Copper affects biofilm inductiveness to larval settlement of the serpulid polychaete Hydroides elegans (Haswell)

Copper (Cu) contamination is a potential threat to the marine environment due to the use of Cu-based antifouling paints. Cu stress on larval settlement of the polychaete Hydroides elegans was investigated, and this was linked to Cu stress on biofilms and on the biofilm development process. The inductiveness of young biofilms was more easily altered by Cu stress than that of old biofilms, indicating the relative vulnerability of young biofilms. This might result from changes in bacterial survival, the bacterial community composition and the chemical profiles of young biofilms. Cu also affected biofilm development and the chemical high performance liquid chromatograph fingerprint profile. The results indicate that Cu affected larval settlement mainly through its effect on the process of biofilm development in the marine environment, and the chemical profile was crucial to biofilm inductiveness. It is strongly recommended that the effects of environmentally toxic substances on biofilms are evaluated in ecotoxicity bioassays using larval settlement of invertebrates as the end point.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

The effect of carbon nanotubes and titanium dioxide incorporated in PDMS on biofilm community composition and subsequent mussel plantigrade settlement

This study investigated the effect of carbon nanotubes (CNTs) and titanium dioxide (TiO₂) incorporated in PDMS on biofilm formation and plantigrade settlement of Mytilus coruscus. TiO₂ increased bacterial density, and CNTs also increased bacterial density but reduced diatom density in biofilms after 28 days. Further analysis was conducted between bacterial communities on glass, PDMS, CNTs (0.5 wt%) and TiO₂ (7.5 wt%). ANOSIM analysis revealed significant differences (R > 0.9) between seven, 14, 21 and 28 day-old bacterial communities. MiSeq sequencing showed that CNTs and TiO₂ impacted the composition of 28 day-old bacterial communities by increasing the abundance of Proteobacteria and decreasing the abundance of Bacteroidetes. The maximum decreased settlement rate in 28 day-old biofilms on CNTs and TiO₂ was > 50% in comparison to those on glass and PDMS. Thus, CNTs and TiO₂ incorporated in PDMS altered the biomass and community composition of biofilms, and subsequently decreased mussel settlement.     

Effects of different biotic substrata on mussel attachment

Surface colonization by invertebrates can be stimulated or inhibited by cues produced by biofilms, conspecifics or other macroorganisms. To study the effects of living substrata on the attachment of the brown mussel, Perna perna, two different approaches were employed: (1) mussels were distributed in sets of Petri dishes consisting of one sterile set (controls), three sets in which marine biofilms were allowed to develop in aquaria for 1, 7 or 15 days and another set that had been immersed in a natural marine environment for 1-day. There was no significant effect of biofilms on attachment, suggesting that neither age nor the source of the biofilm influenced attachment. (2) Mussels were suspended over PVC panels (controls) and over panels on which Balanus trigonus (Crustacea), Schizoporella errata (Bryozoa), Symplegma rubra or Didemnum speciosum (Ascidiacea) were present. Attachment was significantly higher on the controls and on B. trigonus than on colonial taxa such as S. rubra, S. errata and D. speciosum, probably due to antifouling defenses of these species. The results show that the composition of the biological substratum is an important factor affecting mussel behavior.     

Characterization of Pseudomonas aeruginosa fatty acid profiles in biofilms and batch planktonic cultures

The fatty acid composition of Pseudomonas aeruginosa PAO1 was compared between biofilm and batch planktonic cultures. Strain PAO1 biofilms were able to maintain a consistent fatty acid profile for up to 6 days, whereas strain PAO1 batch planktonic cultures showed a gradual loss of cis-monounsaturated fatty acids over 4 days. Biofilms exhibited a greater proportion of hydroxy fatty acids but a lower proportion of both cyclopropane fatty acids and saturated fatty acids (SAFAs). SAFAs with >=16 carbons, in particular, decreased in biofilms when compared with that in batch planktonic cultures. A reduced proportion of SAFAs and a decline in overall fatty acid chain length indicate more fluidic biophysical properties for cell membranes of P. aeruginosa in biofilms. Separating the biofilms into 2 partitions and comparing their fatty acid compositions revealed additional trends that were not observed in the whole biofilm: the shear-nonremovable layer consistently showed greater proportions of hydroxy fatty acid than the bulk liquid + shear-removable portion of the biofilm. The shear-nonremovable portion demonstrated a relatively immediate decline in the proportion of monounsaturated fatty acids between days 2 and 4; which was offset by an increase in the proportion of cyclopropane fatty acids, specifically 19:0cyc(11,12). Simultaneously, the shear-removable portion of the biofilm showed an increase in the proportion of trans-monounsaturated fatty acids and cyclopropane fatty acids.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Larval settlement of the common Australian sea urchin Heliocidaris erythrogramma in response to bacteria from the surface of coralline algae

Bacterial biofilms are increasingly seen as important for the successful settlement of marine invertebrate larvae. Here we tested the effects of biofilms on settlement of the sea urchin Heliocidaris erythrogramma. Larvae settled on many surfaces including various algal species, rocks, sand and shells. Settlement was reduced by autoclaving rocks and algae, and by treatment of algae with antibiotics. These results, and molecular and culture-based analyses, suggested that the bacterial community on plants was important for settlement. To test this, approximately 250 strains of bacteria were isolated from coralline algae, and larvae were exposed to single-strain biofilms. Many induced rates of settlement comparable to coralline algae. The genus Pseudoalteromonas dominated these highly inductive strains, with representatives from Vibrio, Shewanella, Photobacterium and Pseudomonas also responsible for a high settlement response. The settlement response to different bacteria was species specific, as low inducers were also dominated by species in the genera Pseudoalteromonas and Vibrio. We also, for the first time, assessed settlement of larvae in response to characterised, monospecific biofilms in the field. Larvae metamorphosed in higher numbers on an inducing biofilm, Pseudoalteromonas luteoviolacea, than on either a low-inducing biofilm, Pseudoalteromonas rubra, or an unfilmed control. We conclude that the bacterial community on the surface of coralline algae is important as a settlement cue for H. erythrogramma larvae. This study is also an example of the emerging integration of molecular microbiology and more traditional marine eukaryote ecology.     

Biofilm history and oxygen availability interact to affect habitat selection in a marine invertebrate

In marine systems, oxygen availability varies at small temporal and spatial scales, such that current oxygen levels may not reflect conditions of the past. Different studies have shown that marine invertebrate larvae can select settlement sites based on local oxygen levels and oxygenation history of the biofilm, but no study has examined the interaction of both. The influence of normoxic and hypoxic water and oxygenation history of biofilms on pre-settlement behavior and settlement of the bryozoan Bugula neritina was tested. Larvae used cues in a hierarchical way: the oxygen levels in the water prime larvae to respond, the response to different biofilms is contingent on oxygen levels in the water. When oxygen levels varied throughout biofilm formation, larvae responded differently depending on the history of the biofilm. It appears that B. neritina larvae integrate cues about current and historical oxygen levels to select the appropriate microhabitat and maximize their fitness.     

Marine Biofilms as Mediators of Colonization by Marine Macroorganisms: Implications for Antifouling and Aquaculture

In the marine environment, biofilms on submerged surfaces can promote or discourage the settlement of invertebrate larvae and macroalgal spores. The settlement-mediating effects of biofilms are believed to involve a variety of biofilm attributes including surface chemistry, micro-topography, and a wide range of microbial products from small-molecule metabolites to high-molecular weight extracellular polymers. The settled organisms in turn can modify microbial species composition of biofilms and thus change the biofilm properties and dynamics. A better understanding of biofilm dynamics and chemical signals released and/or stored by biofilms will facilitate the development of antifouling and mariculture technologies. This review provides a brief account of 1) existing knowledge of marine biofilms that are relevant to settlement mediation, 2) biotechnological application of biofilms with respect to developing non-toxic antifouling technologies and improving the operation of aquaculture facilities, and 3) challenges and future directions for advancing our understanding of settlement-mediating functions of biofilms and for applying this knowledge to real-life situations.     

Amino acid composition of epilithic biofilm and benthic animals in a large Siberian river

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A bacterial polysaccharide biosynthesis-related gene inversely regulates larval settlement and metamorphosis of Mytilus coruscus

Abstract

Larval settlement and metamorphosis is essential for the development of marine invertebrates. Although polysaccharides are involved in larval settlement and metamorphosis of Mytilus coruscus, the molecular basis of polysaccharides underlying this progression remains largely unknown. Here, the roles of the polysaccharide biosynthesis-related gene 01912 of Pseudoalteromonas marina ECSMB14103 in the regulation of larval settlement and metamorphosis were examined by gene-knockout technique. Compared with biofilms (BFs) of the wild-type P. marina, Δ01912 BFs with a higher colanic acid (CA) content showed a higher inducing activity on larval settlement and metamorphosis. Deletion of the 01912 gene caused an increase in c-di-GMP levels, accompanied by a decrease in the motility, an increase in cell aggregation, and overproduction of CA. Thus, the bacterial polysaccharide biosynthesis-related gene 01912 may regulate mussel settlement by producing CA via the coordination of c-di-GMP. This work provides a deeper insight into the molecular mechanism of polysaccharides in modulating mussel settlement.     

Metabolic dynamics of Desulfovibrio vulgaris biofilm grown on a steel surface

In this study, a comparative metabolomics approach combining gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS) was applied first between planktonic cells and biofilms and then between pure cultures and biofilms of Desulfovibrio vulgaris. The results revealed that the overall metabolic level of the biofilm cells was down-regulated, especially for metabolites related to the central carbon metabolism, compared to the planktonic cells and the pure culture of D. vulgaris. In addition, pathway enrichment analysis of the 58 metabolites identified by GC-MS showed that fatty acid biosynthesis in the biofilm cells was up-regulated, suggesting that fatty acids may be important for the formation, maintenance and function of D. vulgaris biofilm. This study offers a valuable perspective on the metabolic dynamics of the D. vulgaris biofilm.     

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.     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

Role of polymer complexes in the formation of biofilms by corrosive bacteria on steel surfaces

The composition of exopolymer complexes (EPCs), synthesized by the monocultures Desulfovibrio sp. 10, Bacillus subtilis 36, and Pseudomonas aeruginosa 27 and by microbial associations involved in the corrosion of metal surfaces has been studied. An analysis of the monosaccharide composition of carbohydrate components, as well as the fatty acid composition of the lipid part of EPCs, was carried out by gasliquid chromatography (GLC). It was found that bacteria in biofilms synthesized polymers; this process was dominated by glucose, while the growth of bacteria in a suspension was marked by a high rhamnose content. Hexouronic acids and hexosamine have been revealed as a part of B. subtilis 36 and P. aeruginosa 27 EPCs. Qualitative differences were revealed in the fatty acid composition of exopolymers in biofilms and in a bacterial suspension. It was shown that the transition to a biofilm form of growth led to an increase in the unsaturation degree of fatty acids in the exopolymers of associative cultures. The results can be used to develop methods to control microbial corrosion of metal surfaces.     

Effect of light and temperature on biomass, photosynthesis and capsular polysaccharides in cultured phototrophic biofilms

Phototrophic biofilms seem to be suitable candidates for tertiary wastewater treatment due to their high uptake capacity for nutrients and other pollutants, also taking into account the time and cost savings derived from easy procedures for biomass harvesting. Biomass accrual, structure, and physiology of biofilms affect the efficiency of nutrient removal by its microbial community. Here, we construct a biofilm consisting of a cyanobacterium Synechocystis sp. and the green alga Chlorococcum sp. and determine the effect of combined variations of irradiance and temperature on the biofilm structure and function. The two species were isolated from phototrophic biofilms naturally developing in an Italian wastewater treatment plant and grown in a microcosm designed for biofilm investigations. Phototrophic biomass accumulation, percent species composition, photosynthetic response and the amount and composition of capsular polysaccharides (CPS), including anionic residues, are reported. The results showed that biofilm development required relatively moderate irradiances (60 μmol photons m⁻² s⁻¹) below which development was arrested. Both light and temperature had a strong effect on the composition of each species to the biofilm. The CPS compositions also changed with temperature, light and species composition. The CPS of the green-algal-dominated biofilm had the higher uronic acid content indicating a potential to exploit green algae in the treatment of waste contaminated with heavy metals. Given the knowledge of the response of certain species to light and temperature combinations, it may be possible to construct biofilms of known species and CPS composition to use them for specific applications.     

A three-species biofilm model for the evaluation of enamel and dentin demineralization

Although Streptococcus mutans biofilms have been useful for evaluating the cariogenic potential of dietary carbohydrates and the effects of fluoride on dental demineralization, a more appropriate biofilm should be developed to demonstrate the influence of other oral bacteria on cariogenic biofilms. This study describes the development and validation of a three-species biofilm model comprising Streptococcus mutans, Actinomyces naeslundii, and Streptococcus gordonii for the evaluation of enamel and dentin demineralization after cariogenic challenges and fluoride exposure. Single- or three-species biofilms were developed on dental substrata for 96?h, and biofilms were exposed to feast and famine episodes. The three-species biofilm model produced a large biomass, mostly comprising S. mutans (41%) and S. gordonii (44%), and produced significant demineralization in the dental substrata, although enamel demineralization was decreased by fluoride treatment. The findings indicate that the three-species biofilm model may be useful for evaluating the cariogenic potential of dietary carbohydrates other than sucrose and determining the effects of fluoride on dental substrata.     

Environmental Dissolved Organic Matter Governs Biofilm Formation and Subsequent Linuron Degradation Activity of a Linuron-Degrading Bacterial Consortium

It was examined whether biofilm growth on dissolved organic matter (DOM) of a three-species consortium whose members synergistically degrade the phenylurea herbicide linuron affected the consortium''s integrity and subsequent linuron-degrading functionality. Citrate as a model DOM and three environmental DOM (eDOM) formulations of different quality were used. Biofilms developed with all DOM formulations, and the three species were retained in the biofilm. However, biofilm biomass, species composition, architecture, and colocalization of member strains depended on DOM and its biodegradability. To assess the linuron-degrading functionality, biofilms were subsequently irrigated with linuron at 10 mg liter⁻¹ or 100 μg liter⁻¹. Instant linuron degradation, the time needed to attain maximal linuron degradation, and hence the total amount of linuron removed depended on both the DOM used for growth and the linuron concentration. At 10 mg liter⁻¹, the final linuron degradation efficiency was as high as previously observed without DOM except for biofilms fed with humic acids which did not degrade linuron. At 100 μg liter⁻¹ linuron, DOM-grown biofilms degraded linuron less efficiently than biofilms receiving 10 mg liter⁻¹ linuron. The amount of linuron removed was more correlated with biofilm species composition than with biomass or structure. Based on visual observations, colocalization of consortium members in biofilms after the DOM feed appears essential for instant linuron-degrading activity and might explain the differences in overall linuron degradation. The data show that DOM quality determines biofilm structure and composition of the pesticide-degrading consortium in periods with DOM as the main carbon source and can affect subsequent pesticide-degrading activity, especially at micropollutant concentrations.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

Settlement of Pomatoceros lamarkii (Serpulidae) larvae on biofilmed surfaces and the effect of aerial drying

The settlement responses of Pomatoceros lamarkii (Polychaeta: Serpulidae) larvae to biofilms of varying age on slate surfaces and to dried biofilms on slate surfaces were investigated in the laboratory. Settlement experiments were performed as multi-treatment, still water assays. Larvae did not settle on clean, non-biofilmed slates but settled on biofilms up to 28 days old. Settlement intensity was closely related to the bacterial density of a biofilm. Drying a biofilmed surface for 1-2 h at 20 degrees C to simulate a single tidal emersion completely negated the former inductive effect of the biofilm. Drying also negated the larval settlement-inducing effect that normally results from the presence of conspecific adults. The settlement inhibition lasted for approximately 5 days following a single drying event. Larvae settled readily on biofilms exposed to formalin and antibiotics. Treating biofilms with formalin or antibiotics before or after drying had no effect upon larval avoidance of dried biofilms. Freeze-drying a biofilm had the same effect as aerial drying. The biofilm drying effect could not be mimicked by exposing biofilms to hyper-saline seawater. The finding that P. lamarkii larvae do not settle on dried biofilms could have significance in explaining the natural distribution of this species in the intertidal.