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Organisms of the microalgal genus Nannochloropsis produce high levels of triacylglycerols (TAGs), an efficient raw material for biofuels. A complete understanding of the TAG-breakdown pathway is critical for improving the productivity of TAGs to meet future needs. Among a number of lipases annotated as TAG lipase in the genomes of every organism, Arabidopsis SUGAR-DEPENDENT 1 (AtSDP1) lipases are characterized as a type of crucial TAG lipase in plants, similar to ScTgl3–5 in Saccharomyces cerevisiae. Homologs of the AtSDP1 TAG lipases are universally found in the genomes of plants, fungi, and algae. Here we identified two homologs of AtSDP1 TAG lipases in the oleaginous microalga species Nannochloropsis oceanica , NoTGL1 and NoTGL2. We generated single- and double-knockout strains for these lipases by homologous recombination. Whereas overall TAG content in the NoTGL2 single-knockout mutant was identical to that of wild type, the NoTGL1 knockout showed a two-fold increase in TAG content per cell in early log phase under nutrient-sufficient conditions without affecting growth. Homologs of AtSDP1 in S. cerevisiae are localized to the surface of lipid droplets, and AtSDP1 is transported from peroxisomes to the surface of lipid droplets. In contrast, NoTGL1 localized to the endoplasmic reticulum in both Nannochloropsis and yeast. We suggest that homologs of AtSDP1 lipases in Nannochloropsis modulate de novo TAG biosynthesis in the endoplasmic reticulum, unlike the roles of these lipases in other organisms. These results provide important insights into the mechanisms of TAG metabolism catalyzed by homologs of AtSDP1 lipase, which are highly conserved across species. • Nannochloropsis is one of the best triacylglycerol (TAG) producing genera. • We characterized two TAG lipases in N. oceanica , NoTGL1 and NoTGL2. • tgl1 and tgl1;2 mutants increased the TAG content by two-fold in early log phase cells. • NoTGL1 specifically localized to the ER is distinct from AtSDP1 and ScTgl3–5. • NoTGL1 and NoTGL2 are differently involved in the TAG hydrolysis. [ABSTRACT FROM AUTHOR]

We investigated potential biosynthetic pathways of long chain alkenols (LCAs), long chain alkyl diols (LCDs), and long chain hydroxy fatty acids (LCHFAs) in Nannochloropsis oceanica and Nannochloropsis gaditana , by combining culturing experiments with genomic and transcriptomic analyses. Incubation of Nannochloropsis spp. in the dark for 1 week led to significant increases in the cellular concentrations of LCAs and LCDs in both species. Consistently, 13C-labelled substrate experiments confirmed that both LCA and LCD were actively produced in the dark from C14–18 fatty acids by either condensation or elongation/hydroxylation, although no enzymatic evidence was found for the former pathway. Nannochloropsis spp. did, however, contain (i) multiple polyketide synthases (PKSs) including one type (PKS-Clade II) that might catalyze incomplete fatty acid elongations leading to the formation of 3-OH-fatty acids, (ii) 3-hydroxyacyl dehydratases (HADs), which can possibly form Δ2/Δ3 monounsaturated fatty acids, and (iii) fatty acid elongases (FAEs) that could elongate 3-OH-fatty acids and Δ2/Δ3 monounsaturated fatty acids to longer products. The enzymes responsible for reduction of the long chain fatty acids to LCDs and LCAs are, however, unclear. A putative wax ester synthase/acyl coenzyme A (acyl-CoA): diacylglycerol acyltransferase is likely to be involved in the esterification of LCAs and LCDs in the cell wall. Our data thus provide useful insights in predicting the biosynthetic pathways of LCAs and LCDs in phytoplankton suggesting a key role of FAE and PKS enzymes. [ABSTRACT FROM AUTHOR]

Improving acid tolerance is pivotal to the development of microalgal feedstock for converting flue gas to biomass or oils. In the industrial oleaginous microalga Nannochloropsis oceanica , transcript knockdown of a cytosolic carbonic anhydrase (CA2), which is a key Carbon Concentrating Mechanism (CCM) component induced under 100 ppm CO 2 (very low carbon, or VLC), results in ∼45%, ∼30% and ∼40% elevation of photosynthetic oxygen evolution rate, growth rate and biomass accumulation rate respectively under 5% CO 2 (high carbon, or HC), as compared to the wild type. Such high-CO 2 -level activated biomass over-production is reproducible across photobioreactor types and cultivation scales. Transcriptomic, proteomic and physiological changes of the mutant under high CO 2 (HC; 5% CO 2) suggest a mechanism where the higher pH tolerance is coupled to reduced biophysical CCM, sustained pH hemostasis, stimulated energy intake and enhanced photosynthesis. Thus "inactivation of CCM" can generate hyper-CO 2 -assimilating and autonomously containable industrial microalgae for flue gas-based oil production. • In the industrial oleaginous microalga Nannochloropsis oceanica , a cytosolic carbonic anhydrase (CA2) was identified as a key Carbon Concentrating Mechanism (CCM) component induced in response to lowered CO 2 level. • Knockdown of CA2 resulted in ~40% elevation of biomass accumulation rate under 5% CO 2 (versus the wild type), which is reproducible across photobioreactor types and cultivation scales. • The higher pH tolerance of CA2-knockdown mutant is underpinned by reduced biophysical CCM, sustained pH hemostasis, stimulated energy intake and enhanced photosynthesis. • "Inactivation of CCM" is an effective strategy to generate hyper-CO2-assimilating and autonomously containable industrial microalgae for flue gas-based oil production. [ABSTRACT FROM AUTHOR]

Context: Screening of natural biomolecules from microalgae. Background: The microalgae were recognized for their biological and pharmacological importance of active natural products with high antioxidant and antiproliferative profile. In the preliminary screening, three species Nannochloropsis sp. (NC) (green algae), Amphora sp. (diatom), and Nostoc sp. (blue-green algae) were tested and Nannochloropsis was selected based on their scavenging properties. Objective: The objective of the study is to explore the biological information of microalgal species where the clinical investigation is still quite limited. Materials and Methods: The phytochemical screening of selected NC. primarily comprises saponins, terpenoids, flavonoids, and phenols which were confirmed by high-performance thin-layer chromatography, Fourier transform infrared, and gas chromatography–mass spectra analysis. Results: The ethyl acetate extract Nannochloropsis hexane (EAENH) fraction showed 40.61 mg GAE/g, 68.77 mg QE/g, 5.73 mg/g, and 57.38 mg CHL/g for total phenolic, flavonoid, carotenoid, and sterol content, respectively. Moreover, antioxidant activities were evaluated for the extract showing high flavonoid and phenolic contents after partial purification with hexane. The half inhibitory concentration (IC50) values for EAENH was found to be 13.9, 21.22, and 14.58 μg/mL for 1,1-diphenyl-2-picrylhydrazyl radical, hydrogen peroxide, and reducing power assays, respectively. The antiproliferative activity of EAENH on human non-small lung cancer cell line (A549) IC50 value was 175 μg/mL using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Conclusion: The present study confirmed that the bioactive components present in the EAENH were accountable for excellent antioxidant and cytotoxic properties. [ABSTRACT FROM AUTHOR]

Abstract Translationally controlled tumor proteins (TCTPs) are eukaryote-conserved multifunctional proteins, but their primary functions are not well understood yet. Study on TCTP from unicellular species may provide insight into the primary function of the TCTP family. Bioinformatic analysis indicated that the TCTP from Nannochloropsis oceanica (No TCTP), a model unicellular microalga for biodiesel and polyunsaturated fatty acid production, has low sequence homology to other structure-known TCTPs and two TCTP signature patterns are not detected in NoTCTP. Hence, we determined the solution structure of NoTCTP. The overall structure of NoTCTP, including a long flexible loop, a β-barrel subdomain, and a helical subdomain, is generally similar to other TCTP structures. NoTCTP has a eukaryote-conserved surface for the binding of eukaryotic elongation factor 1B, confirming that TCTP is involved in protein synthesis, which is one of the primary functions of TCTP. Additionally, NoTCTP has distinct features different from other TCTPs. NoTCTP structure lacks a short α-helix which exists in all other known TCTP structures. The helical subdomain and some loops of NoTCTP also have distinct conformations among the TCTP family proteins. Therefore, our study on NoTCTP revealed not only conserved structural features but also the structural diversity of TCTP family proteins. Graphical abstract Image 1 Highlights • TCTP from Nannochloropsis oceanica (NoTCTP) lacks two TCTP signature patterns. • NoTCTP has a conserved structure including a long flexible loop and two subdomains. • NoTCTP has the conserved sites for the binding of eukaryotic elongation factor 1B. • NoTCTP lacks a short α-helix which is conserved in other known TCTP structures. • NoTCTP has distinct conformations in the helical subdomain and some loops. [ABSTRACT FROM AUTHOR]

Utilization of algae includes both macroalgae for human consumption dating back to thousands of years, as well as the application of microalgae in health promoting dietary supplements. The autotrophic growth of microalgae is slow, but can be accelerated by optimizing their cultivation conditions. Efficiency optimizations for time and economy should be performed in many parallel experiments. A new high-throughput microalgae cultivation method is presented here, applying 24-low-well microplate with varying illumination, in which the cell growth is followed via evaluation of scanned images. A strain of the genus Nannochloropsis and two Chlorella vulgaris species have been chosen as well described and frequently applied model organisms in order to test the recently developed cultivation system. In these scaled down experiments, the custom design lighting panel was tested by studying the effect of the colour of illumination on cell growth kinetics. RGB LEDs (i.e. light emitting diodes, red: 622 nm, green: 528 nm, and blue: 467 nm) were used individually or together providing red, green, blue, and white colours. While the effect of light's colour on algae growth was evaluated, also the new system was proven to be suitable for comparing maximal growth rates for different microalgae strains. While the tested two Chlorella isolates reached 1.2–1.4 g l–1 concentrations, the Nannochloropsis strain reached 1.4 g l–1 final cell dry weight, and specific growth rates were observed between 0.58–0.62 day–1. [ABSTRACT FROM AUTHOR]

Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res 136:49-61, 2017). This complex contained stress-related protein, Lhcx, as a major component of LHC (Protein ID is Naga_100173g12.1), suggesting that non-photochemical quenching activities may be taking place in the PSII-LHC complex. In this study, we examined the energy transfer dynamics in the isolated LHCs and PSII-LHC complexes, and found substantial quenching capacity. In addition, the LHCs contained low-energy chlorophylls with fluorescence maxima at approximately 710 nm, which may enhance the quenching efficiency in the PSII-LHC. Delayed fluorescence analysis suggested that there was an approximately 50% reduction in energy trapping at the PSII reaction center in the PSII-LHC supercomplex under low-pH condition compared to neutral pH condition. Enhanced quenching may confer a survival advantage in the shallow-water habitat of Nannochloropsis. [ABSTRACT FROM AUTHOR]

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica. We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea·+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea·+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica. [ABSTRACT FROM AUTHOR]

The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a ~ 1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology. [ABSTRACT FROM AUTHOR]

Microalgae are microscopic organisms that are usually identified based on the morphological features. However traditional classification are complicated by high levels of morphological plasticity and convergence that will lead to uncertain identification especially at the species level. Therefore, molecular marker is advantageous to distinguish and correctly identify microalgae at the lowest taxonomic level based on the genetic information and phylogenetic analyses. In the present study, we have characterized five strains of microalgae that were deposited in Borneo Marine Research Institute based on 18S rDNA gene sequences. The BLAST results of microalgae sequences showed high percentage similarities that revealed the species as Chaetoceros gracilis (99.77%), Thalassiosira weissflogii (99.70%), Nannochloropsis oceanica (100%) and Isochrysis sp. (99.97%). Combination of both classical and genetic approach is vital to clearly identify microalgae at generic and species levels. [ABSTRACT FROM AUTHOR]

Abstract As a result of a secondary endosymbiosis of a red alga, stramenopiles retained β-1,3 glucans as the primary soluble storage carbohydrate, in contrast to the starch and glycogen of green plants and animals. This storage carbohydrate has been identified and characterized in representative diatoms, brown algae, and oomycetes, but has not been biochemically characterized in species of Nannochloropsis , an industrially relevant genus of algae favored for its lipid content and productivity. In this study, the soluble storage carbohydrate of Nannochloropsis is characterized by 1H NMR, linkage analysis, and size exclusion chromatography. The putative genes encoding enzymes required for synthesis of the carbohydrate oligosaccharide from glucose 6-phosphate are identified. A glycogenin-like glycosyltransferase family 8 (GT8) protein was also identified to be conserved among all species of Nannochloropsis, despite the lack of glycogen in the genus. Homologs were identified in available genomes of brown algae, diatoms, and oomycetes, all documented to utilize β-1,3 glucans for carbon storage. Finally, a set of three likely laminarinases are highlighted from the glycosyl hydrolases by phylogenetic analyses. Highlights • N. gaditana stores a 1,3-beta glucan carbohydrate with rare 1,6-branching. • Genus-conserved glycogenin-like protein may nucleate glucan polymerization. • Heterokont-wide phylogenetic analysis identifies likely laminarinases. [ABSTRACT FROM AUTHOR]

Marine and freshwater algae and their products are in growing demand worldwide because of their nutritional and functional properties. Microalgae (unicellular algae) will constitute one of the major foods of the future for nutritional and environmental reasons. They are sources of high-quality protein and bioactive molecules with potential application in the modern epidemics of obesity and diabetes. They may also contribute decisively to sustainability through carbon dioxide fixation and minimization of agricultural land use. This paper reviews current knowledge of the effects of consuming edible microalgae on the metabolic alterations known as metabolic syndrome (MS). These microalgae include Chlorella, Spirulina (Arthrospira) and Tetraselmis as well as Isochrysis and Nannochloropsis as candidates for human consumption. Chlorella biomass has shown antioxidant, antidiabetic, immunomodulatory, antihypertensive, and antihyperlipidemic effects in humans and other mammals. The components of microalgae reviewed suggest that they may be effective against MS at two levels: in the early stages, to work against the development of insulin resistance (IR), and later, when pancreatic -cell function is already compromised. The active components at both stages are antioxidant scavengers and anti-inflammatory lipid mediators such as carotenoids and -3 PUFAs (eicosapentaenoic acid/docosahexaenoic acid; EPA/DHA), prebiotic polysaccharides, phenolics, antihypertensive peptides, several pigments such as phycobilins and phycocyanin, and some vitamins, such as folate. As a source of high-quality protein, including an array of bioactive molecules with potential activity against the modern epidemics of obesity and diabetes, microalgae are proposed as excellent foods for the future. Moreover, their incorporation into the human diet would decisively contribute to a more sustainable world because of their roles in carbon dioxide fixation and reducing the use of land for agricultural purposes.

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.

The growing world population is in need for new sustainable resources for healthy food, animal feed and chemicals. Algae produce a variety of compounds via conversion of CO 2 , nutrients, and light, and offer potential as renewable feedstock. However, producing microalgae is costly. Therefore, new economically viable supply chains must be developed. A variety of techno-economic assessments (TEA) has been performed to gain insight in the economic feasibility of algae production systems and refineries. While a TEA provides insights relevant to the technical and economic feasibility of a process, geographical conditions must be considered since the regional characteristics influence process feasibility and supply chain design. This paper presents a spatio-temporal TEA by combining a dynamic TEA model and a supply chain design optimisation model, MooV. The dynamic TEA outlines the conditions of the processes to reach a techno-economic feasible case as well as mass balances and OPEX and CAPEX of the different potential processes in the chain. MooV combines these parameters with geographic specific parameters such as the locations of activities and the characteristics of the transport network to define the optimal supply chain configuration (e.g., location and process type and capacity) based on minimal cost. The approach is demonstrated by year-round cultivation of Nannochloropsis gaditana in a horizontal tubular photobioreactor in 3 regions in North-West Europe. The analysis indicates that the cost per kg DM algae output can vary considerably (±40 %) depending on the region and the set-up of the supply chain. Including geographic characteristics as well as comparing different supply chain design options are essential to come to a viable business case. The approach can also be applied to different algae strains, different regions and different process options. • Decision model for spatio-temporal techno-economic assessment of algae value chains • The objective is to minimize cost from farm over processing towards formulator. • Supports designing new algae-based value chains at low Technology Readiness Level • Regional characteristics impact process feasibility and supply chain configuration. [ABSTRACT FROM AUTHOR]

[Display omitted] • Enrichment with Nannochloropsis shows oxidative instability. • Enrichment with Phaeodactylum shows oxidative stability. • Impact of carotenoid content and type rather than presence only. • N–3 LC–PUFA analysis is a useful addition for detecting lipid oxidation. Microalgae rich in omega-3 long-chain polyunsaturated fatty acids (n–3 LC–PUFA) have already shown their potential for developing functional food rich in these healthy fatty acids. Not only could they offer a more sustainable alternative for the fish stock that is currently relied upon but is unable to keep up with the demand, enrichment with certain microalgae also leads to oxidatively stable products. Although the reason for this stability has been attributed to the presence of endogenous carotenoids, further insight into their antioxidative role is missing and would be clarifying for selecting the proper microalgae for food enrichment. In trying to further accomplish this, a storage experiment (4 weeks, 37 °C) was set up with the parallel analysis of both oxidation products (primary and secondary) and carotenoids of two aqueous model systems of different (promising) microalgae (Nannochloropsis and Phaeodactylum). The results showed a clear difference in oxidative stability despite both microalgae containing endogenous carotenoids: Nannochloropsis led to oxidatively unstable and Phaeodactylum to oxidatively stable products. This was clearly confirmed by the analysis of n–3 LC–PUFA throughout storage which showed a breakdown of half of the n–3 LC–PUFA for Nannochloropsis. All carotenoids (violaxanthin, zeaxanthin and β-carotene for Nannochloropsis , and fucoxanthin and β-carotene for Phaeodactylum) acted as an antioxidant as shown by their degradation throughout storage, but the difference in oxidative stability pointed out an impact of carotenoid content and (possibly) type. The presence of a sufficient amount of carotenoids seems to be an important factor for perceiving oxidative stability. Phaeodactylum has shown to be more potent for food enrichment. [ABSTRACT FROM AUTHOR]

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.

In microalgae, photosynthesis provides energy and sugar phosphates for the biosynthesis of storage and structural carbohydrates, lipids, and nitrogenous proteins. The oleaginous alga Nannochloropsis salina does not preferentially partition photoassimilates among cellulose, chrysolaminarin, and lipids in response to nitrogenous nutrient deprivation. In the present study, we investigated whether genetic impairment of the cellulose synthase gene (CesA) expression would lead to protein accumulation without the accumulation of storage C polymers in N. salina. Three cesA mutants were generated by the CRISPR/Cas9 approach. Cell wall thickness and cellulose content were reduced in the cesA1 mutant, but not in cesA2 or cesA4 cells. CesA1 mutation resulted in a reduction of chrysolaminarin and neutral lipid contents, by 66.3% and 37.1%, respectively, but increased the soluble protein content by 1.8-fold. Further, N. salina cells with a thinned cell wall were susceptible to mechanical stress, resulting in a 1.7-fold enhancement of lipid extractability. Taken together, the previous and current studies strongly suggest the presence of a controlling mechanism that regulates photoassimilate partitioning toward C and N metabolic pathways as well as the cellulose metabolism as a potential target for cost-effective microalgal cell disruption and as a useful protein production platform.

Microalgae are promising feedstock for the production of biodiesel and diverse medium- and high-value products such as pigments and polyunsaturated fatty acids. The importance of strain selection adapted to specific environments is important for economical purposes. We characterize here two microalgal strains, isolated from wastewater of shrimp cultivation ponds in Vietnam. Based on the 18S rDNA-ITS region, one strain belongs to the Eustigmatophyceae class and is identical to the Nannochloropsis salina isolate D12 (JX185299.1), while the other is a Chlorophyceae belonging to the Desmodesmus genus, which possesses a S516 group I intron in its 18S rDNA gene. The N. salina strain is a marine and oleaginous microalga (40% of dry weight (DW) at stationary phase) whole oil is rich in saturated fatty acids (around 45% of C16:0) suitable for biodiesel and contains a few percent of eicosapentaenoic acid (C20:5). The Desmodesmus isolate can assimilate acetate and ammonium and is rich in lutein. Its oil contains around 40%−50% α-linolenic acid (C18:3), an essential fatty acid. Since they tolerate various salinities (10% to 35‱), both strains are thus interesting for biodiesel or aquaculture valorization in coastal and tropical climate where water, nutrient, and salinity availability vary greatly depending on the season.

Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.

Microalgae have potential as a chemical feed stock in a range of industrial applications. Nannochloropsis salina was subject to EMS mutagenesis and the highest lipid containing cells selected using fluorescence-activated cell sorting. Assessment of growth, lipid content and fatty acid composition identified mutant strains displaying a range of altered traits including changes in the PUFA content and a total FAME increase of up to 156% that of the wild type strain. Combined with a reduction in growth this demonstrated a productivity increase of up to 76%. Following UV mutagenesis, lipid accumulation of the mutant cultures was elevated to more than 3 fold that of the wild type strain, however reduced growth rates resulted in a reduction in overall productivity. Changes observed are indicative of alterations to the regulation of the omega 6 Kennedy pathway. The importance of these variations in physiology for industrial applications such as biofuel production is discussed.

Microalgae often undergo different CO 2 experiment in their habitat. To adapt to low CO 2 , carbon concentrating mechanism (CCM) could be launched in majority of microalgae and CCM are regulated at RNA level are well known. However, epigenetic modifications and their potential regulation of the transcription of masked genes at the genome level in response to CO 2 fluctuation remain unclear. Here epigenetic regulation in response to CO 2 fluctuation and epigenome-association with phenotypic plasticity of CCM are firstly uncovered in marine microalga Nannochloropsis oceanica IMET1. The result showed that lysine butyrylation (Kbu) and histone H3K9m2 modifications were present in N. oceanica IMET1. Moreover, Kbu modification positively regulated gene expression. In response to CO 2 fluctuation, there were 5,438 and 1,106 genes regulated by Kbu and H3K9m2 in Nannochloropsis, respectively. Gained or lost histone methylations were closely associated with activating or repressing gene expressions. Differential modifications were mainly enriched in carbon fixation, photorespiration, photosynthesis, and lipid metabolism etc. Massive genome-wide epigenetic reprogramming was observed after N. oceanica cells shifted from high CO 2 to low CO 2 . Particularly, we firstly noted that the transcription of the key low CO 2 responsive carbonic anhydrase (CA5), a key component involved in CCM stress signaling, was potentially regulated by bivalent Kbu-H3K9m2 modifications in microalgae. This study provides novel insights into the relationship between gene transcription and epigenetic modification in Nannochloropsis, which will lay foundation on genetic improvement of CCM at epigenetic level., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Microalgae oil can be converted into aviation fuel to reduce dependence on fossil fuels and decrease the carbon footprint. This could be a significant step toward sustainable energy resources that have the potential to produce 'drop‐in' fuels. Algae‐based fuels are potential substitutes for fossil fuels due to the quality of their crude oil. However, this is only possible if appropriate conversion processes are undertaken. Conversely, microalgae species have low lipid content and biomass harvesting is still an energy‐demanding process. In this study, a conceptual design is developed for the conversion of microalgae oil to jet fuel. It is based on a process undertaken in the laboratory using a species named Nannochloropsis sp. Nutrients and CO2 were supplied to the growing culture for effective growth. Biomass harvesting was completed on the tenth day of the growth cycle. It was followed by physiological modification to improve lipid content. Finally, crude oil extraction was followed by bio‐oil hydrocracking at 350 °C, and fractionation of cracked bio‐oil between 70 and 300 °C to separate light‐, middle‐, and heavy‐end hydrocarbons for use in the production of jet fuel. During simulation, reforming and upgrading processes were added to the design to enhance the quality of the jet fuel to be produced on large scale in the future. The study, including the results, suggests that it is technically feasible to convert microalgae oil into jet fuel because of the similarity between algae bio‐oil and petroleum crude oil. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

Nannochloropsis is a genus of fast-growing microalgae that are regularly used for biotechnology applications. Nannochloropsis species have a high triacylglycerol content and their polar lipids are rich in the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid. Placed in the heterokont lineage, the Nannochloropsis genus has a complex evolutionary history. Genome sequences are available for several species, and a number of transcriptomic datasets have been produced, making this genus a facile model for comparative genomics. There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a chassis for synthetic biology. Recently, techniques for gene stacking, and targeted gene disruption and repression in the Nannochloropsis genus have been developed. These tools enable gene-specific, mechanistic studies and have already allowed the engineering of improved Nannochloropsis strains with superior growth, or greater bioproduction. [ABSTRACT FROM AUTHOR]

The increasing use of energy in the world is leading to the exhaustion of fossil fuels, so novel alternative solutions have to be found to meet our needs. One solution is renewable raw materials extracted from algae. The use of microalgae is widespread, in addition to energy formation, their biomass can also be utilized as food and other valuable components of them, e.g. amino acids, vitamins and minerals can be used in drugs and cosmetics. Due to their boundless diversity and components, they have become the focus of an ever-increasing number of research areas. Different processes can induce changes in their nutritional content, so optimizing the conditions used during their cultivation is important to produce the desired product. In our study different isolates of microalgae, namely Nannochloropsis sp. and Chlorella vulgaris, were studied using Fourier-transform infrared (FT-IR) spectroscopic analysis. Variations in the spectra of a given species were studied under different cultivation conditions. [ABSTRACT FROM AUTHOR]

Microalgae is projected as a promising third generation biomass feedstock for the production of biofuel and fine chemical intermediates. This study is focused on the selective production of chemicals from Nannochloropsis oculata microalga via catalytic fast pyrolysis technique using Co-Mo/γ-Al 2 O 3 catalyst. Non-catalytic and catalytic fast pyrolysis (CFP) experiments were conducted in an analytical micropyrolyzer coupled with gas chromatograph/mass spectrometer to study the effects of temperature, catalyst-to-algae ratio, and metal loading on support on pyrolysate composition. The catalyst, Co-Mo/γ-Al 2 O 3 , was prepared using wetness incipient impregnation method, and characterized for its structure and pore size distribution. Catalyst-to-algae mass ratios used to study the pyrolysate composition and quality were 1:3, 1:1 and 2:1. The major organic compounds from both non-catalytic fast pyrolysis and CFP of the microalga were long chain nitriles, long chain alkanes and alkenes, polyaromatic and monoaromatic hydrocarbons. The optimum fast pyrolysis temperature under non-catalytic conditions that promoted the selectivity of aliphatic and aromatic hydrocarbons was 500 °C. The incorporation of Co-Mo/γ-Al 2 O 3 catalyst promoted the formation of specific organic compounds like 1‑isocyanobutane and dimethylketene to the tune of 35% selectivity. The formation pathways for these compounds are proposed to involve dehydration, isomerization, ketonization and CH addition reactions of the amide and carboxylic acid moieties generated from the protein and lipid fractions of the alga. Owing to the low oxygen content in the pyrolysates from CFP, the estimated calorific value of organics in the pyrolysates was higher (33–39 MJ kg −1 ) than that of the microalga (18 MJ kg −1 ). [ABSTRACT FROM AUTHOR]

Microalgae are potential sources of bioactive compounds that have health promoting effects due to the abundant presence of flavonoids and polyphenols. The goal of this study was to investigate the chemical profiles of microalgae extracts from Nannochloropsis sp. and Spirulina sp. Total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activity (DPPH) were measured. Nannochloropsis sp. had a significantly higher TPC value than Spirulina sp. (58.43 ± 0.85 mg GAE/g DW vs. 19.64 ± 0.52 mg GAE/g DW, p < 0.01) and a significantly higher TFC value than Spirulina sp. (79.87 ± 0.12 mg QE/g DW vs. 11.19 ± 0.07 mg QE/g DW, p < 0.01). Nannochloropsis sp. also exhibited a greater percentage of DPPH inhibition compared to Spirulina sp. (EC50 0.195 ± 0.007 mg/mL vs. 0.613 ± 0.003 mg/mL, p < 0.01). TPC and TFC were negatively correlated with the EC50 of DPPH antioxidant inhibition activity (-0.956, p = 0.003, and -0.899, p = 0.015, respectively). These negative correlations indicate that polyphenol compounds are the major contributors to the antioxidant activity observed in this study. Further analysis is needed to determine how to utilize the health benefits of both microalgae. [ABSTRACT FROM AUTHOR]

Mixotrophic growth is an ability of microalgae to concurrently assimilate CO 2 and organic carbon. In the present study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrate availability. Under nitrate sufficient conditions, the biomass productivities of Nannochloropsis salina (CCMP 1776) and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol thereby indicating the importance of the combined effects of carbon and nitrogen. Additionally, a carbon mass balance was conducted based on glycerol utilisation and biomass production in N. salina cultures, which revealed a net release of CO 2 during mixotrophic growth. In the presence of glycerol, the proliferation of bacteria was consistently observable in these non-axenic cultures. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to α- and γ-subclass of Proteobacteria ( Paracoccus sp. MBIC4036, and Marinobacter alkaliphilus NBSL03 and Marinobacter lipolyticus SM-19, respectively) in the cultures of N. salina . Among these strains, M. alkaliphilus was shown to have denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor. This provides an explanation for the utilisation of nitrate and glycerol that was observed in heterotrophic cultures despite the absence of microalgal growth. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for algae production. The present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation. [ABSTRACT FROM AUTHOR]

Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation. [ABSTRACT FROM AUTHOR]

The microalga Nannochloropsis produces high-value omega-3-rich fatty acids and carotenoids. In this study the effects of light intensity and wavelength on biomass, fatty acid, and carotenoid production with respect to light output efficiency were investigated. Similar biomass and fatty acid yields were obtained at high light intensity (150 μmol m −2  s −1 ) LEDs on day 7 and low light intensity (50 μmol m −2  s −1 ) LEDs on day 11 during cultivation, but the power efficiencies of biomass and fatty acid (specifically eicosapentaenoic acid) production were higher for low light intensity. Interestingly, low light intensity enhanced both, carotenoid power efficiency of carotenoid biosynthesis and yield. White LEDs were neither advantageous for biomass and fatty acid yields, nor the power efficiency of biomass, fatty acid, and carotenoid production. Noticeably, red LED resulted in the highest biomass and fatty acid power efficiency, suggesting that LEDs can be fine-tuned to grow Nannochloropsis algae more energy-efficiently. [ABSTRACT FROM AUTHOR]

The effects of an enzymatic treatment with cellulase and mannanase on the properties of marine microalgae Nannochloropsis sp. were investigated. The combined use of these enzymes synergistically promoted the recovery of lipids from the microalgae, increasing the extraction yield from 40.8 to over 73%. Untreated and enzymatically treated microalgae were characterized by chemical analysis and by TGA/DTG, FTIR, XRD and SEM. Significant changes were observed in the chemical composition and thermal behavior of the microalgae. The enzymatic treatment also resulted in an increase of the crystalline-to-amorphous cellulose ratio. SEM images revealed dramatic changes in cell morphology, extensive cell damage and release of intracellular material. Overall, the results obtained indicate that the enzymes used are capable of disrupting the microalgal cell wall and that a combination of common analytical techniques can be used to assess the enzyme-induced damage. [ABSTRACT FROM AUTHOR]

Microalgal oils, depending on their degree of unsaturation, can be utilized as either nutritional supplements or fuels; thus, a feedstock with genetically designed and tunable degree of unsaturation is desirable to maximize process efficiency and product versatility. Systematic profiling of ex vivo (in yeast), in vitro , and in vivo activities of type-2 diacylglycerol acyltransferases in Nannochloropsis oceanica (NoDGAT2s or NoDGTTs), via reverse genetics, revealed that NoDGAT2A prefers saturated fatty acids (SFAs), NoDGAT2D prefers monounsaturated fatty acids (MUFAs), and NoDGAT2C exhibits the strongest activity toward polyunsaturated fatty acids (PUFAs). As NoDGAT2A , 2C , and 2D originated from the green alga, red alga, and eukaryotic host ancestral participants of secondary endosymbiosis, respectively, a mechanistic model of oleaginousness was unveiled, in which the indigenous and adopted NoDGAT2s formulated functional complementarity and specific transcript abundance ratio that underlie a rigid SFA:MUFA:PUFA hierarchy in triacylglycerol (TAG). By rationally modulating the ratio of NoDGAT2A : 2C : 2D transcripts, a bank of N . oceanica strains optimized for nutritional supplement or fuel production with a wide range of degree of unsaturation were created, in which proportion of SFAs, MUFAs, and PUFAs in TAG varied by 1.3-, 3.7-, and 11.2-fold, respectively. This established a novel strategy to simultaneously improve productivity and quality of oils from industrial microalgae. [ABSTRACT FROM AUTHOR]

Microalgae are promising feedstocks for the production of biofuels thanks to their high biomass yield and the lack of competition with food crops for arable land. Algal industrial exploitation, however, still needs to address several technological challenges to reach energetic and economic sustainability. Genetic improvement of microalgae strains is seminal to fully exploit the potential of these organisms. In this work, we investigated how key environmental parameters affected productivity of a promising genetically modified algal strain cultivated in industrially relevant conditions. We observed that intensive growth conditions strongly influenced algae biology as evidenced by molecular and functional analyses. We also showed that specific operational conditions significantly affected performances, enhancing or reducing the advantages of the strains genetic modification, such as the chlorophyll content per cell and the abundance of photosynthetic apparatus components. This work demonstrates the presence of a strong influence of cultivation environment on the phenotype of improved strains, suggesting that operational parameters have a seminal influence on algae performances and must be taken into full account during strains development efforts. [ABSTRACT FROM AUTHOR]

There is an increasing interest in algae-based biomass produced outdoors in natural and industrial settings for biotechnological applications. To predict the yield and biochemical composition of the biomass, it is important to understand how the transcriptome of species and strains of interest is affected by seasonal changes. Here we studied the effects of Nordic winter and summer on the transcriptome of two phytoplankton species, namely the diatom Skeletonema marinoi (Sm) and the eustigmatophyte Nannochloropsis granulata (Ng), recently identified as potentially important for biomass production on the west coast of Sweden. Cultures were grown in photobioreactors in simulated Nordic summer and winter, and the gene expression in two phases was quantified by Illumina RNA-sequencing. Five paired comparisons were made among the four conditions. Sm was overall more responsive to seasons since 70 % of the total transcriptome (14,783 genes) showed differential expression in at least one comparison as compared to 1.6 % (1403 genes) for Ng. For both species, we observed larger differences between the seasons than between the phases of the same season. In summer phase 1, Sm cells focused on photosynthesis and polysaccharide biosynthesis. Nitrate assimilation and recycling of intracellular nitrogen for protein biosynthesis were more active in summer phase 2 and throughout winter. Lipid catabolism was upregulated in winter relative to summer to supply carbon for respiration. Ng favored lipid accumulation in summer, while in winter activated different lipid remodeling pathways as compared to Sm. To cope with winter, Ng upregulated breakdown and transport of carbohydrates for energy production. Taken together, our transcriptome data reveal insights into adaptive seasonal responses of Sm and Ng important for biotechnological applications on the west coast of Sweden, but more work is required to decipher the molecular mechanisms behind these responses. [Display omitted] • Skeletonema and Nannochloropsis transcriptomes were analyzed in two Nordic seasons. • Skeletonema was overall more transcriptionally responsive than Nannochloropsis. • Skeletonema had more PUFAs and used energy from lipid catabolism in winter. • Nannochloropsis had more MUFAs and used energy from carbohydrate breakdown in winter. • The adaptive responses to Nordic seasons help design biotechnological applications. [ABSTRACT FROM AUTHOR]

La producción de microalgas vivas para cultivar rotíferos constituye uno de los mayores costos de operación en la larvicultura de peces, por lo que se están desarrollando sustitutos comerciales de microalgas para la alimentación y producción de rotíferos. Se desconoce el efecto que tienen las formulaciones comerciales sobre la dinámica poblacional de rotíferos nativos del noroeste de México y en este estudio se evaluó el efecto de cuatro dietas comerciales (Espirulina, RotiMac®, Nanno 3600®, RotiGrow-Plus®) y la microalga viva Nannochloropsis sp., sobre el crecimiento poblacional y fecundidad del rotífero Brachionus ibericus (GenBank KJ949043), aislado de una granja de camarón blanco (Litopenaeus vannamei). Se realizaron cinco réplicas por tratamiento alimenticio utilizando recipientes con volumen de 12 L de agua, con temperatura de 29 ± 1°C y salinidad de 35 ± 1, respectivamente. Los resultados mostraron que durante los periodos experimentales, las dietas probadas resultaron adecuadas para alimentar a B. ibericus, con tasas de crecimiento, tiempos de duplicación, densidades máximas y fecundidades que fluctuaron entre 0.20 a 0.27 rotíferos día-1, 2.60 a 3.42 días, 215 a 344 rotíferos mL-1 y de 0.16 a 0.39 huevos hembra-1, respectivamente. Las diferencias entre tratamientos fueron significativas (P < 0.05); los menores tiempos de duplicación y las mayores tasas de crecimiento, fecundidad y densidad poblacional fueron obtenidas con RotiGrow-Plus®, pero el tiempo de duplicación y la tasa de crecimiento con RotiGrow-Plus® no fueron significativamente diferentes (P > 0.05) a las obtenidas con Nanno 3600®.

Instead of sole nutrient starvation to boost algal lipid production, we addressed nutrient limitation at two different seasons (autumn and spring) during outdoor cultivation in flat panel photobioreactors. Lipid accumulation, biomass and lipid productivity and changes in fatty acid composition of Nannochloropsis oculata were investigated under nitrogen (N) limitation (nitrate:phosphate N:P 5, N:P 2.5 molar ratio). N. oculata was able to maintain a high biomass productivity under N-limitation compared to N-sufficiency (N:P 20) at both seasons, which in spring resulted in nearly double lipid productivity under N-limited conditions (0.21 g L−1 day−1) compared to N-sufficiency (0.11 g L−1 day−1). Saturated and monounsaturated fatty acids increased from 76% to nearly 90% of total fatty acids in N-limited cultures. Higher biomass and lipid productivity in spring could, partly, be explained by higher irradiance, partly by greater harvesting rate (~30%). Our results indicate the potential for the production of algal high value products (i.e., polyunsaturated fatty acids) during both N-sufficiency and N-limitation. To meet the sustainability challenges of algal biomass production, we propose a dual-system process: Closed photobioreactors producing biomass for high value products and inoculum for larger raceway ponds recycling waste/exhaust streams to produce bulk chemicals for fuel, feed and industrial material.

Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.