*2.3. Microalgae*

Microalgae are unicellular photosynthetic microorganisms that have attracted significant interest in the field of nanomanufacturing [62] (see Table 3). Microalgae like *Tetraselmis kochinensis*, *Scenedesmus* and *Desmodesmus* have been used for the biosynthesis of noble metal nanoparticles with good antimicrobial activity, useful for applications in biomedical tool designing but also in drug delivery, catalysis and electronics [63–65]. For these microalgae, mechanisms described for nanoparticles biosynthesis include phenomena of nucleation, control of dimension, and stabilization of nanoparticle structure, mediated by reducing agents [64], enzymes present in the cell wall cytoplasmic membrane [63], biomolecules like polysaccharides, proteins, polyphenols and phenolic compounds [65]. Mechanisms behind biological mineralization (or biomineralization), i.e., the in vivo inorganic minerals formation, have been extensively studied for possible development of new nanomaterials. Diatoms are unicellular microalgae with very peculiar biomineralized silica cell wall called frustules. Diatom frustules possess a highly periodic and hierarchical 3D-porous micro-nanostructure of di fferent morphology (pennate and centric). Hypotheses about their natural functions include mechanical protection, biological protections, filtration, DNA protection from UV and optimization of light harvesting [66–68]. Compared to analogous synthetic mesoporous silica materials, e.g., MCM-4, diatom frustules possess di fferent advantages, including higher biocompatibility, reduced toxicity and easily purification. Diatom frustules also exhibit interesting optical and optoelectronic properties [66,69–74]. The abundance of silanol groups (Si-OH) make the diatom frustules surface easily functionalizable (also in vivo), thus allowing to fully exploit the potentiality of structural nanopatterning of diatom frustules [75]. Functionalization with antibodies [72–77] and gold nanoparticles [78,79] has been described for di fferent sensitive materials in optical or electrochemical immunosensors. Diatom frustules have showed potential application in drug delivery systems [80–82]. Diatomaceous earth (or diatomite) is a large available microfossil material from diatom frustules with extensive commercial use in abrasives or filters. Recently, diatomite—gold nanoparticle nanocomposites have been described for on-chip chromatography and surface-enhanced Raman scattering-based sensoristic devices for the detection of cocaine in biological samples [83] and of histamine in salmon and tuna samples [84]. The "diatom nanotechnology" is a rapidly evolving research field which aims to fully exploit the unique properties of diatom frustules and the grea<sup>t</sup> potential of silica biomineralization cellular pathway for the development of new functionalized nanomaterials for emerging applications in sensing, photonic and drug delivery [85]. Another characteristic of diatom frustules is the presence of xanthophyll pigment fucoxanthin. Recent studies have highlighted the active role of fucoxanthin as photo-reducing agen<sup>t</sup> of metal ions to stabilize silver nanoparticles. These silver nanoparticles have showed a significant in vitro antimicrobial activity against *Escherichia coli*, *Bacillus stearothermophilus*, and *Streptococcus mutans* [86] and possible application in optical chemosensing of dissolved ammonia in water samples [87]. Compared to diatoms, microalgae coccolithophores have received less attention. Coccolithophores are calcifying nanoplankton that produces CaCO3 microparticles (coccoliths), in form of arrays of nanoscaled substructures. Interesting optical properties (e.g., light scattering) of coccoliths have been described but also some drawbacks including low electrical conductivity, dissolution at low pH values and scarcity of surface functional groups. Despite these, coccoliths morphologies have showed a grea<sup>t</sup> applicative potential for nanodevices fabrications, especially following appropriate in vivo or in vitro modification and functionalizations [10,78]. In very recent work the fabrication of an electrochemical aptamer-based sandwich-type biosensor for the detection of type 2 diabetes biomarker Vaspin has been described [88].


**Table 3.** Nanomaterials synthesized by microalgae.

#### *Nanomaterials* **2020**, *10*, 11



#### **3. Towards a Large-Scale Applicability: Knowledge, Issues, and Potentiality**

The in vivo microbial nanobiosynthesis and possible control and tuning of nanomaterial properties represent a concrete opportunity for future development and promising uses in biosensoristics and biomedical fields. Despite all the advantages, microbial nanotechnology still has very limited uses [89]. Bacteria have showed the ability to synthetize nanomaterials either by extracellular or intracellular mechanisms. These mechanisms generally produce opposite advantages and disvantages in terms of metal nanoparticles dispersity and purification. Extracellularly produced nanoparticles are generally more polydispersed (i.e., with a grea<sup>t</sup> variability in size) than intracellularly produced nanoparticles. By contrast, in extracellular nanomaterial productions less downstream extraction/purification steps (e.g., ultrasound treatment and detergent uses) are required. Thus, the extracellular mediated synthesis described for yeas<sup>t</sup> and molds can greatly simplify the purification steps, besides being an advantage for a possible reuse of microorganisms for more biosynthesis cycles. However, the characterization and identification of the enzymes responsible for nanobiosynthesis in molds is still uncomplete. The photoautotrophic metabolism of microalgae and cyanobacteria is based on carbon dioxide (as carbon source), light (as energy source), inorganic nutrients and water. This condition generally reduce the costs of culture media (compared to culture media used for the growth of bacteria, yeasts, and molds) and it can strongly spur the future scaling-up from the laboratory to the industrial scale, also through the design and the development of solar photobioreactors for the fixation (and reduction) of atmospheric carbon dioxide.

#### *3.1. Nanoparticles Dispersion and Capping Layers*

One of the main challenges in microbial nanobiosynthesis is the control of dispersity of nanostructure materials, which heavily influence electronic and optical properties, and the isolation and purification of plural form. Dispersity, i.e., the size distribution of the nanoparticle population, is a key property that strongly influences the particle's behavior in fluids. Improvement and optimization of extraction and purification protocols are required, both for intracellular and extracellular biosynthesis: methods like freeze-thawing, osmotic shock and centrifugation could lead to changes in nanoparticle structures as well as aggregation and precipitation phenomena. Through the adoption of suitable strategies, microbial biosynthesis of nanoparticles could be improved. Selection of appropriate microbial strains (in terms of growth rate and biocatalytic activities), optimization of culturing conditions and uses of genetic engineering tools could help to overcome drawbacks linked to slower producing rate and polidispersity (compared to chemical-based nanomanufacturing) [90]. Microbial biosynthetic nanoparticles are characterized by the presence of a capping layer of biomolecules adsorbed on the surface that act as stabilizing agen<sup>t</sup> and biological active layer of nanoparticles [21]. A deep knowledge of capping characteristics, a clear identification of capping agents (mainly peptides like glutathione, metallothioneins, membrane associated proteins etc.), and possible purification of nanoparticles [23] are fundamental for future in vivo medical applications [15,91].

#### *3.2. Cell Culture Conditions*

For future large-scale productions, costs of culture media for microbial growth should be seriously considered to not limit the applications of microbial biosynthetic nanomaterials. One current example is bacterial nanocellulose whose applications are still limited to a few biomedical devices, mainly because of costs of culture medium [92]. In addition, optimization and standardization of microbial cell culture growth protocols and modifications culture conditions are pivotal for control, tune, and to improve characteristics of microbial biosynthesized nanomaterials. The influence of physico-chemical parameters of cell culture operational set up on nanomaterials biosynthesis have been previously highlighted. These factors include (i) microbial cell concentration; (ii) precursor concentration; (iii) pH; and (iv) temperature. The optimum conditions of pH, temperature, and NaCl concentration have been studied to achieve high purity and high synthesis rate of cadmium selenide nanoparticles by bacterium

*Pseudomonas aeruginosa* strain RB. Interestingly, the results of this work have showed that optimum conditions for nanoparticles synthesis did not match with optimum growth conditions for *Pseudomonas aeruginosa* strain RB [93]. E ffects of precursor concentration, temperature, and pH on silver nanoparticle synthesis and particle sizes have been described for the bacterium *E. coli* strain DH5 α [94]. Other recent examples include: (i) the temperature-dependence of size and monodispersity of silver nanoparticles biosynthesis by mold *Trichoderma viride* [95], (ii) the influence on the type of gold nanostructures synthetized (nanoparticles or nanoplates) and them relative size in yeas<sup>t</sup> *Yarrowia lipolytica* strain NCIM 3589 by changing the proportion of cell concentration and precursor gold salt concentration, (iii) the e ffect of temperature on gold nanostructures release from cell wall into the aqueous phase [96] and control of bacterial growth kinetics of the bacterium *Morganella psychrotolerans* to achieve shape anisotropy of silver nanoparticles [97]. Elsoud et al. (2018) observed an improvement in tellurium nanoparticles production by a 1 kGy of gamma irradiation of mold *Aspergillus welwitschiae* KY766958 broth culture (compared to the non-irradiated broth culture control). These results have been ascribed to the activation of enzyme (s) involved in biosynthetic pathway [59]. Although the controlling of biomineralization process still remains a challenge, the optimization of frustules morphological properties (e.g., pore sizes and pore density) has been explored by changing operational parameters of experimental setup (e.g., pH, salinity, temperature, nutrient concentration, precursor Si(OH)4 concentration, and light regime) [98]. Interestingly Townley et al. (2007) reported alteration in pore sizes of *Coscinodiscus wailesii* frustules when exposed to sublethal concentration of nickel [99]. The possibility of in vivo chemical modification of frustules or other biomineralized structures has been recently described. These in vivo chemical modifications lead to the inorganic elements/compounds-doping of biomineralized structures through the addition to the culture medium of given precursors at sublethal concentrations. Several works described the doping of diatom *Pinnularia* sp. frustules or diatoms *Thalassiosira weissflogii* frustules with titania (TiO2) [100,101] nanobiosynthesis containing Si-Ge oxides nanocomb in diatom *Nitzschia* and *Pinnularia* sp. by adding Ge(OH)4 or GeO2 in the diatom culture medium at photobioreactor scale-productions [102–105]. Compared to diatoms, possible nanotechnological applications of the calcareous-based shell of marine protozoa foraminifera have not been so widely explored. A recent work described the in vivo preparation of a bionic material through the inclusion of fluorescent magnetite nanoparticles within calcite skeletal structure of the unicellular organism foraminifer *Amphistrigina lesson*. Such in vitro synthetic approach exploited the natural biomineralization process of growth in the presence of magnetic nanoparticles functionalized with a hydroxylated dextran shell [106].

#### *3.3. Biochemistry, Molecular Biology, and Genetics*

A deeper knowledge of molecular biology and genetic aspects behind microbial nanobiosynthetic pathways is strongly required. For instance, the characterization of not fully understood biochemical mechanisms and a complete identification of extracellular enzymes secreted by filamentous fungi could lead to an improved control in chemical compositions, shapes, and sizes of nanoparticles [55]. The availability of microorganism genome sequences could considerably increase the range of possibilities in genetic manipulation of microorganism to implement the nanoparticles biosynthesis and the in vivo tuning of nanoparticle characteristics. A recent example has come from the study of Zhang et al. (2017), which showed how CdSe quantum dots biosynthesis can be improved through genetic modification of the ATP metabolism pathway in yeas<sup>t</sup> *S. cerevisiae* [107]. Biotechnological approaches based on genetic engineering and recombinant technologies could allow the identification of sequences of gene involved in nanoparticle synthesis and a possible heterologous expression (i.e., controlled expression of one or more gene sequences in a host organism) to enhance nanomaterial production e fficiency [91]. The bacterium *E. coli* is a highly e fficient model host microorganism that has been exploited as heterologous expression system for phytochelatin synthase and/or metallothionein for the in vivo synthesis of various metal nanoparticles (e.g., CdSeZn, PrGd, CdCs, and FeCo) never synthesized before by chemical methods [108,109] or cadmium selenide quantum dots [110]. Thanks to

recent advances in the characterization of biochemical mechanisms involved in bacterial nanocellulose biosynthesis, the future development of new genetically engineered bacterial nanocellulose-producing strains could be achieved. This could eventually lead to a reduction of production costs, an improvement of production yield, and biosynthesis of nanocellulose with new properties, suitable for broader range of technological applications [92,111]. Concerning genetic manipulation of nanostructure-producing microorganisms, Tan et al. (2016) have reported a 2000-fold increase in electrical conductivity and diameter of nanowires filaments produced by model microorganism *Geobacter sulfurreducens*. In this case, genetic modification has been concerned the modification of aminoacidic composition of the carboxyl end of PilA protein, the structural component of bacterial nanowires [112]. The biomineralization process behind magnetosome biogenesis in bacterial species (*Magnetospirillum* species are the most studied) is very complex and not completely elucidated. The mechanism of magnetosome formation has been shown to be under tight genetic control and induced by growth conditions. To date, six di fferent models have been proposed to elucidate magnetosome formation, but they are still not completed [11]. Delalat et al. (2015) have reported a genetically engineered diatom *Thalassiosira pseudonana* (whose genome has been completely sequenced) to incorporate immunoglubulin G-binding domain of protein G (GB1) into the frustules surface. Such antibody-labelled genetically modified diatom enables an in vitro selectively cell targeting and selectively killing of neuroblastoma and B-lymphoma cells [113]. Furthermore, the role of gene Silicanin-1 in the control of biosilica morphology has been recently highlighted in *Thalassiosira pseudonana*, opening new possibilities for future genetic engineering of frustules architectures [114]. A deeper knowledge of biosilicification process as well as the role of organic components of diatom frustules (i.e., proteins sila ffins and long-chain polyamines) in biogenesis and formation of nanopatterns in diatom frustules are still a challenge. To date, biocalcification system of coccolithophores remains unclear, even though a very recent genetic and proteomic study about expression of transcripts and proteins in coccolithophore *Emiliania huxleyi* will help future identification and more detailed characterization of molecular mechanisms and metabolic pathways underlying calcification in coccolithophores [115].
