## 3 Results and Discussion  
### Sbpa Recrystallization in Fe (II): Chemical Control  
In order to test the potentiality to induce S-layer formation in the presence of Fe\({}^{2+}\) ions, the recrystallization process was firstly attempted without any type of applied electrical (potential) control. Instead, recrystallization was performed by incorporating chemical additives (DTT) which act as retardant of the naturally occurring oxidation process. The usual CaCl\({}_{2}\) was then replaced by FeCl\({}_{2}\) or FeSO\({}_{4}\) and the routinely used Tris buffer was also replaced by HEPES, since formation of precipitates was observed with the former. Furthermore, HEPES had already been reported in literature to prevent auto-oxidation and to stabilize Fe\({}^{2+}\) for a considerable length of time [28]. A first experimental issue to overcome corresponded to the finding of optimal conditions which would allow iron salts to form a transparent, aggregate-free solution in HEPES buffer, as summarized in Figure 1.  
The solubility of iron strongly depends on pH making iron less available at neutral-to-basic pH than at acidic ones. As can be observed, HEPES solutions of both Fe (II) salts show increasing turbidity at pH values above 6.4, together with the formation of large green coloured aggregates (Figure 1a) which sediment and attach onto the underlying silicon wafers. The appearance of such aggregates compromises the formation of a homogeneous film both because of the blocking of large substrate areas and, in addition, the partial conversion of Fe\({}^{2+}\) into Fe\({}^{3+}\) ions taking place, a critical limiting factor for the reaction intended. Therefore, employment of a final pH of 6.3 was approved, attending to the optimal performance of this solution and still being in the range of parameters to maintain SppA protein intact and active (>> pl). The stability of the prepared Fe\({}^{2+}\)/HEPES solution upon oxidation was also tested over a time range of 18 hours, maximum recrystallization time required under some particular conditions [22]. Figure 1b shows the evolution in both colouring and turbidity of an iron sulphate sample (n\({}^{\circ}\) 2-5) in comparison to a positive control (n\({}^{\circ}\)1), featured by its strong yellow colour, where DTT was not incorporated nor the buffer degassed prior to the iron salt addition. Even though the chemistry-driven control turns certainly effective for quite some time, where DTT gets oxidized instead of iron (II), subsequent oxidation remains uncontrolled, leading to Fe\({}^{3+}\) formation over time anyway. Thus, such an oxidation kinetics led to the election of hydrophobic substrates for the follow-up of the process, so SppA recrystallization could take place more rapidly (<3 h, when performed with CaCl\({}_{2}\)) and while the amount of Fe (III) in the recrystallization buffer stayed as low as possible. In such conditions, SppA was recrystallized from both FeCl\({}_{2}\) and FeSO\({}_{4}\) containing solutions over 210 min and the resulting topographies analysed by Atomic Force Microscopy, as shown in Figure 1c,d. In these micrographs, the crystal lattice can be visually determined although in a rather faint manner. However, Fourier domain analysis over each image (see insets) confirms the existence of a repeated pattern, which appears somehow more distorted than for the Ca (II) case (see Appendix A, Figure A1). Figure 1: (**a**) S-layer recrystallization on hydrophobic silicon wafers in the presence of a 2 mM FeSO\({}_{4}\)-7H\({}_{2}\)O solution in degassed 0.1M HEPES buffer (+5 mM DTT) at different pH values. (**b**) Time course (images 2–5) of Fe\({}^{2+}\) auto-oxidation (2 mM FeSO\({}_{4}\)-7H\({}_{2}\)O/5 mM DTT in 0.1 M HEPES at pH 6.3). Pictures were taken after 30 min (2), 180 min (3), 420 min (4) and overnight (5). Sample 1 acts as positive control in the absence of DTT and without previous degassing (image taken after 30 min).