AVS2010 Session MB+BI+AS-MoA: Preventing & Characterizing Marine Biofouling

The protein resistance of galactoside-terminated alkanethiol self-assembled monolayers (SAMs) can be tuned by partial methylation of the terminating saccharides, and has a non-trivial dependence on the degree of methylation [1], and for other mono- and oligosaccharide-terminated SAMs, protein resistance may vary considerably with small changes in sugar structure. We have used such mono- and oligosaccharide-terminated SAMs in a series of assays using marine fouling organisms as biological model systems, representing common micro- and macrofoulers. We investigate to what extent protein resistance properties are related to effective prevention of fouling by the marine model organisms, and discuss the results in terms of physicochemical properties of the SAMs.

[1] Hederos, M.; Konradsson, P.; Liedberg, B., Langmuir 2005, 21(7), 2971-2980. DOI: 10.1021/la047203b

When manmade surfaces are immersed into the ocean, biofouling rapidly occurs. To support the outphase of toxic coating formulations from the market we derive design rules for environmental benign alternatives. Therefore we study the interaction of biofouling organisms such as zoospores of the green seaweed Ulva linza with well defined surfaces and disentangle the influence of wetting, hydration, morphology, and charge. The obtained results are discussed in the context of time depending formation of conditioning layers. Especially because of its motility, the settlement step of Ulva is highly selective and crucial in their life cycle. A detailed investigation of the relevant phases of approach, exploration and eventually settle­ment is desired but challenging due to the quick, three dimensional swimming motions of spores. Digital in-line holography is suited for this application as time lapse holograms recorded with a single detector provide the 3D position of microorganisms with high accuracy and at a high frame rate. From such 4D tracking data, the sensitive response of spores and their interaction with surfaces has been studied. Statistical analysis of the motion pattern occurrence, velocity distributions and turning motions on surfaces with different chemical termination can be correlated with the accumulated biomass. By this we obtain quantitative access to the interaction between single spores and surfaces.

Proteinaceous secretions are widely recognized to be significant contributors to marine biofouling. The resulting interfacial films can be physisorbed or chemisorbed, and have varying degrees of permanency – they may be highly polymerized and cross-linked, or simply sticky enough to allow surface exploration. Conventional approaches to examining interfacial films derived from bioadhesive junctions is forensic in nature – foulant removal (separating the surfaces) followed by ex situ examination of the adhesive composition and surface morphology. While “what” the adhesive is may be gleaned from ex situ approaches, “how” the adhesive is applied and cures cannot. These time dependent changes can’t be examined “after the fact” and instead require real-time measures of interfacial interactions.

At NRL, we have made significant progress in developing in situ methods to demonstrate the chemical, mechanical and rheological processes in interfaces. We are now applying and extending these approaches to examine underwater adhesion in marine organisms, specifically the little striped barnacle, Balanus amphitrite. We are developing in situ and in vivo spectroscopic approaches to determine how protein structure and chemistry influence marine foulant adhesion. We are particularly interested in determining the structure and chemistry of the cement, the biochemical processes influencing polymerization, cross-linking, and water displacement, as well as the physiochemical nature of the adhesion. Our in situ approaches include performing temporally- and spatially-resolved microscopy and spectroscopy through adhesive interfaces transparent at UV, visible, IR, and x-ray wavelengths. I will describe how we have used these tools to develop new understanding of the properties and development of the adhesive interface of barnacles.

Far-UV circular dichroism (CD) is a valuable method for estimating protein structure components. Analysis of protein CD spectra typically requires deconvolution to resolve overlapping bands and standard methods require that the concentration and pathlength of the sample are accurately known. While this is usually not an issue for the solution state, it is sometimes desirable or a necessity to analyze samples as solid films, complicating deconvolution. Barnacle cement is one example of a proteinaceous bioadhesive that is insoluble by standard biochemical methods and of inconsistent thickness in the native state. To analyze such samples by CD, we have applied g-factor analysis,¹ where the CD spectra are normalized by absorption spectra. This has been demonstrated as a valid, concentration independent deconvolution method, but so far has not been widely used. We will present protein secondary structure estimation results of barnacle cement films as determined by g-factor analysis and show how these results compare with those obtained by infrared spectroscopy. Potential issues and further applicability of solid state CD for bioadhesion studies will be discussed.

¹ McPhie, P. Anal. Biochem. 2001, 293, 109.