Aerobically incubated bacterial biomass-promoted formation of disordered dolomite and implication for dolomite formation

Accepted Manuscript Aerobically incubated bacterial biomass-promoted formation of disordered dolomite and implication for dolomite formation

Ya-Rong Huang, Qi-Zhi Yao, Han Li, Feng-Ping Wang, Gen-Tao Zhou, Sheng-Quan Fu PII: DOI: Reference:

S0009-2541(19)30301-8 https://doi.org/10.1016/j.chemgeo.2019.06.006 CHEMGE 19206

To appear in:

Chemical Geology

Received date: Revised date: Accepted date:

11 November 2018 4 June 2019 6 June 2019

Please cite this article as: Y.-R. Huang, Q.-Z. Yao, H. Li, et al., Aerobically incubated bacterial biomass-promoted formation of disordered dolomite and implication for dolomite formation, Chemical Geology, https://doi.org/10.1016/j.chemgeo.2019.06.006

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ACCEPTED MANUSCRIPT

Aerobically incubated bacterial biomass-promoted formation of

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disordered dolomite and implication for dolomite formation

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Ya-Rong Huang1,2, Qi-Zhi Yao3, Han Li1,2*, Feng-Ping Wang4, Gen-Tao Zhou1,2*, Sheng-Quan

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Fu5

CAS Key Laboratory of Crust-Mantle Materials and Environments, School of Earth and

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Space Sciences, University of Science and Technology of China, Hefei 230026, P.R. China. CAS Center for Excellence in Comparative Planetology, Hefei 230026, P. R. China

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School of Chemistry and Materials Science, University of Science and Technology of China,

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Hefei 230026, P.R. China.

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State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology,

Shanghai Jiao Tong University, Shanghai 200240, P.R. China. Hefei National Laboratory for Physical Sciences at Microscale, University of Science and

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Technology of China, Hefei 230026, P.R. China.

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Corresponding author：Prof. Dr. Gen-Tao Zhou Email: [email protected] Tel.: 86 551 63600533 Fax: 86 551 63600533

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ACCEPTED MANUSCRIPT Abstract

With the realization that the formation of low-temperature dolomite could involve the microbial activity, a new pathway to understand the origin and mechanism of dolomite is emerging. Although microbially mediated Ca-Mg carbonates and dolomite occur in some

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aerobic and high-salinity conditions, little information about the exact role of aerobic microbes is available. Herein, a strain of moderately halophilic bacteria, Shewanella

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piezotolerans WP3, was selected to study the involvement of aerobically incubated bacterial

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biomass in the Ca-Mg carbonate mineralization. Different biomass components were isolated from the bacterial cultures by a set of separation techniques, and used to mediate Ca-Mg

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carbonate mineralization under a carbon dioxide diffused system. The experimental results showed that bacterial cells play a dominant role in the formation of disordered dolomite. And

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the mineralization of the disordered dolomite can be attributed to the bare cells and bound extracellular polymeric substances (bound EPS) isolated from the bacterial cells, most likely due to the promotion of high concentration and density of carboxyl and phosphoryl groups on

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the bacterial biomass (i.e., bare cells and bound EPS). Hence, it does imply that bacterial

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biomass, even without the active microbial activities, can also play an important role in the formation of dolomite. Current results can not only extend the insight into the biologically influenced mineralization of Ca-Mg carbonates, but also shed light on the precipitation of

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disordered dolomite/dolomite in modern settings and geological records.

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Keywords: Ca-Mg carbonates; dolomite; disordered dolomite; moderately halophilic bacteria; aerobically incubated bacterial biomass

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1. Introduction

Dolomite, as a kind of Ca-Mg carbonate mineral, is much more abundant in geological records than in modern sediments (e.g., Lippmann, 1973; Warren, 2000; Bontognali et al.,

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2010). Although many aqueous environments, such as modern seawater, certain lake waters and groundwaters, are supersaturated with respect to dolomite and theoretically capable of

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precipitating dolomite, dolomite precipitation is rare in modern carbonate environments (e.g.,

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Hardie, 1987; Warren, 2000). Moreover, many attempts have also been unsuccessful to abiotically synthesize dolomite under the earth-surface conditions (e.g., Hardie, 1987; Land, 1998). Therefore, the origin and formation mechanism of low-temperature dolomite have long

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been a controversy, referred to as the “dolomite problem” (Fairbridge, 1957). In recent

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decades, some studies have ascertained that microorganisms play a fundamental function in low-temperature dolomite formation (e.g., Vasconcelos et al., 1995; Warthmann et al., 2000; Kenward et al., 2009; Spadafora et al., 2010). On the one hand, the microbial dolomite

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precipitation has been discovered in some peculiar environments, such as organic carbon-rich

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deep-sea sediments or some anoxic hypersaline lake settings (e.g., Pisciotto and Mahoney, 1981; Wright, 1999; Spadafora et al., 2010). On the other hand, followed by the field observations, the laboratory syntheses of Ca-Mg carbonates by some anaerobic microbes,

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such as sulfate reducing bacteria (SRB) (e.g., Warthmann et al., 2000; van Lith et al., 2003; Krause et al., 2012), methanogen archaea (Roberts et al., 2004; Kenward et al., 2009), and

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anaerobic fermenting bacteria (Zhang et al., 2015b), have shown that microbial activities can indeed lead to Ca-Mg carbonates with various compositions, like disordered dolomite or high-Mg calcite, and it was proposed that nucleation and growth of the Ca-Mg carbonates commonly occur on the cell walls and/or extracellular polymeric substances (EPS), which can not only act as binding sites and nucleation template, but also favor dehydration of Mg2+ and increase the concentration of available Mg2+, facilitating the formation of high-Mg calcite or disordered dolomite (e.g., Warthmann et al., 2000; Kenward et al., 2009; Krause et al., 2012; Zhang et al., 2015b). Nevertheless, these studies mostly focused on the effects of anaerobic 0

ACCEPTED MANUSCRIPT microbes on dolomite precipitation, but not the aerobic microbial action. In fact, there is a close association between dolomite mineralization and aerobic microbial activity. It has been found that many aerobic bacterial strains isolated from oxic settings, such as moderately halophilic Bacillus sp., Virgibacillus sp., Marinobacter sp., Halomonas marina and Salinivibrio sp., are able to mediate Ca-Mg carbonates including dolomite under aerobic conditions (e.g., Rivadeneyra et al., 1993; Sánchez-Román et al., 2009;

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Deng et al., 2010; Al Disi et al., 2017), and it is proposed that the aerobic bacterial

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metabolism can create chemical environments favoring Ca-Mg carbonate precipitation, such as biodegradation of carbonaceous and nitrogenous compounds accompanied with

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bicarbonate and ammonium release and the consequent CO32- concentration and pH increase (e.g., Dupraz et al., 2009; Bontognali et al., 2010; Sánchez-Román et al., 2008, 2011). These

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culture studies confirmed the ability of aerobic microbes to mediate mineralization of Ca-Mg carbonates and dolomite. However, the precise mechanisms are not fully understood, and the

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specific bacterial components responsible for the enhancement of Mg content in Ca-Mg carbonates remain to be determined. Because of the complexity of bacterial system, bacterial

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cells, metabolites, and even the ingredients of culture medium may all affect the

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mineralization processes (e.g., Tourney and Ngwenya, 2009; Prywer et al., 2012; Zhang et al., 2015b; Li at al., 2017a), it can obscure the recognization to the nature of bacterial mineralization, and significantly limit the interpretation of culture studies meant to simulate

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natural system (Gallaghter et al., 2013), leading to the poor insight into the actual components responsible for Ca-Mg carbonate mineralization. In particular, several studies involving field

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observations unveiled that dolomite can be also found within metabolically non-intensive and inactive microbial mats buried in sediments, and proposed that the extracellular polymeric substances (EPS) within the buried mats act as a nucleation template for the formation of dolomite (e.g., Bontognali et al., 2010; Brauchli et al., 2016). It appears that besides the microbial metabolic activity, the mineral-template property of microbial biomass without biological activity likely also exert a crucial control over dolomite formation. Hence, in this study, by using a series of bacterial biomass separation techniques and a biomimetic mineralization strategy, different bacterial biomass components such as the 1

ACCEPTED MANUSCRIPT cell-free supernatant, bacterial cells, even bare cells and its bound EPS were isolated from the bacterial cultures, and used to influence the Ca-Mg carbonates mineralization. Considering that moderately halophilic bacteria generally exist in saline lakes, lagoons and ocean, such as a highly alkaline playa lake in California where dolomite is still precipitating (e.g., Pisciotto and Mahoney, 1981; Wright, 1999; Spadafora et al., 2010; Meister et al., 2011), Shewanella piezotolerans WP3, a strain of moderately halophilic bacterium separated from the oceanic

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sediments (Xiao et al., 2007), was selected in this study. The experimental results showed that

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both the bare cells and bound EPS separated from the bacterial cells play a dominant role in the formation of disordered dolomite, and the mineralization is predominated by the

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concentration and density of carboxyl and phosphoryl groups on the bacterial biomass. The involved mechanism can provide a less-concerned but significant relationship between

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aerobically incubated bacterial biomass and Ca-Mg carbonate formation, and also have

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valuable insight into the origin of dolomite in modern settings and geologic records.

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2. Materials and methods

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2.1 Materials

Anhydrous calcium chloride (CaCl2), magnesium chloride hexahydrate (MgCl2·6H2O), sodium chloride (NaCl), hydrochloric acid (HCl) and sodium hydroxide (NaOH) were

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purchased from Sinopharm Chemical Reagent Co., Ltd, and are of analytical grade. The

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tryptone and yeast extract are of biotech grade and purchased from Oxoid Ltd. Deionized water was used in all experiments.

2.2 Bacterial cultivation and EPS extraction Shewanella piezotolerans WP3 was used in present study. The cells of S. piezotolerans WP3 were gram-negative rods, 0.5-0.8 μm wide and 2-5 μm long (Xiao et al., 2007; Wang et al., 2009). Strain WP3 was inoculated into 100 mL of sterilized modified marine 2216E medium (5 g/L peptone, 1 g/L yeast extract, 34 g/L NaCl, pH 7.6-7.8) (Wang et al., 2009) by adding 0.1 mL of the seed culture. Subsequently, the strain WP3 was cultured in the open air 2

ACCEPTED MANUSCRIPT for 96 h at 20 °C with constant shaking (200 rpm), and the bacterial cells were harvested by means of centrifugation at 10,380 g for 10 min at 4 °C. The concentrated cells were washed three times with 3.4% (w/w) NaCl solution to remove residual growth medium. The resultant supernatants were filtered through 0.22 µm cellulose acetate membranes to eliminate any remaining cell debris. After that, a portion of the growth media (unseparated liquid culture), the filtered supernatant (cell-free supernatant), and the harvested bacterial cells would be used

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in following biomimetic mineralization experiments.

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For the separation of the intact bound EPS and bare cells, a cation exchange resin (CER) technique (Dowex Marathon C, 20-50 mesh, sodium form, Sigma-Aldrich 91973) was

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specially used (e.g., Sheng et al., 2008; Li et al., 2017a) because the other methods such as the HCHO/NaOH and EDTA routes (e.g., Liu and Fang, 2002; Zhang et al., 2015b; Sheng et al.,

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2005) can bring about the reactions of the function groups (e.g., carboxyl, amino, hydroxyl and sulfhydryl) on bacterial biomass with the added HCHO or the contamination of residual

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EDTA (e.g., Comte et al., 2006). The washed bacterial cells were resuspended into a 3.4% (w/w) NaCl solution, followed by an addition of 20 g CER. After the suspension was stirred

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for 12 h with 500 rpm at 4 °C, the CER was removed by settlement. Subsequently, the

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solution was centrifuged at 10,380 g for 10 min at 4 °C to isolate the cells without EPS (denoted as bare cells) and supernatant. The bare cells were washed three times with 3.4% (w/w) NaCl solution and resuspended for further use. The supernatant was further filtered

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through 0.22 µm cellulose acetate membrane to obtain the filtrate, i.e., the bound EPS.

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2.3 Biomimetic mineralization experiments Mineralization experiments were performed by using a carbon dioxide gas diffusion technique at room temperature (25 oC), as described by Zhou et al. (2010). In a typical protocol, MgCl2·6H2O and CaCl2 were dissolved in 5 mL of deionized water under vigorous stirring to form solution A. Solution B is a series of 20 mL solutions with different bacterial components, i.e., unseparated liquid culture, cell-free supernatant, bacterial cells, bare cells and bound EPS. Then, the solution A was introduced into solution B under continuous stirring to obtain a homogeneous mixture solution in 25-mL beaker, and the initial concentrations of 3

ACCEPTED MANUSCRIPT Ca2+ and Mg2+ in the solutions were 10 mM and 50 mM (Mg/Ca = 5), respectively. The initial pH of the solutions was adjusted to 6.0 using a diluted HCl or NaOH solution. After that, the breaker was covered with parafilm punched with six needle holes and put in a desiccator. A 50-mL breaker containing twenty grams of crushed ammonium carbonate was placed in the center position of the desiccator as the source of CO2, and the 100 mL of H2SO4 (98%) was poured into the bottom of the desiccator to absorb NH3 vapor. The beaker was moved out of

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the desiccator after 10 days of mineralization, and the resulting precipitate was isolated by

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centrifugation, washed with deionized water and absolute alcohol three times, and then dried at room temperature overnight for characterization. Control experiments with deionized water,

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3.4% (w/w) NaCl solution or uninoculated culture medium were also conducted under the same conditions. To avoid microbial contamination, the experimental equipment and solutions

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were UV-sterilized in anticipation, and the experiments were conducted in super clean bench. All the experiments were run in triplicate and the detailed experimental conditions for

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carbonate synthesis are listed in Table 1.

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2.4 Mineral characterization

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Morphological textures of the mineralized products were analyzed after gold sputtering with a JEOL JSM-6700F field-emission scanning electron microscope (FESEM) in LEI mode by using 5.0 kV acceleration voltages. The elemental compositions of the products were

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measured on a field emission SEM microscope (TESCAN MIRA 3) equipped with energy dispersed X-ray spectroscopy (EDX, GENESISAPEX). Micro-Raman spectra were collected

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by a JY Horiba LabRam HR Evolution microprobe with a 532 nm Ar laser excitation. The beam size for Raman spectroscopy was ~1 μm. Monocrystalline silicon and polystyrene were analyzed during the analytical session to monitor the precision and accuracy of the Raman data. X-ray diffraction (XRD) analysis of the samples was conducted on a Japan MapAHF X-ray diffractometer using Cu Kα irradiation (λ= 0.154056 nm), with a scanning rate of 0.01 °s-1 in the 2θ range of 10–70°. The mol% of Mg2+ (Mg content) in Ca-Mg carbonate minerals was calculated by published empirical formula based on its d104 value (i.e., the position of the main peak in the XRD pattern) (Goldsmith et al., 1961). The calculated Mg 4

ACCEPTED MANUSCRIPT contents for various Ca-Mg carbonates are also listed in Table 1. Selected area electron diffraction (SAED) patterns, high-resolution transmission electron microscopy (HRTEM) images, and transmission electron microscopy (TEM) images were obtained on a Hitachi model H-800 transmission electron microscope with an accelerating voltage of 200 kV.

2.5 Characterization of bacterial biomass surface

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Surface site concentrations, densities and pKa values for proton-reactive functional

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groups of bacterial biomass surface were determined from acid-base titrations, which were conducted under a N2 atmosphere at 298 K on suspensions of S. piezotolerans WP3 bare cells

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and the bound EPS in a background electrolyte of 3.4% (w/w) NaCl, as described by Fein et al. (2001). The detailed protocol is as follows: after being washed by 3.4% (w/w) of NaCl

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solution five times, the concentrated bare cells and bound EPS were transferred to an acid washed 250-ml Erlenmeyer flask and brought to a final volume of 50 mL and 200 mL with

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the NaCl solution, respectively. Subsequently, the different bacterial biomass components were acidified to pH 2 with 0.1 M HCl, and the titration was individually performed from pH

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2 to 11 using 0.1 M NaOH solution that had been freshly purged with N2 for 20 min to

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remove CO2. The system was continuously purged with N2 and stirred magnetically at room temperature during the titration process. Finally, the titration data were modeled using PROTOFIT 2.1 software (Turner and Fein 2006), where the ligand pKa values and site

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concentrations are both simultaneously optimized to best fit the data. The corresponding site densities were calculated from the site concentrations using their individual averaged surface

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area from the literature (van der Wal et al., 1997; Kenward et al., 2013). Moreover, the bare cells before and after 12 h of adsorption in solution with 10 mM of Ca2+ and 50 mM of Mg2+ were analyzed by a Thermo ESCALAB 250 X-ray photoelectron spectrometer (XPS) using Al Kα radiation to understand the interactions between the proton-reactive functional groups on the bare cells and Ca2+/Mg2+ ions.

3. Results 5

ACCEPTED MANUSCRIPT After 10 days of mineralization, a white precipitate was obtained in the solution with unseparated bacterial cultures of S. piezotolerans WP3. Typical panoramic FESEM images show that the white precipitate mainly consists of spherical structures (Figures 1a and b). According to their dimensions, however, it is possible to recognize two distinct classes, the large spheres with near one hundred μm in diameter (Figure 1a) and the small spheres with less than or equal to ten μm in diameter (Figure 1b). Higher magnification observations of the

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small spheres show that the spherical surface is granular texture, consisting of much smaller

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spherical structures or nanoglobules, besides a number of bacterial thalli present (e.g., Figure 1d). In contrast, no bacterial thalli can be observed on the surface of the large spheres (e.g.,

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Figure 1c) and the finer-scale texture of the big spheres consists of micron-sized triangular pyramids. The EDX analyses reveal that the big spheres contain O, C and Ca (Figure 1c inset),

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while the small spheres contain O, C, Ca and Mg, and the mole ratio of Mg to Ca is close to 6:5 (Figure 1d inset), where the element Au comes from the sample preparation of FESEM

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analysis (Figures 1c and d insets). These results reveal that the two kinds of spherical structures are significantly different not only in morphological texture but also in composition.

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Further micro-Raman spectroscopy was used to characterize the two kinds of spheres. All the

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Raman spectra collected from the small spheres exhibited clear vibrational bands at around 1092, 720, 290 and 163 cm-1 (Figures 2a and b), which can be well assigned to the internal stretching (ν1) and bending (ν4) vibrations of the C-O bonds in the carbonate groups, and the

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external translational (T) and librational (rotatory) (L) modes of calcite, respectively (Perrin et al., 2016). However, the Raman spectra from different areas of the big sphere all show the

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bands centered at 1066, 694 and 720 cm-1 (Figures 2c and d), which are the symmetric stretching and the in-plane bending modes of carbonate groups in monohydrocalcite (Zhang et al., 2015a). Based on the analyses of EDX and Raman spectroscopy, it can be concluded that the small spheres are magnesian calcite (Mg-calcite), while the big spheres are monohydrocalcite. Furthermore, the XRD analyses also demonstrate that the white precipitate contains monohydrocalcite and Mg-calcite (e.g., Figure 3a). The strong and sharp peaks belonging to monohydrocalcite indicate that the monohydrocalcite is well crystallized, in accordance to the perfect developed habit (i.e., exposed triangular pyramid) of 6

ACCEPTED MANUSCRIPT monohydrocalcite with a space group P31 (Figure 1c). Nevertheless, note that the characteristic (104) diffraction peak of Mg-calcite is significantly shifted from standard calcite d104 = 3.07 Å (JCPDS file 86-2343) to its d104 = 2.90 Å, and the Mg2+ content in Mg-calcite, as estimated by the shift of the (104) diffraction peak in the XRD spectrum (Figure 3a), are ca. 49 mol% (Table 1), close to ideal dolomite stoichiometry [CaMg(CO3)2)]. Because of no appearance of superstructure reflections with indexes (h0l) and (0kl) (l = odd) of dolomite,

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such as (015), (021) and (101) in Figure 3a, the Mg-high calcite is defined as disordered

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dolomite.

It is noteworthy that the Ca-Mg carbonates with such high Mg contents does not appear

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in the control experiments with the uninoculated culture medium, deionized water, or 3.4% (w/w) NaCl solution after the same mineralization time (Figures 3b-d and Table 1). In

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particular, monohydrocalcite and Mg-calcite with only 8.9 mol% of MgCO3 form in the uninoculated culture medium (Figure 3b and Table 1), Mg-calcite and minor aragonite in

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deionized water or the NaCl solution (Figures 3c and d, and Table 1). Moreover, the Mg content in Mg-calcite increases to from 27 mol% in deionized water to 38 mol% in the NaCl

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solution (Figures 3c and d, and Table 1), indicating that the high salinity is favorable to the

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formation of high-Mg calcite. However, the conspicuous gap in the Mg content of obtained Ca-Mg carbonates between the unseparated bacterial culture (49 mol% MgCO3) (Figure 3a and Table 1) and the 3.4% (w/w) NaCl solution (38 mol% MgCO3) (Figure 3d and Table 1)

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reveals that the bacterial biomass from aerobically cultured S. piezotolerans WP3 could significantly promote the incorporation of Mg into the Ca-Mg carbonate.

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In this context, the effects of different components in unseparated bacterial culture on the Mg content in Ca-Mg carbonates were investigated under the same biomimetic mineralization conditions. The XRD pattern of the product obtained from the cell-free supernatant shows a mixture phase consisting of massive Mg-calcite with only 20 mol% of MgCO3 and minor aragonite (Table 1 and Figure 4a). The FESEM analyses reveal that the product mainly exhibit the pillow-shaped morphology (Figure 4c), which is different from the precipitate obtained from unseparated bacterial culture (e.g., Figure 1). These results indicate that the soluble bacterial metabolites in the supernatant are not the key components responsible for the 7

ACCEPTED MANUSCRIPT formation of the spherical disordered dolomite in unseparated bacterial culture. In the case of bacterial cells, however, the XRD result indicates that the mineralized product contains high-Mg calcite with 49 mol% of MgCO3 and minor monohydrocalcite (e.g., Figure 4b and Table 1). The presence of a large amount of Mg in the calcite crystals is also indicated by the anomalous position of the d104 peak, which is 2.90 Å (49 mol% of MgCO3) relative to standard calcite 3.07 Å, and represents a considerable shift towards the stoichiometric dolomite peak

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(2.88 Å) (e.g., Figure 4b and Table 1). Further FESEM observations reveal that the product is

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composed of spherical nanoparticle aggregates from 5 to 10 μm in diameter (Figure 4d), and a number of bacterial thalli can be also observed on the product surface (Figure 4e), similar to

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the disordered dolomite obtained in the unseparated bacterial culture (Figures 1b and d). It appears that the bacterial cells exert crucial control on the mineralization of Ca-Mg carbonate

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close to dolomite composition.

In order to further understand the role of the bacterial cells in the Ca-Mg carbonate

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mineralization, the bacterial cells were firstly separated into bare cells and bound EPS, and then their effects on the mineralization of Ca-Mg carbonate were systematically investigated

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at different time intervals (1, 3, 7, and 10 days). The XRD results show that the precipitates

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from the solution with the bare cells mainly consist of Mg-calcite, but the (104) peak positions of the earlier precipitates are not shifted as much to the high 2θ angle as the later precipitates, as highlighted by the dash line (Figures 5a-d and Table 1), indicating that the

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earlier precipitates have less Mg2+ incorporation, and will evolve to Ca-Mg carbonates with higher Mg contents over time. In particular, after 10 days, the Ca-Mg carbonate with ~49 mol%

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of MgCO3 has been obtained (Figure 5d and Table 1). In addition, the morphological textures of the precipitates were also analyzed by FESEM technique. The FESEM results reveal that the precipitates have a typical transition from dumbbells (with many vestiges of bacteria cells on their surfaces) (1day, Figure 6a) through dumbbells and spheres (3 and 7 days, Figures 6b and c) to compact spheres (10 days, Figure 6d). A broken part from the compact sphere (Figures 6d and its inset) exhibits a radial growth pattern in its inner part. Moreover, the observed vestiges of cells on the mineral surfaces indicate a direct relationship between bare cells and the mineralization (e.g., Figures 6a-c insets). Meanwhile, the Ca-Mg carbonates with 8

ACCEPTED MANUSCRIPT high contents of Mg2+ (16-45 mol%) can also be precipitated out of the solution with bound EPS (e.g., Table 1 and Figure 5e), indicating that the bound EPS can also enhance Mg2+ incorporation into precipitating Ca-Mg carbonates. After mineralization for 1 day, however, there was no visible precipitate production in the presence of bound EPS (Table 1). By contrast, the Ca-Mg carbonate with high crystallinity can always be acquired with the bare cells, and the Mg content in the Ca-Mg carbonates has a coherent increase relative to the case

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and promotion to the Ca-Mg carbonates with high Mg content.

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with bound EPS (Table 1), indicating that the bare cells should play a greater role in catalysis

The microstructure of the spherical Ca-Mg carbonates with near dolomite stoichiometry,

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which were precipitated in the presence of the unseparated bacterial cultures, bacterial cells or bare cells, was further investigated by TEM, respectively. Figure 7a depicts a representative

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TEM image of a subsample from the spherical Ca-Mg carbonate with 49 mol% of MgCO3 harvested from the unseparated bacterial culture (Figure 1a and b). Clearly, this thin section is

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composed of nanocrystals, conferring a polycrystalline nature to the subsample. TEM-based EDX analyses (Figure 7a inset) show that the subsample contains ~ 53 mol% MgCO3,

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confirming that the subsample should originate from the Ca-Mg carbonate spheres. The

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HRTEM image further reveals that it is made of an assemblage of nano-crystals with the clearly resoluble lattice fringes (Figure 7b). The lattice spacing of 0.289 nm and the indexed SAED pattern (Figures 7b and its inset) further confirm that the Ca-Mg carbonate is close to

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the stoichiometry of ideal dolomite, in agreement with the XRD result (e.g., Figure 3a and Table 1). The dispersed and prolonged diffraction dots also indicate that more than one

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nanocrystal may contribute to the diffraction, and the crystallographic directions of the nano-crystals in the Ca-Mg carbonate spheres are not fully random but are roughly local co-oriented. As for the spherical Ca-Mg carbonates from bacterial cell or bare cell solution (e.g., Figures 4d and 6d), the similar microstructure characteristics can be observed, and their typical HRTEM images and SAED patterns are exhibited in Figure 7c and d, respectively. However, due to the lack of characteristic superstructure reflections of dolomite [e.g., (101), (015) and (021)] in the SAED patterns (Figures 7b-d insets), the synthetic Ca-Mg carbonates can only be regarded as disordered dolomite. In a word, according to the experimental results, 9

ACCEPTED MANUSCRIPT it can be summarized that the aerobically bacterial biomass can promote the incorporation of Mg into calcite, facilitating the formation of disordered dolomite. Moreover, to better clarify how the bacterial biomass influence the disordered dolomite mineralization at the molecular functional group level, the proton-reactive sites on the bacterial biomass were determined by acid-base titrations. The titration dataset are modeled by using PROTOFIT 2.1 based on a four-site non-electrostatic surface complexation model

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(Turner and Fein, 2006) to determine the proton binding constants (pKa values) and surface

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functional group concentrations for each of the biomass. The model simulations yield the optimized fits to the raw titration data (Supplemental Figure S1), suggesting that the estimated

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values are reasonable and reliable. Supplemental Table S1 summarizes the pKa values of ligands present on the bare cell and bound EPS surface, which can be assigned to carboxyl

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(pKa 4.38 and 4.69) and phosphoryl (pKa 8.00 and 8.62) (Sokolov et al., 2001), and corresponding concentration and density of the two kinds of functional groups. However, the

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pKa values corresponding to thiol (pKa 7.0) and amino groups (pKa 9.0) (Sokolov et al., 2001; Braissant et al., 2007) are absent, indicating that the proton-reactive sites from thiol and

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amino groups are relatively minor. The bare cells are estimated to have a 6.03 × 10–4 mol/g of

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carboxyl groups and a 0.057 carboxyl Å-2, whereas 3.90 × 10–4 mol/g and 0.037 phosphoryl Å-2 for the phosphoryl groups, respectively. As for the bound EPS, the estimated concentration and density are 1.70 × 10–3 mol/g and 0.021 carboxyl Å-2 for carboxyl group, and 2.50 × 10–4

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mol/g and 0.031 phosphoryl Å-2 for phosphoryl group, respectively. The XPS analyses were also further carried out on the bare cells before and after a 12 h

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of incubation in Ca2+-Mg2+ solution. As shown in Figure S2a and b, two new peaks at 50.7 and 347.5 eV appear on the survey spectrum of cation-adsorbed bare cells, assigned to Mg 2p and Ca 2p3 (Demri and Muster, 1995; Ardizzone et al., 1997), indicating that Mg2+ and Ca2+ are absorbed onto the surface of bare cells. Interestingly, more Mg2+ ions (3.19 mol%) are concentrated on cell surfaces relative to Ca2+ ions (0.87 mol%) (Table S2), indicating a preferential Mg2+ adsorption over Ca2+. To better clarify the functional groups involved in the cation adsorption, the high resolution scans of C 1s,

P 2p, and N 1s are deconvoluted

(Figure S2c-e), and corresponding functional groups are assigned and quantified (Table S3). 10

ACCEPTED MANUSCRIPT Specifically, the C 1s peaks of the two bare cell samples can be resolved into three components, i.e., the peak at 284.7 or 284.8 eV corresponds to the C-(C/H), the peak at 286.1 or 286.3 eV to C-OH, the peak at 287.9 or 288.4 eV to O=C-O (Li et al., 2017a). The deconvoluted P 2p peaks can be attributed to P-C at 132.9 or 133.4 eV, O=P(OR)3 at 133.4 or 133.8 eV, and P-O at 134.3 or 134.5 eV (Li et al., 2017b). Similarly, the high resolution N 1s peaks can be assigned to C-NH2 at 399.0 eV and C-NH3+ at 401.1 eV (Ederer et al., 2017).

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These results confirm the presence of carboxyl, phosphoryl, and amino groups on the bare

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cells. Moreover, it can also be found that after Ca2+ and Mg2+ adsorption, the subpeaks O=C-O and O=P(OR)3 have a shift from 287.9 to 288.4 eV and 133.4 to 133.8 eV, and the

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relative area ratios of these groups decrease, confirming that the complexation of the carboxyl and phosphoryl groups with Ca2+ and Mg2+ occurs (Li et al., 2017b). However, the positions

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and relative area ratios for C-NH2 and C-NH3+ groups do not change before and after cation adsorption, indicating that these groups do not contribute to the binding with Mg2+ and Ca2+

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(Costa et al., 2015). Meanwhile, the peak assigned to S 2p around 164.0 eV is invisible on the XPS survey spectra before and after Mg2+ and Ca2+ adsorption (Figure S2a and b), and

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corresponding high-resolution XPS spectra (Figure S2f) do not confer valuable signal of

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element S (Hsu et al., 2014), indicating that the contribution of thiol to binding Mg2+ and Ca2+ is negligible in our case. These results are consistent with the titration-modeled results. Therefore, it follows that the carboxyl and phosphoryl groups can be as central loci for the

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metal cation adsorption as they are deprotonated and thus may have intimate relationship with

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Ca-Mg carbonate precipitation, even dolomite.

4. Discussion

In this study, disordered dolomite can be obtained in the presence of the unseparated bacterial culture, bacterial cells, bare cells, or bound EPS (Figures 3a, 4b and 5d-e, Table 1). However, uninoculated culture medium, 3.4% (w/w) NaCl solution and deionized water, even the cell-free supernatant, all cannot produce disordered dolomite (Figures 3b-d and 4a, Table 1). Considering that all of the mineralization runs were conducted under the same conditions, 11

ACCEPTED MANUSCRIPT but not all precipitated disordered dolomite, it can be concluded that the aerobically incubated bacterial biomass of S. piezotolerans WP3, i.e., bare cells and bound EPS, should be the key factors for promoting disordered dolomite formation. According to our FESEM results (Figures 1b and d, 4e, and 6), the remains of the bacterial cells are conspicuously observed around and on the surface of the disordered dolomite, indicating that these bacterial cells are tightly associated with the precipitation of the disordered dolomite. However, no disordered

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dolomite was found in the case of the cell-free supernatant, which contains various dissolved

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bacterial metabolites including polysaccharides, proteins, peptides, and small molecular amino acids, etc. (e.g., Braissant et al., 2003; Dupraz, et al., 2009; Li et al., 2017a), revealing

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that these dissoluble metabolic components do not exert the controls over the disordered dolomite formation. At the same time, bacterial biomass is basically absent either around or

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on the surface of the monohydrocalcite (Figures 1a and c). These results indicate that the different precipitation processes lead to the different carbonates, and the bacterial biomass

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(i.e., bare cells and bound EPS) should be responsible for the mineralization of disordered dolomite.

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It is well known that all bacterial cells are surrounded by a porous three-dimensional

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network consisting of various macromolecules such as peptidoglycan, teichuronic acid, (lipo-)teichoic acid, (lipo-)polysaccharides, (lipo-)proteins, enzymes and mycolic acids, and most of these macromolecules are polyelectrolytes, because they carry charged groups such as

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carboxyl, phosphoryl or amino groups (e.g., van der Wal et al., 1997). With their deprotonation, these functional groups can not only act as the nucleation sites or structure

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template for mineral precipitation, but also break down the hydration spheres of Mg2+, favoring dehydration of [Mg(H2O)6]2+ (e.g., Kenward et al., 2013; Roberts et al., 2013; Qiu et al., 2017). For example, Kenward et al. (2013) found that the archaeal cell walls with higher density of carboxyl groups can facilitate the formation of Ca-Mg carbonates or dolomite. They proposed that [Mg(H2O)6]2+ can be adsorbed onto the surface carboxyl groups to form a MgCO3(H2O)4(R-CO2) complex concomitant with a water molecule removal, creating a condition for the attachment of a Ca2+ and a second CO32-, and finally leading to a thin Ca-Mg carbonate template for further deposition of dolomite. Here, the modeling results (Table S1) 12

ACCEPTED MANUSCRIPT show that the bare cells exhibit a carboxyl concentration of 6.0 × 10–4 mol/g and a carboxyl density of 0.057 carboxyl Å-2, which are comparable to the cell walls of archaea Methanobacterium formicicum (8.1 × 10–4 mol/g and 0.06 carboxyl Å-2) and Haloferax sulfurifontis (1.6 × 10–3 mol/g and 0.1 carboxyl Å-2) (Kenward et al., 2013). In particular, the disordered dolomite and Mg-calcite could be precipitated by archaea Methanobacterium formicicum and Haloferax sulfurifontis (Kenward et al., 2013). Therefore, it can be

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analogically concluded that the bare cells from the aerobically cultured strain WP3 mineralize

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the disordered dolomite also by means of their high density of carboxyl groups. Indeed, our XPS analyses for the cation-adsorbed bare cells show that both Mg2+ and Ca2+ can be

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adsorbed onto the bare cells (Figure S2a and b, and Table S2). The deconvolution for high-resolution C 1s spectra further unveils that binding energy of the C assigned as O=C-O

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component has shifted from 287.9 to 288.4 eV after Ca2+ and Mg2+ adsorption (Figure S2c and Table S3). These confirm that the interactions between carboxyl groups with Ca2+ and

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Mg2+ occur.

Furthermore, note that S. piezotolerans WP3 is a strain of moderately halophilic

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bacterium, and the salinity in our culture medium is up to 3.4% (w/w). It has been

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demonstrated that the carboxyl density on microbial cell walls increases with the salinity (Kinnebrew, 2012; Voegerl, 2014). For example, the halophilic microbes, for sustaining the structural integrity, can inhibit the positively charged ions (e.g., Na+) into their cells by

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constructing a hexagonal S-layer consisting of acidic proteins or glycoproteins outside their cell membranes (Oren, 1999; Sleytr et al., 2014), and thus increasing the carboxyl density

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(Qiu et al., 2017). It appears that the high carboxyl density on the bare cells is inevitable in our case. By contrast, the cells of S. putrefaciens aerobically cultured in trypticase soy broth with 0.5% yeast, in despite of the same genus of Shewanella, possess a lower carboxyl density of 0.03 carboxyl Å-2, and no dolomite can be precipitated by this strain (Kenward et al., 2013). It further supports that the occurrence of disordered dolomite in the presence of S. piezotolerans WP3 instead of S. putrefaciens can be attributed to the high carboxyl density of moderately halophilic bacterium S. piezotolerans WP3. This can provide a reasonable interpretation of why the modern dolomites mainly occur in a restricted number of 13

ACCEPTED MANUSCRIPT hypersaline environments (e.g., evaporitic lakes and sabkhas) or seawater, which have the high salinity conditions (e.g., Pisciotto and Mahoney, 1981; Wright, 1999; Spadafora et al., 2010; Petrash et al., 2017), and the biomass of a series of halophilic microbes in these environments can facilitate the formation of Ca-Mg carbonates with high-Mg contents, even dolomite. Similar to carboxyl groups, the phosphoryl groups outside the bacterial cells can also

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provide metal cation binding sites (e.g., van del Wal et al, 1997; Fein et al., 2001). In our case,

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part of Ca2+ and Mg2+ can also bind to phosphoryl sites on the bacterial cell surface, which is similarly supported by the XPS analyses (e.g., Figure S2d and Table S3), i.e., the

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deconvolution for high-resolution P 2p spectra donates that the binding energy assigned as phosphoryl group shift from 133.4 to 133.8 eV upon Ca2+ and Mg2+ adsorption. In general, at

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low pH, the metal cation binding could be best modeled as adsorption onto carboxyl sites, whereas phosphoryl sites contributing more with increasing pH (e.g., Fein et al., 1997, 2001;

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Daughney et al., 1998; Texier et al., 2000). Here, the final pH values are high up to 9.1 (Table 1). Therefore, under such high pH conditions, Ca/Mg-phosphoryl binding should become

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more important relative to the Ca/Mg-carboxyl binding. In addition, the outer membrane of

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Gram-negative bacteria, characterized by the lipopolysaccharide and phospholipid compounds, is essentially composed of phosphoryl moieties (e.g., Beveridge and Fyfe, 1985; Texier et al., 2000). Recently, based on the speciation analysis by EXAFS, Du et al. (2017) also found that

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the binding affinity of Cd-phosphoryl seems stronger in Gram-negative Pseudomonas putida than that in Gram-positive Bacillus subtilis, and hence suggested that the phosphoryl-binding

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is more important in Gram-negative bacteria than in Gram-positive bacteria. Combined with the Gram-negative nature of S. piezotolerans WP3 and the high pH conditions achieved in current mineralization systems, it can be believed that the phosphoryl groups on the bacterial cells should significantly contribute to the sorption of cation Mg2+ and Ca2+, thereby promoting the disordered dolomite mineralization. Furthermore, the titration experiments also show that the concentration and density of phosphoryl groups are 3.9 × 10–4 mol/g and 0.037 phosphoryl Å-2 (Table S1), respectively, indicating that the enhancement of high-Mg calcite or disordered dolomite formation may 14

ACCEPTED MANUSCRIPT also be related, to some degree, to the nucleation promotion of phosphoryl groups outside the bare cell surfaces. Such nucleation promotions exerted by phosphoryl groups on the cells have also been found in several mineral precipitation including barite, aragonite, iron oxides, and Ca-Mg carbonates, and a general consensus is the initial formation of P-rich amorphous precursor (e.g., González-Muñoz et al., 2003, 2012; Miot et al., 2009; Rivadeneyra et al., 2010, 2016; Torres-Crespo et al., 2015; Balci and Demirel, 2016). For example, bacterial

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barite precipitation often follows a transformation from the initial amorphous or poorly

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crystalline P-rich precursor to crystalline barite (Gonzalez-Muñoz et al., 2012; Torres-Crespo et al., 2015), and the P-rich precursor is a common step in bacterial biomineralization of iron

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oxides, apatite, aragonite or Ca-Mg carbonates both under laboratory conditions and in natural environments (e.g., Rivadeneyra et al., 2010, 2016 and references therein; Balci and Demirel,

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2016). In particular, our time-course experimental results also show that massive nano-sized amorphous particles are formed around the bare cell surfaces at earlier mineralization stage (6

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h) (Figure S3a and b). Further TEM, SAED, and EDX analyses reveal that the amorphous nanoparticles also contain rich phosphorus (Figure S3c and d). Appearance of the phosphoric

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amorphous precursor can support the significant contribution of phosphoryl groups to

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concentration of Mg2+ and Ca2+ on the bare cell surface. Moreover, note that the adsorbed Mg2+ (3.19 mol%) on the bare cells is significantly higher than the Ca2+ (0.87 mol%) (Table S2), and the EDS analyses for the P-rich amorphous precursor show that the content of Mg2+

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(9.63 mol%) in the amorphous precursor is also higher than the Ca2+ (5.03 mol%) (e.g., Figure S3d), indicating that the phosphoryl groups can preferentially bind with Mg2+.

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Therefore, it demonstrates that the phosphoryl groups outside of bare cell surfaces are also the crucial factor that promotes the formation of Ca-Mg carbonates with high Mg2+ contents or dolomite.

The bare cells in this study are not the only bacterial biomass potentially capable of binding and dehydrating [Mg(H2O)6]2+. In our case, the disordered dolomite with ~45% MgCO3 can be also obtained with the bound EPS (Figure 5e and Table 1). Our titration experiments reveal that the concentration and density of carboxyl groups on the bound EPS are 1.7 × 10–3 mol/g and 0.021 carboxyl Å-2, respectively (Table S1). These are similar to the 15

ACCEPTED MANUSCRIPT values of EPS excreted by Desulfovibrio sp. (1.6 × 10–3 – 2.4 × 10–3 mol/g, 0.02–0.03) that is able to precipitate disordered dolomite (Table 2 listed in Kenward et al., 2013). Moreover, the corresponding concentration and density for phosphoryl groups are 2.5 × 10–4 mol/g and 0.031 phosphoryl Å-2, respectively, which are also comparable to the estimated values of the phosphoryl groups on the bare cells (Table S1). Therefore, it is not difficult to understand that the bound EPS-promoted mineralization of disordered dolomite similarly proceeds by the

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combined effects from their surface phosphoryl and carboxyl groups with negative charge.

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With regard to the morphogenesis of the obtained high-Mg calcites or disordered dolomites, it is mainly spherulitic or dumbbell-shaped (e.g., Figures 1b, 4d, and 6). In fact, the

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spherulitic and dumbbell-shaped disordered dolomite/dolomite has been reported by several studies involved in bacterial mineralization (e.g. Warthmann et al., 2000; Kenward et al., 2009;

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Sánchez-Román et al., 2011). Such morphology and texture features can be conceptually described as a multistep growth process. That is, an amorphous precursor forms prior to

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crystallization, and then the crystallization proceeds via spherulitic growth (Schmidt et al., 2005; Beck and Andreassen, 2010; De Yoreo et al., 2015; Ruiz-Agudo et al., 2017). In our

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experimental system, the products obtained with bare cells show a time-dependent

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transformation from the amorphous aggregates consisting of nanoparticles (Figures S3a-c, Table 1) through more stable crystalline dumbbell-shaped Mg-calcite with different contents of Mg2+ (Figures S4, 5a-c and 6a-c, Table 1) to the spherulitic disordered dolomite (Figures

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5d and 6d, Table 1), which is analogous to the spherulitic growth process that the spherulites grow from a precursor where the edges fan out giving rise to intermediate “dumbbell-like”

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shapes, to ultimately the spherical structures (Gránásy et al., 2005; Beck and Andreassen, 2010). Interestingly, a kind of radially shaped growth morphology has also been observed in the inner of the spherical product (e.g., Figure 6d inset), and our HRTEM and SAED analyses also reveal that the high-Mg calcite nanocrystals in the spherical structures are locally co-oriented, indicative of a particle aggregation-based pathway toward the crystallization and growth of the disordered dolomite (e.g., Zhou et al., 2009; 2010; De Yoreo et al., 2015; Ruiz-Agudo et al., 2017). In short, a multistep process may be responsible for the crystallization and growth of the dumbbell-shaped or spherical disordered dolomite. 16

ACCEPTED MANUSCRIPT Moreover, the precipitation of disordered dolomite with bacterial biomass (i.e., bare cells and bound EPS) could provide the extended and deeper insights into the mineralization of microbial dolomite. First, because the S. piezotolerans WP3 used in this study is a strain of moderately halophilic bacteria separated from the oceanic sediments (Xiao et al., 2007), our experimental results can be used to interpret the modern dolomite precipitation, which still occurs in the ocean or some hypersaline lake settings rich in halophilic microbes (e.g.,

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Pisciotto and Mahoney, 1981; Wright, 1999; Spadafora et al., 2010; Meister et al., 2011).

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Second, we propose that except for bacterial metabolic activities, the bacterial necromass can also exert significant control over the mineralization of disordered dolomite. It can be used to

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reconcile the recent filed observations that low-temperature dolomite has formed, and even is still forming on the devitalized bacterial biomass in metabolically non-intensive or inactive

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microbial mats (e.g., Bontognali et al., 2010; Sadooni et al., 2010; Brauchli et al., 2016). For example, Bontognali et al. (2010) found that dolomite continues to precipitate nowadays

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within the buried microbial mats that no longer show signs of intensive microbial activity in the supratidal zone of the coastal sabkha in Abu Dhabi (United Arab Emirates). Brauchli et al.

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(2016) studied the correlation between dolomite and total organic carbon within the vertical

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profiles of the Dohat Faishakh sabkha in Qatar, and proposed that the presence of living and decaying microbial mats comprising EPS may be the key factor for dolomite formation in the Dohat Faishakh sabkha, especially no need of active microbial metabolism. Similar scenarios

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have also been observed in the Tarim Basin, NW China (You et al., 2018). Furthermore, it has been proposed that global oxygenation events existed during the

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Ediacaran period which belongs to the Precambrian when the extensive sedimentary dolomite strata were formed (e.g., Sahoo et al., 2016; Wang et al., 2017). Hence, it leads us to speculate that if the assertion of microbial Ca-Mg carbonates or dolomite precipitation scenario is correct, the ancient dolomite deposits may be also formed by a mechanism as proposed here, regardless of whether ancient dolomite formation occurred via other processes. In a word, our study not only helps explain the precipitation of disordered dolomite/dolomite in modern settings, but also have important geological implications for shedding light on the mineralization of ancient dolomite in the geological record. 17

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5. Conclusions

In summary, this study adopted a biomimetic mineralization strategy to investigate the influence of different bacterial components on Ca-Mg carbonate mineralization. The

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experimental results shows that the disordered dolomite can be precipitated with aerobically incubated bacterial biomass (i.e., bared cells and bound EPS), and the mineralization process

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can be ranged as the biologically influenced mineralization. Furthermore, the molecular functional group level analyses reveal that the carboxyl and phosphoryl groups on the

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bacterial biomass act as preferential adsorption loci of Mg2+ and Ca2+, thereby as the nucleation or structure template for the disordered dolomite precipitation. Therefore, the high

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concentration and density of carboxyl and phosphoryl groups on the bacterial biomass without metabolic activity are the key factors to promote the formation of disordered dolomite. Our

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results extend the understanding of the biologically influenced mineralization of Ca-Mg carbonates, and provide deeper insights into the origin of disordered dolomite/dolomite in

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modern settings and geological records.

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Acknowledgements

This study was partially supported by the Natural Science Foundation of China

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(41572026, 41372053), and the Fundamental Research Funds for the Central Universities. We thank Benjamin Francis Turner for assistance with the titration data model analysis by PROTOFIT 2.1 software.

18

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Table 1. Final pH, mineral phase of precipitate, and Mg content in the Ca-Mg carbonate after different mineralization time intervals Final

(day)

pH

10

9.1

Deionized water

10

8.9

3.4% NaCl solution

10

8.7

Cell-free supernatant

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(d104) of Ca-Mg

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carbonate (Å) 2.90

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on d104 (mol%)

3.01

8.9

Mg-calcite and

minor aragonite

2.96

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Mg-calcite and

minor aragonite

2.93

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Mg-calcite and

minor aragonite

2.98

20

High-Mg calcite and

minor monohydrocalcite

2.90

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2.98

20

Amorphous phase Mg-calcite and minor aragonite

1

9.1

Mg-calcite

2.97

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3

9.0

Mg-calcite

2.97

23

7

9.0

Mg-calcite

2.95

31

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8.9

High-Mg calcite

2.90

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1

9.0

No precipitate

3

9.0

Mg-calcite

2.99

16

7

9.1

Mg-calcite

2.98

20

10

8.8

2.91

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* Mg content of

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d-spacing of the

High-Mg calcite and monohydrocalcite

* The Mg content of synthetic Ca-Mg carbonates based on the empirical curve (Goldsmith et al., 1961) correlating the Mg content and the shift of carbonate (104) peak toward dolomite.

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ACCEPTED MANUSCRIPT Figure captions Figure 1. Typical FESEM images of the products mineralized for 10 days in the presence of the unseparated bacterial cultures of aerobically incubated S. piezotolerans WP3: the panoramic (a) and regional (b) images, and the detailed views of the big sphere surface (inset, the EDX spectrum of big spheres) (c) and the small sphere surfaces (inset, the

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EDX spectrum of small spheres) (d). Figure 2. Raman spectra collected from the precipitate formed in the solution bearing the

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unseparated bacterial cultures after 10 days: the optical microscopy images and

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corresponding Raman spectra of small spheres (a, b) and the big spheres (c, d). Figure 3. XRD patterns of the samples after 10 days obtained from the unseparated bacterial

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monohydrocalcite.

Figure 4. XRD patterns of the products after 10 days from the cell-free supernatant (a) and bacterial cells (b). Peaks correspond to: A = aragonite, C = high-Mg calcite, and M =

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Figure 5. XRD patterns of the samples produced in the presence of bare cells after 1 (a), 3 (b),

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solutions containing bare calls shift to the higher 2θ angle as the mineralization time increase, as highlighted by the dash line. Figure 6. FESEM images of the samples formed in the presence of bare cells after 1 (a), 3 (b), 7 (c) and 10 (d) days. The spheres are indicated by arrows. Figure 7. Representative TEM image (a) and the inserted TEM-based EDX spectrum (from the boxed region). HRTEM images (inset the corresponding SAED patterns) of the Ca-Mg carbonates after 10 days obtained from the unseparated bacterial cultures (b), bacterial cells (c) and bare cells (d). 28

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• Disordered dolomite formation can be promoted by aerobically incubated bacterial biomass. • Bare bacterial cells and the bound EPS can both exert a crucial role in the formation of disordered dolomite. • Phosphoryl and carboxyl groups with high density on the biomass are responsible for the disordered dolomite formation. • Bacterial biomass may be crucial in the precipitation of modern and ancient dolomite in sedimentary settings.

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