Microalga Nannochloropsis Oceanica Research Articles

High-efficiency nuclear transformation of the microalgae Nannochloropsis oceanica using Tn5 Transposome for the generation of altered lipid accumulation phenotypes

BackgroundOne of the major problems in the production of lipids for biotechnological purposes using microalgae is maintaining a high productivity of these molecules without reducing cellular biomass. High production rates are usually obtained by cultivating microalgae under different stress conditions. However, many of these changes usually result in lower biomass productivity. Therefore, the optimization of the culture conditions and genetic modification techniques in these organisms is needed to generate robust new strains for profitable economic use.ResultsIn this work, we describe a new strategy for random mutation of genomic DNA in the microalgae Nannochloropsis oceanica by insertion of a Transposome complex Tn5. This complex contains an antibiotic-resistance cassette commanded by a CMV viral promoter that allows high efficiency of transformation and the generation of mutants. This strategy, complemented with a large-scale identification and selection system for mutants, such as flow cytometry with cell selection, allowed us to obtain clonal cultures of mutants with altered phenotypes in the accumulation of intracellular lipids. The characterization of some of these mutants uncovered new genes that are likely to be involved in the regulation of lipid synthesis, revealing possible cellular responses that influence the intracellular homeostasis of lipids.ConclusionThe strategies proposed here are easy to implement in different types of microalgae and provide a promising scenario for improving biotechnological applications.

Pef-assisted Supercritical CO2 Extraction of Pigments from Microalgae Nannochloropsis Oceanica in a Continuous Flow System

This study aimed at evaluating the effect of pulsed electric fields (PEF) pre-treatment on the extractability of pigments (total carotenes, chlorophyll a) from microalgae Nannochloropsis oceanica by supercritical CO2 (scCO2) extraction. Aqueous microalgal suspensions (1% w/w) added with 10% ethanol were treated in a continuous flow unit with either PEF at different field strengths (10–30 kV/cm) and total specific energy inputs (20–100 kJ/kg), or scCO2 at different pressures (8-20 MPa), with fixed temperature (35°C), CO2/biomass ratio (53.3 kgCO2/kgDW) and contact time (7 min). Extracts from conventional solvent extraction process and PEF-assisted scCO2 extraction were analyzed and compared with those achieved upon the application of single PEF and scCO2 extraction processes. Results showed that in PEF-assisted extraction the recovery yield of the target compounds significantly increased with increasing the treatment severity, even though the effect of the field strength appeared less important than the energy input. After PEF treatment at 10 kV/cm and 100 kJ/kg, the extraction yield for total carotenes and chlorophyll a increased by 1.6 and 1.4 times, respectively. During single scCO2 extraction process at 14 MPa the extraction yield for total carotenes and chlorophyll a increased up to 10.3 and 13.6 times, respectively, as compared to the untreated samples. The use of PEF prior to scCO2 extraction remarkably increased the extractability of the two pigments showing a clear synergistic effect. The results obtained in this work suggested that the application of PEF prior to scCO2 extraction could represent a suitable approach for the efficient recovery of pigments from microalgal biomass.

Foam fractionator as a tool to remove dissolved organic matter and improve the flocculation of the marine microalga Nannochloropsis oceanica

Microalgae have great biotechnological potential, yet they are difficult to harvest. Flocculation is a promising technology, but the dissolved organic matter (DOM) released by microalgae during growth interacts with the flocculant, hindering the process. This DOM is composed mostly of proteins and carbohydrates which may present a polarity ranging from hydrophobic to hydrophilic. In aquaculture, foam fractionators are routinely employed to remove DOM from cultures, improving the water quality. However, this equipment has never been employed in microalgae cultures. Therefore, the present study aimed to evaluate the use of the foam fractionator to remove DOM from a Nannochloropsis oceanica culture to improve the flocculation process. First, DOM accumulation was monitored for 33 days in a 330 L outdoor culture to characterize the dissolved organic carbon (DOC) and protein and carbohydrate contents. Samples from logarithmic (day 13) and stationary (day 29) growth phases were fractionated by polarity (hydrophobic, transphilic, and hydrophilic), the DOC and protein and carbohydrate contents of each fraction were measured, and flocculation was performed. Then the culture was treated with a foam fractionator at bench (11 L) and pilot scales (1600 L), and the composition and flocculation were compared with the non-treated culture. Proteins strongly hindered flocculation, and the foam fractionator was effective in removing this element and significantly improved the flocculation efficiency. Furthermore, N. oceanica cell integrity was not affected by the foam fractionator. These results indicate that DOM can be efficiently removed by foam fractionation, improving the microalgae harvesting process.

CRISPR\u2013Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1

BackgroundMicroalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR–Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.ResultsIn this study, we report for the first time an alternative approach for CRISPR–Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.ConclusionWe show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Fish welfare, fast muscle cellularity, fatty acid and body-composition of juvenile spotted wolffish (Anarhichas minor) fed a combination of plant proteins and microalgae (Nannochloropsis oceanica)

Spotted wolffish (Anarhichas minor) is a promising candidate for diversification of cold-water aquaculture. An increased knowledgebase is needed concerning the capacity of spotted wolffish to utilize a variety of feed ingredients such as microalgae and terrestrial plants. The aim of the study was therefore to investigate the effect of incorporating graded levels of microalgae (Nannochloropsis oceanica) on fish welfare indicators (growth, hepasomatic index, hematological parameters), fast muscle cellularity, chemical and fatty acid composition. Three isonitrogenous and isocaloric diets were formulated; one control diet (0% N. oceanica) and two diets with low (7.5% N. oceanica) and high (15% N. oceanica) levels of microalgae replacing fishmeal and wheat in the diets. After 12 weeks of feeding, the fish showed low growth compared to previous wolffish studies (0.26% SGR). However, this effect was not treatment dependent as there were no differences in growth or fast muscle cellularity among the three treatment groups and hematological parameters showed no indications of stress in the fish fed microalgae. Hepatosomatic index decreased over the course of the experiment for all treatment groups; a significantly larger reduction was noted in the algae-fed fish compared to the control. The omega-3 fatty acid EPA increased in the whole body of the fish fed diets containing microalgae. The results suggest that spotted wolffish has potential to utilize inclusions of up to 15% of the microalgae N. oceanica.

Knockdown of carbonate anhydrase elevates Nannochloropsis productivity at high CO2 level

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 CO2 (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% CO2 (high carbon, or HC), as compared to the wild type. Such high-CO2-level activated biomass over-production is reproducible across photobioreactor types and cultivation scales. Transcriptomic, proteomic and physiological changes of the mutant under high CO2 (HC; 5% CO2) 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-CO2-assimilating and autonomously containable industrial microalgae for flue gas-based oil production.

Chlorophyll–carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis

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.

Evaluation of a native oleaginous marine microalga Nannochloropsis oceanica for dual use in biodiesel production and aquaculture feed

Screening of native microalgal species for different biotechnological applications is a key step to achieve a strong algal database with successful commercial applications. In the present study, a new strain namely Nannochloropsis oceanica NIOF15/001 was isolated and identified phylogenetically using rbcL and the newly designed Cyt-c as an alternative molecular marker. The late exponential phase of the isolate was recorded at the 10th day of growth in f/2 medium, with a maximum cellular dry weight (CDW) of 0.722 g L−1 and relatively high lipid content of 37.7% of CDW. The isolate showed biomass and lipid productivities of 75.6 and 28.6 mg L−1 day−1, respectively. The recorded biomass productivity in the current study was 1.4, 3.0, 3.6 and 2.0 times higher than that recorded in the previous studies for N. oceanica IMET, N. oceanica CASACC201, N. oceanica DUT01 and N. oceanica CCNM 1081, respectively. In general, values of cetane number (52.3), iodine value (94.7 g I2/100 g oil), cold filter plugging point (4.7 °C) and kinematic viscosity (3.97 mm2 s−1) of the produced fatty acid methyl esters complied with those recommended by international standards. Furthermore, application of 0.1 g L−1 of lipid-free dry biomass enhanced Artemia survival by 500% and growth by 40%. The present study suggested the new isolate N. oceanica NIOF15/001 as a promising marine microalga for dual use in aquaculture and biodiesel production.

The Protective Effect of Violaxanthin from Nannochloropsis oceanica against Ultraviolet B-Induced Damage in Normal Human Dermal Fibroblasts.

Skin photoaging, which is mainly induced by ultraviolet B (UVB) radiation, is prevented by the application of UV-protective agents. The microalga Nannochloropsis oceanica (N.oceanica) has been primarily reported as a potential biofuel; however, in this study, we investigated whether N.oceanica extracts exerted photoprotective effects against UVB-irradiated human dermal fibroblasts (HDFs) and which single component was responsible for the protective effect of the extracts. Two extracts-pigment and nonpigment-were prepared from N.oceanica biomass. WST-1 assay and expression analysis of interleukin genes showed that the pigment extracts were not significantly cytotoxic to HDFs. Further experiments revealed that treatment with the pigment extract upregulated the expression of collagen genes and significantly blocked UVB-induced damage such as decreased cell viability and increased ROS production. Next, to investigate the pigment composition of the extracts, HPLC analysis was conducted and violaxanthin was identified as the major pigment. The UVB photoprotective effect of the pigment extracts was confirmed in violaxanthin-treated HDFs. In addition, violaxanthin significantly attenuated UVB-induced G1 phase arrest, senescence-associated β-galactosidase activation, p16 and p21 upregulation, ERK phosphorylation and the downregulation of ECM molecules in HDFs. Therefore, we concluded that violaxanthin was a potential antiphotoaging agent.

Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochl oropsis oceanica LAMB0001 were domesticated (~730 generations, ~two days each) to adapt freshwater BG11 medium. A number of freshwater medium adapted colonyderived strains were obtained. The strains were verified phylogenetically to be N. oceanica LAMB0001 based on the 18S ribosomal RNA gene. Freshwater-medium adapted strain (FA1) grew faster in the BG11 medium prepared with freshwater than wild-type N. oceanica grew in f/2 medium prepared with seawater. We assumed that (1) the expression patterns of the genes that expressed differentially between FA1 and the wild-type N. oceanica exposing to the BG11 medium (WT-F) have been reprogrammed; (2) the physiological processes in which these genes involved have been modified; and (3) a Gene Ontology (GO) term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up- or down-regulated if it was enriched simultaneously by up- or down-regulated DEGs between FA1 and WT-F, respectively. Under these assumptions, we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion, membrane and membrane integrity, material transportation, cell movement, and cellular signaling network. These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium. It is feasible to domesticate marine microalgae to a freshwater habitat, which may aid to modify their cultivation performances.

The action of small bubbles in increasing light exposure and production of the marine microalga Nannochloropsis oceanica in massive culture systems

The production of a cost-effective, massive microalgae cultivation system relies on a highly concentrated culture produced with minimal energy input. A previous study demonstrated that reducing air bubble diameters from 9 mm to 6.5 mm through nozzle diameter reduction from a single injector with a 3 mm diameter to a set of nine injectors with diameter of 1 mm resulted in higher biomass yields of the marine microalga Nannochloropsis oceanica without any extra energy demand. However, the main differences in microalgae growth were only observed after 4 to 6 days of cultivation. We hypothesize that this difference may be triggered either by improved nutrient supply and/or higher light exposure of microalgae cells; both provided by the smaller bubbles improved the hydrodynamic mixing performance. In this study, nutrient concentration (Ammonium, Nitrate and Phosphate) and irradiance measurements were conducted over 10 days when N. oceanica was cultivated in 330 L cylindrical photobioreactors and a 1600 L open circular tank with different air bubble sizes (6.5 mm and 9 mm). A higher biomass was obtained in the treatments with smaller bubbles, but this difference was only significant after at least 4 days. The results show that the nutrient concentration did not differ significantly between treatments. However, the main differences in biomass production occurred when light was considerably reduced (<50 μmol photons m−2 s−2), indicating that light is a limiting growth factor; however, this limitation can be partially surpassed by the improved mixing condition created by the higher mass transfer coefficient of the smaller bubbles. This finding is supported by Lagrangian simulations, which show that the trajectories of the microalgae particles/cells presented higher light exposure frequency in the treatment with the smaller air bubbles.

Bioavailability and Safety of Nutrients from the Microalgae Chlorella vulgaris, Nannochloropsis oceanica and Phaeodactylum tricornutum in C57BL/6 Mice

Microalgae are rich in macronutrients and therefore, they have been proposed as a potential future food source preserving natural resources. Here, we studied safety and bioavailability of algae nutrients in mice. Three microalgae species, Chlorella vulgaris, Nannochloropsis oceanica and Phaeodactylum tricornutum, were studied after ball mill disruption at different doses (5%, 15% and 25% dry weight) for 14 days. In response to all three algae diets, we observed a weight gain similar or superior to that in response to the control diet. No substantial differences in organ weights nor gut length occurred. Protein bioavailability from the algae diets did not differ from the control diet ranging from 58% to 77% apparent biological value. Fat absorption was lower for microalgae compared to soy oil in control diets, albeit still substantial. High liver eicosapentaenoic acid levels were measured following feeding with N. oceanica, the algae richest in omega-3 fatty acids. Neither histological nor serum analyses revealed any heart, kidney or liver toxicity induced by any of the algae diets. Algae-rich diets were thus well accepted, well tolerated and suitable for the maintenance of body weight and normal organ function. No toxicological effects were observed.

Studies on the catalytic behavior of a membrane-bound lipolytic enzyme from the microalgae Nannochloropsis oceanica CCMP1779

The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol−1. The enzyme’s enthalpy (ΔΗ*), entropy (ΔS*) and Gibb’s free energy (ΔG*) were in the range of 65.86–66.27 kJ mol−1, 132.38–140.64 J mol−1 K−1 and 107.80–115.81 kJ mol−1, respectively. Among p-nitrophenyl esters of fatty acids tested, MBL-Enzyme exhibited the highest hydrolytic activity against p-nitrophenyl palmitate (pNPP). The Km and Vmax values were found 0.051 mM and of 0.054 mmole pNP mg protein−1 min−1, respectively with pNPP as substrate. The presence of Mn2+ increased lipolytic activity by 68.25%, while Fe3+ and Cu2+ ions had the strongest inhibitory effect. MBL-Enzyme was stable in the presence of water miscible (66% of the initial activity in ethanol) and water immiscible (71% of the initial activity in n-octane) solvents. Myristic acid was found to be the most efficient acyl donor in esterification reactions with ethanol. Methanol was the best acyl acceptor among the primary alcohols tested.

Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica

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.

Betaine Lipid Is Crucial for Adapting to Low Temperature and Phosphate Deficiency in Nannochloropsis

Diacylglyceryl-N,N,N-trimethylhomo-Ser (DGTS) is a nonphosphorous, polar glycerolipid that is regarded as analogous to the phosphatidylcholine in bacteria, fungi, algae, and basal land plants. In some species of algae, including the stramenopile microalga Nannochloropsis oceanica, DGTS contains an abundance of eicosapentaenoic acid (EPA), which is relatively scarce in phosphatidylcholine, implying that DGTS has a unique physiological role. In this study, we addressed the role of DGTS in N. oceanica We identified two DGTS biosynthetic enzymes that have distinct domain configurations compared to previously identified DGTS synthases. Mutants lacking DGTS showed growth retardation under phosphate starvation, demonstrating a pivotal role for DGTS in the adaptation to this condition. Under normal conditions, DGTS deficiency led to an increase in the relative amount of monogalactosyldiacylglycerol, a major plastid membrane lipid with high EPA content, whereas excessive production of DGTS induced by gene overexpression led to a decrease in monogalactosyldiacylglycerol. Meanwhile, lipid analysis of partial phospholipid-deficient mutants revealed a role for phosphatidylcholine and phosphatidylethanolamine in EPA biosynthesis. These results suggest that DGTS and monogalactosyldiacylglycerol may constitute the two major pools of EPA in extraplastidic and plastidic membranes, partially competing to acquire EPA or its precursors derived from phospholipids. The mutant lacking DGTS also displayed impaired growth and a lower proportion of EPA in extraplastidic compartments at low temperatures. Our results indicate that DGTS is involved in the adaptation to low temperatures through a mechanism that is distinct from the DGTS-dependent adaptation to phosphate starvation in N. oceanica.

Hydrodynamic performance of two air nozzles diameters on the massive microalgae culture: Computational and experimental approaches

The scaling of microalgae production in culture systems depends on preventing cells to settle down and assure a well-mixed water column. Doing so, each cell would have better exposure to light and nutrients. Computational Fluid Dynamics (CFD) can be an effective tool for understanding and predicting the multiphase flow inside tanks. In this study, we have used CFD to simulate the velocity field and the turbulent kinetic energy inside a computational tank to find best hydrodynamic performance for two nozzles configurations: case 1) one single 3mm air nozzle; case 2) a set of nine 1mm air nozzles. Computational results showed that smaller air nozzles are more efficient in reducing dead zones inside culturing tanks. Further, mean turbulent kinetic energy field suggested a more homogeneous dissipation, and higher intensities of turbulence were observed in the smaller nozzle simulation. The application of present numerical observations on culture systems was investigated by several experiments in 300L cylindrical photobioreactors and 1600L open circular tanks, both with different air nozzles sizes (1 and 3mm) where the marine microalga Nannochloropsis oceanica was grown. Microalga growth was determined by measurements of water absorbance at 750nm. Higher cell densities were determined in the photobioreactors and tanks with 1mm nozzles. Thus, smaller nozzle was efficient to improve the microalga production even with increasing volume and different culture system designs, supporting the numerical analyses predictions by CFD.

Enhancing photosynthetic biomass productivity of industrial oleaginous microalgae by overexpression of RuBisCO activase

Eukaryotic microalgae are promising cell factories for photosynthesis-driven CO2 sequestration and oil production, however one key challenge has been to genetically enhance their biomass productivity. Here we showed that the biomass productivity of a model industrial oleaginous microalga Nannochloropsis oceanica can be elevated by overexpression of a nuclear-encoded, cbbX-homologous, candidate RuBisCO activase (g1915 or ‘nNoRca-like’) that is transcriptionally induced under air-level CO2 (i.e., 0.04% v/v) as compared to 5% CO2 in wild-type cells. In the mutants, under air-level CO2, growth rate was enhanced by ~32%, biomass accumulation by ~46% and lipid productivity by ~41%, which were also accompanied by ~28% elevation in photosynthesis; furthermore, abundance of RuBisCO large subunit protein increased by ~45%, suggesting nNoRca-like overexpression enhanced microalgal photosynthesis by upregulating the level and activity of RuBisCO. Interestingly, under 5% CO2, no significant enhancement of RuBisCO protein expression was observed. Thus a genetic strategy that activates RuBisCO by augmenting its functional partners was proposed to increase biomass production of industrial microalgae. Our work expands the reservoir of potential photosynthetic gene targets for plant biotechnology and provides a basis for constructing advanced microalgal cell factories for carbon sequestration and biofuel production.

Identification of a malonyl CoA-acyl carrier protein transacylase and its regulatory role in fatty acid biosynthesis in oleaginous microalga Nannochloropsis oceanica.

Oleaginous microalgae hold great promises for biofuel production. However, commercialization of microalgal biofuels remains impracticable due to the lack of suitable industrial strains with high growth rate and lipid productivity. Engineering of metabolic pathways is a potential strategy for the improvement of microalgal strains for the production of lipids and also value-added products in microalgae. Malonyl CoA-acyl carrier protein transacylase (MCAT) has been reported to be involved in fatty acid biosynthesis. Here, we identified a putative MCAT in the oleaginous marine microalga Nannochloropsis oceanica. NoMCAT overexpressing N. oceanica showed a higher growth rate and photosynthetic efficiency. The neutral lipid content of engineered lines showed a significant increase by up to 31% compared to wild type. Gas chromatography-mass spectrometry analysis revealed that NoMCAT overexpression significantly altered the fatty acid composition. The composition of eicosapentaenoic acid (C20:5), which is a polyunsaturated fatty acid necessary for animal nutrition, increased by 8%. These results demonstrate the role of MCAT in enhancing fatty acid biosynthesis and growth in microalgae, and also provide an insight into metabolic engineering of microalgae with high industrial potential.

Occurrence forms of key ash-forming elements in defatted microalgal biomass

This study reports occurrence forms of key ash-forming elements in a defatted microalga, and for comparison, its corresponding raw microalga. Freeze-dried powders of a marine microalga (Nannochloropsis oceanica) were sieved to a size fraction of <75μm and used as raw microalga. The raw microalga was then extracted with hexane to remove crude lipids and prepare a defatted microalga. The raw and defatted microalgae were subjected to chemical fractionation analysis, i.e., sequential leaching in H2O, 1.0M ammonium acetate (NH4Ac), and 1.0M hydrochloric (HCl) acid. The results demonstrate that, whereas the contents of Fe and Al in the raw and defatted microalgae are extremely low, those of other ash-forming elements follow a sequence of Cl>K>P>Mg>Na>Ca. Chemical fractionation results suggest that virtually all of the Na, K, and Cl in the raw and defatted microalgae are water-soluble. While majority of P in the two fuels are water-soluble and acid-soluble, most of Mg and Ca are leached in water and NH4Ac solution. As determined via chemical fractionation analysis, lipid extraction leads to the content of water-soluble Ca in the defatted microalga being ∼69.6% higher than that in the raw microalga counterpart, which is accompanied by a reduction in the amount of Ca leached in NH4Ac solution. Similar trend is also observed for Mg, but to a lesser extent.

Diel oscillation in the optical activity of carotenoids in the absorption spectrum of Nannochloropsis.

In this paper we show that the absorption spectrum of the microalgae Nannochloropsis oceanica exhibits changes in response to the modulation of incident light. A model was used to analyze the contribution of different active pigments to the total absorption in the photosynthetically active radiation region and suggested consistent diel oscillations in the optical activity of carotenoids.