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 for 10 min.</li>
			<li>Decant the water, add ethanol to cover the glass slides, and sonicate in a bath sonicator for 10 min.</li>
			<li>Dry the slides and the glassware with a stream of nitrogen gas and place them in an air plasma cleaner for 10 min at 130 W.</li>
		</ol>
		</li>
		<li>Optimize the concentration of the CaCl2 used in the calcification experiments performed under the desired experimental conditions to achieve a sample rich with smooth-faceted calcite crystals (without or at least with a scarce number of vaterite crystals).
		<ol>
			<li>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. 
			CAUTION: Ammonium carbonate irritates nose and lungs; use only inside the fume hood.</li>
			<li>Prepare a stock solution of 0.5 M CaCl2. This stock solution will be used to prepare a gradient of concentrations of CaCl2 solutions in the multi-well plate. 
			NOTE: A 10 mL stock solution is sufficient for the whole experiment.</li>
			<li>Place the previously cut and cleaned glass pieces into five different wells. Use the closest wells to the center.</li>
			<li>Fill each well bearing a glass piece with 100 &#181;L of a CaCl2&#160;solution16. Mix TDW and 0.5 M CaCl2 (stock) to achieve an increasing concentration gradient of CaCl2 across the different wells. If a different sized well-plate is used, adjust the concentration of CaCl2 to achieve separate calcite crystals (step 1.1.2.10, and see Discussion section). 
			NOTE: An increasing CaCl2 gradient of 10, 20, 30, 40 ,50 mM concentrations in separate wells is used in this protocol. To increase the concentration range or the number of concentrations tested, use additional wells.</li>
			<li>Puncture the cover of each of the wells containing ammonium carbonate 3x with a needle.</li>
			<li>Put back the lid, seal the borders with paraffin film and keep it at 18 &#176;C in an incubator for 20 h.</li>
			<li>After the incubation, open the lid carefully inside a fume hood and remove the crystals formed at the water/air interface with a loop.</li>
			<li>Use a tweezer to transfer the glass pieces into a beaker containing double distillated water (DDW). Remove the samples from the beaker and use a double-sided tape to fix the glass pieces onto the bottom of the Petri dish.</li>
			<li>Dry excessive water touching the borders of the slide with tissue wipes. Cover the petri dish and place it in a desiccator for 24 h.</li>
			<li>Observe the crystals formed on the glass pieces with a stereoscope (3.5x magnification) and/or an upright optical microscope (10x-40x magnification). If the control solutions are clean, rhombohedral crystals (most likely calcite) will be observed with an optical microscope (Figure 2A).</li>
			<li>If in addition to the rhombohedral crystals, the control contains spherical crystals (most likely vaterite, Figure 2B), or if scanning electron microscope (SEM) images show rhombohedral crystals with rough rather than smooth faces (Figure 3A,B), repeat the crystallization protocol making sure that the cleaning step (1.1.1) is performed correctly. Furthermore, make better care that there is no ammonium carbonate in areas on the plate other than the dedicated wells. Otherwise, continue to the next step.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Crystallization in the presence of the additives
	<ol>
		<li>To study the effect of the additives on the crystallization of CaCO3, set up a multi-well plate that contains (in different wells), a control CaCl2 solution without the additives, and CaCl2 solutions with the additives. Use the optimal concentration of CaCl2 found in section 1.1.2 for the experiment. 
		NOTE: The protocol below uses optimal conditions as those reported in a previous study16.</li>
		<li>Repeat step 1.1.2.2.</li>
		<li>Place ammonium carbonate powder in the corners of the plate as described in step 1.1.2.1.</li>
		<li>In each well where precipitation will occur, place a glass piece that was cut and cleaned as described in section 1.1.1.</li>
		<li>To prepare control wells, pipette 90 &#181;L of TDW into the control wells. Prepare at least one replicate of each well including the control. If the additive used is in a buffer solution, then pipette 90 &#181;L of buffer instead of TDW water.</li>
		<li>Prepare the additive-containing wells. Repeat step 1.2.5 by adding 90 &#181;L of the additive solution in water. If the additive is in buffer (instead of TDW), pre-adjust the concentration of the additive with buffer to meet the desired final concentration. Keep a total volume of 90 &#181;L; pipette first the additive, then the buffer. 
		NOTE: A final concentration of 10 &#181;M of the protein TapA in 100 mM NaCl, 25 mM Tris pH 8.0 buffer16 is used in this protocol.</li>
		<li>Add 10 &#181;L of the 0.5 M CaCl2 stock solution (prepared in step 1.2.2) to both the controls and the additives-containing wells to reach a final concentration of 50 mM CaCl2.</li>
		<li>Repeat steps 1.1.2.5-1.1.2.9.</li>
	</ol>
	</li>
</ol>
2. Characterization of calcium carbonate crystals
<ol>
	<li>With a scanning electron microscope, observe the calcium carbonate crystals formed in the presence of the additives at a higher resolution than that obtained by optical microcopy (see step 1.1.2.10).
	<ol>
		<li>Mount the glass pieces containing the crystals on an aluminum stub with double-sided carbon tape.</li>
		<li>Coat with a layer of Au/Pd for 40-50 s.</li>
		<li>Acquire the images at 5 kV acceleration voltage. 
		NOTE: Figure 3A shows a representative SEM image of calcium carbonate crystals formed in a proper control experiment, while Figure 4 shows representative images of calcium carbonate crystals formed in the presence of the protein TapA.</li>
	</ol>
	</li>
	<li>Perform micro Raman spectroscopy to determine the calcium carbonate polymorphs formed. Micro Raman allows the collection of a Raman spectrum from single crystals rather than from a whole powder.
	<ol>
		<li>Use a 20x objective of the microscope to choose the crystal of interest.</li>
		<li>Collect the Raman spectrum in a range of 100&#8722;3200 cm-1 using a 514 nm argon laser. 
		NOTE: Figure 5 shows representative spectra of calcite (A) and vaterite (B). For the spectrum of aragonite, refer to reference18.</li>
	</ol>
	</li>
	<li>Quantification of the mass percentage of the additives in the CaCO3 precipitates
	<ol>
		<li>Verify/measure the extinction coefficient (&#949;) of the additive used. The extinction coefficient of a protein can be given by online servers19. If the extinction coefficient is unknown, measure the absorbance of the additive at different concentrations, plot the absorbance vs. concentration and calculate the extinction coefficient from the slope of the curve.</li>
		<li>Weigh the glass pieces where the crystals formed, preferably use a microbalance.</li>
		<li>Scrap the crystals off the glass into 1.2 mL of 0.1 M acetic acid solution, vortex and sonicate the sample. Store the sample at room temperature for 24 h. 
		CAUTION: Acetic acid is very hazardous in case of skin or eye contact; handle with caution and dispose following the regulations.</li>
		<li>Weigh the glass slide after scraping off the crystals.</li>
		<li>Measure the UV/vis absorbance (A) spectrum of the solution. If the additive is a protein, measure the absorbance at 280 nm and calculate its concentration (C), using the Beer-Lambert equation: 
		<img alt="Equation 1" src="/files/ftp_upload/59638/59638eq1.jpg" /> 
		where l is the optical path inside the cuvette.</li>
		<li>Use the concentration (C) found in 2.3.5 and the volume used (V = 1.2 mL) to calculate the mass (m) of the additives in/on the crystals. If the concentration is in mg/mL, use the equation C &#9679; V = m.
		<ol>
			<li>If the concentration is in mol/L, then calculate the moles (n) applying C &#9679; V = n. Then use the molecular weight (Mw) to calculate the mass (m) of the additives (m = n &#9679; Mw).</li>
		</ol>
		</li>
		<li>Calculate the weight percentage of the additives in/on the crystals using the equation: <img alt="Equation 2" src="/files/ftp_upload/59638/59638eq2.jpg" />, where m is the mass of the additives, and &#916;ms is the mass of the calcium carbonate crystals that were scrapped off the glass piece.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Blakemore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetotactic+bacteria.">Magnetotactic bacteria.</a> Science. 190 (4212), 377-379 (1975).</li><li>Politi, Y., Arad, T., Klein, E., 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=Sea+Urchin+Spine+Calcite+Forms+via+a+Transient+Amorphous+Calcium+Carbonate+Phase.">Sea Urchin Spine Calcite Forms via a Transient Amorphous Calcium Carbonate Phase.</a> Science. 306 (5699), 1161-1164 (2004).</li><li>Nudelman, F., Lausch, A. J., Sommerdijk, N. A. J. M., Sone, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+models+of+collagen+biomineralization.">In vitro models of collagen biomineralization.</a> Journal of Structural Biology. 183 (2), 258-269 (2013).</li><li>Sigel, A., Sigel, H., Sigel, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomineralization: from nature to application. 12, (2008).</li><li>Nielsen, M. H., Lee, J. R. I., De Yoreo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 532, 209-224 (2013).</li><li>Page, M. G., C&#246;lfen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Control+of+CaCO3+Precipitation+by+Direct+Carbon+Dioxide+Diffusion:+Application+in+Mesocrystal+Assembly.">Improved Control of CaCO3 Precipitation by Direct Carbon Dioxide Diffusion: Application in Mesocrystal Assembly.</a> Crystal Growth &amp; Design. 6 (8), 1915-1920 (2006).</li><li>Wang, H., Huang, W., Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion-reaction+compromise+the+polymorphs+of+precipitated+calcium+carbonate.">Diffusion-reaction compromise the polymorphs of precipitated calcium carbonate.</a> Particuology. 11 (3), 301-308 (2013).</li><li>Sedl&#225;k, M., Antonietti, M., C&#246;lfen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+a+new+class+of+double-hydrophilic+block+copolymers+with+calcium+binding+capacity+as+builders+and+for+biomimetic+structure+control+of+minerals.">Synthesis of a new class of double-hydrophilic block copolymers with calcium binding capacity as builders and for biomimetic structure control of minerals.</a> Macromolecular Chemistry and Physics. 199 (2), 247-254 (1998).</li><li>Kitano, Y., Park, K., Hood, D. 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. L., McConnaughey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geochemical+Perspectives+on+Coral+Mineralization.">Geochemical Perspectives on Coral Mineralization.</a> Reviews in Mineralogy and Geochemistry. 54 (1), 151-187 (2003).</li><li>Erez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+effect+on+stable-isotope+composition+seen+in+foraminifera+and+coral+skeletons.">Vital effect on stable-isotope composition seen in foraminifera and coral skeletons.</a> Nature. 273, 199 (1978).</li><li>Azulay, D. N., 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=Biopolymers+from+a+Bacterial+Extracellular+Matrix+Affect+the+Morphology+and+Structure+of+Calcium+Carbonate+Crystals.">Biopolymers from a Bacterial Extracellular Matrix Affect the Morphology and Structure of Calcium Carbonate Crystals.</a> Crystal Growth &amp; Design. 18 (9), 5582-5591 (2018).</li><li>Abbasi, 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=The+Bacterial+Extracellular+Matrix+Protein+TapA+Is+a+Two-Domain+Partially+Disordered+Protein.">The Bacterial Extracellular Matrix Protein TapA Is a Two-Domain Partially Disordered Protein.</a> ChemBioChem. , (2018).</li><li>Gauldie, R. W., Sharma, S. K., Volk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-raman+spectral+study+of+vaterite+and+aragonite+otoliths+of+the+coho+salmon,+Oncorhynchus+kisutch.">Micro-raman spectral study of vaterite and aragonite otoliths of the coho salmon, Oncorhynchus kisutch.</a> Comparative Biochemistry and Physiology Part A: Physiology. 118 (3), 753-757 (1997).</li><li>Gasteiger, E., 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> The Proteomics Protocols Handbook. , 571-607 (2005).</li><li>Gunasekaran, S., Anbalagan, G., Pandi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raman+and+infrared+spectra+of+carbonates+of+calcite+structure.">Raman and infrared spectra of carbonates of calcite structure.</a> Journal of Raman Spectroscopy. 37 (9), 892-899 (2006).</li><li>Trushina, D. 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. M., Tuross, N., Addadi, L., Weiner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mollusc+larval+shell+formation:+amorphous+calcium+carbonate+is+a+precursor+phase+for+aragonite.">Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite.</a> Journal of Experimental Zoology. 293 (5), 478-491 (2002).</li><li>Yamamoto, Y., Nishimura, T., Saito, T., Kato, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaCO3/chitin-whisker+hybrids:+formation+of+CaCO3+crystals+in+chitin-based+liquid-crystalline+suspension.">CaCO3/chitin-whisker hybrids: formation of CaCO3 crystals in chitin-based liquid-crystalline suspension.</a> Polymer Journal. 42, 583 (2010).</li><li>Magnabosco, 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=Insights+on+the+interaction+of+calcein+with+calcium+carbonate+and+its+implications+in+biomineralization+studies.">Insights on the interaction of calcein with calcium carbonate and its implications in biomineralization studies.</a> CrystEngComm. 20 (30), 4221-4224 (2018).</li></ol>

The authors have nothing to disclose.

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><tbody><tr><td>Acetic acid</td><td>Gadot</td><td>64-19-7</td><td></td></tr><tr><td>Ammonium carbonate</td><td>Sigma-Aldrich</td><td>506-87-6</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Merck KGaA</td><td>10035-04-8</td><td></td></tr><tr><td>Ethanol Absolute</td><td>Gadot</td><td>64-17-5</td><td></td></tr><tr><td>Micro-Raman</td><td>Renishaw</td><td></td><td>inVia Reflex spectrometer coupled with an upright Leica optical microscope</td></tr><tr><td>Microscope</td><td>Nikon</td><td></td><td>Eclipse 90i model</td></tr><tr><td>Nis elements Br software</td><td>Nikon</td><td></td><td>For microscope imaging</td></tr><tr><td>Scanning Electron Microscope</td><td>ThermoFisher Scientific</td><td></td><td>FEI Sirion microscope</td></tr><tr><td>Spectrophotometer</td><td>JASCO</td><td></td><td>V-670 model</td></tr><tr><td>Sputter coater</td><td>Polaron</td><td></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 ...

calcium-carbonate-formation-in-the-presence-of-biopolymeric-additives

Research

JoVE Journal

Chemistry

16.7K Views.  The Hebrew University of Jerusalem. We describe a protocol for the precipitation and characterization of calcium carbonate crystals that form in the presence of biopolymers.

Video: Calcium Carbonate Formation in the Presence of Biopolymeric Additives

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

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 ...

Engineering

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 ...

Environment

Additive Manufacturing of Functionally Graded Ceramic Materials by Stereolithography

An additive manufacturing technology is applied to obtain functionally graded ceramic parts. This technology, based on digital light processing/stereolithography, is developed within the scope of the CerAMfacturing European research project. A three-dimensional (3-D) hemi-maxillary bone-like structure is 3-D printed using custom aluminum oxide polymeric mixtures. The powders and mixtures are fully analyzed in terms of rheological behavior in order to ensure proper material handling during the printing process. The possibility to print functionally graded materials using the Admaflex technology is explained in this document. Field-emission scanning electron microscopy (FESEM) show that the sintered aluminum oxide ceramic part has a porosity lower than 1% and no remainder of the original layered structure is found after analysis.

This manuscript describes the processing of single multifunctional ceramic components (e.g., combinations of dense-porous structures) additively manufactured by stereolithography.

An additive manufacturing technology is applied to obtain functionally graded ceramic parts. This technology, based on digital light ...

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

Bioengineering

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

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.

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.

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

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 ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

Calcium Carbonate Formation in the Presence of Biopolymeric Additives

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Rozdziały w tym wideo

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Preparation of Biopolymer Aerogels Using Green Solvents

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.

Additive Manufacturing of Functionally Graded Ceramic Materials by Stereolithography

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

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

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications