Everything Is Everywhere: Physiological Responses of the Mediterranean Sea and Eastern Pacific Ocean Epiphyte Cobetia Sp. to Varying Nutrient Concentration.

Bacteria are essential in the maintenance and sustainment of marine environments (e.g., benthic systems), playing a key role in marine food webs and nutrient cycling. These microorganisms can live associated as epiphytic or endophytic populations with superior organisms with valuable ecological functions, e.g., seagrasses. Here, we isolated, identified, sequenced, and exposed two strains of the same species (i.e., identified as Cobetia sp.) from two different marine environments to different nutrient regimes using batch cultures: (1) Cobetia sp. UIB 001 from the endemic Mediterranean seagrass Posidonia oceanica and (2) Cobetia sp. 4B UA from the endemic Humboldt Current System (HCS) seagrass Heterozostera chilensis. From our physiological studies, both strains behaved as bacteria capable to cope with different nutrient and pH regimes, i.e., N, P, and Fe combined with different pH levels, both in long-term (12 days (d)) and short-term studies (4 d/96 h (h)). We showed that the isolated strains were sensitive to the N source (inorganic and organic) at low and high concentrations and low pH levels. Low availability of phosphorus (P) and Fe had a negative independent effect on growth, especially in the long-term studies. The strain UIB 001 showed a better adaptation to low nutrient concentrations, being a potential N2-fixer, reaching higher growth rates (µ) than the HCS strain. P-acquisition mechanisms were deeply investigated at the enzymatic (i.e., alkaline phosphatase activity, APA) and structural level (e.g., alkaline phosphatase D, PhoD). Finally, these results were complemented with the study of biochemical markers, i.e., reactive oxygen species (ROS). In short, we present how ecological niches (i.e., MS and HCS) might determine, select, and modify the genomic and phenotypic features of the same bacterial species (i.e., Cobetia spp.) found in different marine environments, pointing to a direct correlation between adaptability and oligotrophy of seawater.

Higher biomolecules yield in phytoplankton under copper exposure.

Copper is an important metal for industry, and its toxic threshold in natural ecosystems has increased since the industrial revolution. As an essential nutrient, it is required in minute amounts, being toxic in slightly increased concentrations, causing great biochemical transformation in microalgae. This study aimed at investigating the physiology of Scenedesmus quadricauda, a cosmopolitan species, exposed to copper concentrations including those that trigger intracellular biochemical modifications. The Cu exposure concentrations tested ranged from 0.1 to 25 µM, thus including environmentally important levels. Microalgae cultures were kept under controlled environmental conditions and monitored daily for cell density, in vivo chlorophyll a, and photosynthetic quantum yield (ΦM). After 24 h growth, free Cu2+ ions were determined, and after 96 h, cellular Cu concentration, total carbohydrates, proteins, lipids, and cell volume were determined. The results showed that both free Cu2+ ions and cellular Cu increased with Cu increase in culture medium. Microalgae cell abundance and in vivo chlorophyll a were mostly affected at 2.5 µM Cu exposure (3.8 pg Cu cell-1) and above. Approximately 31% decrease of photosynthetic quantum yield was obtained at the highest Cu exposure concentration (25 µM; 25 pg Cu cell-1) in comparison with the control. However, at environmentally relevant copper concentrations (0.5 µM Cu; 0.4 pg Cu cell-1) cell volume increased in comparison with the control. Considering biomolecules accumulation per unit cell volume, the highest carbohydrates and proteins yield was obtained at 1.0 µM Cu (1.1 pg Cu cell-1), while for lipids higher Cu was necessary (2.5 µM Cu; 3.8 pg Cu cell-1). This study is a contribution to the understanding of the effects of environmentally significant copper concentrations in the physiology of S. quadricauda, as well as to biotechnological approach to increase biomolecule yield in microalgae production.

Cu and Cd affect distinctly the physiology of a cosmopolitan tropical freshwater phytoplankton.

Copper and Cd are natural constituents of freshwater ecosystems, both cycling influenced by microbial communities. The present research examined the impacts of environmentally relevant concentrations of Cu and Cd on the growth, viability, cell size, chlorophyll a (Chl a) content and photochemical efficiency of the tropical freshwater phytoplankton Chlorolobion braunii. Cell growth was significantly impaired by Cu and Cd, with EC50 occurring at 33.6 and 1.6µM, respectively. At sublethal levels (< EC50), cell death was already induced at 5µM Cu and 1µMCd. Average cell volume significantly increased as metal concentrations increased, as did the Chl a content per cell, although the Chl a content per unit volume decreased. Copper did not affect both the photosystem II (PSII) maximum quantum yield (ΦM) or the operational quantum yield (ΦE), while Cd significantly impacted ΦE, with EC50 occurring at 18.4µM. Different responses for Cu and Cd were obtained whether the photochemical fluorescence quenching (Qp) or non-photochemical quenching (Qn) were considered. Qp decreased after Cd addition, but was not altered after Cu addition. Qn values significantly increased after the addition of either metal. Non-photochemical quenching due to heat dissipation (NPQ) significantly increased in response to both metals, but it was more pronounced in the case of Cd. Overall, Cd was more toxic to C. braunii than Cu.

Airborne organic pollutants impact microbial communities in temperate and Antarctic seawaters.

Airborne Organic Pollutants (AOPs) reach remote oceanic regions after long range atmospheric transport and deposition, incorporating into natural microbial communities. This study investigated the effects of AOPs on natural microbial communities of the Mediterranean Sea, the Atlantic Ocean and the Bellingshausen Sea, by assessing the impact of both non-polar and polar AOPs on cell abundances, chlorophyll a concentrations and cell viabilities of different microbial groups. Our results indicate that almost all groups, except flagellates in the Bellingshausen Sea, were significantly affected by AOPs. While no significant differences in chlorophyll a concentrations were observed between non-polar and polar AOPs, significant variations in cell abundances were noted. Cell death occurred at AOP concentrations as low as five times the oceanic field levels, likely due to their high chemical activity. Cyanobacteria in temperate waters exhibited the highest sensitivity to AOPs, whereas medium and larger diatoms in the Bellingshausen Sea were more affected than smaller diatoms or flagellates, contrary to the expected size-related sensitivity trend. Additionally, microorganisms in temperate waters were more sensitive to the polar fraction of AOPs compared to the non-polar fraction, which showed an inverse sensitivity pattern. This differential sensitivity is attributed to variations in the ratio of polar to non-polar AOPs in the respective environments. Our findings underscore the varying impacts of AOPs on marine microbial communities across different oceanic regions.

Toxicity of inorganic nanoparticles and commercial sunscreens on marine bacteria.

The Balearic Islands, a top tourist destination for sunny beaches, face physical and chemical pressures from human activities, impacting keystone species like the endemic seagrass Posidonia oceanica and its associated microbiome. This study evaluated the effects of ZnO and TiO2 nanoparticles and three commercial sunscreens with varying protection factors (50 or 90) and chemical complexities (1- SPF50_E "eco-friendly"; 2- SPF50 not "eco-friendly"; 3- SPF90 not "eco-friendly") on five heterotrophic bacteria (Pseudomonas azotifigens, Marinobacterium litorale, Thiothrix nivea, Sedimenticola thiotaurini and Cobetia sp) and two autotrophic cyanobacteria (Halothece sp. and Fischerella muscicola) associated to P. oceanica, as well as a natural leaf epiphytic community. Results indicated that TiO2 affected all heterotrophic bacteria, while ZnO was toxic to only two species, while autotrophs were unaffected. Commercial sunscreens impacted three heterotrophs and the natural epiphytic community, while autotrophs were only affected by SPF50. SPF50_E reduced phosphorus uptake, and both SPF50 and SPF90 decreased alkaline phosphatase activity. Reactive oxygen species production was mainly induced by SPF90, followed by SPF50_E and SPF50. Generally, the smallest bacteria were most sensitive to UV-filters (UVFs). This study indicates that UVFs exposure may alter the epiphytic community structure of P. oceanica.

Physiological responses of Humboldt current system diatoms to Fe and Cu co-limitation.

Diatoms account for ∼20% of global primary production, often limited by the availability of Fe and other trace nutrients such as Cu. The present study examined the role of both metals in the physiology of two diatoms isolated from the Humboldt Currents System, the centric Chaetoceros c.f. dicipiens and the pennate Nitzschia c.f. draveillensis. Under Fe limitation, a decrease in specific growth rates and sizes of both species was observed, especially in Chaetoceros. However, regarding different photosynthetic parameters, Nitzschia was more impacted. The increase in Cu concentrations improved the physiology of both diatoms, mostly of Chaetoceros. When grown in mixed cultures and under co-limiting conditions, both species remained competive due to morphological advantages (i.e., lower cell size). These results may suggest that the increase of Cu under Fe limitation benefited C. c.f. dicipiens over N. c.f. draveillensis.

Cell size matters: Nano- and micro-plastics preferentially drive declines of large marine phytoplankton due to co-aggregation.

Marine plastic pollution represents a key environmental concern. Whilst ecotoxicological data for plastic is increasingly available, its impact upon marine phytoplankton remains unclear. Owing to their predicted abundance in the marine environment and likely interactions with phytoplankton, here we focus on the smaller fraction of plastic particles (~50 nm and ~2 µm polystyrene spheres). Exposure of natural phytoplankton communities and laboratory cultures revealed that plastic exposure does not follow traditional trends in ecotoxicological research, since large phytoplankton appear particularly susceptible towards plastics exposure despite their lower surface-to-volume ratios. Cell declines appear driven by hetero-aggregation and co-sedimentation of cells with plastic particles, recorded visually and demonstrated using confocal microscopy. As a consequence, plastic exposure also caused disruption to photosynthetic functioning, as determined by both photosynthetic efficiency and high throughput proteomics. Negative effects upon phytoplankton are recorded at concentrations orders of magnitude above those estimated in the environment. Hence, it is likely that impacts of NPs and MPs are exacerbated at the high concentrations typically used in ecotoxicological research (i.e., mg L-1).

"The Good, the Bad and the Double-Sword" Effects of Microplastics and Their Organic Additives in Marine Bacteria.

Little is known about the direct effects of microplastics (MPs) and their organic additives on marine bacteria, considering their role in the nutrient cycles, e.g., N-cycles through the N2-fixation, or in the microbial food web. To fill this gap of knowledge, we exposed marine bacteria, specifically diazotrophs, to pure MPs which differ in physical properties (e.g., density, hydrophobicity, and/or size), namely, polyethylene, polypropylene, polyvinyl chloride and polystyrene, and to their most abundant associated organic additives (e.g., fluoranthene, 1,2,5,6,9,10-hexabromocyclododecane and dioctyl-phthalate). Growth, protein overproduction, direct physical interactions between MPs and bacteria, phosphorus acquisition mechanisms and/or N2-fixation rates were evaluated. Cyanobacteria were positively affected by environmental and high concentrations of MPs, as opposed to heterotrophic strains, that were only positively affected with high concentrations of ~120 µm-size MPs (detecting the overproduction of proteins related to plastic degradation and C-transport), and negatively affected by 1 µm-size PS beads. Generally, the organic additives had a deleterious effect in both autotrophic and heterotrophic bacteria and the magnitude of the effect is suggested to be dependent on bacterial size. Our results show species-specific responses of the autotrophic and heterotrophic bacteria tested and the responses (beneficial: the "good," deleterious: the "bad" and/or both: the "double-sword") were dependent on the type and concentration of MPs and additives. This suggests the need to determine the threshold levels of MPs and additives concentrations starting from which significant effects can be observed for key microbial populations in marine systems, and these data are necessary for effective environmental quality control management.

Differences in the sensitivity to Cu and ligand production of coastal vs offshore strains of Emiliania huxleyi.

Copper is an essential trace metal for different physiological processes in phytoplankton, being either a limiting or toxic element depending on its bioavailability, which may induce local physiological adaptations. Atmospheric Cu deposition to the oceans can negatively impact phytoplankton growth, with the most Cu-sensitive phytoplankton exhibiting differences based on coastal vs oceanic origin. The goal of this work was to analyze sensitivity to Cu toxicity of the cosmopolitan marine calcifying phytoplankton, Emiliania huxleyi, exploring what factors determine intraspecific variability in sensitivity. We compared 17 strains isolated from coastal and open ocean waters of the Eastern South Pacific (ESP), the Mediterranean Sea, and the Tasman Sea. Offshore strains were as sensitive to Cu than coastal strains. Sensitivity to Cu was explained well by predicted depositional inputs of atmospheric Cu in the ESP both for coastal and offshore strains, but not when considered globally. The variability in Cu-sensitivity was also due to the production of organic Cu-ligands (CL), being the most productive strains the most tolerant to Cu at constitutive levels. When exposed to 100nM Cu, E. huxleyi produced significantly higher amounts of CL, especially coastal strains, but CL production did not correlate to observed EC50s.

Toxicity of natural mixtures of organic pollutants in temperate and polar marine phytoplankton.

Semivolatile and persistent organic pollutants (POPs) undergo atmospheric transport before being deposited to the oceans, where they partition to phytoplankton organic matter. The goal of this study was to determine the toxicity of naturally occurring complex mixtures of organic pollutants to temperate and polar phytoplankton communities from the Mediterranean Sea, the North East (NE) Atlantic, and Southern Oceans. The cell abundance of the different phytoplankton groups, chlorophyll a concentrations, viability of the cells, and growth and decay constants were monitored in response to addition of a range of concentrations of mixtures of organic pollutants obtained from seawater extracts. Almost all of the phytoplankton groups were significantly affected by the complex mixtures of non-polar and polar organic pollutants, with toxicity being greater for these mixtures than for single POPs or simple POP mixtures. Cocktails' toxicity arose at concentrations as low as tenfold the field oceanic levels, probably due to a higher chemical activity of the mixture than of simple POPs mixtures. Overall, smaller cells were the most affected, although Mediterranean picophytoplankton was significantly more tolerant to non-polar POPs than picophytoplankton from the Atlantic Ocean or the Bellingshausen Sea microphytoplankton.

Cell size dependence of additive versus synergetic effects of UV radiation and PAHs on oceanic phytoplankton.

Polycyclic Aromatic Hydrocarbons' (PAHs) toxicity is enhanced by the presence of ultraviolet radiation (UVR), which levels have arisen due to the thinning of the ozone layer. In this study, PAHs' phototoxicity for natural marine phytoplankton was tested. Different concentrations of a mixture of 16 PAHs were added to natural phytoplankton communities from the Mediterranean Sea, Atlantic, Arctic and Southern Oceans and exposed to natural sunlight received in situ, including treatments where the UVR bands were removed. PAHs' toxicity was observed for all the phytoplankton groups studied in all the waters and treatments tested, but only for the pico-sized group a synergetic effect of the mixture and UVR was observed (p=0.009). When comparing phototoxicity in phytoplankton from oligotrophic and eutrophic waters, synergy was only observed at the oligotrophic communities (p=0.02) where pico-sized phytoplankton dominated. The degree of sensitivity was related to the trophic degree, decreasing as Chlorophyll a concentration increased.

Cell size dependent toxicity thresholds of polycyclic aromatic hydrocarbons to natural and cultured phytoplankton populations.

The toxicity of pyrene and phenanthrene to phytoplankton was studied by analyzing the effect on the growth, abundance and cell viability of cultured species and natural communities of the Atlantic Ocean and the Mediterranean Sea. A decrease in cell abundance, and growth rate was observed as concentration of PAHs increased, with catastrophic cell mortality induced at the highest PAH concentration tested. A strong positive linear relationship was observed between the LC50 (the PAH concentration at which cell population will decline by a half), and the species cell volume, for both phenanthrene and pyrene. Natural communities were however significantly more sensitive to PAHs than cultured phytoplankton, as indicated by the lower slope (e.g. 0.23 and 0.65, respectively, for pyrene) of the relationship LC50 vs. cell volume. The results highlight the importance of cell size in determining the phytoplankton sensitivity to PAHs identifying the communities from the oligotrophic ocean to be more vulnerable.

Decrease in the abundance and viability of oceanic phytoplankton due to trace levels of complex mixtures of organic pollutants.

Long range atmospheric transport and deposition is a significant introduction pathway of organic pollutants to remote oceanic regions, leading to their subsequent accumulation in marine organisms. Persistent organic pollutants (POPs) bioconcentrate in planktonic food webs and these exert a biogeochemical control on the regional and global cycling of POPs. Therefore, an important issue is to determine whether the anthropogenic chemical perturbation of the biosphere introduced by the myriad of organic pollutants present in seawater influences phytoplankton abundance and productivity. The results reported here from five sets of experiments performed in the NE Atlantic Ocean show that there is a toxic effect induced by trace levels of complex mixtures of organic pollutants on phytoplankton oceanic communities. The levels of single pollutant, such as phenanthrene and pyrene, at which lethality of phytoplankton is observed are high in comparison to field levels. Complex mixtures of organic pollutants, however, have an important toxic effect on phytoplankton abundances, viability and concentrations of Chlorophyll a at pollutant concentrations 20-40 folds those found in the open ocean. The toxicity of these complex mixtures of organic pollutants exceeds by 10(3) times the toxicity expected for a single pollutant. Therefore, our results point out the need for a systematic investigation of the influence of complex mixtures of organic hydrophobic pollutants to oceanic phytoplankton communities, a perturbation not accounted for on previous assessments of anthropogenic pressures in the marine environment.