**1. Introduction**

During the period of 2016–2022 the global nanomaterials market is expected to grow with a compound annual growth rate of about 20% or more [1]. One of the major challenges for the global advancement of nanomaterials market is the environmental sustainability of nanomanufacturing processes. Indeed, traditional top-down or bottom-up chemical and physical nanomanufacturing approaches have a greater energy-intensity compared to manufacturing processes of bulk materials. Further, they are often characterized by low process yields (using acidic/basic chemicals and organic solvents), generation of greenhouse gases, and they require specific facilities, operative conditions (e.g., from moderate to high vacuum), and high purity levels of starting materials [2–4]. The principles of green chemistry ("the invention, design and application of chemical products and processes to reduce

or to eliminate the use and generation of hazardous substances") combined with white biotechnology ("biotechnology that uses living cells—yeasts, molds, bacteria, plants, and enzymes to synthesize products at industrial scale") can really contribute to the development of more sustainable industrial processes [5], also for nanomanufacturing. The microbial-mediated biosynthesis of nanomaterials is a promising biotechnological-based nanomanufacturing process that represents a 'green' alternative approach to physical and chemical strategies of nanosynthesis [6,7]. The microbial-mediated biosynthesis of metallic (also as alloys), non-metallic, or metal oxides nanoparticles have been reported for many microbial strains of bacteria, yeast, molds, and microalgae [8] (Figure 1).

**Figure 1.** Schematic comparing average sizes of the microorganisms described in this review.

In addition, some microorganisms have shown the capability to biosynthesize unique nanostructured materials, i.e., biomineralized nanostructures like silicified frustules [9], calcified coccoliths [10], magnetosomes [11], and organic nanomaterials like bacterial nanocellulose [12] exopolysaccharide nanoparticles [13] and bacterial nanowires [14]. The microbial-mediated biosynthesis of nanomaterials has been extensively explored showing different advantages and features including the following: (i) synthetized nanomaterials have defined chemical composition, size and morphology, (ii) biosynthesis is performed at mild physico-chemical conditions, (iii) easily handling and cultivation of microbial cells and possibility of cell culture scale-up, (iv) possibility of in vivo tuning nanomaterial characteristics by changing key parameters of cell culture operational set up or through genetically engineering tools [15]. In order to enable a broad applicability of microbial-mediated biosynthesis of nanomaterials as a real alternative to 'traditional' synthetic approaches to nanomanufacturing, many hurdles still need to be overcome: a reduction of polidispersity of nanoparticles, a more complete characterization of biocapping layer agents, effectiveness of removal procedures of biocapping layer and nanomaterials purifications, standardization of microbial cell culture protocols for reproducibility of nanosynthesis processess, as well as production costs and yields. Overeaching the challenge for the development of reliable eco-friendly nanotechnologies for nanomaterial synthesis is of utmost importance for future exploitations of broad-impact nanostructured-based technologies and applications, like innovative optical and electrochemical (bio) sensoristic devices [16] and therapeutic and diagnostic applications of nanostructured materials e.g., for drug delivery, in vivo/in vitro imaging and development of antimicrobial and antitumoral drugs [17,18]. In the first part of this review, we reported an overview of scientific literature (mainly from the last ten years) about in vivo microbial biosynthesis of nanomaterials that have been used for (bio) sensoristic and biomedical purposes. We focused on works that have covered several key aspects of nanomaterials: (i) type of biosynthesis (in some cases post-biosynthesis functionalization), (ii) biosynthetic pathways (presumptive or demonstrated), (iii) characterization, (iv) applications. In the second part, main acquired knowledge, challenges, and potentiality of microbial-mediated biosynthesis has been described.

#### **2. Microbial-Mediated Biosynthesis of Nanomaterials for Sensoristic and Biomedical Applications**

## *2.1. Bacteria*

In the last ten years, bacteria have been used to synthesize inorganic nanomaterials (mainly selenium, gold, and silver nanoparticles) with interesting properties for the development of voltammetric sensoristic devices [19], and third-generation biosensors [20], for possible diagnostic applications [21] like cell imaging and biolabeling [22] and for applications where no surface coat is required, like annealing and thin film formation [23] (Table 1). Bacterial-biosynthesized nanoparticles have mainly shown in vitro antimicrobial activity against some pathogenic bacterial strains [24–28] and properties i.e., antioxidant [29], anti-proliferative, anti-migration [30], anticoagulant [31], and anticancer [26–33]. Biochemical mechanisms which mediate the bacterial biosynthesis of nanoparticles have been proposed or they are currently under investigation. Many of these biochemical mechanisms have been described as part of microbial resistance mechanisms for cellular detoxification which involves changes in solubility of inorganic ions by enzymatic reduction and/or precipitation of soluble toxic to insoluble non-toxic nanostructures. Both extracellular and intracellular biocatalytic synthesis (with and possible excretion) mainly involves oxidoreductase enzymes (e.g., NADH-dependent nitrate reductase, NADPH-dependent sulphite reductase flavoprotein subunit α, and cysteine desulfhydrase) and cellular transporters. Physicochemical processes like biosorption, complexation, nucleation, growth, and stabilization mediated by biomolecules (e.g., proteins and carbohydrates) have also been described. In addition to inorganic nanomaterials, some bacteria genera have shown the ability to biosynthesize very peculiar organic nanostructures. Bacterial nanocellulose is a 3-D network of cellulose nanofibrils produced by aerobic acetic bacteria like those belonging to the genus *Gluconacetobacter*, the most e fficient bacteria for nanocellulose biosynthesis. Compared to the nanocrystalline cellulose and nanofibrillated cellulose, bacterial nanocellulose shows higher purity, crystallinity and mechanical stability [34]. Therefore, bacterial nanocellulose is a nanomaterial which has attracted grea<sup>t</sup> attention for use in biomedical applications (e.g., as antimicrobial agent, for drug delivery systems and sca ffolds for tissue engineering) and on biosensoristic platforms (as nanocomposite and as support for the immobilization of biological recognition elements) [35–37]. Exopolysaccharides are microbial extracellular biopolymers with di fferent roles in adhesion of bacterial biofilms and as protection agents. In a recent work a novel self-assembled and spherical nanosized non-glucan exopolysaccharide has been described for bacteria *Lactobacillus plantarum*-605. Results have showed its reducing actions for rapid (30 min.) biosynthesis of good monodispersed gold and silver nanoparticles without any pretreatment or modification [38]. Bacterial nanowires are conductive proteinaceous pilus-like nanostructures involved in extracellular electron transport processes of anaerobic dissimilatory metal-reducing bacteria like *Geobacter* and *Shewanella* genera [39], aerobic bacteria like *Pseudomonas aeruginosa* [40] and aerobic photosynthetic cyanobacteria like *Microcystis* and *Synechocystis* genera [41]. Metallic-like conductivity (due to aromatic amino acids-richness in PilA proteic fibers) and a redox-based conductivity (mediated by cytochrome OmcS present on fibers surface) have been hypothesized for bacterial nanowires in *G. sulfurreducens* [39]. Studies on nanowires *Shewanella oneidensis* MR-1 strain have showed a p-type, tunable electronic behavior with electrical conductivities comparable to moderately doped inorganic semiconductors used in synthetic organic semiconductor-based devices like field-e ffect transistors [42]. The bacterium *S. oneidensis* have been also described for biosynthesis of gold and silver nanomaterials [23,24]. Bacterial nanowires are also very promising nanostructures in the bioelectronic field for the development of new biomaterial for microbial fuel cells and electrochemical (bio) sensoristic devices i.e., as direct electron transfer mediator between bacteria biofilm and the solid-state electrode surfaces. Di fferent silicon-based electrodes for rapid biochemical oxygen demand (BOD) determination and water integral toxicity monitoring have been described in recent literature [43–45]. Bacterial magnetosomes are organic-coated intracellular nanocrystals of Fe3O4 and/or Fe3S4, biosynthesized by both magnetotactic and non-magnetotactic

bacteria. The composition of magnetic inorganic part is species-specific, and the external organic coating layer is derived from bacterial phospholipid bilayer membrane. The putative functions of protein component of the external organic coating layer in the magnetosome biomineralization process have been hypothesized [11]. Bacterial Fe3O4 magnetosomes are stable single-magnetic domains permanently magnetic at ambient temperature, possessing peculiar characteristics of high chemical purity, a narrow size range and consistent crystal morphology [46]. Some recent applications include molecular imaging [47], cancer therapy [48], and the development of a chip-based whole-cell biosensor for toxicity assessment [49].

#### *2.2. Yeasts and Molds*

The research focused on biosynthesis of nanomaterials by fungi, like yeasts and molds, have brought to the coinage of the term 'myconanotechnology', in order to refer to a newly emerging domain of nanotechnology. Yeasts are unicellular fungi mainly known in nanosynthesis for their ability to produce semiconductor nanoparticles [8]. Biosynthesis of high water-soluble and biocompatible cadmium telluride quantum dots by model organism yeas<sup>t</sup> *Saccharomyces cerevisiae* have been reported in literature. These cadmium telluride quantum dots have showed interesting characteristics of size-tunable (changing culture time and temperature) emission and photoluminescence quantum yield as good candidate for bio-imaging and bio-labelling applications [50]. *S. cerevisiae* have been also used for biosynthesis of Au–Ag alloy nanoparticles for electrochemical sensors fabrication [51,52], aimed to the determination of paracetamol in tablet samples and vanillin in vanilla bean and vanilla tea sample, respectively (see Table 2). Possible biosynthesis mechanisms of nanoparticles by *S. cerevisiae* could involve membrane bound and cytosolic oxidoreductases as well as extracellular 1,3-β-glucan synthase-mediated formation and growth of nanoparticles [50–53]. Molds are a large group of microscopic filamentous fungi that include many genera like to *Penicillum*, *Aspergillus*, and *Fusarium*. Compared to bacteria, molds possess many distinctive advantages for biosynthesis of nanomaterials: (i) higher metal tolerance, (ii) higher metal binding and uptake capabilities, (iii) easy culturing and fast growing; (iv) higher extracellular nanosynthesis (mediated by extracellular enzyme, reductive proteins, and secondary secreted metabolites). Extracellular biosynthesis of nanomaterials poses advantages in terms of a possible reuse of cell cultures for new biosynthesis (cell lysis not required) and reduced nanoparticle downstream purification processes [54]. Proposed mechanisms behind fungal synthesis of nanoparticles hypothesized a possible involvement of biomolecules secreted in formation and stabilization of nanoparticles [55], secreted reductases [56,57] and possible trapping of metal ions by electrostatic interaction with positively charged groups in enzymes present in cell wall of the mycelia [58]. In the last ten years, several works have described mold-based biosynthesis of nanoparticles (silver, gold, and tellurium) and quantum dots (zinc sulfide, zinc sulfide with gadolinium, and lead sulfide). These nanoparticles have shown both antibacterial activity [54,57,59] and antitumoral activity [55,56,58] beside possible employment in optical detection of heavy metals and arsenic in water [60,61] (Table 2).


**Table 1.** Nanomaterials synthesized by bacteria.


**Table 1.** *Cont.*

 *10*, 11




**Table 2.** Nanomaterials synthesized by yeasts and molds.

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