## 3 Frustule Modification: Techniques and Applications
### Frustule Metabolic Doping
At the end of the process, titanium is mainly localized as a nanophase surrounding the base of each frustule pore. The obtained biogenic titanate can be easily converted into nanocrystalline anatase TiO\({}_{2}\) by thermal annealing in air at 720 \({}^{\circ}\)C.  
In recent years, diatom frustules-TiO\({}_{2}\) composites have been extensively tested and exploited to increase the efficiency of dye-sensitized solar cells (DSSCs) [20; 37; 38; 39; 40]. In a traditional DSSC, a photosensitive dye is bound to a photoanode comprised of anatase TiO\({}_{2}\) nanoparticles. When exposed to light, the dye absorbs photons and in its photoexcited state injects electrons into the conduction band of the semiconducting TiO\({}_{2}\). Finally, the electrons diffuse through the mesoporous titania layer  
Figure 3: (**a**) Conceptual scheme for metabolic insertion of germanium into a diatom frustule: (1) uptake of Si and Ge by Si transporters (SITs); (2) valve development within the silica deposition vesicle (SDV); (3) cell division. (**b**) Simplified representation of the frustule of the newly-divided cell, showing intracellular Ge located in the new hypo-valve, Ge-rich nanoparticles, and the developing girdle band. Reproduced with permission from Ref. [30]. to the working electrode while the dye is regenerated by a liquid electrolyte [37]. In Ref. [20] titania was substituted by diatom frustules enriched with titania nanoparticles by means of plasma treatment (thus avoiding any linking agent usually exploited in silica functionalization). The high effective surface area of the resulting three-dimensional hybrid structures and the multiple scattering events induced by frustules pores ensure an enhanced interaction of the incoming light with dye electrons. After only three cycles of plasma treatment, an increase of conversion efficiency of about 30% with respect to conventional DSSCs has been achieved. A further improvement in efficiency (up to about 35%) and in the stability of the cell has been very recently obtained by replacing the liquid electrolyte by a gel polymer electrolyte and avoiding the use of volatile solvents for its preparation [39]. In this case the hybrid layers have been obtained by spin coating of a proper mixture of TiO\({}_{2}\) P90 powder and diatom frustules. An alternative way to obtain silica-titania nanostructured hybrids useful in DSSCs is of course given by metabolic insertion, as described in Ref. [38]. The authors also proposed a model for the realization of \"living DSSCs\" to generate electrical pulses aimed at the extraction of oil from diatom cultures without sacrificing the cells. Titania is metabolically inserted by the two-stage cultivation process described in [36] and the culture medium supplemented with an electrolyte and ruthenium dye as a photoactive dye. Electric pulses generated after exposure to light can fracture the frustule, causing lipid drops to ooze from the cells to the culture medium. The diatoms can heal their frustules when fed with nutrient media. The use of electric pulses has been tested for the extraction of proteins from _Chlorella vulgaris_ and _Haematococcus pluvialis_ microalgae, characterized by hard cell walls healed after a suitable incubation time. Other metal and semimetal ions tested in metabolic insertion comprise aluminium in _Stephanopixis turris_[41], with potential applications in catalysis; europium in _Navicula_ sp. [42], which gives frustule biosilica photoluminescence properties with red light emission (614 nm) after excitation at 394 nm; calcium in _Thalassiosira weissflagii_[43] with potential applications in biomedicine of the obtained hybrid frustules as substrates for fibroblasts and osteoblasts growth; tin in _Synedria acus_[44], which decreases the mechanical strength of the frustule; zirconium in _Phaeodactylum tricornutum_[45] for applications in electrochemical sensing aimed at detection of methyl parathion. Metabolic incorporation of rhodamines into diatom frustules by means of _in vivo_ fluorochromation has been exploited by [PERSON] et al.