*8.8. Growth Phase of Organisms Used for NP Fabrication*

The effect of the growth phase on the fabrication process of NPs was studied by Sweeney et al. [182]. Cadmium sulfide nanocrystal production varies dramatically depending on the growth phase of *E. coli*. The formation of NPs increased approximately 20-fold in the stationary phase of *E. coli*, compared with that grown in the late logarithmic phase.

#### **9. The Mechanism of Biological Synthesis of NPs**

Different speculations about the mechanism of NP synthesis using living organisms were reported, but until now, the exact mechanism remained unclear. However, each organism was found to have its own synthesis mechanism against different metals [4,183]. One hypothesis is the ability of living organisms to synthesize NPs occurs via two general steps—(i) metal ions are trapped on the surface of an organism and/or inside their cells and (ii) these ions are reduced to NPs aided by biomolecules such as enzymes, proteins, pigments, or polysaccharides, or by the union effect of different molecules [4,67,183,184]. These biological molecules are responsible for the electron shuttle reduction and stabilization of NPs. Sneha et al. reported that metal ions, particularly Au or Ag ions, are captured on the fungal cell surface through the electrostatic force between the metal ions and cell wall, which carries a negative charge from the enzyme carboxylate groups. The enzymes then fabricate the metals into Au or Ag nuclei, which sequentially grow by reduction and accumulation [185]. Kalishwaralal et al. reported that nitrate reductase produced by *Bacillus licheniformis* facilitates the bioreduction of Ag-NPs. The nitrate ions of silver nitrate were found to activate the nitrate reductase enzyme, resulting in reducing silver ions to metallic silver via an electron shuttle enzymatic metal reduction process [186]. During the biosynthesis of MNPs, NADH and NADH-dependent nitrate reductase enzymes (especially nitrate reductase) are essential factors [187]. These enzymes are known to be secreted from *B. licheniformis* and may be linked to the biofabrication of Ag<sup>+</sup> to Ag<sup>0</sup> and subsequent synthesis of Ag-NPs. Divya et al. reported that the existence of NADH and NADH-based reductases in the supernatant of *Alcaligenes faecalis* was responsible for the reduction of silver nitrate into Ag-NPs [188]. Hamedi et al. studied the synthesis of Ag-NPs using *Fusarium oxysporum66* cell-free culture filtrates [189]. They reported that a surge in C:N ratio resulted in the enhancement of the nitrate reductase activity, causing an increase in the Ag-NPs fabrication process rate. Furthermore, they obtained small Ag-NPs with a narrow size distribution.

Exopolysaccharides (EPSs) are predominantly composed of carbohydrates (such as Dglucose and D-mannose) and noncarbohydrate components (such as carboxyl, phosphate, and sulfate), which characterize them with anionic properties. These organic molecules enhance the lipophilicity of EPSs and directly influence their interaction with polysaccharides and cations. It was found that EPSs chelate metal ions. Then, sugar molecules of EPSs reduce metal ions into NPs to be capping with different functional groups of EPSs [190–192].

Kang et al. synthesized Ag-NPs using EPSs in an *E. coli* biofilm. They reported that EPSs aldehyde and hemiacetal groups of rhamnose sugars were responsible for the reduction and stabilization of NPs [193].

Moreover, the synthesis of heavy MNPs can be due to the genetic and proteomic responses of a metallophilic microorganism to toxic environments [194]. Heavy metal ions such as mercury, cadmium, zinc, and copper ions are a hazard to microbial survival and consequently, microorganisms have developed genetic and proteomic responses to tackle these threats [195]. Microorganisms contain many gene clusters of metal resistance that enable cell detoxification via different mechanisms, such as complexation, efflux, or reductive precipitation [196]. Recently, a mechanism for the synthesis of magnetites using *Shewanella oneidensis* was suggested and comprised both passive and active processes [197]. Initially, Fe2+ is actively produced when bacteria use ferrihydrite as a terminal electron acceptor, accompanied by elevation in the pH round the cells due to the bacterial metabolism of amino acids. The passive mechanism then uses the localized concentration of Fe2+ and Fe3+ at the net negatively charged cell wall, cell structures, and/or cell debris, which enhances a local rise of supersaturation of the system with respect to magnetite, resulting in precipitation of the magnetite phase.

Another hypothesis discussed the role of c-type cytochromes redox proteins for electron transfer during the reduction of metals. Ng et al. synthesized Ag-NPs using a mutant strain of *Shewanella oneidensis* missing cytochrome genes (MtrC and OmcA) and a wild-type strain of *S. oneidensis*. They found that c-type cytochromes aid in electrons transfers to metal ions outside the cells. Similarly, Liu et al. reported that c-type cytochrome protein complexes (ombB, omaB, and omcB) in the outer membrane of metal-reducing bacterium Geobacter sulfurreducens PCA was responsible for the extracellular reduction of Fe (III) citrate and ferrihydrite (Figure 7).

**Figure 7.** The potential mechanism of biological synthesis of NPs.

#### **10. Toxicity of Nanoparticles**

Although nanotechnology is rapidly growing with a wide range of applications in different areas such as industry, agriculture, medicine, biotechnology, etc., there remain many barriers with this technology such as the toxicological effects of NPs on ecology and living organisms. Many in vitro and in vivo investigations revealed that metallic and non-metallic NPs have serious side effects on human health and the environment. Senut et al. reported that mercaptosuccinic acid-capped Au-NPs (1.5 nm) at a concentration of 0.1 µg/mL enhanced cell death of human embryonic stem cells (ESCs). However,

other Au-NPs (4 and 14 nm) at the same concentration showed almost no toxic effect on ESCs [198]. Chen et al. studied the relation between the toxicity of Ag-NPs and their size against fresh red blood cells. The scholars used three different characteristic sizes (15, 50, and 100 nm) of Ag-NPs [199]. They reported that smaller sizes of Ag-NPs enhancing the hemolysis and membrane damage of blood cells, compared with that of other sizes. Wan et al. investigated the genotoxicity of the chemically synthesized cobalt NPs in vivo by utilizing guanine phosphoribosyltransferase delta transgenic mice [200]. The authors reported that cobalt NPs induced oxidative stress, lung inflammation and injury, DNA damage, and mutation.

The toxic effects of TiO<sup>2</sup> NPs against human cells, vertebrates, and invertebrate animals might be attributed to their potency to form free radicals with water in the presence of sunlight. TiO<sup>2</sup> NPs caused DNA damage with or without light and induced the cell death pathway in hamster fibroblasts with stretched micronuclei [201,202]. Although biological synthesis processes have become a simple, eco-friendly, low-cost alternative to traditional methods of NPs fabrication, there are few studies in the literature discussing their toxicity on humans and the environment. Some investigations reported the biocompatibility of green NPs, compared with that synthesized by chemical and physical methods. This could be assumed to the synergetic effect between synthesized NPs and their biological coats [203]. Devasena et al. reported that Mg-NPs synthesized by *Cladonia rangiferina* extract exhibited a better antimicrobial activity and lower toxicity [161]. Khorrami et al. reported that Ag-NPs synthesized by walnut green husk (as reducing and capping agents) showed high selectivity toward breast cancer cells (MCF-7) than normal cells (L-929) [203]. However, the chemically synthesized Ag-NPs showed high toxicity against L-929 cells, compared with biologically synthesized Ag-NPs. Ag-NPs formed by biological synthesis using *Lycium chinense* fruit extract showing low cytotoxicity against normal murine macrophage RAW264.7 cells [204]. On the other hand, Krishnaraj et al. exhibited that Ag-NPs synthesized by *Malva crispa* plant caused morphological alterations in adult zebrafish gills and reduced the biological connection and the homogenous distribution of their liver parenchyma cells [205]. To fill the gap in knowledge regarding this association, many additional in vitro and in vivo investigations are required to test the toxicity of green NPs against normal cells, explore the biocompatibility of biogenic NPs, and determine the precise lethal mechanism of NPs against living cells to increase the potentiality of using these NPs as FDA-approved drugs.
