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The regulation of carbon partitioning in diatoms is key to understanding mechanisms related to their high productivity, and the mechanisms controlling carbon flux towards different metabolic fates are currently not well understood. A unique variant of 6-phosphofructo-2-kinase/fructose 2,6 bisphosphatase (PFK2/F2BP), a regulator of glycolytic flux previously uncharacterized in single-cell photosynthetic eukaryotes, was identified in diatom genomes and overexpressed in Thalassiosira pseudonana. Overexpression resulted in increased activity of the glycolytic rate-determining enzyme phosphofructokinase (PFK) and altered carbon partitioning among intracellular carbohydrate, lipid, and protein levels. Higher PFK activity in transformant lines correlated with reduced growth rate and an extension of the G1 phase of the cell cycle, demonstrating a link between carbon metabolism and the control of cell cycle processes. While the relationship between growth and carbon flux under environmental stress (such as nutrient limitation) is well documented in diatoms, our data demonstrate that manipulation of carbon metabolism, independent of an environmental trigger (such as nutrient limitation), is sufficient to delay cell cycle progression. The inverse relationship between growth and PFK activity (as an indicator of glycolytic flux) differs from those described for other unicellular eukaryotes and supports an alternate organization and regulation of central carbon metabolism in diatoms, including a prominent regulatory role for PFK2/F2BP.

Although algae are often grown at pH values between 6 and 8, shifting to more alkali or acidic conditions may benefit CO 2 delivery to algal cultures. To assess the impact of culture pH on growth rate and uptake of CO 2, we grew three relatively fast growing acidophilic (Coccomyxa sp., Euglena mutabilis and Euglena gracilis) and three alkaliphilic (Thalassiosira pseudonana, Phaeodactylum tricornutum, Chlamydomonas sp.) algal species at pH values near neutral and near the extreme of their growth range. All six species showed similar growth and CO 2 uptake ability at extreme as at neutral pH values. Cultures of the alkaliphilic species captured a higher proportion of CO 2 from the gas stream than the acidophilic species; removing 50 to 65% of CO 2 from air compared to only 38% removed by acidophilic species (or 10–24% from CO 2 enriched air). Alkaliphilic species did not become carbon limited when fed CO 2 at the concentration provided by air (0.04% CO 2), but produced less biomass and captured less total CO 2 (0.06 to 0.08 g CO 2 per day) than the acidophilic species (0.6–0.8 g CO 2 day −1) which required CO 2 enriched air to avoid carbon limitation. Bicarbonate feeding reduced the loss of CO 2 to the environment, compared to feeding gaseous CO 2, but with a potential cost in reduced specific growth rate or biomass production.

Microalgae can synthesize their own active substances by absorbing nitrogen, phosphorus in wastewater to provide the nourishing bait for the aquatic organisms, when purifying the aquaculture wastewater. This research Thalassiosira pseudonana (Tp) and Isochrysis galbana (Ig) were selected to treat fishery wastewater and then evaluation of their active components. The results showed that the highest biomass productivity of Tp and Ig was 0.11 g·L−1·day−1 and 0.14 g·L−1·day−1, respectively, at the optimal initial inoculum biomass of 0.4 g·L−1. Furthermore, the two strains were co-cultured in different initial inoculum volume ratios, and the highest biomass was at the volume ratio of 1:1. With the most suitable inoculum biomass and volume ratio, the removal rates of NH4+-N, TN and TP were 80.65%, 85.29% and 92.75%, respectively. Moreover, the cells after wastewater treatment showed high contents of carbohydrate, protein, and lipid, which were 36.62%, 8.72% and 23.11%, respectively. This study showed that co-culturing Thalassiosira pseudonana with Isochrysis galbana to treat fishery wastewater is feasible, and algae cells have high nutritional value, which may provide a feasible technology for ecological protection and resource utilization of aquaculture.

Both Thalassiosira pseudonana and Chaetoceros calcitrans are important species of marine microalgae. Their adaptive capacity to light intensity significantly varies during actual cultivation. We investigated the responses of these two species to different light intensities at 25 °C through analyses of photoacclimation, antioxidant ability as well as many physiological and biochemical parameters. The growth rate of T. pseudonana was positively correlated with light intensity in the first 8 days. However, after that, the greatest growth rate of T. pseudonana was found at a light intensity of 40 μmol m− 2 s− 1. For C. calcitrans, the growth rate at 200 μmol m− 2 s− 1 was the highest among the three light intensities in the first 10 days, while the growth rate at 120 μmol m− 2 s− 1 became the highest after 10 days. Both algae could yield the most chlorophyll a at 40 μmol m− 2 s− 1, and β-carotene content was increased in T. pseudonana but decreased in C. calcitrans with the increase of light intensity. Moreover, maximal photochemical efficiency of PSII (Fv/Fm) and effective photochemical efficiency of PSII (Fv′/Fm′) in T. pseudonana were decreased with the increase of light intensity, while non-photochemical quenching (NPQ) and superoxide dismutase (SOD) activities were increased. However, Fv/Fm and Fv′/Fm′ in C. calcitrans under all light intensities remained relatively constant, and Fv/Fm was maintained at around 0.6. NPQ was increased and SOD activity was decreased in C. calcitrans with the increase of light intensity. These findings suggested that T. pseudonana employed a more positive physiological strategy for adaptation to environmental stress under low light intensity, while C. calcitrans possessed a good physiological strategy for adaptation to environmental stress under high light intensity. Taken together, we provided a better understanding of the growth effects of these two microalgae under different light intensities and offered novel insights into their mass culture.

In all organisms, the flux of carbon through the fundamental pathways of glycolysis, gluconeogenesis and the pyruvate hub is a core process related to growth and productivity. In unicellular microalgae, the complexity and intracellular location of specific steps of these pathways can vary substantially. In addition, the location and chemical nature of storage carbohydrate can be substantially different. The role of starch storage in green algae has been investigated, but thus far, only a minimal understanding of the role of carbohydrate storage in diatoms as the β-1,3-glucan chrysolaminarin has been achieved. In this report, we aimed to determine the effect of specifically reducing the ability of Thalassiosira pseudonana cells to accumulate chrysolaminarin by knocking down transcript levels of the chrysolaminarin synthase gene. We monitored changes in chrysolaminarin and triacylglycerol (TAG) levels during growth and silicon starvation. Transcript-level changes in genes encoding steps in chrysolaminarin metabolism, and cytoplasmic and chloroplast glycolysis/gluconeogenesis, were monitored during silicon limitation, highlighting the carbon flux processes involved. We demonstrate that knockdown lines accumulate less chrysolaminarin and have a transiently increased TAG level, with minimal detriment to growth. The results provide insight into the role of chrysolaminarin storage in diatoms, and further discussion highlights differences between diatoms and green algae in carbohydrate storage processes and the effect of reducing carbohydrate stores on growth and productivity.

Karenia mikimotoi is an important toxin-producing dinoflagellate, frequently forming blooms along the coast of China. It has been shown that K. mikimotoi has allelopathic effects on other organisms, which benefited it over other microalgal species in competition. However, the underlying allelopathy mechanisms remain largely unclear. Thalassiosira pseudonana, a worldwide-distributed planktonic diatom, is extensively used as a model in diatom physiology studies. Here, we investigated the responses of T. pseudonana to K. mikimotoi allelopathy when they were co-cultured under laboratory condition. We found that there were reciprocal inhibitory effects between K. mikimotoi and T. pseudonana, and the cell-free filtrate of K. mikimotoi also deeply suppressed the growth of T. pseudonana, corroborating that K. mikimotoi allelopathy mainly accounted for the inhibition of K. mikimotoi against T. pseudonana. After exposed to K. mikimotoi allelopathy, the photosynthetic efficiency, contents of neutral lipid and malondialdehyde (MDA), as well as superoxide dismutase (SOD) activity in T. pseudonana were significantly changed. Meanwhile, the expression of genes involved in photosynthesis, oxidative phosphorylation, detoxifying system and nutrients uptake including PsbO, chlorophyll synthase (ChlG), risp1, ferredoxin-NADP+ reductase (PetH), ABCB1, nitrate transporters 1 (NRT1), phosphate transporter (PHT), alkaline phosphatase (ALP) and ascorbate peroxidase (APX) in T. pseudonana dramatically altered. These results suggested that K. mikimotoi could highly affect the efficiency of photosynthesis and energy metabolism of T. pseudonana, and also inhibit the nutrients uptake and assimilation. Our findings might provide insight information for the K. mikimotoi allelopathy.

To address challenges in early detection of pond pests, we have extended a spectroradiometric monitoring method, initially demonstrated for measurement of pigment optical activity and biomass, to the detection of algal competitors and grazers. The method relies upon measurement and interpretation of pond reflectance spectra spanning from the visible into the near-infrared. Reflectance spectra are acquired every 5 min with a multi-channel, fiber-coupled spectroradiometer, providing monitoring of algal pond conditions with high temporal frequency. The spectra are interpreted via numerical inversion of a reflectance model, in which the above-water reflectance is expressed in terms of the absorption and backscatter coefficients of the cultured species, with additional terms accounting for the pigment fluorescence features and for the water-surface reflection of sunlight and skylight. With this method we demonstrate detection of diatoms and the predator Poteriochromonas in outdoor cultures of Nannochloropsis oceanica and Chlorella vulgaris, respectively. The relative strength of these signatures is compared to microscopy and sequencing analysis. Spectroradiometric detection of diatoms is then further assessed on beaker-contained mixtures of Microchloropsis salina with Phaeodactylum tricornutum, Thalassiosira weissflogii, and Thalassiosira pseudonana, respectively, providing an initial evaluation of the sensitivity and specificity of detecting pond competitors.

Microalgae are primary producers in aquatic environments, and their growth is often accompanied and affected by bacteria. Although there are some studies about the relationship between microalgae and bacteria, few studies focused on bait-microalgae. Based on 16S rDNA amplicon sequencing, bacterial community structure and composition associated with seven common bait-microalgae, including Isochrysis galbana 3009, I. galbana 3010, I. galbana 3011, Thalassiosira pseudonana 9005, T. pseudonana 9006, Nannochloropsis oceanica 0001 and N. oceanica 0141, were comprehensively analyzed. Our results indicated that bacteria affiliated with phylum Alphaproteobacteria and Gammaproteobacteria and genera Marinobacter, Oceanicaulis and Dinoroseobacter dominated in all these seven bait-microalgae. Based on alpha and beta diversity analysis, we were interested to found that there were significant differences in the bacterial community between different microalgal species, while the differences were relatively small among the same species. When further searching for putative effect of dominant bacteria on the growth of microalgae, we found that the growth of all these seven microalgae was all improved significantly during co-cultivation with Marinobacter sp. When I. galbana 3011 was co-cultured with other three dominant bacteria, including Algoriphagus sp., Dinoroseobacter sp., and Oceanicaulis sp., the growth was improved significantly to different degrees. Taken together, the present work fully characterized the bacterial community associated with seven bait-microalgae and had a tentative exploration of algae-bacteria interaction, which will help improve the yield and quality of bait-microalgae during their cultivation processes.

Diatoms play a key role in assessing the ecotoxicology of metals and are already part of several national and international guidelines and regulations. Data on metal uptake and its correlation with a natural metal composition of the diatoms are mostly lacking on a cellular basis - mainly due to the lack of a suitable method on both the preparation and detection side. Therefore, within this work a fully automated approach based on the on-line coupling of a high performance liquid chromatograph (HPLC) and an inductively coupled plasma-time of flight-mass spectrometer (ICP-ToF-MS) was applied to analyze single cells of the alga Cyclotella meneghiniana multi-elementally in order to provide a deeper insight into the metal composition and its response to environmental stress. Multi-elemental analysis in single diatoms also enables assessment of combined toxicity of a set of metals. A set of four fingerprint elements, characteristically for diatoms (Mg, P, Si, Fe), were identified and hence the investigation of environmental stress onto the cells was enabled by performing incubation experiments with environmentally relevant toxic elements. It could be shown at moderate environmental stress caused by increasing the metal concentration in the medium (zinc) that the fingerprint element concentrations remained stable and thus the suitability of the selected elements for algae tracing was demonstrated. With regard to further ecotoxicological assessments, a multivariate approach was successfully applied allowing for cell classification upon different incubation concentration levels. This multivariate approach also facilitated an effective identification of three different diatom species (Cyclotella meneghiniana, Thalassiosira weissflogii and Thalassiosira pseudonana).

Diatoms produce a wide diversity of sterols among different species, the biosynthesis and conservation of which is not yet fully understood. To investigate the conservation and divergence of sterol biosynthesis pathways among diatoms, we performed comparative metabolic profiling and transcriptomics for a centric diatom (Thalassiosira pseudonana), a pennate diatom (Phaeodactylum tricornutum) and a chaetocerid (Chaetoceros muelleri) in response to inhibitors of enzymes involved in sterol biosynthesis. These three model diatoms, which are representative of distinct clades, share a unique core phytosterol biosynthesis pathway that relies on a terbinafine-insensitive alternative squalene epoxidase and the cyclization of 2,3-epoxysqualene into cycloartenol by a conserved oxidosqualene cyclase. Lineage-specific divergence in the synthesis of sterol precursors was found in the species analyzed. Cholesterol synthesis in diatoms seems to occur via cycloartenol rather than lanosterol. The diversification of natural sterols produced by each species appears to occur downstream of all experimentally targeted enzymes, suggesting adaptive specialization in terminal synthesis pathways.

Diatoms are dominant phytoplankton accounting for up to 20% of annual global primary production. Diatoms are also one of the first model organisms amongst secondary endosymbionts, which have a highly chimeric genome structure accommodating genes from multiple endosymbiosis partners in their evolutionary history. Despite their ecological importance and evolutionary uniqueness, molecular-level studies have lagged behind other model organisms, hindered by difficulties in controlling their ploidy and homologous recombination system. Recently, genome editing tools, such as clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and transcription activator-like effector nucleases (TALEN), have been established in some diatoms. However, the improvement of CRISPR/Cas9 precision technology in diatoms is still underway, and the experimental complexity of TALEN limits their application in diatom molecular genetics. Cas9 nickase, a mutated version of Cas9 with a paired-single guide RNAs (sgRNAs), was recently developed to introduce a precise double cut of target DNA, suppressing the off-target effects of native Cas9 nuclease. In this study, we developed the Cas9 nickase (D10A) system for genome editing of the marine diatom Thalassiosira pseudonana. Using this system, we successfully introduced mutations into a gene encoding a putative θ-type carbonic anhydrase, a recently discovered class of carbonic anhydrase widespread in Stramenopiles. The results showed that this system introduced precise and relatively short biallelic indels to the diatom genome with minimal off-target effects.

Batch culture is the most common method for microbial cultivation, of which its growth dynamics is classified into distinct phases. Importantly is the phase of declining growth (but known as the “linear growth phase” during shade-limited growth for algae) that follows exponential growth and precedes the stationary growth phases. During this phase, under nutrient-replete and light (shade)-limiting conditions, an increasing algal biomass occurs concurrent with a decreasing growth rate (culture quality) that is historically thought to generate a peak of maximum productivity. Dense batch cultures (to > 2 g dry weight l − 1 ) of the marine diatom Thalassiosira pseudonana were grown under a range of incident scalar irradiances (2–26 Ein m − 2 d − 1 ) using both continuous light and 12-h day length in an integrated light environment. Under these conditions, a light-limiting, transition-state dynamic phase between exponential and stationary growth was examined. Growth rate varied to the power of biomass during the phase of linear growth. As a consequence, maximum productivity was provided a broad plateau, not a peak, throughout this phase as a function of incident irradiance, during which culture quality changed, and needs consideration in the engineering, design, and operation of mass algal cultivation systems.

Diatoms utilize various mechanisms to enhance heat dissipation when they are subjected to drastic fluctuations in the light intensity. The activation of the xanthophyll cycle (XC) leading to non-photochemical fluorescence quenching (NPQ) is one of the important mechanisms. We used the model diatom Thalassiosira pseudonana to investigate the factors controlling the kinetics of NPQ and XC. By adding chemicals to cells exposed to excess light, we found that the increase in NPQ during high light (HL) exposure could not be inhibited by NH4Cl and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), while the increase in diatoxanthin (Dt) could be prevented by DCMU and not by NH4Cl, suggesting that the characteristics of NPQ do not solely depend on the presence of the XC. During HL exposure, the up-regulation of the light harvesting complex protein × 6 (Lhcx6) and Lhcx genes suggests that they are involved in photoprotection. The addition of DCMU and NH4Cl significantly elevated the transcript levels of three of the Lhcx genes during HL treatment, especially Lhcx6. The results suggest that reactive oxygen species (ROS) generated by DCMU and transthylakoid ΔpH changes elicited by NH4Cl may contribute to the development of NPQ during HL exposure by inducing the gene expression of Lhcx instead of controlling the XC. Among these, Lhcx6 protein may play a key role in managing light responses in T. pseudonana. Proteomic data demonstrated that the elevated Calvin cycle and increased synthesis of antioxidants, pseudouridines and plastoglobulins may raise the capability of diatoms to cope with light stress.

Considerable investment through public/private consortia has been towards increasing the productivity from mass algal cultivation for making the industries involved more economically viable. Although high-tech solutions have been popular in recent years, I questioned whether low-tech answers through the method that algae are cultivated could shed light on increasing productivity in mass cultivation systems. Shade-limited growth was studied for Thalassiosira pseudonana (marine diatom) cultures exposed to 85 Ein m − 2 d − 1 under continuous light, and mixed through a 20 cm water column (a standard depth for open pond cultivation), to compare ash-free dry weight (AFDW) and chlorophyll-a (Chl-a) concentrations, net primary productivity ( P ) and specific growth rates ( μ ) obtained by batch culture, continuous culture, and semi-continuous culture. Under shade-limiting conditions, both AFDW and Chl-a concentrations varied inversely with μ for the three cultivation methods. During the linear growth phase of batch culture, P did not vary in relation to μ or biomass ( B ), therefore μ = P / B , and growth rate varied as a power for biomass. For continuous culture methods, B is a function of μ (as dilution rate), therefore B = P μ m , and m = − 1 if linear growth is modeled. Net primary production did vary in relation to μ for both continuous and semi-continuous cultures, and m > − 1. Therefore continuous and semi-continuous culture methods did not reproduce a linear growth phase as found for shade-limited growth. For growth rate > 0.5 doubling d − 1 , semi-continuous culture had highest, and continuous culture had lowest P for the three methods compared. These results provide evidence that the method of cultivation introduces large variability to net primary productivity under shade-limiting conditions, and needs consideration in the design of cultivation systems and microbiological experiments.

Improving the ability of microalgae cells to accumulate triacylglycerol (TAG) without a negative effect on growth or biomass accumulation is desirable for economically-viable biofuels production. Metabolic engineering is one method to increase TAG yields, provided that appropriate gene target(s) are identified. Microalgal lipid and TAG synthesis is incompletely characterized, but it is clear that it differs in fundamental ways from plants and other eukaryotes. We characterize a DGAT2 homolog from the diatom Thalassiosira pseudonana, and show that it shifts intracellular location from the chloroplast during exponential growth, to the endoplasmic reticulum in stationary phase when large cytoplasmic lipid droplets accumulate. This is the first direct localization of a chloroplast DGAT2, which confirms the ability of these organelles to accumulate TAG in evolutionarily-diverse classes of microalgae. The enzyme is also not directly localized to cytoplasmic lipid droplets, suggesting that TAG synthesis and lipid droplet formation are separate processes. Overexpression of the enzyme resulted in significantly increased TAG accumulation without a negative effect on growth or biomass accumulation. Overexpression lines had a distinct shift in fatty acid composition relative to wild-type. The results shed light on fundamental processes of algal lipid metabolism and a practical benefit of improved productivity in transgenic lines.

article Biophotovoltaic(BPV) devicesemploy thephotosynthetic activity of microalgae orcyanobacteriato harvest light energy and generate electrical current directly as a result of the release of electrons from the algal cells. NADPH oxidases (NOX) are plasma-membrane enzymes that transport electrons from the cytosol to generate extracel- lular superoxide anions, and have been implicated in BPV output. In this study, we investigated NOX activity in the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana in an attempt to understand and enhance NOX and BPV function. We found that NOX activity was linked to defined growth regimes and growth phases, and was light dependent. Crucially, current output in a BPV device correlated with NOX activity, and levels of up to 14 μ Ap er 10 6 cells (approximately 500 mA·m �2 ) were obtained. Expression of two putative P. tricornutum NOX genes (PtNOX1 and PtNOX2) was found to correspond with the observed growth patterns of superoxide anion production and power output, suggesting that these are responsible for the observed pat- terns of NOX activity. Crucially, we demonstrate that NOX activity levels could be enhanced via semi- continuous culturing, pointing to the possibility of maintaining long-term power output in BPV devices. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).