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The development of metagenomics has opened up a new era in the study of marine microbiota, which play important roles in biogeochemical cycles. In recent years, the global ocean sampling expeditions have spurred this research field toward a deeper understanding of the microbial diversities and functions spanning various lifestyles, planktonic (free-living) or sessile (biofilm-associated). In this review, we deliver a comprehensive summary of marine microbiome datasets generated in global ocean expeditions conducted over the last 20 years, including the Sorcerer II GOS Expedition, the Tara Oceans project, the bioGEOTRACES project, the Micro B3 project, the Bio-GO-SHIP project, and the Marine Biofilms. These datasets have revealed unprecedented insights into the microscopic life in our oceans and led to the publication of world-leading research. We also note the progress of metatranscriptomics and metaproteomics, which are confined to local marine microbiota. Furthermore, approaches to transforming the global ocean microbiome datasets are highlighted, and the state-of-the-art techniques that can be combined with data analyses, which can present fresh perspectives on marine molecular ecology and microbiology, are proposed. [ABSTRACT FROM AUTHOR]

3D printing has become widely used to rapidly prototype and manufacture novel or bespoke objects or replacement components in a wide range of marine industries, engineering, and research. 3D-printed objects are subject to marine biofouling, impacting their operation and longevity. Application of antifouling paints or coatings adds costly and time-consuming steps and may interfere with the function of fine surface features, counteracting some of the benefits of 3D-printing technology. We measured the antifouling performance of two 3D-printing thermoplastics embedded with antifouling biocides to create 3D-printed materials with inherent antifouling properties: 1) polycaprolactone (PCL) mixed with the organic biocide dichlorooctylisothiazolinone (DCOIT) and extruded as 3D-printing filament, and 2) a commercial polylactic acid (PLA) 3D-printing filament with embedded copper powder. Settlement plates printed from these thermoplastics (“PCL-DCOIT” and “PLA-Cu”, respectively) and deployed in temperate, coastal marine water for 17 weeks during summer remained free of macrofouling. A biofilm developed, and 16S and 18S rRNA metabarcoding analyses revealed that early stage biofilms (at 5 and 12 weeks) had dramatically altered assemblage structures of both prokaryotes and eukaryotes compared to natural biofilms. The assemblage on PCL-DCOIT had reduced microbial diversity, strong dominance of Proteobacteria and chlorophytes, and almost complete absence of Flavobacteriia, Cyanobacteria, and diatoms. In contrast, the biofilm on PLA-Cu had a dominance of Flavobacteriia over Proteobacteria, and resistance to chlorophytes, yet similar to PCL-DCOIT it resisted Cyanobacteria and diatoms. Such alterations to biofilm microbial assemblages could influence microbial dynamics, biofilm growth, and settlement cues to which biofouler propagules respond. At 17 weeks, the two biocide-embedded thermoplastics completely resisted macrofouling, equally well as three commercial antifouling coatings (Intercept 8500, Hempaguard X7, Hempasil X3); however, PCL-DCOIT was more extensively covered by a microalgal film (79%, evidently chlorophytes) than were the commercial coatings, and PLA-Cu had the most settled detritus (100% cover). Biofilm assemblages on the commercial coatings were investigated for comparison, with PCL-DCOIT standing out due to its almost complete resistance to Flavobacteriia. Thermoplastic 3D-printing filaments with embedded biocides show promise for producing 3D-printed objects with inherent antifouling properties, avoiding or lessening the need to apply antifouling coatings, and possibly extending their service lifetime.

Marine biofilms were newly revealed as a giant microbial diversity pool for global oceans. However, the cyanobacterial diversity in marine biofilms within the upper seawater column and its ecological and evolutionary implications remains undetermined. Here, we reconstructed a full picture of modern marine cyanobacteria habitats by re-analyzing 9.3 terabyte metagenomic data sets and 2,648 metagenome-assembled genomes (MAGs). The abundances of cyanobacteria lineages exclusively detected in marine biofilms were up to ninefold higher than those in seawater at similar sample size. Analyses revealed that cyanobacteria in marine biofilms are specialists with strong geographical and environmental constraints on their genome and functional adaption, which is in stark contrast to the generalistic features of seawater-derived cyanobacteria. Molecular dating suggests that the important diversifications in biofilm-forming cyanobacteria appear to coincide with the Great Oxidation Event (GOE), "boring billion" middle Proterozoic, and the Neoproterozoic Oxidation Event (NOE). These new insights suggest that marine biofilms are large and important cyanobacterial factories for the global oceans., Importance: Cyanobacteria, highly diverse microbial organisms, play a crucial role in Earth's oxygenation and biogeochemical cycling. However, their connection to these processes remains unclear, partly due to incomplete surveys of oceanic niches. Our study uncovered significant cyanobacterial diversity in marine biofilms, showing distinct niche differentiation compared to seawater counterparts. These patterns reflect three key stages of marine cyanobacterial diversification, coinciding with major geological events in the Earth's history.

The development of metagenomics has opened up a new era in the study of marine microbiota, which play important roles in biogeochemical cycles. In recent years, the global ocean sampling expeditions have spurred this research field toward a deeper understanding of the microbial diversities and functions spanning various lifestyles, planktonic (free-living) or sessile (biofilm-associated). In this review, we deliver a comprehensive summary of marine microbiome datasets generated in global ocean expeditions conducted over the last 20 years, including the Sorcerer II GOS Expedition, the Tara Oceans project, the bioGEOTRACES project, the Micro B3 project, the Bio-GO-SHIP project, and the Marine Biofilms. These datasets have revealed unprecedented insights into the microscopic life in our oceans and led to the publication of world-leading research. We also note the progress of metatranscriptomics and metaproteomics, which are confined to local marine microbiota. Furthermore, approaches to transforming the global ocean microbiome datasets are highlighted, and the state-of-the-art techniques that can be combined with data analyses, which can present fresh perspectives on marine molecular ecology and microbiology, are proposed.

Marine biofilms are diverse microbial communities and important ecological habitats forming on surfaces submerged in the ocean. Biofilm communities resist environmental disturbance, making them a nuisance to some human activities ("biofouling"). Antifouling solutions rarely address the underlying stability or compositional responses of these biofilms. Using bulk measurements and molecular analyses, we examined temporal and UV-C antifouling-based shifts in marine biofilms in the coastal western North Atlantic Ocean during early fall. Over a 24-day period, bacterial communities shifted from early dominance of Gammaproteobacteria to increased proportions of Alphaproteobacteria, Bacteroidia, and Acidimicrobiia. In a network analysis based on temporal covariance, Rhodobacteraceae (Alphaproteobacteria) nodes were abundant and densely connected with generally positive correlations. In the eukaryotic community, persistent algal, protistan, and invertebrate groups were observed, although consistent temporal succession was not detected. Biofilm UV-C treatment at 13 and 20 days resulted in losses of chlorophyll a and transparent exopolymer particles, indicating biomass disruption. Bacterial community shifts suggested that UV-C treatment decreased the biofilm maturation rate and was associated with proportional shifts among diverse bacterial taxa. UV-C treatment was also associated with increased proportions of protists potentially involved in detritivory and parasitism. Older biofilm communities had increased resistance to UV-C, suggesting that early biofilms are more susceptible to UVC-based antifouling. The results suggest that UV-C irradiation is potentially an effective antifouling method in marine environments in terms of biomass removal and in slowing maturation. However, as they mature, biofilm communities may accumulate microbial members that are tolerant or resilient under UV treatment. [ABSTRACT FROM AUTHOR]

Microbes use signal transduction systems in the processes of swarming motility, antibiotic resistance, virulence, conjugal plasmid transfer, and biofilm formation. However, the signal transduction systems in natural marine biofilms have hardly been profiled. Here we analyzed signal transduction genes in 101 marine biofilm and 91 seawater microbial metagenomes. The abundance of almost all signal transduction-related genes in biofilm microbial communities was significantly higher than that in seawater microbial communities, regardless of substrate types, locations, and durations for biofilm development. In addition, the dominant source microbes of signal transduction genes in marine biofilms were different from those in seawater samples. Co-occurrence network analysis on signal communication between microbes in marine biofilms and seawater microbial communities revealed potential inter-phyla interactions between microorganisms from marine biofilms and seawater. Moreover, phylogenetic tree construction and protein identity comparison displayed that proteins related to signal transductions from Red Sea biofilms were highly similar to those from Red Sea seawater microbial communities, revealing a possible biological basis of interspecies interactions between surface-associated and free-living microbial communities in a local marine environment. Our study revealed the special profile and enrichment of signal transduction systems in marine biofilms and suggested that marine biofilms participate in intercellular interactions of the local ecosystem where they were seeded. [ABSTRACT FROM AUTHOR]

Microbes use signal transduction systems in the processes of swarming motility, antibiotic resistance, virulence, conjugal plasmid transfer, and biofilm formation. However, the signal transduction systems in natural marine biofilms have hardly been profiled. Here we analyzed signal transduction genes in 101 marine biofilm and 91 seawater microbial metagenomes. The abundance of almost all signal transduction-related genes in biofilm microbial communities was significantly higher than that in seawater microbial communities, regardless of substrate types, locations, and durations for biofilm development. In addition, the dominant source microbes of signal transduction genes in marine biofilms were different from those in seawater samples. Co-occurrence network analysis on signal communication between microbes in marine biofilms and seawater microbial communities revealed potential inter-phyla interactions between microorganisms from marine biofilms and seawater. Moreover, phylogenetic tree construction and protein identity comparison displayed that proteins related to signal transductions from Red Sea biofilms were highly similar to those from Red Sea seawater microbial communities, revealing a possible biological basis of interspecies interactions between surface-associated and free-living microbial communities in a local marine environment. Our study revealed the special profile and enrichment of signal transduction systems in marine biofilms and suggested that marine biofilms participate in intercellular interactions of the local ecosystem where they were seeded.

Microbes respond to environmental stimuli through complicated signal transduction systems. In microbial biofilms, because of complex multiple species interactions, signals transduction systems are of an even higher complexity. Here, we performed a signal-molecule-treatment experiment to study the role of different signal molecules, including N-hexanoyl-L-homoserine lactone (C6-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), Pseudomonas quinolone signal (PQS), and cyclic di-GMP (c-di-GMP), in the development of marine biofilms. Comparative metagenomics suggested a distinctive influence of these molecules on the microbial structure and function of multi-species biofilm communities in its developing stage. The PQS-treated biofilms shared the least similarity with the control and initial biofilms. The role of PQS in biofilm development was further explored experimentally with the strain Erythrobacter sp. HKB8 isolated from marine biofilms. Comparative transcriptomic analysis showed that 314 genes, such as those related to signal transduction and biofilm formation, were differentially expressed in the untreated and PQS-treated Erythrobacter sp. HKB8 biofilms. Our study demonstrated the different roles of signal molecules in marine biofilm development. In particular, the PQS-based signal transduction system, which is frequently detected in marine biofilms, may play an important role in regulating microbe-microbe interactions and the assemblage of biofilm communities.

Marine biofilms are known to influence the corrosion of metal surfaces in the marine environment. Despite some recent research, the succession of bacterial communities colonizing artificial surfaces remains uncharacterized in some temporal settings. More specifically, it is not fully known if bacterial colonizers of artificial surfaces are similar or distinct in the different seasons of the year. In particular the study of early biofilms, in which the bacterial cells communities first adhere to artificial surfaces, are crucial for the development of the subsequent biofilm communities. In this work, we used amplicon-based NGS (next-generation sequencing) and universal 16S rRNA bacterial primers to characterize the early biofilm bacterial communities growing on 316 L stainless steel surfaces in a Northern Portugal port. Sampling spanned 30-day periods in two distinct seasons (spring and winter). Biofilm communities growing in steel surfaces covered with an anti-corrosion paint and planktonic communities from the same location were also characterized. Our results demonstrated that distinct temporal patterns were observed in the sampled seasons. Specifically, a significantly higher abundance of Gammaproteobacteria and Mollicutes was found on the first days of biofilm growth in spring (day 1 to day 4) and a higher abundance of Alphaproteobacteria during the same days of biofilm growth in winter. In the last sampled day (day 30), the spring biofilms significantly shifted toward a dominance of photoautotrophic groups (mostly diatoms) and were also colonized by some macrofouling communities, something not observed during the winter sampling. Our results revealed that bacterial composition in the biofilms was particularly affected by the sampled day of the specific season, more so than the overall effect of the season or overall sampling day of both seasons. Additionally, the application of a non-fouling-release anti-corrosion paint in the steel plates resulted in a significantly lower diversity compared with plates without paint, but this was only observed during spring. We suggest that temporal succession of marine biofilm communities should be taken in consideration for future antifouling/anti-biofilm applications.

Microbes respond to environmental stimuli through complicated signal transduction systems. In microbial biofilms, because of complex multiple species interactions, signals transduction systems are of an even higher complexity. Here, we performed a signal-molecule-treatment experiment to study the role of different signal molecules, including N-hexanoyl-L-homoserine lactone (C6-HSL), N-dodecanoyl-L-homoserine lactone (C12-HSL), Pseudomonas quinolone signal (PQS), and cyclic di-GMP (c-di-GMP), in the development of marine biofilms. Comparative metagenomics suggested a distinctive influence of these molecules on the microbial structure and function of multi-species biofilm communities in its developing stage. The PQS-treated biofilms shared the least similarity with the control and initial biofilms. The role of PQS in biofilm development was further explored experimentally with the strain Erythrobacter sp. HKB8 isolated from marine biofilms. Comparative transcriptomic analysis showed that 314 genes, such as those related to signal transduction and biofilm formation, were differentially expressed in the untreated and PQS-treated Erythrobacter sp. HKB8 biofilms. Our study demonstrated the different roles of signal molecules in marine biofilm development. In particular, the PQS-based signal transduction system, which is frequently detected in marine biofilms, may play an important role in regulating microbe-microbe interactions and the assemblage of biofilm communities. [ABSTRACT FROM AUTHOR]

Marine biofilms are known to influence the corrosion of metal surfaces in the marine environment. Despite some recent research, the succession of bacterial communities colonizing artificial surfaces remains uncharacterized in some temporal settings. More specifically, it is not fully known if bacterial colonizers of artificial surfaces are similar or distinct in the different seasons of the year. In particular the study of early biofilms, in which the bacterial cells communities first adhere to artificial surfaces, are crucial for the development of the subsequent biofilm communities. In this work, we used amplicon-based NGS (next-generation sequencing) and universal 16S rRNA bacterial primers to characterize the early biofilm bacterial communities growing on 316 L stainless steel surfaces in a Northern Portugal port. Sampling spanned 30-day periods in two distinct seasons (spring and winter). Biofilm communities growing in steel surfaces covered with an anti-corrosion paint and planktonic communities from the same location were also characterized. Our results demonstrated that distinct temporal patterns were observed in the sampled seasons. Specifically, a significantly higher abundance of Gammaproteobacteria and Mollicutes was found on the first days of biofilm growth in spring (day 1 to day 4) and a higher abundance of Alphaproteobacteria during the same days of biofilm growth in winter. In the last sampled day (day 30), the spring biofilms significantly shifted toward a dominance of photoautotrophic groups (mostly diatoms) and were also colonized by some macrofouling communities, something not observed during the winter sampling. Our results revealed that bacterial composition in the biofilms was particularly affected by the sampled day of the specific season, more so than the overall effect of the season or overall sampling day of both seasons. Additionally, the application of a non-fouling-release anti-corrosion paint in the steel plates resulted in a significantly lower diversity compared with plates without paint, but this was only observed during spring. We suggest that temporal succession of marine biofilm communities should be taken in consideration for future antifouling/anti-biofilm applications. [ABSTRACT FROM AUTHOR]

Planctomycetes are bacteria that were long thought to be unculturable, of low abundance, and therefore neglectable in the environment. This view changed in recent years, after it was shown that members of the phylum Planctomycetes can be abundant in many aquatic environments, e.g., in the epiphytic communities on macroalgae surfaces. Here, we analyzed three different macroalgae from the North Sea and show that Planctomycetes is the most abundant bacterial phylum on the alga Fucus sp., while it represents a minor fraction of the surface-associated bacterial community of Ulva sp. and Laminaria sp. Especially dominant within the phylum Planctomycetes were Blastopirellula sp., followed by Rhodopirellula sp., Rubripirellula sp., as well as other Pirellulaceae and Lacipirellulaceae, but also members of the OM190 lineage. Motivated by the observed abundance, we isolated four novel planctomycetal strains to expand the collection of species available as axenic cultures since access to different strains is a prerequisite to investigate the success of planctomycetes in marine environments. The isolated strains constitute four novel species belonging to one novel and three previously described genera in the order Pirellulales, class Planctomycetia, phylum Planctomycetes.

The cyclic peptides portoamides produced by the cyanobacterium Phormidium sp. LEGE 05292 were previously isolated and their ability to condition microcommunities by allelopathic effect was described. These interesting bioactive properties are, however, still underexplored as their biotechnological applications may be vast. This study aims to investigate the antifouling potential of portoamides, given that a challenge in the search for new environmentally friendly antifouling products is to find non-toxic natural alternatives with the ability to prevent colonization of different biofouling species, from bacteria to macroinvertebrates. A multi-bioassay approach was applied to assess portoamides antifouling properties, marine ecotoxicity and molecular mode of action. Results showed high effectiveness in the prevention of mussel larvae settlement (EC50 = 3.16 µM), and also bioactivity towards growth and biofilm disruption of marine biofouling bacterial strains, while not showing toxicity towards both target and non-target species. Antifouling molecular targets in mussel larvae include energy metabolism modifications (failure in proton-transporting ATPases activity), structural alterations of the gills and protein and gene regulatory mechanisms. Overall, portoamides reveal a broad-spectrum bioactivity towards diverse biofouling species, including a non-toxic and reversible effect towards mussel larvae, showing potential to be incorporated as an active ingredient in antifouling coatings.

Temperature variability is a major driver of ecological pattern, with recent changes in average and extreme temperatures having significant impacts on populations, communities and ecosystems. In the marine realm, very few experiments have manipulated temperature in situ, and current understanding of temperature effects on community dynamics is limited. We developed new technology for precise seawater temperature control to examine warming effects on communities of bacteria, microbial eukaryotes (protists) and metazoans. Despite highly contrasting phylogenies, size spectra and diversity levels, the three community types responded similarly to seawater warming treatments of +3°C and +5°C, highlighting the critical and overarching importance of temperature in structuring communities. Temperature effects were detectable at coarse taxonomic resolutions and many taxa responded positively to warming, leading to increased abundances at the community-level. Novel field-based experimental approaches are essential to improve mechanistic understanding of how ocean warming will alter the structure and functioning of diverse marine communities. [ABSTRACT FROM AUTHOR]

Periphyton communities are complex phototrophic, multispecies biofilms that develop on surfaces in aquatic environments. These communities harbor a large diversity of organisms comprising viruses, bacteria, algae, fungi, protozoans, and metazoans. However, thus far the total biodiversity of periphyton has not been described. In this study, we use metagenomics to characterize periphyton communities from the marine environment of the Swedish west coast. Although we found approximately ten times more eukaryotic rRNA marker gene sequences compared to prokaryotic, the whole metagenome-based similarity searches showed that bacteria constitute the most abundant phyla in these biofilms. We show that marine periphyton encompass a range of heterotrophic and phototrophic organisms. Heterotrophic bacteria, including the majority of proteobacterial clades and Bacteroidetes, and eukaryotic macroinvertebrates were found to dominate periphyton. The phototrophic groups comprise Cyanobacteria and the alpha-proteobacterial genus Roseobacter, followed by different micro- and macro-algae. We also assess the metabolic pathways that predispose these communities to an attached lifestyle. Functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances, mainly in the form of complex sugars such as starch and glycogen-like meshes together with chitin. Genes for 278 different transporter proteins were detected in the metagenome, constituting the most abundant protein complexes. Finally, genes encoding enzymes that participate in anaerobic pathways, such as denitrification and methanogenesis, were detected suggesting the presence of anaerobic or low-oxygen micro-zones within the biofilms. [ABSTRACT FROM AUTHOR]

Biofilm formation is the initial step of the complex marine biofouling process, limiting the performance of submerged surfaces in numerous applications. No technology is known up to now capable to stop the marine biofilm development even on biocidal paints coated surfaces. Several non-toxic approaches for control over marine biofilms based on physical, chemical or/and biological methods, most of them inspired by the nature, are currently on search. Surface patterning, enzyme alternatives of toxic biocides and nature derived anti-biofilm agents (natural biocides, bio-surfactants/dispersants and quorum sensing inhibitors) are in the scope of this review. [ABSTRACT FROM AUTHOR]

• Nutrient amendment of natural waters for evaluating of multispecies biofilms control. • Biofilm growth was more pronounced in seawater than freshwater. • [C 12 MIM][Cl] and [C 16 MIM][Cl] retarded growth of multispecies biofilms in nutrient amended natural waters. • [C 16 MIM][Cl] showed biofilm dispersal action on pre-formed multispecies biofilms. • [C 16 MIM][Cl] prevented biofilm formation in a re-circulating flow-cell reactor. Biofilm associated problems and inherent resistance of biofilm-bacteria to antimicrobial agents drives the need for alternate agents with potent antibiofilm activities. Alkylimidazolium ionic liquids were reported as novel antimicrobial and antibiofilm agents for preventing single species biofilms. However, naturally occurring biofilms are multispecies in nature constituting diverse organisms and have high prevalence, significance in medical, industrial and ecological perspective. In this study, the effect of three alkylimidazolium ionic liquids ([CnMIM]+[X]−, n = 4, 12 and 16) were evaluated against formation of natural and multispecies biofilms in freshwater and seawater. In 96-well biofilm assays, [C 4 MIM][Cl] with –butyl alkyl group did not exert antibiofilm activity even at 500 µM. [C 12 MIM][I] with –dodecyl alkyl group showed antibiofilm activity at 32 and 250 µM, in natural freshwater and seawater, respectively. Among the tested ionic liquids, [C 16 MIM][Cl] with –hexadecyl group showed superior antibiofilm activity. Presence of [C 16 MIM][Cl] in freshwater and seawater, respectively, at 8 and 125 µM caused complete inhibition in biofilm formation. Among the tested compounds, only [C 16 MIM][Cl] showed biofilm dispersal activity on pre-formed biofilms. Biofilm dispersal was near complete at 125 and 250 µM for freshwater and seawater biofilms, respectively. Potent antimicrobial ionic liquid ([C 16 MIM][Cl]) was further evaluated for biofilm prevention under dynamic conditions in a laboratory scale recirculation system. Over a period of 12 d of operation, biofilm formation was retarded in the presence of 100 μM and 250 μM of [C 16 MIM][Cl], in freshwater and seawater, respectively. The results suggest that antimicrobial [C 16 MIM][Cl] may find potential application for preventing multispecies biofilms in cooling water systems. [ABSTRACT FROM AUTHOR]

Planctomycetes are bacteria that were long thought to be unculturable, of low abundance, and therefore neglectable in the environment. This view changed in recent years, after it was shown that members of the phylum Planctomycetes can be abundant in many aquatic environments, e.g., in the epiphytic communities on macroalgae surfaces. Here, we analyzed three different macroalgae from the North Sea and show that Planctomycetes is the most abundant bacterial phylum on the alga Fucus sp., while it represents a minor fraction of the surface-associated bacterial community of Ulva sp. and Laminaria sp. Especially dominant within the phylum Planctomycetes were Blastopirellula sp., followed by Rhodopirellula sp., Rubripirellula sp., as well as other Pirellulaceae and Lacipirellulaceae , but also members of the OM190 lineage. Motivated by the observed abundance, we isolated four novel planctomycetal strains to expand the collection of species available as axenic cultures since access to different strains is a prerequisite to investigate the success of planctomycetes in marine environments. The isolated strains constitute four novel species belonging to one novel and three previously described genera in the order Pirellulales , class Planctomycetia , phylum Planctomycetes .

Settlement of pelagic cypris larvae is crucial for barnacle biofouling which causes severe operational problems and economic penalties in maritime activities and seawater cooled power plants. Marine biofilms formed on submerged surfaces play a key role in settlement of larvae of barnacles and a promising source for environmentally benign antifouling strategies. To determine inhibitory action of biofilms, bacterial strains were isolated from natural biofilms, screened for their biofilm formation potential and interference in settlement of cyrpis larvae of Amphibalanus reticulatus. Monoculture biofilms of three bacterial strains i.e. Alteromonas sp. 1, Alteromonas sp. 2 and Bacillus cereus inhibited cypris settlement by ~67%–93% and 67%–78% in choice and no choice experiments, respectively. Biofilms of two bacterial strains (B. pumilus and Halomonas aquamarina) had no significant effect and one strain (Brevibacterium casei) showed marginal induction in cypris settlement. In case of inhibitory biofilms, the inhibitory action on cypris could be ascertained to the loosely bound extracellular polymeric substances (LB EPS). The effects of LB EPS included settlement inhibition, larval mortality, altered larval behaviour and interrupted moulting. Crude fractionation and testing of individual LB EPS fractions showed that the inhibitory activity was associated with the carbohydrate components. This study shows the importance of further studies on probing marine biofilm-larval interactions, identification and characterization of inhibitory compounds for developing newer environmentally benign antifouling strategies. Image 1 • Monospecies bacterial biofilms showed inhibitory effect or no effect on barnacle larvae. • Alteromonas and Bacillus biofilms prevented settlement of barnacle larvae. • Loosely bound extracellular polymeric substances (LB EPS) of biofilms inhibited barnacle larval settlement. • Extracted LB EPS induced mortality and precocious metamorphosis in barnacle larvae. • The inhibitory activity of LB EPS was assigned to carbohydrate components. [ABSTRACT FROM AUTHOR]

The cyclic peptides portoamides produced by the cyanobacterium Phormidium sp. LEGE 05292 were previously isolated and their ability to condition microcommunities by allelopathic effect was described. These interesting bioactive properties are, however, still underexplored as their biotechnological applications may be vast. This study aims to investigate the antifouling potential of portoamides, given that a challenge in the search for new environmentally friendly antifouling products is to find non-toxic natural alternatives with the ability to prevent colonization of different biofouling species, from bacteria to macroinvertebrates. A multi-bioassay approach was applied to assess portoamides antifouling properties, marine ecotoxicity and molecular mode of action. Results showed high effectiveness in the prevention of mussel larvae settlement (EC50 = 3.16 µM), and also bioactivity towards growth and biofilm disruption of marine biofouling bacterial strains, while not showing toxicity towards both target and non-target species. Antifouling molecular targets in mussel larvae include energy metabolism modifications (failure in proton-transporting ATPases activity), structural alterations of the gills and protein and gene regulatory mechanisms. Overall, portoamides reveal a broad-spectrum bioactivity towards diverse biofouling species, including a non-toxic and reversible effect towards mussel larvae, showing potential to be incorporated as an active ingredient in antifouling coatings. [ABSTRACT FROM AUTHOR]