Improvement on lipid production by Scenedesmus obliquus triggered by low dose exposure to nanoparticles

Carbon nanotubes (CNTs), α-Fe2O3 nanoparticles (nano Fe2O3) and MgO nanoparticles (nano MgO) were evaluated for the effects on algae growth and lipid production. Nano Fe2O3 promoted cell growth in the range of 0–20 mg·L−1. CNTs, nano Fe2O3 and nano MgO inhibited cell growth of Scenedesmus obliquus at 10, 40 and 0.8 mg·L−1 respectively. Neutral lipid and total lipid content increased with the increasing concentration of all tested nanoparticles. The maximum lipid productivity of cultures exposed to CNTs, nano Fe2O3 and nano MgO was observed at 5 mg·L−1, 5 mg·L−1 and 40 mg·L−1, with the improvement by 8.9%, 39.6% and 18.5%. High dose exposure to nanoparticles limited increase in lipid productivity, possibly due to the repression on cell growth caused by nanoparticles-catalyzed reactive oxygen species (ROS) generation, finally leading to reduction in biomass and lipid production. Reduced accumulation of fatty acids of C18:3n3, C18:3n6 and C20:2 was observed in cells exposed to nanoparticles.

resulting in cell damage and death at low concentrations, the oxidative stress induced by NPs usually exceeds the antioxidant defense of algae cells, thus putting the utilization of NPs under careful assessment and precise control to prevent oxidative damage to the cells.
The aim of this study is to test the positive effects of various nanoparticles on algal growth and accumulation of valuable biochemical products. Improvement on the biomass production by crop has been demonstrated by application of carbon nanotubes (CNTs) 10 , but little is known about the possible application of CNTs in microalgae, thus was selected as the first candidate material in this study. Two metal oxide NPs, hematite (α-Fe 2 O 3 ) and magnesium oxide (MgO) were also selected considering their lower ecotoxicity 15,16 and that the disassociated ions from the metal oxide NPs (Fe 3+ and Mg 2+ ) are essential to cell growth. Iron is essential to the metabolism and growth of all organisms and is especially important in phytoplankton because of its presence in iron-sulfur and cytochrome proteins involved in photosynthetic electron transport 17 . Appropriate elevated Fe concentration promotes cell growth and lipid production in Chlorella sorokiniana 18 and carbohydrates accumulation in Dunaliella tertiolecta 19 . Magnesium is one of the key element required for chlorophyll synthesis and the co-factor for several important cellular processes 20 . Excessive Mg concentration and MgSO 4 nanoparticles were found to enhance the lipid accumulation in Chlorella vulgaris cultivated in wastewater 21 .
The idea is to employ suitable nanoparticles, which are less toxic to organisms, as stimuli to alter cell metabolisms to boost accumulation of valuable biochemical compounds under controlled concentrations. Typical nanomaterials, CNTs, nano Fe 2 O 3 and nano MgO were selected and their effects on biofuel production by a potential green algae Scenedesmus obliquus 22 were evaluated, with respect to cells growth, pigments, photosynthetic activity, soluble sugar and protein contents, and lipid production. Potential toxic evaluation of nanoparticles is also undertaken to explore a proper way to utilize nanoparticles in algal biotechnology.

Results
Effects of nanoparticles on cell growth of S. obliquus. Cells of S. obliquus grown under low concentrations of CNTs (2.5 and 5 mg·L −1 ) showed similar growth profiles as the untreated cells ( Fig. 1A). High concentrations of CNTs (10, 15 and 40 mg·L −1 ) did not impose significant effect on cell growth within 96 h, but inhibited cell propagation after then. Promotion on cell growth was observed in cultures grown under tested nano Fe 2 O 3 concentrations of 2-20 mg·L −1 (Fig. 1B). Concentrations higher than 40 mg·L −1 nano Fe 2 O 3 led to slight inhibition on cell growth, with the reduction of 8.0%, 14.7% and 16.9% in the final cell density on 7 d at 40, 60 and 100 mg·L −1 , respectively. Incubation of algae cells in medium containing nano MgO resulted in significant repression on cell growth even at lower concentration of 0.8 mg·L −1 at 72 h (Fig. 1C). The cell density was reduced by 22 Table 1, a suitable amount of CNTs (5 mg·L −1 ) increased the chlorophyll content, while excessive CNTs repressed chlorophyll synthesis at 15 and 40 mg·L −1 . Concentration of 2.5 and 10 mg·L −1 did not impose a significant effect. A similar increase in chlorophyll was observed in nano Fe 2 O 3 treatment at 5, 10 and 20 mg·L −1 , which was in agreement with the cell growth curves. Increasing concentration above 40 mg·L −1 nano Fe 2 O 3 caused reduction in chlorophyll contents. Chlorophyll contents of cultures were greatly reduced by 73.3%, 84.5%, 80.6% and 87.5% when exposed to nano MgO of 0.8, 8, 40 and 100 mg·L −1 , respectively. Protein contents were insignificantly different between treatments of various concentration of CNTs, while those in cultures exposed to nano Fe 2 O 3 increased significantly at tested concentrations. Nano MgO induced noticeable increases in protein content at any tested concentrations, with the maximum protein content obtained at 100 mg·L −1 . Increases in total soluble sugars were observed in all treatments with every tested NPs, resulting to the maximum content for CNTs, nano Fe 2 O 3 and nano MgO at 15, 60 and 100 mg·L −1 , respectively. Lipid and fatty acids profiles. Lipid production parameters and fatty acids profiles of S. obliquus were determined under several representative NPs concentrations ( Table 2). The neutral lipid fluorescence intensity was significantly promoted by increasing CNTs concentration. The total lipid content and lipid productivity was slightly promoted in cultures exposed to 5 and 40 mg·L −1 CNTs, compared to the untreated cells. The neutral lipid fluorescence gradually increased with increasing nano Fe 2 O 3 concentrations, resulting a maximum value at 100 mg·L −1 of nano Fe 2 O 3 . Higher lipid content was observed in cultures under the exposure to 40 mg·L −1 and 100 mg·L −1 of nano Fe 2 O 3 , which was promoted by 27.8% and 44.8% when compared with the content in normal cultures. Nevertheless, the maximum lipid productivity was obtained at 5 mg·L −1 nano Fe 2 O 3 , with an increase by 39.6%. The lipid productivity of cultures treated with 40 and 100 mg·L −1 nano Fe 2 O 3 was lower than that of 5 mg·L −1 but still higher than untreated cultures, probably due to the slight inhibition on biomass accumulation when the concentration of NPs increased from 5 mg·L −1 to 40 and 100 mg·L −1 . Similar to the trend of nano Fe 2 O 3 , the neutral lipid fluorescence intensity of cells treated with nano MgO was greatly promoted with the increase in tested concentrations. A significant increase in total lipid content was observed in all treatments of nano MgO, which was 27.8% 32.2% and 53.4% higher than the control at the concentration of 0.8, 40 and 100 mg·L −1 , respectively. A slight improvement in lipid productivity was observed in cultures treated with 0.8 mg·L −1 of nano MgO, while 100 mg·L −1 nano MgO resulted in lower lipid productivity, owing to the loss of biomass.

NPs induced oxidative stress and cellular antioxidant defenses.
To evaluate the oxidative stress induced by NPs, hydrogen peroxide, an important marker of ROS, was determined after 48 h of exposure to NPs. Although, insignificant differences in H 2 O 2 contents of treatments exposed to low doses of CNTs (2.5, 5 and 10 mg·L −1 ) were observed ( Fig. 2A), the H 2 O 2 content was elevated at 15 mg·L −1 and 40 mg·L −1 CNTs treatment. Unlike CNTs, the H 2 O 2 contents in cultures exposed to nano As presented in Fig. 2D, the catalase (CAT) and superoxide dismutase (SOD) activities were greatly enhanced under 15 and 40 mg·L −1 CNTs. A substantial enhancement on the CAT activities of cells exposed to nano Fe 2 O 3 was observed when the concentration increased from 10 to 60 mg·L −1 (Fig. 2E). The CAT activities decreased when the nano Fe 2 O 3 was further increased to 100 mg·L −1 , which was still higher than the control. The SOD activity of cells exposed to nano Fe 2 O 3 increased greatly at concentrations higher than 10 mg·L −1 , which maintained a relatively higher activity at 100 mg·L −1 . The peroxidase (POD) activities of treatments with nano MgO (Fig. 2F) showed a remarkable enhancement at very low concentration (0.8 mg·L −1 ). The POD activities among the treatments with various nano MgO concentrations showed negligible differences between treatments. Unlike CNTs and nano Fe 2 O 3 , nano MgO triggered an increase in SOD activities at 0.8 mg·L −1 , which then declined with the increasing nano MgO concentrations (  cell-free solution, which might be one of the possible reasons for lower toxicity of nano Fe 2 O 3 than nano MgO. Substantial decreases in F v /F m of algal cells exposed to tested nanoparticles at inhibiting concentrations indicated that the photochemical activity was repressed and occurrence of photoinhibtion was inevitable 23 . Simultaneous reduction in rETR max suggested the block of photosynthetic electron transport 24 . Several studies reported for the toxicity of CNTs to green algae. Schwab et al. 7 found the inhibition on algae cell growth by aggregates of CNTs was highly correlated with the shading of CNTs and the agglomeration of algal cells, suggesting that the reduced algal growth might be caused mainly by indirect effects like limited light availability. Demir et al. 25 Table 2. Lipid production and fatty acid composition (%) of S. obliquus exposed to different concentrations of CNTs, nano Fe 2 O 3 and nano MgO. Fatty acid percentage was calculated as the ratios of the total fatty acids. UFA and SFA represent unsaturated and saturated fatty acids respectively. Data are represented as mean ± SD from triplicate samples (n = 3). Significant difference (p < 0.05) between treatments was indicated by asterisks. Data for neutral lipid fluorescence were obtained from different biological repletion experiments, therefore the valves of the control groups of three tested NPs were different due to different cell density of independent experiments.  such as CAT, SOD, POD, and production of the antioxidants including carotenoids and ascorbic, proteins and sugars 27 . The balance between ROS level and antioxidant defense capacities determines the occurrence of oxidative stress response and cell damage degree 6,27 . Remarkable increases in ROS levels and increased MDA contents (Fig. 3A-C), which is an important indicator for lipid peroxidation under oxidative stress 27 , were observed in cultures exposed to all tested NPs at cell growth repressing concentrations repressing cell growth. Although amounts of protein become the targets of ROS, oxidative stress could induce protein accumulation at low ROS level. The observation of increase in soluble protein content is usually considered as an evidence of active defense mechanism to prevent algae cells from damaging by abiotic stress 28 . The important components of soluble protein, antioxidant 29 and biotransformation enzymes 30 , which are involved in the active defense mechanisms, are usually regarded as biomarkers for identification for xenobiotics. In addition, the elevation in protein content could be related to heat shock protein formation 31 and elevation of specific mitochondria enzymes, such as alternative oxidase and uncoupling proteins 30 in forestalling ROS production. Polak N reported that metallproteins and phytochelatin synthase may play an important role in response to nanoparticles 32 . The protective effects of soluble sugars against oxidative stress have been mostly attributed to signaling, triggering the production of specific ROS scavengers, and even acting as direct ROS scavengers at higher concentrations 33 . Glucose and sucrose play a central role in ROS signaling 33 . The longer water-soluble oligo-and polysaccharides might be effective candidates for capturing ROS and scavenging the free radicals in microalgal cells exposed to a wide range of environmental  34 . The enhancement in the synthesis of sugars might represent another protective mechanism of environmental stress 35 , which is in accordance with the increase of cellular soluble sugars exposed to NPs in this study. In a word, the enhancement on CAT, SOD or POD, and increases in soluble proteins and sugars of cultures exposed to nano Fe 2 O 3 and nano MgO signify the involvement of both antioxidant enzymes and biochemical compounds in the antioxidant defense against the oxidative stress of NPs. When the oxidative stress induced by NPs exceeds the scavenging capacity of the cellular antioxidant defenses, oxidative damage is occurred finally leading to cell growth inhibition. In addition, large aggregates formed by NPs on cell surfaces might cause a shading effect on cell growth at high NPs concentrations by reducing the light availability 7 . The loss of the flagellums of S. obliquus and agglomerating with the NPs (Fig. 4), leading to serious limitation of cell motility, could also contribute to the toxicity at high NPs concentrations.
Interestingly, nano Fe 2 O 3 at a concentration < 20 mg·L −1 promoted cell growth of S. obliquus (Fig. 1B), as well as the chlorophyll, soluble protein and sugar contents (Table 1). However, photosynthetic parameters were not enhanced under such conditions. Nano Fe 2 O 3 at a concentration of 10 mg·L −1 induced oxidative stress while inhibition on cell growth occurred at a concentration higher than 40 mg·L −1 . This indicates that the cells could somehow overcome the oxidative stress and maintain stimuli on cell growth in the range of 0-20 mg·L −1 nano Fe 2 O 3 . This promotion on algae growth might be related to the disassociated trace ions of nanoparticles (Fig. 5), which represents a suitable source of trace elements 36 . The dissociated Fe 3+ from nano Fe 2 O 3 especially at low concentration (<10 mg·L −1 ), might impose a positive effect on cell growth and biochemical compounds accumulation. Similar promotion on algae growth was reported by Pádrová et al. 12 ; the trace concentrations of zero-valent iron nanoparticles (5.1 mg·L −1 ) caused overproduction of biomass during cultivation of cyanobacteria and microalgae. At high NPs concentrations, algal cell growth was repressed due to the NPs-induced oxidative injury. The promoting effect imposed by dissociated Fe 3+ might be eliminated by the inhibition on cell growth, when Fe 2 O 3 NPs concentration increased from low (5 mg·L −1 ) to high concentration (>40 mg·L −1 ). In summary, nano Fe 2 O 3 imposed a stimulating effect on cell growth, while high concentrations of tested NPs imposed inhibition, or even toxicity (100 mg·L −1 nano MgO).
Majority of microalgae accumulate neutral lipids up to 20-50% of dry cell weight as a storage energy source under photo-oxidative stress or other adverse environmental conditions 13 . In this trial to explore the potential of CNTs, it was found they impose a positive effect at low concentration (5 mg·L −1 ) on neutral lipid accumulation (Table 2). However, the neutral lipid accumulation was not significantly improved when the CNTs concentration increased from 5 mg·L −1 to 40 mg·L −1 (p = 0.268). The increase in total lipid content at 40 mg·L −1 CNTs might be resulted from the increase in other types of lipids such as glycol lipids (GLs) and phospholipids (PLs), which are important components of external and chloroplast membrane, along with the endoplasmic reticulum 37 .
On the contrary, increasing concentrations of nano Fe 2 O 3 and nano MgO greatly promoted the neutral lipids accumulation ( Table 2). The optimal concentration of nano Fe 2 O 3 and nano MgO for lipid productivity was 5 and 0.8 mg·L −1 , respectively. Further increase of NPs concentration could not improve the lipid productivity. This could possibly be due to the less biomass accumulation as a result of repression on cell growth. The optimum concentration of ferric ions for biomass and lipid accumulation in green algae, such as Chlorella vulgaris, Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis, was studied 38,39 . Very low Fe 3+ concentration (0.0012 μM-1.2 μM) promoted cell growth, but only high levels of Fe 3+ > 12 μM greatly enhanced both biomass and lipid production. Supplement of high concentrations of Fe could also increase the proportion of saturated fatty acids, which meets the requirements of qualified biodiesel production 39 .
As an essential element of chlorophylls, the beneficial effect of magnesium on biomass and lipid production by microalgae has been reported in mixotrophic conditions 40 . Saurabh et al. synthesized MgSO 4 nanoparticles with sizes ~100 nm was found to greatly promote both biomass and lipid production at 1 g/L using crude glycerol as a substrate 21 . Nanoparticles of Mg aminoclay was reported to improve the lipid productivity of a mixotrophically grown Chlorella sp. by ~25% 41 . On the contrary to the above results obtained under mixotrophic growth condition, our results showed that the nano MgO can not promote both biomass and lipid production under Comparing the fatty acid profiles of cells exposed to different nanoparticles with untreated cells, a common increase in C16:0, C16:1, C16:2, C18:1n9 and C18:2nc and decrease in contents of C18:2nt and C18:3n6 and C18:3n3, C20:2 was observed in treatments exposed to high concentrations of NPs. The UFA/SFA ratios declined slightly under high concentrations of NPs. The converse observation of decreased UFA contents in this study was against the reports of the accumulation of polyunsaturated fatty acids (PUFAs) 12,14 , which is known to have a radical scavenging potential and contribute to cell protection against increased ROS level 42 . One possible explanation is that the efficient involvement of UFAs in ROS scavenging might result in a metabolic interconversion between different FAs. Reduced accumulation of PUFAs such as C18:3n3, C18:3n6 and C20:2 in cells exposed to NPs is beneficial for controlling the oxidation stability of the downstream biodiesel products.
Although the improvement on lipid production by the tested NPs was not as effective as other environmental factors such as nutrients deprivation and UV treatment, the results are still promising that the tested NPs could improve lipid productivity at lower concentrations. Despite the fact that CNTs, nano Fe 2 O 3 and nano MgO repressed cell growth at high concentrations, which might be related to ROS generation and agglomeration of algae cells and NPs, S. obliquus was able to overcome those adverse oxidative stresses typically at lower doses of NPs and diverted the metabolisms to lipid accumulation. Although the improvements achieved are still not soaring, nanoparticles can be considered as an auxiliary tool for enhancing lipid production by combining with other stimuli. Further improvements could be focused on combining various environmental stimuli with NPs to improve lipid production. Another advantage of the application of nanoparticles in algal biotechnology relates to the agglomeration of magnetic NPs and algae cells, facilitating the precipitation of algae cells from the medium, which is economically beneficial for cell harvesting. With respect to lipid extraction process, aminoclay-based nanoparticles, such as Fe-(3-aminopropyl)-triethoxysilane (Fe-APTES) clay imposed positive effects on lipid extraction yield, FAME content and its productivity of Chlorella sp. KR-1, through destabilization of algal cell walls via solubilized aminoclays and disruption in cell walls by hydroxyl radicals (OH·) originated from the Fenton-like reaction induced by Fe 3+43 .
In summary, nano Fe 2 O 3 is found to be a promising candidate for improving both biomass and lipid production by S. obliquus at an appropriate concentration (<20 mg·L −1 ). Further increasing nano Fe 2 O 3 concentrations caused gradual inhibition on cell growth, but still can facilitate lipid accumulation without limiting the lipid productivity. CNTs and nano MgO imposed a more severe repression on cell growth than nano Fe 2 O 3 . Exposure of S. obliquus to low doses of CNTs (5 mg·L −1 ) and nano MgO (0.8 mg·L −1 ) can promote lipid productivity while the lipid productivity was limited by reduction in biomass accumulation under high dose exposure.

Materials and Methods
Nanoparticles. Carbon nanotubes (CNTs, purity > 90%, diameter < 2 nm, length < 20 μm) was provided by Nanjing University of Science and Technology. Nano ferric oxide (α-Fe 2 O 3 , purity > 99.5%, particle size < 30 nm), nano MgO (purity > 99%, particle size < 50 nm), which was purchased from Aladdin Chemistry Co. Ltd, China, was used to prepare nanoparticle suspensions. Prior to every assay, fresh NPs stock suspensions (100 mg·L −1 ) were prepared by sonication for 60 min and then filtering through 0.22 μm pore size PC membranes (Millipore). Transmission electron microscopy images of nanoparticles were provided in the supplement materials.
Strains and culture conditions. Scenedesmus obliquus was obtained from the Freshwater Algae Culture Collection, Institute of Hydrobiology, Chinese Academy of Sciences. The strain was pre-cultured aseptically in 250 mL Erlenmeyer flasks with 100 mL of BG11 medium 44  Algae cells were grown in medium containing various NPs concentration for 7-8 days and algal growth was monitored daily. Cell density was measured by cell counting using a hemocytometer and an optical microscope (Leica, Germany). Growth inhibition (%) was calculated from difference of the specific growth rate in untreated and treated cultures at 96 h. EC 30 and EC 50 for algal growth in response to NPs were calculated from the growth inhibition by nonlinear regression using the cumulative distribution function pnorm of the statistical software SPSS 16.0. The 95% confidence intervals for the EC 30 and EC 50 values were calculated using bootstrap resampling.

Determination of biochemical compounds and antioxidant enzyme activities.
Chlorophyll determination was performed by 100% methanol extraction and the absorbance of supernatant was measured at the wavelengths of 653 nm and 666 nm 45 . Cells at 96 h were harvested and analyzed for total sugar, soluble protein and antioxidant enzyme activity analysis. Total sugar content were measured by phenol-sulfuric acid method 46 . Total soluble protein contents were determined by Bicinchoninic acid method using the total protein assay kit (Nanjing Jiancheng Biology Engineering Institute, China), and the results were expressed as micrograms of protein per 10 7 cells (μg/10 7 cell). The CAT and POD activities were measured by assay kits (Nanjing Jiancheng SCiENtifiC REPORTS | 7: 15526 | DOI:10.1038/s41598-017-15667-0 Biology Engineering Institute, China). The SOD activity was determined according to the method of Mishra 47 . The activity of SOD was defined as the quantity of SOD required to inhibit 50% the photochemical reduction of nitroblue of tetrazolium.
Neutral lipid content was determined via monitoring the nile red fluorescence intensity of the algae samples as described previously 48 . Total lipid was exacted using methanol: chloroform mixture (2:1 v/v) according to Gao et al. 49 and the content was expressed as percentage per dry cell weight (DCW). The lipid composition was determined as fatty acid methyl esters through direct transesterification with potassium hydroxide (KOH) in methanol. The crude lipid sample was analyzed using a gas chromatograph (Thermo Trace GC ULTRA) equipped with a flame ionization detector (FID) as described previously 49 . Measurement of MDA and hydrogen peroxide. MDA was measured spectrophotometrically based on the thiobarbituric acid method 50 . Algae samples of 50 mL after 48 h exposure were harvested and homogenized in an ice bath by ultrasonication as described above, then resuspended in 4 mL 80% ethanol. The supernatants were collected and the MDA content was determined by a commercial kit (Nanjing Jiancheng Biology Engineering Institute, China) following the manufacturer's instruction. MDA content was expressed as nmol MDA/mg protein. The amount of hydrogen peroxide was determined using the TiCl 4 method based on the reaction between hydrogen peroxide and TiCl 4 that forms colored Ti complexes. The absorbance was measured at 410 nm. The H 2 O 2 content was expressed as mmol H 2 O 2 /mg protein.

Photocatalytic Degradation of methylene blue (MB).
Methylene blue is a widely used model dye to assess the photo catalytic potential of nanoparticles. It is also used to confirm intended oxidative stress caused by the photocatalyst arises in a microalgae cultivation system 14 . Typically 10 mg of tested NPs (α-Fe 2 O 3 and MgO) were added to 100 mL of methylene blue dye solution (10 mg·L −1 ). The mixtures were well mixed by magnetically stirring for 30 min to equilibrate the working solution before illumination. The suspensions were then exposed to illumination and the absorbance at 668 nm was monitored at a 60 min interval. Percentage of dye degradation was estimated by the following formula: Measurement of photosynthetic parameters. The chlorophyll fluorescence parameters were measured by using a multi-wavelength phytoplankton pulse-amplitude-modulated fluorometer (Phyto-PAM, Walz, Germany) 51 . The maximal efficiency of photosystem II photochemistry, F v /F m , was measured after dark adaption of the samples for 10 min. Rapid light curves (RLCs) were performed according to a pre-installed software routine. rETR max (the relative maximum Electron Transport Rate) was calculated from the RLCs 52 .
Scanning electron microscopic (SEM) imaging. After exposure to NPs for 48 h, the surface of the algal cells was observed by Field emission SEM (Hitachi S-4800, Japan). Aliquots of 10 mL algal suspensions under treatments with 40 mg·L −1 CNTs, 60 mg·L −1 nano Fe 2 O 3 , and 40 mg·L −1 nano MgO were withdrawn and cells were harvested at 1000 g for 3 min. The pellets were then chemically fixed for ~24 h using 4% (w/v) glutaraldehyde. Then the samples were subjected to a dehydration procedure by graded series of ethanol before air-drying. Finally, the sample films were coated with gold and loaded for SEM analysis. Each sample was analyzed for at least five view fields at different magnifications.
Measurement of Fe 3+ concentration in the culture media. An aliquot of 2 mL culture was sampled from treatments exposed to different nano Fe 2 O 3 concentrations for 3 days, then filtering through a 0.45 μm membrane. The filtrate was collected for analyzing Fe 3+ concentration by an inductively coupled plasma -mass spectrometer (ICP-MS, Agilent 710).
Statistical analysis. SPSS PASW Statistics 16 software was used for all statistical analyses. The mean values, confidence intervals, and standard deviation values for all treatments (triplicates) were calculated. Data from three replicates (n = 3) were analyzed using one-way ANOVA, and p < 0.05 was considered statistically significant.
Data availability statement. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.