Sonochemical synthesis and characterization of aluminum tungsten oxide nanoparticle and study its impact on the growth of microalga

2.1 Materials and method

With the assistance of scanning electron microscopy (SEM) model TESCAN mira3 (TESCAN Company, Czech Republic), the Morphological features of NPs were studied. X-ray diffraction (XRD) patterns analysis was accomplished by a Philips-X’pertPro (Philips Company, Netherlands), Copper source and Kα radiation were used to perform this analysis. A USA-made Nicolet Magna-550 device was used for Fourier transform infrared (FT-IR) analysis. In this analysis, the samples were turned into tablets with the use of KBr. The transmission electron microscope (TEM) model Philips EM208S (Philips company, Netherlands) analysis was used to better analyze particle morphology. All chemical was analyzed by GC-2550TG (Teif Gostar Faraz Company, Iran). The whole chemicals used in this study were of analytical grade: Ethylenediamine (En), Tetraethylenepentamine (TEPA), Hexamethylenetetramine (HMTA), Cetyltrimethylammoniumbromide (CTAB), ammonia, Al(NO₃)₃·9H₂O, and Na₂WO₄·2H₂O, from Merck.

2.2 Method of preparation AWO NPs

Firstly, 0.2 g Na₂WO₄·2H₂O and 0.21 g Al(NO₃)₃·9H₂O precursors were dissolved entirely in distillate water in a separate container. The solution of the precursors was stirred on a magnetic stirrer for 10 min until completely clear. Then, the pH of the solution containing the tungstate ion was adjusted up to 7.5 by adding the pH adjustment factor (NH₃, En, and TEPA). Next, a clear solution containing aluminum cation (Al(NO₃)₃ dissolved in water) was added to the prior solution. After mixing the solution for 10 min, it was subjected to ultrasonic waves to advance the reaction. Also, the synthesis process for the microwave method was followed, as mentioned above. The obtained solution was transferred to a Teflon-lined autoclave of 150 ml capacity in the hydrothermal procedure. The autoclave was held at 180 °C for ten hours and then cooled down to room temperature naturally. Obtained precipitates were centrifuged and washed with distilled water twice. After drying at 60 °C, the sediments were calcined in a furnace at 700 °C for 4 h. All manners of experiments were mentioned in Table. 1 with particularities

2.3 Algae cultivation method and NPs treatment

In this study, Algae with a label (UTX LB 2538) was purchased from algae collective culture in UTEX, USA. The modified Johnson medium as an appropriate algae medium was used to maintain and cultivate these algae (Johnson et al., 1968). 500 ml of medium was added in a 1000 ml Erlenmeyer flasks to prepare the stock culture. The flasks were placed on a rotating shaker at room temperature; at the same time, white light with an irradiance of 100 μmol·m⁻²·s⁻¹ was shone on them. After the number of algae cells reached the exponential phase (about 5 × 10⁴ cells ml⁻¹), algae suspensions with equal numbers of cells were used to investigate the impact of NPs on algae. Different concentrations of AWO NPs (40, 80, and 160 ppm) were added to the treatments, and NP-free treatment was considered as control. The treatments were placed in identical conditions aforesaid and monitored until the last day of the test process (i.e., the tenth day). At the end of the culture period, sampling from algal suspension was performed to check the desired parameters.

2.4 Evaluating biological parameters

A hemocytometer under an Olympus light microscope at 400× was employed to count the number of algae cells. 10 ml of the sample was first filtered with a pre-weighed 45 mm glass filter to record the dry biomass, and then washed with double distilled water and dried at 70 °C in an oven.

The Specific growth rate (μ) was computed by $\mu =\left(ln{N}_2-ln{N}_1\right)/\left(t_2-t_1\right)$ ; In this equation, the natural logarithm of N₁ and N₂ is the natural logarithm of the number of cells counted from the algae at the time of t₁(first day) and t₂ (tenth day) (Fogg and Thake, 1987).

From each treatment, 10 ml was removed to measure chlorophyll-a and b as well as the carotenoid, and each sample was centrifuged at 5000 rpm for 15 min. The top solution was discarded after centrifugation, and the solids that remained were homogenized in 20 ml chilled acetone 80% (v/v) in the dark condition. After homogenization, the samples were again centrifuged at 5000 rpm for 10 min; subsequently, the supernatant adsorption at 663, 646, and 470 nm was measured to obtain the amount of pigment by the equation derived by Lichtenthaler (1987).

The 5 ml of trichloroacetic acid (TCA) solution 10% was used to wash 10 ml of the algae sample, then the solution was centrifuged. Centrifugation was performed for 10 min at 10000 rpm. 1 ml 0.5% thiobarbituric acid (TBA) in 20% TCA was surcharged to 1 ml of the centrifuged supernatant. The resulting samples were heated to 96 °C for 30 min, and then an ice bath was used to cool them. The adsorption of the cooled solution was used to calculate MDA using the equation provided by Heath and Parker. (Heath and Packer, 1968). For this purpose, the cooled samples were centrifuged for 5 min at 10,000 rpm. After that, the adsorption of the supernatant was measured at 532 and 600 nm.

2.5 Ft-IR spectroscopy and data analysis of microalgae

To prepare the samples for FT-IR analysis, after centrifugation of 1 ml of the treatment (5 min at 1000 rpm), the supernatant is thrown away, and the residue is again dispensed in 100 μl Milli-Q water. Then, the number of cells in the samples is adjusted by adding Milli-Q water to 50 × 10⁴ cells per ml. 100 μl of the solution was removed and dried on a glass slide in the oven at 40 °C overnight. The samples were used for FT-IR analysis. Essential FT-IR Spectroscopy software (ver. 3.50) was employed for baseline correction and matching of the spectra. The carbohydrate and lipid to amide ratios were calculated by dividing the maximum peak intensity at specific areas. These areas are (range 1200–950 cm⁻¹) for carbohydrate band, (range 2850–2900 cm⁻¹) for lipid band, and (range 1510–1560 cm⁻¹) for amide II (Kosa et al., 2017).

2.6 Statistical analyses

The studies were performed in each sample with three replications. The one-way analysis of variance (ANOVA) was applied for statistical analysis, and the means were compared using the Fisher LSD test via the Graph Pad Prism 6 software. The selected level of significance for all statistical tests was p < 0.05. The ClustVis online tool was used for hierarchical clustering analysis (HCA) (Metsalu and Vilo, 2015). Heatmap function with correlation-based clustering was used to cluster treatments.

3.1 Nanoparticles characterization

In this work, AWO NPs were prepared by three methods, and the effect of different conditions on the morphology and size of products was investigated. One of the appropriate techniques for recognizing the crystal phase and sincerity of the materials is the XRD (Epp, 2016). Fig. 1 illustrates the XRD patterns of the AWO NPs at multiple methods. The pattern includes the Orthorhombic phase of AWO (JCPDS No. 70–1041, space group: Pbcn) without impurities. As seen from the XRD pattern in all three methods, AWO NPs were synthesized. The crystallite size of the AWO NPs was calculated to be around 15, 18, 27 nm for ultrasonic (sample No. 2), hydrothermal (sample No. 9), and microwave (sample No. 8) methods, respectively; the Scherrer's equation was used for these calculations, Dc = Kλ/βCosθ (Salavati-Niasari et al., 2009).

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Fig. 1 XRD powder patterns of the prepared AWO NPs at several methods (a) ultrasonic, (b) microwave, and (c) hydrothermal.

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Some different mechanisms have been reported for the effects of waves on ultrasonic-assisted reactions (Salavati-Niasari et al., 2008; Salavati-Niasari et al., 2004). Bubble production and explosion due to high temperatures cause the progress of the reactions; this is one of the well-known mechanisms that occur when using ultrasonic (Pollet, 2019). The likely mechanism in the synthesis of AWO NPs is bubble formation. The formation and explosion of these bubbles by generating high heat will accelerate the reaction. On the other hand, the NPs do not have the opportunity to grow due to the continuous wave application, so they are highly morphologically uniform. The general reactions are as follows:

Fig. 2 illustrates the SEM images of samples No. 2–7 prepared with the ultrasonic method. As shown, when used En and TEPA as pH adjustment agents at (a) 5 min in comparison with (b) 10 and (c) 15 min ultrasonic irradiation, particle size is smaller than the others. As shown in Fig. 2 (d, e, and f), the mentioned occurrence also occurred for reactions that TEPA as a pH adjustment. In comparison, for two pH adjustment agents, when used En, the particle size was smaller and more monodisperse than TEPA. Because of this event, En was chosen as a suitable pH adjustment agent. The particle size distribution in sample No.2 shows that this sample is more dispersed and smaller than the other samples. The smaller size distribution in sample No. 2 could be attributed to the 5 min ultrasonic time as the optimal time. As the ultrasonic application increases, the reaction ambient temperature increases, which causes the particles to agglomerate.

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Fig. 2 SEM images prepared by (a) 5, (b) 10, and (c) 15 min under ultrasonic irradiation and used En as pH adjustment and (d) 5, (e) 10, and (f) 15 min under ultrasonic irradiation and used TEPA as pH adjustment.

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Two hydrothermal and microwave methods were used to investigate the effect of other nanoparticle synthesis methods on size and morphology. Fig. 3 (a, b) illustrates the SEM images samples No. 9 and 10, prepared with the hydrothermal method using two different surfactants. Fig. 3 (a) shows a sample that was prepared with HTMA as the surfactant. As can be seen, NPs are monodispersed and with reasonable size, but when used CTAB as a surfactant (Fig. 3 (b)) particle size because agglomeration is rising. In Fig. 3 (c), the microwave method at the cyclic reaction was employed. The purpose of the applied cyclic reaction was to get different morphology, but this did not happen, and NPs used the cut-off radiation time and grown. Fig. 3 (d) illustrates NPs prepared by the ultrasonic-assisted method with NH₃ as pH adjustment. The particles are stuck together, and the particle size is too large.

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Fig. 3 (a, b) SEM images sample No. 9 and 10, prepared by the hydrothermal method, in which (a) HTMA and (b) CTAB were used as surfactants, (c, d) SEM images sample prepared with (c) microwave method and (d) ultrasonic-assisted method with NH₃ as pH agent, and (e, f) TEM images of AWO NPs sample No. 2.

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After reviewing all SEM images, samples No. 2 and 9 were selected as optimum because of their size and monodisperse morphology. Eventually, sample No. 2 was chosen for later use due to the speed and convenience of preparation and lower energy consumption at the synthesis step.

In examining the TEM images obtained from nanoparticles, what was obtained from the in-depth of these sample shows that NPs have a spherical shape and are adhered together. Furthermore, Fig. 3 (e, f) illustrated NPs sample No. 2 with diameters about 15–20 nm.

FT-IR spectroscopy can be a beneficial analysis to recognize and understand functional groups of organic materials. Fig. 4 depicts the FT-IR spectrum of AWO NPs (sample No.2) before and after calcination. Peaks are seen in the 1650–1350 cm⁻¹ can be attributed to ethylenediamine. These peaks disappear after the calcination process at 700 °C. The water adsorbed on the surface of the NPs causes peaks to appear at 3437 and 1633 cm⁻¹ (Mortazavi-Derazkola et al., 2015). The peaks at 820–1050 cm⁻¹ range can be assigned to stretching modes of the WO₄ tetrahedra. The bands in about 430 cm⁻¹ were assigned to stretching vibrations of the AlO₆ octahedral (Prisco et al., 2013).

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Fig. 4 FT-IR of as-synthesized NPs (before and after calcination at 700 °C) sample No. 2.

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Fig. 5 shows the Energy Dispersive X-Ray Spectroscopy (EDS) analysis results applied to the synthesized AWO NPs. As shown, the synthesized NPs are formed of O, Al, and W elements without any impurity that corroborated AWO NPs formation. DRS analysis at room temperature was used to evaluate the optical properties of the manufactured product. For materials with direct band energy absorption coefficients, the Tauc equation was used, which is calculated as follows: (αhυ)n = A(hυ − Eg), where A is a constant, Eg is the energy band, and n is 1/2 for a direct gap and 2 for an indirect gap (Tauc et al., 1966; Hassanpour et al., 2021). The DRS spectrum of sample No. 2 was given in Fig. 6, which illustrates optical absorption capability in the region from 300 to 700 nm. The corresponding NPs energy bandgap (Eg) value is 3.67 eV.

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Fig. 5 EDS spectra obtained from sample No. 2.

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Fig. 6 Diffuse reflectance spectra (DRS) of AWO NPs, sample No. 2.

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3.2 Results of nanoparticle applications

Optimal samples of the NPs with three concentrations (40, 80, 160 ppm) were utilized to investigate the effect of NPs on algae compared to NPs- free conditions as a control. The result of NPs treatment on the growth rate of D.salina was illustrated in Fig. 7 (a, b, c). A similar trend for (a) cell number, (b) biomass, and (c) specific growth rate of algae were observed at all three concentrations of NPs. A mild concentration of NPs (80 ppm) significantly increased growth. In this respect, at the concentration of 160 ppm, growth rates were more sensitive to NPs and were considerably lower than the control sample.

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Fig. 7 Effects of different concentrations of NPs on growth of D.salina, (a) number of cells, (b) specific growth number, (c) biomass, (d) total carotenoid, (e) chlorophyll-a, and (f) chlorophyll-b.

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Fig. 7 (d, e, f) shows the effect of NPs on the carotenoid and chlorophyll-a, b content of the alga. The content of chlorophylls and carotenoids was not significantly different from that of the control sample at concentrations 40 ppm NPs. An increasing concentration of NPs at the second concentration (80 ppm) induced an increase of chlorophylls and carotenoids compared to the control sample. However, at higher concentrations of NPs (160 ppm), a considerable decrement of chlorophylls and carotenoids were observed. An increase of carotenoids in the samples containing NPs might be a defensive response; the carotenoids are known to be a powerful controller of ROS, particularly singlet oxygen (Li et al., 2007). It has also been reported that the reduction of chlorophylls can be related to the correction in the metabolic system of algae. Under stress situations, the CO₂ and nutrient usage rates are decreased by an enhanced rate of the produce of nicotinamide adenine dinucleotide phosphate (El-Sheekh et al., 2003). There are numerous reports of decreased levels of chlorophylls and carotenoids at high concentrations of stress factors; for example, the results of cobalt used as a stress factor on algae showed that when used low content of cobalt affected a considerable increase in chlorophyll-a and carotenoids in Nitzchia perminuta. In contrast, higher concentrations reduced pigment contents (Oukarroum et al., 2012). Therefore, it could be concluded that the effect of stress factor on different organisms is species-dependent and shows a dose-dependent manner. The NPs high reactivity because of the high surface area to volume ratio may lead to particle-algae interactions. The formation of cell-NPs agglomerate was a common interaction of colloidal particles with algal cells (Peralta-Videa et al., 2011). Since cellular aggregation might reduce the availability and use of light or nutrients for growth (De Fillipis et al., 1981), it could be possible that cell-NPs agglomerate at high concentrations of NPs decreased the growth-related parameters. It was also reported that the reduction of chlorophyll-a in the cells was a common symptom of heavy metals and NPs toxicity (Aruoja et al., 2009).

Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in the cell membranes. It was reported that an enhance in free radicals can increase MDA (Gaweł et al., 1960). In general, the amount of MDA can be used as a sign of cell damage (Duong et al., 2015). Graph of existing data (Fig. 8 (a)) indicated that the D.salina treatment with NPs altered the MDA amount compared with the control sample. The amount of MDA increased significantly at a high concentration of NPs (160 ppm) than other treatments. Membrane instability is universally related to lipid peroxidation due to ROS enhancement (Reactive Oxygen Species) during a wide range of stresses (Li et al., 2007; Driver et al., 2015). According to our results, it might be concluded that by increasing volume concentrations of NPs, free radical generation is triggered within the algal cells. The presence of NPs in the medium could be attributed to the electron-hole production by these NPs due to the light irradiation, which leads to the induction of a higher amounts radical species in the aqueous medium (Hassanpour et al., 2017).

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Fig. 8 Effects of different concentrations of NPs on (a) MDA, and ratio of (b) lipid and (c) carbohydrate to amide II, and (d) Heatmaps obtained from hierarchical clustering analysis (HCA) of the parameters under control and samples treated with NPs condition.

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The ratios of lipid and carbohydrate to amide II were shown in Fig. 8 (b) and (c), respectively. Lipid is one of the main biochemical products that microalgae can produce along with starch under nutrient-deprived conditions during light supplementation (Li et al., 2008). As shown in Fig. 8 (b), the lipid to amide II ratio at low concentrations of NPs (40 or 80 ppm) did not change in contrast with control.

Interestingly, the amount of lipid increased in the presence of high concentrations of NPs (160 ppm) compared to control and other treatment groups. As indicated in Fig. 8 (c), a similar trend was observed for the carbohydrate to amid II ratio; however, this ratio was slightly higher at the second concentration of NPs (80 ppm). The increase in lipid and carbohydrate ratios to amide II at a higher concentration of NPs (160 ppm) could be due to the increased free fatty acids and soluble sugars in the algal cells, respectively. According to literature, in previous studies, when the stress factor increased relatively mildly, a higher intracellular lipid accumulation might have occurred. In contrast, a further application of stress factors above a threshold in terms of concentration or exposure time may significantly reduce the synthesis of total lipids, which eventually led to reductions in cell growth (Takagi and Yoshida, 2006; Xia et al., 2014). It has also been reported that lipids, particularly neutral lipids, had essential roles in quelling the accumulated ROS (Srivastava and Goud, 2017). Such defensive strategies frequently occurred in plant or algal cells to enhance cell tolerance against ROS-related damages during stress and countering with stress conditions (Driver et al., 2015). Another hypothesis may be that the increase in the lipid peak in the sample contains the third concentration of NPs (160 ppm) compared to other samples due to an increase in the MDA because of increased lipid peroxidation in the presence of high concentrations of NPs.

Fig. 8 (d) showed the heatmap obtained from hierarchical clustering analysis (HCA) of the parameters under control and different NPs concentrations. The HCA analysis results showed two general categories for the growth, chemical and physiological parameters. The MDA content, carbohydrate, and lipid ratios to amide II were clustered in one group and separated from other parameters. MDA has placed in a separate batch from carbohydrate and lipid to amide II ratios in this cluster. These parameters reached the maximum value at the third concentration of NPs (160 ppm). Other parameters, including the amount of chlorophyll-a, biomass, and total carotenoid, were clustered in another group, which was subdivided into different subgroups. These parameters were in equilibrium in the sample without the NPs and the treatment containing the first concentration of the NPs (40 ppm); in the sample containing 80 ppm of NPs, these parameters reached their maximum value, but in algae treated with 160 ppm of NPs the minimum intensity of these parameters were observed. Chlorophyll-b is also separated by cell number and specific growth rate in the following subgroup: they peaked at 80 ppm of NPs. In the other concentrations, they were in an equilibrium state. As can be seen from the clustering, the parameters at the second concentration of NPs showed a favorable state for better growth and physiology of algae than control and other treatments containing NPs. From a biotechnological perspective, such a concentration of AWO NPs may be applicable in open and close algal culturing systems to induce a higher algal growth and biomass in a shorter period.

CRediT authorship contribution statement

Mohammad Hassanpour: Investigation, Formal analysis, Software, Methodology, Writing – original draft. Masood Hamadanian: Supervision, Software, Visualization. Omid Amiri: Software, Writing – review & editing. Seyed Ali Hosseini Tafreshi: Resources, Validation, Supervision, Investigation. Masoud Salavati-Niasari: Writing – review & editing, Methodology, Conceptualization, Supervision, Project administration, Visualization, Data curation, Validation, Resources.