Biopolymers from a Bacterial Extracellular Matrix Affect the

Subscriber access provided by UNIV OF DURHAM

Article

Biopolymers from a Bacterial Extracellular Matrix Affect the Morphology and Structure of Calcium Carbonate Crystals David N. Azulay, Razan Abbasi, Ilanit Ben Simhon Ktorza, Sergei Remennik, Amarendar Reddy M, and Liraz Chai Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00888 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on August 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Biopolymers from a Bacterial Extracellular Matrix Affect the Morphology and Structure of Calcium Carbonate Crystals David N. Azulay1,2, Razan Abbasi1,2, Ilanit Ben Simhon Ktorza1,2, Sergei Remennik2, Amarendar Reddy M.1,2 and Liraz Chai1* 1

Institute of Chemistry, The Hebrew University of Jerusalem

2

The Harvey M. Krueger Family Center for Nanoscience and Nanotechnology, The Hebrew

University of Jerusalem, Edmond J. Safra Campus, Jerusalem 9190401, Israel

* To whom correspondence should be addressed: Dr. Liraz Chai, Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra campus, Jerusalem 91904, Israel. Telephone: +972-2-6585303, Fax. +972-2-5660425. Email. [email protected]

1 Environment ACS Paragon Plus

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT

Biomineralization is a mineral precipitation process occurring in the presence of organic molecules and used by various organisms to serve a structural and/or a functional role. Many biomineralization processes occur in the presence of extracellular matrices that are composed of proteins and polysaccharides. Recently, there is growing evidence that bacterial biofilms induce CaCO3 mineralization and that this process may be related with their extracellular matrix (ECM). In this study we explore, in vitro, the effect of two bacterial ECM proteins, TasA and TapA and an exopolysaccharide, EPS, on calcium carbonate crystallization. We have found that all the three biopolymers induced the formation of complex CaCO3 structures. The crystals formed in the presence of the EPS were very diverse in morphology and they were either calcite or vaterite in structure. However, more uniformly sized calcite crystals were formed in the presence of the proteins; these crystals were composed of single crystalline domains that assembled together into spherulites (in the presence of TapA) or dumbbell – like shapes (in the presence of TasA). Our results suggest the EPS affects the nucleation of calcium carbonate when it induces the formation of vaterite crystals and that unlike EPS, the proteins stabilize pre-formed calcite nuclei and induce their aggregation into complex calcite structures.

Biomineralization processes induced by bacterial ECM macromolecules make biofilms more robust and difficult to remove when they form, for example, on pipes and filters in water desalination systems or on ship hulls. Understanding the formation conditions and mechanism of formation of calcium carbonate in the presence of bacterial biopolymers may lead to the design of suitable mineralization inhibitors.


Page 2 of 31

Page 3 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.

INTRODUCTION

Living organisms have developed the ability to form inorganic minerals from soluble precursors in a process termed biomineralization. Some biomineralization process occur intracellularly,1-3 with a common example being the formation of magnetite particles within

magnetotactic

bacteria.4 However, in many cases biomineralization processes occur outside cells, where the growing minerals are exposed to extracellular matrix macromolecules, mainly polysaccharides and proteins.2, 3, 5, 6 Particular examples in eukaryotes include the protein collagen that is related with bone formation from hydroxyapatite7 and the protein amelogenin that is related with enamel formation in teeth.8 Common polysaccharides related with mineralized tissues include chitin, a linear chain of N-acetylglucosamine

9, 10

and glycosaminoglycans (GAGs), linear chains of

amino disaccharides.11, 12 A common example for extracellular biomineralization in prokaryotes is the formation of stromatolites, layered calcium carbonate rocks that are formed in the presence cyanobacteria biofilms in a biomineralization process that is possibly induced by the bacterial extracellular matrix.13,

14

In addition, previous reports have shown that calcite is deposited by most soil

bacteria under calcium and CO2 - rich conditions.15 Recently, a few reports have related CaCO3 mineralization with the extracellular matrix (ECM) in bacterial biofilms.16-19 Biofilms are aggregates of cells that form surface and interface – associated communities.20 Similarly to eukaryotic tissues, the cells in a biofilm also secrete extracellular polymers that form a network that is composed of proteins, polysaccharides and sometimes nucleic acids.20, 21 The ECM provides the architecture of biofilms,22, 23 grants them mechanical stability20 and it has a protective role against pathogens.24 Indeed, for many years extracellular biomineralization



research has been dominated by eukarya - related processes, however recently there has been a growing interest in bacterial - related CaCO3 formation. One model for biofilm formation is the soil bacterium, Gram positive, Bacillus subtilis. It has been shown that CaCO3 precipitates at the surface of Bacillus subtilis biofilms grown in the laboratory in a calcium and CO2 - rich environment.15 In particular, calcite crystallites form at the periphery of the biofilms only in strains that harbor the extracellular matrix genes.16 The goal of this study has been to study the direct effect of isolated ECM components on CaCO3 biomineralization, rather than in the biofilm context where there are various other factors that may influence the CaCO3 crystallization. We therefore isolated the major ECM components made by the soil bacterium Bacillus subtilis, the proteins TasA and TapA and the polysaccharide EPS and studied their effect on CaCO3 morphology and structure. TasA is a fiber - forming, functional amyloid protein that we purify in the form of oligomers;25 TapA anchors TasA to the bacterial cell - surface and possibly also serves as a branching agent along the TasA fibers.21 Bacterial exopolysaccharides are multifunctional. They are related with cell-surface and cell-cell adhesion, protection from pathogens and they also serve as a nutrient source.26 The polysaccharide used in this study is composed of the monosaccharides glucose and mannose27 (Figure 1a) and it is therefore neutral in solution. The proteins TasA and TapA (pI 5, 6, respectively28) are negatively charged under our experimental conditions (see Experimental section); the sequences of the proteins are shown in Figure 1 with the negatively charged amino acids marked red.


Page 4 of 31

Page 5 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The composition of the Extracellular matrix components used in this study. The Exopolysaccharide (EPS), composed of Glucose and Mannose (a) and the amino acid composition of the extracellular matrix proteins, TasA29 (b) and TapA (c). Negatively charged amino acids are highlighted in red.

We have found that the bacterial biopolymers affected the formation of calcium carbonate crystals, in particular complex crystals that are composed of microcrystallites were formed. On a structural level, the proteins do not change the thermodynamic preference of CaCO3 to form calcite, but the polysaccharide induces the formation of both calcite and vaterite. Our findings demonstrate that bacterial extracellular biopolymers affect the formation of CaCO3 crystals and they span several possible morphologies and structures of CaCO3 that are formed in solution in a non - biologically controlled manner. Comparison of our results with future experiments that will be performed in real biofilm settings may contribute to the differentiation between biologically induced and controlled processes in biofilm - related CaCO3 mineralization. Biofilm formation poses a significant problem, for example when their formation leads to blockage of water desalination systems or when they form on ship hulls and lead to increased viscous drag.30, 31 Removal of biofilms in these environments becomes even more difficult and



therefore expensive if mineralization is induced at their presence. Our study is therefore important to the understanding of biofilm mineralization prevention in real settings and to the possible design of suitable mineralization inhibitors.

2.

EXPERIM ENTAL SECTION

Calcium Carbonate Crystallization Experiments. CaCO3 was precipitated by diffusion of ammonium carbonate (Sigma-Aldrich) into 100 µl calcium chloride (CaCl2•2H2O, Merck millipore) aqueous solutions in Costar polystyrene 96 well plates ( 24 well plates with 600 µl -1 ml for the XRD and molecule quantification measurements), as previously described by the fast precipitation process in Politi et al32 with minor changes. Glass slides were cut with a diamond pen and cleaned using 10 minutes sonication in deionized water (2 ppb, resistivity = 18.2 MΩ cm, Barnstead, Thermo Scientific), 10 minutes sonication in ethanol, and drying with a nitrogen stream. The glass slides were then air plasma cleaned (Zepto, Diener) for 10 minutes at 130 watt power. The clean glass slides were afterwards placed in the multiwall plate. We used 50 mM calcium chloride solutions either without (control) or with additives. The organic additives were 10 mg/ml (in deionized water) EPS unless otherwise indicated in the text, 10 µM TasA (in 50 mM NaCl, 20 mM Tris buffer pH 8.2) or 10 µM TapA (in 100 mM NaCl, 25 mM Tris pH 8.0). Control experiments were conducted with CaCl2 solutions in water (control for the experiments with EPS) or buffer (control for the protein additives). Ammonium carbonate was placed in the four corners of the plate covered by aluminum foil and parafilm and punctured three times with a needle. Finally, the plate was closed and sealed with a parafilm. The experiments were


Page 6 of 31

Page 7 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


performed at 18 ˚C inside an incubator (Firocell, MMM group). After 20 h of incubation the glass slides were removed, washed with DDW and dried in a vacuum desiccator for 24 h. Crystal characterization. Scanning Electron Microscopy (SEM). Samples were mounted on an aluminum stub with double-sided carbon tape and coated with a layer of Au/Pd using a Polaron SC7640 sputter coater. SEM images were obtained using a FEI Sirion microscope (ThermoFisher Scientific Company) at 5 kV acceleration voltage. Micro-Raman spectroscopy. Micro-Raman spectroscopy was performed using a Renishaw inVia Reflex spectrometer coupled with an upright Leica optical microscope equipped with a 514 nm argon laser that was focused on a single crystal through an X20 objective. Each Raman spectrum was acquired typically for 10 seconds. All spectra were acquired at room temperature and the measurement range was from 100 cm-1 to 3200 cm-1. The Raman data acquisition was performed using the Renishaw WiRE 3.4 (Windows-based Raman Environment) software. FTIR spectroscopy. CaCO3 crystals were scrapped from the glass pieces into a diamond ATR/FT-IR module (Smart iTX) and their FTIR spectrum was recorded using a Nicolet iS50 ThermoFisher spectrometer in the range 525 cm-1 to 4000 cm-1. 256 scans were collected at room temperature with a resolution of 4 cm-1 using a DTGS detector. XRD analysis. Glass pieces were taken from the multiwell plate, washed and dried (see above) and placed in a D8 Advance diffractometer (Bruker AXS, Karlsruhe, Germany) with a goniometer radius 217.5 mm and a secondary Graphite monochromator.

X - ray powder

diffraction patterns were recorded from 5° to 75° 2θ at room temperature using CuKα radiation



(λ= 1.5418 Å) with the following measurement conditions: tube voltage of 40 kV, tube current of 40 mA, step-scan mode with a step size of 0.02° 2θ and a counting time of 1 sec per step. Molecule quantification in CaCO3 precipitates. The CaCO3 crystals were scrapped from the glass pieces and dissolved in a solution of 1.2 ml of 0.1 M acetic acid, vortexed, briefly sonicated and kept at room temperature for 24 h. Afterwards the absorbance was measured with a Jasco V670 UV-Vis spectrophotometer in the range of 200 nm to 800 nm. Focused ion beam thin sectioning. We used a Helios Nanolab 460F1 FIB-SEM (ThermoFisher Scientific Company) to section the crystals. Two types of sectioning were performed. 1. In order to image the interior of the crystals, platinum protected (800 nm) crystals were thinned to the middle using a 30 kV ion beam and a current of 790 pA. SEM images of the cut crystals were then collected using a 1kV voltage for the EPS –induced vaterite crystals and 3 kV for the rest of the crystals. 2. In order to prepare ultrathin TEM lamellae of the crystals produced in the presence of the proteins, the crystals were coated with platinum as described above. Then, a block at the interior of the crystal was removed (30 KV, 21 nA), lifted and mounted on a holder. Thinning of the block was then conducted in two steps obtaining 1 µm thickness (30 KV, 790 pA) and then 150 nm (30 KV, 40 pA) slices. Thereafter, 25 nm slices were removed from both sides until a sample of 100 nm thickness was obtained (5 KV, 15 pA). Finally, in order to get a better result from the TEM measurements, part of the sample was thinned until a thickness of 30-50 nm was obtained. Transmission Electron Microscopy. Samples mounted on a FIB lift out grid were characterized using a FEI Tecnai 12 G2 TWIN TEM operated at 120 kV (Thermo Fisher Scientific Company).


Page 8 of 31

Page 9 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The images were recorded by a 4Kx4K FEI Eagle CCD camera. For the selected area electron diffraction (SAED), various regions of the lamellae were scanned. Measurement of crystal size distribution. SEM images were analysed by ImageJ. The diameter was measured along the axis shown in Figure S2. Measurement of vaterite percentage out of the total number of crystals formed in the presence of EPS. We used our observation that only the vaterite crystals are spherical in the presence of EPS to differentiate the vaterite from the rest of the calcite crystals. We have counted the number of vaterite crystals relative to the total number of crystals in three different samples and a total of N = 4513 crystals. We have chosen to report on the percentage of the vaterite crystals by number since using the XRD or FTIR results would be inaccurate due to the different size distribution on the calcite and vaterite crystals. Extracellular matrix molecules purification. EPS purification. Wild type (WT) Bacillus Subtilis culture was grown overnight in LB medium and transferred to SYN medium at a 1:100 dilution. Pellicles were collected after 24 h at 37 ˚C, transferred into fresh SYN medium and washed twice by centrifugation at 5000 rpm for 15 min at 25 ˚C. The pellet was collected probe sonicated (1518J, 5 sec pulse and 2 sec pulse off) (Sonics, Vibra cell CV334) in fresh medium for 150 sec. The supernatant was separated from the pellet after an additional sonication and centrifugation of 5000 rpm for 30 min at 25 ˚C. The EPS was precipitated by adding x3 ice-cold isopropanol (J.T. Baker) following an overnight at -20 ˚C. The supernatant was discarded and the precipitate was washed twice with ice-cold isopropanol and was kept at -20 ˚C for additional 2 h. The precipitant was collected, dissolved in 0.1 M MgCl2 (J.T. Baker) and extracted twice by phenol-chloroform (Fisher Chemical) on a separating funnel. The aqueous phase was collected,



dialyzed for 48 h against DDW (with a Cellu Sep T3, 12000-14000 MWCO.) and lyophilized to dryness. For purification from DNA and proteins, EPS was dissolved in a 10 mM Tris buffer (VWR) containing 50 mM NaCl, 10 mM MgCl2 pH 7.5 and incubated with DNase (SigmaAldrich) at 37 ˚C for 1 h. Proteinase K (Sigma-Aldrich) was then added and the solution was incubated at 40 ˚C for 1 h. The solution is dialyzed against DDW (with a Cellu Sep T3, 1200014000 MWCO.) for 48 h and lyophilized to dryness. TapA purification. TapA was purified in the laboratory group as described previously.33 Briefly, plasmids containing a His6-TapA gen were used for the production of His6-TapA protein. These plasmids were transformed into Escherichia coli cells. Cultures in LB medium supplemented with ampicillin were grown shaking at 37 ˚C. Cells were transferred to an autoinduction medium (1:100 dilution) and harvested by centrifugation, resuspended in lysis buffer and incubated for 30 min. The lysate was loaded on nickel chelating resin and washed and conditioned as indicated by the manufacturer prior to adding to the samples and incubating with gentle agitation for 1 h at room temperature. The mixture was centrifuged and after decanting the supernatant, the lysate/resin mixture was washed with 5 volumes of binding buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich)), 3 volumes of washing buffer-I (20 mM Tris, 500 mM NaCl, 40 mM imidazole (Sigma-Aldrich), 1 mM PMSF) and 3 volumes of washing buffer-II (20 mM Tris, 500 mM NaCl, 100 mM imidazole, 1 mM PMSF). After eluting the proteins with elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole, 1 mM PMSF), they were further cleaned with gel filtration (Superdex 75). Purified proteins were stored at

-20 ˚C until used.

TasA purification. TasA was purified in the laboratory group as described previously.25 Briefly, Bacillus Subtilis liquid cultures were grown in MSgg broth, at a 1:100 dilution, under shaking


Page 10 of 31

Page 11 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conditions (225 rpm). After 16 h of growth at 37 ˚C the cells were pelleted (10,000 g, 15 min, 4 ˚C) and extracted once with a saline extraction buffer.34 The sample was probe sonicated (1518J, 5 sec pulse and 2 sec pulse off). The supernatant was then collected after an additional centrifugation (10,000 g, 15 min, 4 ˚C) and filtered through a 0.45 µm polyethersulfone (PES) bottle-top filter. The supernatant was collected after centrifugation (20,000 g, 10 min), concentrated with Amicon centrifugal filter tubes and passed through a HiLoad 26/60 Superdex S200 sizing column that was preequilibrated with a 50 mM NaCl, 20 mM Tris solution at pH 8. Purified proteins were stored at -20 ˚C until use. EPS composition analysis. Composition analysis of the polysaccharide was done by GCMS as TMS derivative of methyl-glycosides. Briefly, 50 µg of EPS was spiked with 1 µg of inositol as internal standard. The samples were dried completely and methanolyzed using 1 M methanolic HCl at 80 ˚C for 16 h followed by removal of excess acid by dry nitrogen flush. Afterwards, reN-acetylation was done using a mixture of methanol:pyridine:acetic anhydride (3:1:1 v/v mixture) at 100 ˚C for 1 h and followed by removal of excess reagent. Finally, a TMS derivatization was done by reacting the samples with Tri-Sil reagent (Thermo Scientific) at 80 ˚C for 30 min. The samples were dissolved in hexane and injected on GCMS.

3. RESULTS AND DISCUSSION 3.1 Matrix biopolymers induce the formation of CaCO3 complex structures. In our experimental setup, CaCO3 crystals were formed by diffusion of CO2 into CaCl2 solutions and the crystals were collected from a glass slide that was positioned in the bottom of the



reaction well. This method of CaCO3 formation yielded crystals of uniform size (20 ± 4 µm) and flat faces (Figure 2a); Powder diffraction measurements showed that these crystals were (104) oriented calcite (Figure S1), as expected from crystals that form in solution and land at the bottom of the reaction well with their wide (104) face pointing at the surface. The crystals formed in this control experiment were used as a reference for CaCO3 formation without additives. Exopolysaccharide (EPS). We have recently found that EPS is composed of glucose and mannose (unpublished results), in agreement with reference 27, and that it is a highly polydisperse polysaccharide, with molecular weight ranging between 105 to 109 gr/mole and an average 107 gr/mole molecular weight. In the presence of EPS (10 mg/mL), CaCO3 crystals with different sizes and different morphologies appeared (Figure 2b); the large variability in the calcium carbonate crystal morphology may be accounted for by the presence of a mixture of EPS assemblies (from single chains to aggregates). The different morphologies included pancake like rhombohedral crystals with rounded edges (~ 6 µm in size) (Figure 2c), crystals with spherical geometry of size ~ 35 µm and oval structures of size ~ 8 µm (Figures 2d, 2e). The latter two crystal types were composed of sharp – edged, smooth – faced, microcrystals of a few µm size. Unlike the structures that were described above (Figures 2c-e), additional, spherical structures that were composed of spherical grains were also formed (Figure 2f). The exact size distribution of the different crystals is shown in Figure S3 and the different axes that were used for size determination are shown in Figure S2.


Page 12 of 31

Page 13 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. SEM images of CaCO3 crystals formed in the lack (a) and presence of the polysaccharide EPS (b- f). Control calcite crystals are sharp –edged and smooth – faced (see enlarged view in (a)). Grown in the presence of EPS, CaCO3 crystals exhibit different morphologies in comparison to the control (b). Zooming into the different morphologies (c-f) shows crystals compose of rhombohedra (c,d,e) and spherical structures (f). The insert shows a zoom into (f).

Proteins. In contrast to the diverse CaCO3 structures formed in the presence of EPS (Figure 2), the crystals that formed in the presence of both proteins were rather uniform in morphology (Figures 3a, 3d). In the presence of TasA the crystals were ~ 70 µm in size (see Figure S2 for explanation on size measurement and Figure S3 for size distribution) and exhibited corrugated scale-like, sharp-edged, features at the micron level (Figure 3c). The microcrystals were 1 – 2



µm in size. The presence of TapA induced the formation of spherical macrostructures of size ~ 30 µm (see Figure S2 for explanation on size measurement and Figure S3 for size distribution) that were composed of sharp-edged rhombohedra of 1 – 2 µm in size. These were reminiscent of the control rhombohedral calcite crystals (Figure 2a) but most of them also exposed a new triangular face (Figure 3f, marked with an arrow) that was absent in the control calcite crystals and in the crystals formed in the presence of EPS (Figures 2a and 2d-2e, respectively).

Figure 3. SEM images of CaCO3 crystals formed in the presence of the proteins TasA (a –c), and TapA (d-f); Images are ordered in an increasing magnification order, showing that in the presence of both proteins the crystals are composed of microstructures (c,f). In the presence of TasA, the microstructures are corrugated scale – like (c) and in the presence of TapA, the crystals are composed of truncated rhombohedral microstructures (f). The arrows point at the truncated corners.

To get a better view of the different CaCO3 faces exposed in crystals formed in the presence of TapA, we show an enlarged view of these crystals in Figure 4b together with an enlarged view of the crystals formed in the presence of EPS (Figure 4a) for comparison. In order to understand the


Page 14 of 31

Page 15 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


appearance of the triangular facet in the micro rhombohedra in Figure 4b, we used the software Mercury 3.8 that shows that a triangular face could originate from the intersection of the crystallographic planes (104) and (001). This finding suggests that TapA stabilizes the (001) crystallographic plane.

Figure 4. An enlarged view at the SEM images of the crystals formed in the presence of EPS (a) and TapA (b). The enlarged view shows clearly that in the presence of TapA the crystals expose a triangular facet that is absent in the crystals formed in the presence of EPS. As shown in the diagram in (c), this facet could originate from the intersection of the (104) and (001) crystallographic planes. The diagram was sketched using Mercury 3.8.

3.2 CaCO3 complex structure Raman and FTIR spectroscopy. In order to test whether the bacterial macromolecules affected the crystalline structure of the CaCO3 crystals, we examined them using Raman and FTIR spectroscopy. The Raman measurements (Figure 5) show that the crystals formed in the presence of both TasA and TapA proteins were calcite35 ; The peaks in the range 100 cm-1 - 400 cm-1 correspond to the lattice modes, the peak in the 710 cm-1 region corresponds to the symmetric bending of the CO32- and the peaks in the 1085 cm-1 region correspond to the symmetric stretching of the CO32-. The Raman spectra of the sharp – edged crystals (Figures 2c-e), formed in the presence of EPS, 15 Environment ACS Paragon Plus


showed that they were also calcite in structure. However, the spherical crystals (Figure 2f) were vaterite,36 as shown by the typical split of the υ1 peak (1075 cm-1, 1090 cm-1) in their Raman spectra (Figure 5a). We used the clear morphology of the vaterite crystals in order to estimate their abundance (by number) and found that ~ 6% (number/number) of the total number of crystals are vaterite (see Experimental methods).

Figure 5. Normalized Raman spectra of CaCO3 crystals formed in the presence of the different biopolymers. On the left/ right hand side we show the carbonate (100 – 1250 cm-1)/ C-H (2750 – 3200 cm-1) - related spectra, respectively. In the presence of EPS, two typical spectra appear (a-d): Spectra of spherical crystals (corresponding to Figure 2f) are shown in (a-b) and the non spherical crystals (corresponding to Figures 2 c-e), specified as 'other' are shown in (c-d). The Raman spectra of crystals formed in the presence of TasA and TapA are shown in (e-f), (g-h), respectively.


Page 16 of 31

Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Interestingly, a broad peak at ~ 2950 cm-1 appeared in the spectrum of the crystals formed in the presence of TapA and EPS (spherical structures only, when a concentration of 1mg/ml was used, Figure 5h, supplementary Figure S4). This peak corresponds to the C-H vibration and therefore indicates the inclusion of the organic additives in or on the crystals.37 This broad ~ 2950 cm-1 peak was absent in the Raman spectra of crystals formed with TasA and EPS (10 mg/ml) (Figures 5 a-f), probably due to fluorescence of the crystals with the additives. Autofluorescence of the crystals due to the presence of the additives might also explain the tilted baseline of the spectra in Figure 5, in comparison to the baseline of the control calcite crystals (see Figure S5a). The FTIR spectra of the different samples (Figure S6) supports the Raman measurements. The crystals that formed in the presence of both the proteins TasA and TapA exhibited a typical calcite FTIR spectra (Figures S6c and S6d, respectively) and the FTIR spectrum of the crystals formed in the presence of EPS was a combination of calcite and vaterite (Figure S6b).38 X - Ray Powder Diffraction (XRD). We used X-ray powder diffraction in order to test the effect of the organic additives on the crystallographic preference for certain planes in CaCO3. The control crystals that formed without biopolymers showed a clear (104) preference (Figure 6a) in line with our experimental settings, as we explained above. The presence of the biopolymers induced the formation of structures that, in contrast to the control, lacked a preferable orientation relative to the glass surface. This is observed in the FIB - sectioned crystals in Figure S7 as well as in the XRD of the crystals grown with the biopolymers. Specifically, the XRD of the crystals grown in the presence of EPS shows additional calcite planes ((012), (110), (113), (202), (018), (116)) (PDF-01-081-2027) as well as vaterite peaks (PDF-01-072-0506), marked with a ‘V’ in Figure 6b ((002)V, (100)V, (101)V,



(102)V, (104)V). The XRD results support the micro Raman and FTIR results, showing that the presence of EPS induced the formation of a mixture of vaterite and calcite crystals. The crystals grown in the presence of TasA and TapA show similar calcite planes to those formed in the presence of EPS. In correspondence with the micro Raman and FTIR measurements, no vaterite peaks were observed in these crystals. We note that despite the clearly apparent (001) plane in the SEM images of the crystals grown in the presence of TapA (Figures 3f, 4b), the corresponding (006) peak does not appear in XRD diffractograms. This suggests oriented growth of these planes relative to the glass piece that, together with the small (006) intensity (see PDF01-081-2027), leads the (006) to be below our detection limit.

Figure 6. X ray powder diffraction of CaCO3 crystals formed in the absence of the biopolymers (a) and in the presence of EPS (b), TasA (c), and TapA (d). Additional calcite peaks to the (104) appear in the presence of the biopolymers; Additional vaterite peaks appear in the presence of EPS.


Page 18 of 31

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3 Matrix biopolymers reside in/on the CaCO3 crystals. Acknowledging the effect of the organic additives on the morphology and structure of the CaCO3 crystals, we asked whether the organic additives were incorporated on/in the CaCO3 crystals. To test the organic content of the crystals, we dissolved the different CaCO3 crystals in acetic acid and measured the absorbance of the solution at 280 nm that is proportional to the concentration of the proteins and EPS (EPS absorbs at 270 nm, data not shown). Table 1 summarizes the weight percentage of the macromolecules in the crystals, showing a large abundance of organic molecules in the crystals that were grown in the presence of TapA (~ 2%) and EPS (~ 25%). We could not measure the weight percent of TasA via absorbance measurements since it forms aggregates that settle in solution.25 The association of TapA with the crystals to a few percent level, indicates that it adsorbs at the surface of the crystals, rather than affecting their nucleation. This is supported by a previous study that showed that organic content of a few percent suggests face stabilization by macromolecules but an organic content of fractions of a percent suggest their effect on nucleation.39 Figure 4b that shows that TapA stabilizes the (001) face strengthens this suggestion.

The organic content of the EPS – induced crystals is rather high and does not

agree with lower percentages that were measured when organic additives influenced the crystals in the nucleation stage. We attribute the high organic content to EPS aggregates that settle on top of the crystals after they are formed (Figure S8). We note that, in addition to the EPS aggregates that appeared at the surface of the crystals (Figure S8), in a few experiments (Figure S8b,c) we observed fibers, that are possibly EPS, connecting the microcrystallites in vaterite crystals.



Page 20 of 31

Table 1. Weight percentage of the biopolymers in the CaCO3 crystals. EPS %

TasA %

TapA %

25 ± 5

N/A

1.8± 0.2

3.4 Internal structure of the protein – induced calcite crystals. The presence of EPS resulted in the formation of calcite (Figures 2 c-e) and vaterite (Figure 2f) crystals and the presence of the proteins resulted in the formation of calcite crystals only. While the EPS –induced CaCO3 crystal formation showed a classical vaterite morphology,40,

41

the

calcite structures that were formed in the presence of the proteins exhibited an interesting morphology. To gain more insight into the mechanism of the formation of these crystals, we prepared thin sectioned lamellae of 30-50 nm thickness using a focused ion beam (FIB). Selected Area Electron Diffraction Patterns (SAEDP) were thereafter collected from the lamellae. Figures 7a, 7b show a TEM image of a thin section of TasA- and TapA- induced calcite crystals, respectively; The thin sections of the former were taken across the shorter axis of the crystal (representative SEM images of whole crystals after the FIB sectioning are shown in Figure S7). The SAED collected from region a1 Figure 7 corresponds to single crystalline calcite, however, the SAED collected from region a2 corresponds to two twins with a (012) twin plane. Such a twin plane has been previously observed and it is one of the five known twin planes in calcite.42


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. TEM micrographs of Focused ion beam thin sections of the TasA – and TapA – induced calcite crystals (a,b, respectively) and SAED patterns (a1,a2 and b1,b2) taken in areas 1,2, in images a, b, respectively. The patterns in a1 and b1 derive from a single crystalline region; The pattern in a2 derives from the diffraction of a twin boundary area, corresponding to a (012) twin plane (two plane sets (m=matrix, t=twin) ((w)=(1,-1,-2) m and (1,1,2)t, (x)=(1,0,-1) m and (0,1,1)t, (y)=(1,1,0)m and (1,1,0)t, (z) = (0,1,1)m and (1,0,-1)t)). The pattern in b2 derives from several single crystals in the middle of the spherulite. The indexing of the SAED patterns shown in Figures a1, a2 and b1 correspond to calcite. The different zone axes are indicated in the relevant Figures.

The TapA – induced crystals grew in a spherulite form, as suggested by the TEM image of the corresponding lamella in Figure 7b, showing single crystalline cones fanning out from the center



of the spherulite (the center is marked in a circle in Figure 7b). The indexing of the SAED pattern from region 1 in Figure 7b correspond to a single crystalline calcite recorded from a [1, 2ത, 0] zone axis. However, the diffraction pattern appears as arcs when it is collected from region 2, at the center of the spherulite, showing an almost randomly distributed crystal orientation. Comparison of the thin sections of the calcite crystals grown in the presence of both proteins (Figure 7) to the SEM surface images of the corresponding crystals (Figure 3) reveals that the core of the crystals is composed of single crystalline domains that are different in morphology than the microcrystals that appear at the surface of the crystals. This suggests a two – stage growth mechanism with the core growing first and then being decorated by a shell of microcrystals on its surface. The core- shell boundary is mostly apparent in the TapA-induced crystals, shown in Figure S7c. Why do the different biopolymers induce different CaCO3 morphologies and structures? Our experimental system includes three different biopolymers, two negatively charged proteins and a neutral polysaccharide (EPS). The EPS induced the formation of various calcite crystal morphologies as well as the less thermodynamically stable vaterite structure, while the proteins induced the formation of complex structures of calcite. For comparison, previous studies have linked the formation of calcite with the extracellular matrix in biofilms of Bacillus subtilis16 and Pseudomonas aeruginosa17. Other studies have shown that purified bacterial exopolymers, induces either the formation of vaterite (e.g. in the presence of Xanthan) or the formation of calcite (e.g. in the presence of amino acids and xanthan). Interestingly, the morphology of both these structures are spherulitic.43


Page 22 of 31

Page 23 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


According to the Ostwald – Lussac law of phases, amorphous CaCO3 nuclei form and reorganize to vaterite and aragonite until they reach the most thermodynamically stable calcite.44 The presence of organic molecules may interfere with this spontaneous process in the nucleation or growth stages (or both). Two growth mechanisms can therefore account for the crystal morphology and structure that we present in this study: One possible scenario is that the biopolymers interact with Ca2+ ions, affecting CaCO3 formation in the nucleation stage. Another possible scenario is the pre-formation of microcrystalline structures that are then stabilized by the adsorption of biopolymers. Aggregation due to interaction between the biopolymers would then lead to the formation of complex structures. The vaterite crystals that were induced by the presence of EPS point at a kinetically – favored process in earlier stages of nucleation in a highly supersaturated solution. This suggests that EPS binds Ca2+ ions, possibly through the multiple hydroxyl groups in the sugars (see Figure 1 for the monosaccharide composition of the polysaccharide), as was previously reported for glucose and inositol.45-47 The proteins TasA and TapA are negatively charged under our experimental conditions. Furthermore, the percentage of negatively charged amino acids (Asp, Glu, marked red in Figure 1) in their sequences is larger than the average value found in common proteins (see Table S5) and therefore these proteins may potentially bind calcium ions and affect the nucleation of the CaCO3 crystals. However, the fact that these proteins affected the crystal morphology but not the structure of calcite suggests that that they did not affect the nucleation stage but rather, they interacted with already-formed calcite microcrystallites during their growth. Our results show



that the proteins interacted with several crystal planes (see the X-ray powder diffraction patterns in Figure 6) but there are two examples of a strong protein-crystal interaction: One example is that of TapA that stabilized the highly (negatively or positively) charged and unstable (001 plane) 48, 49 (Figure 4b). TapA is a disordered protein (data not shown) and therefore, similarly to other disordered proteins50-53 that are related with biomineralization, TapA can adopt a stable structure upon interaction with a certain crystal face thereby lowering its surface energy. This effect, combined with the high (001) surface reactivity and the relatively high organic content of the crystals, support the stabilization of the calcite microcrystallites by the adsorption of TapA. A second example for protein-crystal interaction is the interaction of the negatively – charged TasA with the positively charged (012) plane that may explain the twin formation in these crystals. Given the role of the proteins TasA and TapA in biofilm formation, where their 'natural' tendency is to interconnect the cells and aggregate into fibers,25 it is likely that after they adsorb onto microcrystalline calcite, these microcrystallites further aggregate into complex structures due to protein – protein interactions. In the case where EPS is adsorbed on such microcrystals, the association of microcrystals into complex crystals could be attributed to entanglements between these high molecular weight polymers.

4. Conclusions

We have performed an in vitro study of the effect of a bacterial extracellular polysaccharide (EPS) and proteins on calcium carbonate precipitation. We have found that the proteins TasA and TapA and the polysaccharide EPS – all affected the morphology of calcium carbonate and induced the formation of complex structures. The EPS induced the formation of calcite in 24 Environment ACS Paragon Plus

Page 24 of 31

Page 25 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different morphologies as well as the formation of vaterite. Similarly to previous observations of calcite formation in the presence of bacterial exopolymers16,17,

54

,both the proteins TasA and

TapA did not affect the thermodynamic preference for calcite but they significantly affected the crystals' morphology and induced the formation of complex structures. Our results suggest that the EPS affected the formation of calcium carbonate in the nucleation stage when it induced the formation of vaterite crystals and that the proteins stabilized pre-formed calcite nuclei and induced their aggregation into complex calcite structures.

ASSOCIATED CONTENT A Supporting Information file includes: X-ray powder diffraction of the control and FTIR spectra of the different samples. Size distribution of the crystals and microcrystals, description of the measurements and a summary of the sizes. Entire micro-Raman spectra of all the crystals including those that grew in the presence of EPS 1mg/ml. SEM images of EPS on the vaterite. FIB sections (of the crystals grown in presence of EPS 1mg/ml and the proteins). A comparison of the percentage of negatively charged amino acids of TapA and TasA with standard proteins. AUTHOR INFORMATION Corresponding Author * Liraz chai Tel. +972-2-658-53-05 Email: [email protected]



ACKNOWLEDGMENTS We thank the team of the Hebrew University Center for Nanoscince and Nanotechnology: Atzmon Vakahi for his help with Focused Ion beam thin sections, Yael Kalisman for support with electron diffraction measurements, Vladimir Uvarov for assistance in collecting X-ray powder diffraction data, Anna Radko for microRaman training, and Lia Addadi and Jonathan Erez for fruitful discussions.

REFERENCES (1) Martignier, A.; Pacton, M.; Filella, M.; Jaquet, J. M.; Barja, F.; Pollok, K.; Langenhorst, F.; Lavigne, S.; Guagliardo, P.; Kilburn, M. R.; Thomas, C.; Martini, R.; Ariztegui, D., Intracellular amorphous carbonates uncover a new biomineralization process in eukaryotes. Geobiology 2017, 15, 240-253. (2) Subburaman, K.; Pernodet, N.; Kwak, S.; DiMasi, E.; Ge, S.; Zaitsev, V.; Ba, X.; Yang, N.; Rafailovich, M., Templated biomineralization on self-assembled protein fibers. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14672-14677. (3) Weiner, S.; Dove, P. M., An overview of biomineralization processes and the problem of the vital effect. Rev. Mineral. Geochem. 2003, 54, 1-29. (4) Blakemore, R., Magnetotactic bacteria. Science 1975, 190, 377-379. (5) Dhami, N. K.; Reddy, M. S.; Mukherjee, A., Biomineralization of calcium carbonates and their engineered applications: a review. Front Microbiol 2013, 4, 314. (6) Lowenstam, H., Minerals formed by organisms. Science 1981, 211, 1126-1131. (7) Nudelman, F.; Lausch, A. J.; Sommerdijk, N. A. J. M.; Sone, E. D., In vitro models of collagen biomineralization. J. Struct. Biol. 2013, 183, 258-269. (8) Sigel, A.; Sigel, H.; Sigel, R. K., Biomineralization: from nature to application. Ed.; John Wiley & Sons: 2008; Vol. 12, pp 531-533 (9) Ehrlich, H., Chitin and collagen as universal and alternative templates in biomineralization. Int. Geol. Rev. 2010, 52, 661-699. (10) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L., Control of Aragonite or Calcite Polymorphism by Mollusk Shell Macromolecules. Science 1996, 271, 67-69. (11) Arias, J. L.; Fernández, M. a. S., Polysaccharides and proteoglycans in calcium carbonate-based biomineralization. Chem. Rev. 2008, 108, 4475-4482.


Page 26 of 31

Page 27 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(12) Lopes-Lima, M.; Ribeiro, I.; Pinto, R. A.; Machado, J., Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2005, 141, 319-326. (13) Riding, R., Calcareous algae and stromatolites. ed.; Springer Science & Business Media: 2012. (14) Riding, R., The term stromatolite: towards an essential definition. Lethaia 1999, 32, 321330. (15) Boquet, E.; Boronat, A.; Ramos-Cormenzana, A., Production of Calcite (Calcium Carbonate) Crystals by Soil Bacteria is a General Phenomenon. Nature 1973, 246, 527. (16) Oppenheimer-Shaanan, Y.; Sibony-Nevo, O.; Bloom-Ackermann, Z.; Suissa, R.; Steinberg, N.; Kartvelishvily, E.; Brumfeld, V.; Kolodkin-Gal, I., Spatio-temporal assembly of functional mineral scaffolds within microbial biofilms. npj Biofilms and Microbiomes 2016, 2, 15031. (17) Li, X.; Chopp, D. L.; Russin, W. A.; Brannon, P. T.; Parsek, M. R.; Packman, A. I., Spatial patterns of carbonate biomineralization in biofilms. Appl. Environ. Microbiol. 2015, 81, 7403-7410. (18) Decho, A. W., Overview of biopolymer-induced mineralization: What goes on in biofilms? Ecol. Eng. 2010, 36, 137-144. (19) Phillips, A. J.; Lauchnor, E.; Eldring, J.; Esposito, R.; Mitchell, A. C.; Gerlach, R.; Cunningham, A. B.; Spangler, L. H., Potential CO2 Leakage Reduction through Biofilm-Induced Calcium Carbonate Precipitation. Environ. Sci. Technol. 2013, 47, 142-149. (20) Flemming, H.-C.; Wingender, J., The biofilm matrix. Nat. Rev. Microbiol. 2010, 8, 623. (21) Vlamakis, H.; Chai, Y.; Beauregard, P.; Losick, R.; Kolter, R., Sticking together: building a biofilm the Bacillus subtilis way. Nat. Rev. Microbiol 2013, 11, 157. (22) Costerton, J. W.; Cheng, K.; Geesey, G. G.; Ladd, T. I.; Nickel, J. C.; Dasgupta, M.; Marrie, T. J., Bacterial biofilms in nature and disease. Ann. Rev. Microbiol. 1987, 41, 435-464. (23) Branda, S. S.; Vik, Å.; Friedman, L.; Kolter, R., Biofilms: the matrix revisited. Trends Microbiol. 2005, 13, 20-26. (24) Mah, T.-F. C.; O'toole, G. A., Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9, 34-39. (25) Chai, L.; Romero, D.; Kayatekin, C.; Akabayov, B.; Vlamakis, H.; Losick, R.; Kolter, R., Isolation, characterization, and aggregation of a structured bacterial matrix precursor. J. Biol. Chem. 2013, 288, 17559-17568. (26) Nwodo, U.; Green, E.; Okoh, A., Bacterial Exopolysaccharides: Functionality and Prospects. Int. J. Mol. Sci. 2012, 13, 14002. (27) Argianas, A., Characterization of Exopolysaccharide (EPS) Produced by Bacillus subtilis Mutants. M.Sc. Thesis, Loyola University Chicago, 2015. (28) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Wilkins, M. R.; Appel, R. D.; Bairoch, A., Protein identification and analysis tools on the ExPASy server. In The proteomics protocols handbook, Springer: 2005; pp 571-607. (29) The UniProt Consortium, UniProt: the universal protein knowledgebase. Nucleic acids research 2017, 45, (D1), D158-D169. (30) Townsin, R. L., The Ship Hull Fouling Penalty. Biofouling 2003, 19, 9-15. (31) Nguyen, T.; Roddick, F. A.; Fan, L., Biofouling of water treatment membranes: a review of the underlying causes, monitoring techniques and control measures. Membranes 2012, 2, 804840.



(32) Politi, Y.; Mahamid, J.; Goldberg, H.; Weiner, S.; Addadi, L., Asprich mollusk shell protein: in vitro experiments aimed at elucidating function in CaCO3 crystallization. CrystEngComm 2007, 9, 1171-1177. (33) Romero, D.; Vlamakis, H.; Losick, R.; Kolter, R., Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly. J. Bacteriol. 2014, 196, 1505-1513. (34) Romero, D.; Aguilar, C.; Losick, R.; Kolter, R., Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2230-2234. (35) Gunasekaran, S.; Anbalagan, G.; Pandi, S., Raman and infrared spectra of carbonates of calcite structure. J. Raman Spectrosc. 2006, 37, 892-899. (36) Trushina, D. B.; Bukreeva, T. V.; Kovalchuk, M. V.; Antipina, M. N., CaCO3 vaterite microparticles for biomedical and personal care applications. Mater. Sci. Eng., C 2014, 45, 644658. (37) Jiang, W.; Pacella, M. S.; Athanasiadou, D.; Nelea, V.; Vali, H.; Hazen, R. M.; Gray, J. J.; McKee, M. D., Chiral acidic amino acids induce chiral hierarchical structure in calcium carbonate. Nat. Commun. 2017, 8, 15066. (38) Dupont, L.; Portemer, F., Synthesis and study of a well crystallized CaCO3 vaterite showing a new habitus. J. Mater. Chem. 1997, 7, 797-800. (39) Xu, A. W.; Dong, W. F.; Antonietti, M.; Cölfen, H., Polymorph switching of calcium carbonate crystals by polymerÅcontrolled crystallization. Adv. Funct. Mater. 2008, 18, 13071313. (40) Andreassen, J.-P., Formation mechanism and morphology in precipitation of vaterite— nano-aggregation or crystal growth? J. Cryst. Growth 2005, 274, 256-264. (41) Rieger, J.; Frechen, T.; Cox, G.; Heckmann, W.; Schmidt, C.; Thieme, J., Precursor structures in the crystallization/precipitation processes of CaCO3 and control of particle formation by polyelectrolytes. Faraday Discuss. 2007, 136, 265-277. (42) Pokroy, B.; Kapon, M.; Marin, F.; Adir, N.; Zolotoyabko, E., Protein-induced, previously unidentified twin form of calcite. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 7337-7341. (43) Braissant, O.; Cailleau, G.; Dupraz, C.; Verrecchia, E. P., Bacterially induced mineralization of calcium carbonate in terrestrial environments: the role of exopolysaccharides and amino acids. J. Sediment. Res. 2003, 73, 485-490. (44) De Yoreo, J. J.; Vekilov, P. G., Principles of crystal nucleation and growth. Rev. Mineral. Geochem. 2003, 54, 57-93. (45) Cook, W. J.; Bugg, C. E., Calcium interactions with D-glucans: crystal structure of α, αtrehalose-calcium bromide monohydrate. Carbohydr. Res. 1973, 31, 265-275. (46) Tajmir-Riahi, H.-A., Interaction of d-glucose with alkaline-earth metal ions. Synthesis, spectroscopic, and structural characterization of Mg (II)-and Ca (II)-d-glucose adducts and the effect of metal-ion binding on anomeric configuration of the sugar. Carbohydr. Res. 1988, 183, 35-46. (47) Angyal, S., Complex formation between sugars and metal ions. Pure Appl. Chem. 1973, 35, 131-146. (48) Cölfen, H.; Antonietti, M., Mesocrystals: Inorganic Superstructures Made by Highly Parallel Crystallization and Controlled Alignment. Angew. Chem., Int. Ed. 2005, 44, 5576-5591. (49) Addadi, L.; Weiner, S., Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 4110-4114. (50) Boskey, A. L.; Villarreal-Ramirez, E., Intrinsically disordered proteins and biomineralization. Matrix Biol. 2016, 52-54, 43-59.


Page 28 of 31

Page 29 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(51) Kalmar, L.; Homola, D.; Varga, G.; Tompa, P., Structural disorder in proteins brings order to crystal growth in biomineralization. Bone 2012, 51, 528-534. (52) Delak, K.; Harcup, C.; Lakshminarayanan, R.; Sun, Z.; Fan, Y.; Moradian-Oldak, J.; Evans, J. S., The tooth enamel protein, porcine amelogenin, is an intrinsically disordered protein with an extended molecular configuration in the monomeric form. Biochemistry 2009, 48, 22722281. (53) Michenfelder, M.; Fu, G.; Lawrence, C.; Weaver, J. C.; Wustman, B. A.; Taranto, L.; Evans, J. S.; Morse, D. E., Characterization of two molluscan crystalÅmodulating biomineralization proteins and identification of putative mineral binding domains. Biopolymers 2003, 70, 522-533. (54) Ercole, C.; Cacchio, P.; Botta, A. L.; Centi, V.; Lepidi, A., Bacterially induced mineralization of calcium carbonate: the role of exopolysaccharides and capsular polysaccharides. Microsc. Microanal. 2007, 13, 42-50.



For table of contents only

Biopolymers from a Bacterial Extracellular Matrix Affect the Morphology and Structure of Calcium Carbonate Crystals David N. Azulay, Razan Abbasi, Ilanit Ben Simhon Ktorza, Sergei Remennik, Amarendar Reddy M. and Liraz Chai1*

TOC graphic

Synopsis We describe the effect of three bacterial biopolymers, a polysaccharide and two proteins (TasA and TapA) on the formation of calcium carbonate crystals. The biopolymers induce the


Page 30 of 31

Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


formation of CaCO3 complex structures that are either calcite or vaterite in structure. The crystals' morphology and structure indicate that the EPS affects the crystal's nucleation whereas the proteins induce the aggregation of pre-formed crystallites.


Biopolymers from a Bacterial Extracellular Matrix Affect the

Recommend Documents