Recently, the ZnO-NPs have gained recognition for their application in several industrial areas, including pharmaceuticals, food, photocatalyst, cosmetics, and agriculture, due to their distinctive properties (Jain et al., 2020; Murali et al., 2021a; Akhtar et al., 2022). As mentioned earlier, the large surface area-to-volume ratio of ZnO-NPs makes them more efficient than their counterparts, i.e., bulk ZnO, for their applications. Physical and chemical techniques have traditionally been employed for synthesizing ZnO-NPs, which provide high production rates with control sizes. However, the physical approach uses much energy, pressure, and heat, whereas the chemical method uses toxic and hazardous chemicals that contribute to environmental pollution and adversely affect human and animal health (Basnet et al., 2018). Moreover, the chemically synthesized ZnO-NPs have limited clinical and biological applications because they are toxic, less biocompatible, and may also bind or reside in the final NP products that interfere with the biological applications (Anjum et al., 2021). Hence, it is necessary to develop non-toxic, bio-safe, cost-effective, more eco-friendly, biocompatible ZnO-NPs as a substitute.

The green synthesis of ZnO-NPs, which uses diverse bioactive chemicals from plants and microbes as reductants and stabilizers, has recently come into the spotlight as a replacement for chemically and physically manufactured ZnO-NPs without altering their properties. Apart from plants, both unicellular and multicellular organisms (such as bacteria, actinomycetes, fungi, and yeast) are involved in the biological synthesis of ZnO-NPs (Kundu et al., 2014; Moghaddam et al., 2017; Shamsuzzaman et al., 2017; Kalaba et al., 2021). The green synthesis process is devoid of toxic chemicals and materials, energy-efficient, eco-friendly, and makes their applications in living organisms completely safe (Weldegebrieal, 2020). The plant-mediated synthesis of ZnO-NPs has been extensively reviewed to understand their unique properties and biological applications (Ahmed et al., 2017; Murali et al., 2021a); in contrast, only a few studies have noted the importance of microbial synthesis of ZnO-NPs and their applications. Hence the present study was focused on providing a holistic view of the microbe-mediated synthesis of ZnO-NPs and their potential applications, with specific objectives that include synthesis mechanisms and biological properties apart from their toxicological effects on the environmental ecosystem.

Since the microbes are easily reproducible compared to plants, it offers an advantage over plant-mediated ZnO-NP synthesis. In the new millennium, using microbes, viz., bacteria, actinomycetes, fungi and yeast, has attracted considerable interest in synthesizing the ZnO-NPs. These organisms serve as miniature nano-factories wherein the enzymes, proteins or biomolecules secreted by them help to selectively reduce metal ions into their corresponding metal or metal oxide NPs. The formation of mono- and poly-dispersed NPs with different sizes and shapes is attributed to the numerous organic compounds released into the growing media or suspension cultures (Yusof et al., 2020a). Not all microbes are involved in the NPs synthesis process due to unique enzyme activity and metabolic processes, which differ from organism to organism. Due to these properties, selecting appropriate microbes independent of their enzyme activity or metabolic and biosynthetic pathways (intracellular or extracellular) is critical for forming ZnO-NPs, which remains unexplored. In addition, these microbes must also be able to tolerate heavy metals to synthesize the ZnO-NPs, as it is noted that the high metal stress is well documented to alter various microbial activities (Jain et al., 2020). Under this stress, the microbes reduce metal ions into appropriate metal NPs, demonstrating their potential to act as natural nano-factories (Lahiri et al., 2021). Microbes generally inhabit metal-rich environments and have strong metal resistance owing to their metal chelation and adsorption by intracellular and extracellular proteins (Yusof et al., 2019). As a result, mimicking the natural biomineralization process for synthesizing ZnO-NPs could be considered a promising strategy. Many Zn metal-tolerating microbes have been isolated from native metal-rich soils and mines to synthesize the ZnO-NPs (Jain et al., 2013).

In addition, metal (Zn) precursors (such as zinc acetate, zinc chloride, zinc nitrate, zinc sulphate, etc.) are needed for the microbe-mediated ZnO-NP synthesis. These salts are typically provided as soluble salts precipitating in the microbial cell suspension or its extract with bioactive components during the synthesis, usually taking minutes or hours (Murali et al., 2021b). The successful transformation is indicated by precipitation/ white deposition or gradual changes in the suspension color. Once the synthesis process is complete, the mixture contains microbial cells and ZnO-NPs, separated using various techniques, including centrifugation, filtration, or calcination. In addition, to obtain white crystalline powder of ZnO-NPs, the obtained particles are dried in a hot air oven for an extended time at 60°C after being completely washed with distilled water, followed by ethanol to remove any contaminants that were present (Kundu et al., 2014; Kalpana et al., 2018; Yusof et al., 2019). Different physicochemical methods characterize the synthesized NPs to ascertain their distinctive characteristics, such as shape, particle size, purity, composition, surface charge, and active functional groups. These methods include Ultraviolet–Visible (UV–Vis) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, X-Ray Diffraction (XRD), Dynamic Light Scattering (DLS), Scanning Electron and Transmission Electron Microscopy (Faridvand et al., 2021; Nayeri et al., 2021; Thakur et al., 2022). Furthermore, several factors, like temperature, pH, reaction period and precursor concentration, are essential for determining the production rate, yield, and morphologies of NPs. The microbe-assisted synthesis of ZnO-NPs and their potential biological applications are listed in Table 1.

Usually, the microbes have the inherent ability to synthesize the ZnO-NPs directed via intra or extracellular pathways. Among the two, synthesis of the nanoparticles through extracellular mode is more favorable and has been extensively employed than intracellular synthesis since it can be utilized to synthesize large extent and involves a considerably simple downstream processing protocol that avoids several synthesis steps as well as easy cell separation and industrialization (Kundu et al., 2014; Busi et al., 2016; Sumanth et al., 2020). To obtain NPs with purity, the recovery of intracellular synthesized NPs requires extra purification steps, such as collecting cell biomass and ultrasonication for cell lysis (Yusof et al., 2020b). It has been well documented that the pH affects the synthesis of NPs through surface or intra or extracellular. Although microbial components, such as proteins, enzymes, and other substances, are essential to the synthesis process, only a few chemical elements are involved in NP production (Figure 1).

By giving Pi (π) electrons from carbonyl groups, bioactive chemicals, such as amino acids and polyphenolic compounds, serve as effective reducing agents during ZnO-NPs synthesis. Following the reduction of zinc ions (Zn²⁺) to zero-valent zinc atoms (Zn⁰), the zinc complex is broken down into ZnO-NPs during annealing. The ZnO nano-rods formed are stabilized by certain extracellular proteins secreted by the bacteria to detoxify the stress condition (Tripathi et al., 2014). The investigations showed that the protein’s amino acids interact with Zn²⁺ ions to create NPs, showing that the protein’s natural state is not necessary to create ZnO-NPs (Jain et al., 2013). The interaction between hydrogen bonds may explain the disruption of non-polar hydrophobic molecules during heating. As a result, zinc ions were exposed to amino acids, leading to the formation of ZnO-NPs. Microbial-secreted amino acids have hydroxyl groups that allow the complexation of zinc ions, hydrolysis, and synthesis of ZnO-NPs via thermal decomposition. The amino acids assist in the stability of ZnO-NPs by aggregation inhibition and control of crystal growth (Moghaddam et al., 2017).

With the aid of microbes and water molecules in an acidic milieu, zinc’s complexation commenced the ZnO-NP production process. The development of the zinc aqua-hydroxo complex, which later changed into ZnO-NPs, was caused by the zinc aqua complex accepting an electron from the deprotonated carboxyl group of bioactive molecules (such as enzymes and peptidoglycan) released by the bacteria (Król et al., 2018). In some cases, the specific organic functional groups present on the surface of the cell wall serve as the building blocks for a non-enzymatic synthesis that aids in reducing the zinc ions. The organic molecules (as reducing agents) are released from the microbial cell due to cell membrane rupture and cell lysis during the heat-killed process. The zinc ions made more organic molecules available, leading to a higher reduction (Yusof et al., 2019). The size, chemical composition, shape, solubility, dispersion factor and surface area of NPs have all significantly impacted their biological responses. The NPs synthesis depends on physicochemical factors such as temperature, pH, precursor concentration, age of microbes, reaction time, stirring, and irradiation, which affect the particle size, monodispersity, shape and yield (Jain et al., 2020).

Several microbes have been used for ZnO-NP synthesis, but bacteria are most preferred due to their genetic manipulation capability and ease of handling over other eukaryotic microbes (Jayabalan et al., 2019; Iqtedar et al., 2020; Ahmed et al., 2021). The reproducible bacteria (like lactic acid producing bacteria) have gained more interest in bacterial-mediated NP synthesis because of their high production of diverse enzymes and non-pathogenic nature. The lactic acid bacteria (Gram-positive) has a thick cell wall layer comprising proteins, polysaccharides, lipoteichoic and teichoic acid, etc., which helps in the bioreduction and biosorption of metal ions (Yusof et al., 2020b; Suba et al., 2021). They can also produce exopolysaccharides that defend cells from metal stress and serve as an additional site for metal ion biosorption (Yusof et al., 2020a). Moreover, lactic acid bacteria are recognized as health-beneficial bacteria for their pessimistic electrokinetic potential (Yusof et al., 2020b), which permits them to attract metal ions for the synthesis of NPs under oxidative as well as reductive conditions (Yusof et al., 2020a,b). Selvarajan and Mohanasrinivasan (2013) have reported that moderately stable ZnO-NPs were produced in which the lactic acid bacteria secreted biomolecules acted as capping agents in intracellular synthesis.

Actinomycetes are regarded as superior among commercially important microbial species because of their saprophytic behavior, allowing them to produce various extracellular enzymes and bioactive components. Streptomyces sp. is a member of the actinomycetes, recognized for their soil degrading properties and as the potent source of secondary metabolites, especially antibiotics (Rajivgandhi et al., 2022). Many reports have suggested that actinomycetes can synthesize ZnO-NPs via extracellular and intracellular methods (Shanmugasundaram and Balagurunathan, 2017; Kalaba et al., 2021; Rajivgandhi et al., 2022) and needs to be exploited further to utilize them as an alternative to plants. The advantage of using actinomycetes for NP synthesis lies in their ability to produce specific enzymes and metabolites that can control the size, shape, and stability of the NPs formed. It offers potential advantages in tailoring the properties of ZnO-NPs for specific biological applications.

The mechanical method for producing ZnO-NPs from the fungal culture supernatant or biomass is similar to bacterial-mediated synthesis. But, the fungal-mediated ZnO-NP synthesis is a potential strategy owing to their greater metal tolerance, excellent metal binding capacity, metal bioaccumulation ability and higher productivity over bacteria (Baskar et al., 2013; Gao et al., 2019; Sumanth et al., 2020). In addition, the fungi efficiently secrete an excess of extracellular redox enzymes and proteins as bioactive phytochemicals to the culture media than bacteria, which helps reduce the zinc ions (Zn²⁺) into ZnO-NPs in greater quantities (Fouda et al., 2018; Ganesan et al., 2020). The fungal extracellular enzymes and proteins secreted in the media, which act as reducing and capping agents, are bound and encapsulated on the surface of ZnO-NPs, ensuring their stability without agglomeration (Raliya and Tarafdar, 2013; Sarkar et al., 2014).

The fungal strains are typically grown in sterilized media and incubated at the appropriate temperature to prepare the fungal extracts. The fungal metabolite-enriched cell-free supernatant is then harvested by filtration and centrifugation for further ZnO-NP synthesis (Ibrahem et al., 2017; Abdelhakim et al., 2020; Ganesan et al., 2020). The ability of fungal cell-free extracts to potentially cap and reduce the size of ZnO-NPs formed in a definite size and shape was demonstrated. Thus, the shape of fungal-mediated ZnO-NPs synthesized depends on the species type (Mohamed et al., 2019). The plausible mechanism of fungal-mediated ZnO-NPs synthesis has been postulated that the synthesis process involves the initial transformation of zinc acetate (as a precursor) into ZnO-NPs, followed by capping fungal extracellular proteins on the surface of NPs (Kadam et al., 2019).

Like fungi, yeast has been shown to synthesize the ZnO-NPs because of their better zinc stress tolerance. Specific yeast strains are chosen for their ability to interact with zinc ions and facilitate the reduction of zinc ions to form ZnO-NPs. The yeast cells act as a biological reducing agent, converting the zinc ions in the solution into ZnO-NPs through a reduction reaction. The process is usually carried out under controlled conditions to regulate the size and shape of the NPs. The yeast is preferred due to its availability, ease of handling, and biocompatibility. Moghaddam et al. (2017) have reported that ZnO-NPs (~10–61 nm) formed from Pichia kudriavzevii yeast strain were found to depend on the reaction time, which plays a main role in the size, distribution and shape of NPs. In addition, studies have revealed that ZnO-NP synthesized from P. fermentans extracellularly possessed potent inhibitory effects against various pathogenic bacteria and fungi, thereby contributing to the beneficial effect of the pharmaceutical industry. Due to the toxic metals absorbing and accumulating ability, the diverse yeast species could be used as carriers for synthesizing the ZnO-NPs (Chauhan et al., 2015; Aswathy et al., 2017).

The microbial-mediated ZnO-NPs possess prospective biomedical applications, especially antimicrobial, antioxidant, anticancer, wound healing, drug delivery system, photocatalytic activity, etc., which are described below.

The toxicological effects of microbe-mediated ZnO-NPs are an important area of research and concern. When the NPs are synthesized using microbes such as bacteria, actinomycetes, fungi, or yeast, their potential toxicity can differ from conventionally produced NPs due to size, shape, surface charge, and chemical composition variations. It is important to note that the toxicological effects of NPs can be highly dependent on factors such as specific microbial synthesis methods, distinctive characteristics of NPs, dose, exposure route, and the vulnerability of target organisms. Research on the toxicity of microbial-mediated ZnO-NPs is ongoing, and it is crucial to conduct comprehensive studies to understand and assess their potential risks fully. It was reported that the larger NPs also tend to accumulate in the kidneys for a longer period due to the slower glomerular filtration excretion mechanisms, and this prolonged retention might result in organ damage (Rauf et al., 2017; Sumanth et al., 2021; Faisal et al., 2022). Furthermore, the different morphologies (such as nanorods, nanoflowers, nanosheets and nanoplates) of NPs also contribute to their toxicity due to their larger surface area. The fungal-mediated ZnO-NPs were significantly found to show in vitro cytotoxicity and genotoxicity in human lymphocyte cells, wherein they demonstrated a concentration-dependent decrease in mitochondrial activity at ≥0.5 mg mL⁻¹ and induced DNA fragmentation at 1 mg mL⁻¹ (Sarkar et al., 2014). Many NPs toxicity studies have focused on the plants due to their critical functions in ecosystems as primary producers of organic compounds from atmospheric or aqueous carbon dioxide. Güllüce et al. (2020) have reported that the ZnO-NPs synthesized by Rhodococcus erythropolis using the precursors (such as zinc sulfate heptahydrate, zinc nitrate hexahydrate, zinc chloride and zinc acetate) were found to show their toxicological potentials on Triticum aestivum, where they significantly affected the seed germination and seedling growth along with their genotoxic potentials. If released into the environment, the microbe-mediated ZnO-NPs may interact with living organisms and ecosystems, potentially causing ecological toxicity and disrupting natural processes. Therefore, it is further necessary to investigate any potential long-term toxicological effects of microbe-mediated ZnO-NPs on plants, animals and human health due to these multiple uses in numerous industries. To ensure more effective and safe use of ZnO-NPs, it is also essential to adopt responsible manufacturing practices, perform rigorous toxicity assessments, and follow safety assessments and regulatory guidelines. In addition, Table 7 provides the advantages and disadvantages of microbe-mediated synthesis of ZnO-NPs.

The major drawback of microbe-mediated synthesis of ZnO-NPs is that the microbes may lose their capacity to synthesize NPs because of mutations over time. In addition, not all microbes can produce ZnO-NPs; thus, those potential microbes need to be explored through rigorous screening programs. It is still unknown how exactly microbes synthesize NPs through extracellular and intracellular processes. There is a lack of knowledge regarding the reducing and capping agents involved in bioreduction and stabilization, respectively, during NPs synthesis. Uncertainty exists regarding the identity of reducing and capping agents and their function in influencing both the size and shape of NPs. Particularly because of the requirement of a complete aseptic environment and specific maintenance, the microbe-mediated NPs are not suitable for large-scale production. In the future, extensive optimisation studies are required before scaling up the industrial production of NPs to understand the impact of every aspect completely. Further, cooperation between major stakeholders in basic sciences, chemical engineering, and industry is required to exploit the microbe-mediated ZnO-NPs economically. Future research studies must address the concentration and duration of exposure required for causing the potential toxicologic effects of ZnO-NPs to understand the therapeutic effects better and prevent unintended cytotoxic effects.

The present review highlights the need for green synthesis approaches in producing ZnO-NPs due to the environmental pollution caused by conventional chemical synthesis methods. Using biological sources like plants and microbes makes the process more environmentally friendly and safer. The advantages of microbial synthesis include using bioactive metabolites released by microbes as reducing and stabilizing agents, which enhance the biological properties of the ZnO-NPs. The extracellular biosynthesis method is simpler since the enzymes and proteins outside the cells directly reduce or chelate Zn²⁺ ions. In contrast, the intracellular method requires an additional cell lysis step to release the NPs inside the microbe, making it more expensive and time-consuming. Among the microbial synthesis methods, fungal-mediated synthesis shows promise due to its ability to produce more bioactive compounds, while bacteria have an advantage in cell growth activity.

The review also suggests that microbial synthesis has excellent biotechnological potential and could pave the way for large-scale industrial production of ZnO-NPs. Since the biological components are natural, biodegradable, and safer, this approach is expected to be a promising alternative for plant-mediated ZnO-NPs for future industrial and commercial production. However, further research is still needed to understand the biochemical and molecular mechanisms of NP synthesis among different microbes. Investigating these mechanisms could unlock even more potential applications of microbe-mediated ZnO-NPs in various fields, including medicine, industry, environment, and agriculture. Overall, this review emphasizes the importance of green synthesis methods and highlights the promising role of microbe-mediated synthesis in producing ZnO-NPs with exciting applications in diverse areas. It encourages further research in this area to exploit the full potential of these eco-friendly nanomaterials and extend the next level of their applications.

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