Disturbance frequency directs microbial community succession in marine biofilms exposed to shear.

IMPORTANCE: Disturbances are major drivers of community succession in many microbial systems; however, relatively little is known about marine biofilm community succession, especially under antifouling disturbance. Antifouling technologies exert strong local disturbances on marine biofilms, and resulting biomass losses can be accompanied by shifts in biofilm community composition and succession. We address this gap in knowledge by bridging microbial ecology with antifouling technology development. We show that disturbance by shear can strongly alter marine biofilm community succession, acting as a selective filter influenced by frequency of exposure. Examining marine biofilm succession patterns with and without shear revealed stable associations between key prokaryotic and eukaryotic taxa, highlighting the importance of cross-domain assessment in future marine biofilm research. Describing how compounded top-down and bottom-up disturbances shape the succession of marine biofilms is valuable for understanding the assembly and stability of these complex microbial communities and predicting species invasiveness.

Vibrio cyclitrophicus population-specific biofilm formation and epibiotic growth on marine copepods.

Vibrio spp. form a part of the microbiome of copepods-an abundant component of marine mesozooplankton. The biological mechanisms of the Vibrio-copepod association are largely unknown. In this study we compared biofilm formation of V. cyclitrophicus isolated from copepods (L-strains related to other particle-associated strains) and closely related strains originating from seawater (S-strains), and visualized and quantified their attachment and growth on copepods. The S- and L-strains formed similar biofilms in the presence of complete sea salts, suggesting previously unknown biofilm mechanisms in the S-strains. No biofilms formed if sodium chloride was present as the only salt but added calcium significantly enhanced biofilms in the L-strains. GFP-L-strain cells attached to live copepods at higher numbers than the S-strains, suggesting distinct mechanisms, potentially including calcium, support their colonization of copepods. The cells grew on live copepods after attachment, demonstrating that copepods sustain epibiotic V. cyclitrophicus growth in situ. The results demonstrate that in spite of their 99.1% average nucleotide identity, these V. cyclitrophicus strains have a differential capacity to colonize marine copepods. The introduced V. cyclitrophicus-A. tonsa model could be informative in future studies on Vibrio-copepod association.

Comparison of three-dimensional motion of bacteria with and without wall accumulation.

A comparison of the movement characteristics between bacteria with and without wall accumulation could potentially elucidate the mechanisms of biofilm formation. However, authors of previous studies have mostly focused on the motion of bacteria that exhibit wall accumulation. Here, we applied digital holographic microscopy to compare the three-dimensional (3D) motions of two bacterial strains (Shewanella japonica UMDC19 and Shewanella sp. UMDC1): one exhibiting higher concentrations near the solid surfaces, and the other showing similar concentrations in near-wall and bulk regions. We found that the movement characteristics of the two strains are similar in the near-wall region but are distinct in the bulk region. Near the wall, both strains have small velocities and mostly perform subdiffusive motions. In the bulk, however, the bacteria exhibiting wall accumulation have significantly higher motility (including faster swimming speeds and longer movement trajectories) than the one showing no wall accumulation. Furthermore, we found that bacteria exhibiting wall accumulation slowly migrate from the bulk region to the near-wall region, and the hydrodynamic effect alone is insufficient to generate this migration speed. Future studies are required to test if the current findings apply to other bacterial species and strains.

Nitrogen Fixation Influenced by Phosphorus and Nitrogen Availability in the Benthic Bloom-forming Cyanobacterium Hydrocoleum sp. Identified in a Temperate Marine Lagoon.

The nitrogen-fixing, non-heterocystous cyanobacterium Hydrocoleum sp. (Oscillatoriales) is a common epiphytic and benthic bloom-former in tropical and subtropical shallow water systems but shares high phylogenetic similarity with the planktonic, globally important diazotroph Trichodesmium. Multiphasic observations in this study resulted in unexpected identification of Hydrocoleum sp. in mass accumulations in a coastal lagoon in the Western temperate North Atlantic Ocean. Hydrocoleum physiology was examined in situ through measurements of N2 and CO2 fixation rates and expression of genes involved with N2 fixation, CO2 fixation, and phosphorus (P) stress. Bulk N2 fixation rates and Hydrocoleum nifH expression peaked at night and were strongly suppressed by dissolved inorganic nitrogen (DIN). The expression of high affinity phosphate transporter (pstS) and alkaline phosphatase (phoA) genes of Hydrocoleum was elevated during the night and negatively responded to phosphate amendments, as evidence that these mechanisms contribute to P acquisition during diazotrophic growth of Hydrocoleum in situ. This discovery at the edge of the previously known Hydrocoleum habitat range in the warming oceans raises intriguing questions about diazotrophic cyanobacterial adaptations and transitions on the benthic-pelagic continuum.

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"). 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 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 ranging 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 antifouling 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 light-emitting diode (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, the overall bacterial community composition remained relatively stable, 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.

Practicing Critical Thinking in Undergraduate Microbiology Classes by Presenting News Stories with Data Evidence.

Developing scientific literacy is a key objective in undergraduate biology education. However, finding and accessing important primary research articles and drawing conclusions from original data is often challenging for students. The approach described here aims to alleviate these challenges by using news stories as a starting point for identifying important research findings and having students ultimately explain the findings with original data. The students identify a microbial news story, find and read the original peer-reviewed article the news story was based upon, and finally, present the key findings in class, while explicitly focusing on presenting data evidence, followed by the key conclusions that resulted in the news story. This approach practices scientific literacy and critical thinking and promotes a student-centered learning environment.

Underwater shear-based grooming of marine biofouling using a non-contact Bernoulli pad device.

The biofouling removal ability of a shear-based device was tested on two submerged surface types, Garolite G-10 and Intersleek 1100SR. Each surface was groomed at four frequencies along with a control group. The seven-week grooming study was conducted in Narragansett Bay, Rhode Island. The Bernoulli pad device uses confined radial outflow to generate both surface-normal forces to grip the submerged surface and shear stress to groom the surface without contact. An image-processing algorithm was developed and used to assess the effectiveness of the various grooming protocols, along with direct measurements of chlorophyll a per surface area. The image-processing data showed that the grooming resulted in ∼50% cleanliness on the Garolite at the end of the study whereas the Intersleek was continuously restored to nearly its initial clean state. Chlorophyll a data supported these overall conclusions. These results indicate that surface cleanliness can be maintained effectively on Intersleek using frequent shear-based grooming.

Copepod-Associated Gammaproteobacterial Alkaline Phosphatases in the North Atlantic Subtropical Gyre.

Planktonic organisms may provide a niche to associated bacteria in the oligotrophic ocean. Bacterial fitness strategies in association with copepods - abundant planktonic crustaceans - were examined by sampling and incubation experiments in the North Atlantic Subtropical Gyre (NASG). The bacterial metatranscriptome was dominated by Gammaproteobacteria and showed expression of complete bacterial pathways including chemotaxis, cell signaling, and alkaline phosphatase activity. Quantitative PCR and reverse transcriptase qPCR revealed the consistent presence and expression of alkaline phosphatase genes primarily by Vibrio spp. in the copepod association. Copepod-associated bacteria appear to respond to prevailing phosphorus limitation by using alkaline phosphatases to break down organophosphoesters, presumably originating from the copepods. The results suggest that the basin-wide tendency for phosphorus limitation in the North Atlantic Ocean is occurring at microscales in these nitrogen-enriched copepod microenvironments. The bacterial communities and their fitness strategies supported by associations with these abundant mesozooplankton are unique from the surrounding seawater and could have large-scale implications for biogeochemical cycling, marine food web structuring, and copepod and ecosystem health.

Draft Genome Sequence of Marinobacter hydrocarbonoclasticus NCT7M, Obtained from the Microbiome of a Marine Copepod.

The gammaproteobacterium Marinobacter hydrocarbonoclasticus NCT7M was cultivated from the copepod Acartia tonsa, collected from the coastal western North Atlantic Ocean. The genome was assembled into 45 contigs for a total of 4,128,590 bp, a GC content of 57.3%, and 3,890 protein-coding genes. The genome contains the full gene cluster for denitrification.

Dissolved organic matter stimulates N2 fixation and nifH gene expression in Trichodesmium.

Mixotrophy, the combination of heterotrophic and autotrophic nutrition modes, is emerging as the rule rather than the exception in marine photosynthetic plankton. Trichodesmium, a prominent diazotroph ubiquitous in the (sub)tropical oceans, is generally considered to obtain energy via autotrophy. While the ability of Trichodesmium to use dissolved organic phosphorus when deprived of inorganic phosphorus sources is well known, the extent to which this important cyanobacterium may benefit from other dissolved organic matter (DOM) resources is unknown. Here we provide evidence of carbon-, nitrogen- and phosphorus-rich DOM molecules enhancing N2 fixation rates and nifH gene expression in natural Trichodesmium colonies collected at two stations in the western tropical South Pacific. Sampling at a third station located in the oligotrophic South Pacific Gyre revealed no Trichodesmium but showed presence of UCYN-B, although no nifH expression was detected. Our results suggest that Trichodesmium behaves mixotrophically in response to certain environmental conditions, providing them with metabolic plasticity and adding up to the view that mixotrophy is widespread among marine microbes.

Draft Genome Sequence of the Non-Microcystin-Producing Microcystis aeruginosa Strain KLA2, Isolated from a Freshwater Reservoir in Northern California, USA.

We report a draft genome sequence for Microcystis aeruginosa KLA2. The total draft genome size is 5,213,465 bp with a GC content of 42.5%. The genome does not have genes indicative of microcystin production but does contain genes indicative of production of several other secondary metabolites.

Draft Genome Sequence of Vibrio cyclitrophicus NCT10V, Cultivated from the Microbiome of a Marine Copepod.

Here, we report the draft genome sequence of Vibrio cyclitrophicus NCT10V, cultivated from the copepod Acartia tonsa, collected from coastal surface waters of the western North Atlantic Ocean. The assembly is 5,638,575 bp long and has 44 contigs, a GC content of 43.2%, and 5,044 protein coding sequences.

Copepods promote bacterial community changes in surrounding seawater through farming and nutrient enrichment.

Bacteria living in the oligotrophic open ocean have various ways to survive under the pressure of nutrient limitation. Copepods, an abundant portion of the mesozooplankton, release nutrients through excretion and sloppy feeding that can support growth of surrounding bacteria. We conducted incubation experiments in the North Atlantic Subtropical Gyre to investigate the response of bacterial communities in the presence of copepods. Bacterial community composition and abundance measurements indicate that copepods have the potential to influence the microbial communities surrounding and associating with them - their 'zoosphere', in two ways. First, copepods may attract and support the growth of copiotrophic bacteria including representatives of Vibrionaceae, Oceanospirillales and Rhodobacteraceae in waters surrounding them. Second, copepods appear to grow specific groups of bacteria in or on the copepod body, particularly Flavobacteriaceae and Pseudoalteromonadaceae, effectively 'farming' them and subsequently releasing them. These distinct mechanisms provide a new view into how copepods may shape microbial communities in the open ocean. Microbial processes in the copepod zoosphere may influence estimates of oceanic bacterial biomass and in part control bacterial community composition and distribution in seawater.

Copepod-Associated Gammaproteobacteria Respire Nitrate in the Open Ocean Surface Layers.

Microbial dissimilatory nitrate reduction to nitrite, or nitrate respiration, was detected in association with copepods in the oxygenated water column of the North Atlantic subtropical waters. These unexpected rates correspond to up to 0.09 nmol N copepod-1 d-1 and demonstrate a previously unaccounted nitrogen transformation in the oceanic pelagic surface layers. Genes and transcripts for both the periplasmic and membrane associated dissimilatory nitrate reduction pathways (Nap and Nar, respectively) were detected. The napA genes and transcripts were closely related with sequences from several clades of Vibrio sp., while the closest relatives of the narG sequences were Pseudoalteromonas spp. and Alteromonas spp., many of them representing clades only distantly related to previously described cultivated bacteria. The discovered activity demonstrates a novel Gammaproteobacterial respiratory role in copepod association, presumably providing energy for these facultatively anaerobic bacteria, while supporting a reductive path of nitrogen in the oxygenated water column of the open ocean.

Chasing after Non-cyanobacterial Nitrogen Fixation in Marine Pelagic Environments.

Traditionally, cyanobacterial activity in oceanic photic layers was considered responsible for the marine pelagic dinitrogen (N2) fixation. Other potentially N2-fixing bacteria and archaea have also been detected in the pelagic water column, however, the activity and importance of these non-cyanobacterial diazotrophs (NCDs) remain poorly constrained. In this perspective we summarize the N2 fixation rates from recently published studies on photic and aphotic layers that have been attributed to NCD activity via parallel molecular measurements, and discuss the status, challenges, and data gaps in estimating non-cyanobacterial N2 fixation NCNF in the ocean. Rates attributed to NCNF have generally been near the detection limit thus far (<1 nmol N L-1 d-1). Yet, if considering the large volume of the dark ocean, even low rates of NCNF could make a significant contribution to the new nitrogen input to the ocean. The synthesis here shows that nifH transcription data for NCDs have been reported in only a few studies where N2 fixation rates were detected in the absence of diazotrophic cyanobacteria. In addition, high apparent diversity and regional variability in the NCDs complicate investigations of these communities. Future studies should focus on further investigating impacts of environmental drivers including oxygen, dissolved organic matter, and dissolved inorganic nitrogen on NCNF. Describing the ecology of NCDs and accurately measuring NCNF rates, are critical for a future evaluation of the contribution of NCNF to the marine nitrogen budget.

Seasonal variation in the copepod gut microbiome in the subtropical North Atlantic Ocean.

Characterisation of marine copepod gut microbiome composition and its variability provides information on function of marine food webs, biogeochemical cycles and copepod health. Copepod gut microbiomes were investigated quarterly over two years at the Bermuda Atlantic Time-series Station in the North Atlantic Subtropical Gyre, while assessing seasonal shifts in stable and transient communities. Microbial communities were analysed using amplicon sequencing targeting the bacterial 16S rRNA V3-V4 region and the cyanobacterial ntcA gene. Persistent bacterial groups belonging to Firmicutes, Bacteroidetes and Actinobacteria were present in the copepod guts throughout the year, and showed synchronous changes, suggesting a link to variability in copepod nutritional content. The gut communities were separate from those in the seawater, suggesting the copepod gut hosts long-term, specialized communities. Major temporal variations in the gut communities during the early winter and spring, specifically a high relative abundance of Synechococcus (up to 65%), were attributed to bacterioplankton shifts in the water column, and copepod grazing on these picoplanktonic cyanobacteria. The presence of obligate and facultative anaerobes, including Clostridiales year round, suggests that anaerobic bacterial processes are common in these dynamic microhabitats in the oligotrophic open ocean.

Mesopelagic N2 Fixation Related to Organic Matter Composition in the Solomon and Bismarck Seas (Southwest Pacific).

Dinitrogen (N2) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N2 fixation rates (up to ~1 nmol N L-1 d-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N2 fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N2 fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 103 nifH gene copies L-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.

Stable Associations Masked by Temporal Variability in the Marine Copepod Microbiome.

Copepod-bacteria interactions include permanent and transient epi- and endobiotic associations that may play roles in copepod health, transfer of elements in the food web, and biogeochemical cycling. Microbiomes of three temperate copepod species (Acartia longiremis, Centropages hamatus, and Calanus finmarchicus) from the Gulf of Maine were investigated during the early summer season using high throughput amplicon sequencing. The most prominent stable component of the microbiome included several taxa within Gammaproteobacteria, with Pseudoalteromonas spp. especially abundant across copepod species. These Gammaproteobacteria appear to be promoted by the copepod association, likely benefitting from nutrient enriched microenvironments on copepods, and forming a more important part of the copepod-associated community than Vibrio spp. during the cold-water season in this temperate system. Taxon-specific associations included an elevated relative abundance of Piscirickettsiaceae and Colwelliaceae on Calanus, and Marinomonas sp. in Centropages. The communities in full and voided gut copepods had distinct characteristics, thus the presence of a food-associated microbiome was evident, including higher abundance of Rhodobacteraceae and chloroplast sequences in the transient communities. The observed variability was partially explained by collection date that may be linked to factors such as variable time since molting, gender differences, and changes in food availability and type over the study period. While some taxon-specific and stable associations were identified, temporal changes in environmental conditions, including food type, appear to be key in controlling the composition of bacterial communities associated with copepods in this temperate coastal system during the early summer.

Nitrogenase expression in estuarine bacterioplankton influenced by organic carbon and availability of oxygen.

The genetic capacity to fix gaseous nitrogen (N) is distributed among diverse diazotrophs belonging to the Bacteria and Archaea. However, only a subset of the putative diazotrophs present actively fix N at any given time in the environment. We experimentally tested whether the availability of carbon and inhibition by oxygen constrain N fixation by diazotrophs in coastal seawater. The goal was to test whether by alleviating these constraints an increased overlap between nitrogenase (nifH)-gene-carrying and -expressing organisms could be achieved. We incubated water from a eutrophic but N-limited fjord in Denmark under high-carbon/low-oxygen conditions and determined bacterial growth and production, diazotrophic community composition (Illumina nifH amplicon sequencing), and nifH gene abundance and expression [quantitative PCR (qPCR) and quantitative reverse transcriptase PCR (qRT-PCR)]. Bacterial abundances and production increased under high-carbon/low-oxygen conditions as did the similarity between present and active diazotrophic communities. This was caused by the loss of specific abundant yet non-active gammaproteobacterial phylotypes and increased expression by others. The prominent active gamma- and epsilonproteobacterial diazotrophs did not, however, respond to these conditions in a uniform way, highlighting the difficulty to assess how a change in environmental conditions may affect a diverse indigenous diazotrophic community.

Microbial diversity associated with copepods in the North Atlantic subtropical gyre.

Patchiness of marine microbial communities has an important influence on microbial activities in the ocean, particularly in the oligotrophic open ocean where bioavailable nutrients are otherwise scarce. Such spatial heterogeneity is present in associations with dead and living particles, including zooplankton. The microbial community composition of mesozooplankton was investigated from the Sargasso Sea using 16S rRNA amplicon pyrosequencing. Zooplankton microbiomes were studied on the copepods Undinula vulgaris, Pleuromamma spp., Sapphirina metalina, Pseudocalanus spp. and Tigriopus sp., and an amphipod, Phrosina semilunata. The overall richness was lower in the zooplankton than in the seawater, and zooplankton-specific bacterial communities were distinct from the communities in seawater. Gammaproteobacteria dominated in all zooplankton studied, with Vibrio spp. highly represented. Firmicutes were detected in all copepods, providing evidence for anaerobic conditions present on the copepods. Bacterial groups known to grow on concentrated organic substrates or to prevent biofouling were highly represented in association with copepods, suggesting they benefit from copepod-derived nutrients or carbon. The described copepod microbiome has similarities to communities previously described in coastal copepods, suggesting some aspects of the copepod microbiome are not habitat specific. The communities are distinct of that in seawater, demonstrating significant microbial patchiness in association with marine zooplankton in the oligotrophic open ocean.