*3.3. Biological (Green) Synthesis*

Biological synthesis is a modern alternative to both physical and chemical synthesis processes and is considered a type of bottom-up route [57]. This approach utilizes natural sources such as microorganisms, macroorganisms, and biomolecules (proteins, lipids, polysaccharides, pigments, etc.) to fabricate NPs from their bulk materials without the need for toxic chemicals during the fabrication process [4,58]. Various significant properties of biosynthesis routes such as the absence of poisonous chemical compounds used as reducing or stabilizing agents, no toxic yields generated from the process, the low energy consumption, inexpensive cost, and high scalability have resulted in green synthesis methods becoming more attractive than other traditional methods [4].

Biological synthesis routes are categorized into two main approaches—extracellular and intracellular synthesis routes.

#### 3.3.1. Extracellular Synthesis

In extracellular synthesis routes, the fabrication process occurs outside living cells [57,59]. This process can be achieved via three different patterns:

(i) Cell-biomass-filtrate synthesis of NPs: In this pattern, cells of living organisms are dried with a lyophilizer, oven, or air-based methods and then crushed into fine powders that are mixed with distilled water for boiling. The mixture is cooled, passed through a filtration system such as Whatman filter paper, and then the resulting filtrate is mixed with a defined concentration of bulk material to fabricate it into NPs [28,58,60]. An alternative procedure involves washing the natural sources such as *Streptomyces* sp., algae, etc., then soaking the cells in water for a number of days, followed by centrifugation and use of the resulting supernatant as a reducing and stabilizing agent to synthesize NPs [61,62]. Other methods achieved the extracellular synthesis of NPs by sonicating or boiling the natural sources under certain conditions, then filtering the mixture and using the filtrate in the synthesis process [33,63];

(ii) Cell-free, culture-medium-based synthesis of NPs: This method is suitable for cultured microorganisms. First, the culture is centrifuged and the supernatant used for bioreduction of bulk compounds into their NPs under suitable conditions. Keskin et al. demonstrated that cell-free culture media of *Synechococcus* sp. had a reducible activity that resulted, under light conditions, in the formation of Ag-NPs with an average nanosize of 140 nm [64]. However, this process is sometimes unsuitable for NP synthesis because many types of media used for the culture of microorganisms contain components that act as reductants and stabilizing agents. These compounds interfere with the reducible activity of active biomolecules of the cultured microorganisms [65];

(iii) Biomolecule-mediated synthesis of NPs: This approach uses biomolecules such as pigments, carbohydrates, proteins, enzymes, etc. as reducing and capping materials to produce NPs. Briefly, target biomolecules are extracted from their micro- or macroorganisms, purified, and mixed with a defined concentration of bulk material solutions to start the NP fabrication process under specific conditions of temperature, illumination, and pH [66,67] (Figure 3).

**Figure 3.** Green synthesis methods include intracellular synthesis route (**I**) and extracellular synthesis routes including cell-free, culture-medium-based synthesis of NPs (**II**), cell–biomass-filtrate synthesis of NPs (**III**), and biomolecule-mediated synthesis of NPs (**IV**).

#### 3.3.2. Intracellular Synthesis

The intracellular synthesis method refers to the production of NPs inside living cells, with biological processes such as metabolic activity, respiration, and growth stage, playing crucial roles in the biosynthesis process [68]. Intracellular synthesis can be performed according to two protocols, each composed of three steps: (i) culturing the target living organism, (ii) the reaction between precursor materials and living cells, and (iii) separation and purification of NPs and subsequent characterization using different physicochemical methods [4,69]. The first protocol includes the incubation of bulk materials solution with microbe cultures during their growing period under standard culture conditions until the microbes reach a certain growth [70]. In the second method, living cells in the logarithmic phase are collected by centrifugation, washed multiple times to discard any undesired materials, and then the cleaned microbial biomass is dissolved in water and mixed with a suitable amount of bulk material solution [71].

Intracellular synthesis is more complicated than extracellular fabrication due to the additional steps required to extract and purify NPs from inside the cells [72]. Both types of biological synthesis methods are eco-friendly routes, do not usually need toxic chemical materials, and are easily performed under normal laboratory conditions [4,65] (Figure 3).
