Climate change impacts on the abiotic degradation of acyl-homoserine lactones in the fluctuating conditions of marine biofilms

Marine biofilms are functional communities that shape habitats by providing a range of structural and functional services integral to coastal ecosystems. Impacts of climate change on biological aspects of such communities are increasingly studied, but impacts on the chemicals that mediate key interactions of biofilm organisms have largely been overlooked. Acyl-homoserine lactones (AHLs), crucial bacterial signals within biofilms, are known to degrade through pH and temperature-dependent hydrolysis. However, the impact of climate change on AHLs and thus on biofilm form and function is presently unknown. This study investigates the impact of changes in pH and temperature on the hydrolysis rate, half-life time and quantitative abundance of different AHLs on daily and seasonal timescales for current conditions and future climate change scenarios. We established the mathematical relationships between pH, hydrolysis rates/half-life times and temperature, which revealed that natural daily pH-driven changes within biofilms cause the greatest fluctuations in AHL concentration (up to 9-fold). Season-dependant temperature enhanced or reduced the observed daily dynamics, leading to higher winter and lower summer concentrations and caused a shift in timing of the highest and lowest AHL concentration by up to two hours. Simulated future conditions based on climate change projections caused an overall reduction of AHL degradation and led to higher AHL concentrations persisting for longer across both the daily and seasonal cycles. This study provides valuable quantitative insights into the theoretical natural dynamics of AHL concentrations. We highlight critical knowledge gaps on the scale of abiotic daily and seasonal fluctuations affecting estuarine and coastal biofilms and on the biofilms' buffering capacity. Detailed experimental studies of daily and seasonal dynamics of AHL concentrations and assessment of the potential implications for a suite of more complex interactions are required. Substantial fluctuations like those we show in this study, particularly with regards to concentration and timing, will likely have far reaching implications for fundamental ecosystem processes and important ecosystem services such as larval settlement and coastal sediment stabilisation.

Profiling Signal Transduction in Global Marine Biofilms

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.

Impacts of UV-C irradiation on marine biofilm community succession

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’). Anti-fouling 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-d 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 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 UV-C 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. Importance Marine biofilms regulate processes from organic matter and pollutant turnover to eukaryotic settlement and growth. Biofilm growth and eukaryotic settlement interfering with human activities via growth on ship hulls, aquaculture operations, or other marine infrastructure are called ‘biofouling’. There is a need to develop sustainable anti-fouling techniques by minimizing impacts to surrounding biota. We use the biofouling-antifouling framework to test hypotheses about marine biofilm succession and stability in response to disturbance, using a novel UV-C LED device. We demonstrate strong bacterial biofilm successional patterns and detect taxa potentially contributing to stability under UV-C stress. Despite UV-C-associated biomass losses and varying UV susceptibility of microbial taxa, we detected high compositional resistance among biofilm bacterial communities, suggesting decoupling of disruption in biomass and community composition following UV-C irradiation. We also report microbial covariance patterns over 24 days of biofilm growth, pointing to areas for study of microbial interactions and targeted antifouling.

Coral reef biofilm bacterial diversity and successional trajectories are structured by reef benthic organisms and shift under chronic nutrient enrichment

Work on marine biofilms has primarily focused on host-associated habitats for their roles in larval recruitment and disease dynamics; little is known about the factors regulating the composition of reef environmental biofilms. To contrast the roles of succession, benthic communities and nutrients in structuring marine biofilms, we surveyed bacteria communities in biofilms through a six-week succession in aquaria containing macroalgae, coral, or reef sand factorially crossed with three levels of continuous nutrient enrichment. Our findings demonstrate how biofilm successional trajectories diverge from temporal dynamics of the bacterioplankton and how biofilms are structured by the surrounding benthic organisms and nutrient enrichment. We identify a suite of biofilm-associated bacteria linked with the orthogonal influences of corals, algae and nutrients and distinct from the overlying water. Our results provide a comprehensive characterization of marine biofilm successional dynamics and contextualize the impact of widespread changes in reef community composition and nutrient pollution on biofilm community structure.

Characterization of marine eukaryotic biofilms at offshore wind farm sites: assessment of DNA extraction methods and marker gene used for metabarcoding approaches

Among marine lifestyles, biofilms are considered as diversified communities embedded in complex exopolymers whose development depends on several factors, related to both environmental conditions and physical-chemical characteristics of substrates (Antunes et al. 2019, Bellou et al. 2012). For the maritime industry, bio-colonization and its impact on human activities were well-described (Schultz et al. 2011). However, this phenomenon represents a new challenge in Renewable Marine Energies (RME) due to their specificities (materials, structures, localization…). In particular, macro-organism assemblages appeared to include a wide variety of eukaryotic groups but the literature is sparse considering the sequencing of eukaryotic diversity in comparison to those of bacterial communities (Briand et al. 2018, Dang and Lovell 2000, Salta et al. 2013). As a matter of fact, the very small size of some of the eukaryotes and/or their insufficient morphological discernible features appear to considerably limit their detection and identification, leading to underestimate their diversity (Carugati et al. 2015). When talking about molecular approaches, analysis of eukaryotes also represents a challenge because such organisms possess resilient cellular structures which can give poor DNA extraction yield (Hermans et al., 2018Hermans et al. 2018). In addition, SSU rRNA in eukaryotes fails to be as universal as for prokaryotes (Bik et al. 2012, Medinger et al. 2010). However, the use of marker genes from environmental DNA, when focused on the targeted eukaryotic community, remains critical to decoding the complexity of marine biofilms diversity. In this study, four extraction methods, including a preliminary mechanic cell lysis, both soil and biofilm kits, and global approaches, have been compared. We also examined the coverage and the identification capability of several primers to characterize eukaryotic communities colonizing three plastic surface types (polyvinyl chloride, HD polyethylene, and polyamide) which have been immersed in several locations along the French Mediterranean and Atlantic coasts. Sequence quality and number remain the same whatever the extraction method. However, the richness and community structure were clearly affected regardless of the sample type (Figure 1). Finally, two kits (PowerMaxSoil, and PowerBiofilm kits) evaluated in this study were considered as the most powerful overall. Secondly, we amplified and sequenced short fragments of two genes: one region of the mitochondrial Cytochrome Oxidase subunit I (COI) and five variable regions of the 18S small subunit ribosomal DNA (rDNA) gene (V1V2, V4TAR, V4UNI, V7, and V9). The Chao1 index was considerably lower for the CO1 gene compared to those of the 18S rDNA regions. The V4TAR and V7 regions showed a significant highest richness, followed closely by the V1V2 and V9 regions. The 18S rDNA gene sequences were dominated by microeukaryotes whereas the COI sequences were dominated by macro-organisms. Each of the 18rDNA primer pairs also exhibited dissimilar community structures although the dominant taxa seemed to be common. To conclude, our results provided a global assessment of tools dedicated to the description of the diversity of marine eukaryotes biofilms from three surfaces used in the design of RME. Among the four extraction methods described here, PowerMaxSoil and PowerBiofilm kits allowed recovering the highest diversity. COI and 18S rDNA gene sequencing covered different groups including at high taxonomic levels. Despite limitations, metabarcoding will help in the characterization of marine biofilms diversity on RME. Especially, it may be relevant to use primers targeting these two genes to better cover the eukaryotic diversity.

Using metabarcoding and « Diat.Barcode » database to perfom molecular identification on marine biofilms

Marine benthic diatoms highly contribute to biofilms formation, playing a crucial role on both living and artificial surfaces ‘colonization (Briand 2017, Salta 2013). However, their microscopic morphological identification is time consuming and requires a high expertise in taxonomy. We therefore decided to look toward molecular analyses and especially metabarcoding. In this study, we determine : (i) if the use of the “Diat.barcode” database, mostly developed for freshwater diatoms (Rimet 2019), is relevant to characterize marine biofilm communities, (ii) if the amplification of degenerated primers targeting the rbcL gene (Vasselon et al. 2017) could improve the diversity of marine diatom biofilms, and (iii) if molecular and morphological analyses could be correlated.A large majority of OTUs (>95%) was affiliated using the “Diat.barcode” database and the pipeline FROGS, with coverage and affinity values above 80%. OTUs tables contained 75% of diatom species already reported from marine environment, with 82% belonging to the pennates group. The use of degenerated primers significantly improved richness and diversity. Moreover, it allowed us to identify taxa that were not present before, as Iconella, Sellaphora and Coronia. Finally, we showed higher richness and diversity, but also a higher repeatability (replicates closeness) leading to a better clustering with metabarcoding. We found differences in terms of biomarkers, but more broadly, we were able to correlate significantly (r = 0,404; p<0.0001) diatom assemblages.While the latest version of “Diat.barcode” database contains only 12.4% species referenced as marine, it appears to be a powerful tool, even on biofilm samples from the Mediterranean, Baltic seas and Indian Ocean. Furthermore, we confirmed the relevance of degenerated primers to amplify a higher diversity of diatoms. Finally, beta-diversity similarity using molecular and microscopic analysis appeared positive, leading to the conclusion that the two methods should be used in a complementary way.

Microbial Functional Responses in Marine Biofilms Exposed to Deepwater Horizon Spill Contaminants

Marine biofilms are essential biological components that transform built structures into artificial reefs. Anthropogenic contaminants released into the marine environment, such as crude oil and chemical dispersant from an oil spill, may disrupt the diversity and function of these foundational biofilms. To investigate the response of marine biofilm microbiomes from distinct environments to contaminants and to address microbial functional response, biofilm metagenomes were analyzed from two short-term microcosms, one using surface seawater (SSW) and the other using deep seawater (DSW). Following exposure to crude oil, chemical dispersant, and dispersed oil, taxonomically distinct communities were observed between microcosms from different source water challenged with the same contaminants and higher Shannon diversity was observed in SSW metagenomes. Marinobacter, Colwellia, Marinomonas, and Pseudoalteromonas phylotypes contributed to driving community differences between SSW and DSW. SSW metagenomes were dominated by Rhodobacteraceae, known biofilm-formers, and DSW metagenomes had the highest abundance of Marinobacter, associated with hydrocarbon degradation and biofilm formation. Association of source water metadata with treatment groups revealed that control biofilms (no contaminant) harbor the highest percentage of significant KEGG orthologs (KOs). While 70% functional similarity was observed among all metagenomes from both experiments, functional differences between SSW and DSW metagenomes were driven primarily by membrane transport KOs, while functional similarities were attributed to translation and signaling and cellular process KOs. Oil and dispersant metagenomes were 90% similar to each other in their respective experiments, which provides evidence of functional redundancy in these microbiomes. When interrogating microbial functional redundancy, it is crucial to consider how composition and function evolve in tandem when assessing functional responses to changing environmental conditions within marine biofilms. This study may have implications for future oil spill mitigation strategies at the surface and at depth and also provides information about the microbiome functional responses of biofilms on steel structures in the marine built environment.