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Rapid light curves are one of the most widely used methods for assessing the physiological state of photosynthetic organisms. While the method has been applied in a range of physiological studies over the last 20 years, little progress has been made in adapting it for the new age of multi-parametric phenotyping. In order to advance research that is aimed at evaluating the physiological impact of multiple factors, the Phenoplate was developed: a simultaneous assessment of temperature and light gradients. It was used to measure rapid light curves of three marine microalgae across a temperature gradient and altered phosphate availability. The results revealed that activation of photoprotective mechanisms occurred with high efficiency at lower temperatures, and relaxation of photoprotection was negatively impacted above a certain temperature threshold in Tetraselmis sp. It was observed that Thalassiosira pseudonana and Nannochloropsis oceanica exhibited two unique delayed non-photochemical quenching signatures: in combinations of low light with low temperature, and darkness with high temperature, respectively. These findings demonstrate that the Phenoplate approach can be used as a rapid and simple tool to gain insight into the photobiology of microalgae.

Phytoplankton are key components of the oceanic carbon and sulfur cycles1. During bloom events, some species can emit large amounts of the organosulfur volatile dimethyl sulfide (DMS) into the ocean and consequently the atmosphere, where it can modulate aerosol formation and affect climate2,3. In aquatic environments, DMS plays an important role as a chemical signal mediating diverse trophic interactions. Yet, its role in microbial predator–prey interactions remains elusive with contradicting evidence for its role in either algal chemical defence or in the chemo-attraction of grazers to prey cells4,5. Here we investigated the signalling role of DMS during zooplankton–algae interactions by genetic and biochemical manipulation of the algal DMS-generating enzyme dimethylsulfoniopropionate lyase (DL) in the bloom-forming alga Emiliania huxleyi6. We inhibited DL activity in E. huxleyi cells in vivo using the selective DL-inhibitor 2-bromo-3-(dimethylsulfonio)-propionate7 and overexpressed the DL-encoding gene in the model diatom Thalassiosira pseudonana. We showed that algal DL activity did not serve as an anti-grazing chemical defence but paradoxically enhanced predation by the grazer Oxyrrhis marina and other microzooplankton and mesozooplankton, including ciliates and copepods. Consumption of algal prey with induced DL activity also promoted O. marina growth. Overall, our results demonstrate that DMS-mediated grazing may be ecologically important and prevalent during prey–predator dynamics in aquatic ecosystems. The role of algal DMS revealed here, acting as an eat-me signal for grazers, raises fundamental questions regarding the retention of its biosynthetic enzyme through the evolution of dominant bloom-forming phytoplankton in the ocean. Algal production of dimethyl sulfide plays a role in attracting predators and enhancing predation by zooplankton, thus mediating predator–prey relationships in the ocean.

The importance of microalgal lipids for the survival and growth of shrimp postlarvae has been recognized in a range of studies. Microalgae with fast growth rates and high levels of polyunsaturated fatty acids (PUFA) are considered vital to maximise production and minimise cost in shrimp larviculture. The lipid content and fatty acid composition of microalgae used in shrimp production varies substantially between the algal classes and species being used in Vietnam. This study aims to characterise microalgal lipid and fatty acid (FA) profiles and evaluate the most promising species under growth conditions that are most suitable for shrimp aquaculture. Here, we report that the highest lipid contents were obtained in the Haptophyta microalgae, Tisochrysis lutea and Isochrysis galbana, at 90.3 and 61.1 mg/g, respectively. In contrast, two of the most popular diatom species being used for shrimp larval cultivation in Vietnam, Thalassiosira pseudonana and T. weissflogii, displayed the lowest lipid contents at 16.1 mg/g. Other microalgal species examined showed lipid contents ranging from 28.6 to 55 mg/g. Eicosapentaenoic acid (EPA, 20:5ω3) ranged from 0.6 to 29.9% across the species, with docosahexaenoic acid (DHA, 22:6ω3) present at 0.01 to 11.1%; the two omega (ω)–3 long-chain (LC, ≥C20) LC-PUFA varied between the microalgae groups. Polar lipids were the main lipid class, ranging from 87.2 to 97.3% of total lipids, and triacylglycerol was detected in the range of 0.01 to 2.5%. Saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) increased and PUFA decreased with increasing growth temperatures. This study demonstrated the differences in the lipid contents and FA profiles across 10 microalgal species and the effect of the higher temperature growing conditions encountered in Vietnam.

Temperature is one of the environmental factors that most strongly influence the life of microalgae, from the enzymatic level up to the modification of biosynthetic and degradation potential and, t...

Recent studies indicate that aluminum (Al) could play an important role in the ocean carbon cycle by increasing phytoplankton carbon fixation and reducing organic carbon decomposition. However, how Al may influence the decomposition of organic carbon has not yet been explicitly examined. Here we report the effects of Al on carbon fixation by marine diatoms and their subsequent decomposition. By using radiocarbon as a tracer, the carbon fixation and decomposition of three model marine diatoms were examined in Aquil* media at different concentrations (0, 40, 200, and 2000 nM) of dissolved Al. Addition of Al enhanced net carbon fixation by the diatoms in the declining growth phase (by 9%–29% for Thalassiosira pseudonana, 15%–20% for T. oceanica, 15%–23% for T. weissflogii). Under axenic conditions the decomposition rates (d⁻¹) of the diatom-produced particulate organic carbon (POC) significantly decreased (by 21%–57% for T. pseudonana, 0%–41% for T. oceanica, 29%–58% for T. weissflogii) in the Al-enriched treatments. In the presence of bacteria, the decomposition rates of T. weissflogii-produced POC were still 37%–38% lower in Al-enriched treatments compared to the control. Significant increases in cell size, cellular carbon content (pmol C/cell) and cellular carbon density (pmol C/μm³) of T. weissflogii were also observed in the Al-enriched treatments compared to the control. The Al-related increase in net carbon fixation and cell size, and the decrease in POC decomposition rate may facilitate carbon export to ocean depths. The study provides new evidence for the iron–aluminum hypothesis, which suggests that Al could increase phytoplankton uptake of atmospheric CO₂ and influence climate change.

Background Diatoms contribute 20% of the global primary production and are adaptable in dynamic environments. Diatoms always bloom earlier in the annual phytoplankton succession instead of dinoflagellates. However, how diatoms acclimate to a dynamic environment, especially under changing light conditions, remains unclear. Results We compared the growth and photosynthesis under fluctuating light conditions of red tide diatom Skeletonema costatum, red tide dinoflagellate Amphidinium carterae, Prorocentrum donghaiense, Karenia mikimotoi, model diatom Phaeodactylum tricornutum, Thalassiosira pseudonana and model dinoflagellate Dinophycae Symbiodinium. Diatoms grew faster and maintained a consistently higher level of photosynthesis. Diatoms were sensitive to the specific inhibitor of Proton Gradient Regulation 5 (PGR5) depending photosynthetic electron flow, which is a crucial mechanism to protect their photosynthetic apparatus under fluctuating light. In contrast, the dinoflagellates were not sensitive to this inhibitor. Therefore, we investigate how PGR5 functions under light fluctuations in the model diatom P. tricornutum by knocking down and overexpressing PGR5. Overexpression of PGR5 reduced the photosystem I acceptor side limitation (Y (NA)) and increased growth rate under severely fluctuating light in contrast to the knockdown of PGR5. Conclusion Diatoms acclimatize to fluctuating light conditions better than dinoflagellates. PGR5 in diatoms can regulate their photosynthetic electron flow and accelerate their growth under severe light fluctuation, supporting fast biomass accumulation under dynamic environments in pioneer blooms.

Two contrasting estuarine copepods, Acartia tonsa and Eurytemora carolleeae, the former a broadcast spawner and the latter a brood spawner, were fed a constant carbon-based diatom diet, but which had a variable N:P content, and the elemental composition (C, N, P) of tissue and eggs, as well as changes in the rates of grazing, excretion, egg production and viability were measured. To achieve the varied diet, the diatom Thalassiosira pseudonana was grown in continuous culture at a constant growth rate with varying P supply. Both copepods altered their chemical composition in response to the varied prey, but to different degrees. Grazing (clearance) rates increased for A. tonsa but not for E. carolleeae as prey N:P increased. Variable NH4+ excretion rates were observed between copepod species, while excretion of PO43− declined as prey N:P increased. Egg production by E. carolleeae was highest when eating high N:P prey, while that of A. tonsa showed the opposite pattern. Egg viability by A. tonsa was always greater than that of E. carolleeae. These results suggest that anthropogenically changing nutrient loads may affect the nutritional quality of food for copepods, in turn affecting their elemental stoichiometry and their reproductive success, having implications for food webs.

The efficiency of microalgal biomass production is a determining factor for the economic competitiveness of microalgae-based industries. N-acetylcysteine (NAC) and pluronic block polymers are two compounds of interest as novel culture media constituents because of their respective protective properties against oxidative stress and shear-stress-induced cell damage. Here we quantify the effect of NAC and two pluronic (F127 and F68) culture media additives upon the culture productivity of six marine microalgal species of relevance to the aquaculture industry (four diatoms-Chaetoceros calcitrans,Chaetoceros muelleri,Skeletonema costatum, andThalassiosira pseudonana; two haptophytes-Tisochrysis luteaandPavlova salina). Algal culture performance in response to the addition of NAC and pluronic, singly or combined, is dosage- and species-dependent. Combined NAC and pluronic F127 algal culture media additives resulted in specific growth rate increases of 38%, 16%, and 24% forC. calcitrans,C. muelleri, andP. salina, respectively. Enhanced culture productivity for strains belonging to the genusChaetoceroswas paired with an ~27% increase in stationary-phase cell density. For some of the species examined, culture media enrichments with NAC and pluronic resulted in increased omega-3-fatty acid content of the algal biomass. Larval development (i.e., growth and survival) of the Pacific oyster (Crassostrea gigas) was not changed when fed a mixture of microalgae grown in NAC- and F127-supplemented culture medium. Based upon these results, we propose that culture media enrichment with NAC and pluronic F127 is an effective and easily adopted approach to increase algal productivity and enhance the nutritional quality of marine microalgal strains commonly cultured for live-feed applications in aquaculture.Key points• Single and combined NAC and pluronic F127 culture media supplementation significantly enhanced the productivity of Chaetoceros calcitrans and Chaetoceros muelleri cultures.• Culture media enrichments with NAC and F127 can increase omega-3-fatty acid content of algal biomass.• Microalgae grown in NAC- and pluronic F127-supplemented culture media are suitable for live-feed applications.

Arsenic pollution is a widespread threat to marine life, but the ongoing rise pCO2 levels is predicted to decrease bio-toxicity of arsenic. However, the effects of arsenic toxicity on marine primary producers under elevated pCO2 are not well characterized. Here, we studied the effects of arsenic toxicity in three globally distributed diatom species (Phaeodactylum tricornutum, Thalassiosira pseudonana, and Chaetoceros mulleri) after short-term acclimation (ST, 30 days), medium-term exposure (MT, 750 days), and long-term (LT, 1460 days) selection under ambient (400 µatm) and elevated (1000 and 2000 µatm) pCO2. We found that elevated pCO2 alleviated arsenic toxicity even after short acclimation times but the magnitude of the response decreased after mid and long-term adaptation. When fed with these elevated pCO2 selected diatoms, the scallop Patinopecten yessoensis had significantly lower arsenic content (3.26–52.83%). Transcriptomic and biochemical analysis indicated that the diatoms rapidly developed arsenic detoxification strategies, which included upregulation of transporters associated with shuttling harmful compounds out of the cell to reduce arsenic accumulation, and upregulation of proteins involved in synthesizing glutathione (GSH) to chelate intracellular arsenic to reduce arsenic toxicity. Thus, our results will expand our knowledge to fully understand the ecological risk of trace metal pollution under increasing human activity induced ocean acidification.

Recent development of DNA sequencing technologies has enabled successful construction of thousands of mitochondrial genomes. Nevertheless, only 33 mitochondrial genomes have been reported for species in Bacillariophyta, which includes harmful algal bloom (HAB) species. In this study, we determined the complete mitogenome of the Bacillariophyta HAB species Odontella regia. For comparison, we also constructed the mitogenome of Lithodesmium undulatum, another Bacillariophyta species. Both strains were isolated in the Jiaozhou Bay, an epitome of China’s coastal ecosystem and an ideal site for HAB research. These two mitogenomes were characterized as 37,617 bp and 37,057 bp circular-mapping molecules with AT content of 73.4% and 75.3%, respectively. The phylogenetic tree based on 32 protein-coding genes of the mitogenome encoded in 35 Bacillariophyta species revealed that the class Mediophyceae consisted of multi-phyletic clades. While O. regia formed an independent clade, L. undulatum was closely related to Thalassiosira pseudonana and Skeletonema marinoi. Synteny comparison of O. regia and L. undulatum mitogenomes and mitogenomes of three closely related species displayed substantial differences among lineages in Mediophyceae by a series of gene translocations and/or inversion with many conserved gene blocks such as rpl2-rps19-rps3-rpl16-atp9. These analyses suggested a complex evolutionary relationship in Mediophyceae in which the mitogenome of O. regia was the least conservative compared with the mitogenomes of four other Bacillariophyta species. Further studies are needed to clarify detailed phylogenetic relationships in Bacillariophyta.

Carbonic anhydrases (CAs, EC 4.2.1.1) have been studied for decades and have been classified as a superfamily of enzymes which includes, up to date, eight gene families or classes indicated with the Greek letters α, β, γ, δ, ζ, η, θ, ι. This versatile enzyme superfamily is involved in multiple physiological processes, catalysing a fundamental reaction for all living organisms, the reversible hydration of carbon dioxide to bicarbonate and a proton. Recently, the ι-CA (LCIP63) from the diatom Thalassiosira pseudonana and a bacterial ι-CA (BteCAι) identified in the genome of Burkholderia territorii were characterised. The recombinant BteCAι was observed to act as an excellent catalyst for the physiologic reaction. Very recently, the discovery of a novel ι-CAs (COG4337) in the eukaryotic microalga Bigelowiella natans and the cyanobacterium Anabaena sp. PCC7120 has brought to light an unexpected feature for this ancient superfamily: this ι-CAs was catalytically active without a metal ion cofactor, unlike the previous reported ι-CAs as well as all known CAs investigated so far. This review reports recent investigations on ι-CAs obtained in these last three years, highlighting their peculiar features, and hypothesising that possibly this new CA family shows catalytic activity without the need of metal ions.

© 2020, Springer Nature Switzerland AG. Temperature plays a fundamental role in determining phytoplankton community structure, distribution, and abundance. With climate models predicting increases in ocean surface temperatures of up to 3.2°C by 2100, there is a genuine need to acquire data on the phenotypic plasticity, and thus performance, of phytoplankton in relation to temperature. We investigated the effects of temperature (14–28°C) on the growth, morphology, productivity, silicification and macromolecular composition of the marine diatom Thalassiosira pseudonana. Optimum growth rate and maximum P:R ratio were obtained around 21°C. Cell volume and chlorophyll a increased with temperature, as did lipids and proteins. One of the strongest temperature-induced shifts was the higher silicification rates at low temperature. Our results reveal temperature-driven responses in physiological, morphological and biochemical traits in T. pseudonana; whereby at supra-optimal temperatures cells grew slower, were larger, had higher chlorophyll and protein content but reduced silicification, while those exposed to sub-optimal temperatures were smaller, heavily silicified with lower lipid and chlorophyll content. If these conserved across species, our findings indicate that as oceans warm, we may see shifts in diatom phenotypes and community structure, with potential biogeochemical consequences of higher remineralisation and declines in carbon and silicon export to the ocean interior.

The marine diatom Thalassiosira pseudonana was grown in continuous culture systems to study the interactive effects of temperature, irradiance, nutrient limitation, and the partial pressure of CO2 (pCO2 ) on its growth and physiological characteristics. The cells were able to grow at all combinations of low and high irradiance (50 and 300 μmol photons · m-2 · s-1 , respectively, of visible light), low and high pCO2 (400 and 1,000 μatm, respectively), nutrient limitation (nitrate-limited and nutrient-replete conditions), and temperatures of 10-32°C. Under nutrient-replete conditions, there was no adverse effect of high pCO2 on growth rates at temperatures of 10-25°C. The response of the cells to high pCO2 was similar at low and high irradiance. At supraoptimal temperatures of 30°C or higher, high pCO2 depressed growth rates at both low and high irradiance. Under nitrate-limited conditions, cells were grown at 38 ± 2.4% of their nutrient-saturated rates at the same temperature, irradiance, and pCO2 . Dark respiration rates consistently removed a higher percentage of production under nitrate-limited versus nutrient-replete conditions. The percentages of production lost to dark respiration were positively correlated with temperature under nitrate-limited conditions, but there was no analogous correlation under nutrient-replete conditions. The results suggest that warmer temperatures and associated more intense thermal stratification of ocean surface waters could lower net photosynthetic rates if the stratification leads to a reduction in the relative growth rates of marine phytoplankton, and at truly supraoptimal temperatures there would likely be a synergistic interaction between the stresses from temperature and high pCO2 (lower pH).

Global warming is altering both phytoplankton-experienced temperature and light-exposure duration through shifting their niches from low to high latitudes. We explored the growth, physiology, and compositions of a smaller Thalassiosira pseudonana and a larger Thalassiosira punctigera, temperate marine centric diatoms, in responses to a matrix of temperatures (12, 15, 18, and 21 °C) and photoperiods (light:dark cycles of 4:20, 8:16, 16:8, and 24:0). Both T. pseudonana and T. punctigera grew faster under medium temperature and longer photoperiod, under the expected optimal instantaneous light intensity. The biovolume-based pigments content of T. pseudonana responded largely to temperature, while that of T. punctigera responded more to photoperiod duration than to temperature. In T. pseudonana, shortest photoperiod enhanced cellular protein content and alleviated their temperature dependency. Continuous growth light reduced the photosynthetic capacity of T. pseudonana at the lowest temperature and reduced that of T. punctigera across temperatures. Moreover, we found the increasing temperature linearly increased the dark respiration rate (Rd) and molar ratio of carbon to nitrogen (C:N) of T. pseudonana but decreased that of T. punctigera, with the scattered effects of photoperiod. Our results demonstrated that responses of diatoms Thalassiosira across photoperiods and temperatures vary with species and possibly with cell size, suggesting that the poleward shift of the niches of phytoplankton in nature might cause a change in community structure.

Diatoms are one of the most prominent oceanic primary producers and are now recognized to be distributed throughout the world. They maintain their population despite predators, infections, and unfavourable environmental conditions. One of the smallest diatoms, Chaetoceros tenuissimus, can coexist with infectious viruses during blooms. To further understand this relationship, we sequenced the C. tenuissimus strain NIES-3715 genome. A gene fragment of a replication-associated gene from the infectious ssDNA virus (designated endogenous virus-like fragment, EVLF) was found to be integrated into each 41 Mb of haploid assembly. In addition, the EVLF was transcriptionally active and conserved in nine other C. tenuissimus strains from different geographical areas, although the primary structures of their proteins varied. The phylogenetic tree further suggested that the EVLF was acquired by the ancestor of C. tenuissimus. Additionally, retrotransposon genes possessing a reverse transcriptase function were more abundant in C. tenuissimus than in Thalassiosira pseudonana and Phaeodactylum tricornutum. Moreover, a target site duplication, a hallmark for long interspersed nuclear element retrotransposons, flanked the EVLF. Therefore, the EVLF was likely integrated by a retrotransposon during viral infection. The present study provides further insights into the diatom-virus evolutionary relationship.

International audience; Inorganic carbon availability can limit primary productivity and control species composition of freshwater phytoplankton. This is despite the presence of CO2-concentrating mechanisms (CCMs) in some species that maximize inorganic carbon uptake. We investigated the effects of inorganic carbon on the seasonal distribution, growth rates and photosynthesis of a freshwater diatom, Asterionella formosa, and the nature of its CCM using genomics. In a productive lake, the frequency of A. formosa declined with CO2 concentration below air-equilibrium. In contrast, CO2 concentrations at 2.5-times air-equilibrium did not increase growth rate, cell C-quota or the ability to remove inorganic carbon. A pH-drift experiment strongly suggested that HCO3− as well as CO2 could be used. Calculations combining hourly inorganic carbon concentrations in a lake with known CO2 and HCO3− uptake kinetics suggested that rates of photosynthesis of A. formosa would be approximately carbon saturated and largely dependent on CO2 uptake when CO2 was at or above air-equilibrium. However, during summer carbon depletion, HCO3− would be the major form of carbon taken up and carbon saturation will fall to around 30%. Genes encoding proteins involved in CCMs were identified in the nuclear genome of A. formosa. We found carbonic anhydrases from subclasses α, β, γ and θ, as well as solute carriers from families 4 and 26 involved in HCO3− transport, but no periplasmic carbonic anhydrase. A model of the components of the CCM and their location in A. formosa showed that they are more similar to Phaeodactylum tricornutum than to Thalassiosira pseudonana, two marine diatoms.

The 2010 Deepwater Horizon oil-spill exposed the microbes of Gulf of Mexico to unprecedented amount of oil. Conclusive evidence of the underlying molecular mechanism(s) on the negative effects of oil exposure on certain phytoplankton species such as Thalassiosira pseudonana is still lacking, curtailing our understanding of how oil spills alter community composition. We performed experiments on model diatom T. pseudonana to understand the mechanisms underpinning observed reduced growth and photosynthesis rates during oil exposure. Results show severe impairment to processes upstream of photosynthesis, such as light absorption, with proteins associated with the light harvesting complex damaged while the pigments were unaffected. Proteins associated with photosynthetic electron transport were also damaged, severely affecting photosynthetic apparatus and depriving cells of energy and carbon for growth. Negative growth effects were alleviated when an organic carbon source was provided. Further investigation through proteomics combined with pathway enrichment analysis confirmed the above findings, while highlighting other negatively affected processes such as those associated with ferroxidase complex, high-affinity iron-permease complex, and multiple transmembrane transport. We also show that oxidative stress is not the primary route of negative effects, rather secondary. Overall, this study provides a mechanistic understanding of the cellular damage that occurs during oil exposure to T. pseudonana.

SummaryMorphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica morphogenetic proteins have been identified. Therefore, it is unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species specific silica patterns.To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica and Cyclotella cryptica) by complete demineralization of the cell walls. LC-MS/MS analysis of the extracts identified 92 proteins that we name ‘Soluble Silicome Proteins’ (SSPs).Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In depth bioinformatics analyses revealed that SSPs can be grouped into distinct classes bases on short unconventional sequence motifs whose functions are yet unknown. The results from in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

Carbonic anhydrases (CAs) are a family of ubiquitous enzymes that catalyze the interconversion of CO2 and HCO3−. The “iota” class (ι-CA) was first found in the marine diatom Thalassiosira pseudonana (tpι-CA) and is widespread among photosynthetic microalgae and prokaryotes. The ι-CA has a domain COG4875 (or COG4337) that can be repeated from one to several times and resembles a calcium–calmodulin protein kinase II association domain (CaMKII-AD). The crystal structure of this domain in the ι-CA from a cyanobacterium and a chlorarachniophyte has been recently determined. However, the three-dimensional organization of the four domain-containing tpι-CA is unknown. Using biophysical techniques and 3-D modeling, we show that the homotetrameric tpι-CA in solution has a flat “drone-like” shape with a core formed by the association of the first two domains of each monomer, and four protruding arms formed by domains 3 and 4. We also observe that the short linker between domains 3 and 4 in each monomer confers high flexibility, allowing for different conformations to be adopted. We propose the possible 3-D structure of a truncated tpι-CA containing fewer domain repeats using experimental data and discuss the implications of this atypical shape on the activity and metal coordination of the ι-CA.

Diatoms are a large group of marine algae that are responsible for about one-quarter of global carbon fixation. Light-harvesting complexes of diatoms are formed by the fucoxanthin chlorophyll a/c proteins and their overall organization around core complexes of photosystems (PSs) I and II is unique in the plant kingdom. Using cryo-electron tomography, we have elucidated the structural organization of PSII and PSI supercomplexes and their spatial segregation in the thylakoid membrane of the model diatom species Thalassiosira pseudonana. 3D sub-volume averaging revealed that the PSII supercomplex of T. pseudonana incorporates a trimeric form of light-harvesting antenna, which differs from the tetrameric antenna observed previously in another diatom, Chaetoceros gracilis. Surprisingly, the organization of the PSI supercomplex is conserved in both diatom species. These results strongly suggest that different diatom classes have various architectures of PSII as an adaptation strategy, whilst a convergent evolution occurred concerning PSI and the overall plastid structure.

Diatom cell walls, called frustules, are main sources of biogenic silica in the ocean and their intricate morphology is an inspiration for nanoengineering. Here we show dynamic aspects of frustule biosynthesis involving acidification of the silica deposition vesicle (SDV) by V-type H+ ATPase (VHA). Transgenic Thalassiosira pseudonana expressing the VHA B subunit tagged with enhanced green fluorescent protein (VHAB -eGFP) enabled subcellular protein localization in live cells. In exponentially growing cultures, VHAB -eGFP was present in various subcellular localizations including the cytoplasm, SDVs and vacuoles. We studied the role of VHA during frustule biosynthesis in synchronized cell cultures of T. pseudonana. During the making of new biosilica components, VHAB -eGFP first localized in the girdle band SDVs, and subsequently in valve SDVs. In single cell time-lapse imaging experiments, VHAB -eGFP localization in SDVs precluded accumulation of the acidotropic silica biomineralization marker PDMPO. Furthermore, pharmacological VHA inhibition prevented PDMPO accumulation in the SDV, frustule biosynthesis and cell division, as well as insertion of the silicalemma-associated protein SAP1 into the SDVs. Finally, partial inhibition of VHA activity affected the nanoscale morphology of the valve. Altogether, these results indicate that VHA is essential for frustule biosynthesis by acidifying the SDVs and regulating the insertion of other structural proteins into the SDV.

Volatile organic compounds (VOCs) produced by phytoplankton are molecules with high vapor pressures that can diffuse across cell membranes into the environment, where they become public goods. VOCs likely comprise a significant component of the marine dissolved organic carbon (DOC) pool utilized by microorganisms, but they are often overlooked as growth substrates because their diffusivity imposes analytical challenges. The roles of VOCs in the growth of the photoautotrophic diatom Thalassiosira pseudonana and heterotrophic bacterium Pelagibacter sp. HTCC1062 (SAR11) were examined using co-cultures and proton-transfer reaction time-of-flight mass spectrometry. VOCs at 82 m/z values were produced in the cultures, and the concentrations of 9 of these m/z values changed in co-culture relative to the diatom monoculture. Several of the m/z values were putatively identified, and their metabolism by HTCC1062 was confirmed by measuring ATP production. Diatom carbon fixation rates in co-culture with HTCC1062 were 20.3% higher than the diatom monoculture. Removal of VOCs from the T. pseudonana monoculture using a hydrocarbon trap caused a similar increase in carbon fixation (18.1%). These results show that a wide range of VOCs are cycled in the environment, and the flux of VOCs from phytoplankton to bacterioplankton imposes a large and unexpected tax on phytoplankton photosynthesis.

Estuarine Pettaquamscutt River is a unique habitat 10 km in length with physical and biogeochemical characteristics analogous to a miniature fjord. Its meromictic upper basins are characterized by a permanent oxic–anoxic vertical gradient in which a well-oxygenated upper layer overlies a deeper, anoxic reservoir, with persistent blooms of phototrophic anoxygenic bacteria (Chromatium, Chlorobium) at the oxic–anoxic transition layer. A diverse assemblage of nanoplanktonic, centric diatoms (Cyclotella caspia, Thalassiosira pseudonana, cf. Cyclotella cryptica) dominated the seasonal phytoplankton cycle in the aerobic layer, similar to comparable meromictic habitats elsewhere. This assemblage of nano-centric diatoms appears to be trait-separated from other species clusters and is potentially useful as a functional group flora with ecophysiology diagnostic of marine estuarine rivers and meromictic habitat niche structure. The most conspicuous phytoplankton feature, however, was the year-round occurrence of the photoautotrophic euglenid Euglenaformis (Euglena) proxima, restricted in its upper water column distribution to the O2–H2S boundary layer where it formed a consortium with the photosynthetic green and purple sulfur bacteria community. The association of E. proxima with meromixis, H2S, and phototrophic anoxygenic bacteria is similar to that reported previously in a brackish Norwegian oyster poll and a brackish loch in Scotland.

Diatoms and other phytoplankton groups are exposed to abrupt changes in pCO2, in waters in upwelling areas, near CO2 seeps, or during their blooms; or to more gradual pCO2 rise through anthropogenic CO2 emissions. Gradual CO2 rises have, however, rarely been included in ocean acidification (OA) studies. We therefore compared how small (Thalassiosira pseudonana) and larger (Thalassiosira weissflogii) diatom cell isolates respond to gradual pCO2 rises from 180 to 1000 μatm in steps of ~40 μatm with 5–10 generations at each step, and whether their responses to gradual pCO2 rise differ when compared to an abrupt pCO2 rise imposed from ambient 400 directly to 1000 μatm. Cell volume increased in T. pseudonana but decreased in T. weissflogii with an increase from low to moderate CO2 levels, and then remained steady under yet higher CO2 levels. Growth rates were stimulated, but Chl a, particulate organic carbon (POC) and cellular biogenic silica (BSi) decreased from low to moderate CO2 levels, and then remained steady with further CO2 rise in both species. Decreased saturation light intensity (Ik) and light use efficiency (α) with CO2 rise in T. pseudonana indicate that the smaller diatom becomes more susceptible to photoinhibition. Decreased BSi/POC (Si/C) in T. weissflogii indicates the biogeochemical cycles of both silicon and carbon may be more affected by elevated pCO2 in the larger diatom. The different CO2 modulation methods resulted in different responses of some key physiological parameters. Increasing pCO2 from 180 to 400 μatm decreased cellular POC and BSi contents, implying that ocean acidification to date has already altered diatom contributions to carbon and silicon biogeochemical processes.

Diatoms perform an estimated 20% of global photosynthesis, form the base of the marine food web, and sequester carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the micronutrient cobalamin (vitamin B12), yet the biochemical ramifications of cobalamin limitation are not well understood. In a laboratory setting, we grew the diatom Thalassiosira pseudonana under replete and low cobalamin conditions to elucidate changes in metabolite pools. Using metabolomics, we show that the diatom experienced a metabolic cascade under cobalamin limitation that affected the central methionine cycle, transsulfuration pathway, and composition of osmolyte pools. In T. pseudonana, 5'-methylthioadenosine decreased under low cobalamin conditions, suggesting a disruption in the diatom's polyamine biosynthesis. Furthermore, two acylcarnitines accumulated under low cobalamin, suggesting the limited use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin availability may have consequences for microbial interactions that are based on metabolite production by phytoplankton.

The species-specifically patterned biosilica cell walls of diatoms are paradigms for biological mineral morphogenesis and the evolution of lightweight materials with exceptional mechanical performance. Biosilica formation is a membrane-mediated process that occurs in intracellular compartments, termed silica deposition vesicles (SDVs). Silicanin-1 (Sin1) is a highly conserved protein of the SDV membrane, but its role in biosilica formation has remained elusive. Here we generate Sin1 knockout mutants of the diatom Thalassiosira pseudonana. Although the mutants grow normally, they exhibit reduced biosilica content and morphological aberrations, which drastically compromise the strength and stiffness of their cell walls. These results identify Sin1 as essential for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm. This insight paves the way for genetic engineering of silica architectures with desired structures and mechanical performance., Stefan Görlich et al. investigate the role of a highly-conserved diatom gene Sin1 in the biogenesis of the biosilica cell wall. Analyzing Sin1 knockout mutants of the diatom Thalassiosira pseudonana, they find that this gene is necessary for generating mechanically robust cell walls.

Genome engineering for materials synthesis is a promising avenue for manufacturing materials with unique properties under ambient conditions. Biomineralization in diatoms, unicellular algae that use silica to construct micron-scale cell walls with nanoscale features, is an attractive candidate for functional synthesis of materials for applications including photonics, sensing, filtration, and drug delivery. Therefore, controllably modifying diatom structure through targeted genetic modifications for these applications is a very promising field. In this work, we used gene knockdown in Thalassiosira pseudonana diatoms to create modified strains with changes to structural morphology and linked genotype to phenotype using supervised machine learning. An artificial neural network (NN) was developed to distinguish wild and modified diatoms based on the SEM images of frustules exhibiting phenotypic changes caused by a specific protein (Thaps3_21880), resulting in 94% detection accuracy. Class activation maps visualized physical changes that allowed the NNs to separate diatom strains, subsequently establishing a specific gene that controls pores. A further NN was created to batch process image data, automatically recognize pores, and extract pore-related parameters. Class interrelationship of the extracted paraments was visualized using a multivariate data visualization tool, called CrossVis, and allowed to directly link changes in morphological diatom phenotype of pore size and distribution with changes in the genotype.

Diatoms secrete a significant amount of polysaccharides, which can serve as a critical organic carbon source for bacteria. The 2010 Deepwater Horizon oil spill exposed the Gulf of Mexico to substantial amounts of oil that also impacted the phytoplankton community. Increased production of exopolymeric substances was observed after this oil spill. Polysaccharides make up a major fraction of exopolymeric substances; however, their physiological role during an oil spill remains poorly understood. Here, we analyzed the role of polysaccharides in the growth and physiology of the oil-sensitive diatom Thalassiosira pseudonana and how they shape the surrounding bacterial community and its activity in the presence of oil. We found that inhibition of chrysolaminarin synthesis had a negative effect on the growth of T. pseudonana and intracellular monosaccharide accumulation, which in turn suppressed photosynthesis by feedback inhibition. In addition, by acting as a carbon reserve, chrysolaminarin helped in the recovery of T. pseudonana in the presence of oil. Inhibition of chrysolaminarin synthesis also influenced the bacterial community in the free-living fraction but not in the phycosphere. Exposure to oil alone led to increased abundance of oil-degrading bacterial genera and the activity of exoenzyme lipase. Our data show that chrysolaminarin synthesis plays an important role in the growth and survival of T. pseudonana in the presence of oil, and its inhibition can influence the composition and activity of the surrounding bacterial community.

Although increasing the pCO2 for diatoms will presumably down-regulate the CO2 -concentrating mechanism (CCM) to save energy for growth, different species have been reported to respond differently to ocean acidification (OA). To better understand their growth responses to OA, we acclimated the diatoms Thalassiosira pseudonana, Phaeodactylum tricornutum, and Chaetoceros muelleri to ambient (pCO2 400 μatm, pH 8.1), carbonated (pCO2 800 μatm, pH 8.1), acidified (pCO2 400 μatm, pH 7.8), and OA (pCO2 800 μatm, pH 7.8) conditions and investigated how seawater pCO2 and pH affect their CCMs, photosynthesis, and respiration both individually and jointly. In all three diatoms, carbonation down-regulated the CCMs, while acidification increased both the photosynthetic carbon fixation rate and the fraction of CO2 as the inorganic carbon source. The positive OA effect on photosynthetic carbon fixation was more pronounced in C. muelleri, which had a relatively lower photosynthetic affinity for CO2 , than in either T. pseudonana or P. tricornutum. In response to OA, T. pseudonana increased respiration for active disposal of H+ to maintain its intracellular pH, whereas P. tricornutum and C. muelleri retained their respiration rate but lowered the intracellular pH to maintain the cross-membrane electrochemical gradient for H+ efflux. As the net result of changes in photosynthesis and respiration, growth enhancement to OA of the three diatoms followed the order of C. muelleri > P. tricornutum > T. pseudonana. This study demonstrates that elucidating the separate and joint impacts of increased pCO2 and decreased pH aids the mechanistic understanding of OA effects on diatoms in the future, acidified oceans.

Temperature effects on the fatty acid (FA) profiles of phytoplankton, major primary producers in the ocean, have been widely studied due to their importance as industrial feedstocks and to their indispensable role as global producers of long‐chain, polyunsaturated FA (PUFA), including omega‐3 (ω3) FA required by organisms at higher trophic levels. The latter is of global ecological concern for marine food webs, as some evidence suggests an ongoing decline in global marine‐derived ω3 FA due to both a global decline in phytoplankton abundance and to a physiological reduction in ω3 production by phytoplankton as temperatures rise. Here, we examined both short‐term (physiological) and long‐term (evolutionary) responses of FA profiles to temperature by comparing FA thermal reaction norms of the marine diatom Thalassiosira pseudonana after ~500 generations (ca. 2.5 years) of experimental evolution at low (16°C) and high (31°C) temperatures. We showed that thermal reaction norms for some key FA classes evolved rapidly in response to temperature selection, often in ways contrary to our predictions based on prior research. Notably, 31°C‐selected populations showed higher PUFA percentages (including ω3 FA) than 16°C‐selected populations at the highest assay temperature (31°C, above T. pseudonana's optimum temperature for population growth), suggesting that high‐temperature selection led to an evolved ability to sustain high PUFA production at high temperatures. Rapid evolution may therefore mitigate some of the decline in global phytoplankton‐derived ω3 FA production predicted by recent studies. Beyond its implications for marine food webs, knowledge of the effects of temperature on fatty acid profiles is of fundamental importance to our understanding of the mechanistic causes and consequences of thermal adaptation.

The post-transcriptional regulation involved in the responses of diatoms to silicon is poorly understood. Using a poly(A)-tag sequencing (PAT-seq) technique that interrogates only the junctions of 3'-untranslated region (UTR) and the poly(A) tails at the transcriptome level, a comprehensive comparison of alternative polyadenylation (APA) was performed to understand the role of post-transcriptional regulation in various silicon-related cellular responses for the marine diatom Thalassiosira pseudonana. In total, 23 701 poly(A) clusters and 6894 APA genes, treated with silicon starvation and replenishment, were identified at nine time points. Significant APA was found in numerous genes (e.g. five cingulin genes) closely associated with the silicon-starvation response, girdle bands and valve synthesis, suggesting that many genes participated in the responses to silicon availability and biosilica formation through changes in transcript isoforms. The poly(A) site usage profiles were distinct during various stages of silicon biomineralization responses. Moreover, a correlation between APA and expression levels of APA switching genes was also discovered. This is an interesting study that presents a genome-wide profile of transcript ends in diatoms, which is distinct from that of higher plants, animals and other microalgae. This work provides an important resource to understand a different aspect of cell-wall synthesis.

Diatoms show a special organisation of their plastid membranes, such that their thylakoids span the entire plastid in bands of three. While in higher plants the interaction of the light harvesting complex II and photosystem II with divalent cations (especially Mg2+) was found to take part in the interplay of electrostatic attraction and repulsion in grana membrane appression, for diatoms the key players in maintaining proper membrane distances were not identified so far. In this work, we investigated the changes in the thylakoid architecture of Thalassiosira pseudonana in reaction to different salts by using circular dichroism and fluorescence spectroscopy in combination with other techniques. We show that divalent cations have an important influence on optimal pigment organisation and thus also on maintaining membrane appression. Thereby, monovalent cations are far less effective. The concentration needed is in a physiological range and fits well with the values obtained for higher plant grana stacking, despite the fact that strict protein segregation as seen in higher plant grana is missing.

Background The marine diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum are valuable model organisms for exploring the evolution, diversity and ecology of this important algal group. Their reference genomes, published in 2004 and 2008, respectively, were the product of traditional Sanger sequencing. In the case of T. pseudonana, optical restriction site mapping was employed to further clarify and contextualize chromosome-level scaffolds. While both genomes are considered highly accurate and reasonably contiguous, they still contain many unresolved regions and unordered/unlinked scaffolds. Results We have used Oxford Nanopore Technologies long-read sequencing to update and validate the quality and contiguity of the T. pseudonana and P. tricornutum genomes. Fine-scale assessment of our long-read derived genome assemblies allowed us to resolve previously uncertain genomic regions, further characterize complex structural variation, and re-evaluate the repetitive DNA content of both genomes. We also identified 1862 previously undescribed genes in T. pseudonana. In P. tricornutum, we used transposable element detection software to identify 33 novel copia-type LTR-RT insertions, indicating ongoing activity and rapid expansion of this superfamily as the organism continues to be maintained in culture. Finally, Bionano optical mapping of P. tricornutum chromosomes was combined with long-read sequence data to explore the potential of long-read sequencing and optical mapping for resolving haplotypes. Conclusion Despite its potential to yield highly contiguous scaffolds, long-read sequencing is not a panacea. Even for relatively small nuclear genomes such as those investigated herein, repetitive DNA sequences cause problems for current genome assembly algorithms. Determining whether a long-read derived genomic assembly is ‘better’ than one produced using traditional sequence data is not straightforward. Our revised reference genomes for P. tricornutum and T. pseudonana nevertheless provide additional insight into the structure and evolution of both genomes, thereby providing a more robust foundation for future diatom research.

Acidification of the ocean due to high atmospheric CO2 levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatom Thalassiosira pseudonana under elevated CO2. Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na+(K+)/H+ antiporter that is significantly upregulated at >800 ppm CO2, with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO2 conditions. In future acidified oceanic conditions, dramatic oscillation with >10-fold change between nighttime (high) and daytime (low) transcript abundances of the antiporter was associated with increased resilience of T. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO2 conditions in marine environments.

With global warming, the intensity and frequency of extreme episodic weather events such as typhoons are rising in tropical and subtropical regions, disturbing the water column and shifting phytoplankton therein from deep to surface layers, and exposing them to high light as well as nutrients. To explore how phytoplankton respond to such environmental changes, we tracked the growth, cell compositions and physiology of small Thalassiosira pseudonana and large Thalassiosira punctigera from simulated ambient to upward-shifted light and nitrogen (N) conditions. Shifting to high levels of light caused a limited effect on the growth of small T. pseudonana, but reduced that of large T. punctigera by 36%, with supplemental N alleviating the light-caused growth reduction. The upward-shifted light reduced the cellular pigments contents in small T. pseudonana, but not in large T. punctigera. The upward-shifted light reduced the photosynthetic capability (FV/FM) of both species, as well as the light utilization efficiency (α) and maximal relative electron transport rate (rETRmax), but it enhanced their dark reparations. Moreover, the upward-shifted light did not affect the superoxide dismutase (SOD) activity of small T. pseudonana, but it did enhance that of large T. punctigera. In addition, the supplemental N showed a limited effect on cellular pigments and the dark respiration of T. pseudonana, but it reduced that of T. punctigera. Our results showed that the growth responses of Thalassiosira to upward-shifted light and nitrogen vary with species and possibly with cell size, indicating that the field species composition might change after the occurrence of extreme weather events.

Background CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is an actor of the redox signaling pathway involved in the regulation of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological importance and their complex and still enigmatic evolutionary background. Methods A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance spectroscopy and small X ray scattering, was used to characterize a diatom CP12. Results Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 similarly to its homologues in other species has some features of intrinsically disorder protein family: it behaves abnormally under gel electrophoresis and size exclusion chromatography, has a high net charge and a bias amino acid composition. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as suggested by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering revealed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12 has a high content of α-helices, and nuclear magnetic resonance spectroscopy suggested that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled coil and disordered regions. Conclusions These findings bring new insights into the large family of dynamic proteins containing disordered regions, thus increasing the diversity of known CP12 proteins. As it is a protein that is more abundant in many stresses, it is not devoted to one metabolism and in particular, it is not specific to carbon metabolism. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle. Choregraphy of metabolism by CP12 proteins in Viridiplantae and Heterokonta. While the monomeric CP12 in Viridiplantae is involved in carbon assimilation, regulating phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through the formation of a ternary complex, in Heterokonta studied so far, the dimeric CP12 is associated with Ferredoxin-NADP reductase (FNR) and GAPDH. The Viridiplantae CP12 can bind metal ions and can be a chaperone, the Heterokonta CP12 is more abundant in all stresses (C, N, Si, P limited conditions) and is not specific to a metabolism.

After the Deepwater Horizon oil spill in 2010, toxicity tests were conducted using 4 microalgae (Dunaliella tertiolecta, Skeletonema costatum, Isochrysis galbana, and Thalassiosira pseudonana) and one macroalga (Ectocarpus siliculosus) to study potential impacts on phytoplankton and other primary producers in the Gulf of Mexico and characterize species sensitivity. Tests were performed with Corexit 9500 and fresh source oil and weathered oil samples collected from the field during the Deepwater Horizon oil spill. Because crude oils are mixtures of poorly water-soluble hydrocarbons, dosing was performed using water-accommodated fractions (WAFs) and chemically enhanced (CE) WAFs with the addition of dispersant at a 1:20 dispersant:oil ratio using standard toxicity testing protocols. Exposure media were analyzed for volatile organic compounds, parent and alkylated polycyclic aromatic hydrocarbons, and saturated hydrocarbon compounds. Toxicity was reported as no-observable effect concentration and median effect concentration (EC50) values for average specific growth rate based on nominal percent dilution of stock solution WAFs and sum of dissolved oil toxic units for WAF/CEWAF tests. The macroalga and green alga D. tertiolecta were largely unaffected by any WAF or CEWAFs tested. Isochrysis galbana was found to be the most sensitive species overall with significant growth rate inhibitions for dispersant and all the WAFs/CEWAFs tested. Physically dispersed source oils were generally more toxic than weathered oils. The protectiveness of the chronic toxic units was effective at identifying observed algal growth rate inhibitions across algal species and oil types despite the impact of dispersants. Environ Toxicol Chem 2021;40:2742-2754. © 2021 SETAC.

Diatoms are unicellular phototrophic organisms with huge ecological impact. Characteristic for these organisms is their peculiar cell wall, which is composed of inorganic and organic components. Cell wall formation is a highly complex and orchestrated process, and in the last years has been studied intensively, also on the molecular level. Here, we review on the cell wall proteins of diatoms, with a focus on the species Thalassiosira pseudonana. We report on the expression patterns of these proteins in synchronized cultures, as well as their modifications and intracellular targeting.

Anthropogenic activities release effluents containing nutrients and pathogenic bacteria that change the characteristics of coastal ecosystems. An important type of marine pollution which has occurred in 3 different states in India during 2019 is sea foam. Sea foam was found on Hole beach, Goa (Lat: 15.404° N, Long: 73.787° E), where nutrients (NO3- = 137 μM and organic nitrogen = 121 μM) from a garbage dumpyard are released directly via streams/gutters to coastal waters. This resulted in a bloom of the diatom Thalassiosira pseudonana, associated with high concentration of total organic carbon and fucoxanthin. Decay of this bloom along with strong agitation due to rocks and wave action resulted in sea foam. We isolated foam-associated bacteria and identified pathogenic bacteria including Enterobacter cancerogenus through 16S rRNA gene sequencing. Such foam-associated pathogenic bacteria, could be antibiotic resistant, and may have adverse effects on human health. This can also hamper the tourism industry of a small state like Goa that relies heavily on tourism.

The mixotrophic cultivation of microalgae often couples high biomass production with lipid accumulation, and thus it is considered a useful approach to obtain high quantities of added value microalgal biomass. In this study, the marine diatom Thalassiosira pseudonana was supplied with glycerol to determine the potential of the mixotrophic alga for the accumulation of lipids. In preliminarily experiments, T. pseudonana was cultivated in the presence of increasing concentrations of glucose, acetate and glycerol in order to select the best organic carbon source for growth promotion, and glycerol was selected for further experiments. The best glycerol dose for both biomass productivity and lipid production was 2.5 g L−1. To overcome the costs of the pure glycerol supply, the crude glycerol derived from a biodiesel production plant was also tested, as a low-cost alternative. Beside growth and photosynthetic parameters useful to monitor the algal conditions, lipid amount and profile were compared between autotrophic and pure/crude glycerol-treated algae. Interestingly, an almost doubled lipid content was accumulated in mixotrophic algae compared to the autotrophic ones. Moreover, an evident increase in a relatively short-chain fatty acid (C14:0, myristic acid), potentially useful for bio-jet fuels, and in a long-chain omega3-polyunsaturated fatty acid (C18:4; stearidonic acid), interesting for nutritional purposes, was highlighted in mixotrophic samples, especially when cultivated with crude glycerol. Differently, the fatty acid profile obtained from the autotrophic cultures suggested a preferable employment of the algal biomass for biodiesel production, owing to the prevalence of palmitic and palmitoleic acids. This study highlights that the mixotrophic cultivation of T. pseudonana drives the algal metabolism towards the production of different lipids, suitable for diversified applications. Moreover, the use of biodiesel-derived crude glycerol could make mixotrophy economically advantageous, in particular considering a bio-refinery approach.

This study investigated the biosorption efficiency of Mn (II) by algae (Thalassiosira pseudonana) and membrane performance in a hybrid algae-membrane system. The presence of Mn (II) at 2–10 mM could influence (i.e., at lower algal inoculation ratio of OD = 0.1) or showed negligible effect (i.e., at higher inoculation ratio of OD = 0.17), on algal growth, cell morphology, pigment content, and extracellular organic matter production. The Mn (II) removal was mainly attributed to the algal uptake mechanisms; and higher removal efficiency (>66%) was achieved at higher algal inoculation ratio. Although increasing the Mn (II) concentration could reduce the algal uptake ratio of Mn (II), the membrane performance was improved. The optical coherence tomography (OCT) analysis revealed that the specific volume of cake layer increased gradually over time, however, the irreversible fouling was predominant and associated the presence of building blocks in soluble organics. Economic Development Board (EDB) The Economic Development Board (EDB) of Singapore is acknowledged for funding the Singapore Membrane Technology Centre (SMTC), Nanyang Environment & Water Research Institute, Nanyang Technological University.

Background CP12 is a small chloroplast protein that is widespread in various photosynthetic organisms and is often involved in the redox metabolic on/off switch of the Calvin Benson Bassham (CBB) cycle. The gene encoding this protein is conserved in many diatoms, but the protein has been overlooked in these organisms, despite their ecological predominance and their complex and still enigmatic evolutionary background. Methods A combination of biochemical, bioinformatics and biophysical methods including electrospray ionization-mass spectrometry, circular dichroism, nuclear magnetic resonance and small X ray scattering spectroscopy, was used to characterize a diatom CP12. Results Here, we demonstrate that CP12 is expressed in the marine diatom Thalassiosira pseudonana constitutively in dark-treated and in continuous light-treated cells as well as in all growth phases. This CP12 behaves abnormally under gel electrophoresis, is heat resistant and lacks a structural core, all features of intrinsically disorder family similarly to its homologues in other species. By contrast, unlike other known CP12 proteins that are monomers, this protein is a dimer as shown by native electrospray ionization-mass spectrometry and small angle X-ray scattering. In addition, small angle X-ray scattering showed that this CP12 is an elongated cylinder with kinks. Circular dichroism spectra indicated that CP12, though it has features of disordered proteins, has a high content of α-helices. Nuclear magnetic resonance spectroscopy showed that these helices are unstable and dynamic within a millisecond timescale. Together with in silico predictions, these results suggest that T. pseudonana CP12 has both coiled-coil and disordered regions. Conclusions These findings bring new insights into the large family of intrinsically disordered proteins increasing the diversity of known CP12 proteins. This raises questions about the role of this protein in addition to the well-established regulation of the CBB cycle.

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes, however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24&rsquo, )-dien-3&beta, ol was observed in all P. tricornutum overexpression lines, and campesterol increased three-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

Acidification of the ocean due to high atmospheric CO2 levels may increase the resilience of diatoms causing dramatic shifts in abiotic and biotic cycles with lasting implications on marine ecosystems. Here, we report a potential bioindicator of a shift in the resilience of a coastal and centric model diatom Thalassiosira pseudonana under elevated CO2. Specifically, we have discovered, through EGFP-tagging, a plastid membrane localized putative Na+(K+)/H+ antiporter that is significantly upregulated at > 800 ppm CO2, with a potentially important role in maintaining pH homeostasis. Notably, transcript abundance of this antiporter gene was relatively low and constant over the diel cycle under contemporary CO2 conditions. In future acidified oceanic conditions, dramatic oscillations of >10-fold change between nighttime (high) and daytime (low) in transcript abundances of the antiporter gene were associated with increased resilience of T. pseudonana. By analyzing metatranscriptomic data from the Tara Oceans project, we demonstrate that phylogenetically diverse diatoms express homologs of this antiporter across the globe. We propose that the differential between night- and daytime transcript levels of the antiporter could serve as a bioindicator of a shift in the resilience of diatoms in response to high CO2 conditions in marine environments.

Background The nitrogen-containing polysaccharide chitin is the second most abundant biopolymer on earth and is found in the cell walls of diatoms, where it serves as a scaffold for biosilica deposition. Diatom chitin is an important source of carbon and nitrogen in the marine environment, but surprisingly little is known about basic chitinase metabolism in diatoms. Results Here, we identify and fully characterize 24 chitinase genes from the model centric diatom Thalassiosira pseudonana. We demonstrate that their expression is broadly upregulated under abiotic stresses, despite the fact that chitinase activity itself remains unchanged, and we discuss several explanations for this result. We also examine the potential transcriptional complexity of the intron-rich T. pseudonana chitinase genes and provide evidence for two separate tandem duplication events during their evolution. Conclusions Given the many applications of chitin and chitin derivatives in suture production, wound healing, drug delivery, and other processes, new insight into diatom chitin metabolism has both theoretical and practical value.

As oceans warm, the depth of the upper mixed layer is predicted to decrease, resulting in insufficient nutrient supply and higher solar radiation for phytoplankton. In order to understand the photophysiological responses of the key eukaryotic phytoplankton diatoms to high light and nutrient limitation, we grew two diatoms, Thalassiosira weissflogii and Thalassiosira pseudonana under N starvation conditions and exposed them to high visible light. It showed that the large-sized diatom T. weissflogii can maintain photosynthetic activity for a longer period of time under nitrogen starvation as compared with the small-sized diatom T. pseudonana. The electron transfer reaction was inhibited in both diatoms and the fast closing of reaction centers promoted the development of QB non-reducing PSII centers, thus facilitated the rapid induction of NPQ, however, the induction of NPQ depended on the degree of N starvation. N starvation exacerbated the photoinhibition caused by high light. The smaller-sized T. pseudonana had a higher σi value and was more sensitive to high-light, but its PSII repair rate was also higher. In contrast, T. weissflogii was more tolerant to high light with a lower σi value, but the tolerance was severely reduced under N-starvation. This study provides helpful insight into how climate change variables impact diatom's photosynthetic physiology.

Diatoms are unicellular photosynthetic algae known to secrete organic matter that fuels secondary production in the ocean, though our knowledge of how their physiology impacts the character of dissolved organic matter remains limited. Like all photosynthetic organisms, their use of light for energy and reducing power creates the challenge of avoiding cellular damage. To better understand the interplay between redox balance and organic matter secretion, we reconstructed a genome-scale metabolic model of Thalassiosira pseudonana strain CCMP 1335, a model for diatom molecular biology and physiology, with a 60-year history of studies. The model simulates the metabolic activities of 1,432 genes via a network of 2,792 metabolites produced through 6,079 reactions distributed across six subcellular compartments. Growth was simulated under different steady-state light conditions (5-200 μmol photons m−2 s−1) and in a batch culture progressing from exponential growth to nitrate-limitation and nitrogen-starvation. We used the model to examine the dissipation of reductants generated through light-dependent processes and found that when available, nitrate assimilation is an important means of dissipating reductants in the plastid; under nitrate-limiting conditions, sulfate assimilation plays a similar role. The use of either nitrate or sulfate uptake to balance redox reactions leads to the secretion of distinct organic nitrogen and sulfur compounds. Such compounds can be accessed by bacteria in the surface ocean. The model of the diatom Thalassiosira pseudonana provides a mechanistic explanation for the production of ecologically and climatologically relevant compounds that may serve as the basis for intricate, cross-kingdom microbial networks. Diatom metabolism has an important influence on global biogeochemistry; metabolic models of marine microorganisms link genes to ecosystems and may be key to integrating molecular data with models of ocean biogeochemistry.

Ferredoxins are iron–sulfur proteins essential for a wide range of organisms because they are an electron transfer mediator involved in multiple metabolic pathways. In phytoplankton, these proteins are active in the mature chloroplasts, but the petF gene, encoding for ferredoxin, has been found either to be in the chloroplast genome or transferred to the nuclear genome as observed in the green algae and higher plant lineage. We experimentally determined the location of the petF gene in 12 strains of Thalassiosira covering three species using DNA sequencing and qPCR assays. The results showed that petF gene is located in the nuclear genome of all confirmed Thalassiosira oceanica strains (CCMP0999, 1001, 1005, and 1006) tested. In contrast, all Thalassiosira pseudonana (CCMP1012, 1013, 1014, and 1335) and Thalassiosira weissflogii (CCMP1010, 1049, and 1052) strains studied retained the gene in the chloroplast genome, as generally observed for Bacillariophyceae. Our evolutionary analyses further extend the dataset on the localization of the petF gene in the Thalassiosirales. The realization that the petF gene is nuclear-encoded in the Skeletonema genus allowed us to trace the petF gene transfer back to a single event that occurred within the paraphyletic genus Thalassiosira. Phylogenetic analyses revealed the need to reassess the taxonomic assignment of the Thalassiosira strain CCMP1616, since the genes used in our study did not cluster within the T. oceanica lineage. Our results suggest that this strains’ diversification occurred prior to the ferredoxin gene transfer event. The functional transfer of petF genes provides insight into the evolutionary processes leading to chloroplast genome reduction and suggests ecological adaptation as a driving force for such chloroplast to nuclear gene transfer.

Simple Summary One of the challenges in the emerging field of synthetic biology is engineering organelle genomes. Creating synthetic organelle genomes can open the door to a wide range of applications, such as improving crop yields, treating mitochondrial diseases, or manufacturing high-value chemicals in an environmentally sustainable way. Organelles are tiny biological machines that work inside of living cells. Mitochondria, for example, are responsible for harvesting sugar to create energy for the cell. In previous work, we demonstrated a method to make copies of an alga mitochondrial genome using yeast and bacteria. Algae are of industrial interest for their potential to produce and store large quantities of biofuels and nutritional ingredients. Here, we applied the same approach to copy the mitochondrial genome of a related alga. Although the cloning of this mitochondrial genome in yeast using the previously developed method was possible, the properties of this genome may make it more susceptible to mutations during propagation in bacteria. This work expands our understanding of potential hurdles that can be encountered when cloning and propagating synthetic organelle genomes in host organisms. Abstract Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.