Synergistic antimicrobial mechanisms of silver-doped zinc oxide for water treatment: a systematic review Open Access

In the quest to provide clean and safe drinking water in marginalized societies, point-of-use water treatment (POUWT) is vital (Venis & Basu 2021). POUWT involves the filtration of particulate and organic matter as well as the elimination of harmful microorganisms. The latter can be achieved through a number of processes, including the use of nanoparticles (NPs) with microbial elimination properties. This review paper, therefore, focuses on reviewing the use and mechanisms of metal and metal oxide NPs as microbial elimination materials during water treatment as attributed to their nano size (Jinrun et al. 2019; Murcia et al. 2019; Asamoah et al. 2020; Castro-Muñoz 2020; Al-Gaashani et al. 2023; Ghaffari & Sarrafzadeh 2023; Zhang et al. 2023). NPs are materials with small particle sizes normally ranging from 1 to 100 nm (Makauki et al. 2023). Silver nanoparticles (AgNPs) are among the noble metal NPs that are reported to have excellent antimicrobial properties. They are, therefore, recommended and utilized in water treatment, considering their low toxicity against human cells. Even though AgNPs have low toxicity to human cells, they may cause health hazards if their intake is in large doses. Their concentration in drinking water must not exceed the limits of the World Health Organization (WHO); which should be below 0.1 mg/L (Alherek & Basu 2023).

On the other hand, ZnO, as an eco-friendly semiconductor metal oxide, has been recognized as a non-toxic chemical listed by the US Food and Drug Administration (21CFR182.8991) as a ‘generally recognized as safe’ material (Raj Kumar & Gopinath 2017; Motelica et al. 2020). ZnO is characterized by a wide band gap of about 3.37 eV and a high exciton-binding energy of (60 meV), which is the energy required to dissociate from excited electron-hole pair into free charge carriers. Its broader absorption range and higher quantum efficiency under solar light influence its unique antimicrobial properties (Oualid et al. 2017; Murcia et al. 2019). Due to its wide band gap, ZnO is used as a heterogenous photocatalyst and antimicrobial material (Agnihotri et al. 2015; Bednář et al. 2019). When ZnO is exposed to ultraviolet (UV) light or sunlight with a wavelength of less than 385 nm, it absorbs the light energy and causes its valence electrons to jump (excited) to a higher energy level referred to as the conduction band leaving behind positive holes (h⁺). In this state, the electrons (e⁻) and the positive holes (h⁺) create electronhole pairs that react with oxygen and hydroxyl groups in water to generate reactive oxygen species (ROS). ROS create oxidative stress in bacteria, which damages their cells and eventually leads to their death (Gupta et al. 2017; Jha et al. 2023).

Although ZnO is an outstanding antimicrobial material due to its wide band gap, the photoexcited electrons in the conduction band recombine quickly, lowering its microbial elimination efficiency. This is also caused by its inefficiency in absorbing and utilizing energy in visible light as it favours UV light absorption (Ma et al. 2019). Different attempts to improve ZnO microbial elimination efficiency have been made to maximize the existence of the electron–hole pair by extending its recombination time. Among the techniques to achieve this is doping ZnO with metals to form metalmetal oxide nanocomposites (NCs), which improves the antimicrobial activity of ZnO compared with its standalone application (Li et al. 2014; Bednář et al. 2019; Alherek & Basu 2023). For instance, AgNPs doped in the ZnO lattice provide surface plasmon resonance (SPR) in the ZnO lattice and reduce the recombination of the ZnO charge carriers/lone pairs. This creates more room for ZnO to absorb visible light (Manna et al. 2015; Ma et al. 2019; Zyoud et al. 2023). The reduced recombination time also creates enough time for the positive holes (h⁺) and electrons (e⁻) to react with oxygen and hydroxyl radicals and generate ROS (Bazant et al. 2014; Li et al. 2014; Dutta et al. 2023). Different studies have reported a narrowing down in the ZnO band gap doped by AgNPs. Asamoah et al. (2020) reported a reduction in the ZnO band gap to 2.88 eV from 3.36 eV for the silver-doped ZnO. Jha et al. (2023) reported the decrease of the ZnO band gap to 3.23 eV when it was doped by Ag. The narrowing down of the ZnO band gap correlates with improved antimicrobial efficiency, a good sign for enhanced photocatalytic and antimicrobial activity. Some studies have reported a reduction of a tremendous amount of antimicrobial material when used as composites while achieving improved antimicrobial activity (Sher et al. 2021; Zhang et al. 2023). The improvement of microbial elimination, narrowing down of the band gap, and the reduced number of materials used in elimination clearly state the importance of doping Ag into ZnO. This brings the use of these materials to a sustainable level while bringing about improved microbial elimination to prevent unnecessary waterbone disease infection.

Although Ag/ZnO has a higher antimicrobial activity than Ag and ZnO used individually, their antimicrobial mechanisms are not fully understood. Different mechanisms have been reported by different researchers, including the involvement of ROS, silver (Ag⁺) and zinc (Zn²⁺) ions, as well as the direct contact of the microbial cells with the NCs, with each species having different attack mechanisms. Moreover, the microbial elimination relationship between Ag and ZnO needs more investigation. This will ensure an efficient utilization of these materials during water treatment. It will also help researchers develop proper Ag: ZnO ratios for the optimized microbial elimination in terms of time and amount of composite to be used. Therefore, this systematic review aims to provide a comprehensive overview of current knowledge on the existing relationship between Ag and ZnO as microbial elimination agents when used together. This review provided the depth to which the study is covered and highlighted gaps that still need to be studied. Additionally, the review reports on the mechanism of Ag/ZnO NCs on the antimicrobial activity against gram-negative and gram-positive bacteria.

Standard Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) was used to collect relevant information required in this review (Mwalongo et al. 2024). Six core systematic literature review procedures listed below were implemented in the process to eliminate bias, recognize irrelevant information, and improve information repeatability and reliability.

• Developing a proper research question.

• Formating search keywords using Boolean operators.

• Choosing reliable search databases.

• Limiting the article's publication to the selected time interval.

• Importing the articles' abstracts to Excel for relevant screening.

• Extraction, grouping, and analysing the data.

This systematic review was based on the research question ‘What are the antimicrobial mechanisms of silver-doped zinc oxide?’. This question established three key concepts: antimicrobial mechanism, silver-doped zinc oxide, and water. A Boolean operator was used to connect the keywords and generate the strings for the first two concepts (Bramer et al. 2018). The third concept was used as it is. PubMed, Scopus, and Web of Science databases were used as search engines. The strings were set according to the search engine requirements and used accordingly.

The search string for PubMed database was ‘Protein Synthesis Inhibitors’[Mesh] OR ‘antimicrob* mechanism*’[tw] OR ‘antibacter* mechanism*’[tw] OR ‘microb* inhibition*’[tw] OR ‘bacter* inhibition*’[tw] OR ‘microbi* remov*’[tw] OR ‘bacter* remov*’[tw] OR ‘disinfect* mechanism*’[tw] OR ‘disinfect*’[tw] AND ‘silver doped zinc oxide’[tw] OR ‘silver zinc oxide’[tw] OR ‘Ag ZnO’[tw] OR ‘Ag/ZnO’[tw] OR ‘ZnO/Ag’[tw] OR ‘Ag–ZnO’[tw] OR ‘AgZnO’[tw] OR ‘ZnAgO’[tw] OR ‘AgZn’[tw] AND water[tw].

‘MeSH’ is the abbreviation for Medical Subject Headings as used in PubMed during the search, retrieving all publications on such particular subjects even in terminology other than those used by the authors (Terwee et al. 2009; Bramer et al. 2018). ‘tw’ is the abbreviation for ‘text words’ as used in PubMed. This instructs the search engine to search the word in published articles (Terwee et al. 2009).

The search string for the Scopus database was ‘Protein Synthesis Inhibitors’ OR ‘antimicrob* mechanism*’ OR ‘antibacter* mechanism*’ OR ‘microb* inhibition*’ OR ‘bacter* inhibition*’ OR ‘microbi* remov*’ OR ‘bacter* remov*’ OR ‘disinfect* mechanism*’ OR ‘disinfect*’ AND ‘silver doped zinc oxide’ OR ‘silver zinc oxide’ OR ‘Ag ZnO’ OR ‘Ag/ZnO’ OR ‘ZnO/Ag’ OR ‘Ag–ZnO’ OR ‘AgZnO’ OR ‘ZnAgO’ OR ‘AgZn’ AND water.

The search string for the Web of Science database was ((‘Protein Synthesis Inhibitors’ OR ‘antimicrob* mechanism*’ OR ‘antibacter* mechanism*’ OR ‘microb* inhibition*’ OR ‘bacter* inhibition*’ OR ‘microbi* remov*’ OR ‘bacter* remov*’ OR ‘disinfect* mechanism*’ OR ‘disinfect*’) AND (‘silver doped zinc oxide’ OR ‘silver zinc oxide’ OR ‘Ag ZnO’ OR ‘Ag/ZnO’ OR ‘ZnO/Ag’ OR ‘Ag–ZnO’ OR ‘AgZnO’ OR ‘ZnAgO’ OR ‘AgZn’) AND (water)).

The PubMed, Scopus, and Web of Science database searches included 251 articles. During the first screening process, the number of possibly relevant publications was reduced to 208. The second filtering eliminated 42 more articles that were repetitions from different databases. The third screening excluded 112 articles, including 1 retracted article. Therefore, only 53 articles were used for this review work. The inclusion and exclusion criteria used were:

• Available full text for review.

• The article should contribute to the knowledge of the antimicrobial mechanism and synergy of silver zinc oxide NCs.

• The document type must be a peer-referred or -reviewed article.

• The article should have been published in 10-year intervals from December 2023 (2014–2023).

• The subject area was not restricted.

• The article is published in the English language.

The articles acquired based on inclusion and exclusion criteria were further evaluated. Each article was independently reviewed by each author, taking into account the information of interest. The data collected were recorded in the data collection sheet created with the following items:

• Names of the authors.

• Article titles.

• Year of publication.

• Ag/ZnO synthesis method and route.

• The morphology of the NC.

• The average particle size of the NC.

• The efficiency of the NC on microbial inhibition.

• Mechanisms of microbial inhibition.

• The effect of Ag on ZnO antimicrobial efficacy.

The ⁠, ⁠, and H₂O₂ are referred to as ROS and are the main ZnO microbial elimination route.

Although ZnO produces ROS as its main microbial elimination species, its efficiency can be limited as it favours the absorption of UV light to that of visible light utilized due to the rapid recombination of its photoexcited electronhole pairs (Ma et al. 2019). This can be improved by extending the recombination time of the photoexcited hole pairs and increasing their charge separation time (González-Penguelly et al. 2017; Jinrun et al. 2019). This phenomenon allows full utilization of visible light by ZnO and improves its ability to generate ROS and microbial elimination. Doping of noble metals, such as silver (Ag), gold (Au), and lead (Pb) in the ZnO lattice, assists in ZnO, improving its microbial elimination with Ag coming out as the most efficient dopant metal.

Asamoah et al. (2020) doped silver into ZnO and realized a reduction of the ZnO band gap to 2.88 eV and improved its antimicrobial efficiency. They reported the total elimination of Escherichia coli and Staphylococcus aureus as a result of doping silver in the ZnO lattice compared with the undoped ZnO, which had negligible elimination on both bacteria strains after 24 h of treatment (Asamoah et al. 2020). Jha et al. (2023) reported a decrease in the ZnO band gap to 3.23 eV when it was Ag-doped. The antimicrobial study reported negligible bacteria elimination (9.85%) by ZnO against E. coli compared with that of Ag–ZnO, which had 99.9% E. coli elimination after 8 h of treatment. Both treatments were conducted under UV irradiation (Jha et al. 2023). Generally, narrowing down the ZnO band gap improved the antimicrobial efficiency of both gram-negative and gram-positive bacteria (Sher et al. 2021; Zhang et al. 2023).

A study by Chang et al. reports ROS generation of Ag/ZnO to be more than twice when compared to Ag and ZnO separately (Chang et al. 2019). Darwish et al. further emphasized the synthesized Sm-doped ZnO had a 3.16 eV energy band gap with limited photoactivated water disinfection linked by low ROS formation. Furthermore, when Ag was doped to form Ag (Ag-Sm-doped ZnO), a narrower 2.12 eV energy band showed an increase in 100% ROS generation and improved disinfection (Darwish et al. 2018). The energy band becomes narrower with an increase in Ag concentration, indicating the importance of Ag in ROS formation (Gao et al. 2020; Niu et al. 2020; Sher et al. 2021). These findings clearly show the importance of Ag in narrowing down the zinc oxide band gap. It is necessary to further study how the amount of Ag added to ZnO can affect the narrowing down of this band gap and its efficiency in microbial elimination.

The ROS formed are the main Ag–ZnO microbial elimination routes through a number of reactions. ROS attack the cell wall lipopolysaccharide layer of bacteria by introducing oxidative stress that causes the degeneration of the peptidoglycan layer. This further causes peroxidation of the lipid membrane, resulting in the oxidation of the membrane proteins followed by dysfunction of the potassium channels, deregulation of cell signalling, and accelerated K⁺ ion leakage. On the other hand, it causes peroxidation of polyunsaturated phospholipid components of the cell membrane, which adds to the deterioration of cell functionalities, causing cell death (Das et al. 2017; Primo et al. 2022). The ROS also causes Deoxyribonucleic acid (DNA) and enzyme damage, cell rupture (Mohamed et al. 2021), bacterial filamentation (Tian et al. 2017), and cavity formation on the cell wall (Ma et al. 2019). Cell rupture contributes to the leakage of intracellular components and disrupts cell membrane's integrity (Das et al. 2017; Raj Kumar & Gopinath 2017). The destruction of the E. coli cell morphology was observed within 120 min (Ma et al. 2019) as ZnO ROS formation was synergistically affected by Ag (Mohamed et al. 2021). Progressive cell membrane disintegration through ROS was observed through an increase in Malondialdehyde (MDA) levels, a biomarker of lipid peroxidation (Das et al. 2017) causing irreversible cell damage (Sinha et al. 2021).

⁠, h⁺, and are the main ROS-contributing species to microbial elimination (Chatterjee et al. 2017; Niu et al. 2020; Ismail et al. 2023). The higher the amount of ROS, the higher the microbial elimination. For instance, a study reported more ROS generation by Ag/ZnO nanowires (Ag/ZnO-NW) compared to ZnO-NW, leading to superior antimicrobial performance, with 100 and 95% removal of E. coli and S. aureus, respectively. In contrast, ZnO-NW achieved 95 and 50% removal rates (Tian et al. 2017). Similarly, it was further observed that the production of higher levels of hydroxyl radicals by AgZnO NCs than ZnO correlates with its significantly improved microbial elimination (65%) compared to that of ZnO (10%) on E. coli (Zhang et al. 2023). The results are further supported as silver-doped ZnO impregnated in activated carbon (CAZ) exhibited superior antibacterial performance than ZnO and Ag⁺ ion DNA (Sinha et al. 2021). Ma et al. highlighted h⁺ as a primary contributor to microbial disinfection, followed by ⁠, with minimal involvement of (Ma et al. 2019).

Furthermore, ROS formation is affected by the treatment conditions. Some studies conducted under dark environments reported silver-doped ZnO NCs to demonstrate remarkable microbial elimination (Ying et al. 2022), while other studies presented negligible elimination (Jinrun et al. 2019). The microbial elimination is pronounced up to 100% under UV light, as UV introduces photocatalysis, an added mechanism for the improvement of ROS formation (Manna et al. 2015; Jinrun et al. 2019; Niu et al. 2020; Panchal et al. 2020).

Overall, the studies emphasize the importance of using silver to increase the ability of zinc oxide to generate ROS. It further emphasizes the importance of ROS in microbial elimination through a number of routes. In some cases, conditions affecting the AgZnO microbial elimination efficiency reported contradicting results. This highlights the need for more investigation into factors influencing antimicrobial efficacy in varying environmental conditions.

The Ag–ZnO composite can eliminate microbes through photocatalytic (related to light) and non-photocatalytic (not related to light) antibacterial activities. The non-photocatalytic microbial eliminations include attacks through silver (Ag⁺) and zinc (Zn²⁺) ions (Niu et al. 2020). This microbial elimination takes place even during water storage (Huang et al. 2020). Ag⁺ and Zn²⁺ ions have a cytotoxic effect on the bacteria cells as they can inhibit the enzymatic system of the respiratory chains and damage them (Patel et al. 2019). The metallic ions interact with the bacterial cell membrane, making it more porous and permeable for the NCs to enter the microbial cell (Gupta et al. 2017).

The metallic ions can change DNA synthesis and compromise cell integrity (Sinha et al. 2021). Zn²⁺ ions enter the microbial cell through Zn²⁺ import channels and then bond with Mn importers, leading to Mn starvation. This causes DNA and respiration damage to the cell (Alherek & Basu 2023). On the other hand, Ag⁺ ions can bind to the metabolic enzymes of the bacterial cell through their sulphydryl groups, causing their inactivation and cell death as they interfere with the bacterial electron transport chain (Nakhjavani et al. 2017). The positive Ag⁺ ions easily react with electron-rich cellular biomolecules such as nitrogen, phosphorus, and sulphur to destabilize significant cell processes (Mendes et al. 2013; Bednář et al. 2019). Since Ag⁺ and Zn²⁺ ions have different microbial elimination mechanisms, they contribute to synergetic antimicrobial activities resulting in improved outcomes (Li et al. 2021). Ag⁺ and Zn²⁺ ions in treated water results in microbial elimination after treatment during storage. It has been reported that, at low ionic concentrations, there was more than 99.7% bacterial elimination on both E. coli and S. aureus after 48 h in water (Alherek & Basu 2023). There was an increase of microbial elimination with time increase due to prolonged interaction between the metallic ions and the bacteria cells (Asamoah et al. 2020). This necessitates further investigation into materials to determine the optimal treatment time required for complete microbial elimination in water. Enhancing the quality and efficiency of materials is also critical to achieving 100% microbial elimination within the designated treatment timeframe.

In a synergetic effect study by Venis and Basu, the effluent water from the ceramic water filters impregnated with Ag and ZnO NPs was confirmed to have Ag⁺ and Zn²⁺ ions. These ions had an antimicrobial effect on E. coli for more than 48 h during storage. This is an added advantage as the ions prevent the secondary microbial infection during storage. This study tested 100% AgNPs, 67% AgNPs:33% ZnO, 67% ZnO:33% AgNPs, and 100% ZnO against E. coli-contaminated water. After 24 h, the log removal values (LRVs) were 1.6 ± 0.63, 1.9 ± 0.25, and 2.9 ± 0.25 for the first three samples, respectively. Approximately 100% ZnO had no significant bacterial removal. Ionic concentrations in the effluent water had a positive effect on the LRVs. The 0.5 ppb Ag⁺ and 9.4 ppb Zn²⁺ had an LRV of 4.5, while 1.4 ppb Ag and 15.3 ppb Zn achieved an LRV of 5.6. ZnO, which had insignificant bacterial removal, had low Ag concentrations, indicating a synergy between the two. O.15 ppb Ag with 13.2 ppb Zn achieved 4.3 LRV in 48 h. Moreover, O.21 ppb Ag with 19.1 ppb Zn achieved 5.5 LRV in 48 h. Sorption of Zn²⁺ by the bacterial cell reduces the bacterial cell's replication. Zn²⁺ also weakens the cell, making it vulnerable to an Ag⁺ attack (Venis & Basu 2023).

Silver zinc oxide doped in chitosan-bentonite composite had a lower release of Ag⁺ and Zn²⁺ ions compared with chitosan-bentonite composite doped by the individual NPs. From this study, the composite helps to control ions released. The ionic concentration in effluent water was below WHO Ag⁺ and Zn²⁺ ion limits. Ag⁺ and Zn²⁺ ions in effluent water indicated the involvement of the metallic ions as one of the disinfection mechanisms. It also saved the secondary microbial elimination during storage (Bazant et al. 2014).

Particle size significantly impacts the antimicrobial activity of the composites as smaller particles enhance the release of metallic ions, facilitating their diffusion into bacterial cells (Rajaboopathi & Thambidurai 2019). A study reported how uniform distribution of Ag NPs on ZnO nanowires controlled Ag NP particle size and release of the Ag⁺ ions which prolonged the antimicrobial effect of the nanomaterials and also provided an after-treatment effect (Li et al. 2014; Agnihotri et al. 2015; Tian et al. 2017). Ag NPs with 4.1 ± 1.9 nm size evenly distributed on ZnO nanowires have a higher antimicrobial efficacy due to the high and controlled release of Ag⁺ ions, caused by the high surface area and small particle size (Li et al. 2014).

Ma et al. (2019) reported cell integrity destruction by Ag/ZnO/g-C₃N₄. The normal rod-shaped morphology of E. coli cells with a smooth and intact surface membrane changed with treatment time. In the first 15 min of exposure, the cells exhibited wrinkles, small bulges, and pits developing into small pits after 30 min, and extensively cracked cell walls and membranes after 60 min. After 90 min, the cell membranes were extensively fractured and destroyed after 120 min, as it was difficult to identify the cell membranes. It was further reported by Ma et al. (2019) that Ag/ZnO/g-C₃N₄ had a more bactericidal effect than ZnO/g-C₃N₄ and Ag/g-C₃N₄ as evidenced by the fluorescent-based cell live/dead test investigations. Most of the bacteria eliminated by the ZnO/g-C₃N₄ and Ag/g-C₃N₄ were in direct contact with the composite; the free cells were still alive, unlike with Ag/ZnO/g-C₃N₄, where all the bound and free bacterial cells were eliminated due to the synergetic effect between Ag and ZnO in the composite (Ma et al. 2019). Pit formation observed from the SEM images indicated cell damage through NP internalization. As reported by Agnihotri et al., the internalized NPs significantly accumulate as aggregates in the periphery of the cell membrane and inside the bacterial cells, inhibiting bacterial growth through multiple mechanisms, such as ROS generation, blocking cell respiration, and inhibiting DNA replication. In this study, Zn was not detected in any of the treated bacterial cells, suggesting it is a carrier for Ag NPs (Agnihotri et al. 2015).

Although silver zinc oxide NC has high efficiency in microbial elimination, it can be affected by a number of factors such as particle size, concentration and surface morphology. The particle size of NCs determines how easily will the NPs be internalized into the bacteria cell through the cell membrane (Bazant et al. 2014; Patel et al. 2019; Makauki et al. 2023). The smaller the NPs, the higher the number of atoms on their surface, the easier the internalization and the higher antibacterial activity (Asamoah et al. 2020). Studies reported high efficiency of ZnO and ZnO-CA against S. aureus and E. coli than Ag–ZnO-CA due to their small particle size. From this study doping of Ag into the composite had no significant effect as the Ag NPs agglomerated and reduced the contact sites to the bacteria (Motelica et al. 2020; Mousa et al. 2024). The same condition was observed when ZnO:Ag 1%, ZnO:Ag 3%, and ZnO:Ag 5% exhibited 95, 94, and 91% microbial elimination as the particle size increased, respectively (González-Penguelly et al. 2017).

The NCs' surface morphology can also contribute to microbial elimination through physical contact. Doping of the Ag/ZnO on other materials has also shown a positive effect as it tends to affect the surface structure of the NPs. Ag/ZnO/Ch beads have rough surfaces, aiding microbial disinfection through cell rapture through physical contact (Chatterjee et al. 2017). Decoration of the ZnO/Ag NPs into the graphene oxide (GO) upgrades the dispersion, roughness, and hydrophilicity of the GO, which then eliminates the bacteria through physical contact. Second, the release of the NP is optimized due to the improved dispersion and control of NP size (Ghaffari & Sarrafzadeh 2023).

The concentration of the NPs has an antimicrobial effect on the inhibition process. Different studies have reported an increase in Ag concentration as a dopant in the AgZnO composite to have increased microbial elimination due to the increased release of Ag⁺ and ROS generation (Oualid et al. 2019; Mtavangu et al. 2022; Saratovskii et al. 2022; Makauki et al. 2023). An AgZnO composite synthesized with different Ag doping concentrations, 0.84, 1.68, and 2.98 wt%, had a relative microbial inhibition increase on E. coli and S. aureus. (Oualid et al. 2017). Gupta et al. (2017) also reported increased E. coli cell growth inhibition by increased silver doping concentration. In this study, 1, 5, and 10 AgNO₃ wt% were used. 1% silver-doped composite had no significant bacterial inhibition but the 5 and 10% had significant inhibition with the lowest used masses of 12.5 μg mL⁻¹ (Gupta et al. 2017). The study observed an increase in cell membrane deformity and disruption with the increasing Ag–ZnO NC concentration. On the other hand, the higher inhibition concentrations of Ag/ZnO, especially in the light environment, can decrease the disinfection rate by reducing the percentage transmittance of sunlight (Das et al. 2015).

It is, therefore, crucial to consider key factors in the synthesis of NCs to optimize their performance. Parameters, such as doping concentrations, particle size, surface morphology, and treatment concentrations, must be carefully monitored to ensure efficient material synthesis and effective water treatment applications.

Studies in this review demonstrate that Ag-doped ZnO composites have improved microbial inhibition compared to the individual application of the same NPs. This is caused by the different mechanisms through which Ag and ZnO attack microbial cells. Ag is responsible as an electron sinker to increase the efficiency of ZnO to generate ROS, while Zn weakens and reduces the cell's capacity to replicate making it vulnerable to Ag⁺ attack (Venis & Basu 2023). Zn²⁺ ions cause Mn starvation in the microbial cells, while Ag⁺ causes the inactivation of the sulphhydryl groups of the metabolic enzymes (Nakhjavani et al. 2017; Alherek & Basu 2023). The combination of the different attack routes brings improved antimicrobial activity. The combined use of these nanomaterials in water treatment requires careful consideration to prevent adverse effects on users. Compliance with national and international standards, such as WHO guidelines, is essential to safeguard public health. Therefore, it is necessary to quantitatively evaluate the synergy between Ag and ZnO in water treatment applications. This analysis would help determine optimal ratios that ensure both effective treatment and adherence to established standards.

This study found a synergetic relationship when Ag and Zn were co-applied on E. coli cells. When 10 ppb of Ag and 50 ppb of ZnO were individually used, they had no significant disinfection against E. coli in the 48 h but elimination when co-applied (Kaur et al. 2019; Alherek & Basu 2023).

In this study, when the composites were used, the antimicrobial agent mass was reduced up to 54.80, 29.55, 55.07, and 55.07% compared to the mass used when the individual NPs were used. This explains a great synergy between the Ag and Zn when used together (Bednář et al. 2019). The composite reduces the use of the individual NCs and their concentration in the treated water. By doing so, the material costs are also reduced.

Venis & Basu (2021) reported the 2:1 ratio AgZnO (0.67 Ag and 0.33 ZnO mg/L) had 3.18 ± 0.23 LRV after 300 min and 7.4 pH. This LRV was 670.0% (1.4 LRV) of that of silver and 173.3% (0.66 LRV) of that of ZnO when individually applied. The results indicate a great synergy when the two metallic nanomaterials were used together. In addition, the authors reported the higher the dissolved oxygen, the higher the LRV, highlighting the importance of oxygen in ROS generation. The LRV decreased in natural water treatment due to the presence of organic matter, which binds around the NPs, reducing their chances of interacting with the cell membrane and the metallic ion release rate (Venis & Basu 2021). The SEM images of untreated E. coli cells were intact with strong visible contrast with the background surface. The treated E. coli cells had a lower contrast with elongated and twisted structures. They also reported pit formation on the edges of E. coli-treated cells, indicating further damage to the cells. The number of pits formed was higher on cells treated with Ag and ZnO, indicating their synergetic relationship (Venis & Basu 2021). The treated cells' SEM images by Ag, ZnO, or Ag–ZnO have demonstrated the strength and synergy of Ag–ZnO on bacterial destruction. ZnO-treated cells were in a poor living state with some deformation. The cell walls remained intact. Ag treatment caused cell walls to shrink significantly with cytoplasmic leakage (Li et al. 2021).

Another study by Venis and Basu on ceramic water filters impregnated with Ag with/without ZnO reported a synergetic effect of the two metallic NPs in the disinfection process: 100% AgNPs, 67%AgNPs:33% ZnO, 67% ZnO:33% AgNPs, and 100% ZnO were used for the study. After 24 h of interaction with 10⁵ colony forming units per mL (CFU/mL) E. coli the LRVs were 1.6 ± 0.63, 1.9 ± 0.25, and 2.9 ± 0.25 for the first three, respectively, while 100% ZnO had no significant bacterial removal. Ag⁺ and Zn²⁺ concentrations in the effluent water had a positive effect on the LRVs as 0.5 ppb Ag⁺ and 9.4 ppb Zn²⁺ had LRV equal to 4.5, while 1.4 ppb Ag and 15.3 ppb Zn achieved LRV of 5.6. Even with the low disinfection capacity of ZnO, it demonstrated a positive effect in the presence of low concentrations of Ag, indicating their synergy. Zn sorption weakens and reduces the cell's capacity to replicate, making it vulnerable to an Ag⁺ attack (Venis & Basu 2023). In the CFU study with 1 × 10⁶ mg/mL E. coli, ZnO eliminated 58.1% while AgZnO eliminated 99.5% in the first 1 h. Zn achieved 99.6% elimination after 5 h, while AgZnO had elimination after 2 h. The antibacterial activity of AgZnO is synergistically two to four orders higher than that of ZnO (Li et al. 2014).

The two studies have significantly presented the synergetic effect brought by Ag into the ZnO lattice. This reduces not only the amount of ZnO used but also that of Ag. Very small amounts of these materials with no microbial elimination effects when used separately would bring noticeable and remarkable outcomes when used together.

ZnO is a metal oxide that is a good microbial-killing agent, especially on exposure to sunlight. Its exposure to sunlight leads to the formation of ROS, which are responsible for microbial elimination. To improve its ROS formation, ZnO is doped with silver (Ag). The resulting composite (silver zinc oxide) has higher efficiency for microbial elimination than ZnO itself. The microbial elimination mechanism by silver zinc oxide (Ag–ZnO) can take place through the generated ROS, which is the main route. It can also take place through its Ag⁺ and Zn²⁺ ions as well as the direct and physical contact of the NCs with the microbial cell. These materials affect the microbial cell through increased cell membrane porosity, internalization of the NPs, DNA damage, enzyme damage, cell rupture, and blocked cell functions, which lead to cell death. The effectiveness of these NCs can be affected by particle size, treatment, and doping concentrations, as well as cell morphology.

The studies have reported a positive (synergetic) relationship between Ag and ZnO when applied together, and the quantitative presentations are provided through the Bliss model. The relationship has led to the application of fewer materials then they would be used individually. Moreover, amounts that did not show microbial elimination when individually used had significant microbial elimination up to six times (670%).

Apart from the strengths presented, there is a lack of studies that explain how the quality of water under study would affect the NCs’ microbial elimination synergy. This would bring a meaningful and optimized use of the Ag/ZnO NCs, including customization according to the water characteristics.

Natural water research is lacking as many studies reported lab test studies using either plate (inhibition) experiments or synthetic water. More studies are to be conducted on natural water to evaluate real treatment challenges and improve the use of the materials.

There is limited research on the green/biological synthesis of Ag/ZnO NCs, unlike chemical synthesis. Among the 53 studies involved, only 7 studies had the materials green/biologically synthesized. Green synthesis produces clean and non-toxic NCs that can be used for human consumption, such as water treatment. This study encourages more research on green synthesis for a sustainable supply of clean and safe Ag/ZnO NCs for water treatment to avoid health challenges accompanied by the chemically synthesized materials.

The African Development Bank (AfDB) and Higher Education Economic Transformation (HEET) projects through the Nelson Mandela African Institution of Science and Technology (NM-AIST) for a PhD scholarship. The Queen Elizabeth Scholarship-Advanced Scholars program (QES-AS) through the Canadian Queen Elizabeth II Diamond Jubilee Scholarship program funded by Canada's International Development Research Centre [IDRC]. The Erasmus Plus International Credit Mobility Action – student mobility program under the collaboration of the Nelson Mandela African Institution of Science and Technology (NM-AIST) and the University of Milano Bicocca Milan, Italy for the mobility program to the University of Milano Bicocca Milan. Canada Scholarship (SICS) program – Student Exchange Program (SEP) under Global Affairs Canada (GAC) and Carleton University, Canada for the visiting scholarship to Carleton University.

E. M., O. B., M. R., and R. M. conceptualized the process; E. M., O. B., M. R., and R. M. developed the methodology; E. M. rendered support in formal analysis; E. M. investigated the work; L. S., A. L., and M. M. provide the resources; L. S., A. L., M. M. rendered support in data curation; E. M. wrote the original draft; O. B., M. R., and R. M. reviewed and edited the article; E. M., O. B., M. R., and R. M. visualized the work; O. B., M. R., and R. M. supervised the study; E. M., O. B., M. R., and R. M. rendered support in project administration. All authors read and approved the final manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.