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 p...

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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 border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Petridish</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:255px;">Petridishes being used as Agar plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:224px;">Pyrex Flasks</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S63268</td>
			<td style="width:255px;">Corning Erlenmeyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tris-Base</td>
			<td style="width:108px;">Promega</td>
			<td style="width:119px;">H5133</td>
			<td style="width:255px;">being used to make Tris-Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Hydrochloric Acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9892</td>
			<td style="width:255px;">1.0 N, Bioreagent, suitable for cell culture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Agar Powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td style="width:255px;">microbiology tested, plant cell culture tested, cell culture tested, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulphate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4418</td>
			<td style="width:255px;">for Molecular Biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Yeast extract powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">51475</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Measuring Cylinder</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">CP08559GC</td>
			<td style="width:255px;">Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC;1000ml,1/Pk</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Analytical Balance</td>
			<td style="width:108px;">OHAUS</td>
			<td style="width:119px;">AX124E</td>
			<td style="width:255px;">being used to measure weight of reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave</td>
			<td style="width:108px;">Brinkmann</td>
			<td style="width:119px;">58619000</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave Tape</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">52428864</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Aluminum Foil</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z185140</td>
			<td style="width:255px;">being used to seal the flask before placing it in Autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Bacterial Stock</td>
			<td style="width:108px;">Cedarlane</td>
			<td style="width:119px;">11859</td>
			<td style="width:255px;">-80&#176;C stock of S. pasteurii, ATCC No. is mentioned against Cat. No.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">Mline Single-Channel Mechanical Pipettors, Variable Volume</td>
			<td style="width:108px;">Biohit</td>
			<td style="width:119px;">725010</td>
			<td style="width:255px;">Marketed by VWR under catalog number 14005976</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Micropipette Tip</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">212772B</td>
			<td style="width:255px;">Used for scratching Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Incubator</td>
			<td style="width:108px;">Binder</td>
			<td style="width:119px;">80079098</td>
			<td style="width:255px;">Microbiology Incubator,BF Series</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Shaking Incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">14004300</td>
			<td style="width:255px;">VWR Signature Benchtop Shaking Incubators</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS)&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">BD Falcon Express Pipet-Aid Pipetting Device</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">357590</td>
			<td style="width:255px;">Marketed by VWR under catalog number 53106220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Parafilm</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793</td>
			<td style="width:255px;">Being used to seal Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Urea</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">U1250</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Sodium Bicarbonate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8875</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Calcium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">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...

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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 ...

The overall goal of this protocol is to induce calcite precipitation in a stagnant culture of Sporosarcina pasteurii. This method can help us answer key biophysical questions about Sporosarcina pasteurii-mediated calcite precipitation, like the pertinent length and time scales. The main advantage of this technique is the fact that it standardizes the complex steps involved in suitably culturing and enriching Sporosarcina pasteurii that ensures reliable calcite precipitation.To begin, assemble Petri dishes, flasks, Tris base, HCl, agar, Millipore water, and a pH meter. Sterilize all containers by autoclaving at 121 degrees Celsius before use. Prepare one liter of a 0.13 molar aqueous solution of Tris buffer by mixing 15.75 grams of Tris base with one liter of Millipore water.Add approximately 2.8 milliliters of 50%HCl to adjust the buffer to a pH of nine. Once the culture is ready, it must be suitably enriched with the proper buffer. This step is critical to the success of the experiment, because a failure to provide the proper chemical environment can lead to very long time scales of precipitation or even a complete lack thereof.Next, divide the one liter of buffer into two parts as follows. In one 400 milliliter aliquot, dissolve eight grams of ammonium sulfate. In a second 400 milliliter aliquot, dissolve 16 grams of yeast extract.In 100 milliliters of the buffer, mix two grams of ammonium sulfate, and in the last 100 milliliters of buffer, add four grams of yeast extract and four grams of agar. After wrapping the flasks in aluminum foil, autoclave the four solutions. Immediately after autoclaving, set the two 400 milliliter solutions aside.Combine the two 100 milliliter solutions, mix well, and divide between 10 to 12 Petri dishes. To culture bacterial samples, remove the bacterial stock from the minus 80 degree Celsius freezer and allow it to thaw properly. Then place it along with an agar plate in the biosafety hood.Select a micropipette of the lowest possible volume and dip a micropipette tip into the Sporosarcina pasteurii stock before using the tip to streak an agar plate. Incubate the plate at 31 degrees Celsius for 48 hours, then visually examine for the presence of single colonies. If none are present, incubate for an additional 24 hours.Repeat the check and incubations until single colonies are detected. To prepare the final bacterial culture samples, mix together the two 400 milliliter solutions autoclaved earlier in a sterilized beaker, and transfer 125 milliliters into a flask. Perform a visual examination of the agar plate to identify regions with high concentrations of single colonies.With a micropipette tip, gently nudge and break one of the colonies. Then dip the same tip into the 125 milliliter flask and stir it thoroughly to ensure that a sufficient number of cells are transferred. Place the flask in a shaking incubator at 30 degrees Celsius and 150 rpm for two to three days.To determine a final cell count, draw seven parallel equidistant bold lines on the bottom of one of the agar plates so that they are visible from the top. Place three small drops of about ten microliters of non-diluted culture into one segment. Add one milliliter of non-diluted culture to nine milliliters of sterilized PBS to obtain a one-to-ten dilution.Then place three small drops of the dilution onto a second segment of the plate. Transfer the diluted culture to a new flask, and use PBS to dilute an additional ten times to ten to the minus two. Repeat the plating and dilution process until generating a dilution of at least ten to the minus seven to ensure single colonies will be obtained.Incubate the plate at 31 degrees Celsius for one to two days before counting the colonies in each section of the plate. Transfer nine milliliters of the prepared liquid culture into several sterilized ten milliliter centrifuge tubes. Prepare 100 milliliters of the external enrichment stock solution by using an analytical balance to carefully measure out two grams per liter of urea, one gram per liter of ammonium chloride, 212 milligrams per liter of sodium bicarbonate, and 280 milligrams per liter of calcium chloride.Into a beaker with fresh medium, combine all the ingredients except the urea, and autoclave. After autoclaving, mix the urea with one milliliter of fresh medium and pass the solution through a syringe fitted with a 0.2 micrometer filter before adding to the enrichment medium. Add one milliliter of the enrichment medium with additives to the sterilized centrifuge tubes containing nine milliliters of the prepared culture.Vortex each tube and incubate in a non-shaking incubator at 30 degrees Celsius. Monitor all the units regularly for initiation of precipitation. Once precipitation is detected with the naked eye, transfer a small volume of fluid to a high magnification microscopy chambered cover glass system.Initially view under 4X magnification to observe a wide area of coverage before progressively increasing the magnification. Perform staining and SEM experiments according to the text protocol. When S.pasteurii is cultured following the protocol demonstrated in this video, the bacteria lead to the precipitation of calcium carbonate that settles to the bottom of the tube over time.This figure shows phase contrast images of a clearly distinguished, rod-like bacterial population within the medium. Here, the well-defined cleavage lines, a hallmark of crystallinity, can be seen. This X-ray photoelectron spectroscopy graph for the same sample contains peaks corresponding to carbonate groups that pinpoint the existence of calcium carbonate.Shown in this panel is an untreated sponge that easily compresses when subjected to a one kilogram weight. When a sponge was immersed in a calcite precipitant solution with S.pasteurii for seven days and then dried, it hardened and displayed enhanced compressive strength due to pore clogging from calcite precipitation. Once mastered, this technique can be completed in a few hours if all the steps are done properly.However, although the individual steps take just a few minutes each, there are longer idle periods in between where the sample must be incubated for hours or even days. Following this procedure, several other techniques like additional characterization can be performed to answer other questions about, for example, the statistical distribution of polymorphous forms in calcium carbonate. After watching this video, you should have a proper and clear understanding of how to suitably culture Sporosarcina pasteurii to enrich it properly that ensures calcite precipitation, and also how to observe the process through multi-mode microscopy.Don't forget that working with high pressure autoclaves can be extremely hazardous and hence, all precautions like complete venting of the chamber and wearing of PPEs must be ensured before performing this procedure.

microbiologically-induced-calcite-precipitation-mediated-sporosarcina

Research

JoVE Journal

Bioengineering

21.3K vistas.  University of Alberta. The overall goal of this protocol is to induce calcite precipitation in a stagnant culture of Sporosarcina pasteurii. This method can help us answer key biophysical questions about Sporosarcina pasteurii-mediated calcite precipitation, like the pertinent length and time scales. The main advantage of this technique is the fact that it standardizes the complex steps involved in suitably culturing and enriching Sporosarcina pasteurii that ensures reliable calcite precipitation.To begin, a...