## 3 Frustule Modification: Techniques and Applications  
### Frustule Metabolic Doping  
The possibility to insert semiconducting or metallic elements in dielectric nanomaterials in order to modify their morphological, photo- and electro-luminescent properties is of great interest in several fileds, e.g., in sensing, optoelectronics, solar energy harvesting, catalysis, and biomedicine [29]. In this context, diatom cultures can be viewed as potential biofactories for massive bottom-up production of hierarchical, nanostructured hybrid materials. An example is given by the metabolic insertion of germanium in _Pinnularia_ sp. frustules as described in Ref. [30] and whose principle is schematized in Figure 3. In the first stage of the process, a diatom cell suspension is grown under silicon starvation, then silicon and germanium are co-added to the culture allowing the uptake of germanium by the dividing cells and its incorporation in the frustules of the newly divided ones. As long as the initial Si:Ge molar ratio is at least 14:1, no inhibition of cell division has been observed. By ion-coupled plasma (ICP) analysis, germanium resulted alloyed into the silica matrix of the frustule in the form of Ge-oxide nanoclusters or germanosilicate (Si-O-Ge) and not simply adsorbed or randomly precipitated onto the surface of the cells. Even though the overall shape of the frustule at the micron scale resulted unaltered after germanium metabolic insertion, geometry of the _areolae_ was modified by the thickening of the frustule silica induced by germanium oxides. Metabolic insertion of germanium can thus represent a way to obtain controlled modification of the pore shape. Figure 1: (a) 3D schematic model of an _Actinotypchus senarius_ frustule with indication of the single components. (b) Scanning electron microscope (SEM) image of the whole frustule. (c) Detail of the valve. Reproduced with permission from Ref. [23]. Figure 2: (a–c) SEM images of the different layers of a single valve of a _Coscinodiscus_ sp. diatom frustule: cribellum (a), cribrum (b), and foramen (c). (d) a detail of the girdle band area; (e) sectional view of the valve. Arrows indicate : cribellum (1), cribrum (2), and the internal plate (3). Adapted with permission from Ref. [28]. Copyright 2007 American Chemical Society. It is known that diatom biosilica is characterized by visible photoluminescence under UV excitation, with a peak in the blue spectral range [31]. This is mainly induced by surface defects, including silanol groups (=Si-OH), and organic residuals incorporated in the frustule matrix [15; 32]. _Pinnularia_ sp. frustules containing metabolically inserted germanium are characterized by a more intense photoluminescence emission if compared to non-doped frustules [33]. Indeed, also Ge-O defects in amorphous silica are known to induce blue photoluminescence, which thus superimposes on that of diatom biosilica. Besides photoluminescence, Ge-doped frustules present also electroluminescence whose spectrum is characterized by narrow peaks consistent with the calculated resonance modes of the diatom frustule modeled as a photonic crystal (PhC) slab (for a wide treatise of photonic and optical properties of diatom frustules, refer to [15; 34]). Conversely, the metabolic doping of _Coscinodiscus wailesii_ frustules with nickel [35] resulted in quenching of the photoluminescence detected at 544 nm after excitation with blue light, probably due to overlapping of the biosilica emission spectrum with the asbsorption of the complexes formed by nickel ions within the hydrated frustule matrix. The modification of the optical properties of the frustule after nickel insertion, together with changes in its morphology (in particular the enlargement of the pores), allows one to consider diatom frustules as potential active component of an aquatic pollution sensing system. A two-stages approach similar to the one described above for germanium was adopted by the same authors for the metabolic insertion of titanium [36].