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Biomimetic Regulation of Microbially Induced Calcium Carbonate Precipitation Involving Immobilization of S. pasteurii by Sodium Alginate Jun Wu, and Raymond J Zeng Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b01813 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 13, 2017

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Crystal Growth & Design

Biomimetic Regulation of Microbially Induced Calcium Carbonate Precipitation Involving Immobilization of S. pasteurii by Sodium Alginate

Jun Wu, Raymond J. Zeng*

CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology of China, Hefei 230026, PR China

* Corresponding author. E-mail: [email protected]; Tel/Fax: +86 551 63600203

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Abstract Many researchers in the past decade have explored the controlled synthesis of calcium carbonate with specific size, morphology, and polymorphism. This study explored the biomimetic regulation of microbially-induced calcium carbonate precipitation (MICP) via employing immobilization technology. Calcium alginate gel was used to immobilize Sporosarcina pasteurii, a urea-positive microorganism. CO was generated driven by ureolysis and reacted directly with Ca2+ that was cross-linked in sodium alginate to produce CaCO3 precipitation. Based on SEM, TEM, XRD, HRTEM, and SAED results, amorphous calcium carbonate, vaterite, and calcite appeared in order. This evolution of CaCO3 morphology and polymorphism apparently conforms to Ostwald’s rule. Various concentrations (1%-3%) of sodium alginate caused different alginate molecules to form due to the collapse of calcium alginate gel carrying negative charges and exerting a significant influence on the morphology of CaCO3 from hexagonal vaterite to capsule-shaped vaterite. The techniques discussed here can also be applied to other polysaccharides on CaCO3, which implies that they are valuable in regards to polymorphic regulation because abundant polysaccharide apparently favors the vaterite polymorph.

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Introduction Calcium carbonate (CaCO3), a mineral that is common in nature, has been extensively applied to many industrial areas for hundreds of years; it is used in ceramics, plastics, coatings, and papermaking among other fields and is favored for its sophisticated architectures and superior mechanical performance.1,2 It has three anhydrous crystalline polymorphs, namely, calcite, aragonite, and vaterite, plus one amorphous calcium carbonate (ACC).3,4 Among them, calcite is the most thermodynamically stable under ambient conditions; aragonite and vaterite are generally metastable and rarely seen in the naturally occurring mineral. Metastable phases tend to have lower surface energies and convert slowly to calcite.5 Based on a thermodynamic view of nucleation and growth, however, calcium carbonate initially precipitates in the hydrous form of ACC, which is the precursor of anhydrous crystalline polymorphs according to the Ostwald stepping rule.6,7 Because different polymorphs impart different structures and mechanical properties, regulating over the crystal form is of importance to end-users. Many previous researchers have attempted to control the formation of CaCO3 minerals, often by using additives to modify CaCO3 growth. The mechanism is a topic of considerable interest. Surfactant aggregates, such as sodium dodecylsulfate, sodium dodecylsulfonate, and sodium dodecylbenzene sulfonate, have been used to influence the crystalline phase and morphology of the precipitated crystals by acting as nucleators for inorganic substances and inducing the oriented nucleation of complementary crystal faces.8,9 Amino acids, mostly aspartic acid, glutamic acid, and

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glycine, are regarded as effective additives to control the crystal phase, shape, and size. Amino acids trapped onto the surface of matrices and carboxyl providing abundant Ca2+ ion bonding sites are known to play an important role in the control of CaCO3 polymorphs.2, 10,11 Sodium alginate, a biotic polysaccharide widely used in the food industry, also exerts an important influence on the crystal growth of CaCO3.12,13 Although the natural occurrence of metastable vaterite and aragonite are rare relative to stable calcite, biogenetic vaterite widely seen in ﬁsh otoliths,14 freshwater cultured pearls from mussels,15 and larval snail shells16 shows high stability compared to its abiotic counterpart due to accurate regulation by polysaccharides or proteins in living organisms.17 Beyond this, microbial calcium carbonate precipitation extensively

occurs

in

the

bacterial

kingdom

and

can be

divided

into

microbially-controlled and microbially-induced CaCO3 precipitation (MICP).18 During MICP, microbial metabolic activities result in an increase in environmental alkalinity thereby promoting calcium carbonate precipitation.19 As a microorganism of MICP, Sporosarcina pasteurii can secrete urease to hydrolyze urea to ammonia and carbon dioxide; the production of ammonia causes an increase in the pH of the environment, which facilitates CaCO3 precipitation in calcium-rich environments.20 MICP has extensive applications including cultural heritage remediation,21 cement repair,22,23 heavy metal removal,24,25 and CO leakage reduction.26,27 Recent studies have suggested that bacterial cells and extracellular polymeric substances (EPS) can influence the morphology and polymorphism of CaCO3 so as to potentially provide a way of distinguishing MICP from abiotic precipitates.28,29,30

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MICP is often structurally ill-defined, physically heterogeneous, and spatially disorganized, however, due to the occurrence of extruded metabolic

products (ions,

gases, polypeptides, electrons) co-precipitating with extraneous metal ions in the surrounding environment.17 Be that as it may, organic matters existing commonly throughout nature (like polysaccharide) may be applicable in regulating the morphology and polymorphism of MICP. The goal of this study was to investigate the influence of sodium alginate, a biotic polysaccharide, on MICP with special focus on the morphology and polymorphism of CaCO3. Immobilization technology was employed by mixing sodium alginate, S. pasteurii, urea, and nutrients evenly, then the mixture was dropwise added to the calcium chloride solution. The microorganisms were immobilized immediately once the calcium alginate gel formed. CO was generated as-driven by ureolysis after a cultivation period and combined directly with the Ca2+ cross-linked in the sodium alginate to precipitate CaCO3. Due to the absence of Ca2+, the formed sodium alginate wrapped around the CaCO3 and affected the morphology and polymorphism of CaCO3. We hope that this work will provide a new insight into the roles of polysaccharide on biominerals, particularly MICP.

Experimental Section Chemicals All chemicals were of analytical grade and used as-received without further purification. Sodium alginate, calcium chloride (CaCl ), ammonium chloride (NH Cl),

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sodium chloride (NaCl), urea, peptone from casein, and peptone from soymeal were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Deionized water was used in all experiments.

Preparation of microbial immobilization system and CaCO3 harvest Sporosarcina pasteurii (ATCC 11859), a ureolytically active microorganism, was activated overnight to the stationary phase at 30°C with the medium consisting of 15.0 g/L peptone from casein, 5.0 g/L peptone from soymeal, 5.0 g/L NaCl, and 20.0g/L urea then its pH was adjusted to 7.3. The microorganism was washed via centrifugation (7000 rpm, 15 min) and resuspended in the fresh sterile medium (30 g/L Difco Nutrient Broth, 200 g/L urea and 100 g/L NH4Cl) to achieve an OD

of 2 prior to the immobilization experiment. The medium was prepared as described in detail in our references.27 The microbial immobilization system was prepared by adding 1 g sodium alginate concentration to the 50 mL microorganism suspension (e.g. 2% w/v) and stirring it magnetically until it dissolved completely. A 10 mL syringe was used to absorb the microorganism suspension containing 1-3% w/v sodium alginate and add it to the 3% w/v CaCl solution. Pellets formed immediately when the drops contacted the solution. The pellets were taken out after crosslinking for 10 min and rinsed three times with pH 7.0 phosphate buffer. The preparation of 1% and 3% w/v sodium alginate concentration microbial immobilization system followed the same steps. All the above operations were performed in a super clean bench. The prepared

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microbial immobilization system was then placed into an Erlenmeyer flask containing sterile deionized water and cultivated in a shaker at a speed of 160 rpm. The CaCO3 was obtained via centrifugation at 12000 rpm for 10 min and gently rinsed with deionized water to remove the alginate molecules.

Characterization The microorganism distributions were observed under a fluorescence microscope (Olympus BX51, Japan). Prior to microscopy, the microorganisms immobilized in gels were fixed with 4% (w/v) paraformaldehyde at 4 °C for 2 h then washed with 1× phosphate-buffered saline (PBS). The microorganism immobilization system was stained with 4’, 6-diamidino-2-phenylindole (DAPI) and cut into slices with a microtome (Leica CM1950，Germany).31 The slice was then transferred onto the well of a microscope slide just before observation. A phase contrast microscope (Olympus IX81, Japan) was used to analyze the gel structures in the slices. Field emission scanning electron microscope (FE-SEM) images were taken on a JEOL JSM-6700F SEM. A thin film of gold was used to increase the conductivity of samples for SEM analysis. Powder X-ray diffraction (XRD) patterns were recorded with a Philips X’Pert ProSuper X-ray diffractometer equipped with graphite monochromatized Cu Kα irradiation (λ =1.54178Å), employing a scanning rate of 0.02°·s−1 in the 2θ range 10−70°. FTIR spectra were recorded on a Nicolet Impact 400 FTIR spectrometer from 4000 to 400 cm−1 at room temperature. Transmission electron microscopy (TEM) and selected area electron diffraction (SAED) were

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conducted on a JEOL-2010 (JSM) microscope at an acceleration voltage of 200 kV. HRTEM was carried out with a JEOL JEM-ARF200F. Thermogravimetric analysis was performed with TA Instruments SDT Q600 under a stream of air at a heating rate of 10 °C/min. Nitrogen sorption data were gathered from a Micromeritics Tristar II 3020M automated gas adsorption analyzer utilizing Barrett−Emmett−Teller (BET) calculations for surface area and Barrett−Joyner−Halenda (BJH) calculations for pore size distribution for the adsorption branch of the isotherm. The particle size distribution analysis from the SEM images was conducted in ImageJ software (National Institute of Health). The zeta potential of each sample was recorded using a Zetasizer Nano ZS Instrument (Malvern Co., U.K.) at 25 °C.

Results Distribution of microorganisms and the gel structure Many dendritic-like pores were evenly distributed throughout the interior of the gel; the pore diameter was approximately 100 µm (Figure S1A). The porosity of the gel provided an outstanding capacity to immobilize microorganisms. Dyed microorganisms were uniformly distributed in the gel according to the fluorescence microscope images, demonstrating the remarkable microorganism immobilization capability of the gel (Figure S1B).

CaCO3 mineralization process Time-resolved observation of CaCO3 mineralization process was carried out in a

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2% w/v sodium alginate. No calcium carbonate was found initially, but a small amount of white calcium carbonate was produced in the gel after a period of cultivation (Figure S2). SEM images of CaCO3 particles after different mineralization periods are shown in Figure 1. Spherical CaCO3 with 2 to 3 µm in diameter was formed to which some smaller particles were adhered after 4 h of mineralization (Figure 1A, red arrow). The XRD pattern showed three intense peaks at 2θ = 24.8, 27.0, and 32.7° corresponding to the (110), (111), and (112) planes, respectively, and less intense peaks centered at 2θ = 43.98, 49.19, and 50.12° corresponding to (200), (202), and (114) planes (JCPDS: 74-1867) that are attributable to vaterite (Figure 2). The vibrational bands of 745 cm-1, 875 cm-1, and 1085 cm-1 found in the FTIR results can unambiguously be assigned to the carbonate symmetric stretching (ν1 mode), carbonate out-of-plane bending (ν2 mode), and in-plane bending (ν4 mode) vibrations of vaterite, respectively (Figure S3A). These results are consistent with the XRD results. The occurrence of a 696 cm-1 band indicated the existence of ACC (Figure S3A), thus we speculated that the small particles attached to the CaCO3 surface might be ACC. Further experimentation was conducted to verify this, as described below. After 6 h mineralization, the CaCO3 sphere became bigger and the surface became rough (Figure 1B). The size of CaCO3 was distributed in a narrow range; 75% of the CaCO3 spheres were 3.5 to 6.5 µm in diameter (Figure S4). As opposed to the results after 4 h mineralization, there were no small particles found on the CaCO3 surface. XRD and FTIR results altogether indicated that the crystal phase of CaCO3

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was still vaterite (Figure 2 and Figure S3A). Well-defined rhombohedral particles mixed with spherical particles were observed after 12 h of crystallization (Figure 1C inset). The spherical CaCO3 particles were elongated and about 10 µm in diameter, i.e., considerably larger than those observed in the early stage (Figure 1C). In the XRD pattern, the most intense peak was located at 2θ = 29.4° assigned to the (104) plane, with other peaks centered at 23, 35.9, 39.4, 43.15, 47.5, and 48.5° that clearly corresponded to calcite. Vaterite peaks were also observed (Figure 2). FTIR results showed 876 cm-1, 712 cm-1, and 745 cm-1 bands corresponding to calcite and a 1085 cm-1 band corresponding to vaterite (Figure S3A). XRD and FTIR results altogether revealed a mixed CaCO3 phase. The spheres disappeared and were replaced by dumbbell-shaped

CaCO3

consisting

of

rhombohedral crystals after 24

h

mineralization (Figure 1D). The XRD patterns were accordance with the FTIR results: The CaCO3 phase was calcite. Microorganisms were observed in all CaCO3 samples, as-marked by blue arrows in the SEM images (Figure 1). TEM tests were conducted to confirm our speculation that the small particles attached on the CaCO3 surface were ACC after 4 h crystallization. The TEM images indicated that this section was constructed of spherical CaCO3. A remarkable number of particles were seen around the bulk, as well (Figure 3). The HRTEM of the marked area showed that the d-spacing was 3.57 Å corresponding to the (110) faces of vaterite (Figure 3A). The single-crystalline SAED pattern, with sharp faculae, also revealed that the crystal had high crystallographic orientation; the hexagonal prism can be indexed as vaterite as-viewed from the [001] zone axis (Figure 3A). This result

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is consistent with the XRD pattern. The HRTEM and SAED of the marked area at the edge of the spherical vaterite indicate the emergence of ACC (Figure 3B). In short: We were correct in our speculation that some small ACC particles adhered to the surface of spherical vaterite. Based on a thermodynamic view of nucleation and growth, the CaCO3 initially precipitates in the hydrous form of ACC that is the precursor of anhydrous crystalline polymorphs. We investigated the CaCO3 produced at the early stage to explore this. The CaCO3 was harvested once a small quantity of white solid emerged so as to obtain the ACC; the small particles (200 nm in diameter and with rough surfaces) were observed under SEM (Figure 4A). The XRD patterns clearly indicated that the product was composed of pure ACC (Figure 4B). This result demonstrates the transformation of crystalline CaCO3 from ACC. In our observation of time-resolved CaCO3 mineralization, we unexpectedly discovered that the cross-sections had different morphologies after different mineralization periods (Figure 5). Figure 5A displays a loose and porous interior structure composed of fine particles after 4 h mineralization. The pore diameter was mainly distributed at approximately 80 to 150 Å and nitrogen adsorption−desorption isotherms show that the maximum quantity of nitrogen adsorbed was about 50 cm3/g (Figure 6A and Figure 6B). Compared to 4 h mineralization, the cross-section was much more compact after 24 h mineralization and rhombohedral particles accumulated in the surrounding regions. The particles sizes gradually increased as distance increased from the interior to the edge (Figure 5B). Smaller pore diameter

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(80 to 150 Å) and fewer maximum quantity of adsorbed nitrogen (20 cm3/g) were observed in pore size distribution and nitrogen adsorption−desorption isotherms (Figure 6C and Figure 6D). These results are in accordance with the SEM observations.

Effect of sodium alginate concentration on CaCO3 morphology Sodium alginate commonly was used as immobilization material and previous researches demonstrated the concentration between 0% and 3% was suitable for the microbial immobilization.32-35 Therefore, the concentrations of sodium alginate between 0% and 3% in this study were chosen to investigate the effect of sodium alginate concentration on the morphology of CaCO3 after 6 h crystallization. The SEM images showed a layered structure constructed of multiple rhombohedral particles when the concentration of sodium alginate was 0% w/v, namely, microorganisms were not immobilized (Figure 7A). Hexagon CaCO3 was generated with 2 µm in side length and 1 µm in thickness when 1% w/v sodium alginate was used (Figure 7B). As sodium alginate concentration increased to 2% w/v, spherical CaCO3 4 to 5 µm in diameter and with a rough surface were generated and distributed uniformly (Figure 7C). Transformation in CaCO3 morphology from spherical to capsule-shaped particles occurred when 3% w/v sodium alginate was used to immobilize the microorganisms (Figure 7D). According to our analysis of XRD patterns and FTIR curves, the crystal phase of CaCO3 was composed of calcite when sodium alginate was not utilized (Figure 8 and Figure S3B). Remarkably, vaterite

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dominated the CaCO3 morphology as-demonstrated by XRD and FTIR results regardless of sodium alginate concentration (Figure 8 and Figure S3B). The vaterite produced in different concentrations of sodium alginate was also characterized by TG-DSC; the corresponding results are shown in Figure 9. Three stages of weight loss can be observed in the thermogram curve of 1% w/v sodium alginate. The first, slow weight loss with a total of 1.18 wt%, occurred from room temperature to 130 °C corresponding to the evaporation of adsorbed water. The second noticeable weight loss, 3.32 wt% at 475 °C, in the TG curve can be attributed the decomposition of sodium alginate. The final weight loss, ∼38.77 wt% from 475 to 760 °C, can be ascribed to the decomposition of CaCO3 to CaO and CO2. CaCO3 generated in 2% and 3% w/v sodium alginate; this tendency was similar for 1% concentration though the specific amount of weight loss differed. The second weight loss assigned to the decomposition of sodium alginate increased from 3.32% to 5.82% as the concentration of sodium alginate increased. In effect, more sodium alginate was absorbed on the surface of CaCO3 when a higher concentration of sodium alginate was utilized. The zeta potential of CaCO3 obtained with different concentrations of sodium alginate was measured to find that it was consistently negative whether or not sodium alginate was utilized at all (Figure S5). When no sodium alginate was used to immobilize microorganism, the zeta potential of CaCO3 was -63 V. This value increased gradually to -90 V as sodium alginate concentration increased from 1% to 3% w/v as-attributed to the negative charges carried by alginate molecules.

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We tried to increase the concentration and 5% sodium alginate was used to immobilize microorganisms. The obtained CaCO3 was characterized with SEM and XRD, and the results were shown as follows. The capsule-shaped particles were formed (Figure S6A.) and pure vaterite was demonstrated by XRD (Figure S6B.). Comparing with 3% sodium alginate used, the crystalline polymorphs of CaCO3 in 5% sodium alginate didn’t show much difference, but the surface of vaterite was apparently smoother. When the concentration of sodium alginate continued increasing, we found that the sodium alginate was difficult to dissolve in water. Basing on the 3% and 5% sodium alginate used, we assume that more sodium alginate beyond 3% won’t bring obvious changes as for the morphology and polymorph of obtained CaCO3 concerned if we don’t consider the solubility of sodium alginate.

Discussion Crystallization mechanism According to the Ostwald’s step rule, the emergence of crystalline polymorphs and different phases of CaCO3 can be attributed to the energy barrier to nucleation for this phase.36 A probable crystallization mechanism for the growth of CaCO3 combining with above results is proposed accordingly in Figure 10. ACC, usually the precursor of crystalline CaCO3,37 with 200 nm in diameter (Figure 4) was formed first by binding with alginate molecules that were released due to the collapse of calcium alginate gel. The high density of alginate charges then stabilized the amorphous particles and prolonged their lifetime.38,39 This would allow the capture of ACC in the

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early stages of CaCO3 crystallization. Due to the higher interfacial energy possessed by smaller nanoparticles, nanoparticles aggregation was likely favored due to reduction in chemical potential; alginate molecules in this system then drove these nanoparticles to grow into orderly architectures. These small nanoparticles subsequently transformed into spherical vaterite while some remained attached to the vaterite surface (Figure 1A). As reaction time increased, the CO in the solution was evidently enhanced resulting in more and more CaCO3 being formed along with the amorphous particles transforming into more, bigger, and more uniform spherical vaterite (Figure 1B). Even though the formation of vaterite occurred due to the existence of the alginate stabilizer, the transformation of vaterite into calcite continued due to the thermodynamic instability of vaterite. Rough spheres composed of rhombohedral particles were generated (Figure 1C); the rhombohedral particles preferentially stacked to form spheres under the control of the functional groups on alginate. The crystal phase of these spheres changed from vaterite to calcite, as evidenced by our XRD and FTIR results. This was not the end of transformation, however, until the appearance of dumbbell-shaped calcite particles (Figure 1D). We speculate that spherical calcite composed of rhombohedra still possessed high interfacial energy, thus bigger CaCO3 particles were produced as a results of the fusion of two spherical calcites. The effect of various sodium alginate concentrations on the morphology of CaCO3 was also investigated here, as depicted in Figure 10B. Hexagonal vaterite was formed as 1% sodium alginate was used to immobilize microorganisms producing

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CO and NH driven by urealysis (Figure 7B). As stated by Pouget et al., the unfavorable (001) plane of vaterite can be stabilized through the presence of NH - or NH -bearing additives interacting with the carbonate ions in this crystal plane giving rise to hexagonal tablet-like crystals.7 When 1% sodium alginate was used, the high concentration of NH neutralized the negative charge carried by fewer alginate molecules allowing excess NH to occupy the CaCO3 surface. As sodium alginate concentration increased to 2%, spherical vaterite dominated as far as the morphology and polymorphism of CaCO3 (Figure 7C). As shown in Figure S5, the zeta potential increased as of sodium alginate concentration increased, indicating that more negative charges attached to the CaCO3 surface and the effect of NH was offset. Capsule-shaped CaCO3 was formed under the condition of 3% sodium alginate concentration, suggesting that negative charges attached to the surface of CaCO3 predominated the CaCO3 morphology.

Advantages of this study The influence of bacterial cells and EPS on the morphology and polymorphism of MICP have been fairly extensively researched to date.28,29,30 In addition to the self-regulation by microorganisms, factors in the outer environment (e.g., polysaccharide) exert a notable influence on MICP, though said influence has been rarely researched. Immobilization technology has been extensively applied to various fields such as the natural environment,40 fuel cells,41 and photocatalysts,42 but is not often

utilized

to

regulate

morphology

and

polymorphism.

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Immobilized

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microorganisms are randomly distributed in the microcavities of immobilization gel due to its spongy structure.43 In addition, the stable network matrices of immobilization gel can create a biocompatible environment to provide mechanical and chemical stability.44 These factors make the potential application of immobilization technology to regulate MICP morphology and polymorphism very attractive. Even though many previous researchers have attempted to control the formation of CaCO3 minerals, often by using organic additives to modify CaCO3 growth and some spherical particles are obtained. The features of CaCO3 are not characteristic in this study, but the methodology of regulation is novel. In recent years, the prevalence of hydrogel-like organic matrices in biomineralization has gained attention as a route to synthesize a diverse range of crystalline structures.45-47 The structural features of biopolymers can further influence the structure of the network formed in physical gels. However, microbial immobilization technology (calcium alginate gel) utilized to regulate CaCO3 morphology and polymorphism is firstly reported. Moreover, there are three advantages to the biomimetic regulation of MICP through microorganism immobilization by sodium alginate that reach beyond traditional regulatory methods. First, calcium alginate gel provides a residence for microorganisms that can reduce the outer interference: The only factor that can affect the MICP is the calcium alginate gel itself. Second, calcium sources of CaCO3 are provided in situ that crosslink with sodium alginate and are released gradually from the calcium alginate gel. This means that the CO is always situated under the condition of saturation, in accordance with

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the real-world microenvironment. Third, alginate molecules (a common biosynthetic polysaccharide) can serve as the organic additive to regulate the morphology and polymorphism of CaCO3 resulting from the collapse of calcium alginate gel. Though the effect of alginate molecules on CaCO3 have been reported by previous researchers,12,13 the influence of the microbial immobilization system induced by alginate molecules on MICP has been scarcely examined. Biomineralization is the process that produces variety of inorganic phases such as carbonates, phosphates, and sulfides by living organisms (microorganisms, unicellular, and mammalian etc.).48 Many of the compositions that form as biominerals can exist in several different structural modifications, for example, calcite, aragonite, and vaterite for CaCO3.49 Among them, calcite is the most thermodynamically stable under ambient conditions; aragonite and vaterite are generally metastable and rarely seen in the naturally occurring mineral.5 According to the Ostwald stepping rule that a crystallizing phase first will form a sequence of available transitional metastable phases before finally forming the stable phase, metastable vaterite is formed before final calcite.50 However, it is hard to harvest vaterite due to thermodynamically unstable under ambient conditions. Thus, a strategy for making a given polymorph in the laboratory (typically by controlling organic and inorganic additives, concentration of reactants, and ionic strength), and one that may be used by organisms as well, is to precipitate the desired polymorph especially the metastable products (e.g. vaterite).36 In this study, different CaCO3 morphologies and polymorphs were obtained from microbially induced mineralization through the

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microbial immobilization technology. The calcite and vaterite with different morphologies were obtained by regulating the mineralization and sodium alginate concentration. Due to numerous carboxyl and hydroxyl possessed by sodium alginate, the interfacial energy of CaCO3 could be effectively reduced. As a consequence, metastable vaterite could be obtained under ambient conditions. The obtained CaCO3 was induced from microbial synthesis, which resulted in some microorganisms attached on the CaCO3. In order to acquire pure CaCO3 as soon as possible, a reliable method to separate microorganisms from the synthesised CaCO3 should be utilized.

Centrifugal washing may be a suitable method to achieve

this goal. When the CaCO3 is obtained, some deionized water is added and then centrifuged at 5000 rpm for 5 min. The supernatant is poured out and same procedure is used again except for the absolute ethyl alcohol instead of deionized water. The pure CaCO3 can be obtained after 5 cycles. However, due to adhesion of extracellular polymeric substances (EPS) secreted by microorganisms, some microorganisms still adhere to the surface of CaCO3 (Data not shown). Therefore, it is difficult to separate the microorganisms completely from the synthesised CaCO3.

Implications In this study, the effects of alginate molecules on MICP morphology and polymorphism were explored through the use of immobilization technology. The influence of EPS mainly composed of polysaccharide on CaCO3 was demonstrated through complexation of Ca2+ by EPS in biofilms.30 Earth scientists and

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astrobiologists have made numerous efforts to distinguish microbial signatures in the rock record, but have been largely unsuccessful. The technique we utilized here uses simplified, biosynthetic polysaccharide (alginate molecules) to simulate the effect of polysaccharide in EPS on CaCO3. Our finding that artificially adding polysaccharide (alginate molecules) can influence the morphology and polymorphism of precipitated mineral phases indicates the potential effect of EPS on CaCO3. Natural organic matter (NOM) composed of polysaccharide, protein, and humus is widely distributed throughout nature. The mineral saturation index, the driving force for precipitation, decreases as NOM binds to dissolved metal ions. The interfacial properties of new phases that form from the solution are altered due to the adsorption of NOM to the surface of nucleated particles.51 This effect of NOM on CaCO3 may be slow, however, biosynthetic polysaccharide can considerably accelerate this process. To this effect, the characteristic crystal morphologies observed here raise the possibility that microbially-mediated precipitates may be identified in the geological record.

Conclusions In this study, the biomimetic regulation of MICP was successfully achieved by employing immobilization technology. There are three main advantages to this process: 1) The calcium alginate gel provides a residence for microorganisms; 2) calcium resources are slowly released and offered for precipitation in situ; and 3) alginate molecules regulate the morphology of CaCO3 due to the collapse of the gel.

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Our results indicate that the evolution of CaCO3 morphology and polymorphism conforms to Ostwald’s rule. The negative charges carried by alginate molecules likely exert a significant influence on the morphology and polymorphism of CaCO3 according to our utilization of different sodium alginate concentrations. Alginate molecules, a biosynthetic polysaccharide, can be used to simulate the effect of polysaccharide on CaCO3. From the perspective of microbial signatures, our study also shows promise in terms of polymorphic regulation because abundant polysaccharide apparently favors the vaterite polymorph.

Acknowledgments The authors would like to acknowledge the financial support of the Collaborative Innovation Center of Suzhou Nano Science and Technology, the Program for Changjiang Scholars and Innovative Research Team in University, and the Fundamental Research Funds for the Central Universities.

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Firgure captions Figure 1. SEM images of CaCO3 particles after different mineralization periods: (A) 4 h (B) 6 h (C) 12 h (D) 24 h. Figure 2. XRD patterns of CaCO3 after different mineralization periods. Figure 3. TEM images of CaCO3 particles generated at 4 h. (A) and (B) HRTEM images of the marked area. Insets show SAED patterns of each sample. Figure 4. ACC of (A) SEM image (B) XRD patterns. Figure 5. SEM images of CaCO3 cross-section after different mineralization periods (A) 6 h (B) 24 h. Figure 6. Pore size distribution and nitrogen adsorption−desorption isotherms after different mineralization periods: (A) and (B) 4 h, (C) and (D) 24 h. Figure 7. SEM images of CaCO3 generated in different concentrations of sodium alginate: (A) 0% (B) 1% (C) 2% (D) 3%. Figure 8. XRD patterns of CaCO3 generated in different concentrations of sodium alginate. Figure 9. TG curve (black line) and heat flow curve (blue line) of CaCO3 generated in (A) 1% (B) 2% (C) 3% sodium alginate concentration. Figure 10. Schematic drawing of (A) CaCO3 formation after different mineralization periods; (B) effect of different sodium alginate concentrations on CaCO3 morphology

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Figure 1. SEM images of CaCO3 particles after different mineralization periods: (A) 4 h (B) 6 h (C) 12 h (D) 24 h.

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Figure 2. XRD patterns of CaCO3 after different mineralization periods.

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Figure 3. TEM images of CaCO3 particles generated at 4 h. (A) and (B) HRTEM images of the marked area. Insets show SAED patterns of each sample.

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Figure 4. ACC of (A) SEM image (B) XRD patterns.

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Figure 5. SEM images of CaCO3 cross-section after different mineralization periods (A) 6 h (B) 24 h.

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Figure 6. Pore size distribution and nitrogen adsorption−desorption isotherms after different mineralization periods: (A) and (B) 4 h, (C) and (D) 24 h.

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Figure 7. SEM images of CaCO3 generated in different concentrations of sodium alginate: (A) 0% (B) 1% (C) 2% (D) 3%.

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Figure 8. XRD patterns of CaCO3 generated in different concentrations of sodium alginate.

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Figure 9. TG curve (black line) and heat flow curve (blue line) of CaCO3 generated in (A) 1% (B) 2% (C) 3% sodium alginate concentration.

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Figure 10. Schematic drawing of (A) CaCO3 formation after different mineralization periods; (B) effect of different sodium alginate concentrations on CaCO3 morphology

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For Table of Contents Use Only Biomimetic Regulation of Microbially Induced Calcium Carbonate Precipitation Involving Immobilization of S. pasteurii by Sodium Alginate

Jun Wu, Raymond J. Zeng*

CAS Key Laboratory for Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology of China, Hefei 230026, PR China

* Corresponding author. E-mail: [email protected]; Tel/Fax: +86 551 63600203

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Graphical abstract 391x178mm (72 x 72 DPI)


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Biomimetic Regulation of Microbially Induced ... - ACS Publications

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