where OD540 is the absorbance at 540 nm (measured using a Helios Omega UV–Vis spectrophotometer, Thermo Scientific, Horsham, England). The photosynthetic efficiency of Photosystem II (Fv/Fm ) for the freely-suspended cells in the culture broth was recorded with a pulse amplitude modulation (PAM) chlorophyll fluorometer (PAM-2500 chlorophyll fluorometer, Heinz Walz GmbH, Effeltrich, Germany), as described elsewhere (López-Rosales et al., 2014).Fv/Fm is unanimously considered to be an indicator of cell stress. All determinations were performed in triplicate and the average value was used.
Regarding the attachment of the cells to the coupons, once the cultivation experiments were finished, the coupons were carefully detached from the vessels and transferred into a flask filled with plenty of sterile Mediterranean seawater to remove non-adherent cells and debris. The microalgae adhesion intensity on the coupons was evaluated using a procedure based on the Chla fluorescence of the attached algae cells, as previously described (Zeriouh et al., 2017a).
2.4.3 Supernatant characterization
The phosphate and nitrate in the supernatants were measured using the 4500-P and 4500-N spectrophotometric methods for the examination of water, as published by the American Public Health Association (APHA, 1995). The protein concentration in the supernatants was quantified using a bicinchoninic acid protein assay kit (Catalog Nº BCA1 and B9643, Merck KGaA, Darmstadt, Germany). The ammonium-nitrogen (NH4+-N) in the supernatants was measured calorimetrically using Nessler’s method (protocol D1426-08, as proposed by the American Society for Testing and Materials (D1426-08, 2008)). The total concentration of amino acids was quantified as described elsewhere (Nielsen et al., 2001). All determinations were performed in triplicate and the average value was used.
2.4.4 Other analytical measurements.
The total phosphorous and nitrogen contained in the biomass and supernatants were determined as phosphate (4500-P) and nitrate (4500-N) after applying a modified chemical wet-oxidation method, as previously reported (Molina-Miras et al., 2018). The protein content in the biomass was measured, as described by González-López et al. (2010). All determinations were performed in triplicate and the average value was used.
2.4.5 Toxicity assessment of the different materials and coatings
To evaluate the potential toxicity of the different types of coupons assayed in the study, 21 coupons of the same rigid material or coating were affixed to the same transparent polypropylene conical-frustum vessel (14 on the side wall and 7 on the bottom), as described in Section 2.4.1. The control vessel consisted of a vessel that did not contain coupons.
N. gaditana was grown in batch mode in all the vessels for 15 days. The culture conditions and procedure were the same as those detailed in Section 2.4.1. The toxicity was assessed at the beginning and at the end of the culture.
Coupon toxicity was evaluated using the two methods described earlier (Zeriouh et al., 2017a): (i) the maximum photochemical photosystem II (FV/FM) yield; and (ii) the rapid light curve (RLC) technique for measuring the photosynthesis-light response curves (White & Critchley, 1999). After dark-cell acclimation for 30 minutes, the Fv/Fm was measured, and subsequently the RLCs were obtained with a 27 s exposure at each actinic light level. The photosynthetic activity estimated as the relative electron transport rate (rETR) at incremental irradiances,E (0, 10, 60, 140, 242, 411, 661, 997, 1381, and 1963 μmol photons m−2 s−1), allowed us to obtain a complete RLC, fitting the rETR vs E data to the Eilers and Peeters’ model (Eilers and Peeters, 1988). The photosynthetic parameters calculated were α (the photosynthetic rate in the light-limited region), Ek (the saturating irradiance) and rETRmax (the relative maximum electron transport rate). All experiments were carried out in duplicate vessels, with duplicate sampling in each vessel, and the average value was used. The values (data not shown) of the above parameters did not vary significantly compared to the controls throughout the cultivation period, indicating that these materials and coatings are not toxic toN. gaditana , even after long‐term exposure.
2.5 Fluid-dynamic characterization in the culture vessels
Computational fluid dynamics were used to simulate and characterize the flow field, and to map the velocity and strain-rate fields.
The time-dependent simulations were performed using ANSYS Fluent® v2020R1 (www.ansys.com) software. As we were interested in the velocity and strain rate values close to the coupons, the SST k-ω turbulence model was used. An implicit two-phase VOF model was employed to track and locate the liquid free surface. The liquid velocity at the solid walls was taken to be zero (i.e. non-slip conditions). In all the simulations, the liquid was seawater (density=1025 kg·m−3, viscosity=1.012×10-3 Pa s) and the air was in the gas phase (density=1.225 kg·m−3, viscosity=1.789×10-5 Pa s). The viscosity of the cell suspension at the inoculation time was the same as seawater. The viscosity was checked throughout the culture period and no measurable variations in the culture viscosity were observed over this time. Therefore, seawater properties were maintained in all the simulations. A water-air surface tension of 72.5×10-3 N·m-1 was used.
The simulations utilized a pressure-based model under transient conditions. Gravity was included in the model in the negative z-direction. The pressure reference was fixed at the mouth of the flask. The other models employed were SIMPLEC for the pressure-velocity coupling, the Least Square Cell-Based for Gradient scheme for spatial discretization, QUICK for momentum, compressive for volume fraction, and second-order upwind discretization for turbulent kinetic energy and the dissipation rate.
The time-step size was fixed at 1×10-3 s with a maximum of 25 iterations per time step to ensure a maximum CFL value of 1, and that the residuals dropped four orders of magnitude. The flow stabilized after 9 seconds.
The optimum mesh contained around 1.6 million computational grids, created using the tetra mesh option of ANSYS®. 15 inflation layers were created on the surface of every coupon. The first cell had a size of 5 µm. The mesh was converted to polyhedral in Fluent to ensure that the cells were aligned with the rotating flow to minimize the numerical error diffusion.
The flask movement was simulated as a moving mesh. The clockwise orbit of the flask
was specified in a user-defined function (UDF) in terms of the orbital angular velocity and orbital radius.
2.6 Statistical analyses
A one-way ANOVA test was used for significant difference analysis. Multifactor ANOVA were performed to determine the effect of the following factors and their interactions on long-term biofouling results: N/P (factor A), coupon (i.e. type of material and coatings; factor B), coupon position in the culture vessels (wall or bottom; factor C) and interactions (A-B; A-C; B-C and A-B-C). Statistically significant differences in the mean response between factors were fixed at a 5.0% significance level threshold (p value < 0.05). The method used to discriminate between the means at the 95.0% confidence level was Fisher’s least significant difference (LSD) procedure. Statistical data analyses were performed using the Statgraphics Centurion XVII (version 17.2.04) statistical software (2014, Statpoint Technologies, Inc., Warrenton, VA).