微生物诱导方解石沉淀通过介导<em> Sporosarcina巴斯德</em
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Petridish | Fisher Scientific | FB0875712 | Petridishes being used as Agar plate |
| Pyrex Flasks | Fisher Scientific | S63268 | Corning Erlenmeyer |
| Tris-Base | Promega | H5133 | being used to make Tris-Buffer |
| Hydrochloric Acid | Sigma-Aldrich | H9892 | 1.0 N, Bioreagent, suitable for cell culture |
| Agar Powder | Sigma-Aldrich | A1296 | microbiology tested, plant cell culture tested, cell culture tested, powder |
| Ammonium Sulphate | Sigma-Aldrich | A4418 | for Molecular Biology |
| Yeast extract powder | Sigma-Aldrich | 51475 | |
| Measuring Cylinder | Cole-Parmer | CP08559GC | Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC;1000ml,1/Pk |
| Analytical Balance | OHAUS | AX124E | being used to measure weight of reagents |
| Autoclave | Brinkmann | 58619000 | |
| Autoclave Tape | VWR | 52428864 | |
| Aluminum Foil | Sigma-Aldrich | Z185140 | being used to seal the flask before placing it in Autoclave |
| Bacterial Stock | Cedarlane | 11859 | -80°C stock of S. pasteurii, ATCC No. is mentioned against Cat. No. |
| Mline Single-Channel Mechanical Pipettors, Variable Volume | Biohit | 725010 | Marketed by VWR under catalog number 14005976 |
| Micropipette Tip | Fisher Scientific | 212772B | Used for scratching Agar plates |
| Incubator | Binder | 80079098 | Microbiology Incubator,BF Series |
| Shaking Incubator | VWR | 14004300 | VWR Signature Benchtop Shaking Incubators |
| Phosphate Buffer Saline (PBS) | Sigma-Aldrich | P7059 | |
| BD Falcon Express Pipet-Aid Pipetting Device | BD Biosciences | 357590 | Marketed by VWR under catalog number 53106220 |
| Parafilm | Sigma-Aldrich | P7793 | Being used to seal Agar plates |
| Urea | Sigma-Aldrich | U1250 | Enrichment for nutrient medium |
| Sodium Bicarbonate | Sigma-Aldrich | S8875 | Enrichment for nutrient medium |
| Calcium chloride | Sigma-Aldrich | C1016 | Enrichment for nutrient medium |
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