Metabolic engineering of omega-3 long chain polyunsaturated fatty acids in plants using different ∆6- and ∆5-desaturases co-expressed with LPCAT from the marine diatom Phaeodactylum tricornutum.

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆6-desaturases from diatom Phaeodactylum tricornutum, fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆5-desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp. The in planta agroinfiltration of essential ∆6-desaturases, ∆6-elongases and ∆5-desaturases allowed for successful introduction of eicosapentaenoic acid (20:5∆5,8,11,14,17) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called 'substrate dichotomy' for improving the transgenic production of VLC-PUFAs in plants.

Chromosome-level genome assembly of marine diatom Skeletonema tropicum.

Skeletonema tropicum is a marine diatom of the genus Skeletonema that also includes many well-known species including S. marinoi. S. tropicum is a high temperature preferring species thriving in tropical ocean regions or temperate ocean regions during summer-autumn. However, mechanisms of ecological adaptation of S. tropicum remain poorly understood due partially to the lack of a high-quality whole genome assembly. Here, we report the first high-quality chromosome-scale genome assembly for S. tropicum, using cutting-edge technologies including PacBio single molecular sequencing and high-throughput chromatin conformation capture. The assembled genome has a size of 78.78 Mb with a scaffold N50 of 3.17 Mb, anchored to 23 pseudo-chromosomes. In total, 20,613 protein-coding genes were predicted, of which 17,757 (86.14%) genes were functionally annotated. Collinearity analysis of the genomes of S. tropicum and S. marinoi revealed that these two genomes were highly homologous. This chromosome-level genome assembly of S. tropicum provides a valuable genomic platform for comparative analysis of mechanisms of ecological adaption.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Temperature-dependent toxicity of fluoxetine alters the thermal plasticity of marine diatoms.

Anthropogenic activities have led to the emergence of pharmaceutical pollution in marine ecosystems, posing a significant threat to biodiversity in conjunction with global climate change. While the ecotoxicity of human drugs on aquatic organisms is increasingly recognized, their interactions with environmental factors, such as temperature, remain understudied. This research investigates the physiological effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, on two diatom species, Phaeodactylum tricornutum and Thalassiosira weissflogii. Results demonstrate that fluoxetine significantly reduces growth rate and biomass production, concurrently affecting pigment contents and the thermal performance curve (TPC) of the diatoms. Fluoxetine reduces the synthesis of chlorophyll a (Chl a) and carotenoid (Car), indicating inhibition of photosynthesis and photoprotection. Furthermore, fluoxetine decreases the maximum growth rate (µmax) while increasing the optimum temperature (Topt) in both species, suggesting an altered thermal plasticity. This shift is attributed to the observed decrease in the inhibition rate of fluoxetine with rising temperatures. These findings emphasize the physiological impacts and ecological implications of fluoxetine on phytoplankton and underscore the significance of considering interactions between multiple environmental drivers when accessing the ecotoxicity of potential pollutants. The present study provides insights into crucial considerations for evaluating the impacts of pharmaceutical pollution on marine primary producers.

Preliminary investigation on the causes of red tides in Qinhuangdao coastal areas in 2022.

To explore the causes of red tides in Qinhuangdao coastal water, we conducted surveys on both water quality and red tides during April to September of 2022 and analyzed the relationships between main environmental factors and red tide organisms through the factor analysis and canonical correspondence analysis. The results showed that there were eight red tides along the coast of Qinhuangdao in 2022, with a cumulative blooming area of 716.1 km2. The red tides could be divided into three kinds based on the major blooming organisms and occurrence time, Noctiluca scintillans bloom, diatom-euglena (Skeletonema costatum, Eutreptiella gymnastica, Pseudo-nitzschia spp.) bloom, and dinoflagellate (Scrippsiella trochoidea and Ceratium furca) bloom. Seasonal factor played roles mainly during July to September, while inorganic nutrients including nitrogen and phosphorus influenced the blooms mainly in April and July. The canonical correspondence analysis suggested that N. scintillans preferred low temperature, and often bloomed with high concentrations of ammonium nitrogen and dissolved inorganic phosphorus. S. costatum, E. gymnastica, and Pseudo-nitzschia spp. could tolerate broad ranges of various environmental factors, but favored high temperature and nitrogen-rich seawater. C. furca and S. trochoidea had higher survival rate and competitiveness in phosphate-poor waters. Combined the results from both analyses, we concluded that the causes for the three kinds of red tide processes in Qinhuangdao coastal areas in 2022 were different. Adequate diet algae and appropriate water temperature were important factors triggering and maintaining the N. scintillans bloom. Suitable temperature, salinity and eutrophication were the main reasons for the diatom-euglena bloom. The abundant nutrients and seawater disturbance promoted the germination of S. trochoidea cysts, while phosphorus limitation caused the blooming organism switched to C. furca and maintained the bloom hereafter.

Resting cells of Skeletonema marinoi assimilate organic compounds and respire by dissimilatory nitrate reduction to ammonium in dark, anoxic conditions.

Diatoms can survive long periods in dark, anoxic sediments by forming resting spores or resting cells. These have been considered dormant until recently when resting cells of Skeletonema marinoi were shown to assimilate nitrate and ammonium from the ambient environment in dark, anoxic conditions. Here, we show that resting cells of S. marinoi can also perform dissimilatory nitrate reduction to ammonium (DNRA), in dark, anoxic conditions. Transmission electron microscope analyses showed that chloroplasts were compacted, and few large mitochondria had visible cristae within resting cells. Using secondary ion mass spectrometry and isotope ratio mass spectrometry combined with stable isotopic tracers, we measured assimilatory and dissimilatory processes carried out by resting cells of S. marinoi under dark, anoxic conditions. Nitrate was both respired by DNRA and assimilated into biomass by resting cells. Cells assimilated nitrogen from urea and carbon from acetate, both of which are sources of dissolved organic matter produced in sediments. Carbon and nitrogen assimilation rates corresponded to turnover rates of cellular carbon and nitrogen content ranging between 469 and 10,000 years. Hence, diatom resting cells can sustain their cells in dark, anoxic sediments by slowly assimilating and respiring substrates from the ambient environment.

Distribution patterns and co-occurrence network of eukaryotic algae in different salinity waters of Yuncheng Salt Lake, China.

The community structure and co-occurrence pattern of eukaryotic algae in Yuncheng Salt Lake were analyzed based on marker gene analysis of the 18S rRNA V4 region to understand the species composition and their synergistic adaptations to the environmental factors in different salinity waters. The results showed indicated that the overall algal composition of Yuncheng Salt Lake showed a Chlorophyta-Pyrrophyta-Bacillariophyta type structure. Chlorophyta showed an absolute advantage in all salinity waters. In addition, Cryptophyta dominated in the least saline waters; Pyrrophyta and Bacillariophyta were the dominant phyla in the waters with salinity ranging from 13.2 to 18%. Picochlorum, Nannochloris, Ulva, and Tetraselmis of Chlorophyta, Biecheleria and Oxyrrhis of Pyrrophyta, Halamphora, Psammothidium, and Navicula of Bacillariophyta, Guillardia and Rhodomonas of Cryptophyta were not observed in previous surveys of the Yuncheng Salt Lake, suggesting that the algae are undergoing a constant turnover as the water environment of the Salt Lake continues to change. The network diagram demonstrated that the algae were strongly influenced by salinity, NO3-, and pH, changes in these environmental factors would lead to changes in the algal community structure, thus affecting the stability of the network structure.

Effect of seasonality and estuarine waters on the phytoplankton of the Guamá River (Belém, Amazon, Brazil).

This study aimed to analyze the application of the Phytoplankton Community Index-PCI and Functional Groups-FG in determining the water quality of the Guamá River (Pará, Amazônia, Brazil). Samplings occurred monthly for analyses of phytoplankton and physical and chemical parameters, for two years, at the station where water was collected for human supply consumption. Seasonality influenced electrical conductivity, total suspended solids, dissolved oxygen, transparency, winds, true color, and N-ammoniacal. The ebb tide showed high turbidity and suspended solids. The density varied seasonally with the highest values occurring in September and December (61.1 ind mL-1 and 60.2 ind mL-1, respectively). Chlorophyll-a was more elevated in December (21.0 ± 4.7 µg L-1) and chlorophyll-c higher in relation to clorophyll- b indicated the dominance of diatoms. Functional Group P prevailed in the study months. Through the PCI índex the waters of Guamá River varied from reasonable to excellent and the TSI ranged from oligo to mesotrophic. The use of Functional Groups proved to be a promising tool in the determination of water quality since it covered the most abundant species in the Environment, but the PCI is not adequate to characterize Amazonian white-waters rivers, which have diatoms as the leading dominant group.

Development of a new kappa-carrageenan hydrogel system to study benthic diatom vertical movements.

Benthic diatom vertical movement has been investigated mainly through indirect measurements based on chlorophyll a fluorescence and spectral reflectance signals. The presence of sediment hinders direct imaging and grazers activity renders the work under controlled conditions very difficult. This study provides a tool to study diatoms movement in a 3D hydrogel matrix. Synthetic and natural hydrogels were tested to find the best 3D transparent scaffold where diatoms could grow and freely move in all directions. Polyamidoamines (PAAm) hydrogels were no-cytocompatible and hyaluronic acid (HA) only allowed diatoms to survive for 2-days. Natural hydrogels made of gelatin/Na-alginate, Na-alginate and kappa-carrageenan (KC) were cytocompatible, with KC showing the best properties for diatom growth and movement on a long term (up to 2 months). Comparing Nitzschia spathulata, Gyrosigma limosum and Navicula phyllepta growth in liquid media vs in KC gels, we found that diatoms reached a significantly higher final biomass in the hydrogel condition. Hydrogels were also useful to isolate large size diatom species e.g., Nitzschia elongata, that did not survive in suspension. Finally, we showed three ways to study diatom species-specific movement in KC hydrogels: 1) controlled species mix; 2) natural diatom assemblages with grazers; and 3) natural diatom assemblages without grazers. With our system, single diatoms could be imaged, identified, and counted. In addition, different stimuli, e.g., light intensity and light composition can be applied and their effects on movement and physiology studied without being masked by sediment or impaired by meiofauna.

Microbial community structure and diversity attached to the periphyton in different urban aquatic habitats.

Periphyton is a complex community composed of diverse prokaryotes and eukaryotes; understanding the characteristics of microbial communities within periphyton becomes crucial for biogeochemical cycles and energy dynamics of aquatic ecosystems. To further elucidate the community characteristics of periphyton across varied aquatic habitats, including unpolluted ecologically restored lakes, aquaculture ponds, and areas adjacent to domestic and industrial wastewater treatment plant outfalls, we explored the composition and diversity of prokaryotic and eukaryotic communities in periphyton by employing Illumina MiSeq sequencing. Our findings indicated that the prokaryotic communities were predominantly composed of Proteobacteria (40.92%), Bacteroidota (21.01%), and Cyanobacteria (10.12%), whereas the eukaryotic communities were primarily characterized by the dominance of Bacillariophyta (24.09%), Chlorophyta (20.83%), and Annelida (15.31%). Notably, Flavobacterium emerged as a widely distributed genus among the prokaryotic community. Unclassified_Tobrilidae exhibited higher abundance in unpolluted ecologically restored lakes. Chaetogaster and Nais were enriched in aquaculture ponds and domestic wastewater treatment plant outfall area, respectively, while Surirella and Gomphonema dominated industrial sewage treatment plant outfall area. The alpha diversity of eukaryotes was higher in unpolluted ecologically restored lakes. pH and nitrogen content ( NO 2 - - N , NO 3 - - N , and TN) significantly explained the variations for prokaryotic and eukaryotic community structures, respectively. Eukaryotic communities exhibited a more pronounced response to habitat variations compared to prokaryotic communities. Moreover, the association networks revealed an intensive positive correlation between dominant Bacillariophyta and Bacteroidota. This study provided useful data for identifying keystone species and understanding their ecological functions.

Multi-scale fractal Fourier Ptychographic microscopy to assess the dose-dependent impact of copper pollution on living diatoms.

Accumulation of bioavailable heavy metals in aquatic environment poses a serious threat to marine communities and human health due to possible trophic transfers through the food chain of toxic, non-degradable, exogenous pollutants. Copper (Cu) is one of the most spread heavy metals in water, and can severely affect primary producers at high doses. Here we show a novel imaging test to assay the dose-dependent effects of Cu on live microalgae identifying stress conditions when they are still capable of sustaining a positive growth. The method relies on Fourier Ptychographic Microscopy (FPM), capable to image large field of view in label-free phase-contrast mode attaining submicron lateral resolution. We uniquely combine FPM with a new multi-scale analysis method based on fractal geometry. The system is able to provide ensemble measurements of thousands of diatoms in the liquid sample simultaneously, while ensuring at same time single-cell imaging and analysis for each diatom. Through new image descriptors, we demonstrate that fractal analysis is suitable for handling the complexity and informative power of such multiscale FPM modality. We successfully tested this new approach by measuring how different concentrations of Cu impact on Skeletonema pseudocostatum diatom populations isolated from the Sarno River mouth.

Unveiling the ecological resilience and industrial potential of Skeletonema marinoi through mixotrophic cultivation in Nordic winter condition.

Mixotrophy, the concurrent use of inorganic and organic carbon in the presence of light for microalgal growth, holds ecological and industrial significance. However, it is poorly explored in diatoms, especially in ecologically relevant species like Skeletonema marinoi. This study strategically employed mixotrophic metabolism to optimize the growth of a strain of Skeletonema marinoi (Sm142), which was found potentially important for biomass production on the west coast of Sweden in winter conditions. The aim of this study was to discern the most effective organic carbon sources by closely monitoring microalgal growth through the assessment of optical density, chlorophyll a fluorescence, and biomass concentration. The impact of various carbon sources on the physiology of Sm142 was investigated using photosynthetic and respiratory parameters. The findings revealed that glycerol exhibited the highest potential for enhancing the biomass concentration of Sm142 in a multi-cultivator under the specified experimental conditions, thanks to the increase in respiration activity. Furthermore, the stimulatory effect of glycerol was confirmed at a larger scale using environmental photobioreactors simulating the winter conditions on the west coast of Sweden; it was found comparable to the stimulation by CO2-enriched air versus normal air. These results were the first evidence of the ability of Skeletonema marinoi to perform mixotrophic metabolism during the winter and could explain the ecological success of this diatom on the Swedish west coast. These findings also highlight the importance of both organic and inorganic carbon sources for enhancing biomass productivity in harsh winter conditions.

Characterization of polyphenols and carbohydrates exuded by Phaeodactylum tricornutum diatom grown under Cu stress.

This study is focused on analysing polyphenols and carbohydrates released by Phaeodactylum tricornutum (P. tricornutum) diatoms cultured in natural seawater enriched with sublethal and lethal Cu doses. Cu concentrations of 0.31, 0.79 and 1.57 µM reduced cell densities by 37, 82 and 91%, respectively, compared to the control. The total sum of all identified polyphenols and total carbohydrates released by cells grown under lethal Cu levels increased up to 18.8 and 107.4 times, respectively, compared to data from a control experiment. Four different in vitro assays were used to estimate the antioxidant activities of the extracellular compounds: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition, cupric ion reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power and Cu complexing ability (CCA). The highest antioxidant activities were observed in the Cu lethal treatments, where the CCA assay exhibited a greater increase (up to 32.2 times higher than that found in the control experiment) to reduce the concentration of free Cu in the medium and its toxicity. The presence of Cu stimulated the release of polyphenols and carbohydrates to the medium as a detoxification mechanism to survive under lethal levels of Cu regulating its speciation.

Multiannual patterns of genetic structure and mating type ratios highlight the complex bloom dynamics of a marine planktonic diatom.

Understanding the genetic structure of populations and the processes responsible for its spatial and temporal dynamics is vital for assessing species' adaptability and survival in changing environments. We investigate the genetic fingerprinting of blooming populations of the marine diatom Pseudo-nitzschia multistriata in the Gulf of Naples (Mediterranean Sea) from 2008 to 2020. Strains were genotyped using microsatellite fingerprinting and natural samples were also analysed with Microsatellite Pool-seq Barcoding based on Illumina sequencing of microsatellite loci. Both approaches revealed a clonal expansion event in 2013 and a more stable genetic structure during 2017-2020 compared to previous years. The identification of a mating type (MT) determination gene allowed to assign MT to strains isolated over the years. MTs were generally at equilibrium with two notable exceptions, including the clonal bloom of 2013. The populations exhibited linkage equilibrium in most blooms, indicating that sexual reproduction leads to genetic homogenization. Our findings show that P. multistriata blooms exhibit a dynamic genetic and demographic composition over time, most probably determined by deeper-layer cell inocula. Occasional clonal expansions and MT imbalances can potentially affect the persistence and ecological success of planktonic diatoms.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

Determining the Subcellular Localization of Proteins in the Different Membranes of Diatom Secondary Plastid.

Diatoms such as Phaeodactylum tricornutum arose through a process termed secondary endosymbiosis, in which red alga-derived plastids are surrounded by a complicated membrane system. Subcellular marker proteins provide defined localizations on the compartmental and even sub-compartmental levels in the complex plastids of diatoms. Here we introduce how to use subcellular marker proteins and in vivo co-localization in the diatom P. tricornutum by presenting a step-by-step method allowing the determination of subcellular localization of proteins in different membranes of the secondary plastid. This chapter describes the materials required and the procedures of transformation and microscopic observation.

Targeted Gene Editing of Nuclear-Encoded Plastid Proteins in Phaeodactylum tricornutum via CRISPR/Cas9.

Genome modifications in microalgae have emerged as a crucial and indispensable tool for research in fundamental and applied biology. In particular, CRISPR/Cas9 has gained significant recognition as a highly effective method for genome engineering in these photosynthetic organisms, enabling the targeted induction of mutations in specific regions of the genome. Here, we present a comprehensive protocol for generating knock-out mutants in the model diatom Phaeodactylum tricornutum using CRISPR/Cas9 by both biolistic transformation and bacterial conjugation. Our protocol outlines the step-by-step procedures and experimental conditions required to achieve successful genome editing, including the design and construction of guide RNAs, the delivery of CRISPR/Cas9 components into the algae cells, and the selection of the generated knockout mutants. Through the implementation of this protocol, researchers can harness the potential of CRISPR/Cas9 in P. tricornutum to advance the understanding of diatom biology and explore their potential applications in various fields.

Influence of the industrial pollutant on water quality, radioactivity levels, and biological communities in Ismailia Canal, Nile River, Egypt.

In the twenty-first century, numerous forms of pollution have adversely impacted freshwater and the entire aquatic ecosystem. The higher population density in urban areas also contributes to increased releases of substances and thermal contaminants, significantly stressing the ecosystem of industrial companies. This study aimed to assess the potential pressure of industrial and municipal activities on water quality, radioactivity levels, and biological diversity, focusing on the consequences of radionuclides on periphytic diatom communities. Furthermore, the environmental impact of pollutants will be evaluated to monitor the ecological condition of the Ismailia Canal. Chemical analyses employed various instruments and methods to identify and quantify matter, with radionuclide elements measured by gamma spectrometry and diatoms counted and identified by inverted microscopy. Our results revealed that the canal was classified as excellent for irrigation, aquatic life, and drinking water based on FAO, CCME, and EWQS water quality indices, with high nutrient levels at Abu Za'baal fertilizer company. The activity concentration of 226Ra-series, 232Th-series, and 40K in the water and sediment samples for two seasons was within the guideline values, except for a few stations in the zone [B] (the industrial zone). Fertilizer samples (raw material) showed a high value of the 226Ra-series activity. Diatom community structure significantly varied across the different canal locations regarding the presence or absence of industrial activities, with no discernible variations between the study seasons. A specific variety of algal species was found to be predominant at the highest radioactive sites. Redundancy analysis (RDA) showed a significant correlation between parameters (pH, Na, TDS, PO4, SO4, SiO2, K, and CO3), radionuclides, environmental conditions, and the composition of the diatom community, especially in the area affected by industrial discharges. Moreover, the radiological hazard index in water and sediment remained below the maximum for two seasons. This research provides valuable data and information for communities and decision-makers, suggesting the strategic use of phycoremediation as a water biotreatment process to protect the valuable economic resources of the Ismailia Canal.

Reactive oxygen species mediated extracellular polymeric substances production assisting the recovery of Thalassiosira pseudonana from polystyrene micro and nanoplastics exposure.

As emerging pollutants in the aquatic environments, micro- and nano-plastics (MNPs) aroused widespread environmental concerns for their potential threats to the ecological health. Previous research has proved that microalgae growth could recover from the MNPs toxicities, in which the extracellular polymeric substances (EPS) might play the key role. In order to comprehensively investigate the recovery process of microalgae from MNPs stress and the effecting mechanisms of EPS therein, this study conducted a series of experiments by employing two sizes (0.1 and 1 µm) of polystyrene (PS) MNPs and the marine model diatom Thalassiosira pseudonana during 14 days. The results indicated: the pigments accumulations and photosynthetic recovery of T. pseudonana under MPs exposure showed in the early stage (4-5 days), while the elevation of reactive oxygen species (ROS) and EPS contents lasted longer time period (7-8 days). EPS was aggregated with MNPs particles and microalgal cells, corresponding to the increased settlement rates. More increase of soluble (SL)-EPS contents was found than bound (B)-EPS under MNPs exposure, in which the increase of the protein proportion and humic acid-like substances in SL-EPS was found, thus facilitating aggregates formation. ROS was the signaling molecule mediating the overproduction of EPS. The transcriptional results further proved the enhanced EPS biosynthesis on the molecular level. Therefore, this study elucidated the recovery pattern of microalgae from MNPs stress and linked "ROS-EPS production changes-aggregation formation" together during the growth recovery process, with important scientific and environmental significance.

Ocean acidification and desalination increase the growth and photosynthesis of the diatom Skeletonema costatum isolated from the coastal water of the Yellow Sea.

Global climate changes induce substantial alterations in the marine system, including ocean acidification (OA), desalination and warming of surface seawater. Here, we examined the combined effects of OA and reduced salinity under different temperatures on the growth and photosynthesis of the diatom Skeletonema costatum. After having been acclimated to 2 CO2 concentrations (400 µatm, 1000 µatm) and 2 salinity levels (20 psu, 30 psu) at temperature levels of 10 °C and 20 °C, the diatom showed enhanced growth rate at the lowered salinity and elevated pCO2 irrespective of the temperature. The OA treatment increased the net photosynthetic rate and biogenic silica (Bsi) contents. Increasing the temperature from 10 to 20 °C raised the net photosynthetic rate by over twofold. The elevated pCO2 increased the net and gross photosynthetic rates by 20%-40% and by 16%-32%, respectively, with the higher enhancement observed at the higher levels of salinity and temperature. Our results imply that OA and desalination along with warming to the levels tested can enhance S. costatum's competitiveness in coastal phytoplankton communities under influence of future climate changes.