**Crystal Growth of Inorganic and Biomediated Carbonates and Phosphates**

Antonio Sánchez-Navas, Agustín Martín-Algarra, Mónica Sánchez-Román, Concepción Jiménez-López, Fernando Nieto and Antonio Ruiz-Bustos

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52062

### **1. Introduction**

[23] Chen, K.; Zhu, J.W.; Ji, L.J.; Wu, B. Dynamic Solventing-out Crystallization Process of Erythromycin. Journal of East China University of Science and Technology (Natural

[24] Chen, K.; Zhu, J.W.; Wu, B.; Ji, L.J. The dynamic control of preparation of erythromy‐

[25] Song, Y.H.; Zhu, J.W.; Chen, K.; Wu, B. Adsorption of erythromycin on macroporous resins and thermodynamic analysis. Journal of Chemical Industry and Engineering

[26] Gmehling, J.; Onken, U. Vapor-liquid equilibrium data collection 1. Frankfurt: DE‐

[27] Gmehling, J.; Onken, U.; Arlt, W. Vapor-liquid equilibrium data collection 1a. Frank‐

cin from erythromycin lactate 2005; China Patent 1219788c.

Science Edition) 2006; 32(8) 897-901.

(China) 2006; 57(4) 715-718.

furt: DECHEMA; 1981.

CHEMA; 1977.

66 Advanced Topics on Crystal Growth

Precipitation of carbonate minerals is tightly linked to water chemistry. After hydration of dissolved carbon dioxide, two pH-dependent partitioning-reactions govern the abundance of chemical species (H2CO3, HCO3 – and CO3 2–) formed in aqueous solution:[1,2]

H2CO3↔HCO3 –+ H+↔CO3 2–+ 2H<sup>+</sup>

where the O-H covalent bond in the oxyacid makes carbonate salts moderately soluble. The most common metal cations forming carbonate minerals are Ca2+, Mg2+, Mn2+, Fe2+, Pb2+, Sr2+, Co2+, Ni2+, Zn2+, Cd2+ and Cu2+. Continental and marine waters are enriched in Ca and Mg and are known to be saturated with respect diverse Ca-Mg carbonates such as calcite (CaCO3), aragonite (CaCO3) and dolomite (MgCa(CO3)2).[3] The concentration of the phosphate species (H3PO4, H2PO4 – , HPO4 2–, and PO4 3–) is also a function of pH, and their respective oxyacids are stronger than those of carbonic acids.[2] Because of this, phosphates are more stable than carbonates at low pH (<5). Chemical composition of phosphate minerals is more variable than that of carbonate minerals, and crystalchemical substitution of the PO4 3– group by CO3 2–, OH– , F– , Cl– , etc, is rather common. In addition, numerous metals as Ca2+, Mg2+, Fe2+, Na+ , Sr2+, Ce3+, La3+, Ba2+, and Pb2+ can be incorporat‐ ed into the structure of the phosphate minerals.

Limestones and dolostones constitute the most important carbonate reservoirs on the Earth, but phosphates are much more diluted within the Earth crust, as phosphate rocks are much

© 2013 Sánchez-Navas et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

less common that carbonate rocks. Nevertheless, many organisms form CaCO3 shells, and Cacarbonates and phosphates are the main constituents of normal bone and of pathologically calcified organic tissues.[4] In many cases, the precipitation of carbonate and phosphate minerals at Earth-surface conditions is a organic matrix- biomediated process related to shell formation. Indeed, carbonate and phosphate crystals form sophisticated composite materials (*e.g.*, shells, spicules, bones, teeth), where they appear embedded within an organic framework, which apparently controls the final texture of the mineralized material. When these organisms die, Ca-carbonate shells accumulate to form limestone. Precipitation of Ca-carbonate often occurs in domestic and industrial installations where untreated natural waters are used..[5]

**2. Material and methods**

from laboratory rats.

solid gel.

**2.3. Bacterially mediated precipitation**

source for bacteria growth.

**2.2. Inorganic experiments**

**2.1. Natural Ca-phosphate samples**

Crystal growth studies of natural Ca-phosphate have been performed on ancient phosphate stromatolites and on modern calcified tissues. Stromatolites come from Upper Jurassic and Lower Cretaceous limestones of the Betic Cordilleras, Southern Spain (Almola Sierra and Peñón del Berrueco, respectively). Calcified tissues correspond to enamel, dentine and bones

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The inorganic synthesis of amorphous calcium phosphate was obtained mixing 300 ml of 0.04 M calcium salt (CaCl2.6H2O) with 0.036 M ammonium phosphate dibasic salt ((NH4)2HPO4) in 400 ml of constantly stirred 0.15 M buffer. All solutions were thermally equilibrated at 25ºC before mixing. The amorphous precursor phase was removed immediately after mixing.

Inorganic growth of carbonates in laboratory experiments was performed by using aqueous solutions of diverse metal cations (mainly Ca2+, Mg2+, Fe2+ and Pb2+) and carbon dioxide or highly soluble bicarbonate/carbonate salts as source for carbonate ions. Inorganic solution growth of carbonates was carried out by bulk crystallization (free drift experiments), where two solutions of relatively easily dissolved salts of the diverse metals and sodium bicarbonate/ carbonate were mixed: Pb(CH3COO)2.3H2O, CaCl2.6H2O, Ca(NO3)2.4H2O, MgCl2.6H2O, FeCl3.6H2O, Fe(ClO4)2.nH2O, Na2CO3 and NaHCO3 (see results for the specific concentrations used for the precipitation of the different obtained carbonate minerals). The resulting solution becomes supersaturated with respect to the less soluble metal carbonate. Temperature was usually 25ºC. Gel growth of Ca-Mg carbonates was performed by the counter-diffusion of carbonate solutions versus solutions containing diverse metal salts through a column of silica gel (stock solutions, 1 M of Na2CO3, 1 M CaCl2.6H2O, and variable concentrations of MgCl2.6H2O; see below). The silica gel with a pH 5.5 was prepared by acidification of sodium silicate solution with 1 M HCl. It was poured into a U-tube and allowed to polymerize to a

A liquid culture medium with a natural bacterial consortium mainly belonging to the genus *Acetobacter* sp., was used for biomediated precipitation of Ca, Fe and Pb phosphates and carbonates. This consortium was obtained from vinegar dregs after natural degradation of untreated wine. Aerobic *Acetobacter* grown in wine makes volatile acetic acid in the form of vinegar, and oxidizes acetic acid to carbon dioxide and water. Bacteria metabolize nitrogenated organic matter (*e.g.* proteins) with the subsequent production of CO2 and NH3. Metal cations were added to the liquid culture as soluble salts (50 mM CaCl2.6H2O, 50 mM FeCl3.6H2O and 5 mM Pb(NO3)2) to obtain phosphate and carbonate minerals. Yeast extract (1%) was the carbon

The study of the origin of life on Earth, and of the possible existence of extraterrestrial life is also tightly associated with carbonate and phosphate precipitation, as it is recorded in microbial accretions, frequently forming stromatolites and oncoids.[6-9] Ca- and Mg- carbo‐ nate and phosphate minerals similar to those found in ancient and modern stromatolites are also usually obtained in laboratory bacterial culture experiments.[10-13]. The occurrence of amazing complex textural features in relation to these minerals in the geologic record has prompted many authors to consider them as biomarkers.[14]

Different formation mechanisms have been proposed for bacterially mediated carbonate and phosphate minerals.[15] The metabolic activity of the bacteria alters the physico-chemical parameters of their surrounding habitats, allowing mineral precipitation. It has been suggested that microbes influence nucleation and that, in general, they control the kinetics of precipitation of carbonate and phosphate minerals, and therefore their morphology. Spheroidal precipitates (spherulitic bioliths) are commonly obtained in bacterial culture experiments.[13]

Some crystal-growth studies have shown that the microstructure of carbonates and phos‐ phate minerals precipitated by bacteria at high supersaturation conditions has particular signatures.[7,13,16] They nucleate as nanocrystal units after the precipitation of amor‐ phous precursors, and then, start to grow, developing spherulites and dumbbells. Singu‐ lar crystal-growth features of aragonite and calcite occur in diverse species of molluscs as result of the inhibition of growth of specific crystallographic faces by organic molecules. [17] However it is well known that the habit of the crystals formed at high supersatura‐ tion is completely different from the crystal growth features observed in precipitates ob‐ tained at lower supersaturation.[18] Hence, spheroidal morphology may results from different origin. In addition, some crystal aggregates that normally occur in shells can be easily explained by competitive crystal growth processes.[17]

In this chapter, we describe similar crystal growth features in inorganic and biogenic carbo‐ nates and phosphates formed in natural environments and in laboratory experiments. We focus on the importance of the kinetics on the crystal habit of carbonates and phosphates precipitated in biological and abiotic systems. We also discuss the influence of the nature and composition of the precipitation medium, as well as the structural control on crystal habit of Ca-Mg carbonates.

### **2. Material and methods**

less common that carbonate rocks. Nevertheless, many organisms form CaCO3 shells, and Cacarbonates and phosphates are the main constituents of normal bone and of pathologically calcified organic tissues.[4] In many cases, the precipitation of carbonate and phosphate minerals at Earth-surface conditions is a organic matrix- biomediated process related to shell formation. Indeed, carbonate and phosphate crystals form sophisticated composite materials (*e.g.*, shells, spicules, bones, teeth), where they appear embedded within an organic framework, which apparently controls the final texture of the mineralized material. When these organisms die, Ca-carbonate shells accumulate to form limestone. Precipitation of Ca-carbonate often occurs in domestic and industrial installations where untreated natural waters are used..[5]

The study of the origin of life on Earth, and of the possible existence of extraterrestrial life is also tightly associated with carbonate and phosphate precipitation, as it is recorded in microbial accretions, frequently forming stromatolites and oncoids.[6-9] Ca- and Mg- carbo‐ nate and phosphate minerals similar to those found in ancient and modern stromatolites are also usually obtained in laboratory bacterial culture experiments.[10-13]. The occurrence of amazing complex textural features in relation to these minerals in the geologic record has

Different formation mechanisms have been proposed for bacterially mediated carbonate and phosphate minerals.[15] The metabolic activity of the bacteria alters the physico-chemical parameters of their surrounding habitats, allowing mineral precipitation. It has been suggested that microbes influence nucleation and that, in general, they control the kinetics of precipitation of carbonate and phosphate minerals, and therefore their morphology. Spheroidal precipitates

Some crystal-growth studies have shown that the microstructure of carbonates and phos‐ phate minerals precipitated by bacteria at high supersaturation conditions has particular signatures.[7,13,16] They nucleate as nanocrystal units after the precipitation of amor‐ phous precursors, and then, start to grow, developing spherulites and dumbbells. Singu‐ lar crystal-growth features of aragonite and calcite occur in diverse species of molluscs as result of the inhibition of growth of specific crystallographic faces by organic molecules. [17] However it is well known that the habit of the crystals formed at high supersatura‐ tion is completely different from the crystal growth features observed in precipitates ob‐ tained at lower supersaturation.[18] Hence, spheroidal morphology may results from different origin. In addition, some crystal aggregates that normally occur in shells can be

In this chapter, we describe similar crystal growth features in inorganic and biogenic carbo‐ nates and phosphates formed in natural environments and in laboratory experiments. We focus on the importance of the kinetics on the crystal habit of carbonates and phosphates precipitated in biological and abiotic systems. We also discuss the influence of the nature and composition of the precipitation medium, as well as the structural control on crystal habit of

(spherulitic bioliths) are commonly obtained in bacterial culture experiments.[13]

prompted many authors to consider them as biomarkers.[14]

68 Advanced Topics on Crystal Growth

easily explained by competitive crystal growth processes.[17]

Ca-Mg carbonates.

### **2.1. Natural Ca-phosphate samples**

Crystal growth studies of natural Ca-phosphate have been performed on ancient phosphate stromatolites and on modern calcified tissues. Stromatolites come from Upper Jurassic and Lower Cretaceous limestones of the Betic Cordilleras, Southern Spain (Almola Sierra and Peñón del Berrueco, respectively). Calcified tissues correspond to enamel, dentine and bones from laboratory rats.

### **2.2. Inorganic experiments**

The inorganic synthesis of amorphous calcium phosphate was obtained mixing 300 ml of 0.04 M calcium salt (CaCl2.6H2O) with 0.036 M ammonium phosphate dibasic salt ((NH4)2HPO4) in 400 ml of constantly stirred 0.15 M buffer. All solutions were thermally equilibrated at 25ºC before mixing. The amorphous precursor phase was removed immediately after mixing.

Inorganic growth of carbonates in laboratory experiments was performed by using aqueous solutions of diverse metal cations (mainly Ca2+, Mg2+, Fe2+ and Pb2+) and carbon dioxide or highly soluble bicarbonate/carbonate salts as source for carbonate ions. Inorganic solution growth of carbonates was carried out by bulk crystallization (free drift experiments), where two solutions of relatively easily dissolved salts of the diverse metals and sodium bicarbonate/ carbonate were mixed: Pb(CH3COO)2.3H2O, CaCl2.6H2O, Ca(NO3)2.4H2O, MgCl2.6H2O, FeCl3.6H2O, Fe(ClO4)2.nH2O, Na2CO3 and NaHCO3 (see results for the specific concentrations used for the precipitation of the different obtained carbonate minerals). The resulting solution becomes supersaturated with respect to the less soluble metal carbonate. Temperature was usually 25ºC. Gel growth of Ca-Mg carbonates was performed by the counter-diffusion of carbonate solutions versus solutions containing diverse metal salts through a column of silica gel (stock solutions, 1 M of Na2CO3, 1 M CaCl2.6H2O, and variable concentrations of MgCl2.6H2O; see below). The silica gel with a pH 5.5 was prepared by acidification of sodium silicate solution with 1 M HCl. It was poured into a U-tube and allowed to polymerize to a solid gel.

### **2.3. Bacterially mediated precipitation**

A liquid culture medium with a natural bacterial consortium mainly belonging to the genus *Acetobacter* sp., was used for biomediated precipitation of Ca, Fe and Pb phosphates and carbonates. This consortium was obtained from vinegar dregs after natural degradation of untreated wine. Aerobic *Acetobacter* grown in wine makes volatile acetic acid in the form of vinegar, and oxidizes acetic acid to carbon dioxide and water. Bacteria metabolize nitrogenated organic matter (*e.g.* proteins) with the subsequent production of CO2 and NH3. Metal cations were added to the liquid culture as soluble salts (50 mM CaCl2.6H2O, 50 mM FeCl3.6H2O and 5 mM Pb(NO3)2) to obtain phosphate and carbonate minerals. Yeast extract (1%) was the carbon source for bacteria growth.

Concerning phosphate, bacterial phosphate precipitates of Fe and Pb were obtained, but with only minor amounts of lead phosphate, since bacterial tolerance to Pb is very low. Concerning carbonates, NH3 production by *Acetobacter* increases the basicity of the precipitating medium (thus favouring the formation of carbonate minerals) but vinager acidity obviously hinders carbonate precipitation. So the culture medium was supplemented with bicarbonate (50 mM NaHCO3), and the pH adjusted to 5.5-6 with 1 M NaOH to favour the precipitation of Cacarbonates.

and appear surrounded by a sediment matrix. These sediments are made of cryptocrystalline carbonate (micrite) formed by bioclastic particles (Figs. 2B-C). Sediment particles were trapped by microbes (heterotrophic bacteria) within the biosedimentary structure. Gelly-like appear‐ ance of arborescent morphologies and other mineralized structures, such as tubular bridges with smooth surfaces connecting phosphatic microcupolae (Figs. 2C and 2D), constitute the

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**Figure 1.** XRD patterns corresponding respectively to Pb-phosphate from biomediated laboratory experiments (A), Ca-phosphate from inorganic precipitates (B) and Ca-phosphate from phosphate stromatolites (C). Arrow in figure C

The authigenic laminae within stromatolites are defined by the occurrence of neoformed minerals containing P, Fe, Mn and Al, as deduced from X-ray maps of figure 3. Amorphous precursors of francolite, calcite, iron oxyhydroxides and clay minerals, mainly smectites are still preserved in small areas within these authigenic laminae as evidenced in TEM images (Figs. 4 and 5). The authigenic minerals within microbial laminae normally surround sediment particles such as terrigenous grains (detrital clay and quartz), micron-sized fossils (benthic and

fossil microbial mat.

corresponds to quartz.

### **2.4. Analytical techniques**

Natural mineral samples and laboratory precipitates (both biotic and inorganic) were analyzed by X-ray-diffraction (XRD) using a PANalytical X'Pert Pro diffractometer (CuKα radiation, 45kV, 40mA) equipped with an X'Celerator solid-state lineal detector and on line connection with a microcomputer. The diffraction patterns were obtained by using a continuous scan between 3-50 º2θ, 0.01 º2θ of step size and 20 s of time step. Data processing was performed using Xpowder®.[19]

Carbonates and phosphates were studied with a high-resolution Field-Emission Scanning Electron Microscope (FESEM) LEO GEMINI 1525, equipped with an energy dispersion X-ray (EDX) spectroscopy microanalysis Inca 350 version 17 Oxford Natural samples and inorganic (abiotic) and bacterial (biotic) precipitates were studied under Transmission Electron Micro‐ scopy (TEM). Representative samples of the natural material (stromatolites and calcified tissues) were ion-milled after extraction from selected areas of thin sections. The same powder samples of bacterial and inorganic precipitates used for XRD analyses were embedded in an epoxy resin, then sectioned by ultramicrotome following the methodology of Vali and Koster for clays,[20] and finally carbon coated. The samples were examined using a LIBRA 120 PLUS EFTEM (Energy Filtered TEM) instrument equipped with in-column corrected OMEGA Filter, operating at an acceleration voltage of 120 kV. Optimum amplitude contrast was achieved using lens aperture of 30 mm, and afterward removing inelastic electrons by zero-loss filtering in the elastic scattering image (ESI) mode. Electron energy loss spectroscopy (EELS) was also performed with this instrument. HRTEM and analytical electron microscopy study was performed using a Philips CM20 instrument (Philips) operated at 200 kV and equipped with an EDX system EDAX for microanalysis.

### **3. Results**

### **3.1. Crystal growth features in phosphate stromatolites and calcified tissues**

Ca-phosphate mineral occurring in stromatolites is francolite (Ca5(PO4)2.5(CO3)0.5F). Francolite is a low crystalline variety of apatite.[7,21] XRD analyses show that francolite from phosphate stromatolites is more crystalline than other phosphate minerals precipitated by us in the laboratory (Fig 1). Secondary electron images of the phosphate stromatolites show the occurrence of accretion structures with columnar and arborescent morphologies defined mainly by francolite (Fig. 2A). Phosphate laminae alternate with particulate sediment layers, and appear surrounded by a sediment matrix. These sediments are made of cryptocrystalline carbonate (micrite) formed by bioclastic particles (Figs. 2B-C). Sediment particles were trapped by microbes (heterotrophic bacteria) within the biosedimentary structure. Gelly-like appear‐ ance of arborescent morphologies and other mineralized structures, such as tubular bridges with smooth surfaces connecting phosphatic microcupolae (Figs. 2C and 2D), constitute the fossil microbial mat.

Concerning phosphate, bacterial phosphate precipitates of Fe and Pb were obtained, but with only minor amounts of lead phosphate, since bacterial tolerance to Pb is very low. Concerning carbonates, NH3 production by *Acetobacter* increases the basicity of the precipitating medium (thus favouring the formation of carbonate minerals) but vinager acidity obviously hinders carbonate precipitation. So the culture medium was supplemented with bicarbonate (50 mM NaHCO3), and the pH adjusted to 5.5-6 with 1 M NaOH to favour the precipitation of Ca-

Natural mineral samples and laboratory precipitates (both biotic and inorganic) were analyzed by X-ray-diffraction (XRD) using a PANalytical X'Pert Pro diffractometer (CuKα radiation, 45kV, 40mA) equipped with an X'Celerator solid-state lineal detector and on line connection with a microcomputer. The diffraction patterns were obtained by using a continuous scan between 3-50 º2θ, 0.01 º2θ of step size and 20 s of time step. Data processing was performed

Carbonates and phosphates were studied with a high-resolution Field-Emission Scanning Electron Microscope (FESEM) LEO GEMINI 1525, equipped with an energy dispersion X-ray (EDX) spectroscopy microanalysis Inca 350 version 17 Oxford Natural samples and inorganic (abiotic) and bacterial (biotic) precipitates were studied under Transmission Electron Micro‐ scopy (TEM). Representative samples of the natural material (stromatolites and calcified tissues) were ion-milled after extraction from selected areas of thin sections. The same powder samples of bacterial and inorganic precipitates used for XRD analyses were embedded in an epoxy resin, then sectioned by ultramicrotome following the methodology of Vali and Koster for clays,[20] and finally carbon coated. The samples were examined using a LIBRA 120 PLUS EFTEM (Energy Filtered TEM) instrument equipped with in-column corrected OMEGA Filter, operating at an acceleration voltage of 120 kV. Optimum amplitude contrast was achieved using lens aperture of 30 mm, and afterward removing inelastic electrons by zero-loss filtering in the elastic scattering image (ESI) mode. Electron energy loss spectroscopy (EELS) was also performed with this instrument. HRTEM and analytical electron microscopy study was performed using a Philips CM20 instrument (Philips) operated at 200 kV and equipped with

**3.1. Crystal growth features in phosphate stromatolites and calcified tissues**

Ca-phosphate mineral occurring in stromatolites is francolite (Ca5(PO4)2.5(CO3)0.5F). Francolite is a low crystalline variety of apatite.[7,21] XRD analyses show that francolite from phosphate stromatolites is more crystalline than other phosphate minerals precipitated by us in the laboratory (Fig 1). Secondary electron images of the phosphate stromatolites show the occurrence of accretion structures with columnar and arborescent morphologies defined mainly by francolite (Fig. 2A). Phosphate laminae alternate with particulate sediment layers,

carbonates.

**2.4. Analytical techniques**

70 Advanced Topics on Crystal Growth

using Xpowder®.[19]

an EDX system EDAX for microanalysis.

**3. Results**

**Figure 1.** XRD patterns corresponding respectively to Pb-phosphate from biomediated laboratory experiments (A), Ca-phosphate from inorganic precipitates (B) and Ca-phosphate from phosphate stromatolites (C). Arrow in figure C corresponds to quartz.

The authigenic laminae within stromatolites are defined by the occurrence of neoformed minerals containing P, Fe, Mn and Al, as deduced from X-ray maps of figure 3. Amorphous precursors of francolite, calcite, iron oxyhydroxides and clay minerals, mainly smectites are still preserved in small areas within these authigenic laminae as evidenced in TEM images (Figs. 4 and 5). The authigenic minerals within microbial laminae normally surround sediment particles such as terrigenous grains (detrital clay and quartz), micron-sized fossils (benthic and

**Figure 2.** Secondary electron images from phosphate stromatolites. (A) General view of arborescent microstromato‐ lites (etched sample). (B) Cupola formed by francolite. (C) Carbonate-rich sediment located among the phosphate cu‐ polas (arrow points to close-up shown in D). D) Gelly-like tubular bridge between two phosphate domes; it is made of poorly crystalline P-rich and Si-Al-Fe-rich substances.

comb-like aggregates of small hexagonal apatite crystals of dental enamel (Fig. 6B) are roughly equivalent to parallel aggregates of prismatic phosphate crystals observed in phosphate stromatolites (Fig. 7 in Sánchez-Navas and Martín-Algarra).[7] Hydroxyapatite crystals in dentine have lower sizes than in enamel, and appear embedded in an organic framework (Fig. 7). They are usually closely packed in subparallel aligments, with the long dimension *c* axis

**Figure 3.** X-ray images of P (A), Fe (B), Mn (C) and Al (D) defining lamination in stromatolites. P-rich laminae are made of francolite. Fe- and Mn-rich laminae correspond to Fe and Mn oxyhydroxides, respectively. Authigenic and detrital clays are represented by the Al-rich areas. Most black areas in these images correspond to carbonate sediment, which

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Precursory phases of the Ca-phosphate have been observed in the studied calcified tissues, as evidenced by TEM images corresponding to the incipient stages of mineralization in dentine, enamel and bone of young rats and embryos (Fig. 8). In the case of enamel from embryos, amorphous calcium phosphate forms rounded masses of more electron dense material within an organic matrix (Fig. 8A). Nanometre-sized acicular crystals can be differentiated from amorphous Ca-phosphate forming electron dense regions with massive texture in TEM

being attached to collagen fibrils.[23,24]

is mainly calcite.

plancktonic foraminifera and coccoliths) and bioclasts that constitute the fine-grained marine, mainly carbonate sediment (Fig. 5). Francolite crystals from these laminae are nanometre-sized prisms with hexagonal basal sections and elongated along the *c* axis. Lattice fringes at 0.8 nm are observed (Fig. 4A).

Crystal growth features described for francolite in stromatolites are very similar to those observed for Ca-phosphates from calcified tissues (enamel, dentine and bone), which mainly correspond to hydroxyapatite. Dentine from adult rats shows accretion patterns defined by alternating layers composed by hydroxyapatite crystals and collagen fibrils (Fig. 6A). Honey‐

**Figure 3.** X-ray images of P (A), Fe (B), Mn (C) and Al (D) defining lamination in stromatolites. P-rich laminae are made of francolite. Fe- and Mn-rich laminae correspond to Fe and Mn oxyhydroxides, respectively. Authigenic and detrital clays are represented by the Al-rich areas. Most black areas in these images correspond to carbonate sediment, which is mainly calcite.

comb-like aggregates of small hexagonal apatite crystals of dental enamel (Fig. 6B) are roughly equivalent to parallel aggregates of prismatic phosphate crystals observed in phosphate stromatolites (Fig. 7 in Sánchez-Navas and Martín-Algarra).[7] Hydroxyapatite crystals in dentine have lower sizes than in enamel, and appear embedded in an organic framework (Fig. 7). They are usually closely packed in subparallel aligments, with the long dimension *c* axis being attached to collagen fibrils.[23,24]

plancktonic foraminifera and coccoliths) and bioclasts that constitute the fine-grained marine, mainly carbonate sediment (Fig. 5). Francolite crystals from these laminae are nanometre-sized prisms with hexagonal basal sections and elongated along the *c* axis. Lattice fringes at 0.8 nm

**Figure 2.** Secondary electron images from phosphate stromatolites. (A) General view of arborescent microstromato‐ lites (etched sample). (B) Cupola formed by francolite. (C) Carbonate-rich sediment located among the phosphate cu‐ polas (arrow points to close-up shown in D). D) Gelly-like tubular bridge between two phosphate domes; it is made of

Crystal growth features described for francolite in stromatolites are very similar to those observed for Ca-phosphates from calcified tissues (enamel, dentine and bone), which mainly correspond to hydroxyapatite. Dentine from adult rats shows accretion patterns defined by alternating layers composed by hydroxyapatite crystals and collagen fibrils (Fig. 6A). Honey‐

are observed (Fig. 4A).

72 Advanced Topics on Crystal Growth

poorly crystalline P-rich and Si-Al-Fe-rich substances.

Precursory phases of the Ca-phosphate have been observed in the studied calcified tissues, as evidenced by TEM images corresponding to the incipient stages of mineralization in dentine, enamel and bone of young rats and embryos (Fig. 8). In the case of enamel from embryos, amorphous calcium phosphate forms rounded masses of more electron dense material within an organic matrix (Fig. 8A). Nanometre-sized acicular crystals can be differentiated from amorphous Ca-phosphate forming electron dense regions with massive texture in TEM

**Figure 4.** (A) HRTEM image of a francolite nanocrystal from an authigenic phosphate stromatolite lamina. It corre‐ sponds to a (001) hexagonal basal section. Three equivalent sets of crystallographic planes can be observed: (100), (010) and (110), intersecting at 120º, with an interplanar spacing of 0.82 nm. (B) Spindle-like packets 20-50 nm thick of smectite (Sm) with layer terminations and wavy layers from the same stromatolite lamina as A. Packets without fringe contrast (Smd) result from slight misorientation of the layers, mostly with 1 nm *d*-spacing. Fe-rich amorphous substances (arrows) are intergrown between smectite packets.

**Figure 6.** (A) TEM image of hydroxyapatite crystals surrounded by collagen fibrils in dentine from adult rat. (B) Honey‐ comb-like aggregate of small hexagonal crystals (50–200 nm in average size) in dental enamel of adult rat. Note the concave-convex boundaries among crystals. Inset corresponds to selected area diffraction pattern of the parallel ag‐

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**Figure 7.** HRTEM images of nanocrystallites of hydroxyapatite in dentine. Crystals appear oriented with their *c* axis normal (A) and parallel (B) to the plane of observation. Lattice-image of figure A corresponds to a hexagonal section of one nanocrystal, for which the three sets of equivalent lattice planes parallel to *c* axis, with interplanar spacings at 0.82 nm, are resolved simultaneously. Crystals have sizes around 10-15 nm in basal section (A) and up to 80 nm in

length following the *c* axis (B).

gregate of hydroxyapatite crystals with their *c* axis parallel aligned to the normal to outer surface.

**Figure 5.** (A) TEM image of a coccolith bioclast surrounded by iron oxyhydroxides within an authigenic phosphate stromatolite lamina (compare this image with the transmission electron micrographs of cocoliths of Figure 17 in de Vrind-de-Jong and de Vrind, 1997).[22]. This bioclast is made of five calcite crystals, each around 1μm in size. (B) Detail of iron oxyhydroxide nanocrystals surrounding the bioclast. The average diameter of these crystals is 30 nm in pseudohexagonal sections, and around 70 nm along directions perpendicular to basal section.

images; this is particularly evident in dentine (Figs. 8B and C), and in bone from one-day-old rats (Fig. 8D).

**Figure 6.** (A) TEM image of hydroxyapatite crystals surrounded by collagen fibrils in dentine from adult rat. (B) Honey‐ comb-like aggregate of small hexagonal crystals (50–200 nm in average size) in dental enamel of adult rat. Note the concave-convex boundaries among crystals. Inset corresponds to selected area diffraction pattern of the parallel ag‐ gregate of hydroxyapatite crystals with their *c* axis parallel aligned to the normal to outer surface.

**Figure 7.** HRTEM images of nanocrystallites of hydroxyapatite in dentine. Crystals appear oriented with their *c* axis normal (A) and parallel (B) to the plane of observation. Lattice-image of figure A corresponds to a hexagonal section of one nanocrystal, for which the three sets of equivalent lattice planes parallel to *c* axis, with interplanar spacings at 0.82 nm, are resolved simultaneously. Crystals have sizes around 10-15 nm in basal section (A) and up to 80 nm in length following the *c* axis (B).

images; this is particularly evident in dentine (Figs. 8B and C), and in bone from one-day-old

hexagonal sections, and around 70 nm along directions perpendicular to basal section.

**Figure 5.** (A) TEM image of a coccolith bioclast surrounded by iron oxyhydroxides within an authigenic phosphate stromatolite lamina (compare this image with the transmission electron micrographs of cocoliths of Figure 17 in de Vrind-de-Jong and de Vrind, 1997).[22]. This bioclast is made of five calcite crystals, each around 1μm in size. (B) Detail of iron oxyhydroxide nanocrystals surrounding the bioclast. The average diameter of these crystals is 30 nm in pseudo-

**Figure 4.** (A) HRTEM image of a francolite nanocrystal from an authigenic phosphate stromatolite lamina. It corre‐ sponds to a (001) hexagonal basal section. Three equivalent sets of crystallographic planes can be observed: (100), (010) and (110), intersecting at 120º, with an interplanar spacing of 0.82 nm. (B) Spindle-like packets 20-50 nm thick of smectite (Sm) with layer terminations and wavy layers from the same stromatolite lamina as A. Packets without fringe contrast (Smd) result from slight misorientation of the layers, mostly with 1 nm *d*-spacing. Fe-rich amorphous

substances (arrows) are intergrown between smectite packets.

74 Advanced Topics on Crystal Growth

rats (Fig. 8D).

lattice fringes at 0.8 nm corresponding to *d*-spacing of (100) crystallographic planes of apatite are observed (Fig. 9C). There exists a marked resemblance between these TEM images and those previously shown for calcified tissues in one-day-old rats (Figs. 8B-D). Thus, needleshaped Ca-phosphate nanocrystals surrounded by amorphous calcium phosphate occur both

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**Figure 9.** (A) FESEM image of inorganically precipitated phosphate sample showing structureless "clouds" of amor‐ phous calcium phosphate including acicular crystals. (B) ESI-mode TEM image of the inorganically precipitated phos‐ phate sample, where nano-sized needle crystals of apatite are included within amorphous calcium phosphate. (C) Lattice-fringe image of needle apatite crystallite with lattice fringes at 0.82 nm corresponding to the *d*-spacing of

(100) or equivalent crystallographic planes.

in the non-mature bone (Fig. 8D) and in the inorganic precipitates (Fig. 9C).

**Figure 8.** TEM images showing incipient mineralization of hard tissues. (A) Amorphous calcium phosphate forming electron dense masses within a lower electron dense organic matrix in enamel from rat embryo. (B) Electron dense regions formed by amorphous calcium phosphate in dentine from one-day-old rat. (C) Magnified TEM image showing thin nano-sized crystals within the mineralized regions shown in (B). (D) TEM image (ESI mode) needle-shaped crystals in bone from one-day-old rat.

#### **3.2. Inorganic phosphate and carbonate minerals obtained by solution and gel growth**

Poorly crystalline Ca-phosphates have been determined after XRD study of inorganic precip‐ itates (Fig. 1B). In the secondary electron images they appear as clumps with a foamy porous structure, where nanometre-sized acicules are visible in the most crystalline areas (Fig. 9A). TEM images confirm the existence of needle-shaped crystallites, which normally appear surrounded by structureless films and "clouds" of amorphous calcium phosphate (Fig. 9B). The acicular crystals are up to 30 nm in length and up to 4 nm in thickness. In some cases, lattice fringes at 0.8 nm corresponding to *d*-spacing of (100) crystallographic planes of apatite are observed (Fig. 9C). There exists a marked resemblance between these TEM images and those previously shown for calcified tissues in one-day-old rats (Figs. 8B-D). Thus, needleshaped Ca-phosphate nanocrystals surrounded by amorphous calcium phosphate occur both in the non-mature bone (Fig. 8D) and in the inorganic precipitates (Fig. 9C).

**Figure 8.** TEM images showing incipient mineralization of hard tissues. (A) Amorphous calcium phosphate forming electron dense masses within a lower electron dense organic matrix in enamel from rat embryo. (B) Electron dense regions formed by amorphous calcium phosphate in dentine from one-day-old rat. (C) Magnified TEM image showing thin nano-sized crystals within the mineralized regions shown in (B). (D) TEM image (ESI mode) needle-shaped crystals

**3.2. Inorganic phosphate and carbonate minerals obtained by solution and gel growth**

Poorly crystalline Ca-phosphates have been determined after XRD study of inorganic precip‐ itates (Fig. 1B). In the secondary electron images they appear as clumps with a foamy porous structure, where nanometre-sized acicules are visible in the most crystalline areas (Fig. 9A). TEM images confirm the existence of needle-shaped crystallites, which normally appear surrounded by structureless films and "clouds" of amorphous calcium phosphate (Fig. 9B). The acicular crystals are up to 30 nm in length and up to 4 nm in thickness. In some cases,

in bone from one-day-old rat.

76 Advanced Topics on Crystal Growth

**Figure 9.** (A) FESEM image of inorganically precipitated phosphate sample showing structureless "clouds" of amor‐ phous calcium phosphate including acicular crystals. (B) ESI-mode TEM image of the inorganically precipitated phos‐ phate sample, where nano-sized needle crystals of apatite are included within amorphous calcium phosphate. (C) Lattice-fringe image of needle apatite crystallite with lattice fringes at 0.82 nm corresponding to the *d*-spacing of (100) or equivalent crystallographic planes.

Rhombohedral micron-sized siderite (FeCO3) crystals and siderite spherulites have been precipitated in oxygen-free water inside an anaerobic chamber from solutions with salt concentrations 50 mM NaHCO3–50 mM Fe(ClO4)2.nH2O and 1 M NaHCO3–1 M Fe(ClO4)2.nH2O, respectively (Fig. 10). Siderite spherulites normally show rough surfaces formed by uniaxial aggregates of skeletal crystals with nanometre size (Figs. 10C and D). Both siderite crystals and spherulites appear surrounded by a poorly-crystalline precursory material formed by iron oxyhydroxides (Figs. 10E and 10F).

Crystallization of Ca-Mg rhombohedral carbonates were conducted through gel-growth experiments to favour the incorporation Mg to the calcite structure. The effect of Mg con‐ tent on the structural parameters and on the crystal habit of calcite was analyzed. Thus, calcite rhombohedrons with dendritic morphologies were formed in Mg-free crystalliza‐ tion experiments (Fig. 11A). Calcite crystals with rough surfaces and crystal morpholo‐ gies corresponding probably with {021} forms (Fig. 11B) and calcite spherulites (Fig. 11C) grew from 0.1 M and 1 M MgCl2.6H2O stock solutions, respectively. The increase of the Mg-content also produced a lattice contraction and a decrease of the crystallinity of the Ca-Mg carbonate, as well as the appearance of metastable phases (monohydrocalcite) as deduced from XRD analyses (Fig. 11D).

Spherulites composed by low-crystalline Mg-rich Ca-Mg carbonates were also studied by SEM and TEM. Secondary images of these spherulites indicate that they grew by adhesion of nanometre-sized particles (Fig. 12A). In addition, TEM study of these samples reveals the occurrence of carbonate nanocrystal building units, with sizes from 40 to 80 nanometres in diameter, which aggregate to form small clusters with approximately the same crystallo‐ graphic nanocrystals orientation (Fig. 12B).

### **3.3. Crystal growth features of bacterially-mediated phosphates and carbonates**

Bacterially-mediated phosphate and carbonate precipitates show analogous crystal growth features to those of some inorganic (abiotic) precipitates (compare Figs. 10B, 11A and 12A with Figs. 13 and 14). Poorly crystalline Fe- and Pb-phosphates occur in relation to bacterial cells, organelles and biofilms during the initial stages of biomineralization (Figs. 13A and 13B).

Fe-phosphate spherulites and capsules with diverse sizes and textural features were formed after several weeks (Figs. 13C-13E). Voids and channels with widths of 200 nanometres and 1 micron in length are normally observed in SEM images of Fe-phosphate spherulites (Figs. 13C and 13D). Fe-phosphate capsules develop a central hollow (Fig. 13E).

Other bacterially-mediated precipitates are constituted by poorly crystalline Pb-phosphates (Fig. 13F). Their low crystallinity is evidenced by the broad peaks observed in the XRD patterns (Fig. 1A). Secondary electron images obtained from of *Acetobacter* Pb cultures show two different types of precipitates (Fig. 13F). Those of largest dimension correspond to Pbphosphate spherulites. The smallest precipitates are constituted by irregular clumps of spheroidal Pb carbonates (hydrocerussite) particles around 0.1-0.3 μm in diameter, formed by aggregation of nanoparticles.

**Figure 10.** (A) Secondary electron image of siderite rhombohedron surrounded by less crystalline precipitates ob‐ tained from solution growth at the following salt concentration: 50 mM NaHCO3, and 50 mM Fe(ClO4)2.nH2O. (B) Side‐ rite spherulites partially covered with a thin poorly crystalline Fe-rich layer obtained from solution growth at the following salt concentration: 1 M NaHCO3, and 1 M Fe(ClO4)2.nH2O). (C) Uniaxial aggregate of crystals at the rough surface of a siderite spherulite. (D) Close-up of (C) showing the triangular tips of the tripod-like dendritic crystals of siderite. (E) TEM image of rhombohedral siderite crystals surrounded by small needle-like iron oxyhydroxydes. (F) TEM

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view of a siderite spherulite within a poorly crystalline Fe-rich matrix.

Rhombohedral micron-sized siderite (FeCO3) crystals and siderite spherulites have been precipitated in oxygen-free water inside an anaerobic chamber from solutions with salt concentrations 50 mM NaHCO3–50 mM Fe(ClO4)2.nH2O and 1 M NaHCO3–1 M Fe(ClO4)2.nH2O, respectively (Fig. 10). Siderite spherulites normally show rough surfaces formed by uniaxial aggregates of skeletal crystals with nanometre size (Figs. 10C and D). Both siderite crystals and spherulites appear surrounded by a poorly-crystalline precursory

Crystallization of Ca-Mg rhombohedral carbonates were conducted through gel-growth experiments to favour the incorporation Mg to the calcite structure. The effect of Mg con‐ tent on the structural parameters and on the crystal habit of calcite was analyzed. Thus, calcite rhombohedrons with dendritic morphologies were formed in Mg-free crystalliza‐ tion experiments (Fig. 11A). Calcite crystals with rough surfaces and crystal morpholo‐ gies corresponding probably with {021} forms (Fig. 11B) and calcite spherulites (Fig. 11C) grew from 0.1 M and 1 M MgCl2.6H2O stock solutions, respectively. The increase of the Mg-content also produced a lattice contraction and a decrease of the crystallinity of the Ca-Mg carbonate, as well as the appearance of metastable phases (monohydrocalcite) as

Spherulites composed by low-crystalline Mg-rich Ca-Mg carbonates were also studied by SEM and TEM. Secondary images of these spherulites indicate that they grew by adhesion of nanometre-sized particles (Fig. 12A). In addition, TEM study of these samples reveals the occurrence of carbonate nanocrystal building units, with sizes from 40 to 80 nanometres in diameter, which aggregate to form small clusters with approximately the same crystallo‐

Bacterially-mediated phosphate and carbonate precipitates show analogous crystal growth features to those of some inorganic (abiotic) precipitates (compare Figs. 10B, 11A and 12A with Figs. 13 and 14). Poorly crystalline Fe- and Pb-phosphates occur in relation to bacterial cells, organelles and biofilms during the initial stages of biomineralization (Figs. 13A and 13B).

Fe-phosphate spherulites and capsules with diverse sizes and textural features were formed after several weeks (Figs. 13C-13E). Voids and channels with widths of 200 nanometres and 1 micron in length are normally observed in SEM images of Fe-phosphate spherulites (Figs. 13C

Other bacterially-mediated precipitates are constituted by poorly crystalline Pb-phosphates (Fig. 13F). Their low crystallinity is evidenced by the broad peaks observed in the XRD patterns (Fig. 1A). Secondary electron images obtained from of *Acetobacter* Pb cultures show two different types of precipitates (Fig. 13F). Those of largest dimension correspond to Pbphosphate spherulites. The smallest precipitates are constituted by irregular clumps of spheroidal Pb carbonates (hydrocerussite) particles around 0.1-0.3 μm in diameter, formed by

**3.3. Crystal growth features of bacterially-mediated phosphates and carbonates**

and 13D). Fe-phosphate capsules develop a central hollow (Fig. 13E).

material formed by iron oxyhydroxides (Figs. 10E and 10F).

deduced from XRD analyses (Fig. 11D).

78 Advanced Topics on Crystal Growth

graphic nanocrystals orientation (Fig. 12B).

aggregation of nanoparticles.

**Figure 10.** (A) Secondary electron image of siderite rhombohedron surrounded by less crystalline precipitates ob‐ tained from solution growth at the following salt concentration: 50 mM NaHCO3, and 50 mM Fe(ClO4)2.nH2O. (B) Side‐ rite spherulites partially covered with a thin poorly crystalline Fe-rich layer obtained from solution growth at the following salt concentration: 1 M NaHCO3, and 1 M Fe(ClO4)2.nH2O). (C) Uniaxial aggregate of crystals at the rough surface of a siderite spherulite. (D) Close-up of (C) showing the triangular tips of the tripod-like dendritic crystals of siderite. (E) TEM image of rhombohedral siderite crystals surrounded by small needle-like iron oxyhydroxydes. (F) TEM view of a siderite spherulite within a poorly crystalline Fe-rich matrix.

**Figure 11.** (A) to (C): Optical images and SEM views (insets) of the Ca-Mg carbonates obtained from gel growth inor‐ ganic precipitation experiments. (D) XRD features of their (104) peaks. (A) Dendritic calcite crystals precipitated in Mgfree medium. (B) Crystal morphologies of magnesian calcites precipitated from 0.1 M MgCl2.6H2O stock solution. (C) Spherulites formed in Mg-richest medium (1 M MgCl2.6H2O stock solution). (D) XRD patterns showing the displace‐ ment and broadening of the (104) peak with increasing Mg content of the Ca-Mg carbonate from calcite (A) to Mgcalcite (B) and to Mg-kutnahorite (C). Monohydrocalcite (Mo) is also formed together with the Mg-kutnahorite.

**Figure 13.** SEM and TEM images of bacterially-mediated phosphate precipitates obtained from precipitation experi‐ ments with vinegar dregs. (A) Secondary electron image corresponding to early stages of mineralization of the vinegar dreg. (B) TEM image of bacteria surrounded by iron phosphate nanoparticles, formed at the initial stages of minerali‐ zation. (C) SEM image of a Fe-phosphate spherulite with voids of 200 nm of size. (D) View of a sectioned spherulite showing a radial growth pattern and a porous texture. (E) Secondary electron image of Fe-phosphate capsules with smooth surfaces. (F) Pb-phosphate spherulites of very different sizes surrounded by irregular clumps of smaller Pb-car‐

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Bacterially-mediated precipitation experiments with CaCl2.6H2O (50 mM), and supplemented with bicarbonate (50 mM NaHCO3), resulted in the formation of calcite spherulites and sheaflike crystals (Fig. 14A). The latter are elongated following the *c* axis of calcite crystals. They are

bonate particles.

**Figure 12.** (A) SEM image and EDX spectra of Mg-kutnahorite spherulites obtained from gel growth inorganic precipita‐ tion experiments. They are formed by aggregation of rounded growth units, less than 100 nm in size. (B) TEM image (ESI mode) of Ca-Mg carbonate nanoparticles forming Mg-kutnahorites. The two main C K edge peaks in the EELS spectrum of the nanoparticles correspond to π\* and σ\* antibonding molecular orbitals of CO<sup>3</sup> 2– cluster (peaks at 290.2 and 301.3 eV re‐ spectively [25]). Less intense C K edge peaks from amorphous carbon coating appears also superposed.

**Figure 11.** (A) to (C): Optical images and SEM views (insets) of the Ca-Mg carbonates obtained from gel growth inor‐ ganic precipitation experiments. (D) XRD features of their (104) peaks. (A) Dendritic calcite crystals precipitated in Mgfree medium. (B) Crystal morphologies of magnesian calcites precipitated from 0.1 M MgCl2.6H2O stock solution. (C) Spherulites formed in Mg-richest medium (1 M MgCl2.6H2O stock solution). (D) XRD patterns showing the displace‐ ment and broadening of the (104) peak with increasing Mg content of the Ca-Mg carbonate from calcite (A) to Mgcalcite (B) and to Mg-kutnahorite (C). Monohydrocalcite (Mo) is also formed together with the Mg-kutnahorite.

**Figure 12.** (A) SEM image and EDX spectra of Mg-kutnahorite spherulites obtained from gel growth inorganic precipita‐ tion experiments. They are formed by aggregation of rounded growth units, less than 100 nm in size. (B) TEM image (ESI mode) of Ca-Mg carbonate nanoparticles forming Mg-kutnahorites. The two main C K edge peaks in the EELS spectrum of

2– cluster (peaks at 290.2 and 301.3 eV re‐

the nanoparticles correspond to π\* and σ\* antibonding molecular orbitals of CO<sup>3</sup>

80 Advanced Topics on Crystal Growth

spectively [25]). Less intense C K edge peaks from amorphous carbon coating appears also superposed.

**Figure 13.** SEM and TEM images of bacterially-mediated phosphate precipitates obtained from precipitation experi‐ ments with vinegar dregs. (A) Secondary electron image corresponding to early stages of mineralization of the vinegar dreg. (B) TEM image of bacteria surrounded by iron phosphate nanoparticles, formed at the initial stages of minerali‐ zation. (C) SEM image of a Fe-phosphate spherulite with voids of 200 nm of size. (D) View of a sectioned spherulite showing a radial growth pattern and a porous texture. (E) Secondary electron image of Fe-phosphate capsules with smooth surfaces. (F) Pb-phosphate spherulites of very different sizes surrounded by irregular clumps of smaller Pb-car‐ bonate particles.

Bacterially-mediated precipitation experiments with CaCl2.6H2O (50 mM), and supplemented with bicarbonate (50 mM NaHCO3), resulted in the formation of calcite spherulites and sheaflike crystals (Fig. 14A). The latter are elongated following the *c* axis of calcite crystals. They are formed by aggregation of rounded nanoparticles, with sizes smaller than 50 nanometres, at their tips (Fig. 14B).

been shown that gelly-like media favour the nucleation and the formation of clusters of nanocrystals within hydrogel networks.[27] High supersaturation occurring in gel media leads to the formation of assemblies of nanometer-sized crystalline particles that actually constitute the building blocks of larger crystalline aggregates. These textural features are less frequently

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Spherulitic morphologies typically form at extremely high growth rates, and result from highly nonequilibrium processes operating both in solution and gel growth.[29] Siderite spherulites have been produced under high supersaturation in solution growth for some inorganic precipitation experiments (Figs. 10B, 10C, 10D and 10 F). In bacterially-mediated precipitation, spherulites of many carbonate and phosphate mineral can be precipitated (Figs. 13 and 14). In this case, the fast attachment of building blocks to the surface of the spherulites frequently preserves bacterial moulds and capsules, and voids are also very common (Figs. 13C, 13D, and 13E). Either obtained in biotic or abiotic experiments, all these spherulites and related mor‐ phologies are characterized by a radial texture of nanocrystal aggregates resulting from a geometrical selection process. In this way, fastest crystal-growth directions are perpendicular to the growing surface (Figs. 6, 10C, 10D, and 14). Honeycomb-like aggregates of small hexagonal apatite crystals of dental enamel (Fig. 6B) and equivalent parallel aggregates of prismatic phosphate crystals observed in phosphate stromatolites also result from competitive

The obtained carbonate and phosphate nanocrystals constitute a finely divided crystalline matter composed by particles that attach each others (Figs 4, 7, 8C, 8D, 9B, 9C and 12B). However, the aggregation of these nanoparticles is not related to atom-by-atom attachment, as they are still very large when compared to the atomic size scale. In nanometre-size crystals, the density of electronic energy levels, related to the energy of valence electrons responsible of the chemical bonds, varies smoothly between the atomic and bulk limits.[30] In very small nanocrystals the energy absorption spectra develop discrete features, similar to van der Waals or molecular crystals, where the bands in the solid are very narrow. Therefore, the interactions that allow the attachment of nanocrystals during the aggregation-based growth-process

producing some of the crystal growth features here described must be very weak.

As shown in this work, and also described elsewhere, [7,31] apatite crystals of the authigenic laminae in phosphate stromatolites are usually surrounded by poorly crystalline smectites and Fe-Si-Al amorphous oxyhydroxides (Figs. 3, 4 and 5). These substances represent the fossil record of a mineralized extracellular mucilaginous bacterial gel that played the same role as bacterial organic matter in biotic laboratory experiments, and an equivalent role to that played by organic framework rich in collagen fibrils of dentine and bone. However, crystal growth features observed in authigenic phosphates (francolite) and smectitic clays of stromatolite laminae are not exclusively explained by the occurrence of gelly-like organic substances in a precursory microbial mat. In addition, the clay-rich material and related amorphous substan‐ ces in phosphate stromatolites play an analogous role to that of gels in inorganic and biome‐ diated experiments. In such media, bulk flow of a grain boundary fluid containing dissolved solutes does not occur. Within clay-rich media, fluid film is restricted to a monolayer of adsorbed molecules onto the clay particles. This suppresses convective and advective mass

formed in crystal-growth from diluted solutions.[28]

crystal growth processes.

**Figure 14.** (A) Bacterially-mediated, shrub-like Ca-carbonate precipitate. (B) Close-up of (A), showing the parallel ag‐ gregate of calcite crystals elongated along the *c* axis. They grow by aggregation of rounded nanoparticles usually less than 50 nm in size.

### **4. Discussion**

### **4.1. Kinetics constraints on crystal growth of carbonate and phosphate minerals**

Metal phosphates and carbonates occurring in the studied natural samples and in the precipitates obtained from biotic and abiotic experiments develop complex crystal mor‐ phologies: from single crystals to crystalline aggregates forming dumbbells and spheru‐ lites. In some inorganic experiments, several tens of microns-sized polyhedral forms have been obtained directly (Fig. 11A). However, crystal growth features observed in most samples indicate that phosphates and carbonates here studied can be considered "meso‐ crystals".[26] These are superstructures formed by the aggregation of nanocrystal build‐ ing units (Figs. 10E, 12 and 14) that are frequently associated with amorphous precursors (Figs. 1, 4, 5, 8, 9, 10E and 10F). A comparison between the morphology of abiotic and biotic precipitates may help to understand nucleation and crystal growth of new organicinorganic hybrid materials that are crystallized by oriented aggregation of nanoparticles instead of by ion-by-ion or single molecule attachment. Actually, the occurrence of nano‐ crystalline particles and aggregation based morphologies within an organic matrix seems be key features of biomineralization.[13, 27]

Precipitation of calcium phosphate and carbonate nanoparticles on diverse substrates in relation to gelly-like substances may be the dominant mechanism of phosphate and carbonate formation in some geologic, microbially-mediated biosedimentary structures (Figs. 2-5)[7, 31] and in some calcified tissues (Figs. 6-8).[23, 24] In phosphate stromatolites, the authigenic clay and phosphate laminae within stromatolites are related to the formation of an extracellular, mucilaginous, bacterial gel, rich in polysaccharides at the living microbial mat.[7, 31] It has been shown that gelly-like media favour the nucleation and the formation of clusters of nanocrystals within hydrogel networks.[27] High supersaturation occurring in gel media leads to the formation of assemblies of nanometer-sized crystalline particles that actually constitute the building blocks of larger crystalline aggregates. These textural features are less frequently formed in crystal-growth from diluted solutions.[28]

formed by aggregation of rounded nanoparticles, with sizes smaller than 50 nanometres, at

**Figure 14.** (A) Bacterially-mediated, shrub-like Ca-carbonate precipitate. (B) Close-up of (A), showing the parallel ag‐ gregate of calcite crystals elongated along the *c* axis. They grow by aggregation of rounded nanoparticles usually less

Metal phosphates and carbonates occurring in the studied natural samples and in the precipitates obtained from biotic and abiotic experiments develop complex crystal mor‐ phologies: from single crystals to crystalline aggregates forming dumbbells and spheru‐ lites. In some inorganic experiments, several tens of microns-sized polyhedral forms have been obtained directly (Fig. 11A). However, crystal growth features observed in most samples indicate that phosphates and carbonates here studied can be considered "meso‐ crystals".[26] These are superstructures formed by the aggregation of nanocrystal build‐ ing units (Figs. 10E, 12 and 14) that are frequently associated with amorphous precursors (Figs. 1, 4, 5, 8, 9, 10E and 10F). A comparison between the morphology of abiotic and biotic precipitates may help to understand nucleation and crystal growth of new organicinorganic hybrid materials that are crystallized by oriented aggregation of nanoparticles instead of by ion-by-ion or single molecule attachment. Actually, the occurrence of nano‐ crystalline particles and aggregation based morphologies within an organic matrix seems

Precipitation of calcium phosphate and carbonate nanoparticles on diverse substrates in relation to gelly-like substances may be the dominant mechanism of phosphate and carbonate formation in some geologic, microbially-mediated biosedimentary structures (Figs. 2-5)[7, 31] and in some calcified tissues (Figs. 6-8).[23, 24] In phosphate stromatolites, the authigenic clay and phosphate laminae within stromatolites are related to the formation of an extracellular, mucilaginous, bacterial gel, rich in polysaccharides at the living microbial mat.[7, 31] It has

**4.1. Kinetics constraints on crystal growth of carbonate and phosphate minerals**

their tips (Fig. 14B).

82 Advanced Topics on Crystal Growth

than 50 nm in size.

**4. Discussion**

be key features of biomineralization.[13, 27]

Spherulitic morphologies typically form at extremely high growth rates, and result from highly nonequilibrium processes operating both in solution and gel growth.[29] Siderite spherulites have been produced under high supersaturation in solution growth for some inorganic precipitation experiments (Figs. 10B, 10C, 10D and 10 F). In bacterially-mediated precipitation, spherulites of many carbonate and phosphate mineral can be precipitated (Figs. 13 and 14). In this case, the fast attachment of building blocks to the surface of the spherulites frequently preserves bacterial moulds and capsules, and voids are also very common (Figs. 13C, 13D, and 13E). Either obtained in biotic or abiotic experiments, all these spherulites and related mor‐ phologies are characterized by a radial texture of nanocrystal aggregates resulting from a geometrical selection process. In this way, fastest crystal-growth directions are perpendicular to the growing surface (Figs. 6, 10C, 10D, and 14). Honeycomb-like aggregates of small hexagonal apatite crystals of dental enamel (Fig. 6B) and equivalent parallel aggregates of prismatic phosphate crystals observed in phosphate stromatolites also result from competitive crystal growth processes.

The obtained carbonate and phosphate nanocrystals constitute a finely divided crystalline matter composed by particles that attach each others (Figs 4, 7, 8C, 8D, 9B, 9C and 12B). However, the aggregation of these nanoparticles is not related to atom-by-atom attachment, as they are still very large when compared to the atomic size scale. In nanometre-size crystals, the density of electronic energy levels, related to the energy of valence electrons responsible of the chemical bonds, varies smoothly between the atomic and bulk limits.[30] In very small nanocrystals the energy absorption spectra develop discrete features, similar to van der Waals or molecular crystals, where the bands in the solid are very narrow. Therefore, the interactions that allow the attachment of nanocrystals during the aggregation-based growth-process producing some of the crystal growth features here described must be very weak.

As shown in this work, and also described elsewhere, [7,31] apatite crystals of the authigenic laminae in phosphate stromatolites are usually surrounded by poorly crystalline smectites and Fe-Si-Al amorphous oxyhydroxides (Figs. 3, 4 and 5). These substances represent the fossil record of a mineralized extracellular mucilaginous bacterial gel that played the same role as bacterial organic matter in biotic laboratory experiments, and an equivalent role to that played by organic framework rich in collagen fibrils of dentine and bone. However, crystal growth features observed in authigenic phosphates (francolite) and smectitic clays of stromatolite laminae are not exclusively explained by the occurrence of gelly-like organic substances in a precursory microbial mat. In addition, the clay-rich material and related amorphous substan‐ ces in phosphate stromatolites play an analogous role to that of gels in inorganic and biome‐ diated experiments. In such media, bulk flow of a grain boundary fluid containing dissolved solutes does not occur. Within clay-rich media, fluid film is restricted to a monolayer of adsorbed molecules onto the clay particles. This suppresses convective and advective mass transport and favours diffusion and the development of spherulitic instead of polyhedral morphologies (see next section). Therefore clayey material acts as a gel, and its occurrence favours the formation of amorphous precursors and nanocrystals of later precipitates. This is the case of francolite crystallization in phosphate stromatolites, which is preceded by the precipitation amorphous calcium phosphate, as suggested by Sánchez-Navas and Martín-Algarra.[7]

tion carries out nonbonded repulsion between CO3

laboratory experiments are described in this work.

helped with laboratory precipitation experiments.

observed in dentine and enamel as well as in phosphate stromatolites.

within CO3

long-range forces between CO3

based growth-process.

**5. Conclusions**

the structure of calcite.

**Acknowledgements**

rhomboedral carbonates. The more the distance between CO3

the incorporation of the small Mg cation into the structure of calcite, the more repulsion increases. The increase of nonbonded repulsion makes valence electrons feel less potential wells.[34,35] In the case of carbonates, electrons feel less the potential of the oxygen nuclei

polarizability. The improvement of the so-called polarization, correlation, Van der Waals or

Crystal growth features in biogenic and abiotic carbonates and phosphates occurring in natural environments (phosphate stromatolites and calcified tissues -bone and teeth) and formed in

Phosphate and carbonate minerals precipitated from abiotic and biotic experiments form commonly spherulites and related morphologies. In most cases they can be considered mesocrystals because, independent of their biotic or abiotic origin, they are superstructures formed by the aggregation of nanocrystal building units and appear associated with amor‐ phous precursors. Parallel aggregates constituted by Ca-phosphate crystals are formed by competitive crystal growth processes and, together with amorphous Ca-phosphates, are

Gels in laboratory experiments, organic matrix in calcified tissues and equivalent clayrich media in phosphate stromatolites favour diffusive transport and supersaturation of carbonate and phosphate minerals. Supersaturation is responsible for the development of spherulitic morphologies and the occurrence of nanometre-sized crystals and related amorphous substances. Gels also explain the incorporation of some elements as Mg to

We acknowledge support from grants CGL-2009-09249 (DGICYT, Spain) and P11- RNM-7067 of the Junta de Andalucía (Spain), and by the research group RNM-179 (Junta deAndalucía). We would like to thank María José Martínez Guerrero, Alicia González Se‐ gura, María del Mar Abad Ortega, Concepción Hernández, Isabel Guerra Tschuske and Juan de Dios Bueno (Centro de Instrumentación Científica-CIC, University of Granada) for guidance with SEM-EDX and TEM-AEM-EELS studies. Agustín Martín Rodríguez, José Suarez Valera, María Navas Moreno and Elena Beatriz Sánchez Martín significantly

2– anions. The decrease of electron binding energy in O atoms increases oxygen

2– enhances nanocrystals attachment during the aggregation-

Crystal Growth of Inorganic and Biomediated Carbonates and Phosphates

2– anions within the structure of the

2– anions decreases because of

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### **4.2. Considerations related to gel growth and structural control of habit in Mg-Ca carbonates**

Gels and related substances are responsible for high supersaturation producing the crystal growth features observed in the solid porous media. High supersaturation gradients arise from the slow transport of the chemical species present in gel growth experiments, and the degree of supersaturation driving the crystallization depends on the diffusion gradient in solid porous media.[28] Hence, high growth rates are expected in porous media where the critical super‐ saturation for phosphate and carbonate minerals is reached more quickly than in solution growth.

Gel growth also explains why some elements like Mg are incorporated to the structure of calcite. A correlation between Mg-content in calcite and crystallinity may be deduced from the figure 11. Slow diffusive transport media, such as a clay-rich and water deficient environments or porous media, make the transport of reacting material from solution to those of growth be the rate-determining process. Transport properties in porous media controls on the incorpo‐ ration of elements during the crystal growth.[32] To understand it, we may suppose that the partition coefficient between the concentration of magnesium in carbonate crystal and its concentration in bulk medium (*Ccrystal Mg* / *Cmedium Mg* ) is less than 1. When carbonate crystal growth is diffusion controlled, the concentration of Mg at the crystal-medium interface rises above its concentration in the medium. If the partition coefficient remains constant, the Mg concentra‐ tion in the carbonate increases due to the slower diffusion rate and, in the limit, it is equal to the concentration of Mg in the medium. It makes that the effective partition coefficient *Ccrystal Mg* / *Cmedium Mg* equals 1 and, therefore, most of the Mg of the medium is incorporated into the structure of the rhombohedral carbonate.

Crystal chemical substitution of Mg for Ca within the structure of rhombohedral carbonates decreases crystallinity, stabilizes nanocrystals and favours an aggregation-based crystal growth. The different size of the cations involved (Ca2+ and Mg2+) distorts the calcite crystal lattice. Of these two cations the Mg2+ ion has the smallest ionic radius. The lattice contraction due to the substitution of Mg for Ca is evidenced by the displacement of the (104) peak in XRD patterns (Fig. 11). The *d*(104)-spacing and lattice constants of Mg-calcite are shorter than those of calcite, being the *c* axis the most affected by the incorporation of Mg.[33] This lattice contraction causes the CO3 2– anions layers to be pulled closer together along the *c*-axis and produces a lattice deformation around Mg atoms. The incorporation of Mg into the crystal structure increases drastically the volume strain energy. This prevents reduction in free energy for nucleation with size increase and, therefore, further growth of nanocrystals. In addition, lattice contraction also stabilizes attractive interactions among nanocrystals. Lattice contrac‐ tion carries out nonbonded repulsion between CO3 2– anions within the structure of the rhomboedral carbonates. The more the distance between CO3 2– anions decreases because of the incorporation of the small Mg cation into the structure of calcite, the more repulsion increases. The increase of nonbonded repulsion makes valence electrons feel less potential wells.[34,35] In the case of carbonates, electrons feel less the potential of the oxygen nuclei within CO3 2– anions. The decrease of electron binding energy in O atoms increases oxygen polarizability. The improvement of the so-called polarization, correlation, Van der Waals or long-range forces between CO3 2– enhances nanocrystals attachment during the aggregationbased growth-process.

### **5. Conclusions**

transport and favours diffusion and the development of spherulitic instead of polyhedral morphologies (see next section). Therefore clayey material acts as a gel, and its occurrence favours the formation of amorphous precursors and nanocrystals of later precipitates. This is the case of francolite crystallization in phosphate stromatolites, which is preceded by the precipitation amorphous calcium phosphate, as suggested by Sánchez-Navas and Martín-

**4.2. Considerations related to gel growth and structural control of habit in Mg-Ca carbonates**

Gels and related substances are responsible for high supersaturation producing the crystal growth features observed in the solid porous media. High supersaturation gradients arise from the slow transport of the chemical species present in gel growth experiments, and the degree of supersaturation driving the crystallization depends on the diffusion gradient in solid porous media.[28] Hence, high growth rates are expected in porous media where the critical super‐ saturation for phosphate and carbonate minerals is reached more quickly than in solution

Gel growth also explains why some elements like Mg are incorporated to the structure of calcite. A correlation between Mg-content in calcite and crystallinity may be deduced from the figure 11. Slow diffusive transport media, such as a clay-rich and water deficient environments or porous media, make the transport of reacting material from solution to those of growth be the rate-determining process. Transport properties in porous media controls on the incorpo‐ ration of elements during the crystal growth.[32] To understand it, we may suppose that the partition coefficient between the concentration of magnesium in carbonate crystal and its

is diffusion controlled, the concentration of Mg at the crystal-medium interface rises above its concentration in the medium. If the partition coefficient remains constant, the Mg concentra‐ tion in the carbonate increases due to the slower diffusion rate and, in the limit, it is equal to the concentration of Mg in the medium. It makes that the effective partition coefficient

*Mg* / *Cmedium Mg* equals 1 and, therefore, most of the Mg of the medium is incorporated into the

Crystal chemical substitution of Mg for Ca within the structure of rhombohedral carbonates decreases crystallinity, stabilizes nanocrystals and favours an aggregation-based crystal growth. The different size of the cations involved (Ca2+ and Mg2+) distorts the calcite crystal lattice. Of these two cations the Mg2+ ion has the smallest ionic radius. The lattice contraction due to the substitution of Mg for Ca is evidenced by the displacement of the (104) peak in XRD patterns (Fig. 11). The *d*(104)-spacing and lattice constants of Mg-calcite are shorter than those of calcite, being the *c* axis the most affected by the incorporation of Mg.[33] This lattice

produces a lattice deformation around Mg atoms. The incorporation of Mg into the crystal structure increases drastically the volume strain energy. This prevents reduction in free energy for nucleation with size increase and, therefore, further growth of nanocrystals. In addition, lattice contraction also stabilizes attractive interactions among nanocrystals. Lattice contrac‐

*Mg* / *Cmedium Mg* ) is less than 1. When carbonate crystal growth

2– anions layers to be pulled closer together along the *c*-axis and

Algarra.[7]

84 Advanced Topics on Crystal Growth

growth.

*Ccrystal*

concentration in bulk medium (*Ccrystal*

structure of the rhombohedral carbonate.

contraction causes the CO3

Crystal growth features in biogenic and abiotic carbonates and phosphates occurring in natural environments (phosphate stromatolites and calcified tissues -bone and teeth) and formed in laboratory experiments are described in this work.

Phosphate and carbonate minerals precipitated from abiotic and biotic experiments form commonly spherulites and related morphologies. In most cases they can be considered mesocrystals because, independent of their biotic or abiotic origin, they are superstructures formed by the aggregation of nanocrystal building units and appear associated with amor‐ phous precursors. Parallel aggregates constituted by Ca-phosphate crystals are formed by competitive crystal growth processes and, together with amorphous Ca-phosphates, are observed in dentine and enamel as well as in phosphate stromatolites.

Gels in laboratory experiments, organic matrix in calcified tissues and equivalent clayrich media in phosphate stromatolites favour diffusive transport and supersaturation of carbonate and phosphate minerals. Supersaturation is responsible for the development of spherulitic morphologies and the occurrence of nanometre-sized crystals and related amorphous substances. Gels also explain the incorporation of some elements as Mg to the structure of calcite.

### **Acknowledgements**

We acknowledge support from grants CGL-2009-09249 (DGICYT, Spain) and P11- RNM-7067 of the Junta de Andalucía (Spain), and by the research group RNM-179 (Junta deAndalucía). We would like to thank María José Martínez Guerrero, Alicia González Se‐ gura, María del Mar Abad Ortega, Concepción Hernández, Isabel Guerra Tschuske and Juan de Dios Bueno (Centro de Instrumentación Científica-CIC, University of Granada) for guidance with SEM-EDX and TEM-AEM-EELS studies. Agustín Martín Rodríguez, José Suarez Valera, María Navas Moreno and Elena Beatriz Sánchez Martín significantly helped with laboratory precipitation experiments.

### **Author details**

Antonio Sánchez-Navas1 , Agustín Martín-Algarra2 , Mónica Sánchez-Román3 , Concepción Jiménez-López4 , Fernando Nieto1 and Antonio Ruiz-Bustos1

1 Departamento de Mineralogía y Petrología, Facultad de Ciencias, Universidad de Grana‐ da, Granada, Spain

[11] Chivas AR, Torgersen T, Polach HA. Growth rates and Holocene development of stromatolites from Shark Bay, Western Australia. Australian Journal of Earth Science

Crystal Growth of Inorganic and Biomediated Carbonates and Phosphates

http://dx.doi.org/10.5772/52062

87

[12] Walter MR, Golubic S, Preiss WV. Recent stromatolites from hydromagnesite and aragonite depositing lakes near the Coorong Lagoon, South Australia. Journal of

[13] Rivadeneyra MA, Martín-Algarra A, Sánchez-Román M, Sánchez-Navas A, Martín-Ramos D. Amorphous Ca-phosphate precursors for Ca-carbonate biominerals medi‐

[14] Buczybnski C, Chafetz HS. Habit of bacterially induced precipitates of calcium carbonate and the influence of medium viscosity on mineralogy. Journal of Sedimen‐

[16] Sánchez-Navas A, Martín-Algarra A, Rivadeneyra MA, Melchor S, Martín Ramos JD. Crystal-Growth Behavior in Ca−Mg Carbonate Bacterial Spherulites. Crystal Growth

[17] Sunagawa I. Morpholgy of Crystals. Tokyo: Terra Scientific Publishing Company; 1987.

[18] Checa González A, Sánchez-Navas A, Rodríguez-Navarro A. Crystal growth in the foliated aragonite of monoplacophorans (Mollusca). Crystal Growth and Design 2009;

[19] Martín-Ramos J.D., Díaz-Hernández J.L., Cambeses A., Scarrow J.H., López-Galindo A. Pathways for Quantitative Analysis by X-Ray Diffraction. In: Aydinalp C. (ed.) An Introduction to the Study of Mineralogy. Rijeka: InTech; 2012. http://www.intechop‐ en.com/articles/show/title/pathways-for-quantitative-analysis-by-x-ray-diffraction

[20] Vali H, Koster HM. Expanding behaviour, structural disorder, regular and random irregular interstratification of 2–1 layer-silicates studied by high-resolution images of

[21] Jarvis I. Sedimentology, geochemistry and origin of phosphatic chalks: the Upper

[22] De Vrind-de-Jong E.W., de Vrind J.P.M. Algal deposition of carbonates and silicates. In. Banfield J.F., Nealson K.H. (eds.) Geomicrobiology: Interactions between Microbes and Minerals. Reviews in Mineralogy: Mineralogical Society of America; 1997.

[23] Landis WJ, Hodgens KJ, Arena J, Song MJ, McEwen BF. Structural Relations Between Collagen and Mineral in Bone as Determined by High Voltage Electron Microscopic

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2 Departamento de Estratigrafía y Paleontología, Facultad de Ciencias, Universidad de Granada, Granada, Spain

3 Centro de Astrobiología, INTA, Torrejón de Ardoz, Madrid, Spain

4 Departamento de Microbiologia, Facultad de Ciencias, Universidad de Granada, Granada, Spain

Instituto Andaluz de Ciencias de la Tierra. C.S.I.C., Armilla, Granada, Spain

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**Author details**

da, Granada, Spain

Spain

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Antonio Sánchez-Navas1

86 Advanced Topics on Crystal Growth

Granada, Granada, Spain

Concepción Jiménez-López4

, Agustín Martín-Algarra2

1 Departamento de Mineralogía y Petrología, Facultad de Ciencias, Universidad de Grana‐

2 Departamento de Estratigrafía y Paleontología, Facultad de Ciencias, Universidad de

4 Departamento de Microbiologia, Facultad de Ciencias, Universidad de Granada, Granada,

[1] Capewell SG, Hefter G, May PM. Potentiometric investigation of the weak association of sodium and carbonate ions at 25 ºC. Journal of Solution Chemistry 1998; 27: 865-877.

[3] Broecker WS, Peng TS. Tracers in the Sea. New York: Lamont-Doherty Geological

[5] Cowan J, Weintritt D. Water formed scale deposits. Houston: Gulf Publ. Co.; 1976.

[6] Allwood AC, Walter MR, Kamber BS, Marshall CP, Burch IW. Stromatolite reef from

[7] Sánchez Navas A, Martín Algarra A. Genesis of apatite in phosphate stromatolites.

[8] Allen CC, Albert FG, Chafetz HS, Combie J, Graham CR, Kieft TL et al. Microscopic physical biomarkers in carbonate hot springs: implications in the search for life on Mars.

[9] McKay DS, Thomas-Keptra KL, Romanek CS, Gibson Jr EK, Vali H. Evaluating the

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[24] Ziv V, Sabanay I, Arad T, Traub W, Weiner S. Transitional structures in lamellar bone. Microscopy Research and Technology 1996; 33: 203-213.

**Chapter 4**

**Direction Controlled Growth of Organic Single Crystals**

The green lasers are attractive and highly useful for lot of practical applications. It is more than fifty times brighter when compared to a red laser and thus it can be seen from miles away. Due to these features, the green lasers can be used in high-tech weapons for aiming purposes. More‐ over, the green lasers are highly useful for laser televisions and medical applications. Most of the commercially available green lasers are based on the diode pumped solid state frequencydoubled (DPSSFD) laser technology. For the past several decades, researchers and several in‐ dustries were trying to develop the laser diodes based on the compound semiconductors such as Gallium nitride (GaN) and Indium Gallium nitride (InGaN) especially in the blue and green region of wavelengths [1]. However, it is very difficult to grow bulk crystal of these materials with reasonable quality. Moreover, for the preparation of epitaxial thin films of these materi‐ als on substrates, highly sophisticated and expensive techniques like molecular beam epitaxy (MBE) and metal organic chemical vapour deposition (MOCVD) are needed. Apart from these growth aspects, the relatively low power and limited wavelength range restricts their use in important applications [2]. Therefore, laser sources based on Second harmonic generation (SHG) is a better choice for the applications which requires higher powers or longer wave‐ lengths (>400nm). As a consequence, the green laser technology still depends on the nonlinear

In the DPSSFD lasers, a nonlinear optical (NLO) crystal must be placed to halves the wave‐ length of a solid state laser. In the today's market, inorganic NLO crystals of Potassium Titan‐ yl Phosphate (KTP), Lithium triborate (LBO) are used as frequency doublers. For example, the KTP crystal is used to generate green laser at 532 nm by halving the wavelength of Nd:YAG la‐ ser of 1064nm. Organic NLO materials are superior to the inorganic materials both in the speed

and reproduction in any medium, provided the original work is properly cited.

© 2013 Arivanandhan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**by Novel Growth Methods**

K. Sankaranarayanan and Y. Hayakawa

optical phenomena such as frequency doubling.

Additional information is available at the end of the chapter

M. Arivanandhan, V. Natarajan,

http://dx.doi.org/10.5772/53037

**1. Introduction**


**Author details**

Xia Lei1 , Jiayi Pan2 \* and Adam Thomas Devlin2

1 Institute of Space and Earth Information Science, The Chinese University of Hong Kong, Shatin, Hong Kong, China

2 School of Geography and the Environment, Jiangxi Normal University, Nanchang, Jiangxi, China

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*3.1. Dynamic Coalitions*

In open robot teams where the team members are not known a priori at design time, we need support for establishing a temporary team membership. Participants form temporary coalitions in order to solve a problem and achieve a common goal. A traffic intersection in autonomous driving scenarios is an example of a short-lived coalition with continuous team reconfiguration, while Industry 4.0 scenarios would likely imply a longer-lasting coalition. Only members of a coalition should be involved in the teamwork interactions, and agents outside of the coalition should not disturb it. This requires that all agents know about their membership. Moreover, it may require security measures to protect the interactions of a team. Key challenges are as follows:

Many communication paradigms achieve interaction among distributed components based on the identities of the components. Examples are the classical client/server model, the actor model [14], or named channels in channel-based binary communication [15]. On the other hand, broadcast communication [16] may not require identities depending on the capabilities of the underlying communication system, but loses the ability to address a selection of individual agents. However, in open teams in dynamic environments, the identity of robots may not be known at design time, if robots may join and leave a team at run-time. Thus, the concept of identity is not easy to establish and may even be irrelevant [17].

In such environments, we need different ways to determine team membership and to address team members. Note that a single central team manager that monitors and controls team membership is out of the question here because we need to avoid a single point of failure in environments where robots may move out of reach temporarily or break down completely.

One solution is based on attribute-based interaction. It is a variant of publish/subscribe communication, and it was proposed in [17,18] as a paradigm to address collectives of possibly anonymous agents. In attribute-based interaction, robots of a multi-robot system explicitly expose a set of attributes that are relevant for the application at hand. Interaction between robots is based on groupcast communication, whereby sending and receiving messages is determined by predicates over the specified attributes. A *send* command expresses the intention to deliver a message to all robots satisfying the *send predicate*. Likewise, a *receive* command signals willingness to receive messages from team members according to the specified *receive predicate*.

For example, in an Industry 4.0 scenario, one might ask for "components that need to be delivered within the next 15 min" or, in autonomous driving, one might want to address "vehicles that are capable of autonomous driving and are closer than 50 m to the intersection". Thus, attribute-based interaction is a more fine-grained content-based selection of possible receivers and senders. Potentially, it allows a more efficient filtering of messages by the distribution infrastructure and reduces the communication overhead. The drawback is the need for a powerful, rather heavyweight distribution infrastructure. Attribute-based interaction is integrated into the syntax of several programming languages, such as Erlang [19] and Google Go [20].

Other well-known protocols for open coalitions include the JXTA peer-to-peer protocols that target overlay-based communication of peers across public networks, where security issues such as firewall traversal are of utmost importance [21]. An application of JXTA for the control of robots was reported in [22]. However, the JXTA project is no longer officially supported. The FIPA recommendations (http:// www.fipa.org/) offer a conceptual framework for communication and management in multi-agent teams. Their focus is on intelligent (software) agents, not on mechatronic robots. The FIPA standardization is currently not active.


*3.2.1 Aluminum alloy 2025 stress: Strain curve*

The stress–strain curve is obtained by loading objects at a constant speed and measuring the amount of deformation in the tensile test. In this test, a specimen without any crack is installed on the tensile and is pulled at a speed of 1 mm per second. Using the results of this test, the stress–strain curve is plotted and the yield stress and ultimate stress in aluminum alloy 2025 are obtained. After testing and plotting the stress–strain curve, the ultimate stress level was 381.67 MPa and the yield stress was 275 MPa. The ultimate stress level for aluminum alloy 2025-T3 is 400 MPa in Ref. [12]. **Figure 12** shows stress–strain curve of aluminum alloy 2025 specimen that extracted from propeller blade. The difference between the measured stress and the reference stress is due to the life of the specimen used in the test because as the life of aluminum-containing copper alloy increases, this aluminum becomes brittle.

**Table 2.**

*Max. Acoustic emission count and cycle number of start signal changes in each specimen.*

### *Advances in Fatigue and Fracture Testing and Modelling*

#### **Figure 11.**

*Acoustic emission signal amplitude and cumulative count cumulative count vs. standard cycle diagram for all 9 specimens.*

**Figure 12.** *Stress–strain curve of aluminum alloy 2025.*

#### *3.2.2 Acoustic emission count and stress versus time diagram*

After performing the bending fatigue test and recording and analyzing the acoustic emission parameters due to crack initiation, it is necessary to subject the cracked specimens in the bending fatigue test to the tensile load to determine the parameters and characteristics of acoustic emission in fatigue crack growth. After performing the tensile test on 5 of the cracked specimens in the bending fatigue test, it is time to plot the count and stress vs. time diagram. This diagram shows the rate count and stress at each point in time of the test. Because all sources of additional signals and noise are blocked, the received signals are related to the acoustic emission activities inside the specimen. In general, these acoustic emission activities may be the result of plastic deformation or the growth of fatigue cracks created in the specimens. Because the test specimen is aluminum alloy 2025 with long life and brittle material and there is no sign of deformation in the specimen,

## *Determining the Characteristics of Acoustic Emission in the Fatigue Crack Growth of Aluminum… DOI: http://dx.doi.org/10.5772/intechopen.99360*

the signals received by the sensor cannot be the plastic deformation signals so these signals are due to the growth of fatigue cracks.

After examining the count and stress vs. time diagram, it was found that with increasing stress, which is obtained by dividing the tensile force on the cross-section of the specimen, the count also increases. As mentioned in the previous paragraph, this increase in the count is related to the growth of cracks in the aluminum specimen. According to the **Figure 13**, which shows the count and stress vs. time diagram for specimen NO. 1, which is randomly selected from 5 specimens to explain in detail, the count does not increase continuously and the increase in count occurs after increasing the slope of the stress diagram.

**Figure 14** shows the count and stress vs. time diagrams for all 9 specimens. As can be seen from the figure, in all specimens, the count increases sharply at the end of the test time, which indicates the highest crack growth activity during the test or an increase in crack growth rate with increasing force.

The highest increase of count for specimen NO. 1 occurred from 160 seconds to 167 seconds, where the highest rate of crack growth was observed. **Figure 15** shows the condition of the crack in 3 different times. Figure (a) shows the crack condition before the tensile test, when in the bending fatigue test the test is stopped immediately after observing the crack initiation. Figure (b) is after increasing the count

**Figure 13.** *Acoustic emission count, and stress vs. time diagram of specimen NO. 1.*

**Figure 14.** *Acoustic emission count, and stress vs. time diagram for all 9 specimens.*

**Figure 15.** *Crack condition during specimen NO. 1 test (a) before tensile test (b) in 160 second (c) in 167 second.*

at 160 seconds, where the count increases to 106 and the cumulative count to 189. Figure (c) also refers to a time of 167 seconds, where a sharp increase in the count, first at 165 seconds at 530 and then at 166 seconds at 602.

As the crack growth and the count diagram show, as time goes on and the stress and force increase, the count rate increase too, so the internal activity of the material and the crack growth increase, so that the maximum crack growth rate at the end of the test of each specimen.


When my footsteps fall on this road no more I will ferry not, my boat on this pier I will end this buy and sell, I will settle all my dues I won't to and fro to this mart So be it if you recall me or not So be it if gazing up at the stars, you call me or not. When dust accumulates On the strings of the *Tanpura*∗ When barbed fences of vines Creep up doorways of home When the flower garden adorns Ascetic robes of dense green grass When moss surrounds the pond So be it if you recall me or not So be it if gazing up at the stars, you call me or not. Then will the flute still sing thus in this play Days will pass, day after day Just the way it does today

References

Ferryboats will fill up that day Piers full in the very same way In that field, cows will graze, cowherds will play So be it if you recall me or not So be it if gazing up at the stars, you call me or not. Who says then, I am no more, in that morn In all play, this self will play With a new name In a new embrace Come and go I will, This self that is eternal So be it if you recall me or not So be it if gazing up at the stars, you call me or not.

#### **References**

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*2.1. Honey Samples*

Twenty samples of each honey type (local and imported) of diversified botanical origin were collected from different sources in Saudi Arabia during 2020–2021 (Table 1). The honey samples were kept in the dark at room temperature until the subsequent analysis.

**Table 1.** Detail of tested honey samples.