium impurities, according to ICP-MS analysis). Sodium hydroxide is added to neutralize the regenerated acetate. The morphological properties of the resulting calcitic PCC are tuned for its potential application as a filler in papermaking. In parallel, the residual solids from the extraction stage are subjected to hydrothermal conversion in a caustic solution (2 M NaOH) that leads to the predominant formation of a particular zeolitic mineral phase (detected by XRD), namely analcime (NaAlSi2O6&#8729;H2O). Based on its ability to adsorb Ni2+, as reported from batch adsorption experiments and ICP-OES analysis, this product can potentially be used in wastewater treatment or for environmental remediation applications.

A protocol for the parallel production of precipitated calcium carbonate and zeolitic material from blast furnace slag via mineral carbonation and alkaline hydrothermal conversion, respectively, is presented. The performance of the zeolitic material towards nickel adsorption is tested.

高炉矿渣的双向稳定物价:沉淀碳酸钙和沸石重金属吸附剂的合成

The aim of this work is to present a zero-waste process for storing CO2 in a stable and benign mineral form while producing zeolitic minerals with ...

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

监测农业土壤中经修正的硅酸盐矿物风化造成的致病性无机碳积累。

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

极端微生物的生物勘探以解决环境污染问题

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. It is a complex process and therefore a simple, in vitro, system is required to understand the effect of isolated molecules on the biomineralization process. In many cases, biomineralization is directed by biopolymers in the extracellular matrix. In order to evaluate the effect of isolated biopolymers on the morphology and structure of calcite in vitro, we have used the vapor diffusion method for the precipitation of calcium carbonate, scanning electron microscopy and micro Raman for the characterization, and ultraviolet-visible (UV/Vis) absorbance for measuring the quantity of a biopolymer in the crystals. In this method, we expose the isolated biopolymers, dissolved in a calcium chloride solution, to gaseous ammonia and carbon dioxide that originate from the decomposition of solid ammonium carbonate. Under the conditions where the solubility product of calcium carbonate is reached, calcium carbonate precipitates and crystals are formed. Calcium carbonate has different polymorphs that differ in their thermodynamic stability: amorphous calcium carbonate, vaterite, aragonite, and calcite. In the absence of biopolymers, under clean conditions, calcium carbonate is mostly present in the calcite form, which is the most thermodynamically stable polymorph of calcium carbonate. This method examines the effect of the biopolymeric additives on the morphology and structure of calcium carbonate crystals. Here, we demonstrate the protocol through the study of an extracellular bacterial protein, TapA, on the formation of calcium carbonate crystals. Specifically, we focus on the experimental set up, and characterization methods, such as optical and electron microscopy as well as Raman spectroscopy.

We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

生物聚合物添加剂中碳酸钙的形成

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living ...

化学

Sandy Soil Improvement through Microbially Induced Calcite Precipitation (MICP) by Immersion

The goal of this article is to develop an immersion method to improve the microbially induced calcite precipitation (MICP) treated samples. A batch reactor was assembled to immerse soil samples into cementation media. The cementation media can freely diffuse into the soil samples in the batch reactor instead of cementation media being injected. A full contact flexible mold, a rigid full contact mold, and a cored brick mold were used to prepare different soil sample holders. Synthetic fibers and natural fibers were selected to reinforce the MICP-treated soil samples. The precipitated CaCO3 in different areas of the MICP-treated samples was measured. The CaCO3 distribution results demonstrated that the precipitated CaCO3 was distributed uniformly in the soil sample by the immersion method.

Sandy Soil Improvement through Microbially Induced Calcite Precipitation MICP by Immersion

Here, microbially induced calcite precipitation (MICP) technology is presented to improve soil properties by immersion.

通过浸没通过微生物诱导的钙化物沉淀（MICP）改善沙土

The goal of this article is to develop an immersion method to improve the microbially induced calcite precipitation (MICP) treated samples. A batch ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

生物化学

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

生物工程

Production and Analysis of Sporosarcina pasteurii Biocement Bricks Using Custom 3D-Printed Molds for Unconfined Compression Tests

Cement is a key building material used in many structures across the globe, from foundations for homes to historical monuments and roadways. It is a critical and abundant material worldwide. However, the traditional production of cement is a major contributor to man-made atmospheric CO2, leading to greenhouse gas emissions and climate change. Microbially induced calcite precipitation (MICP) is a biological process in which Sporosarcina pasteurii or other bacteria produce a cement material that is as strong as traditional cement, but biocement is carbon-neutral. This MICP method of producing biocement is a promising technology and is currently under active investigation by many companies, countries, and research groups. The protocol presented here employs custom-designed, reusable, 3D-printed molds for flow-through MICP treatment of soil or sand, producing cylindrical bricks that meet standard specifications for unconfined compression tests. The individual, free-standing, reservoir-topped molds allow convenient parallel testing of multiple variables and replicates. This protocol outlines the S. pasteurii MICP reaction and the creation, assembly, and use of the 3D-printed molds to generate biocement cylindrical bricks.

Sporosarcina pasteurii is a ureolytic bacterium that breaks down urea into carbonate and ammonium. The carbonate combines with calcium to form calcium carbonate, creating a crystal lattice that anchors surrounding particles together to produce biocement. This is a convenient protocol for using 3D-printed molds to create biocement bricks suitable for compression testing.

使用定制的 3D 打印模具生产和分析巴 氏 孢子丝菌生物水泥砖，用于无侧限压缩试验

Cement is a key building material used in many structures across the globe, from foundations for homes to historical monuments and roadways. It is a ...

微生物诱导方解石沉淀通过介导

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此视频中的章节

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

高炉矿渣的双向稳定物价:沉淀碳酸钙和沸石重金属吸附剂的合成

监测农业土壤中经修正的硅酸盐矿物风化造成的致病性无机碳积累。

极端微生物的生物勘探以解决环境污染问题

生物聚合物添加剂中碳酸钙的形成

通过浸没通过微生物诱导的钙化物沉淀（MICP）改善沙土

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

使用定制的 3D 打印模具生产和分析巴氏孢子丝菌生物水泥砖，用于无侧限压缩试验

微生物诱导方解石沉淀通过介导

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此视频中的章节

从环境样品中的内生孢子通过无性细胞定向裂解物理隔离

用同步快速反应层析成像动态孔隙规模油藏条件下成像碳酸盐

高炉矿渣的双向稳定物价:沉淀碳酸钙和沸石重金属吸附剂的合成

监测农业土壤中经修正的硅酸盐矿物风化造成的致病性无机碳积累。

极端微生物的生物勘探以解决环境污染问题

生物聚合物添加剂中碳酸钙的形成

通过浸没通过微生物诱导的钙化物沉淀（MICP）改善沙土

一种半自动且可重现的基于生物学的体外定量钙沉积的方法

生物修复和益生菌发育的微生物菌株的勘探，用于生物有机物的研究与保存

使用定制的 3D 打印模具生产和分析巴 氏 孢子丝菌生物水泥砖，用于无侧限压缩试验

使用定制的 3D 打印模具生产和分析巴氏孢子丝菌生物水泥砖，用于无侧限压缩试验