Search

Understanding the maintenance and origin of beta diversity is a central topic in ecology. However, the factors that drive diversity patterns and underlying processes remain unclear, particularly for host-prokaryotic associations. Here, beta diversity patterns were studied in five prokaryotic biotopes, namely, two high microbial abundance (HMA) sponge taxa (Xestospongia spp. and Hyrtios erectus), one low microbial abundance (LMA) sponge taxon (Stylissa carteri), sediment and seawater sampled across thousands of kilometres. Using multiple regression on distance matrices (MRM), spatial (geographic distance) and environmental (sea surface temperature and chlorophyll α concentrations) variables proved significant predictors of beta diversity in all five biotopes and together explained from 54% to 82% of variation in dissimilarity of both HMA species, 27% to 43% of variation in sediment and seawater, but only 20% of variation of the LMA S. carteri. Variance partitioning was subsequently used to partition the variation into purely spatial, purely environmental and spatially-structured environmental components. The amount of variation in dissimilarity explained by the purely spatial component was lowest for S. carteri at 11% and highest for H. erectus at 55%. The purely environmental component, in turn, only explained from 0.15% to 2.83% of variation in all biotopes. In addition to spatial and environmental variables, a matrix of genetic differences between pairs of sponge individuals also proved a significant predictor of variation in prokaryotic dissimilarity of the Xestospongia species complex. We discuss the implications of these results for the HMA-LMA dichotomy and compare the MRM results with results obtained using constrained ordination and zeta diversity.

Microbial communities associated with sponges play important roles in sponge health, defence and nutrient dynamics in coral reef systems. Here, we used a 16S rRNA gene high-throughput sequencing ap...

Dietary glycerol supplementation in aquaculture feed is seen as an alternative and inexpensive way to fuel fish metabolism, attenuate metabolic utilization of dietary proteins and, subsequently, reduce nitrogen excretion. In this study, we evaluated the impact of dietary glycerol supplementation on nitrogen excretion of European seabass (Dicentrarchus labrax) and its effects on metabolite profile and bacterial community composition of gut digesta. These effects were evaluated in a 60-day trial with fish fed diets supplemented with 2.5% or 5% (w/w) refined glycerol and without glycerol supplementation. Nuclear magnetic resonance spectroscopy and high-throughput 16S rRNA gene sequencing were used to characterize the effects of glycerol supplementation on digesta metabolite and bacterial community composition of 6-h postprandial fish. Our results showed that ammonia excretion was not altered by dietary glycerol supplementation, and the highest glycerol dosage was associated with significant increases in amino acids and a decrease of ergogenic creatine in digesta metabolome. Concomitantly, significant decreases in putative amino acid degradation pathways were detected in the predicted metagenome analysis, suggesting a metabolic shift. Taxon-specific analysis revealed significant increases in abundance of some specific genera (e.g., Burkholderia and Vibrio) and bacterial diversity. Overall, our results indicate glycerol supplementation may decrease amino acid catabolism without adversely affecting fish gut bacterial communities. Key points • Glycerol can be an inexpensive and energetic alternative in fish feed formulations. • Glycerol did not affect nitrogen excretion and gut bacteriome composition. • Glycerol reduced uptake of amino acids and increased uptake of ergogenic creatine. • Glycerol reduced putative amino acid degradation pathways in predicted metagenome.

Aim To evaluate the interactive effects of oil contamination and chemical dispersant application on bacterial composition and sediment remediation of an estuarine port environment. Methods and results A multifactorial controlled microcosm experiment was set up using sediment cores retrieved from an estuarine port area located at Ria de Aveiro lagoon (Aveiro, Portugal). An oil spill with and without chemical dispersant addition was simulated. Sediment oil hydrocarbon concentrations and benthic bacterial community structure were evaluated by GC-MS and 16S rRNA high-throughput sequencing respectively. Although initially (first 10 days) chemical dispersion of oil enhanced the concentrations of the heavier polycyclic aromatic hydrocarbons and of the C22 -C30 alkane group, with time (21 days), no significant differences in hydrocarbon concentrations were detected among treatments. Moreover, no significant changes were detected in the structure of sediment bacterial communities, which mainly consisted of operational taxonomic units related to hydrocarbon-contaminated marine environments. We hypothesize that the environmental background of the sampling site preconditioned the communities' response to additional contamination. Conclusion This experimental microcosm study showed that the chemical dispersion of oil did not influence sediment remediation or bacterial community composition. Significance and impact of the study Our study showed that chemical dispersion of oil may not improve the remediation of port sediments. Further studies are needed to investigate the impact of chemical dispersants in combination with bioremediation strategies on the process of sediment remediation in port areas.

Purpose Marine lakes are unique habitats that house diverse assemblages of benthic and planktonic organisms including endemic species. In this study, we aimed to assess to what extent geographical location (Berau versus Papua) and the degree of marine lake connectivity (relatively open versus closed) to the surrounding marine environment structures the prokaryotic community composition of the sponge species Suberites diversicolor. Methods Sponge specimens were sampled in five marine lakes in Borneo and Papua and one open sea habitat in Taiwan. Result Prokaryotic communities of S. diversicolor were dominated by members assigned to the Proteobacteria (particularly Alphaproteobacteria and Gammaproteobacteria) and Cyanobacteria, which together made up from 78 to 87% of sequences in all samples. The dominant operational taxonomic units (OTUs) in most samples, OTUs 1 and 3, were both assigned to the alphaproteobacterial order Rhodospirillales with OTU-1 dominant in the marine lakes of Berau and Papua and OTU-3 in Taiwan. OTU-3 was also largely absent from Papuan samples but present in all Berau samples. Compositionally, S. diversicolor samples clustered according to geographical location with the main axis of variation separating marine lake samples collected in Berau from those collected in Papua and the second axis of variation separating open sea samples collected in Taiwan from all marine lake samples. In addition, our results suggest that the degree of lake connectivity to the open sea also influences prokaryotic composition. Conclusion Although previous studies have shown that sponge-associated microbial communities tend to be stable across different geographical and environmental gradients, in the present study, both geography and local environmental conditions were significant predictors of variation in prokaryotic community composition of S. diversicolor.

Ocean acidification may exacerbate the environmental impact of oil hydrocarbon pollution by disrupting the core composition of the superficial (0-1 cm) benthic bacterial communities. However, at the subsurface sediments (approximately 5 cm below sea floor), the local biochemical characteristics and the superjacent sediment barrier may buffer these environmental changes. In this study, we used a microcosm experimental approach to access the independent and interactive effects of reduced seawater pH and oil contamination on the composition of subsurface benthic bacterial communities, at two time points, by 16S rRNA gene-based high-throughput sequencing. An in-depth taxa-specific variance analysis revealed that the independent effects of reduced seawater pH and oil contamination were significant predictors of changes in the relative abundance of some specific bacterial groups (e.g., Firmicutes, Rhizobiales, and Desulfobulbaceae). However, our results indicated that the overall microbial community structure was not affected by independent and interactive effects of reduced pH and oil contamination. This study provides evidence that bacterial communities inhabiting subsurface sediment may be less susceptible to the effects of oil contamination in a scenario of reduced seawater pH.

The stressful conditions of intensive aquaculture systems, combined with excessive antibiotic use, may have dysbiotic effects on aquaculture microbiomes and promote the spread of opportunistic pathogens. Here, we hypothesized that humic substances (HS), when added to the rearing water of a marine recirculating aquaculture system (RAS), will act as a chemical modulator of fish-associated bacterial communities and suppress potential pathogens. To test this, a 28-day RAS trial for juvenile European sea bass (Dicentrarchus labrax) was conducted with and without HS modulation. High-throughput sequencing of the 16S rRNA gene was used to evaluate role of HS in modulating fish bacterial assemblages (gut and skin mucus). In addition to this, biometric, digestive and oxidative stress parameters were measured to assess the impact of HS on fish performance. HS modulation was associated with an increased bacterial diversity and a distinct bacterial community composition in the fish skin mucus. HS modulation significantly increased the relative abundance of amplicon sequence variants (ASVs) related to the potentially beneficial Roseobacter clade and lowered the abundances of ASVs related to potential fish pathogens (e.g., Acinetobacter jonhsonii and Vibrio harveyi). In line with compositional differences, there was an enrichment of predictive KEGG categories related to antagonism and significantly lower abundances of KEGG categories related to pathogenesis and invasibility. In the fish gut, HS modulation was associated with a lower abundance of the orders Enterobacteriales and Bacillales. HS modulation also was associated with significant reductions in fish oxidative stress parameters and a significant increase in the activity of the digestive enzyme chymotrypsin. For the first time, our results demonstrate the potential of dissolved HS as a modulator of fish microbial communities.

Previously, it was believed that the prokaryote communities of typical ‘low-microbial abundance’ (LMA) or ‘non-symbiont harboring’ sponges were merely subsets of the prokaryote plankton community. Recent research has, however, shown that these sponges are dominated by particular clades of Proteobacteria or Cyanobacteria. Here, we expand on this research and assess the composition and putative functional profiles of prokaryotic communities from LMA sponges collected in two ecosystems (coral reef and hydrothermal vent) from vicinal islands of Taiwan with distinct physicochemical conditions. Six sponge species identified as Acanthella cavernosa (Bubarida), Echinodictyum asperum, Ptilocaulis spiculifer (Axinellida), Jaspis splendens (Tetractinellida), Stylissa carteri (Scopalinida) and Suberites sp. (Suberitida) were sampled in coral reefs in the Penghu archipelago. One sponge species provisionally identified as Hymeniacidon novo spec. (Suberitida) was sampled in hydrothermal vent habitat. Each sponge was dominated by a limited set of operational taxonomic units which were similar to sequences from organisms previously obtained from other LMA sponges. There was a distinct bacterial community between sponges collected in coral reef and in hydrothermal vents. The putative functional profile revealed that the prokaryote community from sponges collected in hydrothermal vents was significantly enriched for pathways related to DNA replication and repair. published

Much recent marine microbial research has focused on sponges, but very little is known about how the sponge microbiome fits in the greater coral reef microbial metacommunity. Here, we present an extensive survey of the prokaryote communities of a wide range of biotopes from Indo-Pacific coral reef environments. We find a large variation in operational taxonomic unit (OTU) richness, with algae, chitons, stony corals and sea cucumbers housing the most diverse prokaryote communities. These biotopes share a higher percentage and number of OTUs with sediment and are particularly enriched in members of the phylum Planctomycetes. Despite having lower OTU richness, sponges share the greatest percentage (>90%) of OTUs with >100 sequences with the environment (sediment and/or seawater) although there is considerable variation among sponge species. Our results, furthermore, highlight that prokaryote microorganisms are shared among multiple coral reef biotopes, and that, although compositionally distinct, the sponge prokaryote community does not appear to be as sponge-specific as previously thought., Here the authors holistically examine prokaryote communities associated with diverse coral reef hosts, including sponges, nudibranchs, sea cucumbers, and corals. The results show that sponges have a relatively low diversity of prokaryotes, most of which are shared across a wide range of host taxa rather than being sponge-specific.

The importance of microbial diversity and their role in the maintenance of fish health in aquaculture systems has been increasingly recognized in recent years. However, there is still a major knowledge gap regarding the ecology, composition and dynamics of microbial plankton assemblages during fish production. In this study, we aimed to investigate the seasonal dynamics and potential interactions of bacterial and microeukaryotic plankton communities in a semi-intensive aquaculture for European sea bass (Dicentrarchus labrax) cultured together with low density of gilthead sea bream (Sparus aurata) over a one-year period (January/2014 – November/2014). While the most abundant bacterial classes were Gammaproteobacteria, Flavobacteriia and Alphaproteobacteria; microeukaryotic communities were dominated by Ochrophyta, Chlorophyta and Ciliophora groups. Temperature and salinity were identified as significant drivers of the overall microbial community composition, which varied congruently along the seasons. However, while the dominant (more abundant) groups of bacteria occurred in the warmest months, the dominant groups of microeukaryotes occurred in the coldest months. There was also an inverse relationship between abundances of grazers and bacterial operational taxonomic units (OTUs). Overall, besides the potential effects of the abiotic parameters on the microbial plankton communities, the correlation between bacteria and microeukaryotic populations observed here may be an indication of trophic and/or metabolic interdependence between these two domains. Future studies should focus on the underlying mechanisms of this interdependence for a better understand of the impact of microeukaryotic communities on aquaculture bacterioplankton structure and function. In addition, this knowledge could be of interest in the development of microbial management strategies for aquaculture systems.

There is a growing consensus that future technological developments of aquaculture systems should account for the structure and function of microbial communities in the whole system and not only in fish guts. In this study, we aimed to investigate the composition of bacterioplankton communities of a hatchery recirculating aquaculture system (RAS) used for the production of Senegalese sole (Solea senegalensis) juveniles. To this end, we used a 16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing analyses to characterize the bacterioplankton communities of the RAS and its water supply. Overall, the most abundant orders were Alteromonadales, Rhodobacterales, Oceanospirillales, Vibrionales, Flavobacteriales, Lactobacillales, Thiotrichales, Burkholderiales and Bdellovibrionales. Although we found a clear distinction between the RAS and the water supply bacterioplankton communities, most of the abundant OTUs (≥50 sequences) in the hatchery RAS were also present in the water supply. These included OTUs related to Pseudoalteromonas genus and the Roseobacter clade, which are known to comprise bacterial members with activity against Vibrio fish pathogens. Overall, in contrast to previous findings for sole grow-out RAS, our results suggest that the water supply may influence the bacterioplankton community structure of sole hatchery RAS. Further studies are needed to investigate the effect of aquaculture practices on RAS bacterioplankton communities and identification of the key drivers of their structure and diversity.

Most existing coral reef studies have focused on a single biotope and a single domain (Archaea or Bacteria). Few coral reef studies have explored the archaeal and bacterial community simultaneously. In this study, we compare the diversity and composition of archaeal and bacterial communities in seawater and two closely related sponge species (Stylissa carteri and Stylissa massa) in the Berau reef system, Indonesia. A 16S rRNA gene barcoded pyrosequencing approach was used to test to what extent seawater, S. carteri and S. massa host compositionally distinct communities of Archaea and Bacteria. Proteobacteria dominated the bacterial communities of all three studied biotopes whereas Euryarchaeota was the most abundant archaeal phylum in seawater and Crenarchaeota the most abundant archaeal phylum in both Stylissa species. Biotopes explained 56% and 53% of the variation in archaeal and bacterial composition respectively and there was significant congruence between the composition of archaeal and bacterial communities. These results suggest that the processes that drive bacterial composition within the studied biotopes may be fundamentally similar to those that drive archaeal composition.

The high/low microbial abundance (HMA/LMA) dichotomy in sponges has been the subject of several studies over recent years, but few studies have analysed this dichotomy in terms of the sponge archaeal community and function. Using a 16S rRNA gene barcoded pyrosequencing approach and predictive functional analysis (PICRUSt) we compared the archaeal composition, richness and predicted function of one HMA sponge (Xestospongia testudinaria), one LMA sponge (Stylissa carteri) and one sponge species of unknown microbial abundance (Aaptos lobata). Although most of the archaeal sequences were assigned to the Crenarchaeota phylum, S. carteri had the highest percentage of sequences assigned to the Euryarchaeota phylum. Variation among sponge species explained >85% of the variation in archaeal operational taxonomic unit (OTU) composition with each sponge species forming a distinct cluster. There were significant differences in predicted PICRUSt profiles among sponge species, suggesting that archaeal communities present in the studied sponge species may perform different functions. X. testudinaria and A. lobata were similar both in terms of OTU and KEGG orthologues composition, which may indicate that A. lobata is a HMA sponge. Additionally, some of the most enriched functions seem to be related to traits associated with high and low microbial abundance sponges. published

Global climate change has the potential to seriously and adversely affect marine ecosystem functioning. Numerous experimental and modeling studies have demonstrated how predicted ocean acidification and increased ultraviolet radiation (UVR) can affect marine microbes. However, researchers have largely ignored interactions between ocean acidification, increased UVR and anthropogenic pollutants in marine environments. Such interactions can alter chemical speciation and the bioavailability of several organic and inorganic pollutants with potentially deleterious effects, such as modifying microbial-mediated detoxification processes. Microbes mediate major biogeochemical cycles, providing fundamental ecosystems services such as environmental detoxification and recovery. It is, therefore, important that we understand how predicted changes to oceanic pH, UVR, and temperature will affect microbial pollutant detoxification processes in marine ecosystems. The intrinsic characteristics of microbes, such as their short generation time, small size, and functional role in biogeochemical cycles combined with recent advances in molecular techniques (e.g., metagenomics and metatranscriptomics) make microbes excellent models to evaluate the consequences of various climate change scenarios on detoxification processes in marine ecosystems. In this review, we highlight the importance of microbial microcosm experiments, coupled with high-resolution molecular biology techniques, to provide a critical experimental framework to start understanding how climate change, anthropogenic pollution, and microbiological interactions may affect marine ecosystems in the future. The authors acknowledge support from Centre for Environmental and Marine Studies (CESAM) and Foundation for Science and Technology (FCT, Portugal) PTDC/AAC -CLI/107916/2008 (http://alfa.fct.mctes.pt) and the European Regional Development Fund (ERDF) through COMPETE-(FCOMP-01-0124-FEDER-008657). Francisco J. R. C. Coelho and Ana L. Santos were supported by Ph.D. scholarships (SFRH/BD/46322/2008 and SFR/BD/ 40160/2007, respectively) funded by Fundação para a Ciência e Tecnologia, Portugal (QREN-POPH – Type 4.1 – Advanced Training, subsidized by the European Social Fund and national funds MCTES). published

The present study assesses the diversity and composition of sediment bacterial and microeukaryotic communities from deep-sea mud volcanoes (MVs) associated with strike-slip faults in the South-West Iberian Margin (SWIM). We used a 16S/18S rRNA gene based pyrosequencing approach to characterize and correlate the sediment bacterial and microeukaryotic communities from MVs with differing gas seep regimes and from an additional site with no apparent seeping activity. In general, our results showed significant compositional changes of bacterial and microeukaryotic communities in sampling sites with different seepage regimes. Sediment bacterial communities were enriched with Methylococcales (putative methanotrophs) but had lower abundances of Rhodospirillales, Nitrospirales and SAR202 in the more active MVs. Within microeukaryotic communities, members of the Lobosa (lobose amoebae) were enriched in more active MVs. We also showed a strong correlation between Methylococcales populations and lobose amoeba in active MVs. This study provides baseline information on the diversity and composition of bacterial and microeukaryotic communities in deep-sea MVs associated with strike-slip faults.

There is growing concern that predicted changes to global ocean chemistry will interact with anthropogenic pollution to significantly alter marine microbial composition and function. However, knowledge of the compounding effects of climate change stressors and anthropogenic pollution is limited. Here, we used 16S and 18S rRNA (cDNA)-based activity profiling to investigate the differential responses of selected microbial taxa to ocean acidification and oil hydrocarbon contamination under controlled laboratory conditions. Our results revealed that a lower relative abundance of sulphate-reducing bacteria (Desulfosarcina/Desulfococcus clade) due to an adverse effect of seawater acidification and oil hydrocarbon contamination (reduced pH-oil treatment) may be coupled to changes in sediment archaeal communities. In particular, we observed a pronounced compositional shift and marked reduction in the prevalence of otherwise abundant operational taxonomic units (OTUs) belonging to the archaeal Marine Benthic Group B and Marine Hydrothermal Vent Group (MHVG) in the reduced pH-oil treatment. Conversely, the abundance of several putative hydrocarbonoclastic fungal OTUs was higher in the reduced pH-oil treatment. Sediment hydrocarbon profiling, furthermore, revealed higher concentrations of several alkanes in the reduced pH-oil treatment, corroborating the functional implications of the structural changes to microbial community composition. Collectively, our results advance the understanding of the response of a complex microbial community to the interaction between reduced pH and anthropogenic pollution. In future acidified marine environments, oil hydrocarbon contamination may alter the typical mixotrophic and k-/r-strategist composition of surface sediment microbiomes towards a more heterotrophic state with lower doubling rates, thereby impairing the ability of the ecosystem to recover from acute oil contamination events.

Bacterial communities of the surface microlayer (SML) of the estuary Ria de Aveiro (Portugal) were characterized in terms of abundance and activity during a 2-year survey at two sites with distinct hydrodynamic properties (marine and brackish water zones). The hydrodynamic conditions were simulated using a bidimensional numerical model and related to the microbiological observations. The pattern of variation of bacterial biomass productivity (BBP) was distinct between the two sampling sites. At the outer site, BBP was significantly lower at the SML, whereas at the inner site, it was significantly enhanced at the SML. Although the total bacterial abundance was similar in the SML and underlying water (UW), the fraction of cells attached to particles was significantly higher at the SML (two to three times). The integration of microbiological results with environmental and hydrological variables shows that strong currents in the marine zone promote the vertical mixing, inhibiting the establishment of an SML bacterial community distinct from that of UW. In contrast, in the brackish water zone, lower current velocities provide conditions for enhancing the bacterial activity in the enriched SML. Estuarine dynamics influence the distribution and activity of microorganisms at the SML and in the water column, with anticipated impacts for the carbon cycle in the estuarine environment.

Photobacterium damselae subspecies damselae and piscicida are known fish pathogens responsible for disease outbreaks in several cultured fish species. Fast detection of these pathogens is important for management and control of disease outbreaks. In this study, we developed a molecular approach for quantification of P. damselae species and detection of its subspecies (piscicida and damselae) based on real time PCR (RT-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of toxR gene fragments. In addition, the efficacy of this molecular methodology was tested against water samples from a natural environment (Ria de Aveiro estuary, Aveiro, Portugal) and from an intensive recirculating aquaculture system (RAS). In the first phase of this study we developed a RT-PCR targeting the toxR gene suitable for detection and quantification of P. damselae species. After the first RT-PCR trial, a nested PCR-DGGE was developed to determine the presence of P. damselae subspecies in the positive samples. Confirmation of the identity of different subspecies was obtained by Sanger DNA sequencing and phylogenetic analysis of toxR gene fragments obtained from excised DGGE bands. Our RT-PCR analysis detected between 1797.85 ± 376.15 (ca 0.71 fg) and 2976.68 ± 1253.63 (ca 1.76 fg) gene copy numbers of P. damselae toxR genes in the aquaculture samples. The DGGE analysis of these samples detected two equally abundant bands near the DGGE reference position for P. damselae subsp. piscicida. The sequence and phylogeny analyses of excised bands revealed the presence of two populations with distinct toxR gene sequences suggesting a close phylogenetic relationship with P. damselae subsp. piscicida. Contrary to aquaculture samples, no RT-PCR signal was obtained for DNA extracted from estuarine water samples. Here we provide evidences that the molecular methodology developed in this study can be used for overall quantification of P. damselae and subsequent detection of its subspecies in natural ecosystems and aquaculture systems.

Saltmarshes, seagrass meadows and mudflats are key habitats in estuarine ecosystems. Despite being involved in key ecological processes, the influence of different estuarine habitats on sediment bacterial communities remains understudied. Few studies have analysed and compared the bacterial communities of more than one estuarine habitat at different depths. Here, we investigated to what extent different habitats (mudflats; mono-specific plots of seagrass [Zostera noltei] and two saltmarsh plants [Juncus maritimus and Spartina maritima]) and sampling depth (0, 5, 10 cm) influence variation in sediment bacterial composition. Our results showed significant differences in the abundance of selected higher taxa amongst habitats and depths. Surface sediment was characterized by bacteria assigned to the Acidimicrobiia, Flavobacteriia, Thiotrichales, Alteromonadales and Rhodobacterales, whereas in deeper sediment Deltaproteobacteria and Anaerolineae were dominant. Juncus sediment, in turn, presented the most distinct bacterial community, with Myxococcales abundant in this habitat. Sampling depth and habitat proved significant predictors of variation in sediment bacterial composition. The compositional dissimilarities amongst habitats and depths suggest functional divergence and complementarity, thus enhancing ecosystem functioning and health. Given the compositionally distinct communities found in different habitats and depths, our study corroborates the importance of conserving a diverse array of estuarine habitats.

For several years, knowledge on the microbiome associated with marine invertebrates was impaired by the challenges associated with the characterization of bacterial communities. With the advent of culture independent molecular tools it is possible to gain new insights on the diversity and richness of microorganisms associated with marine invertebrates. In the present study, we evaluated if different preservation and processing methodologies (prior to DNA extraction) can affect the bacterial diversity retrieved from snakelocks anemone Anemonia viridis. Denaturing gradient gel electrophoresis (DGGE) community fingerprints were used as proxy to determine the bacterial diversity retrieved (H′). Statistical analyses indicated that preservation significantly affects H′. The best approach to preserve and process A. viridis biomass for bacterial community fingerprint analysis was flash freezing in liquid nitrogen (preservation) followed by the use of a mechanical homogenizer (process), as it consistently yielded higher H′. Alternatively, biomass samples can be processed fresh followed by cell lyses using a mechanical homogenizer or mortar & pestle. The suitability of employing these two alternative procedures was further reinforced by the quantification of the 16S rRNA gene; no significant differences were recorded when comparing these two approaches and the use of liquid nitrogen followed by processing with a mechanical homogenizer.

There is growing concern that modifications to the global environment such as ocean acidification and increased ultraviolet radiation may interact with anthropogenic pollutants to adversely affect the future marine environment. Despite this, little is known about the nature of the potential risks posed by such interactions. Here, we performed a multifactorial microcosm experiment to assess the impact of ocean acidification, ultraviolet B (UV-B) radiation and oil hydrocarbon contamination on sediment chemistry, the microbial community (composition and function) and biochemical marker response of selected indicator species. We found that increased ocean acidification and oil contamination in the absence of UV-B will significantly alter bacterial composition by, among other things, greatly reducing the relative abundance of Desulfobacterales, known to be important oil hydrocarbon degraders. Along with changes in bacterial composition, we identified concomitant shifts in the composition of oil hydrocarbons in the sediment and an increase in oxidative stress effects on our indicator species. Interestingly, our study identifies UV-B as a critical component in the interaction between these factors, as its presence alleviates harmful effects caused by the combination of reduced pH and oil pollution. The model system used here shows that the interactive effect of reduced pH and oil contamination can adversely affect the structure and functioning of sediment benthic communities, with the potential to exacerbate the toxicity of oil hydrocarbons in marine ecosystems.

The uppermost millimeter of the water column, the surface microlayer (SML), hosts bacterial communities (bacterioneuston) with potential metabolic adaptations to this unique physical and chemical environment. Hydrolysis and monomer incorporation by bacterioneuston and bacterioplankton communities in the estuarine system, Ria de Aveiro, was investigated and compared during a 2-year survey. The study was conducted at two contrasting sites, typifying the marine and brackish water characteristics of the estuary and with different sources, amounts and composition of organic matter. In the marine zone, bacterioneuston showed higher rates of hydrolysis and lower rates of monomer incorporation than bacterioplankton, whereas in the brackish water zone, neustonic and planktonic microbial communities showed similar activity rates. This pattern may result from the different degree of surface organic and inorganic matter enrichments, which reflect site-specific characteristics, such as hydrodynamics and sources of organic matter composition. In general, the estuarine SML environment favors polymer hydrolysis, but inhibits monomer utilization, in comparison with the subsurface layers. However, the differences between the two communities tend to decrease as autotrophic and heterotrophic activities increase in the brackish-water area.

An experimental life support system (ELSS) was constructed to study the interactive effects of multiple stressors on coastal and estuarine benthic communities, specifically perturbations driven by global climate change and anthropogenic environmental contamination. The ELSS allows researchers to control salinity, pH, temperature, ultraviolet radiation (UVR), tidal rhythms and exposure to selected contaminants. Unlike most microcosms previously described, our system enables true independent replication (including randomization). In addition to this, it can be assembled using commercially available materials and equipment, thereby facilitating the replication of identical experimental setups in different geographical locations. Here, we validate the reproducibility and environmental quality of the system by comparing chemical and biological parameters recorded in our ELSS with those prevalent in the natural environment. Water, sediment microbial community and ragworm (the polychaete Hediste diversicolor) samples were obtained from four microcosms after 57 days of operation. In general, average concentrations of dissolved inorganic nutrients (NO3 (-) ; NH4 (+) and PO4 (-3) ) in the water column of the ELSS experimental control units were within the range of concentrations recorded in the natural environment. While some shifts in bacterial community composition were observed between in situ and ELSS sediment samples, the relative abundance of most metabolically active bacterial taxa appeared to be stable. In addition, ELSS operation did not significantly affect survival, oxidative stress and neurological biomarkers of the model organism Hediste diversicolor. The validation data indicate that this system can be used to assess independent or interactive effects of climate change and environmental contamination on benthic communities. Researchers will be able to simulate the effects of these stressors on processes driven by microbial communities, sediment and seawater chemistry and to evaluate potential consequences to sediment toxicity using model organisms such as Hediste diversicolor. This study was supported by the Centre for Environmental and Marine Studies (CESAM), the Foundation for Science and Technology (FCT, Portugal) PTDC/AAC-CLI/107916/2008 (http://alfa.fct.mctes.pt) and the European Regional Development Fund (ERDF) through COMPETE-(FCOMP-01-0124FEDER-008657). Francisco J.R.C. Coelho and Rui J. M. Rocha were supported by a PhD scholarship (SFRH/BD/46322/2008 and SFRH/BD/46675/2008, respectively) funded by FCT (QREN-POPH – Type 4.1 – Advanced Training, subsidized by the European Social Fund and national funds MCTES). We thank Jos e Ferreira from EpArq, Aveiro University for graphical design of the ELSS. We also wish to thank our anonymous reviewers for their comments and suggestions. published

Bioremediation efforts often rely on the application of surfactants to enhance hydrocarbon bioavailability. However, synthetic surfactants can sometimes be toxic to degrading microorganisms, thus reducing the clearance rate of the pollutant. Therefore, surfactant-resistant bacteria can be an important tool for bioremediation efforts of hydrophobic pollutants, circumventing the toxicity of synthetic surfactants that often delay microbial bioremediation of these contaminants. In this study, we screened a natural surfactant-rich compartment, the estuarine surface microlayer (SML), for cultivable surfactant-resistant bacteria using selective cultures of sodium dodecyl sulfate (SDS) and cetyl trimethylammonium bromide (CTAB). Resistance to surfactants was evaluated by colony counts in solid media amended with critical micelle concentrations (CMC) of either surfactants, in comparison with non-amended controls. Selective cultures for surfactant-resistant bacteria were prepared in mineral medium also containing CMC concentrations of either CTAB or SDS. The surfactantresistant isolates obtained were tested by PCR for the Pseudomonas genus marker gacA gene and for the naphthalene-dioxygenase-encoding gene ndo. Isolates were also screened for biosurfactant production by the atomized oil assay. A high proportion of culturable bacterioneuston was tolerant to CMC concentrations of SDS or CTAB. The gacA-targeted PCR revealed that 64% of the isolates were Pseudomonads. Biosurfactant production in solid medium was detected in 9.4% of tested isolates, all affiliated with genus Pseudomonas. This study shows that the SML is a potential source of surfactant-resistant and biosurfactant-producing bacteria in which Pseudomonads emerge as a relevant group.

Bacterial communities of the surface microlayer (SML) of the estuary Ria de Aveiro (Portugal) were characterized in terms of abundance and activity during a 2-year survey at two sites with distinct hydrodynamic properties (marine and brackish water zones). The hydrodynamic conditions were simulated using a bidimensional numerical model and related to the microbiological observations. The pattern of variation of bacterial biomass productivity (BBP) was distinct between the two sampling sites. At the outer site, BBP was significantly lower at the SML, whereas at the inner site, it was significantly enhanced at the SML. Although the total bacterial abundance was similar in the SML and underlying water (UW), the fraction of cells attached to particles was significantly higher at the SML (two to three times). The integration of microbiological results with environmental and hydrological variables shows that strong currents in the marine zone promote the vertical mixing, inhibiting the establishment of an SML bacterial community distinct from that of UW. In contrast, in the brackish water zone, lower current velocities provide conditions for enhancing the bacterial activity in the enriched SML. Estuarine dynamics influence the distribution and activity of microorganisms at the SML and in the water column, with anticipated impacts for the carbon cycle in the estuarine environment.

Bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) in the estuarine surface microlayer (SML) of the Ria de Aveiro, Portugal—which is chronically polluted with oil hydrocarbons (OH)—were isolated and characterized; Pseudomonas was dominant among the PAH-degrading bacteria. Screening for PAH dioxygenase genes detected almost identical nahAc genes (encoding the alpha subunits of naphthalene dioxygenase) in 2 phylogenetically distinct isolates: Pseudomonas sp. and an unknown species of the family Enterobacteriaceae; this suggested that horizontal transfer of nah genes might be involved in PAH degradation in the SML. We also investigated the effect of PAH contamination on the spatial variability of the bacterioneuston along a gradient of pollution in the estuarine system of the Ria de Aveiro. Culture-independent techniques—fluorescence in situ hy - bridization (FISH) and denaturing-gradient gel electrophoresis (DGGE)—revealed a similar struc- ture among the bacterioneuston communities along the estuary. In contrast, we detected differences in the relative abundance and diversity of organisms of the Gammaproteobacteria, including those of the genus Pseudomonas (which belongs to the Gammaproteobacteria). This is the first insight into the hydrocarbonoclastic bacterial communities in the SML of an estuarine area polluted with hydrocar- bons. Our findings highlight the importance of SML-adapted hydrocarbonoclastic bacterioneuston as a potential source of new PAH-degrading bacteria (including new pseudomonads) with potential use in the bioremediation of hydrocarbon-polluted ecosystems.

The aim of this study was to understand if two species of salt marsh plants, widely distributed in European estuaries (Spartina maritima and Halimione portulacoides) differently influence the distribution, activity, and metabolic physiology of sediment bacterial communities in monospecific banks, in comparison with uncolonized sediment (control). Microbiological descriptors of abundance and activity were assessed along vertical profiles of sediments. Rates of activity of the extracellular enzymes beta-glucosidase, alpha-glucosidase, aminopeptidase, arylsulfatase, and phosphatase were generally higher in the vegetation banks in relation to control sediments where they were also less variable with depth. This is interpreted as an indirect effect related to supply of plant-derived polymeric substrates for bacterial growth. Parameters related to sediment texture (grain size, percent of fines or water content) showed significant relations with cell abundance or maximum hydrolysis rates, pointing to an indirect effect of plant colonization exerted through the modification of sediment physical properties. The profiles of utilization of sole-carbon-source (Biolog Ecoplates) showed that only the communities from the upper sediment layer of the S. maritima and the H. portulacoides banks exhibit consistent differences in terms of physiological profiles. Bacterial communities in control sediments exhibited the lowest physiological variability between surface and sub-surface communities. The results indicate that microbial colonization and organic matter decomposition are enhanced under the influence of salt marsh plants and confirm that plant coverage is a major determinant of the processes of organic matter recycling in intertidal estuarine sediments. published