Diatom attachment inhibition: limiting surface accessibility through air entrapment
© Wu et al.; licensee Springer. 2013
Received: 28 August 2012
Accepted: 7 January 2013
Published: 7 February 2013
Surfaces consisting of sub micron holes (0.420-0.765 μm) engineered into nanoparticle (12 nm) coatings were examined for marine antifouling behaviour that defines early stage settlement. Immersed surfaces were found to be resistant to a 5-hour attachment assay of Amphora coffeaeformis, a marine organism commonly found in abundance on fouled substrates such as foul-releasing paints and self-polishing coatings. Attachment inhibition was attributed to the accessibility of diatoms to the surface. This was governed by the size and morphology of trapped interfacial air pockets measured in-situ using synchrotron small angle x-ray scattering. Surfaces containing larger pores (0.765 μm) exhibited the highest resistance. Macroscopic wettability via contact angle measurements however remained at 160° and sliding angle of < 5° and was found to be independent of pore size and not indicative of early stage fouling behaviour. The balance of hierarchical nano/micro length scales was critical in defining the early stage stability of biofouling character of the interface.
Marine biofouling is the accumulation of marine species onto submerged surfaces within the ocean. It imposes significant cost to the maritime industries [1, 2] and as a result has been the subject of a considerable number of preventative strategies. Until recently all of these have involved toxic coatings containing heavy metals such as copper and tin [3, 4]. More recently, environmental considerations such as bioaccumulation [5, 6] have led to the ban on many of these. This has resulted in a demand for non-toxic alternatives.
Current non-toxic antifouling strategies are driven by interfacial architecture and fall into two main behavioral categories, foul-release (FR) surfaces and attachment-inhibiting (AI) surfaces. FR surfaces seek to reduce the adhesion strength of attached organisms. This facilitates their removal [7–9] in dynamic situations, and thus is optimized for applications on moving vessels. In contrast, attachment-inhibiting (AI) surfaces rely on surface characteristics that completely deter initial attachment of marine organisms .
In both cases variations in chemistry and topography are the key features. Polyethylene glycol (PEG) is a good example of the former [11–13] while advances in analytical techniques such as electron microscopy has provided insights into naturally rough AI surfaces such as sharkskin and barnacle shells . The apparent unique topography has fueled biomimetic designs for antifouling surfaces that exhibit antifouling properties [14, 15]. In conjunction with this, several theories have been developed in order to explain the mechanism of action behind these types of AI surfaces [16–18]. All are focused on adsorption of organisms at the substrate/liquid interface. Attachment point theory describes the surface interactions in terms of the organisms’ relative contact surface area [15, 17, 19] at the water/surface interface, where the size difference dictates adhesion strength. This has been demonstrated in individual species assays . In practice however, the biofouling will eventually occur due to the range of organism sizes.
In contrast to fluid/surface interfaces, liquid/vapour interfaces do not attract adsorption of marine species irrespective of size. Superhydrophobic surfaces contain a blend of chemistry and topography that effectively traps air at the interface and leads to aqueous contact angles greater than 150°. The use of such surfaces for antifouling was first demonstrated experimentally by Zhang et al. . They reported that while contact angle could change it did not correlate necessarily with biofouling behaviour. In a more recent paper  it was shown that reduction in wetted area at the nanoscale was linked to biofouling behaviour. Changes in nanoscale wetting did not, however, impact markedly on the macroscopic contact angle value. Thus, creating surfaces that maintain trapped interfacial air may lead to a new class of antifouling coating.
In this work, the role of air in the attachment behaviour Amphora coffeaeformis, a commonly found organism on fouled surfaces [21–24], on superhydrophobic surfaces was investigated. In-situ synchrotron small angled x-ray scattering was used to examine the lower limits (nanoscale) of air entrapment on superhydrophobic surfaces with varying degrees of surface roughness, to elucidate whether the evolution of the air layer over time is the cause for differences in settlement of diatoms.
2.1 Synthesis of superhydrophobic coatings
Silica nanoparticles (Aerosil 200, av. dia. 12 nm, 0.25 g) was dispersed in ethanol (AR, 95%) by sonication (40 kHz, 10 min). Polymethylmethacrylate (PMMA) latex emulsion (av dia. 420 nm, 0.5 g) was added to the silica dispersion and further sonicated for 10 min. Methyltrimethoxysilane (MTMS) (98%, Sigma-Aldrich, 0.6 g) and concentrated HCl (36% w/v, AR, 60 μL) were added to the solution and allowed to react under sonication for 3 h to form the sol–gel solution .
This was repeated with various PMMA latex particle sizes: 485 nm, 572 nm, 635 nm, 671 nm, and 765 nm.
2.2 Synthesis of PMMA latex particles
PMMA latex particles were synthesized by charging a reaction vessel with ultrapure water (100 mL, Millipore), poly(dimethylsiloxane)-poly(ethylene oxide) block copolymer (5 g, Gelest, 400 cst, 25% non-siloxane), methylmethacrylate (10 g, Sigma-aldrich, 99%), Di(trimethylolpropane) tetraacrylate (1 g, Sigma-aldrich, 99%) and potassium persulfate (0.1 g, Sigma-aldrich, 99%). The mixture was vigorously mixed with mechanical stirring by a hand-held mixer (Braun Multiquick 300 Watts) for 5 minutes. It was then purged with Argon for 10 minutes under constant stirring with a magnetic stirrer set at 700 rpm. The mixture was then heated to 70-75°C for 3 hours with constant stirring. The ready emulsion was allowed to cool and filtered through cotton wool to remove coagulated PMMA.
2.3 Macroscopic wettability measurements of superhydrophobic surfaces
Each coating was subjected to contact angle measurements (Ramé-Hart Inc.) using the sessile drop method. The contact angle of each coating was measured at three different points to obtain a statistical average with reproducibility of ± 5°. Sliding angle was measured in triplicate by gradually tilting each surface by increments of 0.1 degrees, upon which a sessile drop of water is resting, until the water droplet rolls off the surface.
Inverse captive bubble measurements were conducted in a custom-built acrylic container filled with milli-Q water. Surfaces were immersed with the coated side facing down and a bubble (20 μL) was introduced into the chamber via a microsyringe with an inverted needle.
The surface coverage of air on immersed superhydrophobic surfaces was visually inspected by tiling an immersed surface 48° to a digital camera. This is the critical angle for total internal reflection at a water/air interface and is used to visualize changes in the coverage of air on surfaces before and after the attachment assay.
2.4 Nanoscopic wettability measurements using In-situ small-angle x-ray scattering (SAXS)
where φ is the scattering angle and λ is the irradiating wavelength.
Four custom-built fluid cells that allowed remote fluid injection through a peristaltic pump and a reservoir of filtered seawater water were used for the in-situ measurements. The cell was constructed from two sheets of 5 μm-thick kapton separated and sealed by a 1.4 mm-thick neoprene gasket. One of the kapton windows is coated experimentally. The cell could then be filled with fluid without changing its position relative to the incident x-ray beam. As the scan position and the physical morphology are not altered between scans, any change in scattering over time is attributed to a change in the nature of the interface between the coating and liquid.
Multiple SAXS measurements were made on different points on each sample, and the average is used as a representative SAXS profile for each sample. The x-ray beam irradiated a spot approximately 200 μm × 100 μm, with each sampling point spaced 500 μm apart. Ample distance between each measured point prevented overlap between irradiated points.
To measure the amount of entrapped air at the interface, SAXS measurements were first made on dry superhydrophobic surfaces. The fluid cell was then charged with the same diatom media that was used during the attachment assay. This was prepared by culturing a batch of artificial seawater with the same concentration of diatoms as those used in the attachment assay for 5 hours. The mixture was then centrifuged and the supernatant was collected. This minimized the likelihood of diatoms contributing to the x-ray scattering profile, as well as to simulate the maximum possible amount of extracellular polymeric substances (EPS) available within the media during the nanowetting study.
This diatom culture media was then introduced into the fluid cell, and SAXS measurements were made at each point every 10 minutes up to 360 minutes, which corresponds to the length of time required for a diatom attachment assay. To obtain a SAXS profile of immersed superhydrophobic surfaces in the fully wetted regime, the fluid within the cell was exchanged with ethanol, followed by water. The ethanol was used as a wetting agent, to remove air from the interface and thereby forcing the immersed superhydrophobic surfaces into a wetted Wenzel state when water was reintroduced.
2.5 Species attachment assays -- diatoms (amphora coffeaeformis)
Coated glass slides were placed in culture wells with a maximum volume capacity of 40 mL. Diatoms (ϕ ~ 16 μm) were cultured in K+ media for at least 48 hours under a 12-hour day/night cycle under controlled temperature and humidity in a culture fridge. The diatoms were concentrated by centrifugation and the number of diatoms/mL was counted using a haemocytometer. Each well was charged with 20 mL of artificial seawater pre-dispersed with 1 mL of concentrated diatom culture, and the number of diatoms per well is normalized against the haemocytometer count. The immersed surfaces were then left in the culture fridge for 5 hours and were then immersed in 10 L of artificial seawater to dislodge any unsettled diatoms. Care was exercised to keep all surfaces immersed in artificial seawater to prevent disrupting settled diatoms due to dewetting mechanisms.
Settlement of diatoms was characterized using fluorescence microscopy on a Leica DM2500 confocal fluorescence microscope coupled to a Leica DC300F camera. Images produces were 1300 x 1030 pixels. A 5x magnification lens was used to give a field of view (FOV) roughly equal to 17.4 mm2. A mercury arc lamp using a Leica I3 filter cube (blue excitation 450–490 nm) enabled the chlorophyll in Amphora coffeaeformis to fluoresce a very strong shade red. Images were captured on 10 random FOVs per surface using Leica’s camera software, IM50, with the bright field (BF) setting in place. Image analysis was conducted using Adobe Photoshop Elements 2.0, 2002, with a colour tolerance figure of 200 to maximize the fluorescence intensity. This allowed for automatic quantification of red pixels against a dark background.
3.1 Surface properties
Further details and structural characterization of these surfaces can be found elsewhere . The pore size was varied by incorporating latex particles of different sizes during the sol–gel reaction, which were then thermally removed. The latex particles were synthesized using a method described elsewhere , and ranged from 400 nm to 765 nm in diameter. The size range chosen was at least an order of magnitude smaller than the size of marine organisms used in this study to circumvent the effects of attachment point theory.
Wetting characteristics of superhydrophobic surfaces
Surface (Pore size)
Static contact angle (θ)
Sliding angle (θ)
A (420 nm)
166 ± 5
4 ± 2
B (485 nm)
162 ± 5
5 ± 2
C (572 nm)
160 ± 5
4 ± 2
D (635 nm)
164 ± 5
4 ± 2
E (671 nm)
164 ± 5
3 ± 2
F (765 nm)
166 ± 5
2 ± 2
3.2 Effect of Air on attachment behaviour of Amphora coffeaeformis
Fluorescence microscopy was used to characterize the extent of which diatoms were attached onto each superhydrophobic surface. Prior to imaging, each of the surfaces were immersed in fresh filtered seawater and gently perturbed to remove any free-flowing and weakly attached diatoms. Due to the presence of shearing forces at the fluid/air/surface interface that may dislodge bound diatoms during dewetting phenomena, extra care was exercised to ensure none of the surfaces were re-exposed to air until after fluorescence microscope images were taken.
Two distinct features can be observed in the averaged power spectrum plots of all wetted superhydrophobic surfaces (Figure 6b); a prominent peak at approximately 17 μm, representing the size of diatoms, and a decaying oscillation that represents the periodicity of diatom distribution in all directions. This indicates that all settled diatoms are roughly equidistant to one another on the wetted superhydrophobic surface, an observation similar to those of smooth surfaces.
While a similar peak was also observed for fluorescent images of unwetted superhydrophobic surfaces, this peak appeared at around 26 μm, indicating that the diatoms are clustered in pairs or more. Furthermore, the decaying oscillation was not observed. This is because diatoms are no longer evenly distributed on the surface, but rather, exhibit signs of clustering.
3.3 Effect of surface morphology on attachment behaviour of Amphora coffeaeformis
The wettability of all superhydrophobic surfaces was visibly different after a 5-hour immersion in artificial seawater during the diatom species assay. Initially, a reflection at the surface/fluid interface was found at a glancing angle of approximately 48° when the surfaces were immersed. This was almost identical to the reflection at a fluid/air boundary, which suggests that a uniform layer of air resides at the fluid/surface interface of immersed superhydrophobic surfaces. Over the 5-hour assay, emergence of air pockets with a visibly large radius of curvature was observed on all surfaces, and the reflection was no longer uniformly distributed across the entire sample. This suggests that during the assay, localised wetting has occurred, resulting in the partial loss of the glancing reflection at the interface.
3.4 Quantification of trapped air using in-situ synchrotron transmission SAXS
Transmission SAXS measurements based on the technique described previously [28, 29] were used to measure the immersed superhydrophobic surfaces over a scattering q-range of 0.003 < q < 0.1, where the scattering momentum q is a function of the x-ray scattering angle φ and x-ray wavelength λ, given by equation (1). To avoid effects of fluid compressibility at high q and resolution limit at extremely low q, the data analysis range was narrowed to between 0.005 < q < 0.03.
The intensity I AB of x-ray scattering into a given solid angle from a rough interface between two media with average electron densities ρ A and ρ B is proportional to the square of the difference between the densities. This results in high scattering intensity at interfaces with a large electron density difference. So with a dry superhydrophobic surface, x-ray scattering arises from the electron density difference between air and the surface. It has been shown previously that as wetting progresses on an immersed superhydrophobic surface, x-ray scattering intensity decreases as the air/surface interface is replaced by a water/surface interface .
This equation holds a special significance when the exponent of q is equal to −4, which is the case for all measured surfaces in this study. The pre-factor B is directly proportional to the total amount of interface that is illuminated by the x-ray beam. As air/surface interface is replaced with water/surface interface during wetting, the pre-factor B can be used to directly quantify the percentage of surface that remains dry .
Figure 10 shows the % dry interface on superhydrophobic surfaces immersed in diatom culture media over 6 hours. It can be seen that during the wetting study, the percentage surface that remains dry is consistent, suggesting that no further wetting has progressed for both superhydrophobic surfaces.
4.1 Effect of air pockets on attachment of Amphora coffeaeformis
From the attachment assays, it can be seen that the air at the interface of immersed superhydrophobic surfaces prevent attachment of Amphora coffeaeformis. This agrees with the theory proposed by Zhang et al.  whereby a reduction in contact surface area between fluid and surface will induce a reduction in attachment probability of marine organisms. This was further supported by the Fourier transform analysis conducted on each fluorescent image, whereupon the power spectrum highlights the presence of clustering of diatoms around the edges of the air pockets (Figure 6).
4.2 Effect of surface morphology on attachment of Amphora coffeaeformis
It is worth noting that while diatom attachment is reduced significantly on surfaces fabricated using larger pores (Figure 7), on areas where air pockets are absent, the number of diatoms as measured by the % coverage of red pixels, is statistically consistent across all superhydrophobic surfaces (Figure 8). Considering that the fluid/surface interface at the nanoscale remained consistent throughout the diatom assay, and that the contact angle has dropped only on surfaces with smaller pores, this suggests that the settlement of diatoms on superhydrophobic surfaces was significantly inhibited by the presence of larger air pockets, not nanoscopic ones.
The difference resides in the fact while the size of air pockets are large initially on all superhydrophobic surfaces, which gives rise to the high water contact angles, the stability of these larger air pockets can be fine-tuned by surface roughness. According to this theory, it seems to suggest that the significant variation in surface coverage by biofilms between surfaces with different pore sizes can be attributed to the relative stability of larger air pockets retained on the surface. While the mechanism behind this difference is still under investigation, the presence of air pockets and its morphology has a significant influence over the settlement behaviour of Amphora coffeaeformis.
The presence of air is a physical barrier against organism attachment due to reduced fluid/surface contact area. The presence of air at the surface/liquid interface limits the accessibility of diatoms (Amphora coffeaeformis) to the surfaces, thus reducing settlement probability. Through attachment assays of diatoms and direct measurement of the water/air/surface interface of an immersed superhydrophobic surface using in-situ small-angle x-ray scattering, it was found diatom attachment was inhibited on macroscopic air pockets, but not nanoscopic ones. This suggests that the attachment-inhibiting characteristics of superhydrophobic surfaces may depend on the size of the air pockets present on immersed superhydrophobic surfaces rather than a simple measure of surface wettability.
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