Unveiling the Role of the Microalga Nannochloropsis gaditana in the Biogenic Synthesis of Zinc Oxide

Abstract

The increasing demand for environmentally friendly and sustainable approaches to materials synthesis calls, inter alia, for the development of biogenic methods to produce inorganic compounds by exploiting biological macromolecules and organisms. This study focuses on the optimization of a one-pot green synthesis of zinc oxide (ZnO) particles using microalgae extracts as a biogenic agent. Microalgae serve as an environmentally friendly platform for biotechnological applications due to their ability to promote the synthesis of valuable chemicals, thanks to their active components, such as enzymes. A systematic investigation of the experimental parameters revealed that both the reaction temperature and the concentration of microalgae extract significantly influenced the crystallite size of ZnO nanoparticles. In addition, the role of sodium hydroxide as a precipitating agent when used in combination with microalgae extract was addressed and compared with existing literature. The results indicate that microalgae extract can act as a scaffold to promote the controlled growth of ZnO particles. Antimicrobial tests also showed that ZnO particles synthesized with microalgae exhibited comparable antimicrobial activity with respect to ZnO produced by conventional methods. These results highlight the potential of microalgae as biogenic agents for the green synthesis of ZnO particles with tunable structural and antimicrobial properties.

 

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Keywords

Green chemistry - Zinc oxide - Microalgae - Biogenic

Significance

• This study presents a sustainable, water-based method for synthesizing ZnO particles using microalgae extract, eliminating the need for chemical precipitating agents such as sodium hydroxide. This green approach supports the UN Sustainable Development Goal (SDG) 12 on responsible consumption and production.

• Microalgae promote controlled growth and stabilize ZnO, in line with SDG 9 on innovation in green technologies.

• Biogenic synthesis enhances the antimicrobial properties of ZnO, contributing to SDG 3 on health and well-being.

Introduction

In recent years, both the scientific community and the public at large have recognized the need to move toward more sustainable approaches for the synthesis of chemical products and materials to reduce their environmental impact. This shift underlines the urgent need to minimize, if not eliminate, the use of hazardous and polluting chemicals, while promoting the use of alternative, more sustainable energy sources and processes for manufacturing and recycling. In recent times, particular attention has been paid to the optimization of synthetic routes in line with the principles of green chemistry to produce inorganic nanomaterials with relevant functional properties. The term “green chemistry” refers to the “design of chemical products and processes to reduce or eliminate the use and generation of hazardous substances”[1] and has been guided by the Twelve Principles of Green Chemistry introduced by Paul Anastas and John Warner in 1998.[1] These principles are guidelines designed to help scientists strive for sustainability in chemical practice. Among the various synthesis methods, wet chemistry approaches are particularly promising in the context of green chemistry,[2] [3] as they are typically carried out under mild conditions of temperature and pressure. Wet chemical methods for the synthesis of crystalline metal oxide nanoparticles are known for their simplicity and low cost. However, the current challenge is to further reduce processing temperatures to lower energy consumption, a critical factor in achieving sustainable, environmentally friendly, and competitive manufacturing processes with a low carbon footprint. A promising low-temperature strategy to produce metal and metal oxide nanoparticles is biogenic synthesis, which adheres to eight of the twelve principles of green chemistry.[2] This approach minimizes waste, limits the use of hazardous chemicals, and ensures the production of safe, nonpersistent materials, all while reducing energy consumption. Biogenic synthesis relies on naturally occurring biological agents, such as isolated enzymes, microorganisms, yeasts, amino acids, fungi, algae, microalgae, and plant extracts, which serve either as reductants or as green scaffolds in the formation of various inorganic nanoparticles, both metallic as well as binary or ternary compounds.[4] [5] Biogenic synthesis has been widely used for the synthesis of different metallic nanostructures,[6] [7] [8] as well as oxides,[9] [10] [11] sulfides,[12] [13] [14] and other inorganic compounds.[15] In this work, we focus on microalgae extract as a biogenic agent. Microalgae are small photoautotrophic organisms, only a few micrometers in size. They are emerging as valuable cellular factories for producing commercial products and are gaining attention in the synthesis of inorganic nanoparticles due to their diversity and availability.[16] [17] It is worth noting that biogenic synthesis is related to bioremediation but serves a different purpose: while biogenic synthesis focuses on using biological organisms to produce materials in a more sustainable way, bioremediation uses organisms to remove, degrade, or neutralize pollutants in the environment.[18]

Despite the growing interest in biogenic synthesis, the mechanism of formation of metal oxide nanoparticles is not yet fully understood, and conflicting and missing information is widespread in the literature. Concerning the biogenic synthesis of ZnO, many studies report the use of NaOH in combination with microalgae extracts for ZnO synthesis,[10] [19] but it remains unclear whether the reaction is driven by NaOH or by the microalgae. The controlled nucleation and growth of ZnO nanocrystals from zinc solution and NaOH have been well documented,[20] but the specific role of the biogenic agents in these processes requires further clarification. While, as mentioned, there is extensive research on the biogenic synthesis of metal nanoparticles (e.g., gold and silver) via chemical reduction by biogenic agents,[4] [7] the formation mechanisms of metal oxides, such as ZnO and other compounds (e.g., Cu _x O, Fe _x O _y ), are still not fully understood, and a templating role of the biogenic agents is hypothesized.[6]

The aim of this study is to optimize a one-pot approach for the green synthesis of ZnO micro- and nanoparticles, using extracts from the seawater microalga Nannochloropsis gaditana as biogenic agents, and to thoroughly elucidate the role played by the microalgae extract in the biogenic synthesis. In general, microalgae have become a focus in biotechnology due to their potential applications within an economic, circular, and eco-sustainable framework.[16] [21] Nannochloropsis gaditana, in particular, is a eukaryotic microalga that in recent years has received increasing attention for its potential industrial exploitation due to its very high content of triacylglycerides and polyunsaturated fatty acids[22] that makes its biomass suitable for biofuels, food, and feed applications. ZnO is an inorganic system of great interest due to its multiple applications in electronics, optics, and biomedicine, and its synthesis and applications have been recently surveyed by some of us.[23] As a Generally Recognized As Safe (GRAS) material by the US Food and Drug Administration (FDA),[24] ZnO nanoparticles are also used for antimicrobial applications, such as in food packaging.[25] [26] [27]

To better understand the role of microalgae extract in the biogenic synthesis of ZnO micro- and nanoparticles, a systematic exploration of the experimental conditions, including the presence or absence of NaOH and the use of both physiological and non-physiological conditions for the microalgae, was performed. Physiological conditions refer to the conditions of the microalgae’s external or internal environment that may occur in nature (e.g., room temperature and physiological pH). To highlight the role of the biogenic agent, microalgae-free syntheses were also performed as references. With the aim of exploring the extent of the effect of the presence of the biogenic agent, different temperature ranges and different amounts of microalgae were tested. To the best of our knowledge, this is the first comprehensive and systematic study focusing on the influence of these experimental parameters on the crystallite size and morphology of the resulting ZnO obtained from the biogenic synthesis. By exploring these variables and correlating them with the reaction outcomes, the study provides insights into the role of microalgae extract in controlling ZnO particle formation and growth.

In addition, the antimicrobial properties of the biogenically synthesized ZnO nanoparticles were compared with those synthesized without microalgae to understand their potential in biomedical and packaging applications.

 

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Results and Discussion

As a preliminary consideration, it is essential to address the formation of ZnO in an aqueous environment, which involves the simultaneous occurrence of equilibria related to the hydrolysis of Zn²⁺ and the precipitation of Zn(OH)₂. The low solubility of Zn(OH)₂ in water (K _sp = 3.5 × 10⁻¹⁷ at 25 °C)[28] shifts the equilibrium toward the precipitation of zinc hydroxide (K ~ 10⁶).[29] On the other hand, due to the amphoteric character of zinc oxide, the pH conditions strongly influence the reaction equilibria: Zn²⁺ _(aq) ion is stable below pH 6, Zn(OH)₂ precipitates between pH 7 and 11, and the tetrahydroxozincate complex ion, Zn(OH)₄ ²⁻ _(aq), forms above pH 12.[30] The low solubility value of Zn(OH)₂ in water makes the control of its solubility equilibrium the key factor in any synthetic process involving the hydrolysis of a molecular precursor of zinc containing the Zn²⁺ ion. Hydroxo complexes of zinc can decompose to ZnO, releasing hydroxide ions and water.[30]

The temperature and viscosity of the medium, according to the Stokes–Einstein equation[31] ([Eq. (1)]), also play a critical role in the synthesis of nanoparticles by influencing the kinetics of the above-mentioned precipitation reactions.

$$D=\frac{k_{\mathrm{B}}T}{6\mathit{\eta \pi }{R}_{\mathrm{H}}}$$

where D is the diffusion coefficient, η is the solvent viscosity, R _H is the solute radius, k _B is Boltzmann’s constant, and T is the temperature.[31]

The dehydration of solid Zn(OH)₂ to ZnO and H₂O is endothermic at room temperature (ΔH = +9.11 kJ mol⁻¹), and ΔG is slightly negative (ΔG = −1.7 kJ mol⁻¹). From a free energy and a kinetic point of view, the conversion of Zn(OH)₂ to ZnO is increasingly favored at higher temperatures.[28] [32]

In the experiments carried out in this work, the addition of 0.02 equiv of NaOH in the synthesis of ZnO particles did not significantly affect the pH, which remained neutral at around 6.5, due to the buffering effect of the acetate used as a zinc precursor. In the literature, the formation of crystalline ZnO at room temperature in basic or neutral media has been proved. The amphoteric hydroxide species, ZnO _x (OH) _y (OH₂) _z , are initially formed in the colloidal state as amorphous species, and through a dehydration process in their mother liquor, they spontaneously evolve to crystalline wurtzite ZnO.[33]

On the other hand, it is important to consider the reduction potential of Zn²⁺ in water. The standard potential for the Zn²⁺/Zn reduction reaction is −0.760 V vs SHE (standard hydrogen electrode). When considering the Pourbaix diagram for Zn species, which correlates the potential with the pH for aqueous solutions, the reduction of Zn²⁺ occurs when a potential more negative than −0.822 V (for a Zn²⁺ concentration of 10⁻² mol) is applied.[34] At a pH value around 7 (as it is in this study), the reduction potential for Zn²⁺/Zn falls outside the water stability region, meaning that, within the stability region of water, no reduction of Zn²⁺ to Zn occurs without shifting the potential to more negative values. In addition, at pH around 7, the system Zn²⁺ in water is at the boundary line for the acid–base reactions involving the Zn²⁺ _(aq) and ZnO_(s).

To gain a thorough understanding of the role of microalgae extract in the biogenic synthesis of zinc oxide, different key experimental parameters were systematically screened. In particular, the roles of reaction temperature (ranging from 40 to 100 °C) and of NaOH as a precipitating agent, as well as the possibility to obtain ZnO in physiological conditions at room temperature, by using a physiological buffer as solvent, were investigated.

Zinc Oxide Synthesized in Non-physiological Conditions with NaOH

Zinc oxide was synthesized using zinc acetate as the Zn²⁺ precursor, microalgae extract, and sodium hydroxide as a precipitating agent. In this process, called synthesis under non-physiological conditions, the microalgae extract was prepared at 100 °C. The obtained materials were first characterized from the structural point of view by using powder X-ray diffraction (PXRD). The diffraction peaks in the PXRD patterns ([Fig. 1a]) confirm that the synthesized ZnO is in the hexagonal wurtzite phase (space group P63mc (186), PDF 89-1397), indicating its high crystallinity. The average crystallite size was calculated through the Scherrer equation on the (101) diffraction peak at 36° 2θ. As it can be noted from the PXRD patterns, the presence of additional reflections at 33° and 59° 2θ might be related to the formation of hydrozincite species (Zn₅(CO₃)₂(OH)₆, COD 9007481).[35] [36] By varying the reaction temperature from 40 to 80 °C (as detailed in the Experimental Section), it was observed that higher temperatures led to an increase in crystallite size ([Fig. 1b]), varying from 40 nm at 40 °C to 52 nm at 80 °C. Furthermore, decreasing the volume ratio of microalgae extract with respect to the Zn²⁺ precursor solution from 1:25 to 1:50 v/v (calculated as the volume of microalgae extract divided by the volume of Zn²⁺ precursor solution, as detailed in the Experimental Section), resulted in the formation of larger crystallites, suggesting that the biogenic component acts as a scaffolding agent ([Fig. 1b]). A higher concentration of microalgae extract effectively limited the crystallites growth, probably due to steric hindrance and surface interactions between ZnO particles and microalgae extract’s components, thereby enhancing its scaffolding effect. This effect is even more pronounced at lower temperatures, at which ZnO formation is less kinetically favored. For comparison, the data related to the crystallites size of the samples obtained without the microalgae extract, which will be discussed in detail later, is included in the [Fig. 1b]. To the best of our knowledge, this is the first time that the effect of reaction temperature during a biogenic synthesis with microalgae extract is systematically correlated to the variation of the crystallites size.

Fig. 1 (a) PXRD diffractograms of ZnO samples synthesized in non-physiological conditions with NaOH, by varying the reaction temperature (40, 50, 60, 70, 80 °C) and keeping constant the volume ratio between microalgae extract and Zn²⁺ precursor (1:25 v/v). *Diffraction peaks at 33° 2θ and 59° 2θ, ascribable to possible hydrozincite impurities. (b) Crystallite size as a function of reaction temperature for ZnO particles synthesized with varying amounts of microalgae extract (1:50 v/v represented by dark squares, and 1:25 v/v by light circles, microalgae extract to Zn²⁺ precursor) and without microalgae extract, represented by triangles.

Scanning electron micrographs (SEM) show ZnO particles synthesized under non-physiological conditions using NaOH and microalgae extract, with a volume ratio of 1:25 v/v (microalgae extract to Zn²⁺ precursor), at reaction temperatures of 40 °C ([Fig. 2a]) and 80 °C ([Fig. 2b]). A low magnification overview evidences that the sample obtained at 80 °C ([Fig. 2b]) consists of micro-sized zinc oxide particles with visible agglomerates. Interestingly, the presence of hexagonal prism-shaped particles growing from the core of other hexagonal prisms suggests a hierarchical structuring process, probably initiated by a hexagonal seed. ZnO obtained under non-physiological conditions at temperatures lower than 80 °C presents a not well-defined morphology ([Fig. 2a]), probably related to the lower degree of crystallinity and presence of amorphous species in the samples. Higher magnification images (Fig. S1, in SI) provide further evidence of these morphological differences.

Fig. 2 SEM images of ZnO samples synthesized in non-physiological conditions with NaOH, keeping constant the volume ratio between microalgae extract and Zn²⁺ precursor (1:25 v/v) at a reaction temperature of (a) 40 °C and (b) 80 °C.

Syntheses with the same experimental conditions but without microalgae extract were performed to compare these results with those obtained from the microalgae-assisted synthesis. The key scientific question was whether the microalgae play a specific role in the biogenic synthesis of ZnO when NaOH is also involved. An aqueous precipitation method was used, in which NaOH was added dropwise to a zinc salt solution, resulting in a white powder as a product. The synthetic pathway involving the formation of ZnO from zinc acetate with NaOH is well documented in the literature as the Bahnemann reaction.[20]

PXRD patterns (Fig. S2, in SI) of samples synthesized at different reaction temperatures (40, 50, 60, 70, and 80 °C) confirmed the formation of crystalline hexagonal wurtzite ZnO (PDF 89-1397, space group P63mc (186)), with comparable average crystallites sizes of around 45 nm, despite the different reaction temperatures. For samples synthesized at temperatures lower than 80 °C, it is possible to notice the presence of additional reflections at 33° and 59° 2θ, related to the presence of hydrozincite, hinting that the formation of pure-phase ZnO is favored only at high temperatures, both with and without the presence of microalgae.

By considering the variation with temperature of the crystallite size, it is worth highlighting that the presence of microalgae strongly influences the crystallites growth, particularly at lower reaction temperatures ([Fig. 1b]). Indeed, when comparing the syntheses carried out with and without the microalgae extract, the crystallite size increases with increasing temperature when using the microalgae, while it remains constant when performing the reaction without the microalgae extract. This effect became even more pronounced for the samples synthesized with a higher microalgae content (i.e., 1:25 v/v). This difference is an important first result in identifying the role of microalgae in the biogenic synthesis of ZnO. This suggests that the microalgae may act as a scaffold, promoting and driving the controlled growth and stabilization of zinc oxide particles.

The evolution of the zinc speciation throughout the reaction with and without microalgae was evaluated by in situ and time-resolved X-ray absorption spectroscopy (XAS), carried out at Diamond Light Source (Didcot, UK). The reaction was carried out at 60, 80, and 100 °C for ca. 1 h, under continuous suspension flow, with a time resolution of ca. 5 s. The linear combination fitting of the recorded Zn K-edge X-ray absorption near-edge structure (XANES) spectra evidenced the presence of Zn²⁺ ions in solution, together with ZnO only ([Fig. 3]). It should be noted that the XANES spectrum of Zn(OH)₂ is identical to that of ZnO, and therefore, these two species cannot be distinguished. The presence of other species (e.g., zerovalent zinc) is present only to the noise level and is not reported. The presence of the microalgae in the suspension decreases the signal-to-noise ratio of the recorded spectra due to the increased background noise and inhomogeneities.

The behavior of the zinc species under the two considered conditions (with or without microalgae) is very similar at the lower temperatures (60 and 80 °C), evidencing how the presence of the base seems to be the ruling factor leading to the formation of the oxide. The presence of the microalgae, at first sight, does not seem to have a major impact. The effect of the temperature is, on the other hand, evident also from the reported speciation curves. When the temperature is increased from 60 to 80 °C, the amount of ZnO formed rises from approximately 20% (at%) at 60 °C to ca. 35–40% at 80 °C. At 100 °C, the curves show quite different behavior with the ZnO component reaching a plateau in the case of the experiment including the microalgae, while showing a decrease in relative amount after ca. 750 s after NaOH addition for the experiment performed in their absence. On the other hand, the experiment performed without microalgae was affected by enhanced precipitation of the target zinc oxide on the walls of the reactor employed for the in situ measurement, likely leading to an underestimation of the oxide species. On the contrary, the presence of the microalgae seems indeed to be effective in keeping the oxide particles in suspension. The amount of ZnO reaches values similar to those determined at 80 °C.

Fig. 3 Zinc speciation evolution during ZnO particle synthesis, as determined by in situ Zn K-edge XAS. On the left side, syntheses under non-physiological conditions with NaOH only, on the right side, syntheses under non-physiological conditions with microalgae extract (1:10 v/v) and NaOH. Syntheses were performed at 60, 80, and 100 °C.

TEM analysis showed that the zinc oxide particles synthesized without the microalgae extract were rod-shaped ([Fig. 4]). As the temperature increased, the rods became longer and thinner, i.e., increasing their aspect ratio, calculated as the ratio of length and width. For the sample synthesized at 40 °C, the rod lengths ranged from 780 nm to 1.90 μm, with rod widths between 470 and 810 nm, giving an aspect ratio of about 2 ([Fig. 4a]). For the sample synthesized at 70 °C, the rod lengths varied from 830 nm to 3.10 μm, with rod widths between 140 and 530 nm, giving an aspect ratio of about 6 ([Fig. 4b]). These results show that the aspect ratio of the rods increased as the reaction temperature increased, suggesting a possible oriented attachment growth mechanism along the c-axis.

Fig. 4 TEM images of ZnO samples synthesized with NaOH, without microalgae extract, at a reaction temperature of (a) 40 °C and (b) 70 °C.

 

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Zinc Oxide Synthesized in Non-physiological Conditions without NaOH

A key scientific question was whether the addition of sodium hydroxide might hide or replace the role of the biogenic agent in ZnO synthesis. The existing literature on the biogenic synthesis of ZnO reports that zinc oxide is formed via the reduction of Zn²⁺ precursor with NaOH[37] [38] [39] [40] [41]; however, it is difficult to classify this process as a reduction as it is unlikely to change the oxidation state of zinc under the given conditions, and based on the reduction potential of Zn²⁺. One hypothesis in the literature is that Zn²⁺ ions react with NaOH to form Zn(OH)₂, which, upon heating, leads to [Zn(OH)₄]²⁻ complexes that decompose into ZnO nuclei.[42] Another view suggests that Zn²⁺ ions interact with hydroxyl or carbonyl groups in the microalgae extract, which serves as a stabilizing agent in the formation of ZnO.[43] In other cases reported in the literature, the biogenic synthesis product is calcined to produce ZnO, but in these conditions, microalgae no longer play a role in the formation of crystalline ZnO.[40] [44]

To clarify this issue, a series of experiments were carried out under non-physiological conditions without NaOH to investigate the specific role of the biogenic agent in the production of zinc oxide particles.

Without the presence of NaOH as a precipitating agent, but with microalgae extract, a reaction temperature of 100 °C was required to crystallize ZnO. Highly crystalline ZnO, identified as the pure wurtzite phase (PDF 89-1397, space group P63mc (186)), with an average crystallite size of 43 nm was obtained ([Fig. 5]). No crystalline ZnO formation was instead observed at reaction temperatures below 100 °C, i.e., at 40, 50, 70 °C, as only the reflections at 33° and 59° 2θ, possibly related to the formation of hydrozincite species, could be identified.

Fig. 5 PXRD diffractograms of ZnO samples synthesized in non-physiological conditions without NaOH, by varying the reaction temperature (40, 50, 70, 100 °C) and keeping constant the volume ratio between microalgae extract and Zn²⁺ precursor (1:25 v/v). *Diffraction peaks at 33° 2θ and 59° 2θ, ascribable to hydrozincite impurities.

The reaction mechanism responsible for the crystallization of zinc oxide from zinc acetate as the Zn²⁺ precursor and microalgae extract remained unclear. To elucidate this aspect, further in situ XAS investigations were performed. The in situ XAS experiments confirmed that, also in the absence of NaOH, the only species present seem to be Zn²⁺ (as aqueous acetate species in solution/suspension) and ZnO/Zn(OH)₂. The presence of zerovalent zinc, reported by previous studies[37] [39] [40] [41] also as intermediate species, could be ruled out, since also in this case its amount was determined at the noise level. The experiments also confirmed that, in the absence of NaOH, the formation of ZnO/Zn(OH)₂ proceeds at a much slower rate, since in the monitored timeframe (ca. 2 h) only a minor amount of zinc oxide is detected (only ca. 4 at% after 25 min) (see [Fig. 6]). Nevertheless, the solid present in suspension was isolated and further analyzed ex situ, confirming that ZnO was indeed formed (Fig. S3, in SI).

Fig. 6 Zinc speciation evolution during ZnO particle synthesis in the absence of NaOH, as determined by in situ Zn K-edge XAS (microalgae extract (1:10 v/v)). The syntheses were performed at 100 °C.

Considering the results discussed above, it is possible to affirm that the microalgae do not act through enzymatic activity, but probably through a structural role. As the pH of the solution was between 7 and 7.5, the formation of zinc hydroxide was favored. From a thermodynamic and kinetic point of view, the dehydration of Zn(OH)₂ into ZnO is increasingly favored at higher temperatures, as discussed above. A plausible hypothesis is that the ZnO species are stabilized in the reaction environment by the microalgae extract, which acts as a scaffold, stabilizing the ZnO particles and limiting their growth. The possibility of enzymatic involvement of the microalgae extract was ruled out, as the enzymatic components lose their activity during the preparation of the extract under non-physiological conditions. Therefore, the structural role of the extract is better explained by its function as a templating or structure-directing agent, rather than through any redox activity. Nannochloropsis gaditana is known to be rich in lipids (especially polyunsaturated fatty acids), pigments (such as chlorophylls and carotenoids), proteins, and polysaccharides. While thermal treatment at 100 °C may lead to partial denaturation of proteins, many functional groups (e.g., carboxyl, hydroxyl, amine) are likely to remain available for metal ion coordination. In addition, polysaccharides and lipids, which are more thermally stable, may play a crucial role in stabilizing ZnO particles by acting as structure-directing agents or steric barriers during nucleation and growth. These components could explain the observed differences in crystallite size and morphology, supporting the hypothesis that the microalgae extract acts primarily as a nonredox templating or scaffolding agent in the biogenic synthesis of ZnO. The presence of organic capping agents was also confirmed through FTIR-ATR spectroscopy (Fig. S4, in SI). In the sample prepared with microalgae extract, characteristic peaks were observed at around 1400 cm⁻¹ (C–H and N–H bending) and 1550 cm⁻¹ (C=C or C=N stretching), along with a broad band between 3200 and 3400 cm⁻¹ corresponding to O–H stretching, probably arising from hydroxyl-containing molecules or adsorbed water. Although Zn–O stretching typically appears near 450 cm⁻¹, it lies beyond the detection limit of standard ATR-FTIR. These spectral features suggest the presence of biomolecules such as carbohydrates (e.g., cellulose, algaenan) and proteins from the microalgae extract, in agreement with findings reported by Scholz et al., who characterized the cell wall of Nannochloropsis gaditana by ATR-FTIR.[45]

The effect of varying the amounts of microalgae extract without the addition of NaOH was also investigated. Keeping all other conditions constant, syntheses with different volume ratios of microalgae extract to Zn²⁺ precursor (1:50 v/v, 1:25 v/v, and 1:10 v/v) at a reaction temperature of 100 °C were carried out. With the lowest amount of microalgae (1:50 v/v), not enough product was obtained, supporting the hypothesis that the microalgae act as a stabilizing agent. Crystalline ZnO, identified as hexagonal wurtzite phase (space group P63mc (186), PDF 89-1397) (Fig. S5, in SI), was obtained for the 1:25 and 1:10 v/v samples. The average crystallite sizes were approximately 43 and 38 nm, respectively. These results confirm what was observed in the syntheses carried out with NaOH, where increasing the amount of microalgae extract led to a decrease in crystallite size, enhancing its role as a scaffolding and stabilizing agent in limiting the crystallites growth.

The morphology of the particles obtained from the samples synthesized without NaOH but with microalgae extract at 100 °C showed distinctive morphologies. The particles formed hexagonal and hollow hexagonal structures, with sizes ranging from 1.3 to 2.2 μm, as shown in [Fig. 7]. Again, hexagonal prism-shaped particles were observed growing from the core of other hexagonal prisms, suggesting a recurrent hierarchical growth pattern. This type of particle morphology could result from the aggregation of primary particles in an oriented attachment fashion, leading to the formation of hexagonal prismatic structures. Such arrangements are likely to result in mesocrystals,[46] [47] which are formed when nanoparticles align in a highly ordered fashion, to form larger, crystallographically coherent structures.

Comparing the morphologies of ZnO particles synthesized with microalgae extract in the presence of NaOH ([Figs. 2] and S1) and without NaOH ([Fig. 5]), it appears that NaOH promotes a directional growth of the particles. Depending on the ruling growth mechanism, i.e., either thermodynamic or kinetic, different morphologies are formed.[48] The directional growth of the particles could be ascribed to the faster reaction kinetics induced by NaOH, as also supported by in situ XAS results, allowing for anisotropic growth, whereas the slower kinetics in the absence of NaOH may not facilitate such growth within the same reaction time. Alternatively, the microalgae extract itself may hinder growth along one direction, suggesting that both factors, reaction kinetics and the influence of the microalgae extract, may contribute simultaneously to the observed morphological differences, and one does not exclude the other.

Fig. 7 TEM (a, b) and SEM (c, d) images of ZnO samples synthesized in non-physiological conditions without NaOH, at a reaction temperature of 100 °C, by varying the volume ratio between microalgae extract and Zn²⁺ precursor (a, c) 1:25 v/v and (b, d) 1:10 v/v.

Phaeodactylum tricornutum, a commonly used model diatom species, was also tested in this study, performing the synthesis of ZnO without the presence of NaOH at 100 °C. Both P. tricornutum and N. gaditana share a common evolutionary origin,[49] and their use in the biogenic synthesis of ZnO consistently resulted in crystalline zinc oxide in the wurtzite phase with similar particle morphologies (see TEM images in Fig. S6, SI). These results suggest that different species of microalgae could act through the same scaffolding action in the biogenic synthesis of ZnO.

 

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Zinc Oxide Synthesized in Physiological Conditions

To further investigate the role of the microalgae in the biogenic synthesis of zinc oxide particles, the syntheses were carried out under physiological conditions. To preserve the activity of proteins and enzymes in the cell extract, the microalgae extract was prepared by disrupting the cells and separating the soluble fraction from the insoluble fraction. The soluble fraction contained mainly soluble proteins and enzymes, while the insoluble fraction consisted of lipids, membranes, and membrane-associated proteins and enzymes.[50] [51] The aim of this separation was to determine which component of the microalgae extract could chiefly contribute to the biogenic synthesis of zinc oxide particles. To reproduce the physiological environment, zinc acetate was dissolved in a buffer at pH 7.8, and the reaction was carried out for 48 h at room temperature (21–22 °C).

Various parameters were systematically varied by keeping the temperature constant at RT (~20 °C). The following reaction settings were studied: (i) the use of either the soluble or insoluble part of the microalgae extract and (ii) the solvent for the Zn²⁺ precursor (buffer or water).

No crystalline zinc oxide product was obtained from any synthesis carried out under physiological conditions, regardless of whether the soluble or insoluble fraction of the microalgae extract was used. However, powder X-ray diffraction (PXRD) analysis (Fig. S7, in SI) revealed some diffraction peaks, which are likely attributable to the crystallization of salts present in the buffer, such as NaCl. These results suggest that the microalgae components themselves do not play an active role in the formation reaction of ZnO, and most likely, temperature is a crucial factor in this biogenic synthesis. However, increasing the reaction temperature in these experiments would have compromised the physiological conditions under investigation. This finding further supports the hypothesis that the microalgae act more as a stabilizer for the formation of ZnO, which is favored by higher temperatures.

 

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Antimicrobial Properties of ZnO

Both ZnO particles synthesized with microalgae extract at 100 °C without NaOH (with a volume ratio between microalgae extract to Zn²⁺ precursor of 1:25 v/v), and ZnO particles synthesized without microalgae extract at 100 °C with NaOH, were tested for their antibacterial activity against Escherichia coli (ATCC 25922), a representative of Gram-negative bacterium, known to be less sensitive to ZnO treatments compared to Gram-positive bacteria.[52] [53] Both ZnO particles were effective against E. coli. Specifically, the sample synthesized with microalgae extract showed a Minimum Inhibitory Concentration (MIC) value at the ZnO concentration of 0.312% w/v (38 mM), while the ZnO sample synthesized without microalgae extract displayed MIC values at a ZnO concentration of 0.156% w/v (19 mM). Regarding the Minimum Bactericidal Concentration (MBC) analysis, the sample synthesized with microalgae extract showed bactericidal activity at the ZnO concentration of 0.312% w/v (38 mM), whereas the sample synthesized without microalgae extract showed no bactericidal activity at the tested concentrations. Therefore, by comparing the particles synthesized with and without microalgae extract, the bactericidal activity (MBC) was higher for the particles obtained by the biogenic approach. On the other hand, the bacteriostatic activity (MIC) was higher for the particles obtained without microalgae extract (Fig. S8, in SI).

As reported in the state of the art, the antimicrobial activity of ZnO particles is size-dependent, with smaller particles exhibiting enhanced antimicrobial properties due to their increased specific surface area.[54] Padmavathy et al.[55] reported ZnO nanoparticles ranging from 12 to 47 nm, which provided a larger surface area for antimicrobial activity. In contrast, the ZnO particles synthesized in this study, both with and without microalgae extract, were in the micrometer range (Fig. S9, in SI). This difference in particle size likely explains why the bacteriostatic (MIC) and bactericidal (MBC) activities observed in this research required higher ZnO concentrations compared to those reported in the literature.[55]

The ZnO sample synthesized without microalgae extract showed a higher bacteriostatic activity (MIC) compared to the sample synthesized with microalgae extract. However, the bactericidal activity (MBC) was higher for the particles obtained by the biogenic approach. This enhanced bactericidal effect could be attributed to the presence of organic molecules surrounding the biogenic ZnO particles, which may stabilize the suspension in aqueous solution, thereby improving their bactericidal efficiency. It is also possible that residual microalgae extract on the surface of the particles contributed to this enhanced bactericidal activity.

 

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Conclusions

This study successfully optimized a green, biogenic approach for the synthesis of zinc oxide particles using Nannochloropsis gaditana microalgae as a biogenic agent, a process that was previously poorly described in the literature and whose mechanisms are unclear. A systematic investigation of key experimental parameters, including reaction temperature, presence of sodium hydroxide, and concentration of microalgae extract, was carried out to understand their influence on the structural and functional properties of ZnO. The results showed that the microalgae extract plays a crucial role in modulating the crystallite size and morphology of ZnO, likely acting as a scaffold to promote and stabilize particle growth. To better understand the role of microalgae and different synthesis parameters, ZnO particles were synthesized under both sodium hydroxide-assisted and sodium hydroxide-free conditions. Under non-physiological conditions, with the presence of sodium hydroxide, highly crystalline ZnO was formed, and the crystallite size increased with increasing temperature. In contrast, in the absence of sodium hydroxide, crystalline ZnO was only obtained at higher temperatures, i.e., at 100 °C, suggesting that while both the presence of sodium hydroxide and the temperature influence the crystallization of ZnO, the temperature itself plays a particularly critical role when the synthesis is performed without the precipitating agent, i.e., NaOH. Indeed, highly crystalline ZnO was produced at 100 °C, and increasing the concentration of microalgae extract resulted in smaller crystallites. Hexagonal prismatic particle morphologies were observed, suggesting the aggregation of primary particles in an oriented attachment fashion, i.e., mesocrystals. Syntheses carried out at room temperature under physiological conditions failed to yield crystalline ZnO, further supporting the hypothesis that temperature plays a critical role in the kinetics of biogenic ZnO formation. In all cases, an exclusive role of the biogenic agents as scaffolding and structure-directing agents was pointed out, definitely excluding a possible role as reducing agent, as reported in previous studies, also confirmed by in situ XAS investigations. The antimicrobial activity of ZnO particles was tested against Escherichia coli, revealing distinct bacteriostatic and bactericidal properties depending on the synthetic route. ZnO synthesized using microalgae showed higher bactericidal activity, while ZnO produced without microalgae showed stronger bacteriostatic effects. This suggests that the biogenic approach, tuning also the shape and size of the resulting particles, could improve the functional properties of ZnO particles. Future work should investigate the influence of reaction time and explore different species of microalgae to determine their influence on particle morphology. In addition, diatoms with their silicified cell walls should be investigated for their potential and promising role in the biogenic synthesis of ZnO.

 

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Experimental Section

Chemicals and Materials

Zinc(II) acetate dihydrate (Zn(CH₃COO)₂ · 2H₂O) was purchased from Merck. Sodium hydroxide was purchased from VWR. Agar, Luria Bertani Broth, and Ampicillin were purchased from Sigma-Aldrich. All chemicals were used without further purification.

Microalgae biomass of Nannochloropsis gaditana, Phaeodactylum tricornutum, and the bacterial strain Escherichia coli ATCC 25922 were provided by the Department of Biology (University of Padova).

 

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Characterization

X-ray diffraction patterns were recorded using a Bruker D8 Advance diffractometer, fitted with an LYNXEYE detector in 1D mode. Diffraction data were acquired by exposing powder samples to Cu-K_α1,2 X-ray radiation. X-rays are generated from a Cu anode supplied with 40 kV and a current of 40 mA. The data are collected over the 2θ range 20–80° with a step size of 2θ = 0.027° and a nominal time per step of 0.3 seconds. Fixed divergence slits of 0.50° were used together with Soller slits with aperture of 2.5°. The identification of the crystalline phases is carried out through a Search and Match method with the software Bruker Diffrac EVA. The crystallite size was measured through the Scherrer equation, considering an estimation error on the average size of around 10–15%.[56]

TEM images were acquired with a microscopy FEI Tecnai G2 (Department of Biology, University of Padua), working at 100 kV, equipped with an Olympus Veleta camera and a TVIPS F114 camera. The analysis of the dimensions of the nanoparticles is carried out using the software ImageJ.

The SEM analyses were obtained using a Zeiss SUPRE 40VP, coupled with an EDX detector (Department of Chemical Science, University of Padova). FE-SEM images were taken using a primary beam acceleration voltage of 2.0–5.0 kV.

XAS experiments were performed at the Zn K-edge (9659 eV) at the I20-EDE beamline of Diamond Light Source (Didcot, UK). The beamline was operating in energy-dispersive configuration, and measurements were performed in transmission mode. Spectra were acquired in the energy range 9477–10442 eV with a 0.3 eV step size. The acquisition of one spectrum took 5 s, with 0.5 s of deadtime between each spectrum, performing a bidirectional scan to reduce the total deadtime. The starting solutions/suspension were flown through the measuring capillary (Kapton tube with OD 3.2 mm, WT 0.08 mm), positioned perpendicular with respect to the incoming beam, thanks to a peristaltic pump (ISMANTEC REGLO ICC, set on 90 rpm). The residence time from the reaction vessel (round bottom flask) to the measuring capillary was 1 min, with a total residence time for the continuous flow setup of 2 min. The addition of NaOH was controlled remotely through a syringe pump (2.5 mL/min flow rate). The experiments were performed using Zn(CH₃COO)₂ · 2H₂O as precursor (0.2 M, 50 mL for each experiment, 0.01 mol), NaOH 4 M (2.5 mL, total volume added for each experiment, 0.01 mol), and different volumes of microalgae extract. The temperature during the experiments was monitored using two thermocouples, one placed in the reaction vessel and one in the measuring capillary. Experiments at 100 °C were also performed using a reflux, mounted on the round-bottom flask. A detailed scheme of the continuous flow setup is reported in the Supporting Information (Fig. S10, in SI). Prior to analysis, spectra were aligned, normalized and, especially in the case of the dataset collected in the presence of microalgae, smoothed via a Savitzky-Golay smoothing function (window size: 3, polynomial order: 5). Each dataset was investigated for the presence of bubble/inhomogeneity-induced distortions, which usually affected only a few (<10) spectra per dataset. Only in the case of the experiment performed at 100 °C in the absence of microalgae, a series of 15 successive spectra were distorted and removed. For linear combination fitting, the spectra used as references were those of metallic Zn foil, ZnO (measured as a pellet), and Zn acetate solution (measured under flow, in the same conditions used for synthesis). The fitting was performed in the window 9635–9725 eV.

 

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Microalgae Extract (Non-physiological and Physiological Conditions)

For the preparation of the microalgae extract in non-physiological conditions, the microalgae biomass was heated at 100 °C for 20 min under constant stirring. The extract obtained was filtered through Whatman No. 1 filter paper, and the filtrate was stored at 4 °C.

The microalgae extract in physiological conditions was performed under green light (λ = 520–565 nm). Samples of microalgae biomass (0.2 g) were centrifuged at 17,000 rpm for 10 min at 20 °C, and the supernatant was discarded to harvest microalgae cells. Cells were disrupted with a Mini Bead Beater (Biospec Products) and four cycles were performed: rupture at 3500 rpm for 10 sec in the presence of glass beads (Ø = 150–212 μm) and a buffer (0.4 M NaCl, 2 mM MgCl₂ and 20 mM Tricine-KOH, pH 7.8) with three protease inhibitors (1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride and 1 mM 6-aminocaproic acid). After this mechanical cell rupture, the samples were centrifuged at 2500 rcf for 10 min. The supernatant was collected and kept in an ice bath. Then supernatants were centrifuged at 17,000 rcf for 15 min, and the supernatants, containing the soluble part, and the pellets, containing the insoluble part, were collected and stored at 4 °C.

 

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Synthesis of Zinc Oxide with Microalgae and without Microalgae

For the synthesis of ZnO particles in non-physiological conditions, solutions of Zn²⁺ precursors were prepared by dissolving Zn(CH₃COO)₂ · 2H₂O (3.7 g, 20 mmol) in deionized water (100 mL). The microalgae extract was added dropwise to the zinc salt solution under constant stirring. The volume of the microalgae extract was varied to achieve specific volume ratios with the zinc salt solution: 1:50 v/v (2 mL extract in 100 mL zinc salt solution), 1:25 v/v (4 mL extract in 100 mL zinc salt solution), and 1:10 v/v (10 mL extract in 100 mL zinc salt solution). In the syntheses carried out in non-physiological conditions with the addition of sodium hydroxide, NaOH (0.8 g, 20 mmol) was dissolved in deionized water (20 mL), and the NaOH solution was added dropwise under constant stirring to achieve a molar ratio between Zn²⁺ and NaOH of 1:1 mol/mol. The molar ratio of Zn²⁺ and NaOH was kept below 1:2 mol/mol to prevent the quantitative precipitation of Zn(OH)₂. The synthesis time was kept constant at 3 h throughout all syntheses, and the reaction temperatures tested were 40, 50, 60, 70, 80, and 100 °C. The variation of the pH was not significant when NaOH was added, and it remained at about 6.5. The pH value was measured with litmus paper. The green precipitate obtained from the reaction was isolated with three cycles of centrifugation at 12,000 rpm for 5 min and purified by washing with acetone and centrifuging with a further three cycles at 12,000 rpm for 5 min. The solid precipitate was dried overnight in the oven at 90 °C.

For the synthesis of ZnO samples in physiological conditions, a solution of Zn(CH₃COO)₂ · 2H₂O (2.2 g, 10 mmol) was prepared by dissolving the precursor salt in a buffer (0.4 M NaCl, 2 mM MgCl₂ and 20 mM Tricine-KOH pH 7.8) or in deionized water and microalgae extracts were prepared in physiological conditions. The syntheses were carried out by separating the contribution of the soluble and insoluble parts of the microalgae extracts. The soluble or insoluble part of the microalgae extracts was added dropwise to the zinc salt solution under constant stirring. The reaction time was kept constant at 48 h, and the syntheses were performed at room temperature (21 °C). The green precipitate obtained from the reaction was isolated with three cycles of centrifugation at 12,000 rpm for 5 min. To avoid a further decrease in the low quantity of the product obtained in these syntheses, the purification and washing steps were not conducted. The solid precipitate was dried in the oven at 90 °C overnight.

Microalgae-free synthesis was used as a reference to clarify the role of the microalgae in the biogenic synthesis of ZnO. For the synthesis of ZnO particles, a solutions of the Zn²⁺ precursors was prepared dissolving Zn(CH₃COO)₂ · 2H₂O (3.7 g, 20 mmol) in deionized water (100 mL) and sodium hydroxide, NaOH (0.8 g, 20 mmol) was dissolved in deionized water (20 mL), and the NaOH solution was added dropwise under constant stirring to achieve a molar ratio between Zn²⁺ and NaOH as 1:1 mol/mol. The synthesis time was kept constant at 3 h throughout all syntheses, while the temperature was varied in each synthesis; in particular, the reaction temperatures tested were RT, 40, 50, 60, 70, 80, and 100 °C. The variation of the pH was not significant when NaOH was added, and it remained at about 6.5. The pH value was measured with litmus paper. The white precipitate obtained from the reaction was isolated with three cycles of centrifugation at 12,000 rpm for 5 min and purified by washing with deionized water and centrifugation with a further three cycles at 12,000 rpm for 5 min. The white solid precipitate was dried in the oven at 90 °C overnight.

 

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tBacterial Strain and Culture Conditions

The Escherichia coli ATCC 25922 strain was used for evaluating the antimicrobial activity of ZnO particles. The strain was routinely grown in Luria-Bertani (LB) broth or LB agar plate at 37 °C overnight (ON). Bacterial working cultures were obtained from single colonies grown at 37 °C ON in 6 mL LB, with continuous shaking (150 rpm).

 

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Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Tests

As ZnO is nearly insoluble in water, for both biogenic and nonbiogenic ZnO particles, a 1.25% w/v stock suspension was prepared in LB broth and incubated ON at 37 °C at 600 rpm, to facilitate dissolution. 100 μL of each ZnO suspension was plated on LB agar and incubated ON at 37 °C to check for sterility.

ZnO particle suspensions (50 μL) were incubated in triplicate at the final concentrations of 0.625%, 0.312%, 0,156%, 0.078%, and 0.039% with 50 μL of bacterial culture [10⁶ Colony Forming Units (CFU)/mL initial concentration] on sterile polystyrene 96-well plates. Non-ZnO-treated bacteria (positive controls) and those with 50 μg/mL ampicillin (negative controls) were processed in parallel. As the % of ZnO particles affects turbidity per se, for each ZnO particle-bacterial mixture, three blank samples, represented by the ZnO particle suspension alone at the appropriate concentration (from 0.625% to 0%), were also prepared.

Plates were incubated for 24 h at 37 °C (200 rpm) under LED light of 16 μmol m⁻² s⁻¹ (1184 Lux). The change in optical density (OD) at 600 nm (ΔOD₆₀₀), measured using a microplate reader, was calculated by subtracting the OD₆₀₀ recorded at time 0 from that obtained after 24 h. The MIC was considered the lowest concentration resulting in a ΔOD₆₀₀ equal to 0±0.5, as in Romoli et al.[57]

To establish MBC, ZnO suspensions (from 0.625 to 0.039%) were tested in duplicate in 100 μL of bacterial culture (5 × 10⁵ CFU/mL initial concentration) and incubated at 37 °C for 24 h, as described above. Samples were serially diluted 1:10, 1:100, 1:1000, plated on an LB agar plate, and colonies were counted after an incubation of 24 h at 37 °C. The MBC was considered the ZnO concentration able to inhibit 99.9% of bacterial growth. Both MIC and MBC tests were repeated 2–4 times.

 

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Contributorsʼ Statement

Data collection: S. Tinello, C. Mazzariol, C. Pilotto, P. Dolcet, M. Battistolli, F. Sandrelli; Design of the study: G. Perin, T. Morosinotto, S. Gross; Analysis and interpretation of the data: all authors; Drafting the manuscript: all authors; Critical revision of the manuscript: all authors.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

We gratefully thank Dr. Federico Caicci and Dr. Francesco Boldrin (Department of Biology, University of Padova) and Dr. Giulia Bragaggia (Department of Chemical Sciences, University of Padova) for TEM and SEM analyses, respectively. This work was carried out with the support of Diamond Light Source (Didcot, UK), at beamline I20-EDE (proposal SP30680). Dr. Luke Keenan (I20 beamline, Diamond Light Source) is kindly acknowledged for technical assistance. Dr. Francesca Tajoli and Dr. Federico Barbon (Department of Chemical Sciences, University of Padova) are kindly acknowledged for their support during XAS experiments.

• References

• 1 Anastas PT, Warner JC. Green Chemistry: Theory and Practice. Oxford University Press; 1998
• 2 Bretos I, Diodati S, Jiménez R. et al. Chem – Eur J 2020; 26 (42) 9157-9179
• 3 Gross S. Unconventional Green Synthesis of Inorganic Nanomaterials; 2024
• 4 Faramarzi MA, Sadighi A. Adv Colloid Interface Sci 2013; 189: 1-20
• 5 Slocik JM, Knecht MR, Naik RR. Chapter 2: biogenic synthesis of inorganic materials. In Unconventional Green Synthesis of Inorganic Nanomaterials. Gross S. ed Inorganic Materials. Vol 14. 2024: 29-103
• 6 Dahoumane SA, Mechouet M, Wijesekera K. et al. Green Chem 2017; 19 (3) 552-587
• 7 Khanna P, Kaur A, Goyal D. J Microbiol Methods 2019; 163: 105656
• 8 Shankar PD, Shobana S, Karuppusamy I. et al. Enzyme Microb Technol 2016; 95: 28-44
• 9 Brayner R, Yéprémian C, Djediat C. et al. Langmuir 2009; 25 (17) 10062-10067
• 10 Nagarajan S, Kuppusamy KA. J Nanobiotechnol 2013; 11 (39) 1
• 11 Brayner R, Coradin T, Beaunier P. et al. Colloids Surf, B 2012; 93: 20-23
• 12 Lefebvre DD, Kelly D, Budd K. Appl Environ Microbiol 2007; 73 (1) 243-249
• 13 Scarano G, Morelli E. Plant Sci 2003; 165 (4) 803-810
• 14 Rao MD, Pennathur G. Mater Res Bull 2017; 85: 64-73
• 15 Marchegiani F, Cibej E, Vergni P, Tosi G, Fermani S, Falini G. J Cryst Growth 2009; 311 (17) 4219-4225
• 16 Mata TM, Martins AA, Caetano NS. Renewable Sustainable Energy Rev 2010; 14 (1) 217-232
• 17 Gabriella Pasqua GA, Forni C. Botanica Generale e diversità Vegetale. Piccin; 2015
• 18 Mueller JG, C. E. C. Pritchard PH. Bioremediation: Principles and Applications. Cambridge University Press; 1996
• 19 Ebadi M, Zolfaghari MR, Aghaei SS. et al. RSC Adv 2019; 9 (41) 23508-23525
• 20 Bahnemann DW, Kormann C, Hoffmann MR. J Phys Chem 1987; 91 (14) 3789-3798
• 21 Perin G, Bellan A, Bernardi A, Bezzo F, Morosinotto T. Physiol Plantarum 2019; 166 (1) 380-391
• 22 Archibald JM, Keeling PJ. Trends Genet 2002; 18 (11) 577-584
• 23 van Embden J, Gross S, Kittilstved KR, Della Gaspera E. Chem Rev 2022; 1
• 24 U. S. FDA. 2024 https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=182.8991 (accessed)
• 25 Tayel AA, Sorour NM, El-Baz AF, El-Tras WF. Food Preserv 2017; 6: 487-526
• 26 Sirelkhatim A, Mahmud S, Seeni A. et al. Nano-Micro Lett 2015; 7 (3) 219-242
• 27 Pasquet J, Chevalier Y, Couval E. et al. Int J Pharm 2014; 460 1–2 92-100
• 28 Moezzi A, Cortie M, McDonagh A. Dalton Trans 2011; 40 (18) 4871-4878
• 29 Reichle RA, Mccurdy KG, Hepler LG. Can J Chem 1975; 53 (24) 3841-3845
• 30 Charlot G. Qualitative Inorganic Analysis. John Wiley & Sons, Inc.; 1957
• 31 Einstein A. Ann Phys 1905; 322 (8) 549-560
• 32 Goux A, Pauporté T, Chivot J, Lincot D. Electrochim Acta 2005; 50 (11) 2239-2248
• 33 Dolcet P, Latini F, Casarin M. et al. Eur J Inorg Chem 2013; 13: 2291-2300
• 34 Izaki M, Khoo PL, Shinagawa T. J Electrochem Soc 2021; 168 (11) 1
• 35 Molefe FV, Koao LF, Dejene BF, Swart HC. Opt Mater 2015; 46: 292-298
• 36 Yan CL, Xue DF. J Phys Chem B 2006; 110 (23) 11076-11080
• 37 Ahmed S, Annu Chaudhry SA, Ikram S. J Photochem Photobiol B 2017; 166: 272-284
• 38 Selvarajan E, Mohanasrinivasan V. Mater Lett 2013; 112: 180-182
• 39 Hamouda RA, Yousuf WE, Mohammed ABA, Mohammed RS, Darwish DB, Abdeen EE. Microb Pathog. 2020 147
• 40 Rasool A, Kiran S, Gulzar T. et al. J Clean Prod 2023; 398: 1
• 41 Abbas A, Ahmad T, Hussain S. et al. Int J Environ Sci Technol 2022; 19 (11) 11333-11346
• 42 Rao MD, Gautam P. Environ Prog Sustainable Energy 2016; 35 (4) 1020-1026
• 43 Tran GT, Nguyen NTH, Nguyen NTT, Nguyen TTT, Nguyen DTC, Tran TV. J Environ Chem Eng 2023; 11 (5) 1
• 44 Siddique K, Shahid M, Shahzad T. et al. Environ Sci Pollut Res 2021; 28 (22) 28307-28318
• 45 Scholz MJ, Weiss TL, Jinkerson RE. et al. Eukaryot Cell 2014; 13 (11) 1450-1464
• 46 Niederberger M, Cölfen H. Phys Chem Chem Phys 2006; 8 (28) 3271-3287
• 47 Helmut Cölfen MA. Mesocrystals and Nonclassical Crystallization. Wiley; 2008
• 48 Hiemstra T, Mendez JC, Li JY. Environ Sci: Nano 2019; 6 (3) 820-833
• 49 Petroutsos D, Amiar S, Abida H. et al. Prog Lipid Res 2014; 54: 68-85
• 50 Zittelli GC, Lavista F, Bastianini A, Rodolfi L, Vincenzini M, Tredici MR. J Biotechnol 1999; 70 1–3 299-312
• 51 Rocha JMS, Garcia JEC, Henriques MHF. Biomol Eng 2003; 20 4–6 237-242
• 52 Reddy KM, Feris K, Bell J, Wingett DG, Hanley C, Punnoose A. Appl Phys Lett 2007; 90 (21) 1
• 53 da Silva BL, Caetano BL, Chiari-Andréo BG, Pietro RCLR, Chiavacci LA. Colloids Surf, B 2019; 177: 440-447
• 54 Lallo da Silva B, Abucafy MP, Berbel Manaia E. et al. Int J Nanomed 2019; 14: 9395-9410
• 55 Padmavathy N, Vijayaraghavan R. Sci Technol Adv Mater 2008; 9 (3) 035004
• 56 Langford JI, Wilson AJC. J Appl Crystallogr 1978; 11: 102-113
• 57 Romoli O, Mukherjee S, Mohid SA. et al. ACS Infect Dis 2019; 5 (7) 1200-1213

Publication History

Received: 05 November 2024

Accepted after revision: 10 June 2025

Article published online:
04 July 2025