Novel whole cell adhesion assays of three isolates of the fouling diatom Amphora coffeaeformis reveal diverse responses to surfaces of different wettability

Whole cell, strength of adhesion assays of three different isolates of the fouling diatom Amphora coffeaeformis were compared using a hydrophilic surface viz. acid washed glass (AWG), and a hydrophobic surface viz. a self assembled monolayer (SAM) of undecanethiol (UDT). Assays were performed using a newly designed turbulent flow channel that permits direct observation and recording of cell populations on a test surface. Exposure to continuous shear stress over 3 h revealed that the more motile isolate, WIL2, adhered much more strongly to both test surfaces compared to the other two strains. When the response of the isolates to shear stress after 3 h was compared, there was no significant difference in the percentage of cells removed, irrespective of surface wettability. Cells of the three isolates of A. coffeaeformis varied significantly in their response to different surfaces during initial adhesion, indicating the presence of a wide range of ‘physiological races’ within this species.     

Microfluidic accumulation assay probes attachment of biofilm forming diatom cells

Testing of fouling release (FR) technologies is of great relevance for discovery of the next generation of protective marine coatings. In this paper, an accumulation assay to test diatom interaction under laminar flow with the model organism Navicula perminuta is introduced. Using time lapse microscopy with large area sampling allows determination of the accumulation kinetics of the diatom on three model surfaces with different surface properties at different wall shear stresses. The hydrodynamic conditions within the flow cell are described and a suitable shear stress range to perform accumulation experiments is identified at which statistically significant discrimination of surfaces is possible. The observed trends compare well to published adhesion preferences of N. perminuta. Also, previously determined trends of critical wall shear stresses required for cell removal from the same set of functionalized interfaces shows consistent trends. Initial attachment mediated by extracellular polymeric substances (EPS) present outside the diatoms leads to the conclusion that the FR potential of the tested coating candidates can be deducted from dynamic accumulation experiments under well-defined hydrodynamic conditions. As well as testing new coating candidates for their FR properties, monitoring of the adhesion process under flow provides additional information on the mechanism and geometry of attachment and the population kinetics.     

Testing attachment point theory: diatom attachment on microtextured polyimide biomimics

Abstract

This paper explores diatom attachment to a range of laser etched polyimide surfaces to directly test ‘attachment point theory’. Static bioassays were conducted on microtextured polyimide surfaces using four diatom species, Fallacia carpentariae, Nitzschia cf. paleacea, Amphora sp. and Navicula jeffreyi with cell sizes ranging from 1 – 14 μm. The microtextured polyimides were modelled from natural fouling resistant bivalve surfaces and had wavelengths above, below and at the same scale as the diatom cell sizes. Diatoms attached in significantly higher numbers to treatments where the numbers of attachment points was highest. The lowest diatom attachment occurred where cells were slightly larger than the microtexture wavelength, resulting in only two theoretical points of attachment. The results support attachment point theory and highlight the need to address larval/cell size in relation to the number of attachment points on a surface. Further studies examining a range of microtexture scales are needed to apply attachment point theory to a suite of fouling organisms and to develop structured surfaces to control the attachment and development of fouling communities.     

Adhesion and motility of fouling diatoms on a silicone elastomer

Recent demands for non-toxic antifouling technologies have led to increased interest in coatings based on silicone elastomers that ‘release’ macrofouling organisms when hydrodynamic conditions are sufficiently robust. However, these types of coatings accumulate diatom slimes, which are not released even from vessels operating at high speeds ( > 30 knots). In this study, adhesion strength and motility of three common fouling diatoms (Amphora coffeaeformis var. perpusilla (Grunow) Cleve, Craspedostauros australis Cox and Navicula perminuta Grunow) were measured on a polydimethylsiloxane elastomer (PDMSE) and acid-washed glass. Adhesion of the three species was stronger to PDMSE than to glass but the adhesion strengths varied. The wall shear stress required to remove 50% of cells from PDMSE was 17 Pa for Craspedostauros, 24 Pa for Amphora and >> 53 Pa for Navicula; the corresponding values for glass were 3, 10 and 25 Pa. In contrast, the motility of the three species showed little or no correlation between the two surfaces. Craspedostauros moved equally well on glass and PDMSE, Amphora moved more on glass initially before movement ceased and Navicula moved more on PDMSE before movement ceased. The results show that fouling diatoms adhere more strongly to a hydrophobic PDMSE surface, and this feature may contribute to their successful colonization of low surface energy, foul-release coatings. The results also indicate that diatom motility is not related to adhesion strength, and motility does not appear to be a useful indicator of surface preference by diatoms.     

Novel whole cell adhesion assays of three isolates of the fouling diatom Amphora coffeaeformis reveal diverse responses to surfaces of different wettability

Whole cell, strength of adhesion assays of three different isolates of the fouling diatom Amphora coffeaeformis were compared using a hydrophilic surface viz. acid washed glass (AWG), and a hydrophobic surface viz. a self assembled monolayer (SAM) of undecanethiol (UDT). Assays were performed using a newly designed turbulent flow channel that permits direct observation and recording of cell populations on a test surface. Exposure to continuous shear stress over 3?h revealed that the more motile isolate, WIL2, adhered much more strongly to both test surfaces compared to the other two strains. When the response of the isolates to shear stress after 3?h was compared, there was no significant difference in the percentage of cells removed, irrespective of surface wettability. Cells of the three isolates of A. coffeaeformis varied significantly in their response to different surfaces during initial adhesion, indicating the presence of a wide range of 'physiological races' within this species.     

Using textured PDMS to prevent settlement and enhance release of marine fouling organisms

The antifouling efficacy of a series of 18 textured (0.2–1000 μm) and non-textured (0 μm) polydimethylsiloxane surfaces with the profiles of round- and square-wave linear grating was tested by recording the settlement of fouling organisms in the laboratory and in the field by monitoring the recruitment of a multi-species fouling community. In laboratory assays, the diatoms Nitzschia closterium and Amphora sp. were deterred by all surface topographies regardless of texture type. Settlement of propagules of Ulva sp. was lower on texture sizes less than the propagule size, and settlement of larvae of Saccostrea glomerata and Bugula neritina was lower on texture sizes closest to, but less than, the sizes of larvae. After a six month field trial, all textured surfaces lost their deterrent effect; however, the foul-release capabilities of textures were still present. High initial attachment was correlated with most fouling remaining after removal trials, indicating that fouling organisms recruited in higher numbers to surfaces upon which they attached most strongly.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

A simple method to evaluate the potential for degradation of antifouling biocides

A simple method to measure the degradation of antifouling biocides is described which measures the loss of biocidal activity from seawater by bioassay. The bioassay employs either the ship‐fouling diatom Amphora or the brine shrimp Anemia. Loss of bioaclivity from sterile seawater indicates abiotic degradation whilst loss of bioactivity from natural seawater indicates biodegradation. Results are presented for three biocides, viz. the trihalomethylthio compound, N‐dichlorofluoromethylthio‐N’,N'‐dimethyl‐N‐phenyl‐sulphamide (Preventol A4S), di‐n‐octylamine, and the isothiazolone compound 4,5‐dichloro‐2‐n‐octyl‐4‐isothiazolin‐3‐one (Sea‐Nine 211).     

MECHANISMS OF FLUID SHEAR‐INDUCED INHIBITION OF POPULATION GROWTH IN A RED‐TIDE DINOFLAGELLATE1

Net population growth of some dinoflagellates is inhibited by fluid shear at shear stresses comparable with those generated during oceanic turbulence. Decreased net growth may occur through lowered cell division, increased mortality, or both. The dominant mechanism under various flow conditions was determined for the red‐tide dinoflagellate Lingulodinium polyedrum (Stein) Dodge. Cell division and mortality were determined by direct observation of isolated cells in 0.5‐mL cultures that were shaken to generate unquantified fluid shear. Larger volume cultures were exposed to quantified laminar shear in Couette‐flow chambers (0.004–0.019 N·m ^{? 2} shear stress) and to unquantified flow in shaken flasks. In these larger cultures, cell division frequency was calculated from flow cytometric measurements of DNA·cell^?1. The mechanism by which shear inhibits net growth of L. polyedrum depends on shear stress level and growth conditions. Observations on the isolated cells showed that shaking inhibited growth by lowering cell division without increased mortality. Similar results were found for early exponential‐phase cultures exposed to the lowest experimental shear stress in Couette‐flow chambers. However, mortality occurred when a late exponential‐phase culture was exposed to the same low shear stress and was inferred to occur in cultures exposed to higher shear stresses. Elevated mortality in those treatments was confirmed using behavioral, morphological, and physiological assays. The results predict that cell division in L. polyedrum populations will be inhibited by levels of oceanic turbulence common for near‐surface waters. Shear‐induced mortality is not expected unless shear‐stress levels are unusually high or when cellular condition resembles late exponential/stationary phase cultures.     

Reduction of barnacle recruitment on micro‐textured surfaces: Analysis of effective topographic characteristics and evaluation of skin friction

This study investigates five designed micro‐textured surfaces and their effects on barnacle fouling and hydrodynamic drag. Three of the micro‐textures were developed in the present study and evaluated together with two commercial riblet films. All micro‐structures were arranged as longitudinal grooves with different profile depths, widths and angles of inclination. In field tests the recruitment of the barnacle Balanus improvisus on micro‐textured surfaces and smooth controls was evaluated. All micro‐textured surfaces reduced recruitment, and the most efficient texture reduced recruitment by 98%. For some micro‐textures the reduction of recruitment declined as settlement intensity increased. In a correlative analysis, the trigonometric inclination of the micro‐structures explained most of the recruitment reduction. The steepest angle of inclination caused a massive reduction in barnacle settlement. Surface micro‐structures may affect the boundary‐layer flow and the hydrodynamic drag (skin friction) of the surface. The skin friction was empirically measured in a flow channel using a sub‐set of the tested micro‐textures. The measurements of skin friction showed that the orientation of the microstructures is important, with a minimum friction when the grooves are parallel to the flow. For one of the micro‐textures the skin friction was ca 10% lower compared to a hydraulically smooth surface. It is concluded that, depending on the flow speed, micro‐textures will not significantly increase skin friction when arranged parallel to the flow, even at moderate protrusion through the viscous sub‐layer.     

The role of nano-roughness in antifouling

Nano-engineered superhydrophobic surfaces have been investigated for potential fouling resistance properties. Integrating hydrophobic materials with nanoscale roughness generates surfaces with superhydrophobicity that have water contact angles (θ) >150° and concomitant low hysteresis (<10°). Three superhydrophobic coatings (SHCs) differing in their chemical composition and architecture were tested against major fouling species (Amphora sp., Ulva rigida, Polysiphonia sphaerocarpa, Bugula neritina, Amphibalanus amphitrite) in settlement assays. The SHC which had nanoscale roughness alone (SHC 3) deterred the settlement of all the tested fouling organisms, compared to selective settlement on the SHCs with nano- and micro-scale architectures. The presence of air incursions or nanobubbles at the interface of the SHCs when immersed was characterized using small angle X-ray scattering, a technique sensitive to local changes in electron density contrast resulting from partial or complete wetting of a rough interface. The coating with broad spectrum antifouling properties (SHC 3) had a noticeably larger amount of unwetted interface when immersed, likely due to the comparatively high work of adhesion (60.77 mJ m^?2 for SHC 3 compared to 5.78 mJ m^?2 for the other two SHCs) required for creating solid/liquid interface from the solid/vapour interface. This is the first example of a non-toxic, fouling resistant surface against a broad spectrum of fouling organisms ranging from plant cells and non-motile spores, to complex invertebrate larvae with highly selective sensory mechanisms. The only physical property differentiating the immersed surfaces is the nano-architectured roughness which supports longer standing air incursions providing a novel non-toxic broad spectrum mechanism for the prevention of biofouling.     

Microfluidic detachment assay to probe the adhesion strength of diatoms

Fouling release (FR) coatings are increasingly applied as an environmentally benign alternative for controlling marine biofouling. As the technology relies on removing fouling by water currents created by the motion of ships, weakening of adhesion of adherent organisms is the key design goal for improved coatings. In this paper, a microfluidic shear force assay is used to quantify how easily diatoms can be removed from surfaces. The experimental setup and the optimization of the experimental parameters to study the adhesion of the diatom Navicula perminuta are described. As examples of how varying the physico-chemical surface properties affects the ability of diatoms to bind to surfaces, a range of hydrophilic and hydrophobic self-assembled monolayers was compared. While the number of cells that attached (adhered) was barely affected by the coatings, the critical shear stress required for their removal from the surface varied significantly.     

Ultrastructural observations on the marine fouling diatomAmphora

Ecological and Scanning electron microscope (S. E. M.) studies indicated that the diatomAmphora was an important constituent in the initial colonization of test panels coated with a copper antifouling composition.Amphora was also found as the dominant fouling diatom species on paint samples from in-service supertankers and yachts. Associated with the diatom was copious amounts of mucilaginous material, which often encapsulated the cells. Histochemical analysis of the mucilage indicates that it is predominantly polysaccharide in nature. Using the Transmission electron microscope (T. E. M.) and electron microscope cytochemistry the intracellular origin of the adhesive was investigated. T. E. M. and S. E. M. observations of acid-cleaned-cells indicate that the mucilage may be secreted through specialized regions of the frustule. Material isolated from antifouling panels was compared with laboratory culturedAmphora spp. for copper resistance and internal accumulation using TEMSCAN — X ray analytical equipment.     

Interspecific variation in patterns of adhesion of marine fouling to silicone surfaces

Abstract

The adhesion of six fouling organisms: the barnacle Balanus eburneus, the gastropod mollusc Crepidula fornicata, the bivalve molluscs Crassostrea virginica and Ostrea/Dendrostrea spp., and the serpulid tubeworms Hydroides dianthus and H. elegans, to 12 silicone fouling-release surfaces was examined. Removal stress (adhesion strength) varied among the fouling species and among the surfaces. Principal component analysis of the removal stress data revealed that the fouling species fell into two distinct groups, one comprising the bivalve molluscs and tubeworms, and the other the barnacle and the gastropod mollusc. None of the silicone materials generated a minimum in removal stress for all the organisms tested, although several surfaces produced low adhesion strengths for both groups of species. These results suggest that fouling-release materials do not rank (in terms of adhesion strength) identically for all fouling organisms, and thus development of a globally-effective hull coating will continue to require testing against a diversity of encrusting species.     

The effect of turbulent flow and surface roughness on biofilm formation in drinking water

There is considerable interest in both Europe and the USA in the effects of microbiological fouling on stainless steels in potable water. However, little is known about the formation and effects of biofilms, on stainless steel in potable water environments, particularly in turbulent flow regimes. Results are presented on the development of biofilms on stainless steel grades 304 and 316 after exposure to potable water at velocities of 0.32, 0.96 and 1.75 m s⁻¹. Cell counts on slides of stainless steel grades 304 and 316 with both 2B (smooth) and 2D (rough) finishes showed viable and total cell counts were higher at the higher flow rates of 0.96 and 1.75 m s⁻¹, compared to a flow rate of 0.32 m s⁻¹. Extracellular polysaccharide levels were not significantly different (P< 0.05) between each flow rate on all stainless steel surfaces studied. higher levels were found at the higher water velocities. the biofilm attached to stainless steel was comprised of a mixed bacterial flora including Acinetobacter sp, Pseudomonas spp, Methylobacterium sp, and Corynebacterium/Arthrobacter spp. Epifluorescence microscopy provided evidence of rod-shaped bacteria and the formation of stands, possibly of extracellular material attached to stainless steel at high flow rates but not at low flow rates. Received 04 February 1998/ Accepted in revised form 12 February 1999     

Seasonal variations in the fouling diatom community structure from a monsoon influenced tropical estuary

Seasonal variations in the fouling diatom community from a monsoon influenced tropical estuary were investigated. The community composition did not differ significantly between stainless steel and polystyrene substrata due to dominance by Navicula spp. The experimental evidence suggests that Amphora, which is a dominant fouling diatom in temperate waters, ultimately dominates the community in tropical regions when conditions are favourable. These observations reveal that a faster onset of macrofouling interferes with the microfouling community wherein the faster recruiters that have a higher abundance in ambient waters, dominate the community. Seasonal variations were observed in the fouling diatom community. Navicula delicatula dominated during the post-monsoon and tychopelagic diatoms (Melosira and Odontella) were observed during the monsoon. Low diatom abundance was recorded during the pre-monsoon season. The results indicate that although the fouling diatom community composition does not vary between substrata, there is a seasonal change in the community depending on the physical, chemical and biological interactions.     

Frictional drag measurements of large-scale plates in an enhanced plane channel flowcell

Abstract

This paper describes the design of an enhanced, plane channel, flowcell and its use for testing large-scale coated plates (0.6?m × 0.22?m) in fully developed flow, over a wide range of Reynolds numbers, with low uncertainty. Two identical, hydraulically smooth plates were experimentally tested. Uniform biofilms were grown on clean surfaces to test skin friction changes resulting from different biofilm thickness and densities. A velocity survey of the flowcell measurement section, using laser Doppler anemometry, showed a consistent velocity profile and low turbulence intensity in the central flow channel. The skin friction coefficient was experimentally determined using a pressure drop method. Results correlate closely to previously published regression data, particularly at higher speeds. Repeated measurements indicated very low uncertainty. This study demonstrates this flowcell’s applicability for representing consistent frictional drag of ship hull surfaces, enabling comparability of hydrodynamic drag caused by surface roughness to the reference surface measurements.     

Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans

Hydroides elegans is a major fouling organism in tropical waters around the world, including Pearl Harbor, Hawaii. To determine the importance of initial surface characteristics on biofilm community composition and subsequent colonization by larvae of H. elegans, the settlement and recruitment of larvae to biofilmed surfaces with six different initial surface wettabilities were tested in Pearl Harbor. Biofilm community composition, as determined by a combined approach of denaturing gradient gel electrophoresis and fluorescence in situ hybridization, was similar across all surfaces, regardless of initial wettability, and all surfaces had distinct temporal shifts in community structure over a 10 day period. Larvae settled and recruited in higher numbers to surfaces with medium to low wettability in both May and August, and also to slides with high wettability in August. Pearl Harbor biofilm communities developed similarly on a range of surface wettabilities, and after 10 days in Pearl Harbor all surfaces were equally attractive to larvae of Hydroides elegans, regardless of initial surface properties.     

Examination of nanoflow in rectangular slits

This paper presents simulations of 3D nanoscale flow in rectangular channel with molecular dynamics simulation method. Rectangular cross section is a frequently encountered geometric shape for nanoscale flow problems. For a given cross sectional height h, we change the width w of the rectangular cross section and analyze the influence of w/h on the flow characteristics. The distributions of density, temperature, boundary slip and flow velocity inside the rectangular cross section are investigated in detail. Liquid argon material and Lennard-Jones potential are used in the simulations. The simulation results are also compared with Navier–Stokes solutions for rectangular channel flows.     

Effect of shear stress on activity of cellular enzyme in animal cell

The effect of shear stress on the activity of cellular enzyme in an animal cell was discussed by using a flow channel. The activity of lactate dehydrogenase (LDH) in cells exposed to a shear stress of 0.5 Pa for 12 h was about 4-fold greater than in the cells without exposure to shear stress. The relative LDH activity was correlated with the dissipation energy density of the flowing medium. A good correlation was obtained and it was found that the dependency of cellular enzyme activity on the shear stress and the exposure time was related to the transmission of the energy from the flowing medium to attached cells.List of Symbols b m width of flow channel - E J/m² dissipation energy density - h m distance between two plates - Q m³/s volume flow rate - t s exposure time - u m/s velocity of medium - y m distance from wall attached cells - Pa s viscosity - Pa shear stress