Live microbial cells adsorb Mg²⁺ more effectively than lifeless organic matter

Abstract

The Mg²⁺ content is essential in determining different Mg-CaCO₃ minerals. It has been demonstrated that both microbes and the organic matter secreted by microbes are capable of allocating Mg²⁺ and Ca²⁺ during the formation of Mg-CaCO₃, yet detailed scenarios remain unclear. To investigate the mechanism that microbes and microbial organic matter potentially use to mediate the allocation of Mg²⁺ and Ca²⁺ in inoculating systems, microbial mats and four marine bacterial strains (Synechococcus elongatus, Staphylococcus sp., Bacillus sp., and Desulfovibrio vulgaris) were incubated in artificial seawater media with Mg/Ca ratios ranging from 0.5 to 10.0. At the end of the incubation, the morphology of the microbial mats and the elements adsorbed on them were analyzed using scanning electronic microscopy (SEM) and energy diffraction spectra (EDS), respectively. The content of Mg²⁺ and Ca²⁺ adsorbed by the extracellular polysaccharide substances (EPS) and cells of the bacterial strains were analyzed with atomic adsorption spectroscopy (AAS). The functional groups on the surface of the cells and EPS of S. elongatus were estimated using automatic potentiometric titration combined with a chemical equilibrium model. The results show that live microbial mats generally adsorb larger amounts of Mg²⁺ than Ca²⁺, while this rarely is the case for autoclaved microbial mats. A similar phenomenon was also observed for the bacterial strains. The living cells adsorb more Mg²⁺ than Ca²⁺, yet a reversed trend was observed for EPS. The functional group analysis indicates that the cell surface of S. elongatus contains more basic functional groups (87.24%), while the EPS has more acidic and neutral functional groups (83.08%). These features may be responsible for the different adsorption behavior of Mg²⁺ and Ca²⁺ by microbial cells and EPS. Our work confirms the differential Mg²⁺ and Ca²⁺ mediation by microbial cells and EPS, which may provide insight into the processes that microbes use to induce Mg-carbonate formation.

Keywords

microbe / adsorption / magnesium / calcium

Introduction

Mg-bearing calcium carbonates (Mg-CaCO₃) are important minerals on Earth because they constitute a large fraction of the total carbonate reservoir and compose the skeleton of most marine invertebrates (Xu et al., 2013). It is widely accepted that the variation of mineral phases in sedimentary carbonates is highly associated with the fluctuation of oceanic hydrochemical conditions during the Phanerozoic eon (Sandberg, 1983; McKenzie and Vasconcelos, 2009). The Mg/Ca ratio of the ocean (Mg/Ca_solution) is considered to be the most essential hydrochemical variable (Folk and Land, 1975; Mucci and Morse, 1983; Saunders et al., 2014). Low Mg-calcite (Mg<4%, hereafter molar percent) is mainly formed in oceans with low Mg/Ca ratios, and high Mg-calcite (Mg>4%) mainly precipitates in oceans with high Mg/Ca_solution (>2.0). The very high Mg-carbonate, dolomite (Mg= 50%), is also considered to be formed in settings with high Mg/Ca_solution (>5.0; Folk and Land, 1975). Thus, the ratio of Mg/Ca in carbonate minerals was proposed to be indicative of the Mg/Ca ratio in the ocean (Dickson, 2002; Ries et al., 2008).

Nevertheless, the high Mg/Ca_solution alone does not guarantee the formation of high Mg-calcite or dolomite. It has been widely accepted that dolomite cannot be precipitated via pure inorganic processes, despite the extremely high Mg/Ca_solution (99) and high saturation index respected to dolomite (>1000) as well as long reaction time (32 years) (Land, 1998). The incorporation of Mg²⁺ into calcite was found to be very difficult because the high binding energy between Mg²⁺ and H₂O impedes the dehydration of the Mg²⁺–H₂O complex (Chave et al., 1962). In addition, the substitution of Ca²⁺ by Mg²⁺ in calcite would increase the instability of the mineral (Chave et al., 1962) and thus lead to the transformation of high Mg-calcite (Mg>10%) to other relatively stable solid phases, such as low Mg-calcite or aragonite (Reeder and Sheppard, 1984).

High Mg-CaCO₃ has been found in various natural sediments associated with microorganisms, such as organic-rich deposits of the continent margin (50 mol% Mg) (Baker and Burns, 1985), microbialites in the central Pacific Ocean (7–16 mol% Mg) (Camoin et al., 1999), and stromatolite in a Brazil lagoon (40 mol% Mg) (Spadafora et al., 2010). These findings indicated the essential role of microbes and organic matter in the formation of high Mg-CaCO₃. Laboratory studies have also demonstrated that both microbes (Krause et al., 2012) and organic matter (Bontognali et al., 2014) are able to induce the formation of high Mg-CaCO₃. Microbial mineralization processes are highly associated with their metabolic activities, which lead to an increased saturation index of Mg-CaCO₃. For example, cyanobacteria enhance the pH of their surroundings by photosynthesis, while sulfate reducing bacteria (SRB) release Mg²⁺ from the Mg²⁺–SO₄^2- complex by consuming sulfate ions. However, more complex scenarios may exist because the non-metabolizing microbial organism functioned above our expectation in inducing carbonate formation. For example, the amount of carbonate precipitation induced by dead cells of SRB was equal to or even more than that of live cells (Bosak and Newman, 2003). The intact non-metabolizing cells of halophilic archaea or its lysed solution were able to precipitate dolomite, yet metabolizing cells were not capable of doing so (Kenward et al., 2013). These observations suggested something beyond metabolic activities was involved in microbial mineralization that we still did not know.

Both metabolizing microbes and non-metabolizing microbial organisms can adsorb metals in solution via the functional groups with negative charges, such as carboxyl, phosphoryl, and sulfo groups (Braissant et al., 2007). Although the adsorption capacities of functional groups for metals were demonstrated differently by numerous literatures (Wang et al., 2009), it remains unknown whether metabolizing microbes and non-metabolizing microbial organisms behave differently in the adsorption of Mg²⁺ and Ca²⁺. Since adsorption of Mg²⁺ and Ca²⁺ by microbes or EPS was the first step of biotic formation of high Mg calcite and dolomite, answers to this issue will provide more constraints for both the evaluation of microbial effects in the formation of Mg-contained carbonate and the application of Mg/Ca in microbialites as an indicator for paleoenvironment reconstruction (Ries et al., 2008).

In this study, batch culture experiments were conducted to elucidate the different behaviors of live cells and organic matter of microbial mats and bacterial strains in adsorbing Mg²⁺ and Ca²⁺ in solution with varied Mg/Ca ratios. Our results will offer detailed information about the allocation of Mg²⁺ and Ca²⁺ in a system with microbial cells and microbial organic matter, which is beneficial for understanding of microbially mediated carbonate formation.

Materials and methods

Microbial mat and bacterial strains used

The microbial mat was collected from the Dadeng saltern in Xiamen, China (24°33.832′N, 118°17.646′E). The sampling area was oversaturated with salt, and the water-sediment interface was covered with a green mat. The bacterial community in the mat was analyzed with 16S rRNA clone library construction. In total, four bacterial strains were used in our study. Two aerobic heterotrophic bacteria (Staphylococcus sp. and Bacillus sp.) were isolated from the microbial mat and subjected to the subsequent experiments. The other two strains used were Synechococcuselongatus FACHB-410 (a cyanobacterium), purchased from the Institute of Hydrobiology of the Chinese Academy of Science, and Desulfovibrio vulgaris ATCC29579 (a sulfate reducing bacteria), provided by Hailiang Dong from the Miami University.

Subculture of the microbial mat and bacterial strains

The modified artificial seawater medium (MASW) was used to subculture the microbial mat. The components of 1 L MASW were as follows: NaCl 22.33 g, MgCl₂ 5.25 g, CaCl₂ 1.18 g, KCl 0.74 g, Na₂CO₃ 0.32 g, Na₂SO₄ 4.15 g, NaNO₃ 1.00 g, KH₂PO₄ 39.00 mg, Na₂EDTA 4.50 mg, FeCl₃ 0.35 mg, MnCl₂ 0.16 mg, ZnCl₂ 16.70 mg, CoCl₂ 6.50 mg, V_B1 0.12 mg, V_B12 0.10 mg, and 30 mL of soil extraction. The procedures for preparing the soil extraction are described in Stoupin et al. (2012); V_B1 and V_B12 were filtered through a 0.22-mm nitrocellulose membrane before being added to the sterile medium. The ASW, LB, and lactate media were prepared to maintain the cyanobacterium, aerobic heterotrophic bacteria, and SRB, respectively. All media were amended to have a total salinity of 35‰ by altering the dosage of NaCl. The details about the ASW and Lactate media are described in Qiu et al. (2012) and Liu et al. (2012), respectively. Incubation conditions for these cultures are listed in Table 1.

Microbial mat adsorption experiments

The microbial mat adsorption experiments were conducted in MASW with Mg/Ca ratios of 1.5, 2.5, and 5.2. The various Mg/Ca ratios were achieved by changing the dosage of MgCl₂ while fixing the amount of CaCl₂. The salinities were also fixed to 35‰ by varying the amounts of NaCl. Fresh microbial mats were rived into fragments by sterilized tweezers in an ultraclean benchtop. An aliquot of 2.00 mL of this fragment-containing slurry was inoculated into a beaker with 400 mL of MASW medium. The experiments with dead controls (the microbial mat sterilized at 121°C for 30 mins fully covered the bottom of the beaker) and chemical controls (without microbial mat) were conducted simultaneously. The experiments were terminated when the bottom of the beaker was fully covered with newly growing microbial mats, which took 40 days. The solid phase of each group was collected by centrifugation at 9,000 × g for 10 min. After discarding the supernatant, centrifugation was conducted again to thoroughly remove the residual solution. The final pellets were subsequently freeze-dried and stored at 4°C for future use. The morphology and the amounts of Mg²⁺ and Ca²⁺ adsorbed by the microbial mat were subsequently analyzed.

Bacterial strain adsorption experiments

Four bacteria strains were cultured in the media described above with Mg/Ca ratios of 0.5, 1.5, 2.5, 5.2, and 10.0. An aliquot of a 4.0 mL log-phase culture of S. elongatus, Staphylococcus sp., and Bacillus sp. was inoculated into 400 mL fresh medium, and a 15.0 mL culture of D. vulgaris was inoculated into 1,500 mL medium to obtain sufficient biomass. Duplicates were prepared for each group. The duration from the inoculation to a stable phase was 12 days, 3 days, 3 days, and 30 days for S. elongatus, Staphylococcus sp., Bacillus sp., and D. vulgaris, respectively. The native cell (hereafter cell) of the stable phase was collected by centrifugation at 9,000 × g for 10 min. After discarding the supernatant, centrifugation was conducted again to thoroughly remove the residual solution. The soluble EPS (hereafter EPS) in the supernatant was extracted by adding two volumes of precooled (4°C) ethanol, and the mixture was placed in a refrigerator at 4°C for 12 h. The EPS was collected from the bottom of the beaker using the same processes as for the pellet of the cell. Both cell and EPS were freeze-dried for 20 h and then preserved in a drying vessel at room temperature for further use.

Analysis methods

Morphology and elemental distribution

The morphology and Mg²⁺ and Ca²⁺ distributions of the microbial mat were analyzed by scanning electronic microscopy (SEM) and energy diffraction spectra (EDS), respectively. The dried microbial mats were rived into small pieces with sharp tweezers. Several pieces of the mats were then adhered onto SEM stubs with double-sided conductive tape. The stubs were coated with Pt for image observation using a FEI Quanta 450 FEG scanning electron microscope (FEI, Hillsboro, America) and SDD Inca X-Max 50 X-ray energy diffraction spectra (OXFORD, London, Britain). The accelerating voltages of the SEM and EDS were 5 kV and 20 kV, respectively.

Cation concentration analysis

To analyze the amount of Mg²⁺ and Ca²⁺ adsorbed on the cell and EPS, 100 mg of the dried cell and EPS of each strain were individually transferred to 5-mL glass vials, and 3.0 mL concentrated HCl (11.9 M) were added. The vials were covered with lids and heated in a water bath at 95°C for 3 h. The acidolyzed product was completely transferred to a volumetric flask and then diluted with double-distilled water (ddH₂O) to 50.00 mL.

The concentrations of Mg²⁺ and Ca²⁺ in the nitrolysis solution were measured with atomic absorption spectroscopy (AAS, Hitachi, 180-70 polarized Zeeman, Japan). The amounts of Mg²⁺ and Ca²⁺ adsorbed by the cell and EPS per one liter of culture were calculated based on Eq. (1).

(1)M\mathrm{a}\mathrm{d}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{e}\mathrm{d}\hspace{0.17em}\mathrm{b}\mathrm{y}\hspace{0.17em}\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}\hspace{0.17em}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}=\mathrm{2}\times {\mathrm{10}}^{\mathrm{3}}\hspace{0.17em}M\hspace{0.17em}\mathrm{i}\mathrm{n}\hspace{0.17em}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{i}\mathrm{s}\hspace{0.17em}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\hspace{0.17em}\mathrm{o}\mathrm{f}\hspace{0.17em}\mathrm{o}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{i}\mathrm{c}\hspace{0.17em}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r},

M indicates the concentration of Mg²⁺ or Ca²⁺, and organic matter is the cell or EPS.

Functional group analysis

To characterize the functional groups on the surface of the cell and EPS, automatic potentiometric titration was conducted on the S. elongatus cell and EPS because cyanobacteria were the dominant species in the microbial mat used. Cell and EPS were harvested with the methods mentioned in section 2.4. The pellets of the cell were resuspended in NaCl solution with a final wet cell concentration of 1.645 g/L and a final salinity of 35‰ (NaCl concentration of 0.60 mol/L). The final salinity of the resuspension was equal to that of the medium in which S. elongatus were cultured. The pellets of EPS were dissolved in a NaCl-solution with a final wet EPS concentration of 0.724 g/L and a final NaCl concentration of 0.01 mol/L. A volume of 40 mL cell or EPS-suspension was used for the automatic potentiometric titration (Metrohm 902 Titrando, Swiss Land). Hydrochloric acid (HCl) with a concentration of 0.100 mol/L was added into the suspension with an aliquot of 5 ml per step to acidize the solution until a final pH of 2.50 was reached. Then, 0.107 mol/L NaOH solution was added to alkalize the suspension with an aliquot of 5 ml per step until a final pH of 10.50 was reached. The real-time pH of the suspension was recorded during acidification and alkalization. The data obtained from titration were analyzed with Protofit 2.1 under a non-electrostatic adsorption model. The buffer zone and capacity of the material in the solution were automatically estimated with Protofit 2.1 based on the curve between the added volume of acid/base and the change of pH. The buffer zone indicates the possible functional groups and the buffer capacity quantifies the amount of certain functional groups (Turner and Fein, 2006).

Results

Mg²⁺ and Ca²⁺ adsorbed by the microbial mat

Morphology of the cyanobacteria mat

Newly growing microbial mats covered the entire bottom of each beaker after incubation for 40 days. The live microbial mats were characterized by long strips (Fig. 1(a)) with a thick EPS matrix (5–10 µm; Fig. 1(b), black rectangle). These long strips were assemblages of cyanobacterial cells affiliated with Phormidium, as indicated by 16S rRNA sequencing (unpublished data). The sterilized microbial mats were partly degraded; few integrated cyanobacteria assemblages were observed (Fig. 1(c)). EPS matrix and single globular cells (Fig. 1(d), white arrow) were observed. Only a small amount of amorphous solid phase was found under chemical control (Figs. 1(e) and 1(f)).

Mg²⁺ and Ca²⁺ adsorbed by the microbial mat

Figure 2 shows the mole percentage of Mg²⁺ and Ca²⁺ on the dried mat and control samples obtained from EDS analysis. The percentages of Mg²⁺ adsorbed by the live mat (Mg_{live mat}) are discrete, varying from 0% to 19.1%. In contrast, the percentages of Mg²⁺ adsorbed by the dead mat (Mg_{dead mat}) or chemical control (Mg_{chemical control}) show a very limited range of<4%. Overall, most of the percentages of Mg_{live mat} were higher than that of Mg_{dead mat} and Mg_{chemical control} (Figs. 2(a), 2(c), and 2(e)). Furthermore, the ratios of Mg/Ca adsorbed by live mats (Mg/Ca _{live mat}) were higher than that of the dead mats (Mg/Ca _{dead mat}) and chemical control (Mg/Ca _{chemical control}; Figs 2(b), 2(d)_, and 2(f)). Almost all Mg/Ca ratios over 1.0 appeared in the live mat samples, with only two exceptions observed for the dead mats. It should be noted that higher percentages of Mg_{live mat} and ratios of Mg/Ca_{live mat} were observed in samples collected in media with high Mg/Ca ratio (5.2) than in media with low Mg/Ca ratios (2.5 and 1.5). However, this trend was not observed for the dead mats or the chemical controls.

Mg²⁺ and Ca²⁺ adsorbed by the cell and EPS

The content of Mg²⁺ adsorbed by live cells per unit mass is higher than that of Ca²⁺ in all strains (Figs. 3(a), 3(b), 3(c), and 3(d)), yet the reversed phenomenon was observed for EPS (Figs. 3(e), 3(f), 3(g), and 3(h)). The mole ratios of Mg²⁺ to Ca²⁺ adsorbed on the cell are generally above 1.0 (Figs. 3(j), 3(k), 3(l)) and higher than that of the original medium (values were above the dotted lines), with only one exception (Fig. 3(m)). In contrast, nearly all values of Mg/Ca adsorbed on EPS are below 1.0 (Figs. 3(n), 3(o), 3(p), and 3(q)) and lower than the original Mg/Ca ratio of the medium. The cell of S. elongatus shows the highest ability in adsorbing Mg²⁺ (1.55–2.85 mmol/g, Fig. 3(a)). Conversely, the EPS of S. elongatus demonstrates the lowest adsorption capacity of Mg²⁺ (<0.20 mmol/g, Fig. 3(e)). The adsorption capacities of Mg²⁺ and Ca²⁺ by the EPS of Staphylococcus sp. (Fig. 3(g)) and Bacillus sp. (Fig. 3(h)) are similar to those of the organic matter in the LB medium (Fig. 3(i)).

Functional groups on the cell surface and EPS of S. elongatus

Carboxyl, phosphoryl, and amine groups are estimated to be the major functional groups of EPS and the cell surface of S. elongatus. EPS is characterized by acidic (carboxyl) and neutral (phosphoryl) functional groups, with a percentage of 83.08% (Table 2). Conversely, the cell surface is dominated by basic (amine) functional groups, and the total percentage of acidic functional groups (carboxyl and phosphoryl) is estimated to be 12.76% (Table 2).

Discussion

The metabolizing cells adsorb more Mg²⁺ than the non-metabolizing cells

In this study, we found that metabolizing cells adsorbed more Mg²⁺ than Ca²⁺. Although few publications reported this phenomenon before, clues could still be drawn from related studies. It was observed that the increase of the Mg²⁺ concentration from 0.001 M to 0.1 M causes the much larger decrease of the electrophoretic mobilities of the two SRB strains than that from Ca²⁺, which indicated the higher affinity of the cell surface to Mg²⁺ than to Ca²⁺ (Van Lith et al., 2003). The Mg/Ca ratios on the cell surfaces were always higher than 1.0, and also higher than that in the initial solution after suspending the metabolizing cells of Methanobacterium formicicum in the solutions with Mg/Ca ratios varying from 1.0 to 5.2 for 30 min (Kenward et al., 2013). This also suggested that the cell surface of live M. formicicum prefers to adsorb Mg²⁺ rather than Ca²⁺, which coincides with the results in this study conducted on S. elongatus, D. vulgaris, and Staphylococcus sp. (Fig. 3).

In fact, metabolizing cells require more Mg²⁺ than Ca²⁺. Mg²⁺ is largely and approximately equally required by both the outside wall and inside protoplast (Clapham, 2007) for stabilizing the cell wall, surface layer, cell membrane, ribosomes, and neutralizing nucleic acids as well as assisting enzymatic reactions as a cofactor (Groisman et al., 2013). In contrast, Ca²⁺ was generally extruded outside the cells to maintain a low concentration of approximately 100 nM on the inside, which was 10,000 times lower than that of the outside (Michiels et al., 2002; Clapham, 2007). The need of low Ca²⁺ concentration in the cytoplasm may require a low density of Ca²⁺ on the cell surface. Consequently, the relatively high ratio of Mg/Ca appeared on the cell, which was consistent with the observation from live microbe mats in this study (Fig. 2). The ease of Ca²⁺ precipitation with phosphate also limited the concentration of Ca²⁺ on the cell surface and inside the cell. The precipitation of Ca₃(PO₄)₂ may be fatal for the integrity and function of the phospholipid bilayer and nucleic acids (Clapham, 2007). This limitation of Ca²⁺ for live cells no longer exists after the death of the cell, which may facilitate the allocation of Ca²⁺ inside the cell, and subsequently causes the relatively low Mg/Ca ratio of the whole dead cell (Fig. 2).

In addition, the proton pump in the metabolizing cells may also facilitate the adsorption of Mg²⁺. Microbes can exude H⁺ out of the lipid bilayer using the proton pump (Kenward et al., 2013), which resisted the deprotonation of some functional groups on the cell surface, especially carboxyl and phosphate. Deprotonated carboxyl (Wang et al., 2009) and phosphate (Lambert et al., 1975a) prefer to bind to Ca²⁺ rather than Mg²⁺. Therefore, it can be proposed that the release of H⁺ to the outside of the cell would reduce the advantage of Ca²⁺ binding with carboxyl. As a result, this may favor the acceleration of Mg²⁺ on the cell surface. When the cells are dead, the advantage of Ca²⁺ binding to the organisms cannot be restrained, resulting in relatively low Mg/Ca ratios.

The functional groups constrain the adsorption of Mg²⁺ and Ca²⁺

Both the microbial cell wall and EPS can serve as the binding sites for cations due to the abundant functional groups on their surface. The carboxyl groups showed the highest capacity for cation binding (Beveridge and Murray, 1980), and phosphate groups were also found to be effective (Sakaguchi and Nakajima, 1982). Bivalent Mg²⁺ and Ca²⁺ are usually competitors for binding with functional groups. However, compared with Mg²⁺, Ca²⁺ was reported as preferable for teichoic acid (Lambert et al., 1975a, b), lipoteichoic acid (Keith and Hogg, 1995), and amino acids (Wang et al., 2009). This indicates that the affinity of carboxyl to Ca²⁺ is higher than to Mg²⁺.

In this study, we demonstrated that carboxyl and phosphoryl were abundant (83.08%) in the EPS of S. elongatus (Table 2), which resulted in higher amounts of absorbed Ca²⁺ than Mg²⁺. In contrast, amine was dominant (87.24%) on the cell surface of S. elongatus (Table 2), which caused higher amounts of adsorbed Mg²⁺ than Ca²⁺. The results of our work are similar to that of Wilson et al. (2001), who found that EPS had more acidic functional groups than basic functional groups, which may account for the large adsorption capacity of Ca²⁺.

Releasing EPS into water facilitates the adsorption of Mg²⁺ by the cell surface

We propose that the allocation of Mg²⁺ and Ca²⁺ in the cell–EPS–water system goes through three stages. In the first stage, cations are freely dispersed in the water with few microbial cells. In the second stage, the proliferation of the microbes coupled with the release of EPS provides chelating agents for the cations in the water. In the third stage, Ca²⁺ is then largely chelated by EPS, resulting in the elevation of the ratio of free Mg²⁺ to Ca²⁺ in the water, which benefits both the chelation of Mg²⁺ on the cells and the maintenance of their high Mg/Ca ratio (Fig. 4).

Microbes commonly secrete EPS and release them into the water during their growth (De Philippis and Vincenzini, 1998). These EPS can serve as a significant Ca²⁺ sink through cation binding due to the large fraction of EPS in the total biomass (Moreno et al., 1998; Goto et al., 1999; Acuña et al., 2006) and abundant acidic functional groups on the EPS surface (Wilson et al., 2001). The binding of Ca²⁺ by EPS will consequently increase the ratio of free Mg²⁺ to free Ca²⁺ in the solution and benefits the binding of Mg²⁺ onto the cell surface and the maintenance of the high Mg/Ca ratio of the cell. Environmental conditions control microbial diversity and the yield of the cell and EPS. The variation of the total biomass (cell and EPS) and the yield ratio of the cell and EPS may alter the ratio of Mg/Ca in the water, especially in closed locations, such as lagoons and lakes during certain periods without sufficient water exchange, or in open areas with heavy microbe blooms, such as a cyanobacterial bloom. The frequent appearance of high Mg-calcite and dolomite in lagoons and salty lakes (McKenzie and Vasconcelos, 2009) may be related to the preferential adsorption of Ca²⁺ by abundant EPS.

Conclusions

During the growth of microbes and microbial mats, Mg²⁺ and Ca²⁺ are adsorbed to the cell surface and dissolved EPS. More Mg²⁺ is chelated on live cells than on lifeless organic matter, regardless of the variation of the Mg/Ca ratio in the solution. The relatively few acidic functional groups located on the cell surface compared to EPS and the transport of H⁺ to the outer membrane of the live cell are the potential reasons for the larger adsorption capacities of Mg²⁺ compared with lifeless organic matter. Our results may aid in understanding the mechanisms of the microbial-mediated formation of Mg-bearing carbonate minerals.

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