Insight into calcification of Synechocystis sp. enhanced by extracellular carbonic anhydrase

Abstract

Bio-calcification, known as microbiologically induced calcium precipitation, is an important process of the global carbon cycle. A large number of microorganisms exhibit this ability, including cyanobacteria. Even though the process was realized a long time ago, the detailed mechanism is still unclear. In this paper, we investigate the key role of extracellular carbonic anhydrase during bio-calcification of Synechocystis sp. FACHB 898. Detailed studies revealed that the precipitation of CaCO₃ was significantly hindered when the function of extracellular carbonic anhydrase in Synechocystis sp. was inhibited. Furthermore, the reduction of calcium concentration in solution was significantly correlated with the reduction of bicarbonate concentration as 1 : 2. The results suggested that extracellular carbonic anhydrase of cyanobacteria enhanced CaCO₃ precipitation from calcium and bicarbonate through facilitating the proton consumption during transformation of bicarbonate to carbon dioxide.

---

1. Introduction

Microbial carbonate mineralization is widespread in nature.^1–4 Photoautotrophic phytoplankton, including cyanobacteria, are found to be involved in CaCO₃ precipitation.^5,6 Due to its importance in geological environments and applications, the process of cyanobacterial calcification has attracted much attention. The precipitation of CaCO₃ by cyanobacteria is considered to play an important role in many geological processes, such as early marine sediments, karst streams, whiting and the relevant global carbon cycle, etc.^5–8 Especially under the concern of global climate change, growing attention has been drawn to the influence of rising CO₂ concentration in atmosphere and ocean acidification on the cyanobacterial calcification.^9,10 Carbonate minerals induced by cyanobacteria also exhibit many potential applications, including restoration of paleontological landscape,^11,12 remediation of heavy metal contaminated soils,¹³ carbon capture and sequestration (CCS),¹⁴ and so on. Even so, literature on its application are still limited compared to calcification of other microorganisms^15–20 and artificial carbonate nanocrystals^21–24 these years. The progress of learning the influence of environmental changes on it and its applications is slow. The main challenge is that the mechanism of cyanobacterial calcification is still unclear.

According to literature, among all the species of cyanobacteria, Synechococcus sp. and Synechocystis sp. are reported to show high potential of calcification.^25,26 These two species are often chosen for investigation of the mechanism of cyanobacterial calcification, and several different mechanisms have been proposed.^27–31 However, the literature on the process are oftentimes bewildering and occasionally controversial, and the mechanisms of cyanobacterial calcification is still unclear.¹ Studies revealed that the cell surface and extracellular polymeric substances (EPS) may promote the precipitation of CaCO₃ by binding calcium ions or providing nucleation sites.^30–34 It is also considered that the cyanobacteria calcification is a process related to elevated pH on the cell surfaces. The increased pH is resulted from bicarbonate assimilation driven by carbonate concentration mechanism (CCM)-enhanced photosynthesis.^35–37 The CCM involves carbon import in the form of CO₂ and HCO₃⁻. During photosynthesis, HCO₃⁻ is transformed into CO₂ by carbonic anhydrase (CA), and then OH⁻ ions generated and are released to increase pH near the cell. At this alkaline pH, saturation state with respect to CaCO₃ increased.^37,38 Nevertheless, most cyanobacteria are equipped with CCM,³⁹ but not all of them show calcifying abilities, especially under marine environment.²¹ Furthermore, some other bacteria without CCM or photosynthesis are also reported showing calcification process, such as sulphate-reducing bacteria,^40–42 methanogenic archaea^43–45 and fungi.³¹ Considering that CA is a kind of ubiquitous enzymes⁴⁶ and the calcification of cyanobacteria is mainly found outside cells, it seems quite possible that the function of extracellular CA in cyanobacteria is important for calcification process. However, rare studies have been reported about it.

In this article, Synechocystis sp. FACHB 898, which showed the highest capability of calcification among four different cyanobacteria species in our previous study, was chosen to investigate whether the extracellular CA plays a role in the process and how it works. The activity of extracellular CA of Synechocystis sp. FACHB 898 could be inhibited with acetazolamide.⁴⁷ By compared differences between the CA-inhibited groups and CA-activated groups, such as cell yield, solution pH, bicarbonate and calcium concentration, morphology of CaCO₃ precipitation and relations about changes among these parameters, the extracellular CA was proved to play a role of enhancing the calcification process. The mechanism was then proposed that extracellular CA of cyanobacteria enhanced CaCO₃ precipitation from calcium and bicarbonate through facilitating the proton consumption during transformation of bicarbonate to carbon dioxide.

2. Experimental section

2.1. Synechocystis strain and culture conditions

Synechocystis sp. FACHB 898 was chosen to study the role of the extracellular CA in the process of cyanobacterial calcification. The cells were received from Freshwater Algae Culture Collection at Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) (FACHB-Collection) and cultured in BG11 medium (20 g L⁻¹ Na₂CO₃, 1.5 g L⁻¹ NaNO₃, 40 mg L⁻¹ K₂HPO₄, 75 mg L⁻¹ MgSO₄·7H₂O, 36 mg L⁻¹ CaCl₂·2H₂O, 6 mg L⁻¹ citric acid, 6 mg L⁻¹ ferric ammonium citrate, 1 mg L⁻¹ EDTA–Na_2, 2.86 mg L⁻¹ H₃BO₃, 1.86 mg L⁻¹ MnCl₂·4H₂O, 0.22 mg L⁻¹ ZnSO₄·7H₂O, 0.39 mg L⁻¹ Na₂MoO₄·2H₂O, 0.08 mg L⁻¹ CuSO₄·5H₂O and 0.05 mg L⁻¹ Co(NO₃)₂·6H₂O).³⁰ Cultures were maintained at 25 °C, under a photon irradiance of 6000 lux with a 12 h light/dark cycle. Before calcification experiments, cells were cultivated in Z/10 culture medium (168 mg L⁻¹ NaHCO₃, 46.7 mg L⁻¹ NaNO₃, 11.45 mg L⁻¹ Na–EDTA, 5.9 mg L⁻¹ Ca(NO₃)₂·4H₂O, 4.1 mg L⁻¹ K₂HPO₄·3H₂O, 3 mg L⁻¹ FeSO₄·7H₂O, 2.5 mg L⁻¹ MgSO₄·7H₂O, 248 μg L⁻¹ H₃BO₃, 135 μg L⁻¹ MnSO₄·H₂O, 7.2 μg L⁻¹ (NH₄)₆Mo₇O₂₄·4H₂O, 23.2 μg L⁻¹ ZnSO₄·7H₂O, 12 μg L⁻¹, Co(NO₃)₂·6H₂O and 10.4 μg L⁻¹ CuSO₄·5H₂O) to adapt to the oligotrophic condition of calcification experiments.³⁰

2.2. Calcification experiments

Calcification experiments were performed in clear glass serum bottles (120 mL) containing 40 mL solution with butyl rubber stopper secured. The solution contained NaHCO₃, 5.0 mM CaCl₂ and 1–10 × 10⁵ cells mL⁻¹. The initial pH was adjusted to 8.0, which is the optimum pH for cell growth and conducive to minimizing the impact of physicochemical precipitation of CaCO₃, with 1 M HCl or NaOH.⁴⁸ The headspace of bottles was purged with air from gas cylinder. During calcification experiments, the bottles were incubated at 25 °C under continuous illumination of 6000 lux and shaken at moderate intensity with HY-2 Shaker (Guohua, China). All the experiments lasted for 100 h. During the period, duplicate samples were taken for analyzing the cell yield, solution pH, alkalinity, calcium concentration and morphology of CaCO₃ periodically. First of all, solution pH was measured, and cell yield was measured with 5 mL solution. Then cells and precipitates were filtered onto 0.22 μm polycarbonate membrane for SEM. And filtrate through 0.45 μm polyvinyl acetate fiber membrane was collected for calcium concentration and alkalinity. Four millimeter of the filtrate was added 1% HNO₃ for calcium concentration.

Before exploring the role of extracellular CA in the process, suitable concentration range of NaHCO₃ should be screened to minimize the influence of physicochemical precipitation of CaCO₃ as far as possible. The concentration of NaHCO₃ was set as 0, 2.0, 4.0, 6.0 and 8.0 mM. Similar experiments without cyanobacteria were set as the controls.

After determining the range of NaHCO₃ concentration, calcification experiments with extracellular CA activated (Group A) and inhibited (Group B) were conducted. In Group B, 1.0 mM acetazolamide (AZ, an inhibitor of extracellular CA) was added into the solution. The other conditions and operation were the same.

2.3. Analytical methods

During the experiments, cell yield was expressed as solution absorbance measured with a spectrophotometer (722-2000, Caihong, China) at 440 nm. The pH was monitored with a pH meter (Multi 3420, WTW, Germany). Cells and precipitates filtered onto 0.22 μm polycarbonate membrane were observed with scanning electron microscope (SEM) (Apollo-300, Camscan, UK). Calcium concentration was analyzed with inductively coupled plasma optical emission spectrometer (ICP-OES) (IRIS Intrepid II, Thermo, USA). And alkalinity was measured by acid titration (0.01 M HCl). Then the HCO₃⁻ concentration was calculated with solution pH and carbonate equilibrium constants.

After collecting all these parameters, correlation analysis was conducted between the reduction of calcium concentration and other parameters with IBM SPSS Statistics 19, including cell yield, pH, alkalinity and the reduction of HCO₃⁻ concentration. When p < 0.05, parameters were considered to be relevant and the Pearson correlation coefficient was expressed as R.

3. Results and discussion

3.1. Screening range of NaHCO₃ concentration

In order to screen the suitable range of NaHCO₃ concentration, calcification experiments with (experimental groups) and without cells (controls) were carried out. The results showed that cells grew well when NaHCO₃ concentration ranged from 2.0 to 8.0 mM, but grew poorly without NaHCO₃ existed. The degree of CaCO₃ precipitation was indicated with the reduction percentage of calcium concentration, expressed as R_perc(Ca²⁺), which was displayed in Fig. 1.

Fig. 1 Calcium removal from solution at 100 h with different initial HCO₃⁻ concentration. R_perc(Ca²⁺)-reduction percentage of calcium concentration; hollow columns – without cells; shadow columns – with cells.

From Fig. 1, it was clear that the existence of cells could promote calcium precipitation. Without HCO₃⁻, few amount of calcium was precipitated, 5.7% in experimental groups and 0.5% in controls. In experimental groups with HCO₃⁻ concentration ranged from 2.0 to 8.0 mM, R_perc(Ca²⁺) was about 33.7–43.1%. In controls, R_perc(Ca²⁺) with 2.0 and 4.0 mM HCO₃⁻ concentration reached 1.7% and 5.2%, respectively, but increased to 20% with 6.0 mM HCO₃⁻. The results showed that, along with the increase of HCO₃⁻ concentration, calcium precipitation improved. When HCO₃⁻ concentration exceeded 6.0 mM, physicochemical precipitation of CaCO₃ improved significantly.

So in order to ensure cells in good growth condition and also reduce the influence of physicochemical precipitation of CaCO₃, HCO₃⁻ concentration was chosen as 1.0, 2.0 and 4.0 mM in following experiments about the influence of extracellular CA on calcification of Synechocystis sp. FACHB 898.

3.2. Influence of extracellular carbonic anhydrase on calcification of Synechocystis sp. FACHB 898

With the existence of cells, calcification experiments were undertaken with extracellular CA activated (Group A) and inhibited (Group B). Fig. 2 shows the changes of cell yield during experiments, expressed as ln(OD/OD₀), which is logarithm of specific value of absorbance at different sampling time and initial absorbance under 440 nm.

Fig. 2 Cell yield. ln(OD/OD₀) – logarithm of specific value of absorbance in different sampling time (OD) and initial absorbance (OD₀); A – extracellular CA was activated; B – extracellular CA was inhibited; 1, 2 and 3 – HCO₃⁻ concentration was 1.0, 2.0 and 4.0 mM, respectively.

From Fig. 2, it was found that, during the whole experimental period, the cell yield of Group B was lower than that of Group A with the same HCO₃⁻ concentration as a whole. In Group A, cell yield started to increase quickly at the very beginning of experiments, and also increased along with increasing HCO₃⁻ concentration. When HCO₃⁻ concentration was 4.0 mM, ln(OD/OD₀) could even reach near 0.6. But in Group B, cell yield started to increase after 1–2 days. With NaHCO₃ concentration increasing, the adaption period of Group B was shorter, and the difference of cell yield between the two groups also shrank during the experiments. It was obvious that the inhibition of extracellular CA also restrained cell growth. It implied that the activity of extracellular CA was very important for cell growth.

As an important factor on the precipitation of CaCO₃,⁴⁹ changes of solution pH of the two groups are showed in Fig. 3. It was interesting to find that the solution pH of Group B decreased at first and then increased slowly, while the pH of Group A increased directly. In Group A, solution pH increased to different levels, which is related to the concentration of HCO₃⁻. The solution pH of A1 reached up to 10.32 in 43 h and then kept steady, while that of A2 reached 10.09 in 60 h. The solution pH of A3, however, increased slowly during the whole experimental period. In Group B, the solution pH of B1 declined to 6.70 in 60 h and then increased slowly. And for B2 and B3, the pH started to increase at 8 h after decreasing to 7.41 and 7.55, respectively. In general, the lower the HCO₃⁻ concentration, the more obvious the difference between the two groups. This could be attributed to the buffering behavior of HCO₃⁻. As HCO₃⁻ concentration could also serve as pH buffer, the difference between the two groups minimized along with HCO₃⁻ concentration increasing.

Fig. 3 Changes in solution pH. A – extracellular CA was activated; B – extracellular CA was inhibited; 1, 2 and 3 – HCO₃⁻ concentration was 1.0, 2.0 and 4.0 mM, respectively.

The process of CaCO₃ precipitation directly involves in the consumption of calcium and inorganic carbon. During the whole period of experiments, the concentration of CO₃²⁻ remained lower than 0.2 mM, and HCO₃⁻ kept being the major species of dissolved inorganic carbon (DIC). Changes in HCO₃⁻ and Ca²⁺ concentrations are showed in Fig. 4.

Fig. 4 Changes in HCO₃⁻ (a) and Ca²⁺ (b) concentrations. A – extracellular CA was activated; B – extracellular CA was inhibited; 1, 2 and 3 – HCO₃⁻ concentration was 1.0, 2.0 and 4.0 mM, respectively.

It is found that HCO₃⁻ concentration of Group A all decreased significantly to lower than 0.5 mM at the end of experiments, while the HCO₃⁻ concentration of Group B showed slight changes. In Group B, the HCO₃⁻ concentration of B2 and B4 decreased from initial values of 2.0 and 4.0 mM to 1.71 and 3.39 mM, respectively, which might be caused by the carbonate equilibrium and accumulated proton which was discussed in Section 3.4. And the HCO₃⁻ concentration of B1 was nearly unchanged. These results suggested that with the existence of AZ, the assimilation of HCO₃⁻ by Synechocystis sp. was almost completely inhibited.

In addition, the changes in Ca²⁺ concentration showed similar tendency. The reduction of Ca²⁺ concentration of Group A was much higher than that of Group B. Calcium concentration of Group A with 1.0, 2.0 and 4.0 mM HCO₃⁻ decreased from the initial value of 5.0 mM to 4.34, 3.89 and 3.14 mM, respectively. But that of Group B decreased to 4.54, 4.38 and 4.33 mM, respectively. Other than that, the reduction of Ca²⁺ concentration increased for both the two groups with HCO₃⁻ concentration increasing. This proved that the increase of HCO₃⁻ concentration was beneficial to calcification, and the activity of extracellular CA promoted the process greatly.

To further determine the differences between the two groups in CaCO₃ precipitation, SEM was applied to observe the amount, size and morphology of CaCO₃ precipitates. The typical SEM images of CaCO₃ of A2 and A3 at 44 h and B2 and B3 at 42 h, respectively, are shown in Fig. 5.

Fig. 5 Morphology of CaCO₃. (a) and (b) – CaCO₃ precipitates at 44 h of experiments with extracellular CA of cells was activated when there was 2.0 and 4.0 mM HCO₃⁻ concentration in solution, respectively; (c) and (d) – CaCO₃ precipitates at 42 h of experiments with extracellular CA of cells was inhibited when there was 2.0 and 4.0 mM HCO₃⁻ concentration in solution, respectively; A – extracellular CA was activated; B – extracellular CA was inhibited.

In Group A, CaCO₃ precipitates with an average diameter of 1 μm were easily observed at 20 h (not shown here). It was interesting to find that many of them were aggregated together to form big clusters (>10 μm) but still with a clear trigonal outline. By contrast, in Group B, only a few of CaCO₃ precipitates were observed. And the precipitates of Group B were in irregular shape and much smaller compared to that in Group A. Based on the changes of calcium concentration and SEM images of CaCO₃ precipitates, it could be confirmed that CaCO₃ precipitation of Group A was much more significant than that of Group B.

3.3. Enhanced behavior of extracellular carbonic anhydrase in cyanobacterial calcification

The results showed that the inhibition of extracellular CA not only hindered HCO₃⁻ consumption, but also reduced cell yield, solution pH and precipitation of CaCO₃. To further understand the role of CA in the calcification process of Synechocystis sp. FACHB 898, correlation analysis between the reduction of Ca²⁺ concentration, expressed as R(Ca²⁺), and other parameters was conducted. It was found that the reduction of HCO₃⁻ concentration, expressed as R(HCO₃⁻), and cell yield showed better correlation with R(Ca²⁺) in both groups (p < 0.05). In Group A, R² between R(Ca²⁺) and R(HCO₃⁻) was 0.962, and R² between R(Ca²⁺) and cell yield was 0.882. But that in Group B decreased to only 0.524 and 0.669, respectively. This meant that, along with extracellular CA inhibited, the relations between calcium precipitation and HCO₃⁻ consumption and cell growth both weakened. The correlation between R(Ca²⁺) and R(HCO₃⁻) and cell yield is showed in Fig. 6.

Fig. 6 Correlation between R(Ca²⁺) and R(HCO₃⁻) (a) and cell growth yield (b). R(Ca²⁺) – the reduction of Ca²⁺ concentration; R(HCO₃⁻) – the reduction of HCO₃⁻ concentration; ln(OD/OD₀) – logarithm of specific value of absorbance in different sampling time (OD) and initial absorbance (OD₀); A – extracellular CA was activated; B – extracellular CA was inhibited; 1, 2 and 3 – HCO₃⁻ concentration was 1.0, 2.0 and 4.0 mM, respectively.

In Fig. 6, it was found that the ratio of R(Ca²⁺) and R(HCO₃⁻) was approximately 1 : 2, which implied that the main DIC specie participated in calcification of Synechocystis sp. was HCO₃⁻. While with extracellular CA inhibited, the R² decreased drastically, it suggested that the function of extracellular CA was important not only for HCO₃⁻ consumption and cell growth, but also for calcium precipitation. Therefore, it is deserved to investigate the function of extracellular CA of Synechocystis sp. during the calcification process.

3.4. Mechanism of cyanobacterial calcification enhanced by extracellular CA

All the experimental results and correlation analysis show that extracellular CA can promote the calcification. So it is important to understand the function of CA and how it influences the formation of CaCO₃. As reported, the mechanism of CA could be expressed as follows.⁵⁰

E–Zn²⁺OH⁻ + BH⁺ ↔ H⁺–E–Zn²⁺OH⁻ + B (1)

H⁺–E–Zn²⁺OH⁻ ↔ E–Zn²⁺OH₂ (2)

E–Zn²⁺OH₂ + HCO₃⁻ ↔ E–Zn²⁺OH⁻ + CO₂ + H₂O (3)

BH⁺/B represented a pair of buffer molecules. And the reactive hydroxyl group (OH⁻) on the Zn²⁺ reaction center is the active site of CA, E. The reaction of CA results in spontaneous interconversion between CO₂ and HCO₃⁻, which also accompanies proton transfer.

From the reaction mechanism of CA shown above, it is clear that, in the condition of calcification experiments, HCO₃⁻/CO₃²⁻ might work as BH⁺/B, which provide proton for transformation of HCO₃⁻ into CO₂. It could also be found that the transformation of HCO₃⁻ into CO₂ is a process of assimilating proton. This could explain the changes of pH and cell growth in Group A and Group B. As proton consumed, solution pH in Group A increased. And cell yield increased, which was benefited from photosynthesis with accumulated CO₂. But in Group B, cells could not get enough CO₂ from HCO₃⁻ transformation to proliferate, which resulted in less cell yield than Group A.

Considering that extracellular CA mainly transforms HCO₃⁻ into CO₂ with proton consumption, CaCO₃ precipitation with extracellular CA activated can be explained as:

Ca²⁺ + HCO₃⁻ → CaCO₃ + H⁺ (4)

It is clear that the HCO₃⁻ in eqn (4) plays the role of BH⁺ in eqn (1) which transforms into CO₃²⁻ as B in eqn (1) and exists in the form of CaCO₃. The proton from HCO₃⁻ which finally transforms into CaCO₃ is transferred to another molecule of HCO₃⁻ which finally transforms into CO₂ and is assimilated with photosynthesis of cells. From eqn (4) and (5), it could be found that there is two molecules of HCO₃⁻ consumption with one molecule of CaCO₃ generation. This also accord with the ratio of R(Ca²⁺) and R(HCO₃⁻) as 1 : 2 in the correlation analysis. Besides, it is easy to understand why solution pH decreased in Group B. As there was also a few amount of CaCO₃ formed, the generated proton made solution pH decreased.

To sum up, extracellular CA promotes the process of calcification by assimilating proton and generating CO₂, which results in that solution pH and cell yield both increase. But with extracellular CA inhibited (Group B), solution pH decreases with the generated proton stem from CaCO₃ formation with Ca²⁺ and HCO₃⁻. As HCO₃⁻ plays multiple roles in the process of calcification of Synechocystis sp., including both the buffer and reagent in the reaction of CA and CaCO₃ formation, it is confused to understand the process. For better understanding the process, the main reactions in Group A and Group B is showed in Fig. 7.

Fig. 7 Mechanism of calcification of Synechocystis sp. FACHB 898 enhanced by extracellular CA. (a) Extracellular CA was activated; (b) extracellular CA was inhibited. BCT1 is a HCO₃⁻ transporter in cyanobacteria.⁵¹

From Fig. 7, it could be seen that extracellular CA mainly plays a role of enhancing CaCO₃ precipitation by consuming proton stem from CaCO₃ formation with Ca²⁺ and HCO₃⁻. When extracellular CA is inhibited, there could only form a few amount of CaCO₃ as proton accumulation which inhibits the reaction of CaCO₃ precipitation conducted continuously.

By inhibiting extracellular carbonate anhydrase of Synechocystis sp. and comparing the process of calcification with activated and inhibited extracellular carbonate anhydrase, the differences of cell yield, solution pH, calcium and bicarbonate concentration and morphology of CaCO₃ precipitates are found to be remarkable. Along with analyzing the correlation of the reduction of calcium concentration, which indicated the extent of calcification, and changes of the other parameters, it is exciting to find that the reduction of bicarbonate concentration has the highest correlation with the reduction of calcium concentration, and the ratio is about 2 : 1 with extracellular carbonate anhydrase activated. Combined with the mechanism of carbonate anhydrase, the mechanism of cyanobacterial calcification from calcium and bicarbonate is conducted to be enhanced by extracellular carbonic anhydrase of cyanobacteria through facilitating the proton consumption during transformation of bicarbonate to carbon dioxide.

4. Conclusion

The results demonstrated that CaCO₃ precipitation by Synechocystis sp. FACHB 898 decreased obviously when extracellular CA was inhibited. In the CA-inhibited groups, cells grew slowly, solution pH decreased, and calcium concentration decreased a little. The CaCO₃ precipitates were hard to be found and showed irregular shape. When the extracellular CA was activated, cells grew well, solution pH increased, and calcium concentration decreased dramatically. The CaCO₃ precipitates could be found easily at the very beginning of experiments and showed large, trigonal shape. It indicated that the extracellular CA played an important role in the CaCO₃ precipitation. According to the correlation analysis and the reaction mechanism of CA, the mechanism of calcification of Synechocystis sp. FACHB 898 is explored. The ratio between the reduction of calcium and bicarbonate concentration was 1 : 2, indicating that the bicarbonate might be the main carbon source to form CaCO₃. According to the extracellular CA mechanism, the transformation of HCO₃⁻ into CO₂ is a process of proton consumption. Thus, extracellular carbonic anhydrase is conducted to enhance the process of CaCO₃ precipitation by consuming proton released from the transformation of bicarbonate to carbonate, which finally existed in the form of CaCO₃. This mechanism could also provide a new view for calcification of other organisms and their applications.

Acknowledgements

The authors gratefully acknowledge the financial support from the project supported by National Natural Science Foundation of China (Grant No. 51178019, 51373005), National Key Basic Research Program of China (2014CB931800) and Fundamental Research Funds for the Central Universities.

Notes and references

1. N. A. Kamennaya, C. M. Ajo-Franklin, T. Northen and C. Jansson, Minerals, 2012, 2, 338–364.
2. J. Chen and J. Lee, Acta Geol. Sin., 2014, 88, 260–275.
3. C. Dupraz, R. Reid, O. Braissant, A. W. Decho, R. S. Norman and P. T. Visscher, Earth-Sci. Rev., 2009, 96, 141–162.
4. C. R. Miller and N. P. James, J. Sediment. Res., 2012, 82, 633–647.
5. N. Brinkmann, L. Hodač, K. I. Mohr, A. Hodačová, R. Jahn, J. Ramm, C. Hallmann, G. Arp and T. Friedl, Geomicrobiol. J., 2015, 32, 255–274.
6. A. Bissett, A. Reimer, D. de Beer, F. Shiraishi and G. Arp, Appl. Environ. Microbiol., 2008, 74, 6306–6312.
7. N. Planavsky, R. P. Reid, T. W. Lyons, K. L. Myshrall and A. T. Visscher, Geobiology, 2009, 7, 566–576.
8. E. Couradeau, K. Benzerara, E. Gérard, I. Estève, D. Moreira, R. Tavera and P. López-García, Biogeosciences, 2013, 10, 5255–5266.
9. M. Koch, G. Bowes, C. Ross and X. H. Zhang, Global Change Biol., 2013, 19, 103–132.
10. R. G. Zepp, D. J. Erickson, N. D. Paulc and B. Sulzbergerd, Photochem. Photobiol. Sci., 2011, 10, 261–279.
11. E. Couradeau, K. Benzerara, E. Gérard, D. Moreira, S. Bernard, G. E. Brown Jr and P. López-García, Science, 2012, 336, 459–462.
12. K. Benzerara, F. Skouri-Panet, J. Li, C. Férard, M. Gugger, T. Laurent, E. Couradeau, M. Ragon, J. Cosmidis, N. Menguy, I. Margaret-Oliver, R. Tavera, P. López-García and D. Moreira, Proc. Natl. Acad. Sci. U. S. A., 2014, 111, 10933–10938.
13. C. Zhao, Q. Fu, W. Song, D. Zhang, J. Ahati, X. Pan, F. A. Al-Misned and M. G. Mortuza, Ecol. Eng., 2015, 81, 107–114.
14. C. Jansson and T. Northen, Curr. Opin. Biotechnol., 2010, 21, 365–371.
15. V. Merk, M. Chanana, T. Keplinger, S. Gaan and I. Burgert, Green Chem., 2015, 17, 1423–1428.
16. V. Achal, X. L. Pan and D. Y. Zhang, Ecol. Eng., 2011, 3, 1601–1605.
17. V. Achal, X. L. Pan and D. Y. Zhang, Chemosphere, 2012, 89, 764–768.
18. V. Achal, X. L. Pan, D. Y. Zhang and Q. L. Fu, J. Microbiol. Biotechnol., 2012, 22, 244–247.
19. W. E. G. Muller, M. Neufurth, U. Schlossmacher, H. C. Schroder, D. Pisignano and X. Wang, RSC Adv., 2014, 4, 2577–2585.
20. P. C. Sahoo, F. Kausar, J. H. Lee and J. I. Han, RSC Adv., 2014, 4, 32562–32569.
21. S. F. Chen, J. H. Zhu, J. Jiang, G. B. Cai and S. H. Yu, Adv. Mater., 2010, 22, 540–545.
22. H. B. Yao, J. Ge, L. B. Mao, Y. X. Yan and S. H. Yu, Adv. Mater., 2014, 26, 163–188.
23. Y. Boyjoo, V. K. Pareek and J. Liu, J. Mater. Chem. A, 2014, 2, 14270–14288.
24. Y. Boyjoo, K. Merigot, J. F. Lamonier, V. K. Paree, M. O. Tade and J. Liu, RSC Adv., 2015, 5, 24872–24876.
25. B. D. Lee, W. A. Apel and M. R. Walton, Biotechnol. Prog., 2004, 20, 1345–1351.
26. M. Dittrich, P. Kurz and B. Wehrli, Geomicrobiol. J., 2004, 21, 45–53.
27. M. U. E. Merz, Facies, 1992, 26, 81–102.
28. T. A. McConnaughey and J. F. Whelan, Earth-Sci. Rev., 1997, 42, 95–117.
29. R. E. Martinez, E. E. Gardés, O. S. Pokrovsky, J. Schott and E. H. Oelkers, Geochim. Cosmochim. Acta, 2010, 74, 1329–1337.
30. M. Obst, B. Wehrli and M. Dittrich, Geobiology, 2009, 7, 324–347.
31. K. Yasumoto, M. Yasumoto-Hirose, J. Yasumoto, R. Murata, S. M. Baba, K. Mori-Yasumoto, M. Jimbo, Y. Oshima, T. Kusumi and S. Watabe, Mar. Biotechnol., 2014, 16, 465–474.
32. B. Zippel and T. R. Neu, Appl. Environ. Microbiol., 2011, 77, 505–516.
33. A. Liang, C. Paulo, Y. Zhu and M. Dittrich, Colloids Surf., B, 2013, 111, 600–608.
34. D. Ionescu, S. Spitzer, A. Reimer, D. Schneider, R. Daniel, J. Reitner, D. de Beer and G. Arp, Geobiology, 2015, 13, 170–180.
35. F. Shiraishi, T. Okumura, Y. Takahashi and A. Kano, Geochim. Cosmochim. Acta, 2010, 74, 5289–5304.
36. G. Arp, A. Reimer and J. Reitner, Science, 2001, 292, 1701–1704.
37. Y. Xu, M. Zhang, T. Tian, Y. Shang, Z. Meng, J. Jiang, J. Zhai and Y. Wang, NPG Asia Mater., 2015, 7, e215.
38. H. B. Jiang, H. M. Cheng, K. S. Gao and B. S. Qiu, Appl. Environ. Microbiol., 2013, 79, 4048–4055.
39. J. A. Raven, M. Giordano, J. Beardall and S. C. Maberly, Philos. Trans. R. Soc., B, 2012, 367, 493–507.
40. R. P. Reid, P. T. Visscher, A. W. Decho, J. F. Stolz, B. M. Bebout, C. Dupraz, I. G. Macintyre, H. W. Paerl, J. L. Pinckney, L. Prufert-Bebout, T. F. Steppe and D. J. DesMarais, Nature, 2000, 406, 989–992.
41. W. De Muynck, N. De Belie and W. Verstraete, Ecol. Eng., 2010, 36, 118–136.
42. T. R. R. Bontognali, C. Vasconcelos, R. J. Warthmann, S. M. Bernascon, C. Dupraz, C. J. Strohmenger and J. A. Mckenzie, Sedimentology, 2010, 57, 824–844.
43. W. Michaelis, R. Seifert, K. Nauhaus, T. Treude, V. Thiel, M. Blumenberg, K. Knittel, A. Gieseke, K. Peterknecht, T. Pape, A. Boetius, R. Amann, B. B. Jørgensen, F. Widdel, J. Peckmann, N. V. Pimenov and M. B. Gulin, Science, 2002, 297, 1013–1015.
44. G. Arp, G. Helms, K. Karlinska, G. Schumann, A. Reimer and J. Trichet, Geomicrobiol. J., 2012, 29, 29–65.
45. B. P. Burns, F. Goh, M. Allen and B. A. Neilan, Environ. Microbiol., 2004, 6, 1096–1101.
46. S. Tambutté, E. Tambutté, D. Zoccola, N. Caminiti, S. Lotto, A. Moya, D. Allemand and J. Adkins, Mar. Biol., 2007, 151, 71–83.
47. R. Ramanan, K. Kannan, A. Deshkar, R. Yadav and T. Chakrabarti, Bioresour. Technol., 2010, 101, 2616–2622.
48. D. Zhang, X. Pan and J. Zhang, Bull. Mineral., Petrol. Geochem., 2008, 27, 105–111.
49. F. Hammes and W. Verstraete, Rev. Environ. Sci. Bio/Technol., 2002, 1, 3–7.
50. G. D. Price, S. Maeda, T. Omata and M. R. Badger, Funct. Plant Biol., 2002, 29, 131–149.
51. G. D. Price and S. M. Howitt, Biochem. Cell Biol., 2011, 89, 178–188.

---