Zīst/shināsī-i Giyāhī-i Īrān (May 2022)
Physiological responses of Spirulina platensis to nanoparticles of TiO2 and citrate
Abstract
Abstract In order to investigate the stimulating effect of TiO2 nanoparticles on the growth rate and the increase of some metabolites in the microalgae Spirulina platensis, the anatase and rutile forms of the TiO2 nanoparticle were compared to the bulk form, in the presence or absence of citrate, on the microalga. The highest chlorophyll, carotenoid, phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE) were observed on 3rd day. The highest PC and APC/PE were obtained by rutile and anatase, respectively. Rutile NPs could stimulate the increase of dry biomass and maximum specific growth rate (µm) better than anatase and even the bulk form. Carbohydrate production was stimulated by bulk and rutile. Low concentrations of nanoparticles often had a better effect in increasing PC, APC, and PE pigments. The addition of citrate without TiO2 stimulated the production of astheaxanthin, lipid, protein, and ROS. The simultaneous treatment by citrate-TiO2 was able to reduce the negative effect of nanoparticles on dry weight, but totally, its effect was dependent on the form of TiO2 and stress time. Antioxidant activity was stimulated by the bulk form and caused a decrease in Malondialdehyde (MDA), but an increase in MDA was observed by nanoparticles. The highest ROS was observed in the treatment with rutile (with citrate) on the 3rd day. In general, the stimulating effect of TiO2 on the improvement of indices or its negative effects on S.platensis microalga depended on the form and concentration of TiO2, the presence or absence of citrate, and the duration of treatments. IntroductionThe titanium element, in some cases, improves plant metabolic processes (Gohari et al., 2022; Carvajal and Alcaraz 1998). TiO2 nanoparticles in different forms have many applications in industry, and it is possible to find their way into aquatic ecosystems (Yang et al., 2015). The two forms of anatase and rutile have different surfaces, structural, and toxicity properties (Parrino et al., 2021). Some studies have shown that anatase has more cytotoxicity and photocatalytic activity than the rutile form (Clément et al., 2013). Of course, some sources mention TiO2 with low toxicity (Parrino et al., 2021). However, the effects of TiO2 nanoparticles depend on the type of organism under investigation, nanoparticle concentration, physicochemical and morphological characteristics such as its size and crystal structure. S. platensis, a spiral filamentous blue-green microalgae, has attracted the attention of researchers due to its numerous economic applications, including as a fertilizer and enhancer in agriculture, and has valuable minerals, vitamins, and nutritional compounds (Godlewska et al., 2019; Deng and Chow, 2010). Citric acid is required for various biological processes (Mudunkotuwa and Grassian, 2010). The citrate-nanometal complex can facilitate the entry of NPs along with citrate into the cell (Rupasinghe, 2011); hence the citrate form of nanometals has been recommended for the growth of plants (Chandrika et al., 2021). Some studies on plants have also shown that citrate reduces the adverse effects of nanoparticles (Tirani et al., 2018). Citrate can be adsorbed on the oxides of nanoparticles through Van der Waals bonds and prevent particles from aggregation (Park and Shumaker-Parry 2014; Rupasinghe 2011; Mudunkotuwa and Grassian, 2010).The aim of researchThe goal of this research was a comparative evaluation of the possible stimulating or adverse effects of bulk forms and nanoparticles of TiO2 (anatase and rutile), with or without citrate (in concentrations of 12.5, 25, 50, 100 and 200 mg. L-1) on the growth, biomass and intracellular compounds of microalgae S. platensis in 5 days. Materials and Methods Zarrouk nutrient medium was used for the growth of microalgae. Anatase nanoparticles 10-25 nm (Purity 99.9%, Specific surface area, SSA 200-240 m2 g-1), rutile nanoparticles 30 nm, (Purity 99.9%, SSA 35-60 m2 g-1) and bulk sample (diameter <500 nm) were used. Thirty-two (32) treatments were designed as follows: sample S, control culture without any treatment; Samples B1 to B5 treatments containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 of bulk TiO2 respectively; samples A1 to A5 containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 NPs anatase respectively; Samples R1 to R5 containing concentrations of 12.5, 25, 50, 100 and 200 mg L-1 of NPs rutile, respectively; C, samples containing citrate. In samples containing both citrate and TiO2, citrate to titanium dioxide was used in a molar ratio of 3.5 to 1 (Mudunkotuwa and Grassian, 2010). In sample C, which only had citrate, the amount of citrate was considered equal to it in 200 mg/liter TiO2. Then all the samples were placed in controlled conditions of 100 µmol m-2s-1,16/8 h of light/darkness and a temperature of 28 ± 2 °C for five days. Sampling and measurement of indicators were done on days zero (day of inoculation), 3, and 5 experiments. Various parameters were evaluated, including dry weight, maximum specific growth rate (µm), pigments chlorophyll a, total carotenoid, phycobilins, astaxanthin, total soluble sugar, total soluble protein, lipid, lipid peroxidation, antioxidant activity (DPPH radical inhibition), and ROS levels. Results and discussionRutile nanoparticles in the presence of citrate or without it caused an increase in dry biomass and µm index, while anatase nanoparticles caused a decrease. This can be attributed to the difference in particle size and the difference in specific surface area (SSA) of rutile particles (about 30 nm) and Anatase (about 10-25 nm) (Parrino et al., 2021). The high content of ROS was not observed in most of the anatase treatments, but the level of ROS was high on the 5 d in control C, while the simultaneous presence of titanium and citrate caused a significant reduction of it. Surface adsorption of citrate on TiO2 nanoparticles prevents the aggregation of TiO2 and increases cell access to nanometal (Mudunkotuwa and Grassia 2010). However, exopolymers attached to the outer surface of the cells can increase the contribution of citrate-nanometal complex surface adsorption on the cell surface and reduce its entry into the cells (Zhou et al., 2016). On the 3 d, antioxidant system activity increased in anatase treatments, which caused at least a partial reduction of ROS in anatase samples without citrate. The amount of antioxidant activity was often higher in bulk nanoparticles. Sendra et al. (2017), showed that the rate of exopolymers in the treatment with TiO2 nanoparticles was much higher than when they were treated with TiO2 bulk form. The concentration of 100 mg L-1 caused the highest amount of chlorophyll a, carotenoid, and astaxanthin, and the lowest concentration (12.5 mg L-1) caused the highest levels of APC, PC, PE and total phycobilin pigments. Anatase stimulated the production of the highest amount of chlorophyll, APC, and PE, especially on the 3 d, and rutile specifically induced the production of carotenoid, astaxanthin, and PC. The carbohydrate amount was increased by bulk and nano-rutile form. Lipid content was increased by the treatment of citrate, TiO2 (with or without citrate), and bulk form with citrate. ConclusionThe effects of the treatments were observed as stimulating and positive or negative effects depending on the form and concentration of TiO2, the presence or absence of citrate, and the duration of treatment with nanoparticles. Biomass (dry weight) increased by the treatment with rutile nanoparticles and, to some extent, by treatment with a bulk form of rutile. An increase in chlorophyll, carotenoid, astaxanthin, phycobilins, carbohydrate, lipid, and protein was also observed. It is possible that Spirulina with the improved indices containing a certain level of nanoparticles can be used as a fertilizer in agriculture. Citrate did not always show the same effects, and its effects depend on the type of accompanying nanoparticle, TiO2 concentration, and duration of stress.
Keywords
