The Use of 3D Optical Coherence Tomography to Analyze the Architecture of Cyanobacterial Biofilms Formed on a Carbon Nanotube Composite

Abstract

The development of environmentally friendly antifouling strategies for marine applications is of paramount importance, and the fabrication of innovative nanocomposite coatings is a promising approach. Moreover, since Optical Coherence Tomography (OCT) is a powerful imaging technique in biofilm science, the improvement of its analytical power is required to better evaluate the biofilm structure under different scenarios. In this study, the effect of carbon nanotube (CNT)-modified surfaces in cyanobacterial biofilm development was assessed over a long-term assay under controlled hydrodynamic conditions. Their impact on the cyanobacterial biofilm architecture was evaluated by novel parameters obtained from three-dimensional (3D) OCT analysis, such as the contour coefficient, total biofilm volume, biovolume, volume of non-connected pores, and the average size of non-connected pores. The results showed that CNTs incorporated into a commercially used epoxy resin (CNT composite) had a higher antifouling effect at the biofilm maturation stage compared to pristine epoxy resin. Along with a delay in biofilm development, a decrease in biofilm wet weight, thickness, and biovolume was also achieved with the CNT composite compared to epoxy resin and glass (control surfaces). Additionally, biofilms developed on the CNT composite were smoother and presented a lower porosity and a strictly packed structure when compared with those formed on the control surfaces. The novel biofilm parameters obtained from 3D OCT imaging are extremely important when evaluating the biofilm architecture and behavior under different scenarios beyond marine applications.

Keywords: Optical Coherence Tomography; antifouling surfaces; carbon nanotubes; cyanobacterial biofilms; marine biofouling.

Figure 1

Two-dimensional and 3D coordinate systems for 2D and 3D OCT analyses, respectively. X represents the width, Y is the height, and Z refers to the depth.

Figure 2

Two-dimensional AFM (a–c) and SEM (d–f) images of glass, epoxy resin, and CNT composite. The vertical color bars in the AFM images correspond to the z-range (surface height range) of the respective image. The SEM images have a magnification of 100× and the scale bar is equivalent to 100 µm.

Figure 3

Nodosilinea cf. nodulosa LEGE 10377 biofilm development on different surfaces (glass—black, epoxy resin—grey, CNT composite—white). The parameters analyzed refer to biofilm wet weight (a), thickness (b), contour coefficient (c), biovolume (d), porosity (e), and average size of non-connected pores (f). Mean values and SD from two biological assays with two technical replicates each are represented. For each sampling day, different lowercase letters indicate significant differences between surfaces (p ≤ 0.05; one-way ANOVA with Tukey’s multiple comparisons test).

Figure 4

Representative 2D cross-sectional OCT images of Nodosilinea cf. nodulosa LEGE 10377 biofilms developed on glass (a), epoxy resin (b), and CNT composite (c) after 49 days. The empty spaces in the biofilm structure are filled in blue (scale bar = 100 μm).

Figure 5

Representative 3D OCT images of Nodosilinea cf. nodulosa LEGE 10377 biofilms formed on glass (a), epoxy resin (c), and CNT composite (e) after 49 days. The spatial distribution of non-connected pores on the biofilm is also shown (b,d,f). The color scale shows the distance from the substrate surface.

Figure 6

Representative 3D OCT images of the distribution of non-connected pores by size from Nodosilinea cf. nodulosa LEGE 10377 biofilms formed on glass (a), epoxy resin (b) and CNT composite (c) after 49 days. The color scale shows the range of the size of non-connected pores.

Figure 7

SEM images of Nodosilinea cf. nodulosa LEGE 10377 biofilms formed on glass (a), epoxy resin (b), and CNT composite (c) after 49 days of incubation. The red arrows in (c) indicate clusters of CNTs. Magnification = 1000×; scale bar = 10 µm.