Defense responses of the green microalgae Chlorella vulgaris to the vanadium pentoxide nanoparticles
Article Category: Original Research Paper
Publié en ligne: 31 déc. 2023
Pages: 446 - 460
Reçu: 12 avr. 2023
Accepté: 13 juin 2023
DOI: https://doi.org/10.26881/oahs-2023.4.06
© 2023 Hanifeh Akbarian Kalehjahi et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
The improving applications of different types of nanoparticles (NPs) in many industrial products give rise to their undesirable discharge into the ecosystems. Thus, the hazardous impacts of NPs on the biosphere and living organisms have to be regularly assessed (Abbas et al. 2020). In recent times, inquiry into the results and performance of manufactured NMs in the ecosystem has gained growing attention (Malakar et al. 2021). Indeed, it is uncertain whether the advantages of nanotechnologies exceed the threats related to the environmental discharge of NPs. Thus, supplementary toxicological research is needed to identify crucial knowledge gaps in the current understanding of the harmful influences of NPs on the environment (Klaine et al. 2008).
Transition metal oxides have been frequently a research issue because of their variable valence states and broad technological applications (Yuan et al. 2021). Vanadium as a transition metal has the potential to assume multiple oxidation states. It can create different compounds with oxygen such as VO, VO2, V2O3, and V2O5 in various configurations, which determines their chemical properties (Sieradzka et al. 2011). The toxic impact of vanadium oxides on living organisms is influenced by different issues such as their solubility, concentration, length of treatment, and the type of treated cells (Xi et al. 2019; Xi et al. 2021). Vanadium pentoxide-based materials are broadly utilized in industrial products such as gas sensors (Niu et al. 2021), solar cells (Costals et al. 2021), smart glasses (Ma et al. 2008), and smart windows (Li et al. 2023). Notably,
Aquatic ecosystems as the terminal destination of domestic and industrial wastewater have a specific importance for the evaluation of NP toxicity. Accordingly, aquatic organisms were subjected to studies of the unsafe effects of released contaminants into the ecosystems (Walters et al. 2014). However, only a few studies have been performed on the effects of nanoscaled vanadium on algae and cyanobacteria. Recently, the capability of four microalgae in the bioremoval of V2O5 has been assessed, and only
Because of having simple cultivation requirements, a short life cycle, and sensitivity to different classes of contaminants, unicellular green microalgae were widely used as model microorganisms in the risk assessment of pollutants (DeLorenzo et al. 2002; Karimi et al. 2017a,b; Nazari et al. 2018). In this context, the use of the
Here, we examined some of the physiological and cytological criteria of
V2O5 NPs as nanospheres were synthesized by the previously published method (Wu et al. 2013). A mixture of 1 ml H2O2 (30%) and 4 ml H2O was added into a methanol solution of VO (acac)2 (0.7 mmol; 30 ml). After shaking for 1 h, the solution was transferred into an autoclave and subsequently was positioned in an oven at 150°C for 24 h. The obtained precipitates were centrifuged and washed with a small amount of ethanol, and the final product was calcined under air at 400°C for 2 h to acquire spherical V2O5 NPs. The crystal structure and the vibrational spectra analysis of the synthesized NPs was examined by XRD (Tongda, TD-3700, China) and FT-IR (TENSOR 27, Brucker, Germany), respectively. The size distribution and zeta potential of the prepared NPs were demonstrated using DLS (Nanotrac Wave, Microtrac, USA). High resolution images of the V2O5 NPs, indicating their size, morphology and elemental composition, were acquired using TEM (Zeiss LEO 906, kv 100, Germany) and SEM (MIRA3 FEG-SEM, Tescan, Czech Republic), equipped with EDS (Dağlıoğlu et al. 2023).
Algal cells were exposed to V2O5 NPs in the exponential growth stage on the day between 6 to 8 of culture. For this purpose, V2O5 NPs were added to 250 ml flasks holding 200 ml of cell containing media with the final quantities of 50, 100, 150, and 200 mg l-1. Treated cells were collected every 24 h for 4 days by centrifugation at 5000 × g for 10 min and saved at -80°C. The determination of treatment concentration was based on initial experiments and in correlation with the mean range of vanadium quantity in soil, which was reported to be around 150 mg kg-1 and also excess density of its environmental existence because of human activities (Aihemaiti et al. 2020).
The growth of algae was estimated every day, not only by assessing the cell number but also by determining the absorption at 680 nm. A preliminary cell number of 30 × 104 was used for all examinations. Changes in fresh weight were also evaluated after gathering the cells by centrifugation at 5000 × g for 10 min.
The viability of algae after the application of 200 mg l-1 of V2O5 NPs for 96 h was measured by flow cytometric tests. Nearly 1.0 × 106 cells were harvested by centrifugation at 5000 × g for 10 min, rinsed with phosphate buffer (PBS, pH 7.4), and treated with 5 μl of propidium iodide (PI) in the dark for 20 min. The fluorescence emission of the cells was obtained from ~10 000 events per sample in the FACSCalibur FL2 channel (Becton Dickins on Immunocytometry Systems, San Jose, CA, USA). The produced chlorophyll emissions were assembled in the FL3 channel.
Exposed algae to 100 and 200 mg l-1 of V2O5 NPs for 96 h were precipitated at 5000 × g for 5 min using a centrifuge (Eppendorf, 5810R, Germany). Subsequently, the cells were washed three times with phosphate buffered saline (PBS), fixed with formaldehyde and dehydrated sequentially in 20, 40, 60, 80 and 100% ethanol. After freeze-drying and subsequent gold coating, the structure of the treated and control cells was examined under a scanning electron microscope (MIRA3 FEG-SEM, Tescan, Czech Republic) (Ozturk et al. 2019).
The content of chlorophyll
The cells were crushed in 100% methanol prior to be kept at 4°C for 24 h in the dark. After centrifugation of the homogenate at 10 000 × g for 10 min, the supernatant was employed for the experiments. The total phenol quantity was assessed based on the Folin-Ciocalteu technique (Meda et al. 2005). Accordingly, 2.5 ml of distilled water was added to 100 μl of the methanolic algal extract, and then 100 μl of Folin-Ciocalteu was mixed with the solution. After 6 min, 150 μl of sodium carbonate (20%) was mixed and the solution was retained in the dark for 30 min. Subsequently, the absorbance of the solution was read at 760 nm. The overall phenol quantity was expressed as equivalent to a milligram of gallic acid per fresh weight of algae. The assay of total flavonoid was conducted through an adapted aluminum chloride colorimetric process (Chang et al. 2002). A volume of 500 μl aluminum chloride (2%) was added to 500 μl of methanolic extract. Afterwards, the mixture was placed at 4°C for 1 h in the dark, and its absorbance was read at 415 nm. The total flavonoid level of the extracts was reported as equivalent to a milligram quercetin per fresh weight of microalgae.
The attained cells from media were macerated in 2 ml of phosphate buffer (100 mM, pH 7) after adding in liquid nitrogen. The achieved homogenate was centrifuged, and the supernatant was immediately employed for the assessment of the amount of protein content (Bradford 1976), as well as the measurement of antioxidantive enzymes activities, including superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX).
The action of superoxide dismutase (SOD) was calculated by determining the inhibiting the photoreduction of nitro-blue-tetrazolium (NBT) by the extract. This presented the basic quantity of the enzyme for a 50% inhibition of NBT photoreduction as one unit of SOD activity (Winterbourn et al. 1976). The CAT function was noted at 240 nm by the subsequent breakdown of H2O2 for 1 min, where one μmol of reduced H2O2 per minute displayed one unit of enzyme activity (Chance & Maehly 1955). The activity of ascorbate peroxidase (APX) was determined in absorbance at 290 nm regarding the oxidation of ascorbate with the algal extract. The amount of enzyme needed for the oxidation of 1 μmol ascorbic acid in one minute was considered as one unit of enzyme activity (Nakano & Asada 1981).
The measurements used for statistical examinations were conducted in three repetitions. One-way analysis of variance (ANOVA) was conducted with Duncan’s multiple comparison tests by SPSS 18 software. The normality of the data was checked using the Kolmogorov-Smirnov test. The differences were documented as significant when the
The structure, purity, and phase composition of the nanospheres were further studied in detail. The XRD chart of V2O5 NPs is presented in Fig. 1A. All patterns at 2θ = 11.5°, 16.2°, 23.0°, 26.1°, 28.0°, 40.5°, 45.1°, and 48.3° corresponding to the (200), (001), (101), (110), (400), (402), (411) and (302) planes can be signed to the orthorhombic phase of V2O5 (JCPDS no. 41-1426) (Wu et al. 2013) without any extra peaks related to impurities. FT-IR spectra of the vanadium pentoxide particles are demonstrated in Fig. 1B. The V = O stretching of the V2O5 nanospheres appeared at 980 cm-1 (Kera et al. 1967). The bands at 1411 and 1590 cm-1 are caused by the bending vibration of H2O molecules on vanadium oxides, and the broad absorption bands at 3200-3400 cm-1 fit the hydrogen bonds of intermolecular water molecules and the stretching vibration of H2O molecules. An EDS examination confirmed the existence of vanadium and oxygen atoms in the V2O5 NPs (Fig. 1C). To determine the behavior of the synthesized NPs in the aqueous phase, the particle size and zeta potential of the NPs were measured in distilled water. According to the obtained size distribution histogram, hydrodynamic sizes of V2O5 NPs were determined to be 70–289 nm (with a peak below 100 nm), and zeta potentials were measured as +9.8 mV (Fig. 1D). The amount of zeta potential supported the necessary stability of V2O5 NPs in the suspension medium. The structural information of the V2O5 nanospheres was also examined by SEM and TEM observation. The micrographs showed that the V2O5 particles are nanospheres with approximately 100% morphological yields and particle diameter in the range of 60–100 nm (Fig. 2A, B).
Figure 1
Figure 2
The first evaluated growth factor was cell number. The concentration and time dependent influence of V2O5 NPs on the changes in cell number of
Figure 3
The concentration-dependent negative influences of V2O5 NPs on
We managed to explore the extent of cells’ viability by flow cytometry. In this method, propidium iodide (PI) was used as a nucleic acid dye. PI can enter into the perished cells and attach to DNA molecules and produce red fluorescence. In this case, dead fluorescent cells could be distinguished from unstained live cells (Suman et al. 2015). In the graphs, the upper left and right parts expose the ratio of lifeless cells. The lower left and right quadrants specify the number of intact cells and the lower right quadrant signifies the chlorophyll emission (Fig. 4). Cell viability analysis was performed after the treatment of
Figure 4
SEM analysis revealed morphological modification of algal cells after exposure to 100 and 200 mg l-1 of V2O5 NPs as median and final applied concentrations, respectively. Untreated cells displayed the intact turgescent structure in the SEM micrographs (Fig. 5A, B). In comparison, slight plasmolysis of treated cells after treatment with 100 mg l-1 (Fig. 5C, D) and remarkable shrinkage and contraction of cells at 200 mg l-1 V2O5 NPs (Fig. 5E, F) were observed.
Figure 5
SEM micrographs of control and treated algal cells.
For an exploration of the influence of diverse concentrations of V2O5 NPs on algal cells, their photosynthetic pigment content was assessed after 96-hour exposure to NPs. As a result of vanadium oxide treatment, the level of chlorophyll
Figure 6
Effects of different concentrations of V2O5 NPs on photosynthetic pigment contents in
In the current survey, the number of phenols and flavonoids rose in a similar outline at low concentrations of V2O5 NP up to 100 mg l-1 and then lessened intriguingly with the rising concentration of NPs (Fig. 7A, B).
Figure 7
Influence of different concentrations of V2O5 NPs on the
In this section, the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as main antioxidant enzymes of cells, were evaluated after exposure of microalgae to different concentrations of V2O5 NP for 96 h. Based on our results, an increase in the activity of SOD occurred with the elevating concentration of nanoparticles up to 150 and 200 mg l-1 (Fig. 8A). Although the CAT activity did not show significant change at 50 and 100 mg l-1 NP, the enzyme activity rose at concentrations of 150 and 200 mg l-1 of V2O5 NPs (Fig. 8B). The significantly elevated activity of APX was also detected at 150 and 200 mg l-1 of NPs (Fig. 8C).
Figure 8
Activity of
The XRD, FT-IR and EDX examinations established the correct structure, purity, and phase composition of the synthesized V2O5 NPs. The acquired TEM and SEM micrographs revealed the appropriate shape and size of V2O5 NPs, which appeared to fit for the goals of the current investigation. The obtained results of DLS and zeta potential analysis confirmed the suitable size and surface charge of the synthesized NPs in solution that are necessary for their uptake from culture media by cells.
The restrictive influence of V2O5 NPs on the growth of alga in a concentration and time dependent manner was in agreement with some previous findings. The undesirable impact of different transition metal oxide NPs on the growth criteria of plants and algae has previously been reported (Aravantinou et al. 2020; Cardinale et al. 2012; Khan et al. 2021). The lessening in cell number and fresh weight of the algal cell after treatment with metal oxide NPs, such as V2O5 NPs, may occur due to their harm to the cell membrane (Ozkaleli et al. 2018; Xia et al. 2015), inhibiting the photosynthesis by adsorption of NPs on the algal cell wall and shading effect (Chen et al. 2018; Navarro et al. 2008) and intracellular damages by the formation of reactive oxygen species (ROS) caused by NPs (Movafeghi et al. 2019).
Flow cytometry outcome indicated that V2O5 NPs treatment has only a slight effect on
SEM analysis illustrated a modification of the algal cells in NPs exposure. Similar deformation of cells after exposure to metal oxide NPs was formerly affirmed (Li et al. 2020; Movafeghi et al. 2019). The shapes and turgescence of cells are controlled by osmotic force and the function of cell membrane. The first intercourse of NPs with cells occurs in the cell membrane of the target cell. This interaction could cause disruption of membrane integrity and alteration in its hydrophobicity, cationic charge, and permeability (Rai & Biswas. 2018). Furthermore, the exposure of cells to NPs may result in the formation of ROS and resulting changes in metabolic reactions and interruption in the cell membrane function (Abdal et al. 2017; Wei et al. 2017). Subsequently, cell plasmolysis may occur, causing changes in cell morphology (Saxena et al. 2021).
Photosynthetic pigments are recognized as trustworthy indicators for algal cell toxicity (Liu et al. 2009). The cellular metabolic status of algae can be evaluated by determining the content of chlorophylls and carotenoids. Our findings designated the reduction of photosynthetic pigments content. Different studies indicated adverse impacts of metal-based nanoparticles on photosynthetic pigments in algae. Comparable to our finding, the number of photosynthetic pigments in
Exposure to nanoparticles may result in the formation of an excessive amount of ROS that damage cellular structures and inhibit biochemical reactions (Yu et al. 2020). Phenols and flavonoids as non-enzymatic antioxidant molecules in plants and algae scavenge different free radicals or ROS due to their chemical structure (Alghazeer et al. 2013; Ferdous & Balia 2021; Kumar et al. 2008; Melato et al. 2012). Thus, alteration of their content could be considered as a non-enzymatic tolerance mechanism against NP toxicity and induced oxidative stress. Monitoring of phenol and flavonoid content exhibited a rise at a low concentration of NPs and then a drop at high amounts of it. These changes in the content of non-enzymatic defense metabolites in
Beside non-enzymatic antioxidants, cells have developed antioxidant enzymes as powerful ROS scavenging systems to balance the stress-induced ROS (Dvořák et al. 2021; Wang et al. 2019). Superoxide dismutase (SOD) is an essential antioxidant enzyme, which catalyzes superoxide breakdown into oxygen and H2O2, and therefore has a significant function in the tolerance of cells to oxidative stress. In this study, increased activity of the SOD was observed at high concentration of nanoparticles. The role of SOD in the defensive response of algal species to metal oxide nanoparticle toxicity has been previously reported. For instance, it was shown that titanium dioxide and zinc oxide NPs increase the SOD activity in
This research was directed to explore the encouraged cellular toxicity of V2O5 NPs on