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.
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.
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.
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.
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.
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.
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).
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-Kα1,2 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.
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.
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.
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).
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.
