Biomineralization of Monohydrocalcite Induced by the Halophile Halomonas Smyrnensis WMS-3

Article Biomineralization of Monohydrocalcite Induced by the Halophile Halomonas Smyrnensis WMS-3

1,2, 1,2, 3,4 1 1 Juntong Pan †, Hui Zhao † , Maurice E. Tucker , Jingxuan Zhou , Mengzhen Jiang , Yapeng Wang 1, Yanyang Zhao 1, Bin Sun 1, Zuozhen Han 1,2,* and Huaxiao Yan 1,* 1 College of Chemical and Environmental Engineering, College of Earth Science and Engineering, Shandong Provincial Key Laboratory of Depositional Mineralization and Sedimentary Minerals, Shandong University of Science and Technology, Qingdao 266590, China; [email protected] (J.P.); [email protected] (H.Z.); [email protected] (J.Z.); [email protected] (M.J.); [email protected] (Y.W.); [email protected] (Y.Z.); [email protected] (B.S.) 2 Laboratory for Marine Mineral Resources, Center for Isotope Geochemistry and Geochronology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3 School of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK; [email protected] 4 Cabot Institute, University of Bristol, Cantock’s Close, Bristol BS8 1UJ, UK * Correspondence: [email protected] (Z.H.); [email protected] (H.Y.); Tel.: +86-532-86-057-055 (Z.H.); +86-532-86-057-625 (H.Y.) Hui Zhao and Juntong Pan contributed equally and as co-ﬁrst authors. † Received: 1 September 2019; Accepted: 12 October 2019; Published: 15 October 2019

Abstract: The halophilic bacterium Halomonas smyrnensis from a modern salt lake used in experiments to induce biomineralization has resulted in the precipitation of monohydrocalcite and other carbonate minerals. In this study, a Halomonas smyrnensis WMS-3 (GenBank:MH425323) strain was identified based on 16S rDNA homology comparison, and then cultured in mediums with 3% NaCl concentration to induce monohydrocalcite at different Mg/Ca molar ratios of 0, 2, 5, 7, and 9. The growth curve + 2 of WMS-3 bacteria, pH values, NH4 concentration, HCO3− and CO3 − concentration, carbonic anhydrase (CA) activity, and the changes in Ca2+ and Mg2+ ion concentration were determined to further explore the extracellular biomineralization mechanism. Moreover, the nucleation mechanism of monohydrocalcite on extracellular polymeric substances (EPS) was analyzed through studying ultrathin slices of the WMS-3 strain by High resolution transmission electron microscopy (HRTEM), Selected area election diffraction (SAED), Scanning transmission electron microscopy (STEM), and elemental mapping, besides this, amino acids in the EPS were also analyzed. The results show that pH increased to about 9.0 under the influence of ammonia and CA activity. The precipitation ratio (%, the ratio of the mass/volume concentration) of the Ca2+ ion was 64.32%, 62.20%, 60.22%, 59.57%, and 54.42% at Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively, on the 21st day of the experiments, and 6.69%, 7.10%, 7.74%, 8.09% for the Mg2+ ion concentration at Mg/Ca molar ratios 2, 5, 7, and 9, respectively. The obtained minerals were calcite, Mg-rich calcite, aragonite, and hydromagnesite, in addition to the monohydrocalcite, as identified by X-ray diffraction (XRD) analyses. Monohydrocalcite had higher crystallinity when the Mg/Ca ratio increased from 7 to 9; thus, the stability of monohydrocalcite increased, also proven by the thermogravimetry (TG), derivative thermogravimetry (DTG) and differential scanning calorimetry (DSC) analyses. The C=O and C–O–C organic functional groups present in/on the minerals analyzed by Fourier transform infrared spectroscopy (FTIR), the various morphologies and the existence of P and S determined by scanning electron microscope-energy dispersive spectrometer (SEM-EDS), the relatively more negative stable carbon isotope values ( 16.91% to 17.91%) analyzed by a carbon isotope laser spectrometer, − − plus the typical surface chemistry by XPS, all support the biogenesis of these mineral precipitates. Moreover, Ca2+ ions were able to enter the bacterial cell to induce intracellular biomineralization. This study is useful to understand the mechanism of biomineralization further and may provide theoretical reference concerning the formation of monohydrocalcite in nature.

Minerals 2019, 9, 632; doi:10.3390/min9100632 www.mdpi.com/journal/minerals Minerals 2019, 9, 632 2 of 26

Keywords: Halomonas smyrnensis; biomineralization; monohydrocalcite; nucleation site; Mg/Ca ratios

1. Introduction Calcium carbonate is of great scientific significance in biomineralization and the Earth Sciences generally. The formation of calcium carbonate through microbial activities, as well as plankton, acting on a global scale and utilizing huge quantities of CO2, can affect the chemistry of ocean water, the Earth’s atmosphere, and climate [1–5]. Although there is ample evidence that 3.5 Ga stromatolites are biogenic [6,7], the potential role of abiotic processes in the formation of these laminated structures remains controversial [8]. Regardless of stromatolites formed in the past, contemporary microbial mats provide insight into the important role played by microbes in the precipitation of minerals. Understanding microbially-mediated precipitation of carbonate minerals is critical for the interpretation of the rock record and is also one of the fundamental research objectives in the rapidly expanding field of biogeosciences [9–14]. Calcium carbonate minerals at Earth’s surface conditions include the common polymorphs calcite, aragonite, and vaterite, and the less common hydrated phases of monohydrocalcite (CaCO H O) and 3· 2 ikaite (CaCO 6H O) [15–27]. It has been reported that monohydrocalcite is metastable compared with 3· 2 calcite and aragonite at all temperatures and pressures [28]. Maybe due to this reason, monohydrocalcite is not widely distributed in the world. Previously, monohydrocalcite has only been found in fresh-water and terrestrial environments. Dahl and Buchardt (2006) reported that monohydrocalcite is a common mineral (up to 55%) in the debris fragments of ikaite (CaCO 6H O) tufa columns formed in the cold 3· 2 marine Ikka fjord, SW Greenland [28]. In fact, before monohydrocalcite was discovered in the marine environment, it had been induced by the moderately halophilic species Halomonas eurihalina and Bacillus siamensis in both solid and liquid media in the laboratory; at that time, the focus was on the influence of salinity and temperature on the minerals precipitated [29–31]. Later, Halobacillus trueperi bacteria were also used to induce monohydrocalcite in both solid and liquid media at different salt concentrations and different Mg/Ca ratios [32]. Rhodococcus erythropolis [33], Chromohalobacter israelensis [34] and Chromohalobacter marismortui [35] were also used to induce the formation of monohydrocalcite in the laboratory. However, there are fewer studies on the mechanism of monohydrocalcite biomineralization, the nucleation sites, the nucleation mechanism, and intracellular biomineralization. Moreover, using H. smyrnensis bacteria to induce the biomineralization of monohydrocalcite has also been rarely reported. In this study, the H. smyrnensis WMS-3 strain was identified based on 16S rDNA sequence homology analysis, and then used to induce carbonate minerals at different Mg/Ca molar ratios 0, 2, 5, 7, and 9 at a 3% NaCl concentration that was similar to seawater salinity. The extracellular biomineralization mechanism was explored by analyzing the growth curve of WMS-3 bacteria, pH + 2 values, NH4 concentration, HCO3−, and CO3 − concentration, carbonic anhydrase (CA) activity, and the changes in the concentration of Ca2+ and Mg2+ ions. The nucleation mechanism of minerals on extracellular polymeric substances (EPS) was analyzed through studying ultrathin slices of WMS-3 strain by high-resolution transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), scanning transmission electron microscope (STEM), and elemental mapping; in addition, the amino acids in EPS were determined. At the same time, intracellular biomineralization was observed in ultrathin slices. The bacterially-induced minerals were investigated by X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope coupled with energy dispersive spectroscopy (SEM-EDS), stable carbon isotope composition, and X-ray photoelectron spectroscopy (XPS). This study aims to understand the mechanism of biomineralization further and may provide insights into the formation of monohydrocalcite in nature. Minerals 2019, 9, 632 3 of 26

2. Materials and Methods

2.1. Bacterial Strain and Culture Medium The WMS-3 strain used in this study was isolated from the Yinjiashan saltern (Qingdao, China). WMS-3 bacteria were cultured in a liquid medium containing the following components (unit: g L 1, · − pH 7.0): peptone 10, beef extract 5, NaCl 5, and distilled water 1 L. Twenty grams of agar powder were added into the above liquid culture medium to prepare the solid medium.

2.2. Identiﬁcation of H. smyrnensis WMS-3 Bacteria The preserved WMS-3 bacteria were sent to Shanghai Biological Engineering Co., Ltd. (Shanghai, China) for 16S rDNA sequencing, and the ﬁnal 16S rDNA sequences were uploaded to the GenBank/NCBI database to obtain an accession number. The homology between the 16S rDNA sequences of WMS-3 and those of other bacteria in the GenBank/NCBI database was compared by Basic Local Alignment Search Tool (BLAST) analysis. The phylogenetic tree of WMS-3 was constructed by the neighbor-joining method in the MEGA 7 software (Version 7.0, The Pennsylvania State University, Philadelphia, PA, USA).

2.3. Characterization of H. smyrnensis WMS-3 Bacteria

2.3.1. Cell Morphology, Gram Staining, and Ammonia Test The morphology of H. smyrnensis WMS-3 bacteria was analyzed by scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). The detailed steps for performing the SEM and Gram staining experiments were according to the published reference by Han et al. [36]. The ammonia test was performed as reported by Zhuang et al. [37].

2.3.2. Bacterial Growth Curve and pH Changes To determine the optimum salt concentration for the growth of H. smyrnensis WMS-3 bacteria, the culture mediums with 3%, 5%, 10%, 15%, 20%, 25%, and 30% NaCl concentration were prepared, respectively. The bacterial seed (OD600 = 1.0) was inoculated into the above mediums at an inoculation volume ratio of 1%, and then these cultures were cultivated in an oscillating incubator (HZQ-F160, Harbin Electronic Technology Development Co., Ltd., Harbin, China) with a speed of 120 rpm at 37 ◦C, after 24 h, the concentration of H. smyrnensis WMS-3 bacteria was measured by a spectrophotometer (722 s, Shanghai Analysis Instrument Factory, Shanghai, China) at a wavelength of 600 nm. From now on, WMS-3 bacteria were cultured in a medium with the optimum NaCl concentration to perform all the following experiments. The cell concentration was measured according to the above steps to draw the growth curve, and the pH values of the experimental and control groups were measured by a pH indicator (PHS-3C, Shanghai Shengke Instrument Equipment Co., Ltd., Shanghai, China). The diﬀerence between the experimental and control group was that the experimental group was inoculated with a bacterial seed (OD600 = 1.0) at an inoculation volume ratio of 1%, and the control group was inoculated with sterile distilled water at the same volume ratio of 1%. The diﬀerence in pH values between the experimental and control group was analyzed by the SPSS 21 software (Version 21.0, International Business Machines Corporation, Armonk, NY, USA).

+ + 2.3.3. NH4 Concentrations and pH Values Based on NH4 Concentrations + The concentration of NH4 ions in the liquid culture medium inoculated with WMS-3 bacteria + was measured using the method of Zhuang et al. [37]. Meanwhile, the pH values based on NH4 concentrations were also calculated by the method of Zhuang et al. [37]. Minerals 2019, 9, 632 4 of 26

2 2 2.3.4. CA Activity, CO3 − and HCO3− Concentrations, and pH Values Based on CO3 − and HCO3− Concentrations In this study, 1 unit of CA activity (U) was deﬁned as the amount of enzyme required to release 1 µmol of p-nitrophenol per min and 1 L. The detailed steps for analyzing the CA activity and measuring 2 the concentrations of CO3 − and HCO3− ions were all according to the methods of Zhuang et al. [37]. 2 To further determine the role of CO3 − and HCO3− concentrations on pH changes (Na2CO3 + NaHCO3) 2 solutions were prepared based on the diﬀerent CO3 − and HCO3− concentrations, and then the pH values of (Na2CO3 + NaHCO3) solutions were measured.

2.4. Biomineralization Experiments In the bacterially-induced biomineralization experiments, the components of the culture medium were the same as the above 2.1.; besides this, NaCl concentration was 3%, Ca2+ concentration was 0.01 M, and the Mg/Ca molar ratios were set to 0, 2, 5, 7, and 9 by using calcium chloride and magnesium chloride, respectively. The group inoculated with a bacterial seed (OD600 = 1.0) at an inoculation volume ratio 1% was set as the experimental group, and the other group inoculated with sterile distilled water at the same inoculation volume ratio for the control group. The pH values in these two groups were all adjusted to 7.0. All the cultures were incubated in a constant temperature shaker (HZQ-F160, Harbin Electronic Technology Development Co., Ltd., Harbin, China) with a speed of 115 rpm at 37 ◦C. The above experiments were performed in triplicate.

2.5. Concentrations of Ca2+ and Mg2+ Ions The concentrations of Ca2+ and Mg2+ ions in the experimental group were measured by atomic absorption spectrometry (AAS, TAS-986, Zhengzhou Nanbei Instrument Equipment Co., Ltd., Zhengzhou, Henan Province, China) according to the published articles [38,39]. The precipitation ratio of Ca2+ and Mg2+ ions could be calculated by the following formula:

C Ct T = i − 100% (1) Ci ×

Here, T represented the precipitation ratio (%, namely the ratio of the mass/volume concentration), Ci the initial concentration (mg/L), and Ct the ﬁnal concentration (mg/L).

2.6. Characterization of Precipitates The mineral precipitates in the experimental group were taken out from the conical flask and thoroughly washed with distilled water, and then dried at room temperature. The filter membrane with pore size 0.22 µm was used to separate the minerals from the liquid culture medium in the control group without bacteria; no minerals were obtained. Thus, the study on the control group was terminated. The obtained precipitates in the experimental group were analyzed by X-ray diffraction (XRD, D/Max-RC, Rigaku Corporation, Japan) with the scanning angle (2θ) ranging from 10 to 60◦, 1 a step size of 0.02◦ and a count time of 8◦ min− . The XRD data were analyzed by Jade 6.5 software (Version 6.5, Materials Data Ltd., Livermore, CA, USA). The weight ratio of each mineral at different Mg/Ca molar ratios was calculated by Rietveld refinement in Materials Studio 8.0. The full width at half maximum (FWHM) of monohydrocalcite at Mg/Ca molar ratios 7 and 9 could be obtained by Jade 6.0. The mole percent of MgCO3 in Mg-rich calcite was calculated using the following equation [40]:

MMgCO3 xMgCO3 = = 3.6396d104 + 11.0405 (2) MMgCO3 + MCaCO3 −

Here, MMgCO3 and MCaCO3 represented the moles of MgCO3 and CaCO3 per formula unit of Mg-rich calcite, and d104 indicated the d(104) (Å) value of Mg-rich calcite. Minerals 2019, 9, 632 5 of 26

To further test the mineral phase and the functional groups in/on the minerals, the precipitates were further analyzed by Fourier transform infrared spectroscopy (FTIR, Nicolet 380, Thermo Fisher 1 Scientific Inc., Waltham, MA, USA) within the scanning range of 4000–400 cm− with a resolution of 1 4 cm− . The morphology and elemental composition of the precipitates were determined by scanning electron microscopy (SEM, Hitachi S-4800, Japan Hitachi Company, Tokyo, Japan) with an X-ray energy dispersive spectrometer (EDS, EX-450, Japan Horiba, Tokyo, Japan). To determine the stable carbon isotope composition, the obtained precipitates were pretreated using the method of Zhuang et al. [37], and then sent to the Center for Isotope Geochemistry and Geochronology (National Laboratory for Marine Science and Technology, Qingdao, China), and analyzed by a carbon isotope laser spectrometer (Picarro G2121-i, rPicarro Inc., Santa Clara, CA, USA) with the heating and phosphoric acid method. The precipitates at Mg/Ca molar ratios 7 and 9 were also analyzed by a thermogravimetric analyzer (TGA/DSC1/1600LF, Mettler Toledo Co., Schwerzenbach, Switzerland) in the temperature 1 range 50–1000 ◦C at a heating rate of 10 ◦C min− . The chemical characteristics of the mineral surface were analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Scientific Escalab ESCALAB 250XI, Thermo Fisher Scientific, Waltham, MA, USA) fitted with a non-monochromatic Al-Kα X-ray source. The survey (wide) spectra were collected from 1350 to 0 eV with a step size of 0.2 eV, and more detailed scans for elements Ca, O, C, N, S, and P were performed over the regions of interest. Avantage2.16 software (2.16, Thermo Fisher, Shanghai, China) was used to analyze the XPS spectra core-level lines for curve fitting. All spectra were referenced to the O 1s peak of carbonate at 530.9 eV [41].

2.7. Amino Acid Composition of EPS EPS of H. smyrnensis WMS-3 bacteria were extracted with the heating method of Morgan et al. [42] and Zhuang et al. [37], then the obtained EPS solution was dried in a lyophilizer (FD-1A-50, Shanghai Bilang Instrument Manufacturing Co. Ltd., Shanghai, China) at 60 C under vacuum conditions, − ◦ after that, the EPS powder was sent to Jiangsu Coastal Chemical Analysis & Technological Service Ltd. and analyzed by an amino acid analyzer (L-8900, Hitachi, Tokyo, Japan).

2.8. Analyses of Ultrathin Slices of H. smyrnensis WMS-3 Bacteria Ultrathin slices of H. smyrnensis WMS-3 bacteria with a thickness of 70 nm were prepared with the method reported by Han et al. [36], and then observed by high-resolution transmission electron microscopy (HRTEM, JEM-2100, Japan Electronics Company, JEOL, Tokyo, Japan) [43–47], selected area electron diﬀraction (SAED) [48,49], scanning transmission electron microscope (STEM, Tecnai G2 F20, FEI, Hillsboro, OR, USA), and elemental mapping.

2.9. Fluorescence Intensity of Intracellular Ca2+ Ions Fluorescence indicator Fluo-3 AM was used to mark intracellular Ca2+ ions of WMS-3 bacteria cultured at Mg/Ca molar ratios 0, 2, 5, 7, and 9, also including WMS-3 bacterial seed. At the same time, these above cells without being marked by Fluo-3 AM (acetoxymethyl ester form, Molecular Probes) were considered to be control groups. The detailed steps for the use of Fluo-3 AM were according to the method of Han et al. [50]. The ﬂuorescence intensity was measured by a ﬂuorescence spectrophotometer (FluoroMax-4, Horiba Jobin Yvon, France). Statistical analysis was performed by SPSS 21 software (Version 21.0, International Business Machines Corporation, Armonk, NY, USA). Minerals 2019, 9, 632 6 of 26

3. Results and Discussions

3.1. 16S rDNA Identiﬁcation of WMS-3 Bacteria The 16S rDNA sequence of WMS-3 bacteria was detected to be 1449 bp in length and uploaded to MineralstheMinerals 2019 GenBank ,2019 9, x ,FOR 9, withx FORPEER anPEER REVIEW accession REVIEW number MH425323. By BLAST analysis, WMS-3 bacteria shared6 of 626 100%of 26 homology with a large number of bacteria belonging to the genera H. smyrnensis. The phylogenetic relationshiprelationship with with H. smyrnensisH. smyrnensis species. species. Thus, Thus, the theWMS-3 WMS-3 strain strain belongs belongs to ato species a species of H.of H. tree shown in Figure1 indicates that the WMS-3 strain had the closest genetic relationship with smyrnensis.smyrnensis. H. smyrnensis species. Thus, the WMS-3 strain belongs to a species of H. smyrnensis.

FigureFigure 1. Phylogenetic 1. Phylogenetic tree tree of the of H. the smyrnensis H.H. smyrnensis WMS-3 WMS-3 bacteria bacteria constructe constructedconstructed withd with neighbor-joining neighbor-joiningneighbor-joining methodmethod based based on 16S on rDNA 16S rDNA sequence sequence alignment. alignment. 3.2. Characterization of H. smyrnensis WMS-3 Bacteria 3.2. 3.2.Characterization Characterization of H. of smyrnensis H. smyrnensis WMS WMS‐3 Bacteria‐3 Bacteria 3.2.1. Cell Morphology, Gram Staining, and Ammonia Test 3.2.1.3.2.1. Cell Cell Morphology, Morphology, Gram Gram Staining, Staining, and andAmmonia Ammonia Test Test The H. smyrnensis WMS-3 bacterium is about 2 µm in length, and 0.5 µm in width (Figure2a). The H. smyrnensis WMS-3 bacterium is about 2 μm in length, and 0.5 μm in width (Figure 2a). The resultThe H. of smyrnensis Gram staining WMS-3 shows bacterium a red color is about (Figure 22 μb),m indicating in length, that and H.0.5 smyrnensis μm in widthWMS-3 (Figure bacteria 2a). The result of Gram staining shows a red color (Figure 2b), indicating that H. smyrnensis WMS-3 areThe Gram-negative. result of Gram It staining can be clearly shows seen a red that color the liquid(Figure in the2b), experimentalindicating that group H. (thesmyrnensis tube on WMS-3 the left bacteria are Gram-negative. It can be clearly seen that the liquid in the experimental group (the tube inbacteria Figure are2c) Gram-negative. is turbid due to theIt can presence be clearly of H.seen smyrnensis that the liquidWMS-3 in bacteria,the experimental whereas thegroup liquid (the in tube the on the left in Figure 2c) is turbid due to the presence of H. smyrnensis WMS-3 bacteria, whereas the controlon the left group in Figure (the tube 2c) onis turbid the right due in to Figure the presence2c) is transparent of H. smyrnensis before addingWMS-3 thebacteria, Nessler’s whereas reagent. the liquid in the control group (the tube on the right in Figure 2c) is transparent before adding the Afterliquid adding in the thecontrol Nessler’s group reagent, (the tube the on color the of right the experimental in Figure 2c) group is transparent changedto before yellowish-brown adding the Nessler’s reagent. After adding the Nessler’s reagent, the color of the experimental group changed (theNessler’s tube onreagent. the left After in Figure adding2d), the and Nessler’s the control reag groupent, the (the color tube of on the the experimental right in Figure group2d) becamechanged to yellowish-brown (the tube on the left in Figure 2d), and the control group (the tube on the right yellowto yellowish-brown that is the color (the oftube the on Nessler’s the left in reagent. Figure Through2d), and the these control series group of color (the changes, tube on itthe can right be in Figure 2d) became yellow that is the color of the Nessler’s reagent. Through these series of color concludedin Figure 2d) that became the H. smyrnensisyellow thatWMS-3 is the color has the of the ability Nessler’s to release reagent. ammonia. Through these series of color changes,changes, it can it canbe concluded be concluded that thatthe H.the smyrnensis H. smyrnensis WMS-3 WMS-3 has hasthe abilitythe ability to release to release ammonia. ammonia.

Figure 2. Morphology in SEM view (a), Gram staining in a biological microscope view (b), FigureFigure 2. Morphology 2. Morphology in SEM in SEM view view (a), Gram(a), Gram staining staining in a inbiological a biological microscope microscope view view (b), (andb), and and ammonia test (c,d). (c) before adding the Nessler’s reagent (d), after adding the Nessler’s ammoniaammonia test test(c and (c andd). (dc)). before(c) before adding adding the Nessler’sthe Nessler’s reagent reagent (d), (afterd), after adding adding the Nessler’sthe Nessler’s reagent. (left: the experimental group; right: the control group). reagent.reagent. (left: (left: the experimental the experimental group; group; right: right: the control the control group). group).

3.2.2.3.2.2. The TheGrowth Growth Curve Curve of H. of Smyrnensis H. Smyrnensis WMS-3 WMS-3 and andpH VariationpH Variation It wasIt was not notdifficult difficult to find to find from from the growththe growth curve curve of the of H.the smyrnensis H. smyrnensis WMS-3 WMS-3 bacteria bacteria under under differentdifferent salt saltconcentrations concentrations that thatthe optimumthe optimum NaCl NaCl concentration concentration was was 3% (Figure3% (Figure 3a). 3a).As shown As shown in in FigureFigure 3b, the3b, growththe growth curve curve of WMS-3 of WMS-3 bacteria bacteria was wa divideds divided into intofour four periods: periods: the delay/adaptionthe delay/adaption phasephase (0–4 (0–4 h), the h), logarithmicthe logarithmic growth growth phase phase (4–50 (4–50 h), the h), stationarythe stationary phase phase (50–100 (50–100 h), and h), andthe declinethe decline phasephase (100–453 (100–453 h) [51].h) [51]. The ThepH pHvalue value of theof theexpe experimentalrimental group group (the (thegreen gr eencurve) curve) eventually eventually reachedreached around around 9.0, 9.0,while while that thatof theof thecontrol control group group (the (theblack black curve) curve) kept kept at 7.0 at or7.0 so,or almostso, almost unchangedunchanged (Figure (Figure 3b). 3b).The Thedifference difference in pH in pHvalue va luebetween between the theexperimental experimental and andcontrol control group group Minerals 2019, 9, 632 7 of 26

3.2.2. The Growth Curve of H. smyrnensis WMS-3 and pH Variation It was not difficult to find from the growth curve of the H. smyrnensis WMS-3 bacteria under different salt concentrations that the optimum NaCl concentration was 3% (Figure3a). As shown in Figure3b, the growth curve of WMS-3 bacteria was divided into four periods: the delay /adaption phase (0–4 h), the logarithmic growth phase (4–50 h), the stationary phase (50–100 h), and the decline Mineralsphase (100–4532019, 9, x FOR h) [PEER51]. TheREVIEW pH value of the experimental group (the green curve) eventually reached7 of 26 around 9.0, while that of the control group (the black curve) kept at 7.0 or so, almost unchanged was(Figure significant3b). The (p di <ff erence0.01), further in pH valuedemonstrating between thethe experimental important role and played control by group WMS-3 was bacteria. significant In the(p

1.8 10.5 1.4 (b) (a) 10.2

） 1.6 1.2 9.9 1.4 cfu/mL)

9 9.6 cfu/mL 9 1.0 1.2 9.3 9.0 0.8 1.0 8.7 pH 0.6 0.8 8.4 pH of the Na CO +NaHCO 2 3 3 8.1 0.6 pH of the control group 0.4 7.8 pH of the experimental group 0.2 0.4 7.5 Cell concentration 7.2 Cell concentration (0.8×10 Cell concentration

Cell concentration(0.8*10 0.2 0.0 6.9 0.0 6.6 0 5 10 15 20 25 30 0 50 100 150 200 250 300 350 400 450 NaCl concentration (%) Time (h)

8.6 0.25 0.045 - 7 (c) (d) HCO3 concentration 8.4 0.20 0.040 6

mol/L) 2- ) -5 CO concentration 0.035 3 5 8.2 0.15 CA activity mol/L ( 0.030 4 8.0 0.10 0.025 3 pH 7.8 0.05 0.020 2 + 7.6 pH based on NH concentration 0.00 4 0.015 1 + Enzyme (U/L) activity Ion concentration concentration Ion

NH concentration ion concentration (*10

7.4 4 -0.05 + 4 0.010 0

7.2 -0.10 NH 0.005 -1 0 20 40 60 80 100 120 140 160 180 200 0 40 80 120 160 200 240 280 Time (h) Time (h)

FigureFigure 3. 3. PhysiologicalPhysiological and and biochemical biochemical characteristics characteristics of of H.H. smyrnensis smyrnensis WMS-3WMS-3 bacteria. bacteria. ( (aa)) Growth Growth curvecurve of of the the H.H. smyrnensis smyrnensis WMS-3WMS-3 bacteria bacteria at at different diﬀerent NaCl NaCl concentrations concentrations after after 24 24 h h of of cultivation; cultivation; + + ((bb)) GrowthGrowth curve curve and and pH changes;pH changes; (c) NH 4(c) concentrationsNH4+ concentrations and pH curve and basedpH curve on NH 4basedconcentrations; on NH4+ 2 concentrations;(d) CA activity, ( andd) CA HCO activity,3−, and and CO HCO3 − ion3−, and concentrations. CO32− ion concentrations. 3.2.3. Ammonium Concentration and pH Value Based on the Concentration of Ammonium 3.2.3. Ammonium Concentration and pH Value Based on the Concentration of Ammonium According to the results in 3.2.1, ammonia could be released by WMS-3 bacteria, which would According to the results in 3.2.1, ammonia could be released by WMS-3 bacteria, which would then be dissolved in the liquid medium to produce ammonium and hydroxyl ions. The chemical then be dissolved in the liquid medium to produce ammonium and hydroxyl ions. The chemical reaction would be as follows: reaction would be as follows: + NH + H O NH + OH− (3) 3 2 → 4 → + − NH32 + H O NH 4 + OH (3)

The amount of ammonia released by WMS-3 bacteria can be expressed in terms of ammonium concentration. In this study, the concentration of ammonium was almost zero before 24 h and increased sharply in the time range of 24–74 h, eventually reaching a stable state (Figure 3c). The pH value based on ammonium ion concentration increased from 7.3 to the maximum value 8.34 (Figure 3c), while that of the experimental group could reach about 8.9—much higher than the former 8.3 (p < 0.01). That is to say, the quantity of ammonia release by WMS-3 bacteria could not satisfy the requirement of pH increasing to 8.9, suggesting that there was something else affecting the pH increase besides ammonia in the experimental group. Our results are consistent with the Minerals 2019, 9, 632 8 of 26

The amount of ammonia released by WMS-3 bacteria can be expressed in terms of ammonium concentration. In this study, the concentration of ammonium was almost zero before 24 h and increased sharply in the time range of 24–74 h, eventually reaching a stable state (Figure3c). The pH value based on ammonium ion concentration increased from 7.3 to the maximum value 8.34 (Figure3c), while that of the experimental group could reach about 8.9—much higher than the former 8.3 (p < 0.01). That is to say, the quantity of ammonia release by WMS-3 bacteria could not satisfy the requirement of pH increasing to 8.9, suggesting that there was something else aﬀecting the pH increase besides ammonia in the experimental group. Our results are consistent with the conclusions reported by Han et al. [36,50,52] and Zhuang et al. [37]. Early, it is a general recognition that the ammonia released by bacteria is the main reason for the pH increase in an environment with abundant bacteria [50]. Now, there is a much better and more scientiﬁc explanation for why microbes cause pH to rise.

2 3.2.4. CA Activity and the Concentration of CO3 − and HCO3− Ions

CA enzymes have gained considerable attention for their potential use in CO2 capture [53], and recently they have been used as a biocatalyst to sequester CO2 through the transformation of CO2 to 2 HCO3− and CO3 − ions in the CaCO3 mineralization process [54]. At present, a consensus that CA can accelerate the rate of carbonate mineral formation has been reached [55]. Based on this important role played by CA, as mentioned above, CA released by WMS-3 bacteria was also studied. In this study, CA activity sharply increased from almost zero to 5.97 U/L in the time range 0–39 h, and then slowly decreased from 6 U/L to 1.43 U/L in the time range 39–267 h (Figure3d), that is to say, CA enzyme was released by WMS-3 bacteria. The most important point was that CA activity still existed in the decline stage of WMS-3 bacterial growth, indicating that maybe the pH increase in the decline stage was related to CA activity. CA can catalyze the hydration reaction of carbon dioxide to release bicarbonate and carbonate ions in the alkaline condition created by ammonia [37]. The reactions are listed as follows:

CA CO + H O H CO (4) 2 2 → 2 3

H CO + OH− HCO − + H O (5) 2 3 → 3 2 2 HCO − + OH− CO − + H O (6) 3 → 3 2 2 Since CA can catalyze the production of HCO3− and CO3 − ions, the concentration of HCO3− 2 and CO3 − ions were also measured in this study. The concentration of HCO3− ion first increased to 0.04 mol/L at 62 h and then decreased slowly in the time range 62–117 h, and finally kept almost 2 constant at 0.02 mol/L in the time range 117–260 (Figure3d). The concentration of CO 3 − ions could not be detected before 88 h, and then sharply increased to 0.029 mol/L at 237 h, and at last, reached a stable state in the time range 237–260 h (Figure3d). 2 Han et al. [36,50,52] and Zhuang et al. [37] have reported that the resulting HCO3− and CO3 − ions produced by the CA enzyme could lead to an increase in pH values in the culture medium. Thus, according to their methods, (NaCO3 + NaHCO3) solutions were prepared, corresponding to 2 the concentrations of HCO3− and CO3 − ions, and the pH values based on the (NaCO3 + NaHCO3) solutions were also measured. The result showed that the pH values based on the (NaCO3 + NaHCO3) solutions increased from 8.3 to 10.12 during the period 0–260 h (Figure3b), indicating that the changes 2 in the concentration of HCO3− and CO3 − ions also influenced the pH in the medium. Therefore, an increase in the pH values in the experimental group resulted from the presence of ammonia and the catalysis of CA, consistent with the results reported by Han et al. [36,50,52] and Zhuang et al. [37]. It could be there are other factors that increase pH, which need to be explored further.

3.3. Changes in the Concentration of Ca2+ and Mg2+ Ions Precipitates were produced in the experimental group, whereas no minerals were obtained in the control group. Thus, from this step onwards, the control group was not studied anymore. Minerals 2019, 9, 632 9 of 26

The concentrations of Ca2+ ions in the experimental group at Mg/Ca ratios 0, 2, 5, 7, and 9 showed the same trend, and decreased continuously from the original 400 mg/L to 142.74, 151.18, 159.12, 161.73, and 182.31 mg/L in the time range from 0–21 days, respectively (Figure4a). The precipitation ratio (%, from the ratio of the mass/volume concentration) of Ca2+ ion was 64.32%, 62.20%, 60.22%, 59.57%, and 54.42% at the Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively (Figure4c). The Mg 2+ ion 2+ Mineralsconcentration 2019, 9, x changedFOR PEER aREVIEW little (Figure 4b), and the precipitation ratio of Mg ion was 6.69%, 7.10%,9 of 26 7.74%, and 8.09% at Mg/Ca molar ratios of 2, 5, 7, and 9, respectively (Figure4d). 2+ 2+ InIn this this study, thethe CaCa2+ ionion concentration concentration was 0.01 M, farfar belowbelow thethe concentrationconcentration ofof thethe Mg Mg2+ ion, 2+ 2+ while the precipitation ratio of thethe CaCa2+ ionion was much higher than thatthat ofof thethe MgMg2+ ion, suggesting 2+ 2+ thatthat it was much more didifficultfficult forfor MgMg2+ ionsions to to participate participate in in the the bio-precipitation process thanthan CaCa2+ 2+ 2+ ions.ions. It It has has been reportedreported thatthat thethe hydratedhydrated cations, cations, such such as as Mg(H Mg(H2O)2O)6 62+ andand Ca(HCa(H22O)O)662+, ,needed needed to to 2 2 have thethe hydratedhydrated membrane membrane surrounding surrounding them them removed removed if they if they are to are form to Mg-COform Mg-CO3 − and32− Ca-CO and Ca-3 − 2+ COpairs32− [pairs56]. Moreover, [56]. Moreover, the energy the energy required required for the for dehydration the dehydration of Mg(H of2 O)Mg(H6 is2O) 351.862+ is kcal 351.8/mol, kcal/mol, higher 2+ higherthan that than of that Ca(H of2 O)Ca(H6 2(264.3O)62+ (264.3 kcal/ mol)kcal/mol) under under standard standard conditions conditions (298 K,(298 1atm) K, 1atm) [57]. [57]. Thus, Thus, it is 2+ itmuch is much more more difficult difficult for Mg for Mgions2+ toions form to magnesiumform magnesium carbonate carbon minerals,ate minerals, due to due the higherto the energyhigher 2+ energyrequired required for the dehydration.for the dehydration. There are There also are other also opinions other opinions as to why as it to is why diffi cultit is fordifficult the Mg for theion 2+ Mgto be2+ precipitatedion to be precipitated as magnesium as carbonatemagnesium minerals. carbonate Mg minerals.ions with Mg no2+ hydrated ions with membrane no hydrated in dry membraneformamide in still dry do formamide not form magnesium still do not carbonateform magnesium crystals, carbonate and the reason crystals, given and was the that reason the latticegiven 2 2+ 2 waslimitation that the resulting lattice fromlimitation the spatial resulting configuration from the ofspatial CO3 configuration− groups prevented of CO Mg32− groupsand COprevented3 − ions Mgfrom2+ and forming CO32 crystalline− ions from structures forming crystalline [58]. There structures are many [58]. factors There affecting are many themineralization factors affecting of the 2+ mineralizationMg ion, which of needthe Mg to2+ be ion, explored. which need to be explored.

2500 Mg/Ca=9 400 (a) (b)

2000 350 Mg/Ca=7

1500 300 Mg/Ca=5

1000 250 Mg/Ca=0 Mg/Ca=2 concentration (mg/L) concentration

Mg/Ca=2 2+ concentration (mg/L) concentration 2+ 200 Mg/Ca=5 Mg 500 Ca Mg/Ca=7 Mg/Ca=0 150 Mg/Ca=9 0

0 3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 Time (d) Time (d) 70 Mg/Ca=0 y=-0.0288x2+3.7351x-2.34 R2=0.9957 (c) Mg/Ca=0 (d) Mg/Ca=2 y=-0.0113x2+3.3544x-2.5233 R2=0.9924 8 Mg/Ca=2, y=0.0076x2+0.1918x-0.3771, R2=0.9915 Mg/Ca=5 y=-0.0405x2+3.9492x-4.4366 R2=0.9962 Mg/Ca=5, y=0.0119x2+0.1199x-0.4222, R2=0.9872 60 2 2 2 2 Mg/Ca=7 y=-0.0097x +3.2883x-4.2243 R =0.9979 Mg/Ca=7, y=0.0039x +0.3185x-0.4936, R =0.9968 Mg/Ca=9 y=-0.0191x2+3.1688x-3.7637 R2=0.9994 Mg/Ca=9, y=-0.0002x2+0.4532x-1.026, R2=0.9852 50 6

40 4 30

20 precipitation ratio (%)

precipitation ratio (%) ratio precipitation 2 2+ 2+

Ca 10 Mg 0 0

036912151821 0 3 6 9 12 15 18 21 Time (d) Time (d) Figure 4. Changes of Ca2+ concentration (a), Mg2+ concentration (b), Ca2+ precipitation ratio (c), and Figure 4. Changes of Ca2+ concentration (a), Mg2+ concentration (b), Ca2+ precipitation ratio (c), and Mg2+ precipitation ratio (d). Mg2+ precipitation ratio (d).

3.4. Characterization of the Biominerals

3.4.1. XRD Analyses In this study, no precipitates were obtained in the control group; thus, only the experimental group needed to be studied. At Mg/Ca = 0, the biomineral was only calcite, syn (PDF-# 05-0586) (Figures 5 and S1a). At Mg/Ca = 2, Mg-rich calcite (PDF-# 43-0697), aragonite (PDF-# 41-1475), and monohydrocalcite (PDF-# 29-0306) minerals were obtained (Figure 5); the weight percent of Mg-rich calcite, aragonite and monohydrocalcite was 62.93%, 0.01%, and 37.06%, respectively (Figure S1b); 9.09% MgCO3 (molar ratio) was present in the Mg-rich calcite (Figure 5). At Mg/Ca = 5, aragonite (PDF-# 41-1475), monohydrocalcite (PDF-# 29-0306), and hydromagnesite (PDF-# 25-0513) minerals were found (Figure 5); the weight ratio of hydromagnesite was 61.82%, monohydrocalcite 24.38%, Minerals 2019, 9, x FOR PEER REVIEW 10 of 26 and aragonite only 13.80% through Rietveld refinement (Figure S1c). At Mg/Ca molar ratios 7 and 9, the minerals were monohydrocalcite (PDF-# 29-0306) and hydromagnesite (PDF-# 25-0513) (Figure 5). When the Mg/Ca molar ratio changed from 7 to 9, the weight percent of hydromagnesite decreased from 76.15% to 9.99%, and that of monohydrocalcite increased from 23.85% to 90.01% (Figure S1d,e). What is more, the FWHM (o) of monohydrocalcite at Mg/Ca = 7 was higher than that at Mg/Ca = 9 (Table S1), indicating that monohydrocalcite had a much better crystalline structure when the Mg/Ca molar ratio increased from 7 to 9. Monohydrocalcite had previously only been found in fresh-water or terrestrial environments before researchers documented the presence of submarine monohydrocalcite [28]. In this study, the formationMinerals 2019 of, 9, monohydrocalcite 632 induced by WMS-3 bacteria was in a liquid medium with 3% 10NaCl, of 26 which is similar to seawater, also demonstrating the fact that monohydrocalcite can be formed in an environment rich in salt in the lab. Mg plays an important role in the formation and stability of monohydrocalcite3.4. Characterization [59]. of the It Biominerals has been reported that the crystalline monohydrocalcite can be easily prepared at a certain Mg/Ca molar ratio (>1), and that a small amount of Mg2+ ion can be 3.4.1. XRD Analyses incorporated into the lattice structure of monohydrocalcite to stabilize the crystal water [60,61]. In this study,In this study,the concentration no precipitates of werethe Mg obtained2+ ion inslowly the control decreased group; (Figure thus, only 4b,d), the experimentalmaybe due to group the incorporationneeded to be studied.of Mg At2+ Mgion/ Cainto= 0,the the biomineralmonohydrocalcite was only cr calcite,ystals synbesides (PDF-# the 05-0586) formation (Figure of5 hydromagnesite.and Figure S1a). However, At Mg/Ca Han= 2, et Mg-rich al. [36,50] calcite used (PDF-#Bacillus 43-0697),licheniformis aragonite DB1-9 and (PDF-# Bacillus 41-1475), subtilis and J2 bacteriamonohydrocalcite to induce (PDF-#the formation 29-0306) of minerals biominerals were at obtained different (Figure concentrations5); the weight of Mg percent2+ ions, of Mg-richand the obtainedcalcite, aragonite mineral andis aragonite monohydrocalcite and not monohydrocalcite was 62.93%, 0.01%, in the and presence 37.06%, respectivelyof Mg, a different (Figure result S1b); from9.09% ours. MgCO Moreover,3 (molar ratio)Han et was al. present[52] found in the that Mg-rich the obtained calcite (Figuremineral5 ).is Atmonohydrocalcite Mg /Ca = 5, aragonite when using(PDF-# the 41-1475), halophile monohydrocalcite Staphylococcus epidermis (PDF-# 29-0306),Y2 in a medium and hydromagnesite with higher concentrations (PDF-# 25-0513) of Mg minerals2+ ions, thewere same found as (Figure ours. 5Thus,); the weighta very ratio important of hydromagnesite question has was arisen: 61.82%, since monohydrocalcite Mg is present 24.38%,in the environmentand aragonite and only plays 13.80% an through important Rietveld role reﬁnementin monohydrocalcite (Figure S1c). formation, At Mg/Ca why molar is ratiosthe resulting 7 and 9, mineralthe minerals not necessarily were monohydrocalcite monohydrocal (PDF-#cite? In 29-0306) our opinion, and hydromagnesite the formation of (PDF-# monohydrocalcite 25-0513) (Figure is not5). Whenonly related the Mg to/Ca Mg molar but also ratio closely changed tied fromto the 7 micr to 9,obial the weight species. percent It has been of hydromagnesite reported that the decreased release offrom Mg 76.15%2+ ions tocan 9.99%, induce and the that phase of monohydrocalcite transformation of increased monohydrocalcite from 23.85% to toanhydrous 90.01% (Figure aragonite S1d,e). in Whataqueous is more,systems the [62]. FWHM Thus, (o )it of could monohydrocalcite be inferred that at some Mg/Ca bacteria= 7 was would higher make than more that atMg Mg2+ /ionsCa = be9 incorporated(Table S1), indicating into carbonate that monohydrocalcite minerals than other had aba muchcteria. better In this crystalline case, the structureformer was when conducive the Mg/ Cato themolar formation ratio increased of monohydrocalcite from 7 to 9. and the latter to the formation of aragonite.

3 3 Mg/Ca=9 3 3 3 3 3 3 3 5 3 3 5 5-Hydromanesite 3 3 3 Mg/Ca=7 3 3 5 3 3 5 3 3 3 3 3 4-Aragonite 3 5 3 3 3 Mg/Ca=5 5 3 44 4 3 5 3 2 Intensity (cps) Intensity 3-Monohydrocalcite XMgCO3=9.089% Mg/Ca=2 3 3 3 3 3 22 3 2 3 2 2 2 44 33 1 2-Calcite, Mg-rich Mg/Ca =0 1-Calcite, syn

1 1 1 1 1 1 1

10 15 20 25 30 35 40 45 50 55 60 2θ (°)

Figure 5. XRD analyses of biominerals cultured for 30 days.

Monohydrocalcite had previously only been found in fresh-water or terrestrial environments before researchers documented the presence of submarine monohydrocalcite [28]. In this study, the formation of monohydrocalcite induced by WMS-3 bacteria was in a liquid medium with 3% NaCl, which is similar to seawater, also demonstrating the fact that monohydrocalcite can be formed in an environment rich in salt in the lab. Mg plays an important role in the formation and stability of monohydrocalcite [59]. It has been reported that the crystalline monohydrocalcite can be easily prepared at a certain Mg/Ca molar ratio (>1), and that a small amount of Mg2+ ion can be incorporated Minerals 2019, 9, 632 11 of 26 into the lattice structure of monohydrocalcite to stabilize the crystal water [60,61]. In this study, the concentration of the Mg2+ ion slowly decreased (Figure4b,d), maybe due to the incorporation of Mg 2+ ion into the monohydrocalcite crystals besides the formation of hydromagnesite. However, Han et al. [36,50] used Bacillus licheniformis DB1-9 and Bacillus subtilis J2 bacteria to induce the formation of biominerals at diﬀerent concentrations of Mg2+ ions, and the obtained mineral is aragonite and not monohydrocalcite in the presence of Mg, a diﬀerent result from ours. Moreover, Han et al. [52] found that the obtained mineral is monohydrocalcite when using the halophile Staphylococcus epidermis Y2 in a medium with higher concentrations of Mg2+ ions, the same as ours. Thus, a very important question has arisen: since Mg is present in the environment and plays an important role in monohydrocalcite formation, why is the resulting mineral not necessarily monohydrocalcite? In our opinion, the formation of monohydrocalcite is not only related to Mg but also closely tied to the microbial species. It has been reported that the release of Mg2+ ions can induce the phase transformation of monohydrocalcite to anhydrous aragonite in aqueous systems [62]. Thus, it could be inferred that some bacteria would make more Mg2+ ions be incorporated into carbonate minerals than other bacteria. In this case, the former was conducive to the formation of monohydrocalcite and the latter to the formation of aragonite.

3.4.2. FTIR Analyses

1 The characteristic bands of calcite are 712, 875, 1421, and 2514 cm− [36], and those of aragonite 1 1 are 710, 856, 1082, and 1475 cm− [63]. 700, 766, 872, 1060, 1401, and 1492 cm− are the characteristic bands of monohydrocalcite [63], and the characteristic bands 592, 796, 854, 877, 1426, 1488, 3649 belong to hydromagnesite [64]. 1 The bands 712, 875, 1421, and 2514 cm− of minerals at the Mg/Ca ratio 0 indicate the presence of 1 calcite (Figure6a); at Mg /Ca = 2, besides the bands 712, 875, and 2514 cm− of calcite (Mg-rich), there 1 1 1 are the bands 700, 1060, 1401, and 1492 cm− of monohydrocalcite; 712 cm− and 1492 cm− also belong to aragonite (Figure6b). At Mg /Ca = 5, besides the characteristic bands of monohydrocalcite (700, 766, 1 1 1 872, and 1492 cm− ), the bands 710 cm− and 1082 cm− prove the formation of aragonite (Figure6c). 1 592, 796, and 1426 cm− indicate the presence of hydromagnesite (Figure6c). At Mg /Ca = 7 and 9, 1 the characteristic bands prove that the minerals are monohydrocalcite (700, 766, 872, 1492 cm− ) and 1 hydromagnesite (592, 796, 1426 cm− ) (Figure6d,e). The mineral phases obtained by FTIR are consistent with the results of XRD. Moreover, besides these characteristic bands of minerals, organic functional 1 1 groups are also found to be present in/on the minerals, including C=O (1620 cm− , 1638 cm− )[36,65] 1 and C–O–C (1068 cm− )[66] (Figure6a–e). Previously, some scholars have proposed that the C–O–C group plays an important role in biomineralization [66]. Our results shown in Figure6a–e are further confirming the previous opinion. The C–O–C functional group present in/on the minerals also confirms the biogenesis of these minerals. Some functional groups associated with biomacromolecules from bacteria could play important roles in the formation of the peculiar morphology of the biominerals in the biomineralization process. It has been reported that F68, composed of predominant –C–O–C– and a terminal –OH groups, was used as a model organic additive to study the biomimetic mineralization of calcite, and the results showed that the terminal hydroxyl group in F68 had a negligible contribution to the morphogenesis of calcite due to its much lower content, and the –C–O–C– groups (ether or glycosidic group) contributed to the formation of special calcite, like the elongated calcite and the bundle-shaped calcite [67]. In our study, the –C–O–C– groups indeed present in/on the minerals (Figure6a–e) has been confirmed, and the –C–O–C– groups would also result in some changes in the morphology of biominerals according to Zhou’s opinion. Thus, the morphology and elemental composition of the biominerals was also studied. Minerals 2019, 9, x FOR PEER REVIEW 11 of 26

Figure 5. XRD analyses of biominerals cultured for 30 days.

3.4.2. FTIR Analyses

The characteristic bands of calcite are 712, 875, 1421, and 2514 cm−1 [36], and those of aragonite are 710, 856, 1082, and 1475 cm−1 [63]. 700, 766, 872, 1060, 1401, and 1492 cm−1 are the characteristic bands of monohydrocalcite [63], and the characteristic bands 592, 796, 854, 877, 1426, 1488, 3649 belong to hydromagnesite [64]. The bands 712, 875, 1421, and 2514 cm−1 of minerals at the Mg/Ca ratio 0 indicate the presence of calcite (Figure 6a); at Mg/Ca = 2, besides the bands 712, 875, and 2514 cm−1 of calcite (Mg-rich), there are the bands 700, 1060, 1401, and 1492 cm−1 of monohydrocalcite; 712 cm−1 and 1492 cm−1 also belong to aragonite (Figure 6b). At Mg/Ca = 5, besides the characteristic bands of monohydrocalcite (700, 766, 872, and 1492 cm−1), the bands 710 cm−1 and 1082 cm−1 prove the formation of aragonite (Figure 6c). 592, 796, and 1426 cm−1 indicate the presence of hydromagnesite (Figure 6c). At Mg/Ca = 7 and 9, the characteristic bands prove that the minerals are monohydrocalcite (700, 766, 872, 1492 cm−1) and hydromagnesite (592, 796, 1426 cm−1) (Figure 6d,e). The mineral phases obtained by FTIR are consistent with the results of XRD. Moreover, besides these characteristic bands of minerals, organic functional groups are also found to be present in/on the minerals, including C=O (1620 cm−1, 1638 cm−1) [36,65] and C–O–C (1068 cm−1) [66] (Figure 6a–e). Previously, some scholars have proposed that the C–O–C group plays an important role in biomineralization [66]. Our results shown in Figure 6a–e are further confirming the previous opinion. The C–O–C functional group present in/on the minerals also confirms the biogenesis of these minerals. Some functional groups associated with biomacromolecules from bacteria could play important roles in the formation of the peculiar morphology of the biominerals in the biomineralization process. It has been reported that F68, composed of predominant –C–O–C– and a terminal –OH groups, was used as a model organic additive to study the biomimetic mineralization of calcite, and the results showed that the terminal hydroxyl group in F68 had a negligible contribution to the morphogenesis of calcite due to its much lower content, and the –C–O–C– groups (ether or glycosidic group) contributed to the formation of special calcite, like the elongated calcite and the bundle-shaped calcite [67]. In our study, the –C–O–C– groups indeed present in/on the minerals (Figure 6a–e) has been confirmed, and the –C–O–C– groups would also result in some changes in the morphology of biominerals according to Zhou’s opinion. Thus, the morphology and Mineralselemental2019 ,composition9, 632 of the biominerals was also studied. 12 of 26

110 110 (a) Mg/Ca=0 (b) Mg/Ca=2 105 100 100 90 1068 C-O-C 95 2514 1638 C=O 80 2514 700 90 1060 2974 C-H 712 70 85 712 60 1638 C=O Transmittance (a.u.) 80 875 Transmittance (a.u.) 875 1492 1401 1421 75 50 1620 C=O 1620 C=O 1068 C-O-C 70 40 4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 Minerals 2019, 9, x FOR PEER REVIEW -1 12 of 26 Wavenumber (cm-1) Wavenumber (cm ) 110 120 (c) 1082 Mg/Ca=5 (d) Mg/Ca=7 100 766 796 710 105 90 766 1068 3649 90 80 C-O-C 700 872 70 796 75 700

1638 C=O 872 592 1638 C=O 1068 C-O-C

Transmittance (a.u.) Transmittance 60 1620 C=O 60 1492 (a.u.)Transmittance 1492 1426 592 50 1426 1620 C=O 45 40 4000 3500 3000 2500 2000 1500 1000 500 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1) Wavenumber (cm-1) 120 (e) Mg/Ca=9 796 105 766

75 872 700 1068 C-O-C

Transmittance (a.u.) Transmittance 1638 C=O 592 60 14921426 1620 C=O 45

4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1) Figure 6. FTIR analysesanalyses ofof thethe biominerals biominerals cultured cultured for for 30 30 days. days. (a –(ea)), represents (b), (c), (d the), and minerals (e) represents cultured theat Mg minerals/Ca ratios cultured of 0, 2, at 5, Mg/Ca 7 and 9,ratios respectively. of 0, 2, 5, 7 and 9, respectively.

3.4.3. Morphology Morphology and and Elemental Elemental Composition Composition of the Biominerals Biogenic minerals tend to have a wide range of morphologies compared to physicochemical precipitates [[68].68]. The basic formform ofof thethe abioticabiotic calcite calcite is is always always a a rhomb rhomb [37 [37],], while while the the biotic biotic calcite calcite at ata Mga Mg/Ca/Ca ratio ratio of 0of in 0 this in studythis study showed showed several several morphologies, morphologies, including including cauliﬂower cauliflower (Figure (Figure7(a1)), 7(a1)),elongate elongate (Figure (Figure7(a3)) and7(a3)) dumbbell and dumbbell shapes shapes (Figure (Figure7(a4)). In7(a4)). addition, In addition, it would it would appear appear that some that somebacteria bacteria lived lived within within cavities cavities on the on mineral the mineral surface surface (Figure (Figure7(a2)). 7(a2)). The The elements elements of the of the mineral mineral in inFigure Figure7(a4) 7(a4) include include Ca, C,Ca, O, C, P, O, and P, S and (Figure S (Figure7(a5)). Ca,7(a5)). C, andCa, OC, elementsand O elements are within are the within mineral the mineralcalcite, andcalcite, P and and S elements P and mayS elements have come may from ha theve WMS-3come from bacteria, the bacterialWMS-3 metabolites,bacteria, bacterial or the metabolites,organic substances or the organic in the culture substances medium. in the culture medium. At Mg/Ca = 2, the surface of the spheroidal (Figure 7(b1,3)) and dumbbell-shaped minerals (Figure 7(b4)) are different from the spheroidal mineral in Figure 7(b2); the former is composed of a large number of nanometer-scale particles with no regular shape while the latter consist of many larger more regular-shaped minerals. The mineral in Figure 7(b3) contained Ca, C, O, P, S, and Mg elements (Figure 7(b5)). The origin of the Ca, C, O, P, and S is the same as the above, and Mg is from the culture medium. Compared with the Mg/Ca ratio 0, the morphology and elemental composition of minerals at Mg/Ca ratio 2 changed considerably, demonstrating the important role played by Mg. At Mg/Ca = 5, there are dumbbell-shaped minerals composed of many crystallites in the shape of a petal (Figure 7(c1)), spheroidal minerals covered with minute rectangular crystallites (Figure 7(c2)), irregular minerals bearing abundant holes where likely bacteria once lived (Figure 7(c3)), and elongate minerals (Figure 7(c4)). The elongate minerals contain Ca, C, O, P, S, and Mg elements (Figure 7(c5)), and the origin of all these elements is the same as the above. At Mg/Ca = 7, the spheroidal minerals (Figures 7(d1,4)) and elongate minerals (Figure 7(d3)) are all covered with many minute rectangular crystallites. The outer shell of the elongate minerals is composed by larger rectangular crystals, while the inner part consists of smaller rectangular crystals Minerals 2019, 9, x FOR PEER REVIEW 13 of 26

(Figure 7(d3)), indicating that the nucleation rate of minerals within the elongate mineral was much quicker than that of minerals located on the outer surface. The elements of the spheroidal minerals (Figure 7(d1)) include Ca, C, O, P, and S, and the origin of these elements is the same as the above. Too little to be detected perhaps is the reason for the absence of Mg in the EDS analyses. Besides the Mineralsabove 2019morphology,, 9, 632 petal-shaped minerals similar to that in Figure 7(c1) could also be observed13 of 26 (Figure 7(d2)). At Mg/Ca = 9, there are elongate minerals (Figure 7(e1)), dumbbell-shaped minerals (Figure 7(e2)),At and Mg /irregular-shapedCa = 2, the surface mineral of the aggregates spheroidal (Figure (Figure 7(e4)).7(b1,3)) The andaggregates dumbbell-shaped contain Ca, mineralsC, O, P, S, (Figureand Mg7(b4)) elements are di (Figureﬀerent from7(e5)), the and spheroidal the origin mineral of all these in Figure elements7(b2); is the the former same isas composed the above. of The a largeflower-shaped number of or nanometer-scale petal-shaped mineral particles is with still nopresent regular in shapethis culture while system the latter (Figure consist 7(e3)). of many larger moreIn regular-shaped brief, the diversity minerals. of morphology, The mineral complicated in Figure7(b3) elemental contained composition, Ca, C, O, P, S,and and bacteria Mg elements present (Figurewithin7 (b5)).holes Thein the origin minerals of the Ca,(Figure C, O, 7(a2), P, and (b1,4), S is the and same (c3)) as the further above, proved and Mg that is from these the resulting culture medium.minerals Comparedwere biogenic, with not the abiogenic. Mg/Ca ratio Researcher 0, the morphologys have many and different elemental views composition on whether of mineralsminerals atare Mg biogenic/Ca ratio according 2 changed to considerably, the typical morphology. demonstrating Some the researchers important rolehave played suggested by Mg. that spheroidal and Atdumbbell Mg/Ca = shapes5, there reflect are dumbbell-shaped their microbial minerals origin and composed may serve of many as biosignatures crystallites in thein shapethe rock of arecord petal (Figure[69]. However,7(c1)), spheroidal other researchers minerals covered have di withsagreed minute and rectangular proposed crystallitesthat minerals (Figure with7(c2)), such irregularstructures minerals can also bearing be produced abundant under holes abiogenic where likely culture bacteria conditions once lived[70]. (FigureTherefore,7(c3)), it is and necessary elongate to mineralsuse other (Figure detection7(c4)). methods, The elongate not only minerals the morpholo containgy, Ca, to C, determine O, P, S, and whether Mg elements minerals (Figure are biogenic7(c5)), andor not. the origin of all these elements is the same as the above.

Figure 7. SEM and EDS analyses of minerals cultured for 30 days. (a–e), Mg/Ca molar ratios 0, 2, 5, 7, and 9, respectively. The red square shows the area analyzed by EDS.

At Mg/Ca = 7, the spheroidal minerals (Figure7(d1,4)) and elongate minerals (Figure7(d3)) are all covered with many minute rectangular crystallites. The outer shell of the elongate minerals is composed by larger rectangular crystals, while the inner part consists of smaller rectangular crystals Minerals 2019, 9, 632 14 of 26

3.4.4. Stable Carbon Isotope Composition of the Biominerals Microorganisms can utilize the carbon source in the medium to produce metabolites, such as different kinds of organic acid and large amounts of carbon dioxide. The metabolite carbon dioxide is different from atmospheric carbon dioxide—the former originates from organic substances, and the latter comes from the inorganic atmosphere. The stable carbon isotope values of mineral formed by metabolite carbon dioxide produced by chemoheterotrophic microorganisms would likely be more negative than those of mineral formed via atmospheric carbon dioxide [71]. In this study, the δ13C values of the biotic carbonate minerals ranged from 16.91% to 17.91% (Table S2), more negative − − than that of atmospheric CO (δ13C, 8%)[72], but similar to that of beef extract ( 18.4%) and 2 − − tryptone ( 21.3%) reported by Zhuang et al. [37]. Thus, the more negative stable carbon isotope − values of the minerals produced in our experiments further confirms their biogenesis. Sánchez-Román et al. [71] used different aerobic bacterial strains to induce the formation of Mg-rich carbonates at aqueous Mg/Ca ratios (2 to 11.5), and found that the δ13C values for hydromagnesite, huntite, high Mg-calcite, and dolomite ranged from 26.8 to 18.2%. They interpreted this as proving − − that the carbon of the microbial carbonate precipitates was derived from the yeast extract and proteose peptone ( 23.8 and 24.1%, respectively). Thus, microbial metabolism can strongly influence the − − carbon isotope composition of these carbonate biominerals, and the more negative δ13C values proves their biogenesis.

3.4.5. Thermogravimetry-Derivative Thermogravimetry (TG-DTG) and Diﬀerential Scanning Calorimetry (DSC) Analyses of Monohydrocalcite at Mg/Ca Molar Ratios 7 and 9 To compare the thermal stability of monohydrocalcite between Mg/Ca ratio 7 and 9, TG-DTG and DSC analyses were performed. Whether the presence of hydromagnesite could make the TG-DTG and DSC results of monohydrocalcite hard to interpret was an issue that confused us. It has been reported that hydromagnesite (4MgCO Mg(OH) 4H O) can undergo endothermic breakdown from 200 C to 3· 2· 2 ◦ 550 ◦C[73]. The thermal decomposition of hydromagnesite is described by the following reactions:

4MgCO Mg(OH) 4H O 4MgCO Mg(OH) + 4H O (7) 3 · 2 · 2 → 3 · 2 2 4MgCO Mg(OH) 4MgCO MgO + H O (8) 3 · 2 → 3 · 2 4MgCO MgO 5MgO + 4CO (9) 3 · → 2 Minerals 2019, 9, x FOR PEER REVIEW 15 of 26

The DTG results show that there is a great difference in the second peak temperature: 764 °C is the thermal decomposition temperature of the dehydrated monohydrocalcite at Mg/Ca ratio 9, higher than 757 °C of that at Mg/Ca ratio 7 (Figure 8b). Thus, the dehydrated monohydrocalcite at the Mg/Ca ratio of 9 has a higher thermal stability than that at the Mg/Ca ratio of 7. It has been reported that in the decomposition process of monohydrocalcite, vaterite can be formed sometimes, and the vaterite nucleus could be slowly transformed into calcite crystals; moreover, the formation ofMinerals vaterite2019 ,has9, 632 a relationship with Mg content [74]. It could be that more vaterite would be formed15 of 26 in the decomposition process of monohydrocalcite at an Mg/Ca ratio of 7 due to the presence of a higherThat content is to say, of hydromagnesitehydromagnesite, could which be decomposeddetermines the thoroughly lower thermal before the stability temperature shown increases in the second weight loss step. Of course, this is just speculation; further tests are needed. to 700–800 ◦C. It has also been reported that by the thermal dehydration observed in the range from 400 Monohydrocalciteto 560 K, monohydrocalcite at different produces Mg/Ca a molar calcite ratios phase preferentiallyexhibits different [74]. therma In general,l behaviors, the decomposition likely due to the different morphologies and crystallinities. temperature of calcite is higher, 700–800 ◦C at a heating rate of 10 ◦/min [37]. Therefore, the thermal decompositionDSC results characteristics (Figure 8c) show of monohydrocalcite that 1 g of the dehydrated are different monohydrocalcite from that of hydromagnesite. at an Mg/Ca ratio of 9 needed 190.3 J to decompose, higher than the 185 J for that at an Mg/Ca ratio of 7; moreover, From the TG curve, two obvious mass loss phases can be discerned: one was at about 210 ◦C 783.2 °C is the second peak temperature for the decomposition of the dehydrated monohydrocalcite and the other 780–90 ◦C (Figure8a). The first mass loss phase represents the loss of crystalline waterat an Mg/Ca in the ratio minerals of 9, monohydrocalcitealso higher than 773.4 and °C hydromagnesite, for that at an Mg/Ca also overlapping ratio of 7. The the above process DSC of resultsthermal also dehydroxylation reveal that the of thermal dehydrated stability hydromagnesite of the dehydrated [74], andmonohydrocalcite the second indicates at an Mg/Ca the thermal ratio ofdecomposition 9 is higher than of the that dehydrated at an Mg/Ca monohydrocalcite ratio of 7. The [37 reason]. may be closely related to the higher crystallinityThere are of also monohydrocalcite two obvious weight at an loss Mg/Ca phases ratio in Figure of 9 (Table8b: the S1). first From weight the loss above, phase it is appears in the range that Mg2+ ions could promote the formation of monohydrocalcite under the influence of WMS-3 bacteria. of 190–210 ◦C, resulting from the loss of crystalline water from monohydrocalcite and hydromagnesite Theand thechanges thermal in the dehydroxylation crystalline structure of dehydrated and ther hydromagnesite;mal decomposition the secondprocess weight of monohydrocalcite loss phase is in are in need of further study. the temperature range 700–800 ◦C, due to the decomposition of the dehydrated monohydrocalcite.

(a) 100 Mg/Ca=7 0.0 (b) Mg/Ca=9 90 -0.1 ° 80 211.2 C -0.2 211.9°C 70 -0.3 194°C 201°C 60 DTG (%/min) Weight loss (%) Weight 782.3°C -0.4 Mg/Ca=7 ° 50 792.9 C Mg/Ca=9 764°C -0.5 757°C 40 0 200 400 600 800 1000 0 200 400 600 800 1000 Temperature (°C) Temperature (oC)

0.2

0.0

Mg/Ca=7 ΔH=185 J/g

Heat flow (mW) flow Heat -0.2 Mg/Ca=9 ΔH=190.3 J/g ° -0.4 202.8°C 773.4 C 204.8°C 783.2°C 0 200 400 600 800 1000 Temperature(°C)

Figure 8. ThermogravimetryThermogravimetry (TG) (TG) ( (aa),), differential differential thermogravimetry thermogravimetry (DTG) (DTG) ( (bb),), and and differential differential scanning calorimetrycalorimetry (DSC) (DSC) (c )(c analyses) analyses of mineralsof minerals induced induced by H. by smyrnensis H. smyrnensisWMS-3 WMS-3 bacteria culturedbacteria 1 culturedfor 30 days for at 30 a days heating at a rate heating of 10 rate◦C minof 10− °C. min−1. The DTG results show that there is a great difference in the second peak temperature: 764 C 3.4.6. Characterization of Surface Chemistry with XPS ◦ is the thermal decomposition temperature of the dehydrated monohydrocalcite at Mg/Ca ratio 9, higher than 757 ◦C of that at Mg/Ca ratio 7 (Figure8b). Thus, the dehydrated monohydrocalcite at the Mg/Ca ratio of 9 has a higher thermal stability than that at the Mg/Ca ratio of 7. It has been reported that in the decomposition process of monohydrocalcite, vaterite can be formed sometimes, and the vaterite nucleus could be slowly transformed into calcite crystals; moreover, the formation of vaterite has a relationship with Mg content [74]. It could be that more vaterite would be formed in the decomposition process of monohydrocalcite at an Mg/Ca ratio of 7 due to the presence of a higher Minerals 2019, 9, 632 16 of 26 content of hydromagnesite, which determines the lower thermal stability shown in the second weight loss step. Of course, this is just speculation; further tests are needed. Monohydrocalcite at different Mg/Ca molar ratios exhibits different thermal behaviors, likely due to the different morphologies and crystallinities. DSC results (Figure8c) show that 1 g of the dehydrated monohydrocalcite at an Mg /Ca ratio of 9 needed 190.3 J to decompose, higher than the 185 J for that at an Mg/Ca ratio of 7; moreover, 783.2 ◦C is the second peak temperature for the decomposition of the dehydrated monohydrocalcite at an Mg/Ca ratio of 9, also higher than 773.4 ◦C for that at an Mg/Ca ratio of 7. The above DSC results also reveal that the thermal stability of the dehydrated monohydrocalcite at an Mg/Ca ratio of 9 is higher than that at an Mg/Ca ratio of 7. The reason may be closely related to the higher crystallinity of monohydrocalcite at an Mg/Ca ratio of 9 (Table S1). From the above, it appears that Mg2+ ions could promote the formation of monohydrocalcite under the influence of WMS-3 bacteria. The changes in the crystalline structure and thermal decomposition process of monohydrocalcite are in need of further study.

3.4.6. Characterization of Surface Chemistry with XPS XPS analysis was performed to determine the surface heterogeneity of the biomineral further, facilitating the understanding of the important role played by some organic substances in the formation of these carbonate precipitates. Through XPS experiments, the atomic composition can be obtained by analyzing the characteristic peaks of binding energy through high-resolution scan of the elements [75]. The results of XPS clearly show the presence of C, O, Ca, N, S, and P on the surface of the biomineral induced by WMS-3 (Figure9a–g). As shown in Figure9b, two new peaks at 342.7 and 346.3 eV are assigned to Ca 2p [76]. The C1s peak (Figure9c) at 280.86 eV indicates that carbon is bound only to carbon and hydrogen (C–(C, H)), and the peak at 284.86 eV illustrates that carbon is singly-bound to oxygen or nitrogen (C–(O, N)) from ethers, alcohols, amines, and/or amides [77]. Nitrogen appears at a binding energy of 395.56 eV (Figure9d), due to the presence of amine or amide groups of proteins [ 41]. The oxygen peak (O1s) at 527.11 eV (Figure9e) indicates that oxygen is double bonded with carbon or phosphorus (C =O or P=O) from carboxylic acids, carboxylates, esters, carbonyls, amides, or phosphoryl groups [78]. Phosphorus is found at a binding energy of 128.96 eV (Figure9f), attributed to phosphate groups [ 78]. The peak 3/2 ﬁtting results (Figure9g) reveal that S 2p is contributed by the S2P –H at 162.81 eV [79]. The carboxyl groups, phosphoryl groups, and other groups mentioned above can also provide metal cation binding sites [80]. The deconvolution for high-resolution O1s spectra further reveals that the binding energy of O assigned as O=C–O group shifts from 531.5 [41] to 527.11 eV after the adsorption of Ca2+ and Mg2+ (Figure9e), conﬁrming that interactions between carboxyl groups with Ca 2+ and Mg2+ ions have occurred. Moreover, the deconvolution for high-resolution P2p spectra indicate that the binding energy assigned as the phosphoryl group shifted from 133.4 [78] to 128.96 eV due to Ca2+ and Mg2+ adsorption (Figure9f). These functional groups may have come from WMS-3 bacteria and bacterial metabolites. Thus, XPS results further reveal the important role played by WMS-3 bacteria in the formation of biominerals. Minerals 2019, 9, x FOR PEER REVIEW 16 of 26

XPS analysis was performed to determine the surface heterogeneity of the biomineral further, facilitating the understanding of the important role played by some organic substances in the formation of these carbonate precipitates. Through XPS experiments, the atomic composition can be obtained by analyzing the characteristic peaks of binding energy through high-resolution scan of

Mineralsthe2019 elements, 9, 632 [75]. The results of XPS clearly show the presence of C, O, Ca, N, S, and P on17 the of 26 surface of the biomineral induced by WMS-3 (Figure 9a–g).

24.0k 342.71 180.0k (a) O (b) Ca2p 160.0k 20.0k 140.0k 120.0k 16.0k 346.31 100.0k 12.0k Ca

80.0k

60.0k 8.0k Counts(kcps) 40.0k Counts(kcps) 4.0k 20.0k C N P 0.0 0.0 -20.0k 0 200 400 600 800 1000 1200 1400 336 339 342 345 348 351 354 357 Binding Energy (eV) Binding Energy(eV)

4600 (d) N 7000 (c) 284.86 C 395.56 1s 1s 4400 6000 4200 5000 280.86 4000 4000 3800

3000 3600

Counts(kcps) 2000 3400 Counts(kcps) 1000 3200 3000 0 2800 273 276 279 282 285 288 291 294 388 390 392 394 396 398 400 402 404 406 Binding Energy(eV) Binding Energy(eV)

800 48.0k (e) 527.11 O (f) 128.96 P2p 1s 750 40.0k 700 32.0k 650 600

24.0k

550 16.0k Counts(kcps) Counts(kcps) 500 8.0k 450 0.0 522 525 528 531 534 537 540 120 123 126 129 132 135 138 141 Binding Energy(eV) Binding Energy(eV)

600 (g) 162.81 S2p 590

580

570

560

550 Counts(kcps)

540

530 152 154 156 158 160 162 164 166 168 170 172 Binding Energy (eV)

FigureFigure 9. X-ray 9. X-ray photoelectron photoelectron spectroscopy spectroscopy (XPS) (XPS) broad broad survey survey of of minerals minerals at at Mg Mg/Ca/Ca molarmolar ratio of 9 (a) and9 high-resolution(a) and high-resolution of Ca (ofb ),Ca C (b (c),), C N (c (),d ),N O(d (),e O), P(e (),f ),P and(f), and S ( gS). (g). 3.5. Intracellular Biomineralization of H. smyrnensis WMS-3 Bacteria The bacterial seeds cultured in a medium without any Ca2+ and Mg2+ ions were set as the control, and the bacteria growing in a medium with Mg/Ca ratios 0, 2, 5, 7, and 9 were considered as the experimental groups. The growth of the bacterial seeds was normal, and the EPS and cell wall could be clearly observed; moreover, no nanometer-scaled dark inclusions were present inside the cells (Figure 10(a1–3)). However, in the environment with different concentrations of Ca2+ and Mg2+ ions, some cells were deformed and dark inclusions with different sizes (marked with yellow circles and purple arrows) appeared inside the cells (Figure 10b–f), revealing a great difference in the intracellular Minerals 2019, 9, 632 18 of 26 biomineralization between the control and experimental group. In our opinion, the deformation of cells was due to the consequence of the osmotic pressure produced by different concentrations of calcium and magnesium ions. WMS-3 bacteria can tolerate up to 3% NaCl, and the cells dehydrated and died when NaCl concentration increased (Figure3a). In the same way, Ca 2+ and Mg2+ ions can travel down the ion channel into the cell [36,37,50], causing the cell to dehydrate, which may be the cause of cell deformation. Ca2+ and Mg2+ ions that entered the cell’s interior could have accumulated in a regionMinerals to form 2019, a9,series x FOR PEER of nanoscale REVIEW dark inclusions, just as too many Ca2+ ions eaten by humans18 of could26 form stones in a particular part of the body. However, the intracellular inclusions have no crystalline precipitates could be permineralised viruses. The study of intracellular biomineralization has only structurejust begun, as shown and its by mechanism the lack of still diff ractionneeds to spots be explored and circles further. in the SAED images (Figure 10(g1–5)).

FigureFigure 10. 10.High-resolution High-resolution transmission transmission electronelectron microscopymicroscopy (HRTEM) (HRTEM) and and selected selected area area electron electron diﬀractiondiffraction (SAED) (SAED) analyses analyses of of intracellularintracellular biomineralization biomineralization of H. of smyrnensisH. smyrnensis WMS-3WMS-3 bacteria. bacteria. (a), Bacterial seed; (b)–(f), Bacteria at Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively; (g1–5), SAED (a), Bacterial seed; (b–f), Bacteria at Mg/Ca molar ratios of 0, 2, 5, 7, and 9, respectively; (g1–5), analyses of the intracellular inclusions of bacteria at Mg/Ca molar ratios 0, 2, 5, 7, and 9, respectively. SAED analyses of the intracellular inclusions of bacteria at Mg/Ca molar ratios 0, 2, 5, 7, and 9, (The arrows and the circles represent the location of biomineralization). respectively. (The arrows and the circles represent the location of biomineralization). 3.6. EPS Acting as the Nucleation Sites Studies of intracellular biomineralization have been rarely reported due to the limitation of the technology.There Couradeau were no nanometer-scale et al. [81] found minerals that adetected cyanobacterium on/in the EPS growing of WMS-3 in modernbacteria in microbialites a liquid 2+ 2+ in Lakemedium Alchichica without (Mexico) any Ca containsand Mg intracellularions (Figure 10(a1–3)), amorphous while carbonate some nanometer-scale inclusions with minerals calcium, magnesium,appeared in/on strontium, EPS (marked and barium by the blue elements. arrows an Recently,d yellow circles)Candidatus whenGloeomargarita WMS-3 bacteria were lithophora, in a medium with abundant Ca2+ and Mg2+ ions (Figure 11a–d). Moreover, some cells were completely a species of deep-branching cyanobacteria, has been reported to contain amorphous Ca-rich carbonates surrounded by a large number of nanoscale particles in/on the EPS (Figure 11(d2)). The nanoscale inside the cell [82]. There are also some microorganisms that can form intracellular minerals with particles marked by a yellow circle in Figure 11(c3) were proved to be monohydrocalcite by crystallineHRTEM structures. analysis (Figure Keim S2a) et al. due [83 to] foundthe presence that some of (312), magnetosomes (302), (113), and containing (103), and single the minerals prismatic magnetitealso marked crystals by are a yellow present circle in uncultured in Figure marine11d3 were magnetotactic also confirmed bacteria. to be Thus,monohydrocalcite some intracellular by biomineralizationSAED analysis products(Figure S2b) have because amorphous of the structures, occurrence and of other(111), intracellular(211), and (302); biominerals thus, further could be crystalline.revealing The that reported EPS of WMS-3 amorphous bacteria intracellular could act as inclusions the nucleation are mostly sites. amorphous calcium carbonate (ACC), andThe the opinions bacteria on themselvesEPS acting as are the commonly nucleation cyanobacteria;sites for biominerals where has the been intracellular widely accepted minerals by are magnetite,researchers. the bacteria It has been are alwaysreported magnetotactic that aragonite-like bacteria. CaCO As3 forcanH. be smyrnensis nucleated bacteria,within the intracellular EPS of biomineralizationcyanobacteria, not has on rarely the beencell wall, reported. and higher The amorphousconcentrations intracellular of N and P inclusions can result formedin higher in H. smyrnensisamountsbacterial of precipitation cells may in havethe EPS been [85]. a protectionEPS may have mechanism played an for important survival role in the in the face process of adversity. of bacterially-induced biomineralization [70,86–88]. Deng et al. [86] have used the sulfate-reducing bacteria (SRB) and halophilic bacteria to induce the precipitation of dolomite and found that the abundant EPS-like materials may have served as the template for dolomite nucleation while the heat-killed bacteria without EPS materials did not precipitate dolomite. It can be concluded that the EPS of bacteria indeed played an important role in the process of bacterially-induced biomineralization. Minerals 2019, 9, 632 19 of 26

Perri et al. [84] suggested that intracellular precipitates could be permineralised viruses. The study of intracellular biomineralization has only just begun, and its mechanism still needs to be explored further.

3.6. EPS Acting as the Nucleation Sites There were no nanometer-scale minerals detected on/in the EPS of WMS-3 bacteria in a liquid medium without any Ca2+ and Mg2+ ions (Figure 10(a1–3)), while some nanometer-scale minerals appeared in/on EPS (marked by the blue arrows and yellow circles) when WMS-3 bacteria were in a medium with abundant Ca2+ and Mg2+ ions (Figure 11a–d). Moreover, some cells were completely surrounded by a large number of nanoscale particles in/on the EPS (Figure 11(d2)). The nanoscale particles marked by a yellow circle in Figure 11(c3) were proved to be monohydrocalcite by HRTEM analysis (Figure S2a) due to the presence of (312), (302), (113), and (103), and the minerals also marked Mineralsby a yellow 2019, 9 circle, x FOR in PEER Figure REVIEW 11d3 were also conﬁrmed to be monohydrocalcite by SAED analysis (Figure19 of 26 S2b) because of the occurrence of (111), (211), and (302); thus, further revealing that EPS of WMS-3 bacteria could act as the nucleation sites.

Figure 11. HRTEMHRTEM analyses of biominerali biomineralizationzation on the cell surfacesurface/EPS/EPS of H. smyrnensis WMS-3 bacteria atat MgMg/Ca/Ca molar molar ratios ratios 2, 5,2, 7,5, and7, and 9 (a1–3, 9 (a1–3, b1–3, b1–3, c1–3 ,c1–3 and,d1–3 and), d1–3 respectively.), respectively. (The blue (The arrows blue arrowsand yellow and circlesyellow representcircles repres the locationent the location of minerals). of minerals.)

Moreover,The opinions the on activation EPS acting energy as the for nucleation nucleation sites (ΔG forN) biomineralscould be calculated has been by widely the following accepted equationby researchers. [89]: It has been reported that aragonite-like CaCO3 can be nucleated within the EPS of cyanobacteria, not on the cell wall, and higher concentrations of N and P can result in higher 3 16π(ΔG1 ) amounts of precipitation in the EPS [85].ΔG EPSN = may have played an important role in the process 3(kTlog S)2 (10) of bacterially-induced biomineralization [70,86–88]. Denge et al. [86] have used the sulfate-reducing where ΔG1 was the interfacial energy, k the Boltzmann constant, T the temperature, and S the supersaturation. On the one hand, microorganisms could reduce ΔG1 by providing the nucleation sites from EPS. On the other hand, the alkaline CA could catalyze the production of a large number of CO32− and HCO3− ions to increase the supersaturation surrounding the EPS of the bacterial cell (S). As mentioned above these could reduce the nucleation energy barrier (ΔGN) of biominerals and facilitate the nucleation on EPS. In fact, the precipitation of biominerals induced by bacteria involves the controlled nucleation process at interfaces between the template macromolecules and minerals, e.g., the inorganic-organic molecular recognition processes [90]. However, the molecular interactions at the interface between the mineral and organic matrix are not well understood. Thus, the nucleation mechanism on EPS needs to be further explored. In this study, 15 kinds of amino acid were detected in the EPS of WMS- 3 bacteria (Figure 12), among which glycine (Gly, 26.42%), alanine (Ala, 19.3%), glutamic acid (Glu, 17.76%) and aspartic acid (Asp, 10.05%) were the four amino acids with higher content (molar ratio). The pH value could increase to 8.9 under the influence of WMS-3 bacteria, which created an alkaline condition to facilitate the occurrence of amino acid deprotonation that could make the Minerals 2019, 9, 632 20 of 26 bacteria (SRB) and halophilic bacteria to induce the precipitation of dolomite and found that the abundant EPS-like materials may have served as the template for dolomite nucleation while the heat-killed bacteria without EPS materials did not precipitate dolomite. It can be concluded that the EPS of bacteria indeed played an important role in the process of bacterially-induced biomineralization. Moreover, the activation energy for nucleation (∆GN) could be calculated by the following equation [89]: 16π(∆G )3 ∆G = 1 (10) N 2 3(kTlog e S) where ∆G1 was the interfacial energy, k the Boltzmann constant, T the temperature, and S the supersaturation. On the one hand, microorganisms could reduce ∆G1 by providing the nucleation sites from EPS. On the other hand, the alkaline CA could catalyze the production of a large number of 2 CO3 − and HCO3− ions to increase the supersaturation surrounding the EPS of the bacterial cell (S). As mentioned above these could reduce the nucleation energy barrier (∆GN) of biominerals and facilitate the nucleation on EPS. In fact, the precipitation of biominerals induced by bacteria involves the controlled nucleation process at interfaces between the template macromolecules and minerals, e.g., the inorganic-organic molecular recognition processes [90]. However, the molecular interactions at the interface between the mineral and organic matrix are not well understood. Thus, the nucleation mechanism on EPS needs to be further explored. In this study, 15 kinds of amino acid were detected in the EPS of WMS-3 bacteria (Figure 12), among which glycine (Gly, 26.42%), alanine (Ala, 19.3%), glutamic acid (Glu, 17.76%) and aspartic acid (Asp, 10.05%) were the four amino acids with higher content (molar ratio). Minerals 2019, 9, x FOR PEER REVIEW 20 of 26 The pH value could increase to 8.9 under the inﬂuence of WMS-3 bacteria, which created an alkaline conditionadsorbing toof facilitateCa2+ andthe Mg occurrence2+ ions to the of EPS amino become acid deprotonationmuch easier. Is that that could so? The make elemental the adsorbing mapping of 2+ 2+ Ca(Figureand S3) Mg of ionsH. smyrnensis to the EPS WMS-3 become muchproved easier. that Isthere that so?was Thea large elemental number mapping of Ca (Figure2+ (Figure S3) S3 of 2+ 2+ H.(a4,b3,c3)) smyrnensis andWMS-3 Mg2+ provedions (Figure that there S3 was(b2,c2)) a large present number inside of Ca the (FigureEPS, further S3(a4,b3,c3)) illustrating and Mg the ionsimportant (Figure role S3(b2,c2)) played presentby these inside amino the acids EPS, in further the EPS. illustrating Of course, the some important other role organic played substances by these aminocould also acids be in present the EPS. within Of course, the EPS some besides other amin organico acids, substances and other could organi alsoc be substances present within affecting the EPSthe nucleation besides amino should acids, be studied and other in organicthe future. substances aﬀecting the nucleation should be studied in the future.

Gly Ala Glu Asp 10.05% 17.76% 7.15% Pro 3.24% Lys 3.06% Ser 2.45% 2.42% Thr 2.38% Val 2.37%2.19% Leu 19.3% 2.19% 1.17%1.17% Arg 0.62%0.99%0.8% 0.99% Ile 0.62% 0.80% Phe 26.42% His Tyr

Figure 12. Amino acid composition (molar ratio) of the EPS from H. smyrnensis WMS-3. Figure 12. Amino acid composition (molar ratio) of the EPS from H. smyrnensis WMS-3.

3.7. Changes in the Intracellular Ca2+ Concentration The dark nanometer-scale inclusions within the WMS-3 bacteria (Figure 10) demonstrates the occurrence of the intracellular biomineralization; moreover, the results of elemental mapping (Figure S3) reveal the presence of Ca2+ and Mg2+ ions inside the WMS-3 bacterial cells. The concentration of Ca2+ and Mg2+ ions is much higher than that inside the cell; thus, a concentration gradient was formed from the outside to the inside of the cell. Ca2+ and Mg2+ ions could enter the cell by diffusion along the ion channel. The changes of Ca2+ and Mg2+ ion concentration inside the bacterial cells need to be studied. Therefore, the fluorescence intensities of Ca2+ ions inside the WMS-3 bacterial cells marked with the Fluo-3 AM fluorescence indicator were determined, and the results are shown in Figure S4. It can be concluded that the fluorescence intensity of intracellular Ca2+ ion decreases with increasing Mg/Ca ratios, consistent with the results of Zhao et al. [91]. That is to say, Mg2+ ion concentration in the culture medium could strongly influence the diffusion of Ca2+ ions, resulting in a significant decrease in the concentration of intracellular Ca2+ ions (p < 0.01). The reason may be that Mg2+ ions with a smaller radius are able to go through the ion channels more easily than Ca2+ ions. The cell volume would remain unchanged, so that the more Mg2+ ions entered the cell, the less space there would be left for Ca2+ ions. In view of the lack of a fluorescence indicator for the Mg2+ ion, the intracellular Mg2+ ion concentration will be studied in the future.

4. Conclusions The H. smyrnensis WMS-3 strain was identified by 16S rDNA homology comparison and this was used to induce the precipitation of calcium carbonate in a fluid containing 3% NaCl at different Mg/Ca ratios 0, 2, 5, 7, and 9. The results show that WMS-3 bacteria can increase the pH to about 9.0, due to the released ammonia and effects of CA; moreover, a large number of HCO3− and CO32− ions produced by CA catalysis further elevated the supersaturation in the fluid. Higher pH and Minerals 2019, 9, 632 21 of 26

3.7. Changes in the Intracellular Ca2+ Concentration The dark nanometer-scale inclusions within the WMS-3 bacteria (Figure 10) demonstrates the occurrence of the intracellular biomineralization; moreover, the results of elemental mapping (Figure S3) reveal the presence of Ca2+ and Mg2+ ions inside the WMS-3 bacterial cells. The concentration of Ca2+ and Mg2+ ions is much higher than that inside the cell; thus, a concentration gradient was formed from the outside to the inside of the cell. Ca2+ and Mg2+ ions could enter the cell by diffusion along the ion channel. The changes of Ca2+ and Mg2+ ion concentration inside the bacterial cells need to be studied. Therefore, the fluorescence intensities of Ca2+ ions inside the WMS-3 bacterial cells marked with the Fluo-3 AM fluorescence indicator were determined, and the results are shown in Figure S4. It can be concluded that the fluorescence intensity of intracellular Ca2+ ion decreases with increasing Mg/Ca ratios, consistent with the results of Zhao et al. [91]. That is to say, Mg2+ ion concentration in the culture medium could strongly influence the diffusion of Ca2+ ions, resulting in a significant decrease in the concentration of intracellular Ca2+ ions (p < 0.01). The reason may be that Mg2+ ions with a smaller radius are able to go through the ion channels more easily than Ca2+ ions. The cell volume would remain unchanged, so that the more Mg2+ ions entered the cell, the less space there would be left for Ca2+ ions. In view of the lack of a fluorescence indicator for the Mg2+ ion, the intracellular Mg2+ ion concentration will be studied in the future.

4. Conclusions The H. smyrnensis WMS-3 strain was identified by 16S rDNA homology comparison and this was used to induce the precipitation of calcium carbonate in a fluid containing 3% NaCl at different Mg/Ca ratios 0, 2, 5, 7, and 9. The results show that WMS-3 bacteria can increase the pH to about 2 9.0, due to the released ammonia and effects of CA; moreover, a large number of HCO3− and CO3 − ions produced by CA catalysis further elevated the supersaturation in the fluid. Higher pH and supersaturation promoted the biomineralization of calcium carbonate (calcite, Mg-rich calcite, aragonite, and monohydrocalcite) and only a little magnesium carbonate (hydromagnesite). The thermal stability of monohydrocalcite increased when an Mg/Ca ratio of 7 changes to 9, due to the crystallinity of monohydrocalcite at an Mg/Ca ratio of 9 being higher than that at an Mg/Ca ratio of 7. The abundant organic functional groups, a range of mineral shapes, negative stable carbon isotope values, and characteristics of surface chemistry proves the biogenesis of the minerals. Ca2+ ions entered the cell by diffusion to take part in the intracellular biomineralization. EPS could act as the nucleation sites for the precipitation of monohydrocalcite. This study sheds light on the biomineralization mechanism, facilitates the understanding of the formation process of monohydrocalcite in nature, and should useful in interpreting the sedimentary record.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-163X/9/10/632/s1. Table S1: FWHM (o) of monohydrocalcite induced by H. smyrnensis WMS-3 bacteria at Mg/Ca ratios of 7 and 9. 13 Table S2: Stable carbon isotope δ CPDB (%) values of the biominerals. Figure S1: Rietveld refinement of XRD data (a, b, c, d and e represent Mg/Ca molar ratios of 0, 2, 5, 7 and 9, respectively). Figure S2: STEM and elemental mapping of H. smyrnensis WMS-3. (a: HRTEM analysis of minerals marked by the yellow circle in Figure 11c3 image; b: SAED analysis of minerals marked by the yellow circle in Figure 11d3 image) Figure S3: Fluorescence intensity of intracellular Ca2+ ions of H. smyrnensis WMS-3. (a, b and c represent Mg/Ca molar ratios of 0, 2 and 7, respectively). Figure S4: Fluorescence intensity of intracellular Ca2+ ions of H. smyrnensis WMS-3. Author Contributions: Z.H. conceived and designed the experiments; J.P., Y.Z. and Y.W. performed the experiments; Z.H., H.Z., and H.Y. analyzed the results of all the experiments; J.P. and H.Y. wrote the paper; H.Z. and M.E.T. revised the manuscript; J.Z. helped us to analyze the data by using SPSS 21 software; M.J. helped us to modify figures, Y.Z. helped us to do some experiments, Rietveld refinement was performed by Y.Z., and the calculation of SI by using PHREEQC software in response letter was performed by B.S. All authors have read and approved the manuscript. Funding: This work was supported by the National Natural Science Foundation of China (41772095, 41972108, U1663201, 41702131); the Laboratory for Marine Mineral Resources, Qingdao National Laboratory for Marine Science and Technology (MMRZZ201804); Taishan Scholar Talent Team Support Plan for Advanced & Unique Discipline Areas, Major Scientific and Technological Innovation Projects of Shandong Province (2017CXGC1602, Minerals 2019, 9, 632 22 of 26

2017CXGC1603); SDUST Research Fund (2015TDJH101); Natural Science Foundation of Shandong Province (ZR2019MD027, ZR2017BD001); the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (2016ASKJ13); National College Students’ Innovation and Entrepreneurship Training Program (201910424017). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Lv, D.; Li, Z.; Chen, J.; Liu, H.; Guo, J.; Shang, L. Characteristics of the Permian coal-formed gas sandstone reservoirs in Bohai Bay Basin and the adjacent areas, North China. J. Pet. Sci. Eng. 2011, 78, 516–528. [CrossRef] 2. Yu, H.; Yuan, J.; Guo, W.; Cheng, J.; Hu, Q. A preliminary laboratory experiment on coalbed methane displacement with carbon dioxide injection. Int. J. Coal. Geol. 2008, 73, 156–166. [CrossRef]

3. Yu, H.; Zhou, G.; Fan, W.; Ye, J. Predicted CO2 enhanced coalbed methane recovery and CO2 sequestration in China. Int. J. Coal Geol. 2007, 71, 345–357. [CrossRef] 4. Yu, H.; Zhou, L.; Guo, W.; Cheng, J.; Hu, Q. Predictions of the adsorption equilibrium of methane/carbon dioxide binary gas on coals using Langmuir and ideal adsorbed solution theory under feed gas conditions. Int. J. Coal Geol. 2008, 73, 115–129. [CrossRef] 5. Pang, Y.; Guo, X.; Han, Z.; Zhang, X.; Zhu, X.; Hou, F.; Han, C.; Song, Z.; Xiao, G. Mesozoic–Cenozoic denudation and thermal history in the Central Uplift of the South Yellow Sea basin and the implications for hydrocarbon systems: Constraints from the CSDP-2 borehole. Mar. Petrol. Geol. 2019, 99, 355–369. [CrossRef] 6. Allwood, A.C.; Walter, M.R.; Kamber, B.S.; Marshall, C.P.; Burch, I.W. Stromatolite reef from the Early Archaean era of Australia. Nature 2006, 441, 714–718. [CrossRef][PubMed] 7. Allwood, A.C.; Walter, M.R.; Burch, I.W.; Kamber, B.S. 3.43 billion-year-old stromatolite reef from the Pilbara Craton of western Australia: Ecosystem-scale insights to early life on Earth. Precambrian Res. 2007, 158, 198–227. [CrossRef] 8. Lindsay, J.F.; Brasier, M.D.; Mcloughlin, N.; Green, O.R.; Fogel, M.; Mcnamara, K.M.; Steele, A.; Mertzman, S.A. Abiotic earth-establishing a baseline for earliest life, data from the Archean of western Australia. In Proceedings of the Lunar & Planetary Science Conference, League, TX, USA, 17–21 March 2003. 9. Dupraz, C.; Reid, R.P.; Braissant, O.; Decho, A.W.; Norman, R.S.; Visscher, P.T. Processes of carbonate precipitation in modern microbial mats. Earth-Sci. Rev. 2009, 96, 141–162. [CrossRef] 10. Chough, S.K.; Lee, H.S.; Woo, J.; Chen, J.; Choi, D.K.; Lee, S.-b.; Kang, I.; Park, T.-y.; Han, Z. Cambrian stratigraphy of the North China Platform: Revisiting principal sections in Shandong Province, China. Geosci. J. 2010, 14, 235–268. [CrossRef] 11. Lee, J.-H.; Chen, J.; Chough, S.K. Paleoenvironmental implications of an extensive maceriate microbialite bed in the Furongian Chaomidian Formation, Shandong Province, China. Palaeogeogr. Palaeoclimatol. Palaeoecol. 2010, 297, 621–632. [CrossRef] 12. Lv, D.; Chen, J. Depositional environments and sequence stratigraphy of the Late Carboniferous Early − Permian coal-bearing successions (Shandong Province, China): Sequence development in an epicontinental basin. J. Asian Earth Sci. 2014, 79, 16–30. [CrossRef] 13. Yin, S.; Lv, D.; Jin, L.; Ding, W. Experimental analysis and application of the effect of stress on continental shale reservoir brittleness. J. Geophys. Eng. 2018, 15, 478–494. [CrossRef] 14. Xu, Y.; Shen, X.; Chen, N.; Yang, C.; Wang, Q. Evaluation of reservoir connectivity using whole-oil gas 3 chromatographic fingerprint technology: A case study from the Es3 reservoir in the Nanpu Sag, China. Pet. Sci. Technol. 2012, 9, 290–294. [CrossRef] 15. Chen, J.; Van Loon, A.J.; Han, Z.; Chough, S.K. Funnel-shaped, breccia-filled clastic dykes in the Late Cambrian Chaomidian Formation (Shandong Province, China). Sediment. Geol. 2009, 221, 1–6. [CrossRef] 16. Chen, J.; Chough, S.K.; Chun, S.S.; Han, Z. Limestone pseudoconglomerates in the Late Cambrian Gushan and Chaomidian Formations (Shandong Province, China): Soft-sediment deformation induced by storm-wave loading. Sedimentology 2010, 56, 1174–1195. [CrossRef] 17. Chen, J.; Han, Z.; Zhang, X.; Fan, A.; Yang, R. Early diagenetic deformation structures of the Furongian ribbon rocks in Shandong Province of China—A new perspective of the genesis of limestone conglomerates. Sci. China Earth Sci. 2010, 53, 241–252. [CrossRef] Minerals 2019, 9, 632 23 of 26

18. Chen, J.; Chough, S.K.; Han, Z.; Lee, J.-H. An extensive erosion surface of a strongly deformed limestone bed in the Gushan and Chaomidian formations (late Middle Cambrian to Furongian), Shandong Province, China: Sequence-stratigraphic implications. Sediment. Geol. 2011, 233, 129–149. [CrossRef] 19. Chen, J.; Chough, S.K.; Lee, J.-H.; Han, Z. Sequence-stratigraphic comparison of the upper Cambrian Series 3 to Furongian succession between the Shandong region, China and the Taebaek area, Korea: High variability of bounding surfaces in an epeiric platform. Geosci. J. 2012, 16, 357–379. [CrossRef] 20. Lee, J.; Chen, J.; Choh, S.; Lee, D.; Han, Z.; Chough, S.K. Furongian (Late Cambrian) sponge–microbial maze-like reefs in the north china platform. Palaios 2014, 29, 27–37. [CrossRef] 21. Van Loon, A.J.T.; Han, Z.; Han, Y. Origin of the vertically orientated clasts in brecciated shallow-marine limestones of the Chaomidian Formation (Furongian, Shandong Province, China). Sedimentology 2013, 60, 1059–1070. [CrossRef] 22. Woo, J.; Chough, S.K.; Han, Z. Chambers of Epiphyton thalli in microbial buildups, Zhangxia Formation (Middle Cambrian), Shandong Province, China. Palaios 2008, 23, 55–64. [CrossRef] 23. Park, T.Y.; Sang, J.M.; Han, Z.; Choi, D.K. Ontogeny of the middle cambrian trilobite shantungia spinifera walcott, 1905 from north china and its taxonomic significance. J. Paleontol. 2008, 82, 851–855. [CrossRef] 24. Liu, Y.; Jiao, X.; Li, H.; Yuan, M.; Yang, W.; Zhou, X.; Liang, H.; Zhou, D.; Zheng, C.; Sun, Q.; et al. Primary dolostone formation related to mantle-originated exhalative hydrothermal activities, Permian Yuejingou section, Santanghu area, Xinjiang, NW China. Sci. China Earth Sci. 2012, 55, 183–192. [CrossRef] 25. Han, Z.; Zhang, X.; Chi, N.; Han, M.; Woo, J.; Lee, H.S.; Chen, J. Cambrian oncoids and other microbial-related grains on the North China Platform. Carbonates Evaporites 2015, 30, 373–386. [CrossRef] 26. Fan, A.; Yang, R.; van Loon, A.J.; Yin, W.; Han, Z.; Zavala, C. Classification of gravity-flow deposits and their significance for unconventional petroleum exploration, with a case study from the Triassic Yanchang Formation (southern Ordos Basin, China). J. Asian Earth Sci. 2018, 161, 57–73. [CrossRef] 27. Chang, X.; Wang, T.G.; Li, Q.; Cheng, B.; Tao, X. Geochemistry and possible origin of petroleum in Palaeozoic reservoirs from Halahatang Depression. J. Asian Earth Sci. 2013, 74, 129–141. [CrossRef] 28. Dahl, K.; Buchardt, B. Monohydrocalcite in the Arctic Ikka Fjord, SW Greenland: First reported marine occurrence. J. Sediment. Res. 2006, 76, 460–471. [CrossRef] 29. Rivadeneyra, M.A.; Delgado, G.; Ramos-Cormenzana, A.; Delgado, R. Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: Crystal formation sequence. Res. Microbiol. 1998, 149, 277–287. [CrossRef] 30. Rivadeneyra, M.A.; Delgado, G.; Ramos-Cormenzana, A.; Delgado, R. Precipitation of carbonates by Deleya halophila in liquid media: Pedological implications in saline soils. Arid Soil Res. Rehab. 1997, 11, 35–47. [CrossRef] 31. Rivadeneyra, M.A.; Delgado, R.; Delgado, G.; Moral, A.D.; Ferrer, M.R.; Ramos-Cormenzana, A. Precipitation of carbonates by Bacillus sp. isolated from saline soils. Geomicrobiol. J. 1993, 11, 175–184. [CrossRef] 32. Rivadeneyra, M.A.; Párraga, J.; Delgado, R.; Ramos-Cormenzana, A.; Delgado, G. Biomineralization of carbonates by Halobacillus trueperi in solid and liquid media with different salinities. FEMS Microbiol. Ecol. 2004, 48, 39–46. [CrossRef][PubMed] 33. Arias, D.; Cisternas, L.A.; Rivas, M. Biomineralization of calcium and magnesium crystals from seawater by halotolerant bacteria isolated from Atacama Salar (Chile)(Article). Desalination 2017, 405, 1–9. [CrossRef] 34. Han, Z.; Li, D.; Zhao, H.; Yan, H.; Li, P. Precipitation of carbonate minerals induced by the halophilic Chromohalobacter israelensis under high salt concentrations: Implications for natural environments. Minerals 2017, 7, 95. [CrossRef] 35. Rivadeneyra, M.A.; Martín-Algarra, A.; Sánchez-Navas, A.; Martín-Ramos, D. Carbonate and phosphate precipitation by Chromohalobacter marismortui. Geomicrobiol. J. 2006, 23, 89–101. [CrossRef] 36. Han, Z.; Wang, J.; Zhao, H.; Tucker, M.E.; Zhao, Y.; Wu, G.; Zhou, J.; Yin, J.; Zhang, H.; Zhang, X.; et al. Mechanism of biomineralization induced by Bacillus subtilis J2 and characteristics of the biominerals. Minerals 2019, 9, 218. [CrossRef] 37. Zhuang, D.; Yan, H.; Tucker, M.E.; Zhao, H.; Han, Z.; Zhao, Y.; Sun, B.; Li, D.; Pan, J.; Zhao, Y.; et al. Calcite precipitation induced by Bacillus cereus MRR2 cultured at different Ca2+ concentrations: Further insights into biotic and abiotic calcite. Chem. Geol. 2018, 500, 64–87. [CrossRef] Minerals 2019, 9, 632 24 of 26

38. Han, Z.; Meng, R.; Yan, H.; Zhao, H.; Han, M.; Zhao, Y.; Sun, B.; Sun, Y.; Wang, J.; Zhuang, D. Calcium carbonate precipitation by Synechocystis sp. PCC6803 at diﬀerent Mg/Ca molar ratios under the laboratory condition. Carbonates Evaporites 2017.[CrossRef] 39. Han, Z.; Yan, H.; Zhao, H.; Zhou, S.; Han, M.; Meng, X.; Zhang, Y.; Zhao, Y.; Sun, B.; Yao, C.; et al. Bio-precipitation of calcite with preferential orientation induced by Synechocystis sp. PCC6803. Geomicrobiol. J. 2014, 31, 884–899. [CrossRef] 40. Arvidson, R.S.; Mackenzie, F.T. The dolomite problem: Control of precipitation kinetics by temperature and saturation state. Am. J. Sci. 1999, 299, 257–288. [CrossRef] 41. Liu, F.; Csetenyi, L.; Gadd, G.M. Amino acid secretion inﬂuences the size and composition of copper carbonate nanoparticles synthesized by ureolytic fungi. Appl. Microbiol. Biot. 2019, 103, 7217–7230. [CrossRef] 42. Morgan, J.W.; Forster, C.F.; Evison, L. A comparative study of the nature of biopolymers extracted from anaerobic and activated sludges. Water. Res. 1990, 24, 743–750. [CrossRef] 43. Su, T.; Shao, Q.; Qin, Z.; Guo, Z.; Wu, Z. Role of interfaces in two-dimensional photocatalyst for water splitting. Acs. Catal. 2018, 8, 2253–2276. [CrossRef] 44. Ding, Y.; Yang, B.; Hao, L.; Liu, Z.; Xiao, Z.; Zheng, X.; Liu, Q. FePt-Au ternary metallic nanoparticles with

the enhanced peroxidase-like activity for ultrafast colorimetric detection of H2O2. Sensor Actuat. B-chem 2017, 259, 775–783. [CrossRef]

45. Zhang, X.; Wu, Y.; Sun, Y.; Ding, P.; Liu, Q.; Lin, T.; Guo, J. Hybrid of Fe4 [Fe(CN)6]3 nanocubes and MoS2 nanosheets on nitrogen-doped graphene realizing improved electrochemical hydrogen production. Electrochim. Acta. 2018, 263, 140–146. [CrossRef] 46. Wei, C.; Liu, Q.; Zhu, X.; Min, F. One-step in situ growth of magnesium ferrite nanorods on graphene and their microwave-absorbing properties. Appl. Organomet. Chem. 2018, 32, 4011–4019. [CrossRef] 47. Zhang, X.; Sun, Y.; Liu, Q.; Guo, J.; Xiao, Z. FePt nanoalloys on N-doped graphene paper as integrated electrode towards eﬃcient formic acid electrooxidation. J. Appl. Electrochem. 2018, 48, 95–103. [CrossRef]

48. Lin, C.; Hu, L.; Cheng, C.; Sun, K.; Guo, X.; Shao, Q.; Li, J.; Wang, N.; Guo, Z. Nano-TiNb2O7/carbon nanotubes composite anode for enhanced lithium-ion storage. Electrochim. Acta 2018, 260, 65–72. [CrossRef]

49. Pan, D.; Ge, S.; Zhang, X.; Mai, X.; Li, S.; Guo, Z. Synthesis and photoelectrocatalytic activity of In2O3 hollow microspheres via a bio-template route using yeast templates. Dalton Trans. 2018, 47, 708–715. [CrossRef] 50. Han, Z.; Gao, X.; Zhao, H.; Tucker, M.E.; Zhao, Y.; Bi, Z.; Pan, J.; Wu, G.; Yan, H. Extracellular and intracellular biomineralization induced by Bacillus Licheniformis DB1-9 at diﬀerent Mg/Ca molar ratios. Minerals 2018, 8, 585. [CrossRef] 51. Han, Z.; Sun, B.; Zhao, H.; Yan, H.; Han, M.; Zhao, Y.; Meng, R.; Zhuang, D.; Li, D.; Ma, Y.; et al. Isolation of Leclercia adcarboxglata strain JLS1 from dolostone sample and characterization of its induced struvite. Geomicrobiol. J. 2017, 34, 500–510. [CrossRef]

52. Han, Z.; Yu, W.; Zhao, H.; Zhao, Y.; Tucker, M.E.; Yan, H. The signiﬁcant roles of Mg/Ca ratio, Cl− and 2 SO4 − in carbonate mineral precipitation by the Halophile Staphylococcus epidermis Y2. Minerals 2018, 8, 594. [CrossRef] 53. Power, I.M.; Harrison, A.L.; Dipple, G.M. Accelerating mineral carbonation using carbonic anhydrase. Environ. Sci. Technol. 2016, 50, 2610–2618. [CrossRef] 54. Lee, S.W.; Park, S.B.; Jeong, S.K.; Lim, K.S.; Lee, S.H.; Trachtenberg, M.C. On carbon dioxide storage based on biomineralization strategies. Micron 2010, 41, 273–282. [CrossRef][PubMed] 55. Power, I.M.; Harrison, A.L.; Dipple, G.M.; Southam, G. Carbon sequestration via carbonic anhydrase facilitated magnesium carbonate precipitation. Int. J. Greenh. Gas Control 2013, 16, 145–155. [CrossRef] 56. Qiu, X.; Wang, H.; Yao, Y.; Duan, Y. High salinity facilitates dolomite precipitation mediated by Haloferax volcanii DS52. Earth. Planet. Sci. Lett. 2017, 472, 197–205. [CrossRef] 57. Katz, A.K.; Glusker, J.P.; Beebe, S.A.; Bock, C.W. Calcium ion coordination: A comparison with that of beryllium, magnesium, and zinc. J. Am. Chem. Soc. 1996, 118, 5752–5763. [CrossRef] 58. Xu, J.; Yan, C.; Zhang, F.; Konishi, H.; Xu, H.; Teng, H.H. Testing the cation-hydration eﬀect on the

crystallization of Ca-Mg-CO3 systems. Proc. Natl. Acad. Sci. USA 2013, 110, 17750–17755. [CrossRef] 59. Rodriguez-Blanco, J.D.; Shaw, S.; Bots, P.; Roncal-Herrero, T.; Benning, L.G. The role of Mg in the crystallization of monohydrocalcite. Geochim. Cosmochim. Acta 2014, 127, 204–220. [CrossRef] 60. Stoﬀers, P.; Fischbeck, R. Monohydrocalcite in the sediments of Lake Kivu (East Africa). Sedimentology 2010, 21, 163–170. [CrossRef] Minerals 2019, 9, 632 25 of 26

61. Hull, H.; Turnbull, A.G. A thermochemical study of monohydrocalcite. Geochim. Cosmochim. Acta 1973. [CrossRef] 62. Munemoto, T.; Fukushi, K. Transformation kinetics of monohydrocalcite to aragonite in aqueous solutions. J. Miner. Petrol. Sci. 2008, 103, 345–349. [CrossRef] 63. Kalmar, L.; Homola, D.; Varga, G.; Tompa, P. Structural disorder in proteins brings order to crystal growth in biomineralization. Bone 2012, 51, 528–534. [CrossRef][PubMed] 64. Jönsson, M.; Persson, D.; Thierry, D. Corrosion product formation during NaCl induced atmospheric corrosion of magnesium alloy AZ91D. Corros. Sci. 2007, 49, 1540–1558. [CrossRef] 65. Bizani, D.; Motta, A.S.; Morrissy, J.A.; Terra, R.M.; Souto, A.A.; Brandelli, A. Antibacterial activity of cerein 8A, a bacteriocin-like peptide produced by Bacillus cereus. Int. Microbiol. 2005, 8, 125–131. [CrossRef] 66. Wang, Y.; Yao, Q.; Zhou, G.; Fu, S. Formation of elongated calcite mesocrystals and implication for biomineralization. Chem. Geol. 2013, 360, 126–133. [CrossRef] 67. Zhou, G.; Guan, Y.; Yao, Q.; Fu, S. Biomimetic mineralization of prismatic calcite mesocrystals: Relevance to biomineralization. Chem. Geol. 2010, 279, 63–72. [CrossRef] 68. Zhang, C.; Lv, J.; Li, F.; Li, X. Nucleation and growth of mg-calcite spherulites induced by the bacterium Curvibacter Lanceolatus strain HJ-1. Microsc. Microanal. 2017, 23, 1–8. [CrossRef] 69. Buczynski, C.; Chafetz, H. Habit of bacterially induced precipitates of calcium carbonate and the inﬂuences of medium viscosity on mineralogy. J. Sediment. Res. 1991, 61, 226–233. [CrossRef] 70. Sánchez-Román, M.; McKenzie, J.A.; Wagener, A.D.L.R.; Rivadeneyra, M.A.; Vasconcelos, C. Presence of sulfate does not inhibit low-temperature dolomite precipitation. Earth. Planet. Sci. Lett. 2009, 285, 131–139. [CrossRef] 71. Sánchez-Román, M.; Romanek, C.S.; Fernández-Remolar, D.C.; Sánchez-Navas, A.; McKenzie, J.A.; Pibernat, R.A.; Vasconcelos, C. Aerobic biomineralization of Mg-rich carbonates: Implications for natural environments. Chem. Geol. 2011, 281, 143–150. [CrossRef] 72. Jones, M.; Donnelly, A. Carbon sequestration in temperate grassland ecosystems and the inﬂuence of

management, climate and elevated CO2: Tansley review. New Phytol. 2004, 164, 423–439. [CrossRef] 73. Haurie, L.; Fernandez, A.I.; Velasco, J.I.; Chimenos, J.M.; Lopez-Cuesta, J.M.; Espiell, F. Effects of milling on the thermal stability of synthetic hydromagnesite. Mater. Res. Bull. 2007, 42, 1010–1018. [CrossRef] 74. Kimura, T.; Koga, N. Monohydrocalcite in comparison with hydrated amorphous calcium carbonate: Precipitation condition and thermal behavior. Crysl. Growth Des. 2011, 11, 3877–3884. [CrossRef] 75. Ma, K.; Cui, L.; Dong, Y.; Wang, T.; Da, C.; Hirasaki, G.J.; Biswal, S.L. Adsorption of cationic and anionic surfactants on natural and synthetic carbonate materials. J. Colloid Interf. Sci 2013, 408, 164–172. [CrossRef] [PubMed] 76. Ardizzone, S.; Bianchi, C.L.; Fadoni, M.; Vercelli, B. Magnesium salts and oxide: An XPS overview. Appl. Surf. Sci. 1997, 119, 253–259. [CrossRef] 77. Paulo, C.; Kenney, J.P.L.; Persson, P.; Dittrich, M. Effects of phosphorus in growth media on biomineralization and cell surface properties of marine cyanobacteria synechococcus. Geosciences 2018, 8, 471. [CrossRef] 78. Huang, Y.; Yao, Q.; Li, H.; Wang, F.; Zhou, G.; Fu, S. Aerobically incubated bacterial biomass-promoted formation of disordered dolomite and implication for dolomite formation. Chem. Geol. 2019, 523, 19–30. [CrossRef] 79. Hsu, M.; Chuang, H.; Cheng, F.; Huang, Y.; Han, C.; Pao, K.; Chou, S.; Shieh, F.K.; Tsai, F.Y.; Lin, C.; et al. Simple and highly efficient direct thiolation of the surface of carbon nanotubes. RSC Adv. 2014, 4, 14777–14780. [CrossRef] 80. Fein, J.B.; Martin, A.M.; Wightman, P.G.Metal adsorption onto bacterial surfaces: Development of a predictive approach. Geochim. Cosmochim. Acta 2001, 65, 4267–4273. [CrossRef] 81. Couradeau, E.; Benzerara, K.; Gérard, E.; Moreira, D.; Bernard, S.; Brown, G.; Lopez-Garcia, P. An early-branching microbialite cyanobacterium forms intracellular carbonates. Science 2012, 336, 459–462. [CrossRef] 82. Benzerara, K.; Skouri-Panet, F.; Li, J.; Ferard, C.; Gugger, M.; Laurent, T.; Couradeau, E.; Ragon, M.; Cosmidis, J.; Menguy, N.; et al. Intracellular Ca-carbonate biomineralization is widespread in cyanobacteria. Proc. Natl. Acad. Sci. USA 2014, 111, 10933. [CrossRef][PubMed] 83. Keim, C.; Solórzano, I.; Farina, M.; Lins, U. Intracellular inclusions of uncultured magnetotactic bacteria. Int. Microbiol. 2005, 8, 111–117. [CrossRef] Minerals 2019, 9, 632 26 of 26

84. Perri, E.; Tucker, M.; Slowakiewicz, M.; Whitaker, F.; Bowen, L.; Perrotta, I. Carbonate and silicate biomineralization in a hypersaline microbial mat (Mesaieed sabkha, Qatar): Roles of bacteria, extracellular polymeric substances and viruses. Sedimentology 2017, 65, 1213–1245. [CrossRef] 85. Obst, M.; Dynes, J.J.; Lawrence, J.R.; Swerhone, G.D.W.; Benzerara, K.; Karunakaran, C.; Kaznatcheev, K.;

Tyliszczak, T.; Hitchcock, A.P. Precipitation of amorphous CaCO3 (aragonite-like) by cyanobacteria: A STXM study of the inﬂuence of EPS on the nucleation process. Geochim. Cosmochim. Acta 2009, 73, 4180–4198. [CrossRef] 86. Deng, S.; Dong, H.; Lv, G.; Jiang, H.; Yu, B.; Bishop, M.E. Microbial dolomite precipitation using sulfate reducing and halophilic bacteria: Results from Qinghai Lake, Tibetan Plateau, NW China. Chem. Geol. 2010, 278, 151–159. [CrossRef] 87. Kenward, P.A.; Goldstein, R.H.; Gonzalez, L.A.; Roberts, J.A. Precipitation of low-temperature dolomite from an anaerobic microbial consortium: The role of methanogenic Archaea. Geobiology 2009, 7, 556–565. [CrossRef] 88. Bontognali, T.R.R.; Vasconcelos, C.; Warthmann, R.J.; Bernasconi, S.M.; Dupraz, C.; Strohmenger, C.J.; Mckenzie, J.A. Dolomite formation within microbial mats in the coastal sabkha of Abu Dhabi (United Arab Emirates). Sedimentology 2010, 57, 824–844. [CrossRef] 89. Lv, J.; Ma, F.; Li, F.; Zhang, C.; Chen, J. Vaterite induced by Lysinibacillus sp. GW-2 strain and its stability. J. Struct. Biol. 2017, 200, 97–105. [CrossRef] 90. Mann, S.; Heywood, B.R.; Rajam, S.; Wade, V.J. Molecular recognition in biomineralization. In Mechanisms and Phylogeny of Mineralization in Biological Systems; Suga, S., Nakahara, H., Eds.; Springer: Tokyo, Japan, 1991; pp. 47–55. 91. Zhao, Y.; Yan, H.; Zhou, J.; Tucker, M.E.; Han, M.; Zhao, H.; Mao, G.; Zhao, Y.; Han, Z. Bio-precipitation of calcium and magnesium ions through extracellular and intracellular process induced by Bacillus Licheniformis SRB2. Minerals 2019, 9, 526. [CrossRef]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).