(145c) Impact of Titanium Dioxide Nanomaterials On Nitrogen Fixation Rate and Nitrogen Storage Dynamics of Anabaena Variabilis

INTRODUCTION Progress in nanotechnology has raised concerns regarding the potential environmental impact and fate of engineered nanomaterials (NMs). Particularly, titanium dioxide nanomaterials (nTiO2) will unavoidably be released into aquatic habitats from municipal and industrial effluents due to their increasing production rates and utilization in a wide range of applications [1]. So far, limited information is available on the potential impact of NMs on aquatic ecological system and on the primary producers (i.e. algae) and a limited number of studies have demonstrated that exposure to nTiO2 affect algae growth and photosynthetic activity. The potential impact of NMs on nitrogen-fixing algae, such as the cyanobacteria Anabaena variabilis, has not been investigated. Cyanobacteria play an important role on primary production and nitrogen cycle with their ability of fixing atmospheric dinitrogen into ammonia, and have been previously used as a model algae for evaluating environmental stresses [2]. In this study, we quantified the inhibitory effect of nTiO2 exposure on the growth rate, N-fixing activity and intracellular nitrogen-storage structures in the cyanobacteria Anabaena variabilis. Additionally, a morphometric analysis allowed the quantification of intra-structural changes in response to the toxicant. Particularly, the impact on temporal and spatial accumulation on the cyanophycin grana proteins (CGPs), a functionally-relevant biomolecule in Anabaena v. cells involved in nitrogen storage, was assessed, providing insights into the possible alteration of nitrogen metabolic pathways in algae upon nTiO2 exposure. MATERIALS AND METHODS NMs preparation and characterization Nano-TiO2 anatase (nTiO2, NanoStructured&Amorphous Materials, Houston, Texas, USA) was prepared in culture Mes-Volvox medium (stock concentration of 10000 mg/L) and dispersed with the addition of crude 1% bovine serum albumin and sonication in a High energy Cup-sonicator for 20 minutes. Primary size nTiO2 from manufacturer was 10 nm and average size of NM aggregates of 192±0.8 nm was determined through Dynamic Light Scattering after nanomaterials dispersion in culture media. Culture conditions and ecotoxicological tests Anabaena variabilis strain (UTEX #1444) was axenically cultured at 20°C in a nitrogen-free Mes-Volvox media. Cells were cultured in 1L chemostats with 0.15 d-1 dilution rate, incubated under a 12h light/12h dark regime. Growth inhibition tests based on chlorophyll a measurements were performed according to the standard protocols. Anabaena v. nitrogen fixation ability after exposure to nTiO2 was measured using acetylene reduction assay. Results from growth inhibition and nitrogen fixation inhibition tests were fitted in the Hom-type model for inactivation kinetic parameters determination. Observation of intracellular structural modifications via Transmission Electron Microscopy Temporal high-resolution TEM imaging was employed to observe intracellular structural changes in Anabaena variabilis upon exposure to nTiO2. Cells were collected from cultures subjected to growth inhibition tests, and processed as specified in the method by [3]. RESULTS AND DISCUSSION Effect of nTiO2 exposure on growth of Anabaena variabilis The effect of nTiO2 exposure to growth rates of Anabaena v. was evaluated for various nTiO2 concentrations ranging from 0.5 to 500 mg/l, and for different exposure length varied from 24 hours to 6 days. The half maximal effective concentration (EC50) was determined as a function of exposure time and it decreased significantly from 13.98 at 24 hr to 0.15 after 6 days of exposure. The regulatory EC50-96h was determined to be 0.62 mg/l. The application of the Hom model for the inactivation kinetic parameters determination (k, m and n were 0.14, 1.01 and 0.09 respectively) demonstrate that under our experimental conditions, the time of exposure has a greater influence than the nTiO2 concentration in inhibiting Anabaena v. growth. These results suggest that extended toxicity studies beyond the regulatory 72 and 96 hours-endpoints may be necessary when assessing NMs impact in environmental ecosystems. Impact of nTiO2 exposure on nitrogen fixation activity of Anabaena variabilis Figure 1 shows the toxicity effects of nTiO2 on nitrogen fixing activity of the cyanobacteria Anabaena v. at different concentrations. nTiO2 higher than 10 mg/l led to greater than the 50% nitrogen fixation inhibition after short exposure time of 24 hrs. N fixing activity was completely inhibited at nTiO2 concentration of 75 mgTiO2/l after 24 hours exposure and at 1 mgTiO2/l after 6 days exposure, indicating that the inhibition effect of nTiO2 on nitrogenase enzyme activity rates is also Cn∙Tm-dependant, depending on both nTiO2 doses and exposure time. The fitting of the results in a Hom-type model (n and m parameters were 0.72 and 1.93 respectively), again suggests that the time of exposure has a greater power than the nTiO2 concentration in inactivating the Anabaena v. ability of fixing nitrogen. The EC50-96h was found to be 0.4 mgTiO2/l, lower than that determined based on cell growth inhibition, suggesting that N fixation maybe a more sensitive toxicity-indicator endpoint than growth. Observation of intracellular cyanophycin grana protein (CGP) changes in response to nTiO2 TEM observations and a morphometric analysis revealed variations in the dynamics of nitrogen storage of Anabaena v. by exposure to nTiO2 at various concentrations and exposure times, with the increase in the occurrence and size of intracellular cyanophycin grana protein (CGP) (Figure 2). These biomolecules are non-ribosomally synthesized nitrogen-rich storage polymers composed of aspartic acid and arginine. Figure 3 and figure 4 show that there was an increase in both the occurrence and size of the CGPs, which depends on both nTiO2 concentration and exposure time duration. Less than 30% of the cells analyzed contained CGPs in both the control culture with no nTiO2 exposure (at 96 hrs), as well as for those samples exposed to very low nTiO2 concentration (1 mg/l) and very short exposure time (3 hrs) (Figure 3). In exposure to a high concentration of nTiO2 (150 mg/L), more than 80% of cells were found to contain CGPs granules. Increase in the intracellular levels of CGPs (as relative surface area) for cells that contain CGPs were quantified and fitted in lognormal distributions (Figure 4). The average relative area of these granules was found to be 0.87% in cells from controls and sample exposed to low nTiO2 concentration (1 mg/l) and low exposure time (3 hrs). However, distributions characteristic of all other samples were shifted towards higher values of CGPs, ranged from 3 to 4.6% of cells surface to as high as 16.4% (96 hours to 150 mgTiO2/l). Our results suggest that it is possible that elevated cyanophycin granules formation is associated with stress conditions caused by nTiO2 exposure and show that this granule can be immediately-induced rather than form as an accumulative long-term response. One possible hypothesis that supports the dynamics of CGPs observed is that the cell modifies the redistribution of nutrients increasing the diversion of nitrogen into storage products for long-term survival. Additionally, the binding of nTiO2 with intracellular peptides and phosphate species in aqueous solution might have determine the accumulation of CGPs. Lastly, the observed accumulation of CGPs in cells exposed to nTiO2 might be correlated with the inhibition or alteration of the enzymatic activity (cyanophycinase and cyanophycin synthetase) responsible for cyanophycin degradation and synthesis in preparation to cell long term survival. CONCLUSIONS In summary, this study, for the first time, quantitatively assessed the impact of nTiO2 on cell growth and nitrogen-fixing activity of Anabaena variabilis and revealed the possible involvement of intracellular CGPs granules in the stress and detoxifying process in response to nTiO2 exposure. Changes in the cyanophycin grana protein accumulation confirm that exposure to NMs can affect patterns of nitrogen metabolism and potentially other key functional biomolecules in algae. REFERENCES 1. Griffitt, R. J.; Luo, J.; Gao, J.; Bonzongo, J. C.; Barber, D. S., Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms. Environmental Toxicology and Chemistry 2008, 27, (9), 1972-1978. 2. Apte, S. K.; Fernandes, T.; Badran, H.; Ballal, A., Expression and possible role of stress-responsive proteins in Anabaena. Journal of Biosciences 1998, 23, (4), 399-406. 3. Van Hoecke, K.; De Schamphelaere, K. A. C.; Van der Meeren, P.; Lucas, S.; Janssen, C. R., Ecotoxicity of silica nanoparticles to the green alga Pseudokirchneriella subcapitata: Importance of surface area. Environmental Toxicology and Chemistry 2008, 27, (9), 1948-1957.