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.

NOTE: Perform the experimental protocols in the order described below. The bacterial culture protocol is discussed in Section 1 (Also see Figure 1). Section 2 describes the protocol for enriching the culture medium using external additives. Section 3 describes the protocols for multi-mode microscopy. Weights of all the individual components may be measured using an analytical balance. Volume of each solution may be measured using a volumetric cylinder.
NOTE: Proper biosafety protocols must be followed for Sections 1 - 2. Consult the institutional safety office for details.
1. Bacterial Culture
<ol>
	<li>Culture Bacteria - Agar Plate Medium Preparation
	<ol>
		<li>Assemble equipment and ingredients such as Petri dishes, flask, Tris-base, HCl, agar, Millipore water, pH-meter etc.Sterilize all containers by autoclaving at 121 &#176;C before use.</li>
		<li>Prepare 1 L of 0.13 M aqueous solution of Tris-buffer by mixing 15.75 g Tris-base with 1 L of Millipore water. To lower the pH level of the original solution (pH 10.4) add 2,800 &#181;l of HCl (50% concentration). Check continuously using a pH-meter to set pH = 9.</li>
		<li>Divide the 1 L buffer solution into two parts as follows:
		<ol>
			<li>Take 800 ml of this solution. Divide it equally into two parts of 400 ml each. Dissolve 8 g (NH4)2SO4 to one solution and 16 g yeast extract to the other solution.</li>
			<li>Take the remaining (200 ml of) solution and divide it again into two parts of 100 ml each. Mix 2 g (NH4)2SO4 to one. Add 4 g yeast extract and 4 g agar to the other.</li>
		</ol>
		</li>
		<li>Autoclave the 4 solutions separately after wrapping the respective flasks in Al foil and sticking autoclave tapes. 
		NOTE: If a benchtop autoclave unit is used, the volume should be set to 500 ml (temperature and pressure automatically specified as a function of volume).</li>
		<li>After taking them out from the autoclave, set the two 400 ml solutions aside for step 1.3.1 (below). Mix the two 100 ml solutions to have a 200 ml solution. Pour the mixture into 10 - 12 Petri dishes.</li>
	</ol>
	</li>
	<li>Culture Bacteria - Agar Plate Sample Preparation
	<ol>
		<li>Remove the bacterial stock from freezer (-80 &#176;C) and allow it to thaw. After thawing properly, place the bacterial stock and the agar plate inside a biosafety hood.</li>
		<li>Select the micropipette of smallest available dimension (0.5 - 10 &#181;l is a good choice) to infest the tip with the Sporosarcina pasteurii stock. Streak an agar plate with the micropipette tip. Place the streaked agar plate inside a non-shaking incubator at 31 &#176;C for 48 hr.</li>
		<li>After 48 hr, remove the plate from the incubator and visually examine for the existence of single colonies. If there are no single colonies, then place it in the incubator for another 24 hr.</li>
		<li>Repeat the process until single colonies are detected. Do not exceed 7 days of trial. 
		NOTE: If single colonies do not appear even after a week, then it is concluded that the steps have not been followed properly and the entire process must be repeated from step 1.</li>
	</ol>
	</li>
	<li>Culture Bacteria - Final Sample Preparation
	<ol>
		<li>Mix the two 400 ml solutions (Tris buffer + (NH4)2SO4 and Tris buffer + yeast extract (1.5.1) together to obtain an 800 ml solution. Transfer 125 ml of this solution into a flask.</li>
		<li>Perform a visual examination of the surface of the agar plate to identify regions with high concentration of single colonies. Gently nudge and break one of the colonies with a micropipette tip.</li>
		<li>Dip the same micropipette tip into the 125 ml flask and stir it thoroughly to ensure that sufficient number of cells for robust multiplication get transferred. Place the flask in a shaking incubator at 150 rpm, 30 &#176;C for 2 - 3 days. After 2 - 3 days, remove the flask from the incubator.</li>
	</ol>
	</li>
	<li>Culture Bacteria - Final Cell Count
	<ol>
		<li>Perform serial dilution of the non-diluted culture solution using PBS to attain a dilution of at least ten million (10-7) to ensure countable single colonies appear. Draw seven parallel equidistant lines on one of the agar-plates.
		<ol>
			<li>Do this by drawing bold lines on the bottom surface of the Petri dish, prominent enough to be visible from top. Drop 3 little drops of non-diluted solution into one segment. Add 1 ml of non-diluted solution to 9 ml of Phosphate Buffered Saline (PBS) to obtain a 1:10 dilution.</li>
		</ol>
		</li>
		<li>Take a small aliquot (~ 0.1 ml) of this newly diluted solution with a pipette and drop 3 more small drops on the next segment. Transfer the newly diluted solution to a new flask and further diluted ten times (10x) by adding PBS. This brings down the dilution to 10-2 or 1:100.
		<ol>
			<li>Use this 1:100 solution in the next segment. Repeat this he process with small volumes of the freshly diluted solutions by successively transferring them to new flasks and continuously diluting ten-fold (10x) in tandem with PBS to obtain more and more dilute samples from 10-3 or 1:1,000 all the way down to 10-7 or 1:10 million into the last segment.</li>
		</ol>
		</li>
		<li>Perform Colony-Forming Unit (CFU) plate count to count the number of cells present in the agar plate after incubating the plate for 1 - 2 days at 31 &#176;C. This gives a quantitative measure of the bacterial count in the undiluted sample. 
		NOTE: The CFU value is measured based on the ability of the system to give rise to colonies under the specific conditions of nutrient medium, temperature and time assuming that every colony is separate and founded by a single viable microbial cell.</li>
		<li>Seal the Petri dishes with self-sealing film and store remaining items in a refrigerator for future use.</li>
	</ol>
	</li>
</ol>
2. Protocol for Enrichment of Nutrient to Accelerate Precipitation
<ol>
	<li>Transfer 9 ml of the prepared culture liquid (cells + medium) into several sterilized centrifuge tubes, each of 10 ml volume.</li>
	<li>Prepare a 100 ml&#160;stock solution of the external enrichment consisting of four components in the following concentrations in fresh medium: Urea: 2 g/L, Ammonium chloride: 1 g/L, Sodium bicarbonate: 212 mg/L, Calcium chloride: 280 mg/L. Carefully measure all the ingredients using an analytical balance and mix all but urea with fresh medium in a beaker and place in the autoclave (121 &#176;C, 15 psi, 15 min).
	<ol>
		<li>Following autoclave, mix the requisite amount of urea (2 mg) with 1 ml of fresh medium and pass through a syringe fitted with 0.22 &#181;m syringe filter to complete the process of enrichment.</li>
		<li>Add 1 ml of this enrichment medium with additives to the sterilized centrifuge tubes containing 9 ml of the prepared culture liquid (cells + medium) (see step 2.1). 
		NOTE: Of all these components, urea being degradable at elevated temperatures, cannot be sterilized in an autoclave. Hence, after the other components have been autoclaved (121 &#176;C, 15 psi, 15 min), urea is added last through a 0.22 &#181;m syringe filter.</li>
	</ol>
	</li>
	<li>Vortex each tube thoroughly using a mechanical vortexer. Place the enriched liquids (cells + medium + additives) in a non-shaking incubator at 30 &#176;C. Monitor all the units regularly for initiation of precipitation. Using a light microscope, begin microscopic observations once onset of precipitation is detected with the naked eye. This is usually between 30 - 36 hr after start of experiments.</li>
</ol>
3. Protocol for Multi-mode Microscopy
<ol>
	<li>Transfer a small volume (~ 1 ml) of fluid to a high-magnification microscopy-chambered coverglass system to perform initial observations with bright field mode. To begin with, start all observations with the lowest magnification (4X). which provides a wide area of coverage and hence global information about the distribution of precipitates.</li>
	<li>Select particular areas with significant volumes of precipitations and zoom in (20X, 40X, 60X) by progressively increasing the magnification. 
	NOTE: Since it&#39;s impossible to properly resolve the cells from the Extracellular Polymeric Substance (EPS) under bright-field (due to very close refractive indices), switch on phase-contrast mode whenever necessary. In this mode, just the outlines of the cell membranes (being a different phase than the bulk cell) are visible, thus enabling visual differentiation.</li>
	<li>Finally, stain some chambers with Live/ Dead stain that selectively dyes the live components as green while coloring the dead components in red. Refer to standard protocols28.</li>
	<li>Switch on fluorescent mode to properly distinguish between the red and green regions. Red (Red filter cube with Excitation wavelength of 540 - 580 nm, Dichroic mirror of 595 nm and Barrier filter of 600 - 660 nm) and Green (GFP Long-pass Green filter cube with Excitation wavelength of 440 - 480 nm, Dichroic mirror of 505 nm and Barrier filter of 510 nm) filter cubes are used to facilitate fluorescent micrographs.</li>
	<li>Once all multi-mode data have been gathered, select the best images and manually overlay (individual brightfield, phase-contrast and fluorescent images) to obtain the best possible contrast and clarity.</li>
	<li>Perform SEM on the dried solid samples to understand crystal structures. This is a well-established and standard procedure.22 Perform XPS&#160;to identify chemical signatures of functional groups (carbonates). This is a well-established and standard procedure as well.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.

Critical Steps: This manuscript describes in detail the protocols for culturing a viable sample of S. pasteurii. Once the culture has been readied, it must be suitably enriched. This is a key step vital to the success of the experiment because a failure to provide the proper chemical environment leads to either very long time scales of precipitation or a complete lack thereof. S. pasteurii is quite sensitive to several external agencies and must be cultured with a high degree of care an...

Sporosarcina pasteurii is a gram-positive bacterium able to survive in highly alkaline environments (pH~10)1 and is one of the bacterial species that can become a causative agent of a phenomenon called Microbiologically Induced Calcite Precipitation (MICP)2-4. MICP is a process wherein precipitation of calcium carbonate is induced by certain microbes under suitable environmental conditions. S. pasteurii has assumed importance in recent years due to its identification as a possible agent for inducing significant volumes of MICP under certain conditions. This possibility stems from the fact that S. pasteurii has the unique ability to secrete copious amounts of the enzyme urease. This enzyme acts as a catalyst, promoting an accelerated lysis of urea (a naturally occurring biochemical compound with widespread and abundant supply) in the presence of water molecules. Through a cascade of reactions, this process ultimately leads to the generation of negatively charged carbonate ions. These ions, in turn, react with positive metal ions like calcium to finally form precipitates of calcium carbonate (calcite); hence the label MICP5-9.
The process of MICP has been known and studied for several decades10,11. Over the past few years, MICP has been investigated for a wide range of engineering and environmental applications including bottom-up green construction12, enhancement of large-scale structures13,14 and carbon sequestration and storage15,16.
For example, Cunnigham17 et. al designed a high pressure moderate temperature flow reactor containing a Berea sandstone core. The reactor was inoculated with the bacteria S. fridgidimarina and under conditions of high-pressure supercritical carbon dioxide injection, a massive accumulation of biomass inside the pore volumes was observed, which led to more than 95% reduction in permeability. Jonkers and Schlangen18 studied the effect of certain special strains of bacteria on the self-healing process in concrete. External water transported into the pore network entering through the surface pores is expected to activate the dormant bacteria which in turn help structural strength via MICP. Tobler19 et al. have compared the ureolytic activity of S. pasteurii with an indigenous groundwater ureolytic microcosm under conditions favoring large-scale MCIP and found that S. pasteurii has a consistent capability to improve calcite precipitation even when the indigenous communities lacked prior urease activity. Mortensen20 et.al have studied the effects of external factors like soil type, concentration of ammonium chloride, salinity, oxygen concentration and lysis of cells on MICP. Their demonstration that the biological treatment process is very robust with respect to a wide variation in parameter space substantiates the fitness of this process for various large-scale remediation applications provided a proper enrichment process to reinforce the bacteria is undertaken. Phillips21 et. al designed experiments to study the changes in permeability and strength of a sand column and a sandstone core after being injected with S. pasteurii cultures. They found that while the permeability decreased 2 - 4 times while the fracture strength increased three times.
S. pasteurii and its role in MICP are topics of active research and several issues relating to the mechanism of chemical precipitation are still not fully understood. In light of this, it is very important to have a set of consistent standardized protocols to accurately culture a suitably enriched stock of S. pasteurii to achieve MICP. Here, we outline a rigorous protocol that will ensure repeatability and reproducibility. This manuscript describes the detailed protocols for culturing S. pasteurii and suitably enriching the culture medium to induce precipitation. The process is investigated through various microscopic techniques such as optical and Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS). The focus of the manuscript is on the process of MICP. Procedures like SEM and SIMS, being well-established standard protocols, are not described separately.

<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

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

S. pasteurii being an alkaliphile24 can survive relatively harsh conditions. When the above mentioned culture protocol is followed, and S. pasteurii is grown inside a chamber, the bacteria leads to the precipitation of calcium carbonate over time (Figure 2A). Figure 2 (b) shows a phase-contrast optical microscopic image of the bacterial cell population within the culture medium. Individual cells can be clearly distinguished, w...

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.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

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

21.7K Views.  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.

Video: 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.

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

Environment

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.

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

Engineering

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.

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.

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.

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.

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

Chemistry

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.

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.

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

Biochemistry

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.

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

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.

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 ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Summary

Explore More Videos

Chapters in this video

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

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

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

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

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

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

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

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

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