Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

doi:10.4236/jbnb.2012.34041

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Journal of Biomaterials and Nanobiotechnology, 2012, 3, 413-420

http://dx.doi.org/10.4236/jbnb.2012.34041 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)

413

Formation of Calcium Carbonate Polymorphs Induced by

Living Microalgae

Giulia Santomauro*, Johannes Baier, Wanjing Huang, Stefan Pezold, Joachim Bill

Institute for Materials Science, University of Stuttgart, Stuttgart, Germany.

Email: *[email protected]

Received August 7th, 2012; revised September 17th, 2012; accepted September 26th, 2012

ABSTRACT

Calcium carbonate (CaCO3) occurs in the three polymorphs calcite, aragonite and vaterite. The formation of these crys-

tals in inorganic solutions is influenced by parameters like pH, temperature or impurities. Living freshwater microalgae

can also induce the formation of CaCO3 when they live in a suitable environment containing saturated amounts of Ca2+.

Through this biologically induced biomineralization only the formation of the polymorph calcite has been reported yet.

We investigated the precipitates which have been formed in solutions containing the freshwater microalgae Scenedes-

mus obliquus and different zinc amounts (0, 3.27 and 6.53 mg Zn2+/l) by XRD and SEM. As references precipitates

from the same solutions but without algae were investigated. We could show that the presence of living microalgae has

a great influence on the precipitation of calcium carbonate crystals. In algae-containing media without or with a low

zinc amount always calcite and aragonite are formed. In the corresponding medium with 6.53 mg Zn2+/l pure aragonite

crystals were built. In contrast, in the inorganic, algae-free solutions without zinc, pure calcite is precipitated. Both in-

organic solutions with zinc show major calcite precipitation and weak aragonite precipitation. Thus the algae cells ad-

vance significantly the formation of aragonite, which is enhanced by the presence of zinc cations in the media. Possible

mechanisms are discussed.

Keywords: CaCO3 Polymorphs; Biomineralization; Zinc; Microalgae; XRD

1. Introduction

The formation of the three calcium carbonate (CaCO3)

polymorphs calcite, aragonite and vaterite in inorganic

solutions has been the topic of many studies [1-4]. It was

proposed that first an unstable amorphous phase is built

which transforms to the metastable phase vaterite or ara-

gonite, followed by the transformation to the stable phase

calcite [1]. The precipitation of the polymorphs can be

affected by different factors like temperature, pH of the

medium, concentration ratio of individual components,

supersaturation, ionic strength or impurities [3]. The in-

vestigations of Ogino et al. (1987) [1] revealed that ara-

gonite formation is favored at higher temperatures

(>40˚C) in aqueous solution, which has also been sup-

ported in other experiments [4]. At room temperature, the

pH of the solution in a constant-composition environ-

ment is the most important factor for the formation of the

polymorphs [3]. At 24˚C, vaterite is the major product in

the pH range between 8.5 and 10. Aragonite preferably

formed at pH 11; when the pH is higher than 12, calcite

is the dominant product. The precipitation of the different

polymorphs can also be modified by impurities in the

solution. When the ionic radius of the impurities is

smaller than that of the Ca2+ cations, what is the case for

Zn2+ cations [5], aragonite is deposited [6]. This influ-

ence is explained by adsorption phenomena on the crys-

talline faces of the nuclei. Kitano et al. (1976) [7] exam-

ined specifically the adsorption behavior of Zn2+ on

CaCO3 polymorphs in aqueous solutions. They showed

that Zn2+ is strongly adsorbed on aragonite crystals in

comparison to other polymorphs. It was assumed that the

zinc ions inhibit the transformation of aragonite to calcite

[1,7]. The Zn2+ adsorption on the calcite surface occurs

via an exchange of Ca2+ in a surface-adsorbed layer [8].

Also organic additives show an effect on the minerali-

zation of calcium carbonate (reviews in: Meldrum, 2003

[9]; Ren et al., 2011 [10]). Amino acids like glycine,

aspartic acid and glutamic acid or polysaccharides like

cellulose influence the precipitation and morphology of

calcite and vaterite [11-13].

Living organisms like photosynthetic microalgae can

induce the precipitation of CaCO3 through biominerali-

zation. It is known that many freshwater algae build cal-

cite crystals when they live in a supersaturated environ-

ment regarding calcium [14,15]. This is a result of the

*Corresponding author.

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

414

influence of the algae’s metabolism on their aqueous

environment [16]. Through photosynthesis the algae as-

similate CO2 and/or 3, and release OH− [16,17].

This leads to an increase of the pH and the concentration

of carbonate anions within the solution. It is also largely

accepted that the pH affects both number of cell surface

binding sites and metal chemistry within aqueous solu-

tion: in the acidic range, the functional groups of the cell

wall are closely protonated, which limits the approach of

metal cations. The cell walls of green algae consist of

polysaccharides and proteins [18,19]. With increasing pH,

more functional groups, such as carboxyl or amino

groups, are exposed, which increases the biosorption of

metal ions on the negatively charged cell walls. In the

first step, Ca2+ is bound on the cell wall, and afterwards

anions are accumulated and CaCO3 is precipitated

extracellular. This mechanism is named biologically in-

duced [16], because the calcification is not actively con-

trolled by the organisms. In the biologically controlled

biomineralization, different types of biominerals are pro-

duced under the genetic control of the organisms. Up to

now, in the presence of freshwater microalgae only the

extracellular biologically induced formation of calcite is

reported.

HCO

CO 

Positively charged metal ions can also be bound on the

cell surfaces of living organisms [e.g. 20-27]. Zinc is an

essential trace metal for all organisms, but can be toxic

when present in high amounts. Exposure of microalgae to

sublethal Zn2+ concentrations caused inhibition of e.g.

photosynthesis [28]. When the algae are confronted to

toxic elements like Zn2+ in their environment, they de-

toxify it by taking up the ions. The uptake is split in two

steps: the first is a rapid biosorption onto the cell wall,

during the second step the ions are transported into the

cells [29]. Subsequently, the interior of the cells can be

detoxified by chelating the Zn2+ cations to e.g. nontoxic

zinc-phosphate-based nano needles [30].

The aim of this study was to investigate the role of

living algae on the precipitation of different polymorphs

of calcium carbonate. For the experiments the unicellular

alga species Scenedesmus obliquus (Chlorophyta) was

cultivated in media with and without Zn2+ and the results

were compared to the inorganic media without algae.

This alga species was chosen because preliminary ex-

periments have shown that it calcifies readily when cul-

tured in supersaturated media regarding Ca2+.

2. Materials and Methods

2.1. Organisms and Cultures

The experiments were conducted with living Scenedes-

mus obliquus (strain 276-1), obtained from the SAG cul-

ture collection Göttingen. The algae were cultivated in a

modified calcification medium [17], where Na2EDTA,

KH2PO4, (NH4)Mo7O24 and MnCl2 were omitted and

FeCl3 was reduced to 0.01 mg/l to prevent chelate forma-

tion with Zn2+. The solution was saturated regarding Ca2+.

The media contained different amounts of zinc (0 mg

Zn2+/l, 3.27 mg Zn2+/l or 6.53 mg Zn2+/l), added as

ZnSO4·7 H2O (Roth, Karlsruhe). Preparation of the me-

dia: all stock solutions beside Na2CO3 were sterilized

(autoclave Systec V-75) and combined, the pH was ad-

justed to 6.3. The algae were washed in demineralized

water (Millipore), counted with a hemocytometer (Ma-

rienfeld, Lauda-Königshofen) to ensure the same cell

concentration in all cultures and added to the medium

(about 4.0 × 108 cells/l). The culture was held in a rotary

shaker (Infors HT Multitron II, 100 rpm) at 26˚C and

permanent illumination (FL tubes Gro-Lux 15W, 3500

lx). Due to photosynthesis, the pH increased to a value

over 9 during several days. Not till then the Na2CO3 was

added to prevent loss of anions and the pH was

adjusted to 8.5. At this point the experiment started. The

inorganic, algae-free solution was prepared identically;

the pH was adjusted daily according to the organic, al-

gae-containing solution with 0.1 M NaOH. The duration

of the experiment was 72 h. After this time all cultures

were living as revealed by their green color.

CO 

2.2. SEM Measurement

Precipitation of crystals was investigated after 2, 4, 6, 24

and 72 h duration of the experiment. For observation in a

scanning electron microscope (Zeiss DSM 982 GEMINI)

the specimens were washed in demineralized water (Mil-

lipore) using a glass vacuum filter system with 0.2 µm/2

µm pore size filter membrane (Millipore) and then

air-dried. To obtain electron conductivity, the mem-

branes were coated with graphite.

2.3. Powder X-Ray Diffractiometry (XRD)

XRD was used to identify the CaCO3 polymorphs after

72 h duration of the experiment. The XRD patterns were

recorded with a D500 diffractometer (Siemens). Meas-

urements were made in Bragg-Brentano geometry, using

CuKα1 radiation (X-ray tubes setting of 30 mA and 40

kV) employing a primary-beam Johansson monochro-

mator in a 2Θ range of 20˚ to 50˚.

3. Results & Discussion

3.1. pH Trend

At the beginning of all experiments the pH of each cul-

ture was adjusted to 8.5. Due to photosynthesis of the

algae, the pH rose up to 10.8 after 48 h in the culture

without zinc; afterwards it stayed nearly constant (Figure

1). In presence of zinc, the pH-shift was retarded and did

not reach the high values of the culture without zinc. This

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

415

is due to the fact, that Zn2+ cations restrain photosynthe-

sis of algae. The pH-shift is correlated to Zn2+ concentra-

tion within the solution: the culture with 6.53 mg Zn2+/l

only reached pH 10.1 after 72 h, the culture with 3.27 mg

Zn2+/l reached pH 10.5 after 48 h and remained at this

value. According to the observed pH values in cultures

containing algae, the pH in the media without algae were

adjusted daily with a 0.1 M NaOH solution.

in the cultures without zinc (Figure 2(b)). In the next

hours the crystals grew bigger (data not shown). In the

cultures with zinc, no crystallization occurred during the

first 6 h (data not shown).

After 24 h of incubation, in all solutions precipitates

could be found (data not shown). In the media with algae

and with zinc, big crystals occurred with diameters of

about 50 µm. Always many algae were embedded in

these crystals. In the inorganic solutions with Zn2+, only

very small precipitates were found (Ø below 100 nm;

REM image, data not shown).

3.2. Precipitation of Crystals and Identification

of Polymorphs

After 72 h of incubation, in all solutions crystals were

found which differed greatly in their dimensions and

morphology (Figure 3). The precipitates formed in the

organic solutions contained several (Figures 3(a) and (c))

or many algal cells (Figure 3(b)). The largest precipitate,

where always many algal cells were incorporated, were

The formation of precipitates was investigated at the

starting point of the experiments and after 2 h, 4 h, 6 h,

24 h and 72 h in solutions with algae.

At the starting point of the experiments, there was no

precipitation on the algae (Figure 2(a)). After 2 h of in-

cubation, crystals with embedded algae could be detected

8.5

9.0

9.5

10.0

10.5

11.0

0 24487

time [h]

w ithout Zn

with 3.27 mg Zn/l

with 6.53 mg Zn/l

wit h ou t Zn

with 3.27 mg Zn/l

with 6.53 mg Zn/l

Figure 1. pH shift in the cultures with algae and different zinc amounts during the experimental time of 72 h.

(a) (b)

Figure 2. SEM images. (a) Algae (black arrows) without precipitates at the beginning of the experiment; (b) Crystals (white

arrow) with embedded algae were built afte r 2 h in the medium without zinc.

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

416

(a) (b) (c)

(d) (e) (f)

Figure 3. Crystals after 72 h of incubation. (a)-(c) Cultures with algae and different amounts of zinc: (a) Without zinc; (b)

With 3.27 mg Zn2+/l; (c) With 6.53 mg Zn2+/l. (d)-(f) Inorganic solutions with different amounts of zinc: (d) Without zinc; (e)

With 3.27 mg Zn2+/l; (f) With 6.53 mg Zn2+/l. Black arrows—algae, white arrows—crystals.

found in the organic solution with 3.27 mg Zn2+/l (Fig-

ure 3(b)).

These precipitates appear in the SEM images as big

single-crystals, but could also been formed as many

smaller crystals and afterwards baked together. In the

inorganic solution with zinc, two different crystal types

were found (Figures 3(e) and (f)). Due to the crystal

morphology we assume that the needle-like crystals refer

to aragonite and the rhombohedric to calcite [3].

The crystals found after 72 h in the experimental solu-

tions were washed and then analyzed regarding their

modification by XRD. Representative XRD diagrams are

shown in Figures 4 and 5. In the case of solutions with-

out algae cells the dominant (104) reflection of calcite

(JCPDS card No. 01-072-1650) can be found in all sam-

ples at 29.5˚ (3.02 Å) (Figure 4). Furthermore, the

weaker (012)-, (110)-, (113)-, (202)-, (024)-, (018)-, and

(116)-reflection of calcite are present in the sample,

which contains no Zn2+ cations within solution. In former

studies about precipitation in inorganic solutions, vaterite

was found to be the major product in the pH range be-

tween 8.5 and 10 and at 24˚C [3]. In these experiments,

the experimental solution only contained calcium chlo-

ride and sodium carbonate. In our studies, a culture me-

dium for algae with a more complex composition was

used for all experiments, also for preparing the solutions

without algae. This was probably the reason, why calcite

was built and not vaterite.

In the inorganic sample which has been formed in me-

dium with 3.27 mg Zn2+/l weak intensities (near the de-

tection limit) can also be detected at 26.3˚, 27.2˚, and

46.0˚ which can be attributed to the characteristic (111)-,

(021)- and (221)-reflections of aragonite (JCPDS card

No. 00-003-1067). The reflections of aragonite correspond

with SEM observation of a small amount of a second

acerous phase (cf. Figure 3(e)). The precipitate of the

sample, which has the highest amount of Zn2+ cations

within the media, shows only the dominant (104) reflec-

tion of calcite. A small amount of aragonite crystals

could be observed in this medium by SEM observations

(Figure 3(f)), which is below of detection limit of XRD.

The presence of algae cells has a significant influence

on the CaCO3 precipitation within media in comparison

to media without algal cells, which is demonstrated in

Figure 5. In the case of the Zn2+ free media the precipi-

tate is dominated by the reflections of calcite, but small

intensities of aragonite are also present (Please note that

the strong (104) reflection of calcite at about 29.5˚ is cut

at about 1600 a.u. within Figure 5(a)). With increasing

zinc concentration in the solutions the amount of calcite

decreases dramatically. In fact, in the case of media with

6.53 mg Zn2+/l no calcite reflections can be determined

within the corresponding XRD diagram. Only the reflec-

tions of aragonite are presen(Figure 5(c)). Thus, the t

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae 417

(a)

(b)

(c)

Figure 4. XRD diagrams of precipitates in absence of algae (72 h of incubation). (a) Without zinc; (b) With 3.27 mg Zn2+/l; (c)

With 6.53 mg Zn2+/l.

(a)

(b)

(c)

Figure 5. XRD diagrams of precipitates in presence of algae (72 h of incubation). (a) Without zinc; (b) With 3.27 mg Zn2+/l; (c)

With 6.53 mg Zn2+/l.

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae

418

algae cells advance significantly the formation of arago-

nite, which is enhanced by the presence of zinc ions in

the media.

The algae function obviously as heterogeneous nuclea-

tion sites. Since only crystals associated with algae have

been found in the solutions, the cell walls are evidently

directly involved in nucleation. The binding sites of the

cell walls have a great affinity for Ca2+ cations, the

bound Ca2+ attract anions like [16]. Our prelimi-

nary experiments have shown that Scenedesmus obliquus

does not calcify when cultivated in the dark, and the pH

reaches only the maximum of 9. This was also observed

by other authors with different microalga species [17,31].

This shows that the algae do not only act as nucleation

sites for CaCO3 precipitation but are actively involved

through light-dependent photosynthesis and thus alkali-

zation of the medium. The increase of pH was measur-

able in the bulk solution but was probably higher close to

the cells. This was also supposed by other authors

[14,17]. In presence of zinc in the medium the aragonite

modification is favored. In this case, obviously the trans-

formation into the thermodynamically stable calcite mo-

dification is hindered. A possible mechanism that occurs

in micro-environments created by the cells is suggested

in Figure 6. A part of the absorbed Ca2+ on the cell wall

can be exchanged by Zn2+ [18]. The interaction of Zn2+

with the cell wall enables a local enrichment of the ions

which are supposed to stabilize the metastable aragonite

modification [7] and thus prevents its transformation into

thermodynamically stable calcite. Furthermore, Zn2+

ions restrain photosynthesis [28], which is connected

with a lower pH within the micro-environments com-

pared to the algae-containing zinc-free medium. As

mentioned above, the formation of aragonite is favoured

at lower pH compared to the one of calcite [3]. Ac-

cordingly, only aragonite crystals in the media contain-

ing algae and high amounts of zinc are detected. On the

contrary, calcite is produced nearly exclusively in the

CO 

media without algae and high amounts of zinc. Only

traces of aragonite are produced, as revealed by SEM

observations.

4. Conclusion

We compared the precipitates which have been formed in

solutions with and without algae and with and without

different zinc amounts. We could show that the presence

of living microalgae has a great influence on the precipi-

tation of calcium carbonate crystals. Due to photosynthe-

sis, the pH in the algae-containing organic media in-

creased and CaCO3 crystals are precipitated. In organic

media without zinc or with 3.27 mg Zn2+/l always calcite

and aragonite are formed. In the organic media with 6.53

mg Zn2+/l aragonite is obtained even exclusively. In con-

trast, in the inorganic solutions without zinc, pure calcite

is precipitated. Both inorganic solutions with zinc show

major calcite precipitation and weak aragonite precipita-

tion as revealed by XRD measurements and SEM obser-

vations. Thus, the algae cells advance significantly the

formation of aragonite, which is further enhanced by the

presence of zinc cations in the media. The following ef-

fects of algae and zinc in the solution were supposed:

first, as the algae accumulate, a “micro-environment”

around the clusters is formed, where the CaCO3 is pre-

cipitated (Figure 6). When high Zn2+ amounts are pre-

sent in the solution, photosynthesis is inhibited and the

pH in this “micro-environment” is lower compared to the

“micro-environment” around the algae in solution with-

out Zn. The lower pH prefers the building of aragonite

[3]. Second, the algae function as heterogeneous nuclea-

tion sites where in the first step Ca2+ and Zn2+ ions are

absorbed on the negatively charged cell walls and in the

second step anions like are bound (Figure 6).

The presence of Zn2+ leads to the precipitation of arago-

nite [6]. In the “micro-environment” around the cells

more Zn2+ ions are present than in the corresponding in-

CO 

Figure 6. Scheme of possible heterogeneous nucleation of aragonite crystals in presence of algae and zinc. Between several

ggregated cells a “micro-environment” is built leading to a local increase of the pH and Zn2+ ions. a

Formation of Calcium Carbonate Polymorphs Induced by Living Microalgae 419

organic bulk solution, since these ions are attracted from

the cell walls and bound on them. Zn2+ ions inhibit the

transformation from aragonite to calcite [1]. Hence, ara-

gonite is precipitated in the algae-containing media with

high amount of Zn whereas in the corresponding inor-

ganic solution mainly calcite is found.

5. Acknowledgements

We are grateful for financial support provided by the

Deutsche Forschungsgemeinschaft (BI 469/15-1) within

the scope of the project “Biologische Erzeugung von

Oxidkeramiken” (PAK 410). The authors thank M.

Dudek and F. Predel (both MPI-IS) for XRD and SEM

measurements, respectively. Prof. Dr. P. A. van Aken

(MPI-IS) is thanked for providing the SEM.

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