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).