Marine Biofouling of Surfaces:
Morphology, and Nanomechanics of Barnacle Cyprid Adhesion Proteins by AFM

The understanding of biointerfaces in contact with seawater is crucially important in tackling the problems of marine biofouling. Such biointerfaces involve the bioadhesives used by marine organisms to attach temporary or permanently to the surfaces immersed in water.
The aim of this Thesis is to address a particular problem, i.e. barnacle adhesion, to the biointerface and the corresponding fouling process. We try to understand the first steps of the fouling process of this species, and help to set up design criteria for surfaces to suppress, or prevent, corresponding biofouling. The focus in this Thesis is on AFM-based nanoscale characterization of marine bio-interfaces created by barnacle cyprid larva during surface exploration. The application of AFM has the advantages of first visualizing the fouling interface (also in situ), and to measure its nano-scale properties featuring bioadhesives and adhesives in native environments. The morphology and nanomechanical properties of the cyprid temporary adhesive - “footprints” deposited on the surfaces were extensively studied. In addition, bio-interfaces created on surfaces with different wettabilities were used to investigate the settlement behavior of cyprid larvae in laboratory settlement assays and marine field tests. In-depth investigations of barnacle adhesive properties were performed by in situ monitoring of the enzymatic proteolysis degradation of footprint and the in situ curing process of cyprid permanent cement.
Chapter 1 provides a general introduction to this Thesis.
Chapter 2 is a general introduction to marine biofouling and anti-fouling systems. The first part of Chapter 2 focuses on the fouling organisms, particularly barnacles and its life cycle. In addition, the adhesion process and the temporary adhesives used by cyprid larvae are discussed. The second part of Chapter 2 presents the essential elements of the “chain of knowledge” relevant for fouling, from the currently available antifouling systems to the assessment methods of antifouling coating, including nanoscale characterization technique (AFM), laboratory settlement assays, and marine environment field tests to study settlement behavior.
Chapter 3 reveals the first microscopic morphology of native footprints deposited by cyprid larva of Semibalanus balanoide on hydrophobic and hydrophilic surface. The footprint adhesives consisted of nanofibrils and microfibers, and the size of footprints on the micrometer scale corresponded well to the surface texture of the antennular disc of the settling larvae. Footprints showed a greater spreading on hydrophobic surfaces, with three times less volume of material deposited as compared to hydrophilic (-NH2 terminated) glass. Calculations suggested that for the cyprid to prevent detachment during exploration, both “footprint” and “nanohair” (i.e. dry adhesion via van der Waals forces) adhesion mechanisms were required to achieve maximal tenacity of the attachment by the cyprids.
In Chapter 4, the nanomechanical properties of footprints deposited by cyprid larva of Balanus amphitrite are tested by AFM-based force spectroscopy. Characteristic saw-tooth force extension curves and entropy-elastic stretch behavior were observed, depending on the degree of extension and deformation history. Hysteresis behavior was observed in repeated elongation-relaxation cycles. The sacrificial bond model (as introduced by P. Hansma) was proposed to explain the intra/intermolecular loading and unloading process. Delay/recovery time prior to testing of individual fibrils was found to be important. The effective time needed to reform sacrificial bonds was typically between 2 to 5 s, as estimated from stretching experiments with controlled delay. The force-extension curves were simulated by the classical worm-like chain polymer model to estimate the effective persistence and contour lengths. The change in persistence length with repeated testing indicated the breaking of sacrificial bonds between proteinaceous segments connected either in a parallel or in a serial fashion in the protein nanofibrils.
Chapter 5 exploits the interfacial properties of footprint and chemically-functionalized surfaces by AFM-based chemical force microscopy using chemically modified AFM tips. Force extension curves obtained from the footprints deposited on NH2-functionalized glass by commercial untreated Si3N4 tip and CH3-functionalized tips were investigated. All pull-off force histograms showed forces in the range of 0 - 2 nN, with a maximum at ca. 0.9 nN, which was attributed to breaking of sacrificial intermolecular bonds. Functionalized tips with CH3-terminal chemistry gave an additional higher adhesion force as compared to untreated Si3N4 tips. This high force was attributed to changes in water distribution in the local environment of the protein, in contact by hydrophobic surfaces promoting hydrophobic interactions, which consequently affected the footprint protein conformation. The chemical force allowed mimicking and in situ monitoring of the deposition and interactions between footprints and surfaces at the molecular level.
Chapter 6 combined molecular information obtained from morphology and nanomechanical studies with the settlement behavior of cyprid larvae observed in laboratory and marine field tests. The footprint morphology appeared different, and the size of the oval shaped footprints obtained from CH3-glass was five times larger and more porous than those found on NH2-glass. The differences in footprint morphology on surfaces with different wettabilities might indicate a difference in concentration of chemical cues near the different surfaces, “used” by the cyprids during surface exploration. The clearly distinguishable settlement behavior from the laboratory settlement assay and panel immersion test showed that the barnacle cyprids preferred to settle on the CH3-glass rather than on NH2-glass or borosilicate glass surfaces. By combining the observations from all the experiments at different length scales, it is believed that higher concentrations of settlement inducing cues, i.e. settlement inducing protein complex (SIPC), present on the NH2-surface contribute to the preferred settlement of barnacle on amine-terminated surfaces.
Chapter 7 describes a study of the effect of an enzyme-serine protease, Alcalase, on the adhesives of barnacle cyprids. The settlement assay results indicate cyprid preferred settled on surface treated with footprint. However, once these surfaces were treated with Alcalase, the settlement of cyprid reduced. AFM results provided direct evidence of enzymatic proteolysis of cyprid footprints. After introduction of Alcalase, the footprints were removed over the course of 30 mins. Force spectroscopy was used to monitor the in situ enzymatic proteolysis process. After a 16 mins exposure to Alcalase, only a trace of the footprint remained on the substratum with fewer pull-off events from 2000 s onwards. Alcalase showed no effect on the cured permanent cement. In contrast, uncured permanent cement was thinning over the course of 5 hours, which is highly susceptible to Alcalase.
Chapter 8 indicates the first experimental step towards better understanding of the nanomechanical properties of barnacle cyprid permanent cement by AFM. Force extension curves were collected over the time of the permanent cement curing. The results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. It was clear that the properties of the proteinaceous permanent cement change significantly with time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement. This Thesis demonstrates the versatility of AFM in the study of nano-mechanical properties of bioadhesives, which otherwise is inaccessible by other conventional characterization techniques. It also covers a broad range of assessment methods and measurements that would benefit the understanding of barnacle cyprid larva adhesion process. Morphological studies and nanomechanical property measurements uncover the “secrets” of the barnacle cyprid temporary and reversible attachment. We expect that the knowledge acquired in this work will benefit an overall understanding of the fouling biology, the “interface” between materials science and biology, as well as materials science and coatings industry, and will eventually help in the design of a better antifouling surface.