**1. Introduction**

The oil, gas, and maritime industry are significantly impacted by biofouling, either through the costs related to the increased fuel consumption, hull cleaning, material deterioration, repainting, and corrosion [1], or even by the management intervention time and incorrect measurements in the submerged and moored sensors [2]. Ecologically, fouling events in marine environments promote species invasion and the establishment of exotic biofouling species in ports [3]. Moreover, biofouling can be a major concern in health-related problems since contamination of aquaculture facilities, such as fish cages, can occur by toxin accumulation, and air pollution may be increased through greenhouse gas emissions [4].

The non-toxic marine antifouling approaches available in the market are often expensive and not as effective as conventional biocides, which can accumulate in the marine

**Citation:** Romeu, M.J.; Gomes, L.C.; Sousa-Cardoso, F.; Morais, J.; Vasconcelos, V.; Whitehead, K.A.; Pereira, M.F.R.; Soares, O.S.G.P.; Mergulhão, F.J. How do Graphene Composite Surfaces Affect the Development and Structure of Marine Cyanobacterial Biofilms? *Coatings* **2022**, *12*, 1775. https://doi.org/10.3390/ coatings12111775

Academic Editor: Mohor Mihelˇciˇc

Received: 20 October 2022 Accepted: 17 November 2022 Published: 20 November 2022

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environment and affect non-target aquatic organisms. Inorganic ingredient-based coatings have been used for biofouling prevention, including silver nanoparticles [5], carbon nanotubes (CNTs) [6], graphene [7], and metal oxides semiconductors such as zinc oxide (ZnO) [8] and titanium dioxide (TiO2) [9]. Overall, antifouling marine paints containing nanomaterials have been reported to offer superhydrophobicity, microbial resistance, high durability, water repellency, anti-sticking, and anti-corrosive properties [10], being novel solutions for the sustainable development of the maritime industry. Advancements in materials technology have introduced carbon nanomaterials such as CNTs and graphene as a powerful approach for various applications in the marine and shipping industries [11]. Graphene consists of a single layer of carbon atoms arranged in a sp2-bonded hexagonal pattern. It is considered one of the strongest and thinnest materials available, which shows high specific surface area, electrical conductivity, and thermal stability, making it appealing for different applications [12,13]. Moreover, due to its high strength level [14], this material is a breakthrough alternative in the naval industry. All these features make the application of graphene in technical processes of maritime industries attractive, such as in water management systems, desalination, removing toxic pollutants and filtering gasses, and as a coating material [15–17]. As a coating on marine structures, besides the anti-corrosive properties, graphene can also be used in de-icing surfaces for ship operations in extremely low-temperature regions, such as the Arctic and Antarctica, due to its electrical conductivity [15].

Nanotechnology-based technologies can be of great interest in creating novel lowtoxic antifouling coatings [18]. However, analysis of the literature indicates that there is little information about the use of pristine graphene. Indeed, most in situ studies on graphene-based surfaces were performed with functionalized graphene and graphene oxide (GO) coatings [19–23]. Diatom adhesion was completely inhibited after 10 days by surfaces containing 0.36 wt% GO [24]. In turn, GO-silver nanoparticle coatings improved antibacterial and anti-algal properties [25], and showed more than 80% *Halomonas pacifica* biofilm inhibition [26]. Since the current trend is to study the potential of modified and functionalized graphene, the antibiofilm performance of graphene alone is poorly understood. Moreover, most in vitro studies are usually performed for short periods and under hydrodynamic conditions that do not mimic the real marine environment [27]. In fact, some of the in vitro studies have been performed until 24 h [6,23,25,28,29], most of them between days and weeks [7,19,20,24,30–33], but only a study performed by Fazli-Shokouhi et al. [34] extended the assay period for 3 months to evaluate the antifouling potential of graphene-based coatings. Moreover, these studies focus on organisms other than cyanobacteria, namely diatoms, algae, and macrofoulers. Therefore, the main goal of this work was to evaluate the potential of a graphene composite surface to prevent and control the development of biofilms by marine microfoulers over a long-term assay and using an in vitro platform that mimics the hydrodynamic conditions found in real marine scenarios. Cyanobacterial biofilm architecture was evaluated by three different imaging techniques: Optical Coherence Tomography (OCT), Confocal Laser Scanning Microscopy (CLSM), and Scanning Electron Microscopy (SEM). Since the epoxy resin is a commercially available coating generally used to coat the hulls of small recreational vessels [35,36] due to its exceptional physical, chemical, and mechanical properties, no safety issues, and low cost [37], pristine graphene nanoplatelets (GNP) were incorporated into this polymer matrix. Furthermore, epoxy composites exhibited high durability and resistance to fatigue and UV irradiation [38]. Surface characterization was also performed by water contact angle measurements, Optical Profilometry, and SEM.

#### **2. Materials and Methods**

Figure 1 presents the flowchart of the experimental work fully described in the upcoming sections.

**Figure 1.** Scheme of the experimental steps of the present work.
