Calcium carbonate precipitation by cyanobacterial polysaccharides - Tufas and Speleothems: Unravelling the Microbial and Physical Controls - page 59

Fig. 6. Scanning electron microscopy images of agarose beads without additions of extracellular polysaccharides
(blank experiments) after CaCO 3 precipitation experiments. (a) Spherical bead after CaCO 3 experiments without EPS.
(b) Close-up of spherical bead.

precipitates was confirmed using X-ray diffraction
analyses by electron microscopy.
The results of this study showed that extra-
cellular polysaccharides of picocyanobacteria ind-
uced the precipitation of calcium carbonate. All
extracellular polysaccharides have a buffering
capacity at pH values from 3-4, in acidic range.
Therefore, acidic polysaccharides are responsible
for calcium carbonate precipitation in our exper-
iments; they comprise L-glutamic and L-aspatic
acids which were shown to be able to nucleate
calcium carbonate (Braissant et al. 2003). Indeed,
L-glutamic acids pK a 's ¼ 2.23, 4.25, 9.67, L-
glutamine pK a 's ¼ 2.23, 4.42 and 9.95, and
L-aspatic acid have pK a 's ¼ 1.99, 3.9 and 10.02
(Liu & Fang 2002).
Stereo-chemical structure in extracellular poly-
saccharides, which is a result of attaching to solid
surfaces, has been suggested to be an important
factor in calcium carbonate polymorphisms. In our
study, stereo-structures of polymeric substances
were controlled through the attachment of agarose
beads. As it can be seen from our data and previous
studies (Kawaguchi & Decho 2002), calcium car-
bonate nucleation is induced by polysaccharides.
The polysaccharides of three cyanobacterial
strains have similar binding sites as we observed
by the titration experiments and infrared spectra.
In cyanobacterial mats, EPS was shown to affect
the precipitation and dissolution of CaCO 3 in differ-
ent way, even in opposite directions (Dupraz &
Visscher 2005). In cyanobacterial mats, it is a
matter of debate, whether the saturation index of
carbonate is a result of physical (e.g. CO 2 degas-
sing) or photosynthetic activity (Shiraishi et al.
2008). There were suggestions made that the photo-
synthetic activity is the key factor for promoting
carbonate precipitation and EPS was quantitatively

of minor importance with regard to maintaining
CaCO 3 precipitation in calcifying biofilms. Our
studies demonstrated that calcium carbonate pre-
cipitates in the presence of cyanobacterial polysac-
charides, without the photosynthetic activity. The
mechanism behind it shall need to be investigated
in future studies. It is possible that the binding
calcium or carbonates on extracellular polymers
creates templates for crystal nucleation. However,
this hypothesis is needed to be examined as, for
example, Shiraishi et al. (2008) showed that the
EPS-binding Ca plays a minor part on Ca flux.
Cycling of EPS has been shown to be rapid under
oxic and anoxic conditions (Decho et al. 2005).
It was also demonstrated that the EPS pools of
stromatolites are secreted largely by cyanobacteria
(Kawaguchi et al. 2003). During anoxic conditions
EPS is partly decomposed inducing the decrease
of saturation index and dissolution of calcium car-
bonate. Our experiments demonstrated that isolated
polymeric substances from cyanobacteria have a
remarkable buffering capacity and are able to
induced calcium carbonate formation.

Conclusions

In this study, the functional groups of extracellular
polysaccharides of three picocyanobacteria strains
from hardwater lakes were experimentally exam-
ined by potentiomentric titrations and infrared
spectroscopy. The results demonstrated that their
deprotonation constants are very similar. Modelling
and FTIR results are consistent with the presence of
five to six distinct surface sites, corresponding to
carboxyl, phosphoric, sulphydryl, amine/phenol,
and hydroxyl groups, with a total concentration of
3.66-14.97 mM g 21 of bacteria. The carboxyl