Bart Stel1 2 Mina Hong3 Behzad Rad4 Jinghua Guo5 James De Yoreo4 6 Magalí Lingenfelder1 2

1, EPFL, Lausanne, , Switzerland
2, EPFL, Lausanne, , Switzerland
3, George Washington University, Washington, District of Columbia, United States
4, Lawrence Berkeley National Laboratory, Berkeley, California, United States
5, Lawrence Berkeley National Laboratory, Berkeley, California, United States
6, Pacific Northwest National Laboratory, Richland, Washington, United States

Bacterial surface layers (S-layers) are highly ordered 2D protein layers that form the outermost cell wall of many bacterial and cyanobacterial species. Cyanobacteria are known to mineralize CaCO3 on their surfaces, a process which is linked to so-called ‘whiting events’ in lakes where diffusion of CO2 to solution is the main source of carbon. This identifies whiting events as a natural way for atmospheric carbon sequestration.
The formation of CaCO3 on crystalline S-layers (Lysinibacillus sphaericus – ATCC 4525, MW 132 kDa) is analyzed using complementary surface science techniques. Structural information is obtained using continuous flow in situ Atomic Force Microscopy (AFM) whereas chemical information is obtained by collecting X-ray Absorption Spectra (XAS) of the calcium LII and LIII absorption edge. The use of continuous liquid flow cells that are compatible with the Ultra High Vacuum (UHV) conditions necessary during XAS measurements, makes it possible to mimic the typical concentrations of Ca2+ and CO2 in lakes. By using liquid flow cells for both XAS and AFM, their complementary data can be easily integrated.
Experimental results of both ex situ as well as continuous flow in situ XAS show the presence of a stable layer of amorphous CaCO3 on S-layers after exposure to standard concentrations of CaCl2 and NaHCO3. No such CaCO3 formation is found in for S-layers alone, or for S-layers in the absence of CaCl2 and NaHCO3.
These results are consistent with complementary continuous flow in situ AFM measurements that were done under the same experimental conditions. In situ AFM images show the presence of a stable layer of amorphous calcium carbonate (ACC) at the S-layer-liquid interface. As bulk ACC is not stable under these conditions, this indicates the stabilization of ACC by the S-layer biointerface. Furthermore, the growth of crystalline calcite (the thermodynamically most stable form of CaCO3) has been observed in situ on top of this amorphous ACC layer.
Our results indicate that S-layers are able to stabilize ACC and catalyze the formation of crystalline calcite ex vivo, at naturally occurring concentrations. This suggests that the catalytic properties of many cyanobacteria are at least in part due to the specific functional properties of the biointerface expressed at their outer surface.