Chaoqun Wu, Xun Liu,*, Fujun Yao, Xin Yang, Yan Wang, Wenyuan Hu

1 State Key Laboratory of Environment-friendly Energy Materials, Southwest University of Science and Technology, Mianyang 621000, China

2 Materials Science and Engineering, Southwest University of Science and Technology, Mianyang 621000, China

3 National Health Commission Key Laboratory of Nuclear Technology Medical Transformation, Mianyang Central Hospital, Mianyang 621010, China

Keywords:

ABSTRACT

1. Introduction

CaCO3-based biominerals in the form of nacre have attracted extensive attention because of their unique structure and excellent properties[1,2].Research into the mechanism of biomineralization explores the origin of biominerals in order to provide a basis for a biomimetic preparation method[3,4].To date,such studies mainly concentrate on composition induction,biogenesis,or environmental factors. Of these, composition induction has received the most in-depth research, including organic matrices and inorganic ions.There are some reports on the biogenesis, but due to the difficulty of in situ research, progress is relatively slow, and many biological mechanisms remain unclear [5]. Because biomineralization happens at normal temperatures and atmospheric pressure and in waters at near-neutral pH, environmental factors are easily taken for granted and have received the least attention from research into the mechanism of biomineralization.In fact,biomineralization is affected not only by standard environmental factors like temperature,pressure and pH,but also by minor factors like the geomagnetic field and local gravity. For example, although geomagnetic field strength is low (0.5–0.6 Gs) [6], given that biomineralization takes a long time in nature, its cumulative effects should not be dismissed.

The effect of a magnetic field on CaCO3mineralization was first reported in the patent announced by Faunce and Cabell in 1890[7].In the 1950s, Vermeiren [8] reported the effect of a magnetic field on the shape of several crystal precipitates, including calcium carbonate,aluminum sulfate and borax.Since then,the effect of magnetism on the crystallization process has gradually attracted research attention.Due to industrial interest in inhibiting scale formation in circulating cooling water, CaCO3is the compound that has attracted the most research into the effects of magnetism on crystallization. Currently, a consensus has been reached that the presence of a magnetic field causes CaCO3to form small particles,mostly of spherical vaterite or acicular aragonite,rather than large particles of calcite.When studying the effect of a magnetic field on the growth rate of aragonite and precipitation of calcium carbonate scale, Chang et al. [9] found that the strength of a magnetic field and the application time affect the agglomeration of calcium carbonate precipitates and that small spherical calcium carbonate crystals can form under certain magnetic field conditions,reducing scale deposition.Recently,Helal et al.[10]also found that calcite in small particles result when a magnetic field of 6500 Gs is applied,while large spherical crystals form in the absence of a magnetic field.

At present, the magnetic field strength used in industrial-scale inhibition is as high as thousands to tens of thousands of Gausses,and the cooling water is in motion,whereas biomineralization normally occurs in a weak geomagnetic field under static conditions.Indeed, no one has yet reported on the effect of a weak magnetic field on CaCO3biomineralization or the mechanism involved.Therefore, the effect of a weak magnetic field on CaCO3biomineralization in the presence of an organic matrix is studied in this paper. The biomimetic mineralization time for the laboratory is only one hundredth to one thousandth that of natural biomineralization,so,in order to make our research results approximate more closely to those of the natural process, the magnetic field used for this paper was 50–100 times higher than the geomagnetic field[6,11]. That is, two weak magnetic fields of 25 Gs and 50 Gs were studied and compared. As the longest biomimetic mineralization time in the experiment was 362 h,while the shortest natural pearl growth time is 2 years,we have increased the magnetic field intensity by 50–100 times, which is equivalent to the corresponding mineralization time increased to 754-1508 days(about 2–4 years).Therefore, increasing the magnetic field strength and the cumulative effect of time are basically equivalent.The results of this paper provide a useful and needed supplement to the theory of CaCO3biomineralization, and a new technical method for the optimization of biomimetic preparation of CaCO3.

2. Materials and Methods

2.1. Reagents and instruments

The anhydrous sodium carbonate (Na2CO3),anhydrous calcium chloride (CaCl2) used in this study were all analytical grade(Chengdu Cologne Chemicals Co., Ltd.). Fresh eggs used in the experiment were purchased from Mianyang Wal Mart supermarket(no more than 1 week old).The experimental water was ultrapure water (18.25 MΩ?cm, Sichuan You Pu Ultrapure Technology Co., Ltd.).

A uniform magnetic field, generated by two permanent NdFeB magnets with a diameter of 100 mm and a thickness of 10 mm was used in the experiment. The magnetic field strength was regulated by adjusting the distance between the two magnets. The centrifuge and freeze dryer used in the experiment were a highspeed desktop freezer centrifuge(TG16-WS,Jintan Wenhua Instrument Co., Ltd.) and a vacuum freeze-dryer (LGJ-100F, Beijing Songyuan Huaxing Development Technology Co., Ltd.), respectively.

2.2. Mineralization

Biomimetic mineralization was carried out with the participation of egg-white proteins. The main function of egg-white proteins is to simulate the acidic proteins in the process of natural biomineralization, so as to provide nucleation sites, regulate crystal form and morphology. The egg white for this study was first separated by hand, then placed in a beaker and ultrasonically dispersed, and then sealed with a film to keep it fresh.

Direct precipitation method was used for CaCO3biomimetic mineralization.First,100 ml of 0.2 mol?L-1CaCl2solution and Na2-CO3solution were prepared,respectively.The Na2CO3solution was added to the CaCl2solution at a rate of about 2 ml?min-1. 1% (vol)egg white was added to the CaCl2solution for biomimetic mineralization,but none for normal(non-biomimetic)mineralization.The reactions were carried out at room temperature (～20 °C). The device used in this experiment is shown in Fig.1.Once the reaction was complete, the precipitate was separated from the liquid,washed, and freeze-dried to obtain mineralized CaCO3samples.

In order to compare the effects of a magnetic field on mineralization at different points in the process,the field was applied during each of three stages(I)dissolution,when the reaction solutions were placed in the magnetic field and stirred for a certain time;(II)mineralization, when Na2CO3was being added; (III) and aging,when the mixed system was placed after reaction in the field for a certain time.The action time of magnetic field at different stages was not completely consistent. In the first stage, the action time was 1 h;in the second stage,the time was based on the actual time for mixing in the Na2CO3(about 1 h); and in the third stage, the time was 1 h, 1 d and 15 d, respectively. The aging time of stages I and II was fixed at 1 h. In order to avoid the influence of stirring on the magnetic field strength and direction, and in view of the high reaction rate of Ca2+and CO32–, the fast diffusion rate of ions in aqueous solution and the slow dropping rate (about 0.67 drops/s), we did not use stirring in the whole experiment.

2.3. Characterizations

The morphology of mineralized products was observed using a TESCAN MIRA3 (LMU) field emission scanning electron microscope;all sample surfaces were plated with Au to increase conductivity, and the accelerating working voltage was 5 kV. The phase composition of the samples was analyzed with an X-ray diffractometer made by the PANalytical Corporation in the Netherlands at an interval of 3°–80°. The operating voltage and current were 40 kV and 40 mA, respectively. Cu Target Kα Radiation was used with a λ of 0.154187 nm, a step interval of 0.03°, and a scanning speed of 10 (°)?min-1.

3. Results and Discussion

For comparison, two weak magnetic fields of 25 Gs and 50 Gs were used in this experiment. The control sample was the sample mineralized in the absence of magnetic field. The following sections will describe and analyze the crystalline-magnetism action of the 25 Gs and 50 Gs magnetic fields during the three different stages.

3.1. Effects of a magnetic field action on stage I

Fig. 2 gives the XRD and SEM characterization results for the normal mineralization and biomimetic mineralization products under different magnetic field strengths. Comparison of the XRD patterns (Fig. 2(a)) to the standard cards for calcite (PDF#47-1743)and vaterite(PDF#33-0268)shows that in a 25 Gs magnetic field the products of normal mineralization and biomimetic mineralization are all calcite.When the magnetic field strength increases to 50 Gs, the products of both kinds of mineralization change to mixed crystal forms of calcite and vaterite, and the peak intensity of vaterite is significantly stronger than that of calcite. This shows that when the magnetic field acts on the solution during dissolution (i.e., stage I), greater magnetic field strength conduces to the formation of vaterite [12]. Under the two magnetic fields, eggwhite protein has no significant effect on the mineralization results. However, comparison of curves 1 and 2 and of curves 3 and 4 in Fig.2(a)shows that the strongest peak of biomimetic mineralization is slightly stronger than that of normal mineralization,indicating that egg-white protein is more conducive to crystallization [13].

Fig.2. XRD patterns (a) and SEM images of mineralized products prepared under the magnetic fields in stage I:curve 1 & (b),curve 2 &(c), curve 3& (d)and curve 4 & (e)show normal mineralization under 25 Gs, biomimetic mineralization under 25 Gs, normal mineralization under 50 Gs, and biomimetic mineralization under 50 Gs,respectively.

As shown in Fig. 2(b)–(e), product morphology also changes greatly under different magnetic fields. Under a 25 Gs field, the product takes the shape of uniform blocks,but under a 50 Gs field,it is mainly uniform spheres mixed with a few blocks. The blocks and spheres correspond to the characteristic morphology of calcite and vaterite,respectively,so the characterization results from SEM and XRD are consistent. Without egg-white protein, the particle size of samples obtained under the two different magnetic fields is about 4–5 μm,which is significantly larger than that of the sample mineralized in the presence of egg-white protein(about 2 μm).This is mainly because the strong coordination groups on eggwhite protein molecules limit the migration of calcium ions and inhibit the growth of mineralized particles [14–16].

For further comparison, we also carried out mineralization in the absence of a magnetic field, and the results are shown in Fig. 3. Figs. 2 and 3 show that the composition and morphology of a normal sample prepared under a 25 Gs magnetic field are basically consistent with the mineralized sample prepared without a magnetic field, which consists entirely of blocky calcite, though there are a few spherical particles in the sample depicted in Fig.3(c).No characteristic peaks of vaterite appear in the XRD patterns of this sample, but results already presented in this paper show that egg-white protein can induce the formation of vaterite.Therefore,one presumes that the spherical particles visible in Fig.3(c) are vaterite that fails to show up in the XRD patterns because their amount is too small.In contrast,no spherical particles are visible in Fig. 2(c), perhaps because the magnetic field inhibited induction of vaterite by the egg-white protein.In addition,the particle size of the mineralized samples prepared without an applied magnetic field(about 10 μm)(Fig.3(b))is significantly larger than that of the samples prepared under a 25 Gs field (Fig. 2(b)). These results show that, aside from reducing particle size, a 25 Gs magnetic field has little effect on overall mineralization in stage I.The underlying mechanism will be described in Section 3.4.

3.2. Magnetic field effects in the stage II

Fig. 3. XRD patterns (a) and SEM images of control samples prepared in the absence of an applied magnetic field. (b) and (c) are normal mineralization and biomimetic mineralization, respectively.

Fig.4. XRD patterns(a) and SEM images of mineralized products prepared under an applied magnetic field action in stage II:curve 1 &(b),curve 2& (c),curve 3& (d)and curve 4 & (e) are normal mineralization under 25 Gs, biomimetic mineralization under 25 Gs, normal mineralization under 50 G, biomimetic mineralization under 50 Gs,respectively.

XRD patterns and SEM images of normal mineralization and biomimetic mineralization products prepared under an applied magnetic field in stage II (i.e., the mineralization stage) appear in Fig. 4. Under a 25 Gs magnetic field, the normal mineralization samples consist of mixed calcite and vaterite crystals, with calcite dominating(curve 1, Fig. 4(a)). In contrast, the biomimetic mineralization samples (curve 2, Fig. 4(a)) only have the characteristic peak of calcite.This difference is also supported by the corresponding SEM images, which show a few spherical particles among the blocky ones in the normal mineralization samples (Fig. 4(b)) but only blocky particles in the biomimetic mineralization samples(Fig. 4(c)). When the magnetic field strength is increased to 50 Gs,both the normal and the biomimetic mineralized samples contain mixed calcite and vaterite crystals, but the dominant phase changes to vaterite (curves 3 and 4, Fig. 4(a)). The morphological images are also dominated by spherical particles mixed with a small number of blocky particles (Fig. 4(d) and (e)). These results show that in stage II, an applied magnetic field, especially a higher-strength field, conduces to the formation of vaterite.

When a 25 Gs magnetic field is applied to stage II(as opposed to stage I),a small amount of vaterite appears in the normal mineralization samples but not in the biomimetic mineralization samples,indicating that organic matter inhibits the induction of vaterite at this magnetic field strength.In Section 3.1,we suggested that eggwhite protein can induce the formation of vaterite [14]. The fact that such induction does not occur here (curve 2, Fig. 4(a)), indicates that an applied magnetic field may inhibit induction by egg-white protein. Therefore, induction of vaterite by a magnetic field and by egg-white protein may be mutually inhibiting [17].This is further reflected in the effects of an applied 50 Gs magnetic field. When the magnetic field acts alone, the strongest characteristic peak of calcite (curve 3, Fig. 4(a)) is significantly weaker than that of the mineralized products in the presence of egg white(curve 4, Fig. 4(a)). The change in the particle size of the mineralized samples is consistent with the finding in Section 3.1, namely,that egg-white protein and an applied magnetic field both restrict the growth of mineralized particles, resulting in a smaller particle size.

3.3. Magnetic field effects on stage III

In the foregoing experiments, vaterite results from applying a magnetic field to different stages of mineralization, and it dominates under the stronger field. Nonetheless, of all the forms of CaCO3crystals,the most important is aragonite,the basis of nacre’s‘‘brick-mud” structure. In view of the long period biomineralization takes in nature,we applied a magnetic field to the aging stage(i.e.,stage III)for several different lengths of time,namely,0 h,1 h,1 d and 15 d. In the formation of calcium carbonate, amorphous calcium carbonate(ACC)is often the precursor phase and therefore appears first, after which it transforms into unstable vaterite and finally into stable calcite or aragonite (under special conditions).The results in Sections 3.1 and 3.2 show that the influence of 50 Gs magnetic field on crystal transformation is much stronger than that of 25 Gs. The strong influence, however, is not conducive to the discovery of subtle changes in the process of crystallization during aging.Therefore,we chose the 25 Gs magnetic field for performing our aging experiments on the biomimetic mineralized samples prepared at 25 Gs,as shown in Section 3.2,with the aging being carried out in the mother solution without separating the solids from the liquid.

The composition and morphology characterization results for samples aged for the several different times appear in Fig. 5. The XRD patterns (Fig. 5(a)) show that with longer aging times in an applied magnetic field, the samples change from an initial mixed calcite and vaterite(0 h,curve 1 and 1 h,curve 2)to mixed calcite,vaterite and aragonite(PDF#41-1475)(24 h,curve 3)and finally to mixed calcite and aragonite (15 d, curve 4). The SEM images also confirms these changes. The three characteristic morphologies blocky calcite,spherical vaterite and acicular aragonite appear successively in the samples. At first, there are spherical and blocky particles (Fig. 5(b) and (c)), then acicular particles join the spherical and blocky ones(Fig.5(d)),and finally there are only blocky and acicular particles (Fig. 5(e)). Evidently, the applied magnetic field induces vaterite for a short time in the aging stage (stage III), but the vaterite is unstable. As time passes, it gradually changes to more stable calcite or aragonite.Without an applied magnetic field,it inevitably changes to calcite [18]. With an applied magnetic field, however, some of the vaterite transforms into aragonite,showing that an applied magnetic field can induce the formation of aragonite if the aging stage is long enough.

The effects of an applied magnetic field on the different stages of mineralization are summarized in Table 1. From the table and the above experimental results and analysis,the following conclusions can be reached:(i)An applied magnetic field conduces to the crystallization of the unstable CaCO3phases of vaterite or aragonite,and the stronger the magnetic field,the stronger the induction effect; (ii) CaCO3crystallization under an applied magnetic field follows the path,precursor →vaterite →aragonite;(iii)The effects of an applied magnetic field are greatest in stage III,less in stage II,and least in stage I; (iv) The induction effects of an applied magnetic field and egg-white protein are mutually inhibiting, and the effect is more obvious under a low-strength magnetic field; (v)Both an applied magnetic field and egg-white protein restrict the growth of mineralized particles [19,20].

3.4. Mechanism for the effects of an applied magnetic field on CaCO3 mineralization

As shown in the summary table (Table 1), the effects of an applied magnetic field differ depending on the stage when applied.In addition, the presence of an organic matrix changes the field’s effects and their mechanism. In this section, the mechanism in the three different mineralization stages with an organic matrix present will be analyzed. Fig. 6 provides a schematic diagram of the mechanism.

According to the valence bond theory of coordination bonds,Ca2+in solution coordinates with water molecules to form[Ca(H2O)6]2+. The coordination cation has a regular octahedral stereo configuration and a relatively stable structure. Ca2+is paramagnetic,and H2O is diamagnetic. Therefore,a magnetic field will separate Ca2+from coordinated H2O molecules, distorting the regular octahedral configuration of the [Ca(H2O)6]2+and eventually reducing its stability. The decrease in stability will increase Ca2+reactivity,which is equivalent to increasing the Ca2+concentration.The Kspof calcite, aragonite and vaterite are 3.16 × 10-9,4.78 × 10-9and 12.6 × 10-9, respectively [21,22]. Therefore, the amount of [CO32–] remains the same, the more [Ca2+] there is, the more conducive it will be to the formation of vaterite. In addition,it should be noted that the effect of a magnetic field on [Ca(H2O)6]2+is directly proportional to the magnetic field strength.The greater the strength, the more significant its induction effect will be, and the more obvious the formation of vaterite, but when the solution is subjected to a magnetic field in stage I only, the effect of the field on the stability of [Ca(H2O)6]2+turns out to be time-dependent.If the magnetic field is removed,the coordination between H2O and Ca2+will gradually return to normal. Therefore,even if the mineralization reaction is carried out quickly after a magnetic field is applied, the effect of the field is much smaller than when it is directly applied during mineralization (stage II).

Fig.5. XRD patterns and SEM images of biomimetic mineralized samples under a 25 Gs magnetic fieldin stage III after different aging times:curve 1&(b),curve 2&(c),curve 3 & (d) and curve 4 & (e) represent 0 h, 1 h, 24 h and 15 d, respectively.

Table 1 Magnetic field effects on mineralization in different stages

Fig. 6. Schematic of the mechanism of the effects of an applied magnetic field.

When investigating the mechanism of the mineralization of calcium carbonate under a magnetic field, Skytte S?rensenet al. [23]pointed out that the magnetic field has a binding effect on ion movement,and the degree of binding is closely related to the magnetic field strength—i.e.,the greater the magnetic field strength,the stronger the binding force. Due to how quickly mineralization occurs when using the direct precipitation method, theoretically some amorphous CaCO3(ACC) will still be present in the initial mineralization precipitate. Without any intervention, this newly generated ACC will quickly transform into calcite. However,because a magnetic field restricts the migration of ions and increases the difficulty of phase transition, ACC is easier to transform into vaterite due to a low transition potential barrier when aged in a magnetic field. It should be noted that vaterite is also thermodynamically unstable and therefore undergoes spontaneous crystalline transformation. In the absence of a magnetic field, the end result of this transformation will be the most stable phase,namely,calcite, but if a magnetic field is applied,the field’s restrictive effect on the movement of ions means that vaterite can only rearrange its atoms into a configuration closest to its energy state.Of the three crystalline forms of CaCO3,aragonite is definitely closer to vaterite’s energy state than is calcite.Therefore,if aging is prolonged,vaterite will spontaneously transform into aragonite in a magnetic field. Nonetheless, if one compares XRD peak intensities, it appears that only a small fraction of the vaterite is transformed into aragonite, and most of the rest turns into calcite.This may be the result of competition between magnetic field effects and thermodynamic factors.

When there is egg-white protein in the reaction solution,because egg-white protein by itself can induce the formation of vaterite,it will produce vaterite in the absence of an applied magnetic field. Our previous results show that the ability of egg-white protein to induce vaterite formation depends heavily on the speed of mineralization. Generally, the slower that is, the more vaterite will form[24].Because this experiment used a direct precipitation method with a rapid rate of mineralization, the vaterite content was low in the products.In stages I and II of mineralization,active groups such as carboxyl and sulfhydryl groups on the egg-white protein molecule form more stable complexes with calcium ions than hydrated calcium ions, which causes the increase of free[Ca2+] resulting from the applied magnetic field to decrease [25].Indeed, the concentration of free Ca2+will be even lower than in a CaCl2solution with no magnetic field applied, and this decrease in free[Ca2+]inhibits the formation of vaterite.It should be emphasized that a magnetic field can also change the coordination of eggwhite protein molecules with Ca2+.Since egg-white protein has the same diamagnetism as water molecules,the presence of an applied magnetic field will inevitably reduce the stability of this coordination. Furthermore, since the ability of egg-white protein to induce vaterite formation depends on directional nucleation following upon coordination, decreased stability will also inhibit the induction of vaterite formation by egg-white protein to some degree.

4. Conclusions

In conclusion, in a simulation of the geomagnetic field, this paper investigates the effects of 25 Gs and 50 Gs magnetic fields on different stages of calcium carbonate mineralization and analyzes the mechanism underlying those effects. The results show that (as opposed to from normal mineralization) the presence of an applied magnetic field conduces to the formation of vaterite or aragonite crystals.The stronger the magnetic field,the stronger its induction ability.On the whole,under an applied magnetic field,thecrystallizationofCaCO3occursintheseries,precursor →vaterite →aragonite. The effects of a magnetic field differ with the stage of mineralization. Of these, the aging stage shows the most obvious effects,and the dissolution stage the least.The presence of egg-white protein reduces the effects of a magnetic field, and the presence of an applied magnetic field reduces the ability of egg-white protein to induce vaterite formation. In short,the two inhibit each other,more obviously in a weaker magnetic field. An applied magnetic field and egg-white protein also quite clearly limit the growth of mineralized particles. Analysis of the underlying mechanism shows that the opposing magnetism of Ca2+and H2O in the liquid phase are the fundamental reason for the formation of vaterite. The influence of a magnetic field in the aging stage is mainly due to its binding effect on ions,which limits the internal rearrangement of the crystals in mineralized particles to a considerable extent. The results of this paper help one to understand the important role of minor environmental factors in biomineralization.They can also be used as a reference for the biomimetic preparation of a CaCO3nacre-like structure and antiscaling technology for circulating cooling water.

Data Availability

No data was used for the research described in the article.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (12272329), the Sichuan University Student Innovation and Entrepreneurship Training Program (S202110619066),the Project of State Key Laboratory of Environment-friendly Energy Materials, Southwest University of Science and Technology(No.20fksy18), the Undergraduate Innovation Fund Project by Southwest University of Science and Technology (CX21-098) and the NHC Key Laboratory of Nuclear Technology Medical Transformation (Mianyang Central Hospital) (21HYX019).