The authors would like to thank Prof. Lia Addadi, Prof. Jonathan Erez, and Dr. Yael Politi for fruitful discussions. This research has been supported by the Israeli Science Foundation (ISF), grant 1150/14.

1. Calcium carbonate crystallization
<ol>
	<li>Control preparation and optimization
	<ol>
		<li>Prepare clean glass pieces. Use the same cleaning procedure to clean the glassware.
		<ol>
			<li>Use a diamond pen to cut pieces of a glass microscope slide so that they fit in a well of a 96-well plate. 
			NOTE: 5 mm x 5 mm pieces should largely fit.</li>
			<li>Place the glass pieces in a beaker with triple distilled water (TDW) so that water covers the glass slides and sonicate in a bath sonicator ...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pure+aragonite+synthesis.">Pure aragonite synthesis.</a> Journal of Geophysical Research. 67 (12), 4873-4874 (1962).</li><li>Politi, Y., Mahamid, J., Goldberg, H., Weiner, S., Addadi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asprich+mollusk+shell+protein:+in+vitro+experiments+aimed+at+elucidating+function+in+CaCO3+crystallization.">Asprich mollusk shell protein: in vitro experiments aimed at elucidating function in CaCO3 crystallization.</a> CrystEngComm. 9 (12), 1171-1177 (2007).</li><li>Gehrke, N., C&#246;lfen, H., Pinna, N., Antonietti, M., Nassif, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superstructures+of+Calcium+Carbonate+Crystals+by+Oriented+Attachment.">Superstructures of Calcium Carbonate Crystals by Oriented Attachment.</a> Crystal Growth &amp; Design. 5 (4), 1317-1319 (2005).</li><li>Rudloff, 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=Double-Hydrophilic+Block+Copolymers+with+Monophosphate+Ester+Moieties+as+Crystal+Growth+Modifiers+of+CaCO3.">Double-Hydrophilic Block Copolymers with Monophosphate Ester Moieties as Crystal Growth Modifiers of CaCO3.</a> Macromolecular Chemistry and Physics. 203 (4), 627-635 (2002).</li><li>Boquet, E., Boronat, A., Ramos-Cormenzana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Calcite+(Calcium+Carbonate)+Crystals+by+Soil+Bacteria+is+a+General+Phenomenon.">Production of Calcite (Calcium Carbonate) Crystals by Soil Bacteria is a General Phenomenon.</a> Nature. 246, 527 (1973).</li><li>Cohen, A. 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B., Bukreeva, T. V., Kovalchuk, M. V., Antipina, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaCO3+vaterite+microparticles+for+biomedical+and+personal+care+applications.">CaCO3 vaterite microparticles for biomedical and personal care applications.</a> Materials Science and Engineering: C. 45, 644-658 (2014).</li><li>Weiss, I. 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The method described here is aimed at forming calcium carbonate crystals in the presence of organic additives and evaluating the effect of organic biopolymers on the morphology and structure of calcium carbonate crystals in vitro. The method is based on the comparison of the crystals formed in the presence of the organic additives to the calcite crystals formed in the control experiment. We have shown how to use the diffusion method to form the calcium carbonate crystals, how to characterize their morphology using optica...

Biomineralization is the formation of minerals in the presence of organic molecules, often related with functional and/or structural roles in living organisms. Biomineralization may be intracellular, as in the formation of magnetite inside magnetotactic bacteria1, or extracellular, as in the formation of calcium carbonate in sea urchin spikes2, of hydroxyapatite that is related with collagen in bones3 and of enamel that is associated with amelogenin in teeth4. Biomineralization is a complex process that depends on many parameters in the living organism. Therefore, in order to simplify the system under study, it is necessary to evaluate the effect of separate components on the process. In many cases, biomineralization is induced by the presence of extracellular biopolymers. The purpose of the method presented here are as follows: (1) To form calcium carbonate crystals in the presence of isolated biopolymers in vitro, using a vapour diffusion method. (2) To study the effect of the biopolymers on the morphology and structure of calcium carbonate.
Three principal methods to precipitate calcium carbonate in vitro in the presence of organic additives are used5,6. The first method, which we will refer to as the solution method, is based on mixing a soluble salt of calcium (e.g., CaCl2) with a soluble salt of carbonate (e.g., sodium carbonate). The mixing process may be performed in several ways: inside a reactor with three cells that are separated by porous membranes7. Here, each of the outer cells contains a soluble salt and the central cell contains a solution with the additive to be tested. Calcium and carbonate diffuse from the outer to the middle cell, resulting in the precipitation of the less soluble calcium carbonate when the concentrations of calcium and carbonate exceed their solubility product, Ksp = [Ca2+][CO32-]. An additional mixing method is the double-jet procedure8. In this method, each soluble salt is injected from a separate syringe to a stirred solution containing the additive, where calcium carbonate precipitates. Here, the injection and therefore the mixing rate is well controlled, in contrast with the previous method where mixing is controlled by diffusion.
The second method used to crystallize CaCO3 is the Kitano method9. This method is based on the carbonate/hydrogen carbonate equilibrium (2HCO3- (aq) + Ca2+(aq) <img alt="Image 1" src="/files/ftp_upload/59638/59638img01.jpg" /> CaCO3 (s) + CO2 (g) + H2O (l)). Here, CO2 is bubbled into a solution containing CaCO3 in a solid form, shifting the equilibrium to the left and therefore dissolving the calcium carbonate. The undissolved calcium carbonate is filtered and the desired additives are added to the bicarbonate-rich solution. CO2 is then allowed to evaporate, thereby shifting the reaction to the right, forming calcium carbonate in the presence of the additives.
The third method of calcium carbonate crystallization, which we will describe here, is the vapor diffusion method10. In this set-up, the organic additive, dissolved in a solution of calcium chloride, is placed in a closed chamber near ammonium carbonate in a powder form. When ammonium carbonate powder decomposes into carbon dioxide and ammonia, they diffuse into the solution containing calcium ions (e.g., CaCl2), and calcium carbonate is precipitated (see Figure 1 for illustration). The calcium carbonate crystals can grow by slow precipitation or by fast precipitation. For the slow precipitation, a solution containing the additive in CaCl2 solution is placed in a desiccator next to the ammonium carbonate powder. In the fast precipitation, described in length in the protocol, both the additive solution and the ammonium carbonate are placed closer together in a multi-well plate. The slow precipitation method will produce fewer nucleation centers and larger crystals, and the fast precipitation will result in more nucleation centers and smaller crystals.
The methods described above differ in their technical complexity, in the level of control and in the rate of the precipitation process. The mixing method requires a special set-up6 for both the double jet and the three-cell system. In the mixing method, the presence of other soluble counter ions (e.g., Na+, Cl-)6 is inevitable, whereas in the Kitano method, calcium and (bi) carbonate are the only ions in solution and it does not involve the presence of additional counter ions (e.g., Na+, Cl-). Furthermore, the mixing method requires relatively large volumes and therefore it is not suitable for working with expensive biopolymers. The advantage of the double jet is that it is possible to control the rate of solution injection and that it is a rapid process in comparison to other methods.
The advantage of the Kitano method and the vapor diffusion method is that the formation of calcium carbonate is controlled by diffusion of CO2 into/out of a CaCl2 solution, thus allowing to probe slower nucleation and precipitation processes11,12. Furthermore, calcium carbonate formation by diffusion of CO2 may resemble calcification processes in vivo13,14,15. In this method, well-defined and separated crystals are formed16. Last, the effect of single or multiple biopolymers on calcium carbonate formation can be tested. This enables a systematic study of the effect of a series of additive concentrations on calcium carbonate formation as well as a study of mixtures of biopolymers -&#160;all performed in a controlled manner. This method is suitable for use with a large range of concentrations and volumes of additives. The minimal volume used is approximately 50 &#181;L and therefore this method is advantageous when there is a limited amount of the available biopolymers. The maximal volume depends on the accessibility of a larger well-plate, or the desiccator into which the plate or beaker containing CaCl2 are to be inserted. The method described below has been optimized for working in a 96-well plate with a biopolymer chosen to be the protein TapA17.

<table border="0" cellpadding="0" cellspacing="0" style="width:659px;" width="659">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acetic acid</td>
			<td style="width:127px;">Gadot</td>
			<td style="width:119px;">64-19-7</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ammonium carbonate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">506-87-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Calcium chloride dihydrate</td>
			<td style="width:127px;">Merck KGaA</td>
			<td style="width:119px;">10035-04-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol Absolute</td>
			<td style="width:127px;">Gadot</td>
			<td style="width:119px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Micro-Raman</td>
			<td style="width:127px;">Renishaw</td>
			<td style="width:119px;"></td>
			<td>inVia Reflex spectrometer coupled with an upright Leica optical microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Microscope</td>
			<td style="width:127px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Eclipse 90i model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nis elements Br software</td>
			<td style="width:127px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>For microscope imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Scanning Electron Microscope</td>
			<td style="width:127px;">ThermoFisher Scientific</td>
			<td style="width:119px;"></td>
			<td>FEI Sirion microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Spectrophotometer</td>
			<td>JASCO</td>
			<td></td>
			<td>V-670 model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Sputter coater</td>
			<td style="width:127px;">Polaron</td>
			<td style="width:119px;"></td>
			<td>SC7640 model</td>
		</tr>
	</tbody>
</table>

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.

A schematic of the experimental set up is shown in Figure 1. Briefly, the diffusion method is used in order to form calcium carbonate crystals in 96-well plates and test the effect of biopolymers on the morphology and structure of the calcium carbonate crystals. In these experiments, ammonium carbonate is decomposed into ammonia and CO2, which diffuse into calcium carbonate solutions, resulting in the formation of calcium carbonate crystals (Figure 1 ...

Watch this Scientific Journal Video about Calcium Carbonate Formation in the Presence of Biopolymeric Additives at JoVE.com

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

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 ...

Our protocol describes a simple in vitro system, to study the effect of isolated molecules on the morphology and structure of calcium carbonate. This technique is advantageous in cases where the biomolecules I test are expensive, or available in small quantities, as well as when slow calcium carbonate precipitation is required. Furthermore, it allows to probe multiple precipitation experiments under the same conditions at once.First, prepare the control experiment. Use triple distilled water and ethanol to clean glass pieces and glassware. Use a diamond pen to cut pieces of a glass microscope slide, so that they fit in a well of a 96-well plate.Place the glass places in a beaker with triple distilled water, so that the water covers the glass slides. Sonicate in a bath sonicator for 10 minutes. Decant the water.Add ethanol to cover the glass slides, and sonicate again in a bath sonicator for 10 minutes. Then, dry the slides and the glassware with a stream of nitrogen gas, and place them in an air plasma cleaner for 10 minutes at 130 watts. In a fume hood, fill the wells at the corners of a 96-well plate with ammonium carbonate powder, and seal the plate using aluminum foil.Cover the foil with paraffin film. Clean any residual ammonium carbonate using nitrogen gas. Place the previously cut and cleaned glass pieces into five different wells closest to the center.Fill each well bearing a glass piece with 100 microliters of calcium chloride solution prepared with triple distilled water at an increasing gradient of 10, 20, 30, 40, and 50 millimolar concentrations. Next, puncture the cover of each of the wells containing ammonium carbonate three times with a needle. Put the lid back, seal the borders with paraffin film, and keep it at 18 degrees Celsius in an incubator for 20 hours.During the incubation, ammonium carbonate is decomposed into ammonia and carbon dioxide, which diffuse into calcium chloride solutions, resulting in the formation calcium carbonate crystals. After the incubation, open the lid carefully inside a fume hood, and use a loop to remove the crystals formed at the water-air interface. Use tweezers to transfer the glass pieces into a beaker containing double distilled water for a short dip.Then, remove the samples from the beaker, and use double-sided tape to fix the glass pieces onto the bottom of the Petri dish. Dry excessive water touching the borders of the slide, with tissue wipes. Cover the Petri dish, and place it in a desiccator for 24 hours.Observe the crystals formed on the glass pieces with an upright optical microscope at 10 to 40 times magnification. The observed rhombohedral crystals are most likely calcite. If in addition to the rhombohedral crystals, the solution contains spherical crystals which are most likely vaterite, repeat the crystallization protocol, making sure that the cleaning step is performed correctly.Furthermore, make sure there is no ammonium carbonate in areas of the plate other than the dedicated wells. The optimal concentration for calcium chloride is determined according to the sample rich with smooth faceted calcite crystals without vaterite crystals. To begin, clean glass slides and glassware as done previously.In a fume hood, place ammonium carbonate powder in the corners of a 96-well plate. In each well where precipitation will occur, place a glass piece that was cut and cleaned. Prepare the control wells.Into two control wells, pipette 90 microliters of 25 millimolar Tris buffer at pH 8, supplemented with 100 millimolar sodium chloride. Then add 10 microliters of 0.5 molar calcium chloride stock solution. Then, adjust the concentration of additive protein TapA to 10 micromolar TapA, 100 millimolar sodium chloride, and 25 millimolar Tris buffer at pH 8.Prepare the additive containing wells by adding 90 microliters of the additive solution. Add 10 microliters of 0.5 molar calcium chloride stock solution to the additive containing wells to reach the optimal concentration at 50 millimolar calcium chloride determined previously. Prepare the plate with holes on the cover over wells containing ammonium carbonate, and incubate at 18 degrees Celsius as previously done.After incubation, prepare the glass pieces in a Petri dish as done before, and place the dish in a desiccator for 24 hours. To quantify the mass percentage of the additives in the calcium carbonate precipitates, first verify the extinction coefficient of the additive used. Then, use a microbalance to weigh the glass pieces where the crystals formed.After that, scrape the crystals off the glass into an eppe tube with 1.2 milliliters of 0.1 molar acetic acid solution. Vortex briefly, and then place the tube into a sonicator to sonicate the sample until the crystals disappear. Store the sample at room temperature for 24 hours.Weigh the glass slide after scraping off the crystals. Measure the UV absorbance A of the 1.2 milliliter sonicated solution at 280 nanometers for the protein additive. Use the Beer-Lambert equation to calculate its concentration c.L is the optical path inside the cuvette. To calculate the mass of the additives in the crystal, use the equation, c times v equals m, if the concentration is in milligrams per milliliter. If the concentration is in moles per liter, then calculate the moles applying c times v equals n.Then use the molecular weight to calculate the mass of the additives. Calculate the weight percentage of the additives in the crystals. M is the mass of the additives, and delta m s, is the mass of the calcium carbonate crystals that were scraped off the glass piece.SEM images show the comparison between a proper control with smooth calcite faces, and calcite crystals with faces composed of steps. The spherical crystals are vaterite. The successful and unsuccessful control experiments resulted in Raman spectroscopy, showing the typical spectra of calcite and vaterite respectively.The split of the Raman shift at around 1080 per centimeter is the most evident characteristic of vaterite. The crystals of calcium carbonate formed in the presence of TapA, are distinct from the control crystals. A complex spherical calcium carbonate assembly, composed of multiple calcite microcrystals was formed.The Raman spectrum of the crystals formed in the presence of TapA, is similar to the spectrum of calcite. The absorbent spectrums of TapA, and the control without the additive, were measured following dissolution of the crystals in acid. The mass percent of TapA was determined to be 1.8 plus or minus 0.2%It's critical to follow the cleaning steps carefully, and to make sure that the control standards are met before testing the additives'effect on the formation of calcium carbonate.In addition, it is important to remove any excess of ammonium carbonate in powdered form from the wells. Ammonium carbonate decomposes into ammonia and carbon dioxide. Ammonia is toxic if inhaled, and therefore, ammonium carbonate should be handled inside a fume hood only.In order to evaluate the internal morphology and structure of the crystals, you can section them using a focused ion beam, image the sections with a transmission electron microscope, and measure the electron diffraction's patterns from specific locations along the sections. This technique has been previously used to study the effect of various molecules on the morphology and structure of calcium carbonate. We have elaborated this method to biopolymers that are produced by bacterial cells in biofilms.It will be interesting to perform additional studies with other biopolymers produced by different bacterial strains.

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JoVE Journal

Chemistry

16.5K Aufrufe.  The Hebrew University of Jerusalem. Unser Protokoll beschreibt ein einfaches In-vitro-System, um die Wirkung isolierter Moleküle auf die Morphologie und Struktur von Calciumcarbonat zu untersuchen. Diese Technik ist vorteilhaft in Fällen, in denen die Biomoleküle, die ich teste, teuer oder in kleinen Mengen erhältlich sind, sowie wenn eine langsame Calciumcarbonatfällung erforderlich ist. Darüber hinaus ermöglicht es, mehrere Niederschlagsexperimente unter den gleichen Bedingungen gleichzeitig zu untersuchen.Berei...