Effect of Magnesium and Ferric Ions on the Biomineralization of Calcium Carbonate Induced by Synechocystis sp. PCC 6803

: The discovery of cyanobacteria fossils in microbialite prompts the investigation of carbonate biomineralization using cyanobacteria. However, the impact of coexisting magnesium and iron in microbialite on carbonate biomineralization has been overlooked. Here, Synechocystis sp. PCC 6803 was used to induce calcium carbonate in the presence of coexisting magnesium and ferric ions. The ﬁndings demonstrate that cell concentration, pH, carbonic anhydrase activity, and carbonate and bicarbonate concentrations decreased with increasing concentrations of magnesium and calcium ions. Ferric ions yielded a contrasting effect. The levels of deoxyribonucleic acid, protein, polysaccharides, and humic substances in extracellular polymeric substances increased in the presence of separated or coexisting calcium, magnesium, and ferric ions. Magnesium ions inhibited calcium ion precipitation, whereas ferric ions exhibited the opposite effect. Protein secondary structures became more abundant and O-C=O and N-C=O contents increased with increasing ion concentrations by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses. Scanning electron microscopy revealed that ferric ions lead to rougher surfaces and incomplete rhombohedral structures of calcite, whereas magnesium ions promoted greater diversity in morphology. Magnesium ions enhanced the incorporation of ferric ions. This work aims to further understand the effect of magnesium and ferric ions on calcium carbonate biomineralization induced by cyanobacteria.


Introduction
In the early stages of the development of life on Earth, cyanobacteria played significant roles and gave rise to substantial amounts of microbial limestones formed through their activities.Some researchers have suggested that the formation of dendrolite primarily occurred through the process of cyanobacterial calcification during the early Paleozoic and late Devonian periods [1].The origin of carbonates, whether biogenic or abiotic, remains a topic of controversy.There are notable resemblances observed between microbialites found in the Cambrian successions of Shandong Province, China and the bio-precipitates induced by Synechocystis sp.PCC6803 in laboratory experiments, indicating that biomineralization processes play a significant role in both the formation of carbonate minerals and the lithification of microbial mats [2].Synechocystis sp.PCC 6803, a kind of cyanobacteria, is common in freshwater environments [3].In fact, many years ago, numerous researchers utilized Synechocystis sp.PCC6803 in biomineralization experiments to investigate the genetic mechanism of microbialite formation.During the preliminary exploration stage, it was discovered that Synechocystis sp.PCC6803 could induce the formation of calcite in an environment containing high concentrations of calcium ions (Ca 2+ ), and the surfaces of the calcite precipitated exhibited a hexahedral scaly pattern [4,5].It was later discovered that magnesium ions (Mg 2+ ) in microbialites can significantly impact the phase and morphology of carbonate minerals induced by Synechocystis sp.PCC6803.A low concentration of Mg 2+ ions promotes the formation of calcite, whereas a high concentration of Mg 2+ ions favors the formation of Mg-rich calcite and aragonite [6,7].Primary dolomite has been formed in the laboratory through calcification of a freshwater cyanobacterium [8].Hence, cyanobacteria continue to hold a significant role in the field of carbonate sedimentology.There is not only Mg but also iron (Fe) in microbialites.Based on the above research work of Han et al., Synechocystis sp.PCC6803 was selected again here to perform biomineralization experiments in an environment containing multiple metal ions (e.g., Mg 2+ , Fe 3+ , and Ca 2+ ions).In the presence of both Fe 3+ and Mg 2+ ions, how will the process of cyanobacteria inducing carbonate minerals change?Such a study has rarely been considered.The presence of various iron species and concentrations in oilfield-produced water can vary significantly depending on factors such as the composition of the geological formation, the hydrocarbons extracted, and the properties of production wells, resulting in significant variations in Fe levels ranging from a few milligrams per liter to several hundred milligrams per liter [9].According to these data, Fe 3+ concentrations were set at 5.6-11.2mg/L (0.1-0.2 mmol/L) in order to further explore the effect of these cations on CaCO 3 biomineralization; subsequent work will aim to increase the concentration of Fe 3+ ions.
Numerous investigations have been conducted on the ability of bacteria to induce mineral precipitation in both natural environments and controlled laboratory conditions [10][11][12][13][14][15].Microbial metabolism can convert Ca 2+ ions, Mg 2+ ions, and iron ions (Fe 2+ and Fe 3+ ) in the surrounding environment into carbonate, sulfate, sulfide, oxide, and other forms of minerals [16][17][18][19][20][21][22].As for Synechocystis sp.PCC6803, there is little research on what kind of mineral is formed by biomineralization of Ca 2+ , Mg 2+ , and Fe 3+ ions at different concentrations.Microbial mineralization occurs in various environments, including soil, freshwater, marine ecosystems, and oilfield wastewater.Environmental conditions play a crucial role in determining the specific type of calcium carbonate (CaCO 3 ) mineral formed through bacterial precipitation [23][24][25].Since Synechocystis sp.PCC6803 can survive in both liquid and solid BG11 media, and mineral formation is influenced by the environment, a liquid environment was chosen to biomineralize Ca 2+ , Mg 2+ , and Fe 3+ ions in this study, while cyanobacterial mineralization in a solid environment will be examined in the future.Synechocystis sp.PCC6803 can induce CaCO 3 formation, but the effect of separated or coexisting Mg 2+ and Fe 3+ ions on the biomineralization of CaCO 3 under the action of Synechocystis sp.PCC6803 is rarely reported.To perform this research, the survival of Synechocystis sp.PCC6803 in a system containing separated or coexisting Ca 2+ , Mg 2+ , and Fe 3+ ions is crucial.There are few reports on the range of ion concentrations (Ca 2+ , Mg 2+ , and Fe 3+ ions) that support cyanobacteria survival.Therefore, this study aims to investigate the survival of cyanobacteria at different concentrations of Ca 2+ , Mg 2+ , and Fe 3+ ions (i.e., changes in cell concentration) to facilitate subsequent research on the effects of Mg 2+ and Fe 3+ ions on CaCO 3 formation.
Biomineralization involves the organism's metabolic processes and organic matrix, and these can induce alterations in local physical and chemical conditions.These alterations include changes in pH and concentrations of carbonate (CO 3 2− ) and bicarbonate (HCO 3 − ) ions due to the action of carbonic anhydrase (CA) released by bacteria.Some scholars propose that under specific circumstances, bacteria may be capable of triggering the formation of CaCO 3 through CA activity [26].The significance of CA activity in the process of microbial-induced carbonate precipitation cannot be underestimated [27].Yan et al. examined how CA facilitates the hydration process of carbon dioxide (CO 2 ) in the for-Minerals 2023, 13,1486 3 of 24 mation of carbonate minerals [28].Elucidating the fluctuations observed in concentrations of CO 3 2− and HCO 3 − ions within liquid systems holds immense importance.The reaction occurred in accordance with Equation (1): Bacteria have the ability to raise the pH level of the solution and enhance the concentration of OH − ions, thereby promoting the hydrolysis equilibrium of HCO 3 − , which leads to an increase in CO 3 2− concentration [29] within the solution according to the following Equation (2): Synechocystis sp.PCC6803 can release CA at a lower Mg/Ca ratio [6], but the occurrence of changes in CA activity caused by coexisting Ca 2+ , Mg 2+ , and Fe 3+ ions has rarely been documented.In this investigation, Synechocystis sp.PCC 6803 will be cultivated in diverse systems comprising Ca 2+ , Ca 2+ + Mg 2+ , and Ca 2+ + Mg 2+ + Fe 3+ .The alterations in CA activity (including the concentrations of CO 3 2− and HCO 3 − ions as well as pH changes) across these distinct systems will be examined to comprehensively understand the impact of Mg 2+ and Fe 3+ ions on CA activity changes and the formation of CaCO 3 .
It is widely acknowledged that extracellular polymeric substances (EPS) serve as sites for mineral nucleation.Initially, it was believed that polysaccharides formed the primary framework of EPS; however, subsequent exploration has revealed significant proportions of proteins, nucleic acids, and lipids within EPS [30].The successful utilization of three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy has been demonstrated in the detection and characterization of EPS components that resemble proteins, humic acids, and fulvic acids [31,32].Numerous scholars have also conducted investigations on external factors that impact the constituents of bacterial EPS.Their findings indicate that variations in EPS components can occur due to changes in the external environment [33].However, limited research has been conducted on the impact of various metal ions, such as Ca 2+ , Mg 2+ , and Fe 3+ , on changes in the components of EPS from Synechocystis sp.PCC 6803.In this study, Synechocystis sp.PCC 6803 will be cultivated in different systems containing Ca 2+ , Ca 2+ + Mg 2+ , and Ca 2+ + Mg 2+ + Fe 3+ .The changes in EPS components will then be examined.
The presence of organic matter at nucleation sites leads to bacterially induced minerals that exhibit distinct biological features, such as protein secondary structures and chemical bonds in organics.External environmental factors can influence the composition of EPS [33], which acts as nucleation sites, consequently altering these inherent biological traits found within acquired minerals.However, limited investigation has been conducted into the impact of Ca 2+ , Mg 2+ , and Fe 3+ ions on alterations in protein secondary structures and chemical bonds in harvested minerals.Therefore, this study aims to analyze the effect of separated or coexisting Mg 2+ and Fe 3+ ions on changes in the characteristics of CaCO 3 minerals induced by Synechocystis sp.PCC 6803.
In this study, Synechocystis sp.PCC 6803 was cultured in different media containing Ca 2+ , Ca 2+ + Mg 2+ , and Ca 2+ + Mg 2+ + Fe 3+ to investigate the following aspects: (1) How variations of the cations influence the cell's behavior relative to CA activity, pH, concentrations of CO 3 2− and HCO 3 − ions, and EPS composition and (2) how variations of the cations affect the formation of biominerals.This study facilitates a comprehensive understanding of the effect of separated or coexisting Mg 2+ and Fe 3+ ions on CaCO 3 biomineralization under the influence of Synechocystis sp.PCC 6803, which is helpful for comprehending the formation mechanism and process of microbialites.

Bacterial Strain, Culture Medium, and Cultivation Conditions
The experimental strain used in this study was Synechocystis sp.PCC 6803, which was kindly provided by Prof. Song Qin from the Yantai Institute of Coastal Zone Re-search, Chinese Academy of Sciences.The BG11 medium (BR grade) used to cultivate Synechocystis sp.PCC 6803 was purchased from Qingdao Hope Bio-Technology Co., Ltd (Qingdao, China).Different concentrations of Ca 2+ , Mg 2+ , and Fe 3+ ions were added to the BG11 medium to create 16 types of media (Table S1).The Ca 2+ , Mg 2+ , and Fe 3+ ions were obtained from calcium chloride (CaCl 2 ), magnesium chloride (MgCl 2 •6H 2 O), and ferric chloride (FeCl 3 •6H 2 O), respectively.The pH values of the media were adjusted to 7.1 ± 0.1 using hydrochloric acid (1 mol/L) and measured with a pH meter (PHS-3C, Shanghai Shengke Instrument Equipment Co., Ltd., Shanghai, China).Synechocystis sp.PCC 6803 was cultivated in an illumination incubator (GPX-250B, Jiangnan Instrument Company, Ningbo, China) with a light intensity of 5000 Lx and at a temperature range of 15-25  S1 at a volume ratio of 10%, and three parallel samples were prepared for each medium.Control groups were also included, where no bacteria were inoculated.All groups were placed in an illumination incubator.To determine the difference in cell concentration between the different groups (16 types), 2 mL samples were collected every two days using a 5 mL pipettor, and the cell concentration at 730 nm was measured with a visible spectrophotometer (721G, Yi electric Analytical Instrument Co., Ltd., Shanghai, China).The pH values were measured using a pH meter every two days during the biomineralization process.CA activity and CO 3 2− and HCO 3 − concentrations were determined following the methods described in the published paper [29].The CA activity (U/L) was calculated by measuring the amount of enzyme required to release 1 µmol of p-nitrophenol within a time frame of 1 min per liter.

EPS Extraction and Composition of EPS
The EPS extraction method used in this study followed the optimized approach proposed by Yan et al. [34].The levels of DNA, polysaccharides, and proteins within the EPS samples were determined based on Zhuang et al.'s methodology [29].Identification of humic acid and protein substances similar to tyrosine and tryptophan produced in the EPS was performed using a three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectrophotometer (F7000, Hitachi Gaoxin, Tokyo, Japan).

Changes in Cation Concentrations, Precipitation Ratio, and Precipitation Rate under the Influence of Synechocystis sp. PCC 6803 in Different Conditions
The concentrations of different cations were determined using atomic absorption spectrometry (AAS, TAS-986F, Zhengzhou Nanbei Instrument Equipment Co., Ltd., Zhengzhou, China) in various systems while considering the influence of Synechocystis sp.PCC 6803.The subsequent formulas, Equation (3) for calculating the precipitation ratio and Equation ( 4) for calculating the precipitation rate, can be employed: In Equations ( 3) and ( 4), T 1 denotes the percentage of removal (%), whereas T 2 represents the rate of removal (mg/L/d); C 0 refers to the initial concentration (mg/L); C t indicates the final concentration (mg/L); and the variable T signifies time measured in days (d).

Characterization of Biominerals Induced by Synechocystis sp. PCC 6803 at Different Conditions
After a 30-day culture period, the mineral precipitates were transferred to a 10 mL centrifuge tube using a pipettor with a volume of 5 mL and rinsed three times with distilled deionized water.To remove any remaining bacteria and remnants of the culture medium from the mineral surface, anhydrous ethanol and sodium hypochlorite were used for triple washing, respectively, followed by air drying at room temperature.Since no minerals were obtained in the control group, further investigation will solely focus on the experimental group while discontinuing analysis of the control group.The mineral precipitates underwent X-ray diffraction analyses (XRD, D/Max-RC, Rigaku Corporation, Tokyo, Japan) [35].The scanning angle range was set at 10-80 • (2θ), with a step size of 0.02 • and count time of 10 • /min.The Jade 6.5 software (International Centre for Diffraction Data, Newtown Square, PA, USA) was utilized to analyze the obtained XRD data in more detail for mineral phase analysis.In addition to XRD analyses, various tests were conducted on the mineral precipitates to further investigate their biogenesis.Firstly, scanning electron microscopy (SEM, Hitachi S-4800, Hitachi Company, Tokyo, Japan) in conjunction with an X-ray energy-dispersive spectrometer (EDS, EX-450, Horiba, Tokyo, Japan) was employed to explore the morphology and presence of organic elements.Secondly, Fourier transform infrared spectroscopy (FTIR, Nicolet 380, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for analyzing the organic functional groups present in the mineral precipitates.The FTIR parameters included a scanning range of 4000-500 cm −1 and a resolution of 4 cm −1 .The X-ray photoelectron spectroscopy (XPS, ESCALAB 250XI, Thermo Fisher Scientific, Waltham, MA, USA) was finally conducted on these minerals, and the C1s peaks were validated at 284 eV [28].Subsequently, analysis using Avantage 2.16 software was performed to uncover any hidden organic substances on the biomineral surfaces.The Equation ( 5) was utilized for determining the proportion of a specific fitting peak in XPS spectra, as demonstrated below: where A% represents the proportion of a specific fitted peak, A s denotes the size of each individual fitted peak, and A t indicates the total area covered by all fitted peaks.In systems with varying Ca 2+ concentrations, the bacterial concentration reached its maximum value during the stable stage.Among the concentrations of Ca 2+ (400, 800, and 1200 mg/L), an optimal concentration for cyanobacteria growth was found to be 800 mg/L due to the highest cell concentration (OD 730 = 1.33) (Figure 1a).When different concentrations of Mg 2+ ions were added to the medium containing 800 mg/L of Ca 2+ , the bacterial concentration (OD 730 ) decreased from 1.23 to 1.20 and then to 1.18 as the Mg 2+ concentration increased from 0 to 1440 and finally to 2880 mg/L (Figure 1b), suggesting that high concentrations of Mg 2+ ions could impede cell growth.However, when Fe 3+ ions were added to three different media containing 800 mg/L of Ca 2+ (Figure 1c), 800 mg/L of Ca 2+ + 1440 mg/L of Mg 2+ (Figure 1d), and 800 mg/L of Ca 2+ + 2880 mg/L of Mg 2+ (Figure 1e), cell concentrations increased with increasing Fe 3+ ion concentrations (Figure 1c-e).This indicated that Fe 3+ ions promoted cell reproduction under these different conditions.In Figure 1c-e, it can be observed that as the concentration of Mg 2+ ions increases at 800 mg/L of Ca 2+ and 11.2 mg/L of Fe 3+ , cell concentrations decrease from 1.26 to 1.06 and then to 0.99, indicating that these levels of Mg 2+ ions impede the division of cyanobacteria.

Effects of Ca
added to three different media containing 800 mg/L of Ca 2+ (Figure 1c), 800 mg/L of Ca 2+ + 1440 mg/L of Mg 2+ (Figure 1d), and 800 mg/L of Ca 2+ + 2880 mg/L of Mg 2+ (Figure 1e), cell concentrations increased with increasing Fe 3+ ion concentrations (Figure 1c-e).This indicated that Fe 3+ ions promoted cell reproduction under these different conditions.In Figure 1c-e, it can be observed that as the concentration of Mg 2+ ions increases at 800 mg/L of Ca 2+ and 11.2 mg/L of Fe 3+ , cell concentrations decrease from 1.26 to 1.06 and then to 0.99, indicating that these levels of Mg 2+ ions impede the division of cyanobacteria.In systems with varying concentrations of Ca 2+ ions, the pH of the four groups decreased as the concentration of Ca 2+ ions increased on the same day (Figure 2a).The maximum pH value was 8.49, which occurred at the optimal concentration of Ca 2+ ions (800 mg/L) on the 16th day (Figure 2a).At a fixed Ca 2+ concentration (800 mg/L), the pH successively decreased from 8.53 to 8.31 and then to 8.17 with an increase in Mg 2+ ion concentration (Figure 2b), indicating that a sufficiently high Mg 2+ concentration could inhibit the rise in pH.After the addition of Fe 3+ ions (Figure 2c-e), the pH changes follow a similar trend to that of the growth curve in Figure 1c-e.The pH values increase as the concentrations of Fe 3+ ions increase (Figure 2c-e), possibly due to higher cell concentrations with increasing Fe 3+ ion concentrations.In systems with varying concentrations of Ca 2+ ions, the pH of the four groups decreased as the concentration of Ca 2+ ions increased on the same day (Figure 2a).The maximum pH value was 8.49, which occurred at the optimal concentration of Ca 2+ ions (800 mg/L) on the 16th day (Figure 2a).At a fixed Ca 2+ concentration (800 mg/L), the pH successively decreased from 8.53 to 8.31 and then to 8.17 with an increase in Mg 2+ ion concentration (Figure 2b), indicating that a sufficiently high Mg 2+ concentration could inhibit the rise in pH.After the addition of Fe 3+ ions (Figure 2c-e), the pH changes follow a similar trend to that of the growth curve in Figure 1c-e.The pH values increase as the concentrations of Fe 3+ ions increase (Figure 2c-e), possibly due to higher cell concentrations with increasing Fe 3+ ion concentrations.The CA activity decreased with an increase in Ca 2+ ion concentration (Figure 3a).At a Ca 2+ ion concentration of 800 mg/L, the maximum activity of CA was 3.16 U/mL (Figure 3a).The decrease in CA activity with increasing Ca 2+ ion concentrations indicates that a sufficiently high concentration of Ca 2+ can inhibit CA activity.After adding Mg 2+ ions to a system containing 800 mg/L of Ca 2+ (Figure 3b), the CA activity decreased from 1.62 to  The CA activity decreased with an increase in Ca 2+ ion concentration (Figure 3a).At a Ca 2+ ion concentration of 800 mg/L, the maximum activity of CA was 3.16 U/mL (Figure 3a).The decrease in CA activity with increasing Ca 2+ ion concentrations indicates that a sufficiently high concentration of Ca 2+ can inhibit CA activity.After adding Mg 2+ ions to a system containing 800 mg/L of Ca 2+ (Figure 3b), the CA activity decreased from 1.62 to 1.36 U/mL as the concentration of Mg 2+ ions increased from 0 to 1440 mg/L and further decreased to 1.21 U/mL at a concentration of 2880 mg/L (Figure 3b).This indicates that higher concentrations of Mg 2+ ions also inhibit CA activity.After the addition of Fe 3+ ions to a system containing 800 mg/L of Ca 2+ (Figure 3c), CA activity increased from 3.28 to 3.49 and then to 3.83 U/mL as the concentration of Fe 3+ ions increased from 5.6 to 8.4 and finally to 11.2 mg/L (Figure 3c).This indicates that a sufficiently high concentration of Fe 3+ ions can enhance CA activity in the presence of Ca 2+ ions.After Mg 2+ ions were added to the system containing Ca 2+ and Fe 3+ ions (Figure 3d,e), CA activity continued to increase with increasing concentrations of Fe 3+ ion but decreased with increasing concentrations of Mg 2+ ion.The CA activity decreased with an increase in Ca 2+ ion concentration (Figure 3a).At a Ca 2+ ion concentration of 800 mg/L, the maximum activity of CA was 3.16 U/mL (Figure 3a).The decrease in CA activity with increasing Ca 2+ ion concentrations indicates that a sufficiently high concentration of Ca 2+ can inhibit CA activity.After adding Mg 2+ ions to a system containing 800 mg/L of Ca 2+ (Figure 3b), the CA activity decreased from 1.62 to 1.36 U/mL as the concentration of Mg 2+ ions increased from 0 to 1440 mg/L and further decreased to 1.21 U/mL at a concentration of 2880 mg/L (Figure 3b).This indicates that higher concentrations of Mg 2+ ions also inhibit CA activity.After the addition of Fe 3+ ions to a system containing 800 mg/L of Ca 2+ (Figure 3c), CA activity increased from 3.28 to 3.49 and then to 3.83 U/mL as the concentration of Fe 3+ ions increased from 5.6 to 8.4 and finally to 11.2 mg/L (Figure 3c).This indicates that a sufficiently high concentration of Fe 3+ ions can enhance CA activity in the presence of Ca 2+ ions.After Mg 2+ ions were added to the system containing Ca 2+ and Fe 3+ ions (Figure 3d,e), CA activity continued to increase with increasing concentrations of Fe 3+ ion but decreased with increasing concentrations of Mg 2+ ion.In the system containing only Ca 2+ ions (Figure 4a), the concentrations of CO 3 2− and HCO 3 − ions decreased as the concentration of Ca 2+ ions increased, which was consistent with the result that CA activity decreased with increasing Ca 2+ ion concentrations shown in Figure 3a.In addition to this reason, another factor contributing to the decrease in the concentration of CO 3 2− and HCO 3 − ions was their combination with Ca 2+ ions to form calcium carbonate precipitates.The inhibitory effect of Mg 2+ ions on CA activity (as shown in Figure 3b) also resulted in a decrease in the concentrations of CO 3 2− and HCO 3 − ions with increasing concentrations of Mg 2+ ions (Figure 4b).However, the addition of Fe 3+ ions significantly transformed this situation.As observed from Figure 4c-e, the concentrations of CO 3 2− and HCO 3 − ions increased with increasing Fe 3+ ion concentrations, which is consistent with changes in CA activity (Figure 3c-e), pH (Figure 2c-e), and cell concentrations (Figure 1c-e).Therefore, it can be concluded that Fe 3+ ions act as an activator for CA.cium carbonate precipitates.The inhibitory effect of Mg 2+ ions on CA activity (as shown in Figure 3b) also resulted in a decrease in the concentrations of CO3 2− and HCO3 − ions with increasing concentrations of Mg 2+ ions (Figure 4b).However, the addition of Fe 3+ ions significantly transformed this situation.As observed from Figure 4c-e, the concentrations of CO3 2− and HCO3 − ions increased with increasing Fe 3+ ion concentrations, which is consistent with changes in CA activity (Figure 3c-e), pH (Figure 2c-e), and cell concentrations (Figure 1c-e).Therefore, it can be concluded that Fe 3+ ions act as an activator for CA.

Analyses of EPS Components
The EPS underwent further analysis using 3D-EEM, which revealed the presence of four distinct regions (Figure 5).Region I, with an excitation/emission wavelength of 230/298-304 nm, indicated the presence of aromatic protein substances such as tyrosine in the EPS.Region II, with an excitation/emission wavelength of 270/368-374 nm, suggested the presence of amino acids like tryptophan.Regions III indicated the existence of humic acid-like substances in the EPS at an excitation/emission wavelength range of 350-360/445.5-452.5 and 280-320/402-454 nm, respectively, whereas region IV confirmed the presence of fulvic Ca 2+ 800 mg/L+Mg 2+ 0 mg/L+ Fe 3+ 5.6 mg/L Ca 2+ 800 mg/L+Mg 2+ 0 mg/L+ Fe 3+ 8.4 mg/L Ca 2+ 800 mg/L+Mg 2+ 0 mg/L+ Fe 3+

Analyses of EPS Components
The EPS underwent further analysis using 3D-EEM, which revealed the presence of four distinct regions (Figure 5).Region I, with an excitation/emission wavelength of 230/298-304 nm, indicated the presence of aromatic protein substances such as tyrosine in the EPS.Region II, with an excitation/emission wavelength of 270/368-374 nm, suggested the presence of amino acids like tryptophan.Regions III indicated the existence of humic acid-like substances in the EPS at an excitation/emission wavelength range of 350-360/445.5-452.5 and 280-320/402-454 nm, respectively, whereas region IV confirmed the presence of fulvic acid-like substances at an excitation/emission wavelength range of 235-240/417.5-456nm [31,36].At a concentration of 800 mg/L of Ca 2+ ions (Figure 5b), there is an increase in aromatic proteins and tryptophan-like amino acids compared to the liquid seed (Figure 5a).However, at 800 mg/L of Ca 2+ ions and 2880 mg/L of Mg 2+ ions (Figure 5c), there is a decrease in aromatic proteins and tryptophan-like amino acids compared to that observed at 800 mg/L of Ca 2+ ions alone (Figure 5b).The addition of Fe 3+ ions to the system resulted in a significant increase in the content of humic acidlike substances, regardless of the presence of Mg 2+ ions (Figure 5d-g).The contents of DNA, polysaccharides, and protein in EPS from bacteria cultured in systems with Ca 2+ , Ca 2+ + Mg 2+ , Ca 2+ + Fe 3+ , and Ca 2+ + Mg 2+ + Fe 3+ were significantly higher than those in the liquid seed (p < 0.01, Figure 5h, Tables S2-S4), suggesting that the composition and concentration of ions in the surrounding environment would have a significant impact on EPS constituents.
at 800 mg/L of Ca 2+ ions alone (Figure 5b).The addition of Fe 3+ ions to the system resulted in a significant increase in the content of humic acid-like substances, regardless of the presence of Mg 2+ ions (Figure 5d-g).The contents of DNA, polysaccharides, and protein in EPS from bacteria cultured in systems with Ca 2+ , Ca 2+ + Mg 2+ , Ca 2+ + Fe 3+ , and Ca 2+ + Mg 2+ + Fe 3+ were significantly higher than those in the liquid seed (p < 0.01, Figure 5h, Tables S2-S4), suggesting that the composition and concentration of ions in the surrounding environment would have a significant impact on EPS constituents.Under the influence of Synechocystis sp.PCC 6803, the concentration of Ca 2+ continuously decreased with increasing culture time in different systems with initial Ca 2+ concentrations of 400, 800, and 1200 mg/L.On the 40th day (Figure 6a), it reached concentrations of 99.5, 220.6, and 488.5 mg/L, respectively.The control group exhibited no significant change in Ca 2+ concentration, indicating the crucial role played by Synechocystis sp.PCC 6803 in the process of Ca 2+ biomineralization.The precipitation ratio of Ca 2+ increased accordingly and reached 59.3%-75.1% on the 40th day in the experimental groups, whereas it did not exceed 10% in the control groups (Figure 6b).The precipitation rate of Ca 2+ decreased with decreasing concentration levels (Figure 6c).In the later stages of culture,     Under the influence of Synechocystis sp.PCC 6803, the concentration of Ca 2+ continuously decreased with increasing culture time in different systems with initial Ca 2+ concentrations of 400, 800, and 1200 mg/L.On the 40th day (Figure 6a), it reached concentrations of 99.5, 220.6, and 488.5 mg/L, respectively.The control group exhibited no significant change in Ca 2+ concentration, indicating the crucial role played by Synechocystis sp.PCC 6803 in the process of Ca 2+ biomineralization.The precipitation ratio of Ca 2+ increased accordingly and reached 59.3%-75.1% on the 40th day in the experimental groups, whereas it did not exceed 10% in the control groups (Figure 6b).The precipitation rate of Ca 2+ decreased with decreasing concentration levels (Figure 6c).In the later stages of culture, there was a relatively stable precipitation rate for Ca 2+ across different concentrations (Figure 6c).There was a significant difference in the concentration, precipitation ratio, and precipitation rate of Ca 2+ between the experimental and control groups (p < 0.01).In systems containing 800 mg/L of Ca 2+ and varying concentrations of Mg 2+ (Figure 6d-f), as the concentration of Mg 2+ increased from 0 to 1440 and then to 2880 mg/L, the concentration of Ca 2+ ions sharply decreased from 800 mg/L to 239.1, 312.8, and 363.2 mg/L on the 26th day (Figure 6d).Additionally, the precipitation ratios of Ca 2+ ions reached values of 70.1%, 60.9%, and 54.6%, respectively (Figure 6e).Furthermore, the precipitation rates also significantly decreased with increasing concentrations of Mg 2+ ions (Figure 6f).These results indicated that certain concentrations of Mg 2+ ions could inhibit the precipitation of Ca 2+ ions.concentration of Mg 2+ increased from 0 to 1440 and then to 2880 mg/L, the concentration of Ca 2+ ions sharply decreased from 800 mg/L to 239.1, 312.8, and 363.2 mg/L on the 26th day (Figure 6d).Additionally, the precipitation ratios of Ca 2+ ions reached values of 70.1%, 60.9%, and 54.6%, respectively (Figure 6e).Furthermore, the precipitation rates also significantly decreased with increasing concentrations of Mg 2+ ions (Figure 6f).These results indicated that certain concentrations of Mg 2+ ions could inhibit the precipitation of Ca 2+ ions.

Removal of Ca 2+ under the Action of Fe 3+ Ions
The Ca 2+ concentration decreased from 800 mg/L to 182.9, 178.0, and 172.3 mg/L on the 34th day as the Fe 3+ ion concentrations increased from 5.6 to 8.4 and then to 11.2 mg/L (Figure 7a) when different concentrations of Fe 3+ ions were added to systems with a Ca 2+ concentration of 800 mg/L and an Mg 2+ concentration of 0 mg/L under the influence of Synechocystis sp.PCC6803.The corresponding precipitation ratios of Ca 2+ were found to be 77.1%,77.8%, and 78.5% (Figure 7b).This conclusion suggests that the increase in Fe 3+ ion concentration contributed to the removal of Ca 2+ ions (p < 0.01, Table S5), and there was a contrasting effect between Fe 3+ ions and Mg 2+ ions on the removal of Ca 2+ ions.The precipitation rates of Ca 2+ ions also increased with increasing Fe 3+ ion concentrations (Figure 7c), suggesting that the addition of Fe 3+ ions could enhance the removal efficiency of Ca 2+ ions, whereas the presence of Mg 2+ ions had the opposite effect (Figure 6f).

Removal of Ca 2+ under the Action of Fe 3+ Ions
The Ca 2+ concentration decreased from 800 mg/L to 182.9, 178.0, and 172.3 mg/L on the 34th day as the Fe 3+ ion concentrations increased from 5.6 to 8.4 and then to 11.2 mg/L (Figure 7a) when different concentrations of Fe 3+ ions were added to systems with a Ca 2+ concentration of 800 mg/L and an Mg 2+ concentration of 0 mg/L under the influence of Synechocystis sp.PCC6803.The corresponding precipitation ratios of Ca 2+ were found to be 77.1%,77.8%, and 78.5% (Figure 7b).This conclusion suggests that the increase in Fe 3+ ion concentration contributed to the removal of Ca 2+ ions (p < 0.01, Table S5), and there was a contrasting effect between Fe 3+ ions and Mg 2+ ions on the removal of Ca 2+ ions.The precipitation rates of Ca 2+ ions also increased with increasing Fe 3+ ion concentrations (Figure 7c), suggesting that the addition of Fe 3+ ions could enhance the removal efficiency of Ca 2+ ions, whereas the presence of Mg 2+ ions had the opposite effect (Figure 6f).
of Ca 2+ ions sharply decreased from 800 mg/L to 239.1, 312.8, and 363.2 mg/L on the 26th day (Figure 6d).Additionally, the precipitation ratios of Ca 2+ ions reached values of 70.1%, 60.9%, and 54.6%, respectively (Figure 6e).Furthermore, the precipitation rates also significantly decreased with increasing concentrations of Mg 2+ ions (Figure 6f).These results indicated that certain concentrations of Mg 2+ ions could inhibit the precipitation of Ca 2+ ions.

Removal of Ca 2+ under the Action of Fe 3+ Ions
The Ca 2+ concentration decreased from 800 mg/L to 182.9, 178.0, and 172.3 mg/L on the 34th day as the Fe 3+ ion concentrations increased from 5.6 to 8.4 and then to 11.2 mg/L (Figure 7a) when different concentrations of Fe 3+ ions were added to systems with a Ca 2+ concentration of 800 mg/L and an Mg 2+ concentration of 0 mg/L under the influence of Synechocystis sp.PCC6803.The corresponding precipitation ratios of Ca 2+ were found to be 77.1%,77.8%, and 78.5% (Figure 7b).This conclusion suggests that the increase in Fe 3+ ion concentration contributed to the removal of Ca 2+ ions (p < 0.01, Table S5), and there was a contrasting effect between Fe 3+ ions and Mg 2+ ions on the removal of Ca 2+ ions.The precipitation rates of Ca 2+ ions also increased with increasing Fe 3+ ion concentrations (Figure 7c), suggesting that the addition of Fe 3+ ions could enhance the removal efficiency of Ca 2+ ions, whereas the presence of Mg 2+ ions had the opposite effect (Figure 6f).

Removal of Ca 2+ under the Action of Mg 2+ + Fe 3+ Ions
In the groups containing fixed concentrations of Ca 2+ (800 mg/L) and Mg 2+ (1440 mg/L), and variable concentrations of Fe 3+ ions (Figure 8a-c), the concentration of Ca 2+ ions decreased from 800 mg/L to 292.1, 285.9, and 280.2 mg/L as the concentration of Fe 3+ ions increased from 5.6 through 8.4 to 11.2 mg/L on the 34th day (Figure 8a).This indicated that the increasing concentration of Fe 3+ ions could promote the precipitation of Ca 2+ ions, even in the presence of higher concentrations of Mg 2+ ions.The precipitation ratios of Ca 2+ ions were 63.5%, 64.2%, and 65.0% (Figure 8b) (p < 0.01, Table S6), which were significantly lower than those of groups 8-10 without any Mg 2+ ions in Figure 7b, suggesting that the presence of Mg 2+ ions could inhibit the precipitation of Ca 2+ ions even in the presence of Fe 3+ ions.
The precipitation rates of Ca 2+ ions increased with increasing Fe 3+ ion concentrations in the presence of 1440 mg/L of Mg 2+ (Figure 8c), which further supports the notion that Fe 3+ ions could enhance Ca 2+ precipitation.Compared to groups 8-10 (Figure 7c), the precipitation rates of Ca 2+ ions were significantly lower due to the inhibitory effect of Mg 2+ on their precipitation.When the concentration of Mg 2+ ions increased from 1440 to 2880 mg/L (Figure 8d-f), similar conclusions as mentioned above were obtained (p < 0.01, Table S7).Furthermore, both the precipitation ratios and rates of Ca 2+ decreased in the presence of higher concentrations of Mg 2+ ions (p < 0.01, Tables S8-S10).
and variable concentrations of Fe ions (Figure 8a-c), the concentration of Ca ions decreased from 800 mg/L to 292.1, 285.9, and 280.2 mg/L as the concentration of Fe 3+ ions increased from 5.6 through 8.4 to 11.2 mg/L on the 34th day (Figure 8a).This indicated that the increasing concentration of Fe 3+ ions could promote the precipitation of Ca 2+ ions, even in the presence of higher concentrations of Mg 2+ ions.The precipitation ratios of Ca 2+ ions were 63.5%, 64.2%, and 65.0% (Figure 8b) (p < 0.01, Table S6), which were significantly lower than those of groups 8-10 without any Mg 2+ ions in Figure 7b, suggesting that the presence of Mg 2+ ions could inhibit the precipitation of Ca 2+ ions even in the presence of Fe 3+ ions.The precipitation rates of Ca 2+ ions increased with increasing Fe 3+ ion concentrations in the presence of 1440 mg/L of Mg 2+ (Figure 8c), which further supports the notion that Fe 3+ ions could enhance Ca 2+ precipitation.Compared to groups 8-10 (Figure 7c), the precipitation rates of Ca 2+ ions were significantly lower due to the inhibitory effect of Mg 2+ on their precipitation.When the concentration of Mg 2+ ions increased from 1440 to 2880 mg/L (Figure 8d-f), similar conclusions as mentioned above were obtained (p < 0.01, Table S7).Furthermore, both the precipitation ratios and rates of Ca 2+ decreased in the presence of higher concentrations of Mg 2+ ions (p < 0.01, Tables S8-S10).

XRD Analyses of Minerals
To further understand the phase and structural characteristics of minerals induced by Synechocystis sp.PCC 6803 under different conditions, the minerals cultured for 30 days were analyzed using XRD.From Figure 9a, it can be observed that all the minerals formed at Ca 2+ concentrations ranging from 400 to 1200 mg/L are calcite (PDF#86-2334).

Characteristics of Biominerals Induced by Synechocystis sp. PCC 6803 3.4.1. XRD Analyses of Minerals
To further understand the phase and structural characteristics of minerals induced by Synechocystis sp.PCC 6803 under different conditions, the minerals cultured for 30 days were analyzed using XRD.From Figure 9a, it can be observed that all the minerals formed at Ca 2+ concentrations ranging from 400 to 1200 mg/L are calcite (PDF#86-2334).
The results presented in Table S11 demonstrate that the full width at half maximum (FWHM) ( • ) of calcite ( 104), (110), and (113) exhibits an increasing trend with rising concentrations of Ca 2+ ions, implying a reduction in the crystallinity of calcite.The precipitation rates of Ca 2+ ions increased with increasing concentrations (Figure 6c), resulting in a poorly developed crystal structure and subsequently decreased mineral crystallinity.The calcite FWHM result was consistent with the conclusion drawn from the rates of Ca 2+ ion precipitation.
In groups 5-7 (Table S1), the minerals changed from calcite (PDF#86-0174) to Mg-rich calcite (PDF#86-2336) and finally to aragonite (PDF#71-2396) as the concentration of Mg 2+ ions increased from 0 to 1440 mg/L and then to 2880 mg/L (Figure 9b).In the presence of a low concentration of Mg 2+ ions, the formation of Mg-calcite is observed.The FWHM of Mg-rich calcite induced by Synechocystis sp.PCC 6803 increases as the concentration of Mg 2+ ions increases (Table S12), suggesting that the crystal structure of the mineral is compromised due to the substitution of Mg 2+ ions, resulting in a decrease in crystallinity.However, as the concentration of Mg 2+ ions increased, there was a corresponding decrease in the amount of Mg-calcite and an increase in the quantity of aragonite.
When different concentrations of Fe 3+ ions were added to systems containing a fixed Ca 2+ ion concentration (800 mg/L), all the minerals formed were calcite (PDF#  (Figure 9c).As the concentration of Fe 3+ ions increased, the FWHM of calcite ( 104), (110), and (113) also increased (Table S13), indicating a decrease in the crystallinity of calcite.The faster precipitation rates (Figure 7c) suggested a reduced level of crystallinity in calcite.Additionally, the incorporation of Fe 3+ ions also contributed to a compromised crystal structure of calcite.When different concentrations of Fe 3+ ions were added to systems containing a fixed concentration of Ca 2+ ions (800 mg/L) and a fixed concentration of Mg 2+ ions (1440 mg/L), the minerals remained as calcite (PDF#83-0578) (Figure 9d).The FWHM of calcite ( 104), (110), and (113) also increased (Table S14), indicating a decrease in the crystallinity of calcite due to the faster precipitation rates (Figure 8c) and the presence of Mg 2+ and Fe 3+ ions.The results presented in Table S11 demonstrate that the full width at half maximum (FWHM) (°) of calcite (104), (110), and (113) exhibits an increasing trend with rising concentrations of Ca 2+ ions, implying a reduction in the crystallinity of calcite.The precipitation rates of Ca 2+ ions increased with increasing concentrations (Figure 6c), resulting in a poorly developed crystal structure and subsequently decreased mineral crystallinity.The calcite FWHM result was consistent with the conclusion drawn from the rates of Ca 2+ ion precipitation.
In groups 5-7 (Table S1), the minerals changed from calcite (PDF#86-0174) to Mg-rich calcite (PDF#86-2336) and finally to aragonite (PDF#71-2396) as the concentration of Mg 2+ ions increased from 0 to 1440 mg/L and then to 2880 mg/L (Figure 9b).In the presence of a low concentration of Mg 2+ ions, the formation of Mg-calcite is observed.The FWHM of Mg-rich calcite induced by Synechocystis sp.PCC 6803 increases as the concentration of Mg 2+ ions increases (Table S12), suggesting that the crystal structure of the mineral is com-2+

FTIR Analyses of Minerals
The identification of calcite or Mg-rich calcite can be inferred from the distinctive spectral peaks observed at 712, 875, 1421, 1795, and 2514 cm −1 [37].Furthermore, the presence of aragonite is confirmed by bands observed at 713, 862, 1081, 1406, and 1476 cm −1 [37].In Figure 10a-d, the presence of characteristic bands of calcite or Mg-rich calcite can be observed, confirming the presence of these minerals.In Figure 10b, when the concentration of Mg 2+ ions increased to 2880 mg/L, the characteristic bands at 713, 862, 1081, 1406, and 1476 cm −1 provided evidence for the presence of aragonite.These mineral phases correspond well to the XRD results shown in Figure 9.The minerals were also found to contain organic functional groups, such as C-O-C (1033 and 1055 cm −1 ), C-N (1180, 1175, and 1173 cm −1 ), C=C (1649, 1624, and 1626 cm −1 ), and C=O (1722 and 1725 cm −1 ).The presence of O-H (2973, 2974, 2979, and 2981 cm −1 ) and fatty acid C-H bonds (2874, 2869, 2921, and 2920 cm −1 ) indicates the involvement of bacteria and their metabolites in CaCO 3 biomineralization.These functional groups confirm the biotic nature of these minerals.
The identification of calcite or Mg-rich calcite can be inferred from the distinctive spectral peaks observed at 712, 875, 1421, 1795, and 2514 cm −1 [37].Furthermore, the presence of aragonite is confirmed by bands observed at 713, 862, 1081, 1406, and 1476 cm −1 [37].In Figure 10a-d, the presence of characteristic bands of calcite or Mg-rich calcite can be observed, confirming the presence of these minerals.In Figure 10b, when the concentration of Mg 2+ ions increased to 2880 mg/L, the characteristic bands at 713, 862, 1081, 1406, and 1476 cm −1 provided evidence for the presence of aragonite.These mineral phases correspond well to the XRD results shown in Figure 9.The minerals were also found to contain organic functional groups, such as C-O-C (1033 and 1055 cm −1 ), C-N (1180, 1175, and 1173 cm −1 ), C=C (1649, 1624, and 1626 cm −1 ), and C=O (1722 and 1725 cm −1 ).The presence of O-H (2973, 2974, 2979, and 2981 cm −1 ) and fatty acid C-H bonds (2874, 2869, 2921, and 2920 cm −1 ) indicates the involvement of bacteria and their metabolites in CaCO3 biomineralization.These functional groups confirm the biotic nature of these minerals.The presence of protein secondary structures, including β-sheet, α-helix, 310 helix, and β-turn (Table 1), can also be observed in the range of 1600-1700 cm −1 .In Ca 2+ systems, there is only one protein secondary structure, β-sheet, present at a concentration of 400 mg/L of Ca 2+ .However, as the concentration of Ca 2+ ions increases, more protein secondary structures such as α-helix and β-turn appear.This corresponds well to the increased protein content in EPS, as shown in Figure 5h.With increasing concentrations of Mg 2+ and Fe 3+ ions, particularly when all three types of ions coexist in the same system, a greater variety of protein secondary structures would emerge.Therefore, the interaction between Mg 2+ and Fe 3+ ions not only leads to an increase in protein content in EPS (Figure 5h) but also results in a greater diversity of protein secondary structures (Table 1).The interaction between Mg 2+ and Fe 3+ ions alters the protein secondary structures, thereby affecting the biomineralization of CaCO3.The presence of protein secondary structures, including β-sheet, α-helix, 3 10 helix, and β-turn (Table 1), can also be observed in the range of 1600-1700 cm −1 .In Ca 2+ systems, there is only one protein secondary structure, β-sheet, present at a concentration of 400 mg/L of Ca 2+ .However, as the concentration of Ca 2+ ions increases, more protein secondary structures such as α-helix and β-turn appear.This corresponds well to the increased protein content in EPS, as shown in Figure 5h.With increasing concentrations of Mg 2+ and Fe 3+ ions, particularly when all three types of ions coexist in the same system, a greater variety of protein secondary structures would emerge.Therefore, the interaction between Mg 2+ and Fe 3+ ions not only leads to an increase in protein content in EPS (Figure 5h) but also results in a greater diversity of protein secondary structures (Table 1).The interaction between Mg 2+ and Fe 3+ ions alters the protein secondary structures, thereby affecting the biomineralization of CaCO 3 .

SEM-EDS Analyses of Minerals
An analysis was conducted of the minerals using SEM-EDS to examine their microstructures under various circumstances.As shown in Figure 11a-c, the calcite precipi-tated by cyanobacteria exhibits rhombohedral, spherical, and short columnar morphologies in systems with different concentrations of Ca 2+ ions.The area selected by a red circle in Figure 11b was analyzed by EDS, and the results show the presence of organic elements such as N, P, and S in addition to Ca, C, and O elements (Figure 11d).Significant changes occurred in the morphology of calcite when Fe 3+ or Mg 2+ + Fe 3+ ions were added to a system containing 800 mg/L of Ca 2+ ions.The rhombohedral calcite became incomplete (Figure 11e), whereas the surfaces of spherical and short columnar calcite became rough (Figure 11g,j).Unlike the calcite in Figure 11c, the surface of short columnar calcite is no longer adorned with microcrystals shaped like fish scales (Figure 11g,k).The EDS results also indicate the presence of Fe elements (Figure 11h,l) in the minerals selected by a red circle (Figure 11f,j).The minerals contained numerous holes that may have once been occupied by Synechocystis sp.PCC 6803, indicating that the formation of calcium carbonate minerals is not a simple physical and chemical process but occurs under complex conditions.
When different concentrations of Mg 2+ ions are added to a system containing 800 mg/L of Ca 2+ , the addition leads to abundant mineral morphology, including dumbbell, ball, cruciate flower, cauliflower, rod, and rhombohedra (Figure 11m-r).Due to the presence of Mg 2+ ions, the area selected by a red circle in Figure 11n contains not only Ca, C, O, N, P, and S elements but also Mg element (Figure 11s).The observed morphological diversity in aragonite structures further supports the biological origin of aragonite.

XPS Analyses of Biominerals Induced by Synechocystis sp. PCC 6803
The presence of Fe 3+ and Mg 2+ can affect the components of EPS (Figure 5).Since EPS serve as the nucleation sites, any changes in their components would also impact the biomineralization process.To further investigate the effects of Fe 3+ and Mg 2+ ions on CaCO3 biominerals, XPS experiments were conducted.XPS analyses enable the identification of surface chemical properties in carbonate minerals, facilitating the understanding of organic matter involvement during carbonate precipitation.Minerals contain C, Ca, O, N, P, and S at different concentrations of Ca 2+ ions (Figure 12).The C1s spectrum exhibits three  (a-d), Ca 2+ + Fe 3+ (e-h), Ca 2+ + Mg 2+ + Fe 3+ (i-l), and Ca 2+ + Mg 2 (m-s) under the action of free bacteria.The area selected by a red circle is used for EDS analyses (d,h,l,s).

XPS Analyses of Biominerals Induced by Synechocystis sp. PCC 6803
The presence of Fe 3+ and Mg 2+ can affect the components of EPS (Figure 5).Since EPS serve as the nucleation sites, any changes in their components would also impact the biomineralization process.To further investigate the effects of Fe 3+ and Mg 2+ ions on CaCO 3 biominerals, XPS experiments were conducted.XPS analyses enable the identification of surface chemical properties in carbonate minerals, facilitating the understanding of organic matter involvement during carbonate precipitation.Minerals contain C, Ca, O, N, P, and S at different concentrations of Ca 2+ ions (Figure 12).The C1s spectrum exhibits three distinct peaks at 283.9 eV, 285.4 eV, and 288.6 eV (Figure 12a), indicating the presence of C-(C/H), C-O/C-O-C, and O-C=O, which could be derived from alkanes, amino acid side chains, polysaccharides, ethers, alcohols, and carboxylic acids, respectively [38].The O-C=O ratio increased from 9.2% to 20.7% and further to 20.8% with an increasing concentration of Ca 2+ ions, which is consistent with the rising humic acid content in EPS (Figure 5a,b).The two peaks of Ca2p are located at 346.2 eV and 349.9 eV (Figure 12b), respectively, corresponding to Ca2p3/2 and Ca2p1/2 [39,40].The four peaks of O1s are located at 533.0 eV, 532.2 eV, 531.2 eV, and 530.4 eV (Figure 12c), indicating the presence of different oxygen states in carboxylic acids (O=C-OH/O=C-O-C), esters (C=O), carbonyl groups (O-C=O), and amides (N-C=O) [38].The N-C=O content increased from 26.8% to 27.0% and then further to 27.1% (Figure 12c).The presence of the chemical bond N-C=O indicates the presence of protein, as proteins always contain such chemical bonds.The characteristic peaks at 400.4 eV, 399.7 eV, 399.3 eV, and 398.8 eV (Figure 12d) represent NH + , N-C=O, C-NH 2 , and C-N-C [41,42].The N-C=O content increased from 6.5% to 14.2% to 17.5% (Figure 12d), indicating that the protein concentration on the mineral surface increased, resulting in an increase in N-C=O content.The P2p peaks at approximately 133 eV and 131.6 eV (Figure 12e) indicate the presence of P=O and C-PO 3 in DNA, RNA, as well as phospholipids found in biomaterials [43].The S2p spectra reveal four characteristic peaks at 169.3 eV, 168.5 eV, 164.9 eV, and 162.9 eV corresponding to SO 4 2− , SO 2 2− , S-C, and R-SH, respectively (Figure 12f).Cysteine possesses the chemical bond R-SH.These XPS results confirm the involvement of bacterial biomacromolecules in the biomineralization process.
CaCO 3 minerals formed in systems with a fixed concentration of Ca 2+ ions at 800 mg/L and varying concentrations of Mg 2+ ions (0, 1440, and 2880 mg/L) also exhibit the aforementioned characteristics (Figure S1).The O-C=O content in CaCO 3 minerals increases from 19.8% to 30.0% and then to 41.1% as the concentration of Mg 2+ ions increases (Figure S1a).Furthermore, the O-C=O content in the O1s spectra (Figure S1c) also increased from 55.1% to 56.6%, indicating that Mg 2+ ions stimulate the production of significant amounts of carboxylic acids in EPS, which play a crucial role in CaCO 3 biomineralization.The O1s result showed that the N-C=O increased from 27.0% through 27.2% to 28.5% (Figure S1c).Additionally, the N-C=O in the N1s spectra (Figure S1d) also increased significantly from 17.8% to 42.1%.These findings suggest that the increasing concentrations of Mg 2+ ions promoted protein production, which is consistent with the results shown in Figure 5h.In Figure S1g, Mg1s exhibits two characteristic peaks at 1305.6 eV and 1303.2 eV, which represent magnesium carbonate (MgCO 3 ) and Mg-O [44,45].The results reveal that Mg 2+ is involved in mineral formation, and the ions are present as MgCO 3 within CaCO 3 , thereby forming Mg-O bonds.There was a significant difference in S2p spectra between Figure 12f and Figure S1f.The content of SO 2 2− decreased significantly with increasing Mg 2+ ion concentrations (Figure S1f), unlike the result shown in Figure 12f.This indicates that the presence of Ca 2+ ions promotes sulfur reduction, whereas the presence of Mg 2+ ions does not facilitate it.
In systems with a fixed concentration of Ca 2+ ions (800 mg/L) and Fe 3+ ions (11.2 mg/L), as well as varying concentrations of Mg 2+ ions (0 and 1440 mg/L), the levels of O-C=O (Figure S2a,g) and N-C=O (Figure S2f,g) in CaCO 3 minerals also increased.With increasing concentrations of Mg 2+ ions, more Fe2p peaks appear (Figure S2h), indicating that the presence of Mg 2+ ions facilitates the incorporation of Fe ions into CaCO 3 minerals.This effect of Mg 2+ ions on Fe 3+ in CaCO 3 minerals has rarely been reported.In fact, Figure S2h shows that the incorporation of Fe into CaCO 3 minerals includes not only Fe 3+ but also Fe 2+ .The spectrum of Fe2p (Figure S2h) reveals various oxidation states of Fe, including both Fe 3+ and Fe 2+ , on the surfaces of CaCO 3 minerals.The peaks at 710.8, 717.7, and 720.0 eV correspond to the characteristic Fe2p3/2 peaks associated with Fe 3+ , whereas the peaks at 713.6 and 727.4 eV correspond to the typical Fe2p3/2 peaks attributed to Fe 2+ [46].Additionally, the peak at 722.9 eV corresponds to the typical Fe2p1/2 peak related to Fe 2+ [46].The peaks at 713.6 and 727.4 eV correspond to the satellite peaks of Fe 2+ , whereas the peaks at 717.7 and 720.0 eV represent the satellite peaks of Fe 3+ [46].The peak at 722.9 eV corresponds to FeO (Fe 2+ ), while the peak at 710.8 eV represents Fe 2 O 3 (Fe 3+ ).
Minerals 2023, 13, x FOR PEER REVIEW 16 of 25 eV (Figure 12e) indicate the presence of P=O and C-PO3 in DNA, RNA, as well as phospholipids found in biomaterials [43].The S2p spectra reveal four characteristic peaks at 169.3 eV, 168.5 eV, 164.9 eV, and 162.9 eV corresponding to SO4 2− , SO2 2− , S-C, and R-SH, respectively (Figure 12f).Cysteine possesses the chemical bond R-SH.These XPS results confirm the involvement of bacterial biomacromolecules in the biomineralization process.CaCO3 minerals formed in systems with a fixed concentration of Ca 2+ ions at 800 mg/L and varying concentrations of Mg 2+ ions (0, 1440, and 2880 mg/L) also exhibit the aforementioned characteristics (Figure S1).The O-C=O content in CaCO3 minerals increases from 19.8% to 30.0% and then to 41.1% as the concentration of Mg 2+ ions increases (Figure S1a).Furthermore, the O-C=O content in the O1s spectra (Figure S1c) also increased from 55.1% to 56.6%, indicating that Mg 2+ ions stimulate the production of significant amounts of carboxylic acids in EPS, which play a crucial role in CaCO3 biomineralization.The O1s result showed that the N-C=O increased from 27.0% through 27.2% to 28.5% (Figure S1c).Additionally, the N-C=O in the N1s spectra (Figure S1d) also increased significantly from 17.8% to 42.1%.These findings suggest that the increasing concentrations of Mg 2+ ions promoted protein production, which is consistent with the results shown in Figure 5h.In Figure S1g, Mg1s exhibits two characteristic peaks at 1305.6 eV and 1303.2 eV, which represent magnesium carbonate (MgCO3) and Mg-O [44,45].The results reveal that Mg 2+ is involved in mineral formation, and the ions are present as MgCO3 within CaCO3, thereby forming Mg-O bonds.There was a significant difference in S2p spectra between Figure 12f and Figure S1f.The content of SO2   Despite being classified as prokaryotic bacteria, the cyanobacterium Synechocystis sp.PCC 6803 differs from other bacteria in the location of its chlorophyll within its cellular structure.After conducting a comprehensive full-band spectral scan, it has been determined that the maximum absorption peak of cyanobacteria occurs at 730 nm.Therefore, measurements are taken at this specific wavelength to quantify the density or cell concentration of cyanobacteria [47].Excessive levels of Ca 2+ concentration can impede bacterial activity [48].In this study, the cell concentration of Synechocystis sp.PCC 6803 was higher at 800 mg/L of Ca 2+ than at 400 and 1200 mg/L of Ca 2+ , indicating that a certain amount of Ca 2+ ions is required for the growth of Synechocystis sp.PCC 6803.Extremely low or high concentrations of Ca 2+ ions are not conducive to the growth.The optimal concentration of Ca 2+ ions is crucial for maintaining high cyanobacterial activity, thereby providing a significant basis for further research on the influence of Mg 2+ and Fe 3+ ions on calcium carbonate mineralization under the impact of cyanobacteria.In this study, the presence of Mg 2+ ions inhibited the growth of Synechocystis sp.PCC 6803.However, previous research has reported that a culture medium with varying concentrations of Mg 2+ effectively promoted cell proliferation in Rhodopseudomonas faecalis RLD-53 [49].The contrasting conclusions may be attributed to differences in bacterial strains and concentrations of Mg 2+ ions.The acquisition of Fe ions is enhanced by numerous bacteria through the production of siderophores, which are extracellular iron-specific chelators [50,51].Boyer et al. have thoroughly examined the process of iron chelation and uptake in cyanobacteria [50].Within the genome of Synechocystis sp.PCC 6803, the sll1878 gene has been found responsible for producing a distinctive iron transporter protein [52].Optimal growth rate was observed in wild-type Synechocystis sp.PCC 6803 cells at a concentration of 1 µM Fe 3+ , while slower growth was observed in the M-1 mutant (with deactivated transporter genes) at this Fe 3+ concentration [52].In this study, the cell concentrations of Synechocystis sp.PCC 6803 increased with increasing Fe 3+ ion concentrations from 5.6 to 8.4 and then to 11.2 mg/L, indicating that a specific concentration of Fe 3+ ions is favorable for the growth of Synechocystis sp.PCC 6803.
Correlation analysis revealed a significant association between the average activity of CA and the overall quantity of Ca 2+ present in the culture system [53].In this study, high concentrations of Ca 2+ ions (Figure 3a) were found to inhibit CA activity.In addition to Ca 2+ , the presence of Mg 2+ ions also affects CA activity.Previous studies have shown that CA activity is lower at specific Mg/Ca molar ratios (9 and 12) compared to lower ratios (3 and 6) [54].This study also indicates that concentrations of Mg 2+ at 1440 and 2880 mg/L (Figure 3b) could result in a decrease in CA activity.Therefore, a sufficiently high concentration of Mg 2+ ions does indeed inhibit CA activity.It has been reported that CA is a metalloenzyme containing Zn 2+ at its biologically active sites, and this essential metal ion can be substituted with other metals such as Fe 3+ [55].Interestingly, when Fe 3+ replaces the native Zn 2+ , it significantly enhances enzyme activity by modifying the enzymes by metal ion replacement.The complexes formed by Fe 3+ and CA also exhibit higher stability [55].
Seawater pH levels of up to 8.7 have frequently been observed in coastal waters, such as the North Sea and the German Bight, particularly during algal blooms [56,57].In Mariager Fjord, the surface seawater exhibits a pH of around 9 and can even reach as high as 9.75 during July and August when algal blooms occur [58].In this study, the pH values increased from 7.0 to 8.57 under the influence of Synechocystis sp.PCC 6803, resulting in a negatively charged cell surface and thereby, promoting the adsorption of Ca 2+ , Mg 2+ , and Fe 3+ ions.These findings confirm the crucial role of algae in elevating pH, thus creating favorable conditions for CaCO 3 precipitation.The precipitation of CaCO 3 often leads to a depletion of total alkalinity, which is frequently observed in lakes with high mineral content, freshwater springs, and the low-salinity regions of estuaries [59,60].These water bodies typically exhibit a prevalent characteristic of having a low Mg/Ca ratio (Mg/Ca < 2) [59,60].In this study, the presence of Ca 2+ and Mg 2+ ions indeed resulted in a decrease in pH (Figure 2a,b), possibly due to the formation of CaCO 3 or Ca(OH) 2 [61], as well as MgCO 3 or Mg(OH) 2 [62].Regarding Ca(OH) 2 , MgCO 3 , or Mg(OH) 2 , the XRD results do not show their presence (Figure 9), could be due to their extremely low abundance or amorphous nature.The decreased pH values with increasing concentrations of Ca 2+ and Mg 2+ ions may also be attributed to the reduced cell concentrations shown in Figure 1.The decrease in bacterial concentrations resulted in a corresponding reduction in CA activity (Figure 3), leading to decreased levels of HCO 3 − and CO 3 2− ions (Figure 4).Fe, which is a crucial component for bacterial survival, plays a significant role in numerous biochemical reactions within bacterial organisms.The presence of Fe 3+ ions enhances the concentrations of CO 3 2− and HCO 3 − ions (Figure 4c-e) due to an increase in CA activity.The addition of Fe 3+ ions, unlike the impact of Ca 2+ and Mg 2+ ions, can raise pH levels.The precise mechanism by which Fe 3+ ions elevate pH levels remains unclear, and further investigation is needed.
Extracellular polymeric substances (EPS) are synthesized by various microorganisms including bacteria, fungi, microalgae, and cyanobacteria.These substances encompass a range of metabolites, such as polysaccharides, proteins, DNA, and humic substances [63].In this study, the levels of protein, polysaccharides, DNA, and humic-like substances significantly increased in the presence of separated or coexisting Ca 2+ , Mg 2+ , and Fe 3+ ions (Figure 5).It has been documented that the primary constituents of EPS in Synechocystis sp.PCC6803, which are polysaccharides, proteins, DNA, and substances resembling humic compounds, play a crucial role in safeguarding against the infiltration of metal ions [64,65].Therefore, in this study, the increased content of these organic matter was beneficial for the survival of Synechocystis sp.PCC6803 when exposed to three types of metal ions.The potential reason behind the increased protein content could be attributed to the secretion of an active reductase by Synechocystis sp.PCC6803.It has been reported that exposure to chromium ions (Cr 6+ ) induces Synechocystis sp.PCC6803 to release certain enzymatic reducers, including reductase, resulting in enhanced protein detection in EPS [65].Variations observed in the components of EPS following exposure to arsenic (As) indicate the cellular response strategy employed by Synechocystis sp.PCC6803 cells to withstand As-induced stress.The presence of polysaccharides, tyrosine, aromatic tryptophan proteins, and humic-like substances in EPS derived from Synechocystis sp.PCC6803 has been verified in the presence of As [63].Therefore, changes in the environment will lead to changes in the composition of EPS of Synechocystis sp.PCC6803.

Effects of Changes in the Cations, CA Activity, and EPS Composition on the Formation of CaCO 3
Studies have found that Mg 2+ ions play a significant role in inhibiting all aspects of CaCO 3 precipitation, including crystal formation, solubility, and precipitation rate [60].Our results also support this finding.Sun et al. discovered a direct correlation between the surface energy of calcite and the Mg/Ca ratio through thermodynamic model calculations, indicating that Mg 2+ increases the barrier for calcite nucleation [66].When Mg 2+ is present in the solution, it exhibits a preference for binding to specific sites on the calcite surface, such as the kink sites, due to its similar ionic properties to those of Ca 2+ .The attachment of Mg 2+ effectively obstructs the combination of CO 3 2− ions and consequently hinders the nucleation process of CaCO 3 [67].Based on the aforementioned studies, it can be inferred that the presence of Mg 2+ significantly affects various aspects of CaCO 3 precipitation, including crystal formation, solubility, and precipitation rate.The presence of Mg 2+ ions caused a switch from calcite to aragonite.The smaller hydrated ion radius of Mg 2+ ions compared to Ca 2+ ions resulted in the replacement of Ca 2+ by Mg 2+ , thereby influencing the mineral phases and crystallinity.Aragonite was precipitated under the conditions of 800 mg/L of Ca 2+ ions and 2880 mg/L of Mg 2+ ions in this study (Figure 9b).It is widely acknowledged that synthesizing aragonite presents significant challenges due to its tendency to readily convert into calcite, which is thermodynamically more stable [68].This issue has been extensively researched, and numerous efforts have been made to overcome it [68].The concentration of Mg 2+ ions in biotic aragonite has been found to be higher compared to that in inorganically precipitated aragonite [69], and the presence of organic matter facilitates the easier incorporation of Mg 2+ ions into aragonite crystals [70].In this study, only one group of experiments produced aragonite, at a concentration of 2880 mg/L Mg 2+ ions (Figure 9b), which does not meet the requirements for significant difference analysis as it is necessary to compare at least three groups.When the concentration of Mg 2+ ions is increased, either aragonite or monohydrocalcite minerals form.Therefore, appropriate concentrations of Mg 2+ ions should be carefully designed in order to obtain pure aragonite.This process requires a long time.Thus, future studies will focus on comparing the Mg content in biotic aragonite induced by Synechocystis sp.PCC 6803 with that in inorganically precipitated aragonite.
The precipitation ratios and rates of Ca 2+ ions were enhanced in the presence of Fe 3+ ions, with or without Mg 2+ ions (Figures 7 and 8) in this study.In systems containing Cu 2+ , Fe 2+ , Fe 3+ , and Co 2+ ions, only the system with Fe 3+ ions showed the presence of a calcium oxalate ring [71].This observation suggests that the inclusion of Fe 3+ ions enhances the precipitation of Ca 2+ ions and effectively facilitates the formation of concentric rings [71].Our results are consistent with this finding.The research on the impact of iron ions on CaCO 3 precipitation has been limited and, at times, has yielded conflicting findings.The individual effects of Fe 2+ and Fe 3+ on the precipitation kinetics and microstructure of CaCO 3 were examined, and the results show that the presence of Fe 3+ accelerated the rate of CaCO 3 precipitation [72].In this study, in the absence or presence of Mg 2+ ions, both the precipitation ratios and rates increased as the concentration of Fe 3+ ions increased, which is consistent with the aforementioned opinion.This intricate process was further investigated.Fe 3+ ions have the ability to react with OH − ions, resulting in the formation of Fe(OH) 3 .Subsequently, Fe(OH) 3 can promote the initiation of heterogeneous CaCO 3 nucleation by creating a crystalline growth site, where CaCO 3 would selectively precipitate [72].It has been reported that a higher precipitation rate will lead to reduced mineral crystallinity and subsequently result in a decline in thermal stability [39].In this study, the precipitation rates of Ca 2+ increased with increasing concentrations of Fe 3+ ions (Figures 7c and 8c,f), which is consistent with the results indicating a decrease in the crystallinity of calcite (Tables S13 and S14).The acceleration of precipitation rates resulted in a reduction in the crystallinity of calcite.Additionally, the incorporation of Fe 3+ and Mg 2+ ions into calcite also contributed to this phenomenon.The presence of Fe 2+ ions (Figure S2h) in calcite suggests the occurrence of reducing conditions within the oxidation system.Humic substances play a significant role in regulating the iron redox cycle due to their diverse functional groups, which are capable of both accepting and donating electrons [73].Therefore, the presence of Fe 2+ observed in XPS results may be closely associated with the existence of humic substances.Iron reduction is a common phenomenon mediated by microorganisms.Thorne et al. investigated the reduction of Fe 3+ in ferricyanide by Synechocystis sp.PCC 6803 and confirmed its ability to reduce Fe 3+ ions [74].
In this study, unlike the calcite in Figure 11c, the surface of short columnar calcite is no longer adorned with microcrystals shaped like fish scales in the presence of Fe 3+ ions (Figure 11g,k).This conclusion confirms that the surface morphology of cyanobacteriainduced calcite differs when Fe 3+ ions are present or absent.The shape of barite crystal is significantly influenced by the rate of ion precipitation [75].The EDS results show the presence of the element Fe in the calcite formed in the presence of Fe 3+ ions (Figure 11h,l).In this study, the activity of CA could be enhanced with increasing concentrations of Fe 3+ (Figure 3c-e), resulting in a higher concentration of CO 3 2− ions (Figure 4c-e) and an increased precipitation rate of Ca 2+ ions (Figures 7c and 8c,f).The morphology of minerals was influenced by a higher precipitation rate, according to the aforementioned opinion.In addition to the precipitation rate, the presence of organic matter also leads to changes in calcite morphology.The content of polysaccharide, as one component in EPS, significantly increases in the presence of separated or coexisting Mg 2+ and Fe 3+ ions (Figure 5h, Table S4).Glycosidic bonds (C-O-C) are common chemical bonds in polysaccharide molecules.Some scholars have proposed that the C-O-C bond plays a significant role in the process of biomineralization, and the presence of the C-O-C promotes the formation of elongated calcite crystals [76,77].In this study, elongated calcite can be observed (Figure 11c,g,k), possibly also due to the existence of C-O-C (Figures 10, 12a, S1a and S2a).The organic matter in biotic aragonite (Figures 10, 12, S1 and S2 and Table 1) can also influence mineral morphology.Traditionally, aragonite polymorphs exhibit a needle-like shape.However, a distinct aragonite morphology characterized by an enlarged cubic structure was observed when employing a hydrothermal technique with the addition of polyacrylamide, which is abundant in N-C=O groups, and cetyltrimethylammonium bromide as supplementary agents [78].Aragonite with intricate structures was produced using a uniform precipitation method, in which calcium acetate and urea (abundant in N-C=O) were precipitated at a temperature of 90 • C in the presence of polyvinyl pyrrolidone (abundant in C=O) [79].The resulting aragonite exhibited various morphologies, including bundles of rods, formations resembling bouquets, and structures shaped like dumbbells [79].The chemical bonds C=O and N-C=O, which were found in aragonite (Figure S1c,d) in this study, maybe the main reason for the diverse morphology of aragonite shown in Figure 10.
Proteins have the ability to modify the spatial orientation, morphology, and mineral phase of crystals [80].Certain peptides that bind to CaCO 3 induce similar morphological characteristics in calcite as those observed when natural protein mixes were present [81].These processes are regulated by distinct proteins, each exhibiting its own unique secondary structure and undergoing conformational changes [80].In this study, minerals exhibit a diverse range of protein secondary structures (Table 1), which also play a crucial role in mineral morphology as mentioned above.In the presence of separated or coexisting Fe 3+ and Mg 2+ ions, the DNA content in EPS increased in addition to protein (Figure 5h, Table S3).The DNA possesses an impeccable and precise molecular recognition capability, resulting in a remarkably selective assembly process.The surface of DNA molecules exhibits the growth of hydroxyapatite crystallites with diameters ranging from approximately 1 to 14 µm, and scanning electron microscopy analysis successfully reveals diverse morphologies [82].Therefore, changes in the components of EPS would similarly affect the morphology of minerals.The mineral phases can also be influenced by the organic matter in EPS.In this study, the levels of humic acid and polysaccharides (the components of EPS) increased under the influence of Ca 2+ , Mg 2+ , and Fe 3+ cations (Figure 5).These biomacromolecules can also affect the mineral phases.Some researchers utilized humic acid as a representative of humic substances to investigate their impact on CaCO 3 formation and found that the proportion of calcite in the products decreased with increasing concentrations of humic acid [83].Kawano and Hwang conducted an assessment on the impact of polysaccharides on the precipitation of CaCO 3 minerals [84] and found that there was a decrease in the prevalence of aragonite and an increase in the dominance of calcite as the concentration of polysaccharides increased.Therefore, organic matter not only affects the morphology of minerals but also influences their mineral phases.

Conclusions
The effects of Mg 2+ and Fe 3+ cations on the biomineralization of CaCO 3 minerals were studied through the activities of Synechocystis sp.PCC 6803.The presence of Ca 2+ and Mg 2+ ions during the process of CaCO 3 biomineralization resulted in a decrease in cell concentration, pH levels, and CA activity, as well as CO 3 2− and HCO 3 − concentrations.Conversely, Fe 3+ ions promoted an increase in cell concentration, pH levels, and CA activity, as well as CO 3 2− and HCO 3 − concentrations in both the presence and absence of Mg 2+ ions in Ca 2+ -containing systems.The interaction between Mg 2+ and Fe 3+ ions can enhance the release of substances such as DNA, proteins, polysaccharides, and compounds similar to humic acids in EPS.The presence of Mg 2+ ions inhibits the precipitation of Ca 2+ ions, whereas the presence of Fe 3+ ions promotes the precipitation of Ca 2+ ions in both the absence and presence of Mg 2+ ions.The harvested CaCO 3 minerals comprise calcite (groups 2-4, 5, 8-13), Mg-rich calcite (groups 6, 7), and aragonite (group 7).The crystallinity of these minerals decreased as the ion concentration (groups 2-13) increased.Organic functional groups such as C=C, C-O-C, and C=O were found in these CaCO 3 minerals.Additionally, there was an increase in the abundance of protein secondary structures with an increase in both ion species and concentration.The addition of Fe 3+ ions roughens the surface of the crystals and causes incompleteness in their rhombohedral structure.The presence of Mg 2+ ions enhances the diversity of mineral morphology.The content of O-C=O and N-C=O in the CaCO 3 mineral also increases under the influence of mixed Mg 2+ and Fe 3+ ions or with increasing concentrations of Mg 2+ ions.With increasing concentrations of Mg 2+ ions, a greater number of Fe2p peaks appeared, indicating that the presence of Mg 2+ ions facilitated the incorporation of Fe ions into CaCO 3 minerals.This study provides a theoretical basis for further understanding the process of biomineralization induced by Synechocystis sp.PCC 6803 under the interaction of Ca 2+ , Mg 2+ , and Fe 3+ ions.

Figure 7 .
Figure 7. Changes in Ca 2+ concentration (a), Ca 2+ precipitation ratio (b), and Ca 2+ precipitation rate (c) at different conditions under the influence of bacteria.

1 .
Effects of Ca 2+ , Mg 2+ , and Fe 3+ Ions on the Cell's Survival, CA Activity, pH, and EPS Components • C. The illumination time was set to 12 h.The culture was manually shaken every 12 h to prevent bacterial accumulation.The Synechocystis sp.PCC 6803 seed solution (OD 730 = 1.2) was inoculated into the 16 types of media listed in Table 2+ , Mg2+, and Fe 3+ Ions on Cyanobacterial Growth, pH, CA Activity, and

Table 1 .
FTIR analyses of protein secondary structures in biominerals induced by Synechocystis sp.PCC 6803 under different conditions.

Ion concentrations (mg/L) Biominerals Induced by Synechocystis sp. PCC 6803
-, beyond the limit of detection.