2. Materials and Methods
2.1 Preparation of materials and coated coupons
The rigid materials used in the test were polystyrene (PS), borosilicate glass (GS), polymethyl methacrylate (PMMA) and polyethylene terephthalate glycol-modified (PETG); PS was opaque and the remaining materials were transparent. All were cut in the form of rectangular coupons (1.5 cm x 2.5 cm x 0.1 cm and 2 cm x 2.5 cm x 0.1 cm). Since PMMA is a widely used material in the manufacture of PBRs, PMMA coupons were utilized to prepare the following commercially available coatings: (a) a translucent, spray-applied superhydrophobic coating (NeverWet®, NeverWet LLC, USA; NW); (b) a transparent hydrophobic coating with anti-adherent properties based on nanoparticles (Window nano-coating for glass surfaces, Hendlex®, Baltic Nano Technologies, Lithuania; HX); and (c) an opaque non-toxic hydrophobic fouling release coating (FRC) that provides an invisible antibiofouling hydrogel microlayer (Hempasil X3®, Hempel A/S, Denmark; H-X3). The preparation of the three coatings was carried out by strictly following the manufacturers’ instructions. The H-X3 and HX coatings were applied with a wet film thickness of 150 μm using a micrometer adjustable film applicator (3580/3 Universal micrometer applicator, Neurtek, Spain).
The GS coupons was cleaned as described by Ozkan and Berbeglu (2013a). The PMMA, PS and PETG were cleaned as detailed elsewhere (Ruiz-Cabello et al., 2011), except for using acetone on the PS due to chemical incompatibility. The coatings were washed with abundant deionized and sterilized water.
The media surface roughness (Ra ; the arithmetic media deviation from the mean line within the assessment length) was measured with a surface profiler (PCE-RT 11, PCE Ibérica S.L., Albacete, Spain) with a 1mm scan length and a 0.111 μm/sample resolution. Table 1 describes the Ra data for all the surfaces assayed.
2.2 Physicochemical properties of the materials and coatings used
The contact angles on the different surfaces described above were measured by means of the sessile drop method, using a goniometer (Drop Shape Analyzer DSA25, KRÜSS GmbH, Germany). Two polar liquids (water and formamide) and another apolar liquid (diiodomethane) were used as reference liquids. As formamide was chemically incompatible with the HX and NX coatings, it was replaced by glycerol. However, NW was not only incompatible with diiodomethane, but also with a wide variety of apolar liquids commonly used in contact angle measurements (bromonaphthalene, hexadecane, dodecane, decane, ethanol, chloroform and toluene). Consequently, the contact angle for the apolar liquid was not determined for NW. All measurements were performed on duplicate coupons. Before measuring the contact angles, the coupons were rinsed following the washing protocol with abundant deionized water, and then allowed to dry at room temperature.
From the contact angles measured, the different surface energy components (\(\gamma_{s}^{\text{LW}}\), \(\gamma_{s}^{+}\),\(\gamma_{s}^{-}\), \(\gamma_{s}^{\text{AB}}\)) were calculated, as was the change in the free energy of cohesion (ΔG iwi), the water adhesion tension with the surface (τ o) and the critical surface tension (γc ,) for each surface, as described earlier (Zeriouh et al., 2019a). Table 1 shows the values of the above parameters.
The surfaces used vary greatly in terms of hydrophobicity, from hydrophobic surfaces (θw> 65 °), almost even superhydrophobic (θw> 150 °) in the case of the NW coating, to hydrophilic surfaces (θw <65 °), such as glass. Taking the τ0 parameter into account, one can distinguish the purely hydrophobic surfaces as being HX, PS, NW and H-X3. Nevertheless, all the surfaces can be considered smooth (withRa values ranging from 0.02 - 0.06 μm) except for the NW coating, which had an Ra of 5 μm. A surface is considered smooth when it has Ravalues below 0.6 μm. The only non-smooth surface was NW due to the texture effect intentionally introduced by the manufacturer, in an attempt to obtain the lotus effect .
2.3 Short-term BSA batch adhesion test
A model protein, bovine serum albumin (BSA), was used to evaluate the propensity of the different materials and coatings to protein adherence. The BSA adhesion test was carried out as described elsewhere (Wang et al., 2017). Briefly, the coupons were immersed in a BSA solution prepared in PBS at a concentration of 1 mg ml−1 (pH 7.2–7.4) and kept at 25°C for 4 h with orbital shaking at 100 rpm. Transparent 15 ml polypropylene Petri plates, with a 50-mm internal diameter, were used as incubation vessels. Next, the samples were rinsed with fresh PBS and transferred to clean Petri plates where they were washed in an aqueous solution of 1 wt% sodium dodecyl sulfate (SDS). After being shaken for 20 min and sonicated for 10 min, the protein concentration in the SDS solution was determined by spectrophotometric bicinchoninic acid (BCA) assay, measuring the absorbance at 562 nm. The amount of protein adsorbed on the surface was expressed as μg BSA mm-2. The measurements were performed on duplicate coupons for each surface.
2.4 Long-term biofouling experiments
2.4.1 Microalgae, culture conditions and experimental setup
The marine microalga Nannochloropsis gaditana B‐3 was used. It was provided by the Marine Culture Collection at the Andalusian Institute of Marine Sciences (CSIC, Cádiz, Spain). Inoculum was grown in 1 L spherical flasks under a continuous (12:12 h) light-dark illumination regimen at 25 ± 2 ºC, the illumination being provided by 32W fluorescent lamps rendering an average irradiance of 100 μE m−2s−1 on the culture flask surface. The flasks were agitated by filter-sterilized air sparging injected through a sparger nozzle at an aeration rate of 0.5 vv−1 min−1.The culture medium was prepared from natural, filter sterilized (0.22 μm Whatman GF/F 47 mm, Maidstone, The United Kingdom) Mediterranean seawater with 30 psu. The preparation of the culture medium (N-optimized ALGAL medium) and its exact composition is described elsewhere (Camacho-Rodríguez et al., 2013).
Transparent 750 mL polypropylene conical-frustum vessels, with an interior diameter of 62 mm, an exterior diameter of 94 mm and a height of 141 mm, were used as culture systems in the experiments. The seven different materials and coatings were used after being prepared as indicated above. The coupons with the 1.5 cm x 2.5 cm x 0.1 cm dimensions were placed on the wall of each vessel, whereas those of 2 cm x 2.5 cm x 0.1 cm were placed on the bottom. Thus, each vessel had three replicates of each material and coating, fourteen coupons on the wall and seven on the bottom. All the coupons were fixed to the vessels with the help of a harmless hot melt adhesive (Parkside PNKPZ 3 B2, Lidl, Neckarsulm, Germany).
All the vessels were sterilized by rinsing with sodium hypochlorite solution and subsequent neutralization with sodium thiosulfate (Andersen, 2005). The vessels were then held on an orbital shaker with a 3 cm-diameter shaking orbital (OVAN MAXI, OL30-ME, Barcelona, Spain) and maintained at 25 ± 2 ºC in a thermostatic chamber. They were illuminated with cool daylight lamps (Phillips PL-32 W/840/4p) under a 12:12-h light/dark cycle. The average irradiance at the surface of the vessels was 110 μmol·m−2·s−1. The irradiance was measured using a 4π sensor (QSL-2101; Biospherical Instruments, San Diego, CA, USA). The vessels were shaken continuously at 120 rpm.
The N-optimized ALGAL medium was used as a basis for assaying the different molar N/P ratios (5, 15, 45, 60 and 90) by changing the phosphorous (P) concentration and fixing the nitrate concentration (11.3 mM). The experiments were inoculated with exponentially growing cells and acclimated to the irradiance level of the experiments at an initial biomass concentration of 0.12±0.01 g·L−1 and an initial pH of 7.8. The working volume was 200 mL. Two culture modes were assayed in this temporal sequence: batch and fed-batch mode with a pulse-feeding strategy. Fed-batch mode started once the stationary growth phase was reached in batch mode. In fed-batch mode, concentrated medium stocks were added every time a stationary growth phase was reached. For this, 5 mL of culture was replaced by an equal volume containing a nutrient stock equivalent to 200 mL of the medium used. The stationary state was assumed to have been achieved when no significant changes (± 10%) in biomass concentration were recorded over three consecutive days. The culture samples were collected, centrifuged at 7000g for 5 min, washed with a 0.5 M ammonium bicarbonate solution (Zhu et al., 2013) and then freeze-dried. The dry biomass and supernatants were immediately analyzed or stored frozen at − 22 °C. The culture experiments were performed in duplicate.

2.4.2 Monitoring microalgal growth and attachment

The biomass concentration in the culture vessels (Cb , g d.w. L-1) was estimated by applying a calibration curve between Cb andOD540 , as published previously (Camacho-Rodríguez et al., 2013):