Effects of biofilms on zebra mussel postveliger attachment to artificial surfaces

Abstract. The zebra mussel is an introduced fouling organism in North American inland waters. This field study tested whether natural biofilms, formed by covering substrata with a 100-μm mesh that allows microorganisms to attach and films to develop in the absence of postveligers, influenced the attachment of zebra mussel postveligers to artificial surfaces. Low-wettable polycarbonate and wettable glass surfaces were used in the experiments over four field seasons to study biofilm formation (1997–1998) and mussel attachment (1998–2000). The presence of the mesh did not quantitatively affect biofilm development on either substratum as determined by microscopic direct counts and colony-forming units on R2A agar. Natural biofilms on polycarbonate surfaces positively influenced postveliger attachment compared to substrata that initially had no film (ANOVA, p-values ranged from ≤.05 to ≤.001). Biofilms did not influence postveliger attachment to glass surfaces (ANOVA, p>.05). Attachment to both substrata was similar on surfaces with and without previously settled postveligers. Based on these results, we conclude that biofilms can enhance postveliger attachment to some but not all artificial surfaces.     

Hybrid xerogel films as novel coatings for antifouling and fouling release

Hybrid sol-gel-derived xerogel films prepared from 45/55 (mol ratio) n-propyltrimethoxysilane (C3-TMOS)/tetramethylorthosilane (TMOS), 2/98 (mol ratio) bis[3-(trimethoxysilyl)propyl]-ethylenediamine (enTMOS)/tetraethylorthosilane (TEOS), 50/50 (mol ratio) n-octyltriethoxysilane (C8-TEOS)/TMOS, and 50/50 (mol ratio) 3,3,3-trifluoropropyltrimethoxysilane (TFP-TMOS)/TMOS were found to inhibit settlement of zoospores of the marine fouling alga Ulva (syn. Enteromorpha) relative to settlement on acid-washed glass and give greater release of settled zoospores relative to glass upon exposure to pressure from a water jet. The more hydrophobic 50/50 C8-TEOS/TMOS xerogel films had the lowest critical surface tension by comprehensive contact angle analysis and gave significantly greater release of 8-day Ulva sporeling biomass after exposure to turbulent flow generated by a flow channel than the other xerogel surfaces or glass. The 50/50 C8-TEOS/TMOS xerogel was also a fouling release surface for juveniles of the tropical barnacle Balanus amphitrite. X-ray photon electron data indicated that the alkylsilyl residues of the C3-TMOS-, C8-TEOS-, and TFP-TMOS-containing xerogels were located on the surface of the xerogel films (in a vacuum), which contributes to the film hydrophobicity. Similarly, the amine-containing silyl residues of the enTMOS/TEOS films were located at the surface of the xerogel films, which contributes to the more hydrophilic character and increased critical surface tension of these films.     

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.     

Substratum preferences and planulae settling of two red sea alcyonaceans: Xenia macrospiculata Gohar and Parerythropodium fulvum fulvum (Forskl)

The settling behaviour and substratum preferences of the planulae of the Red Sea soft corals Xenia macrospiculata Gohar and Parerythropodium fulvum fulvum (Forskl) were examined in the laboratory. The planulae of the two species have a short pelagic phase and they tend to settle immediately upon leaving the parent colonies. Mucous secretion is used by the larvae for crawling and adhering to the substratum. They exhibit an aggregated pattern of settlement. The developing polyps are found in depressions or pits of the substratum. The planulae preferentially settle on rough substrata and avoid smooth surfaces. They search for substrata covered with an organic coating, composed of turf or crustose coralline algae. Such substrata create better conditions for larval settlement and metamorphosis. The planulae of P. f. fulvum exhibit a striking preference for upside-down attachment on undersides of the substrata, while Xenia macrospiculata utilizes both substratum faces for settlement. Light intensity seems insignificant in determining attachment sites. The findings of the experiments correspond well with the distributional patterns of juveniles of the two species as found in the natural environment. The specific requirements for settling of both species increase their chances of successful development and thus enhance their survival.     

Laboratory studies on adhesion of microalgae to hard substrates

Adhesion of Chlorella vulgaris(chlorophyceae), Nitzschia amphibia(bacillariophceae) and Chroococcus minutus(cyanobacteria) to hydrophobic (perspex, titanium and stainless steel 316-L), hydrophilic (glass) and toxic (copper, aluminium brass and admiralty brass) substrata were studied in the laboratory. The influence of surface wettability, surface roughness, pH of the medium, culture age, culture density, cell viability and presence of organic and bacterial films on the adhesion of Nitzschia amphibia was also studied using titanium, stainless steel and glass surfaces. All three organisms attached more on titanium and stainless steel and less on copper and its alloys. The attachment varied significantly with respect to exposure time and different materials. The attachment was higher on rough surfaces when compared to smooth surfaces. Attachment was higher on pH 7 and above. The presence of organic film increased the attachment significantly when compared to control. The number of attached cells was found to be directly proportional to the culture density. Attachment by log phase cells was significantly higher when compared to stationary phase cells. Live cells attached more when compared to heat killed and formalin killed cells. Bacterial films of Pseudomonas putida increased the algal attachment significantly. %     

Induction of settlement in mussel (Perna canaliculus) larvae by vessel noise

Underwater sound plays an important role in the settlement behaviour of many coastal organisms. Large steel-hulled vessels are known to be a major source of underwater sound in the marine environment. The possibility that underwater sound from vessels may promote biofouling of hulls through triggering natural larval settlement cues was investigated for the mussel, Perna canaliculus. The mussel larvae showed significantly faster settlement when exposed to the underwater noise produced by a 125-m long steel-hulled passenger and freight ferry. Median time to attachment on the substrata (ie settlement) was reduced by 22% and the time taken for all experimental larvae to settle was reduced by 40% relative to a silent control. There was no difference in the survival of the mussel larvae among the various noise treatments. The decrease in settlement time of the mussel larvae appeared to correlate with the intensity of the vessel sound, suggesting that underwater sound emanating from vessels may be an important factor in exacerbating hull fouling by mussels.     

The physico-chemical characterization of casein-modified surfaces and their influence on the adhesion of spores from a Geobacillus species

To gain a better understanding of the factors influencing spore adhesion in dairy manufacturing plants, casein-modified glass surfaces were prepared and characterized and their effect on the adhesion kinetics of spores from a Geobacillus sp., isolated from a dairy manufacturing plant (DMP) was assessed using a flow chamber. Surfaces were produced by initially silanizing glass using (3-glycidyloxypropyl) trimethoxysilane (GPS) or (3-aminopropyl) triethoxysilane to form epoxy-functionalized (G-GPS) or amino-functionalized glass (G-NH₂) substrata. Casein was grafted to the G-GPS directly by its primary amino groups (G-GPS-casein) or to G-NH₂ by employing glutaraldehyde as a linking agent (G-NH₂-glutar-casein). The surfaces were characterised using streaming potential measurements, contact angle goniometry, infrared spectroscopy and scanning electron microscopy. The attachment rate of spores suspended in 0.1 M KCl at pH 6.8, was highest on the positively charged (+14 mV) G-NH₂ surface (333 spores cm^?2 s^?1) compared to the negatively charged glass (?22 mV), G-GPS (?20 mV) or G-GPS-casein (?21 mV) surfaces (162, 17 or 6 spores cm^?2 s^?1 respectively). Whilst there was a clear decrease in attachment rate to negatively charged casein-modified surfaces compared to the positively charged amine surface, there was no clear relationship between surface hydrophobicity and spore attachment rate.     

Bubble Contact Angle Method for Evaluating Substratum Interfacial Characteristics and Its Relevance to Bacterial Attachment

A bubble contact angle method was used to determine interfacial free-energy characteristics of polystyrene substrata in the presence and absence of potential surface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fatty acid-free bovine serum albumin), a bacterial culture supernatant, and a bacterial exopolymer. Clean petri dish substrata gave a contact angle of 90°, but tissue culture dish substrata were more hydrophilic, giving an angle of 29° or less. Bubble contact angles at the surfaces exposed to the macromolecular solutions varied with the composition and concentration of the solution. Modification by pronase enzymes of the conditioning effect of proteins depended on the nature of both the substratum and the protein, as well as the time of addition of the enzyme relative to the conditioning of the substratum. The effects of dissolved and substratum-adsorbed proteins on the attachment of Pseudomonas sp. strain NCMB 2021 to petri dishes and tissue culture dishes were consistent with changes in bubble contact angles (except when proteins were adsorbed to tissue culture dishes before attachment) as were alterations in protein-induced inhibition of bacterial attachment to petri dishes by treatment with pronase. Differences between the attachment of pseudomonads to petri dishes and tissue culture dishes suggested that different mechanisms of adhesion are involved at the surfaces of these two substrata.     

Influence of Substratum Characteristics on the Attachment of a Marine Pseudomonad to Solid Surfaces

The attachment of a marine Pseudomonas sp. to a variety of surfaces was investigated, and the number of bacteria which became attached was related to the surface charge and degree of hydrophobicity of the substratum. Large numbers of bacteria attached to hydrophobic plastics with little or no surface charge [Teflon, polyethylene, polystyrene, poly(ethylene terephthalate)]; moderate numbers attached to hydrophilic metals with a positive (platinum) or neutral (germanium) surface charge; and very few attached to hydrophilic, negatively charged substrata (glass, mica, oxidized plastics). The results suggest that both electrostatic and hydrophobic interactions are involved in bacterial attachment.     

Surface wettability as a determinant in the settlement of the barnacle Balanus Improvisus (DARWIN)

Several studies have shown that the initial surface wettability, is of importance in the settlement of macrofouling larvae such as barnacles, bryozoans and hydroids in the field as well as in laboratory assays. In this study we present results from laboratory assays using hydrophilic and hydrophobic polystyrene (PS) and cyprid larvae of Balanus improvisus (Darwin). The results obtained differ markedly from those reported for the barnacle Balanus amphitrite (Darwin), where a high surface wettability seemed to be preferred for settlement. Our results show that a surface with intermediary wettability (hydrophilic PS) reduced settlement by 38% as compared to surfaces of low wettability (hydrophobic PS) during an 8-day period. During the experiment, the wettability in the hydrophilic PS dishes was not significantly changed as measured by advancing contact angle with mQ water. Over an 8-day period wettability of the hydrophobic PS dishes approached that of the hydrophilic PS surfaces. We further conducted experiments with highly hydrophilic and highly hydrophobic methylsilane-treated glass surfaces with known chemistry. In this experiment, the settlement of cyprid larvae was completely inhibited by the high wettability surfaces. Contact angle measurements revealed that the wettability during the length of the experiment of the hydrophilic glass surfaces was not significantly altered. We conclude by these experiments that even an intermediate wettability can significantly affect the overall settlement success of the barnacle B. improvisus. The mechanism by which the settlement is impeded might be biologically mediated through the recognition by cyprid larvae of the molecular composition of the surface when the cyprid reverts to the settlement phase, i.e. when swimming behaviour is abandoned in favour of surface exploration, or it is mediated by physicochemical forces acting between the surface and the larval body or the larval antennules.     

A comparative study of the non-acidic chemically mediated antifoulant properties of three sympatric species of ascidians associated with seagrass habitats

The present study investigated aspects of the antifoulant properties of three sympatric species of ascidians found in seagrass habitats of the Gulf of Mexico, Southern Atlantic Ocean, and Caribbean. Field observations in Saint Joseph Bay, Florida indicate that all three species are common and that the tunic of the solitary ascidian Molgula occidentalis is often heavily fouled, while the outer surfaces of both the colonial ascidians Amaroucium stellatum and Botryllus planus are free of fouling organisms. Antifoulant activities of a suite of increasing hydrophilic organic extracts prepared from the tunic of M. occidentalis and whole colonies of A. stellatum and B. planus were measured using both sympatric microbial (bacteria) and macroinvertebrate (cyprid larvae of Balanus amphitrite) fouling organisms in laboratory bioassays. In addition, field antifoulant assays were conducted by combining organic extracts with controlled-release resin and subsequently coating this material on to acrylic rods deployed in the field for a 72 h period. Extracts of the tunic of M. occidentalis generally did not inhibit bacterial growth. The exception was the methanol extract, which inhibited growth in one of the six marine bacteria tested. Moreover, only the highest concentrations of hexane and methanol tunic extracts tested prevented attachment of cyprid larvae. Field assays revealed no antifoulant activity on rods coated with resin containing extracts of M. occidentalis. Inhibition of both microbial growth and cyprid settlement were much more pronounced in whole-organism extracts of the two colonial ascidians. Most potent were the aqueous methanol extracts of colonies of B. planus and A. stellatum which inhibited growth in five of the six marine bacteria tested. In addition, hydrophilic and lipophilic extracts of the colonial ascidians significantly inhibited attachment of cyprid larvae, in many instances across a wide range of extract concentrations. Field antifoulant assays indicated that extracts of both colonial ascidians inhibited settlement of bryozoans and barnacles. The findings indicate that the colonial ascidians B. planus and A. stellatum possess chemical antifoulant properties. In contrast, the solitary ascidian M. occidentalis appears to either tolerate fouling or possess other non-chemical mechanisms to cope with the risks associated with epibiont overgrowth.     

The physico-chemical characterization of casein-modified surfaces and their influence on the adhesion of spores from a Geobacillus species

To gain a better understanding of the factors influencing spore adhesion in dairy manufacturing plants, casein-modified glass surfaces were prepared and characterized and their effect on the adhesion kinetics of spores from a Geobacillus sp., isolated from a dairy manufacturing plant (DMP) was assessed using a flow chamber. Surfaces were produced by initially silanizing glass using (3-glycidyloxypropyl) trimethoxysilane (GPS) or (3-aminopropyl) triethoxysilane to form epoxy-functionalized (G-GPS) or amino-functionalized glass (G-NH(2)) substrata. Casein was grafted to the G-GPS directly by its primary amino groups (G-GPS-casein) or to G-NH(2) by employing glutaraldehyde as a linking agent (G-NH(2)-glutar-casein). The surfaces were characterised using streaming potential measurements, contact angle goniometry, infrared spectroscopy and scanning electron microscopy. The attachment rate of spores suspended in 0.1?M KCl at pH 6.8, was highest on the positively charged (+14 mV) G-NH(2) surface (333 spores cm(-2) s(-1)) compared to the negatively charged glass (-22 mV), G-GPS (-20 mV) or G-GPS-casein (-21 mV) surfaces (162, 17 or 6 spores cm(-2) s(-1) respectively). Whilst there was a clear decrease in attachment rate to negatively charged casein-modified surfaces compared to the positively charged amine surface, there was no clear relationship between surface hydrophobicity and spore attachment rate.     

Adhesion of Nongerminated Botrytis cinerea Conidia to Several Substrata

Conidia of the plant pathogenic fungus Botrytis cinerea adhered to tomato cuticle and to certain other substrata immediately upon hydration. This immediate adhesion occurred with both living and nonliving conidia. Adhesion was not consistently influenced by several lectins, sugars, or salts or by protease treatment, but it was strongly inhibited by ionic or nonionic detergents. With glass and oxidized polyethylene, substrata whose surface hydrophobicities could be conveniently varied, there was a direct relationship between water contact angle and percent adhesion. Immediate adhesion did not involve specific conidial attachment structures, although the surfaces of attached conidia were altered by contact with a substratum. Freshly harvested conidia were very hydrophobic, with more than 97% partitioning into the organic layer when subjected to a phase distribution test. Percent adhesion of germinated conidia was larger than that of nongerminated conidia. Evidence suggests that immediate adhesion of conidia of B. cinerea depends, at least in part, on hydrophobic interactions between the conidia and substratum.     

Retention of the Gram-negative bacterium SW8 on surfaces under conditions relevant to the subsurface environment: Effects of conditioning films and substratum nature

Abstract Shallow (5–13 m) and deep (35–65 m) groundwaters were evaluated for their ability to generate conditioning films which affect bacterial adhesion to natural (sandstone, shale, andesite) and man-made substrata (polypropylene, stainless steel). Water contact angles indicated that all water samples produced conditioning films. Most films modified retention of the nonmotile Gram-negative bacterium SW8, but attachment of the organism did not correlate with water contact angles. Each borewater produced conditioning films with a characteristic attachment profile of SW8. Films adsorbed from standing borewaters often retained SW8 in different numbers than coatings derived from pumped bores. Groundwater chemistry was very heterogeneous and microbiological data indicated the presence of a diverse aerobic and anaerobic microbial community. These results indicate that conditioning films derived from dissolved compounds may play a significant role in controlling the interaction of bacteria with substrata in the subsurface.     

Adhesion of algal cells to surfaces

This paper reports the cell–substratum interactions of planktonic (Chlorella vulgaris) and benthic (Botryococcus sudeticus) freshwater green algae with hydrophilic (glass) and hydrophobic (indium tin oxide) substrata to determine the critical parameters controlling the adhesion of algal cells to surfaces. The surface properties of the algae and substrata were quantified by measuring contact angle, electrophoretic mobility, and streaming potential. Using these data, the cell–substratum interactions were modeled using thermodynamic, DLVO, and XDLVO approaches. Finally, the rate of attachment and the strength of adhesion of the algal cells were quantified using a parallel-plate flow chamber. The results indicated that (1) acid–base interactions played a critical role in the adhesion of algae, (2) the hydrophobic alga attached at a higher density and with a higher strength of adhesion on both substrata, and (3) the XDLVO model was the most accurate in predicting the density of cells and their strength of adhesion. These results can be used to select substrata to promote/inhibit the adhesion of algal cells to surfaces.     

Larval behavioral,morphological changes,and nematocyte dynamics during settlement of actinulae of Tubularia mesembryanthemum,Allman 1871 (Hydrozoa: Tubulariidae)

The marine colonial hydroid Tubularia mesembryanthemum produces a morphologically unique dispersive stage, the actinula larva. Detailed observations were made on the behaviors and nematocyte dynamics of actinula larvae during attachment and morphogenesis by employing microscopic and time lapse video techniques. These observations produced four primary results. (1) Actinula larvae demonstrated two forms of attachment: temporary attachment by atrichous isorhiza (AI)-nematocysts discharged from the aboral tentacle (AT) tips-and permanent settlement by cement secretion from the columnar gland cells of the basal protrusion. (2) During larval settlement, numerous AIs were discharged from the AT tips with sinuous movement and rubbing of the tentacles onto the substrata, leading to "nematocyte-printing" around the settlement site. (3) Simultaneous with the discharge of the AIs, migration of stenoteles, desmonemes, and microbasic mastigophores occurred, resulting in a dramatic change of nematocyte composition in the ATs after larval settlement. This was in parallel with changes in larval behavior and the tentacle function. (4) Nematocyte-printing behavior during settlement could be recognized as metamorphic behavior responsible for irreversible changes in AT function, from attachment to feeding and defense.     

The influence of resident adults on larval settlement: experiments with four species of ascidians

Residents within any community can affect the larval settlement of both their own and other species. In marine sessile communities resident adults can affect larval settlement by preying on settling larvae, removing or adding space for the larvae to colonize, or stimulating or prohibiting larval settlement on available substratum nearby. To examine those processes by which residents affect settlement, we exposed experimental substrata with three densities of adults of a single species at a site in eastern Long Island Sound, USA for a 24-h period. Four species of common ascidians, Botryllus schlosseri (Pallas), Botrylloides diegensis Ritter and Forsyth, Diplosoma macdonaldi Herdman, and Molgula manhattensis (De Kay), were used in 11 separate experiments. Few individuals of any species settling attached to the surfaces of these species and this resulted in the main effect of these residents being the usurpation of space and the restricting of settlement to unoccupied areas. A model is also presented to explain the apparent aggregated settlement of several species in open areas adjacent to the resident ascidians. From this model we suggest that the aggregated settlement can result from limited larval mobility such that some larvae that contact and reject the resident species as settlement sites may subsequently contact open surfaces of the same substratum and increase settlement densities there over those observed on control substrata. Finally, settlement data for several species indicate that Molgula may influence settlement by preying on larvae.     

Interactions between microbial biofilms and marine fouling algae: a mini review

Natural and artificial substrata immersed in the marine environment are typically colonized by microorganisms, which may moderate the settlement/recruitment of algal spores and invertebrate larvae of macrofouling organisms. This mini-review summarizes the major interactions occurring between microbial biofilms and marine fouling algae, including their effects on the settlement, growth and morphology of the adult plants. The roles of chemical compounds that are produced by both bacteria and algae and which drive the interactions are reviewed. The possibility of using such bioactive compounds to control macrofouling will be discussed.     

Comparison of fluorinated polymers against stainless steel, glass and polypropylene in microbial biofilm adherence and removal

Biofilm formation is a long-standing problem in ultrapure water and bioprocess fluid transport lines. The standard materials used in these applications (316L stainless steel, polypropylene and glass) have long been known to be good surfaces for the attachment of bacteria and other biological materials. To compare the relative tenacity of biofilms grown on materials used in manufacturing processes, a model system for biofilm attachment was constructed that approximates the conditions in industrial process systems. New fluorinated polymers were compared to the above materials by evaluating the surface area coverage of bacterial populations on materials before and after mild chemical treatment. In addition, contact angle studies compared the relative hydrophobicity of surfaces to suspensions of bacteria in growth media, and scanning electron microscopy and atomic force microscopy studies were used to characterize surface smoothness and surface defects. Biofilm adherence to polymer-based substrata was determined to be a function of both surface finish and surface chemistry. Specifically, materials that are less chemically reactive, as indicated by higher contact angle, can have rougher surface finishes and still be amenable to biofilm removal. Received 20 March 1997/ Accepted in revised form 15 July 1997     

The role of surface energy and water wettability in aminoalkyl/fluorocarbon/hydrocarbon-modified xerogel surfaces in the control of marine biofouling

Xerogel films with uniform surface topogrophy, as determined by scanning electron microscopy, atomic force microscopy (AFM), and time-of-flight secondary ion mass spectrometry, were prepared from aminopropylsilyl-, fluorocarbonsilyl-, and hydrocarbonsilyl- containing precursors. Young's modulus was determined from AFM indentation measurements. The xerogel coatings gave reduced settlement of zoospores of the marine fouling alga Ulva compared to a poly(dimethylsiloxane) elastomer (PDMSE) standard. Increased settlement correlated with decreased water wettability as measured by the static water contact angle, θ_Ws, or with decreased polar contribution (γ_P) to the surface free energy (γ_S) as measured by comprehensive contact angle analysis. The strength of attachment of 7-day sporelings (young plants) of Ulva on several of the xerogels was similar to that on PDMSE although no overall correlation was observed with either θ_Ws or γ_S. For sporelings attached to the fluorocarbon/hydrocarbon-modified xerogels, the strength of attachment increased with increased water wettability. The aminopropyl-modified xerogels did not follow this trend.