Effects of Nannochloropsis salina Fermented Oil on Proliferation of Human Dermal Papilla Cells and Hair Growth.

The hair follicle is the basis of hair regeneration, and the dermal papilla is one of the most important structures in hair regeneration. New intervention and reversal strategies for hair loss may arise due to the prevention of oxidative stress. GC/MS analysis was used to determine the compounds contained in NSO. Then, NSO was applied to DPC for cell proliferation and oxidative stress experiments. RNA-seq was performed in cells treated with NSO and minoxidil. The quantitative real-time polymerase chain reaction (qRT-PCR) was applied to verify the gene expression. The effects of NSO on hair length, weight, the number and depth of hair follicles, and the dermal thickness were also studied. GC/MS analysis showed that the main components of NSO were eicosapentaenoic acid, palmitic acid, and linoleic acid. NSO promotes DPC proliferation and reduces H2O2-mediated oxidative damage. NSO can also activate hair growth-related pathways and upregulate antioxidant-related genes analyzed by gene profiling. The topical application of NSO significantly promotes hair growth and increases hair length and weight in mice. NSO extract promotes hair growth and effectively inhibits oxidative stress, which is beneficial for the prevention and treatment of hair loss.

Heterologous overexpression of the cyanobacterial alcohol dehydrogenase sysr1 confers cold tolerance to the oleaginous alga Nannochloropsis salina.

Temperature is an important regulator of growth in algae and other photosynthetic organisms. Temperatures above or below the optimal growth temperature could cause oxidative stress to algae through accumulation of oxidizing compounds such as reactive oxygen species (ROS). Thus, algal temperature stress tolerance could be attained by enhancing oxidative stress resistance. In plants, alcohol dehydrogenase (ADH) has been implicated in cold stress tolerance, eliciting a signal for the synthesis of antioxidant enzymes that counteract oxidative damage associated with several abiotic stresses. Little is known whether temperature stress could be alleviated by ADH in algae. Here, we generated transgenic lines of the unicellular oleaginous alga Nannochloropsis salina that heterologously expressed sysr1, which encodes ADH in the cyanobacterium Synechocystis sp. PCC 6906. To drive sysr1 expression, the heat shock protein 70 (HSP70) promoter isolated from N. salina was used, as its transcript levels were significantly increased under either cold or heat stress growth conditions. When subjected to cold stress, transgenic N. salina cells were more cold-tolerant than wild-type cells, showing less ROS production but increased activity of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and catalase. Thus, we suggest that reinforcement of alcohol metabolism could be a target for genetic manipulation to endow algae with cold temperature stress tolerance.

Safe-Harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776: Efficient overexpression of transgene via CRISPR/Cas9-Mediated Knock-in at the transcriptional hotspot.

Transgene expression in microalgae can be hampered by transgene silencing and unstable expression due to position effects. To overcome this, "safe harboring" transgene expression system was established for Nannochloropsis. Initially, transformants were obtained expressing a sfGFP reporter, followed by screening for high expression of sfGFP with fluorescence-activated cell sorter (FACS). 'T1' transcriptional hotspot was identified from a mutant showing best expression of sfGFP, but did not affect growth or lipid contents. By using a Cas9 editor strain, FAD12 gene, encoding Δ12-fatty acid desaturase (FAD12), was successfully knocked-in at the T1 locus, resulting in significantly higher expression of FAD12 than those of random integration. Importantly, the "safe harbored" FAD12 transformants showed four-fold higher production of linoleic acid (LA), the product of FAD12, leading to 1.5-fold increase in eicosapentaenoic acid (EPA). This safe harboring principle provide excellent proof of the concept for successful genetic/metabolic engineering of microalgae and other organisms.

Modelling and process optimization for biodiesel production from Nannochloropsis salina using artificial neural network.

In the present investigation, calcium oxide solid nanocatalyst derived from the egg shell and Nannochloropsis salina were used for the production of biodiesel. The morphological characteristics and functional groups of synthesized nanocatalyst was characterized by SEM and FTIR analysis. Process variables optimization for biodiesel production was studied using RSM and ANN. The R2 values for RSM and ANN was found to be 0.8751 and 0.957 which showed that the model was significantly fit with the experimental data. The maximum FAME conversion for the synthesized nanocatalyst CaO was found to be 86.1% under optimum process conditions (nanocatalyst amount: 3% (w/v); oil to methanol ratio 1:6 (v/v); reaction temperature: 60 °C; reaction time 55 min). Concentration of FAME present in biodiesel was identified by GC-MS analysis.

Development and characterization of a Nannochloropsis mutant with simultaneously enhanced growth and lipid production.

BACKGROUND: The necessity to develop high lipid-producing microalgae is emphasized for the commercialization of microalgal biomass, which is environmentally friendly and sustainable. Nannochloropsis are one of the best industrial microalgae and have been widely studied for their lipids, including high-value polyunsaturated fatty acids (PUFAs). Many reports on the genetic and biological engineering of Nannochloropsis to improve their growth and lipid contents have been published. RESULTS: We performed insertional mutagenesis in Nannochloropsis salina, and screened mutants with high lipid contents using fluorescence-activated cell sorting (FACS). We isolated a mutant, Mut68, which showed improved growth and a concomitant increase in lipid contents. Mut68 exhibited 53% faster growth rate and 34% higher fatty acid methyl ester (FAME) contents after incubation for 8 days, resulting in a 75% increase in FAME productivity compared to that in the wild type (WT). By sequencing the whole genome, we identified the disrupted gene in Mut68 that encoded trehalose-6-phosphate (T6P) synthase (TPS). TPS is composed of two domains: TPS domain and T6P phosphatase (TPP) domain, which catalyze the initial formation of T6P and dephosphorylation to trehalose, respectively. Mut68 was disrupted at the TPP domain in the C-terminal half, which was confirmed by metabolic analyses revealing a great reduction in the trehalose content in Mut68. Consistent with the unaffected N-terminal TPS domain, Mut68 showed moderate increase in T6P that is known for regulation of sugar metabolism, growth, and lipid biosynthesis. Interestingly, the metabolic analyses also revealed a significant increase in stress-related amino acids, including proline and glutamine, which may further contribute to the Mut68 phenotypes. CONCLUSION: We have successfully isolated an insertional mutant showing improved growth and lipid production. Moreover, we identified the disrupted gene encoding TPS. Consistent with the disrupted TPP domain, metabolic analyses revealed a moderate increase in T6P and greatly reduced trehalose. Herein, we provide an excellent proof of concept that the selection of insertional mutations via FACS can be employed for the isolation of mutants with improved growth and lipid production. In addition, trehalose and genes encoding TPS will provide novel targets for chemical and genetic engineering, in other microalgae and organisms as well as Nannochloropsis.

Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency.

BACKGROUND: Chlorophylls play important roles in photosynthesis, and thus are critical for growth and related metabolic pathways in photosynthetic organisms. They are particularly important in microalgae, emerging as the next generation feedstock for biomass and biofuels. Nannochloropsis are industrial microalgae for these purposes, but are peculiar in that they lack accessory chlorophylls. In addition, the localization of heterologous proteins to the chloroplast of Nannochloropsis has not been fully studied, due to the secondary plastid surrounded by four membranes. This study addressed questions of correct localization and functional benefits of heterologous expression of chlorophyllide a oxygenase from Chlamydomonas (CrCAO) in Nannochloropsis. RESULTS: We cloned CrCAO from Chlamydomonas, which catalyzes oxidation of Chla producing Chlb, and overexpressed it in N. salina to reveal effects of the heterologous Chlb for photosynthesis, growth, and lipid production. For correct localization of CrCAO into the secondary plastid in N. salina, we added the signal-recognition sequence and the transit peptide (cloned from an endogenous chloroplast-localized protein) to the N terminus of CrCAO. We obtained two transformants that expressed CrCAO and produced Chlb. They showed improved growth under medium light (90 µmol/m2/s) conditions, and their photosynthetic efficiency was increased compared to WT. They also showed increased expression of certain photosynthetic proteins, accompanied by an increased maximum electron-transfer rate up to 15.8% and quantum yields up to 17%, likely supporting the faster growth. This improved growth resulted in increased biomass production, and more importantly lipid productivity particularly with medium light. CONCLUSIONS: We demonstrated beneficial effects of heterologous expression of CrCAO in Chlb-less organism N. salina, where the newly produced Chlb enhanced photosynthesis and growth. Accordingly, transformants showed improved production of biomass and lipids, important traits of microalgae from the industrial perspectives. Our transformants are the first Nannochloropsis cells that produced Chlb in the whole evolutionary path. We also succeeded in delivering a heterologous protein into the secondary plastid for the first time in Nannochloropsis. Taken together, our data showed that manipulation of photosynthetic pigments, including Chlb, can be employed in genetic improvements of microalgae for production of biofuels and other biomaterials.

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines.

Circadian clocks allow organisms to predict environmental changes caused by the rotation of the Earth. Although circadian rhythms are widespread among different taxa, the core components of circadian oscillators are not conserved and differ between bacteria, plants, animals and fungi. Stramenopiles are a large group of organisms in which circadian rhythms have been only poorly characterized and no clock components have been identified. We have investigated cell division and molecular rhythms in Nannochloropsis species. In the four strains tested, cell division occurred principally during the night period under diel conditions; however, these rhythms damped within 2-3 days after transfer to constant light. We developed firefly luciferase reporters for the long-term monitoring of in vivo transcriptional rhythms in two Nannochlropsis species, Nannochloropsis oceanica CCMP1779 and Nannochloropsis salina CCMP537. The reporter lines express anticipatory behavior under light/dark cycles and free-running bioluminescence rhythms with periods of ~21-31 h that damped within ~3-4 days under constant light. Using different entrainment regimes, we demonstrate that these rhythms are modulated by a circadian-type oscillator. In addition, the phase of free-running luminescence rhythms can be modulated pharmacologically using a CK1 ε/δ inhibitor, suggesting a role of this kinase in the Nannochloropsis clock. Together with the molecular and genomic tools available for Nannochloropsis species, these reporter lines represent an excellent system for future studies on the molecular mechanisms of stramenopile circadian oscillators.

Increased biomass and lipid production by continuous cultivation of Nannochloropsis salina transformant overexpressing a bHLH transcription factor.

Microalgae are promising feedstocks for sustainable and eco-friendly production of biomaterials, which can be improved by genetic engineering. It is also necessary to optimize the processes to produce biomaterials from engineered microalgae. We previously reported that genetic improvements of an industrial microalga Nannochloropsis salina by overexpressing a basic helix-loop-helix transcription factor (NsbHLH2). These transformants showed an improved growth and lipid production particularly during the early phase of culture under batch culture. However, they had faster uptake of nutrients, resulting in earlier starvation and reduced growth during the later stages. We attempted to optimize the growth and lipid production by growing one of the transformants in continuous culture with variable dilution rate and feed nitrogen concentration. Relative to wild-type, NsbHLH2 transformant consumed more nitrate at a high dilution rate (0.5 day -1 ), and had greater biomass production. Subsequently, nitrogen limitation at continuous cultivation led to an increased fatty acid methyl ester production by 83.6 mg l -1 day -1 . To elucidate genetic mechanisms, we identified the genes containing E-boxes, known as binding sites for bHLH transcription factors. Among these, we selected 18 genes involved in the growth and lipid metabolism, and revealed their positive contribution to the phenotypes via quantitative real-time polymerase chain reaction. These results provide proof-of-concept that NsbHLH2 can be used to produce biomass and lipids.

Suboptimal Temperature Acclimation Affects Kennedy Pathway Gene Expression, Lipidome and Metabolite Profile of Nannochloropsis salina during PUFA Enriched TAG Synthesis.

In humans, dietary polyunsaturated fatty acids (PUFAs) are involved in therapeutic processes such as prevention and treatment of cardiovascular diseases, neuropsychiatric disorders, and dementia. We examined the physiology, PUFA accumulation and glycerol lipid biosynthesis in the marine microalga Nannochloropsis salina in response to constant suboptimal temperature (<20 °C). As expected, N. salina exhibited significantly reduced growth rate and photosynthetic activity compared to optimal cultivation temperature. Total fatty acid contents were not significantly elevated at reduced temperatures. Cultures grown at 5 °C had the highest quantity of eicosapentanoic acid (EPA) (C20:5n3) and the lowest growth rate. Additionally, we monitored broadband lipid composition to model the occurrence of metabolic alteration and remodeling for various lipid pools. We focused on triacylglycerol (TAG) with elevated PUFA content. TAGs with EPA at all three acyl positions were higher at a cultivation temperature of 15 °C. Furthermore, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, which are polar lipids associated with chloroplast membranes, decreased with reduced cultivation temperatures. Moreover, gene expression analysis of key genes involved in Kennedy pathway for de novo TAG biosynthesis revealed bimodal variations in transcript level amongst the temperature treatments. Collectively, these results show that Nannochloropsis salina is a promising source of PUFA containing lipids.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides.

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina.

Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT-PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae.

Increased lipid production by heterologous expression of AtWRI1 transcription factor in Nannochloropsis salina.

BACKGROUND: Genetic engineering of microalgae is necessary to produce economically feasible strains for biofuel production. Current efforts are focused on the manipulation of individual metabolic genes, but the outcomes are not sufficiently stable and/or efficient for large-scale production of biofuels and other materials. Transcription factors (TFs) are emerging as good alternatives for engineering of microalgae, not only to increase production of biomaterials but to enhance stress tolerance. Here, we investigated an AP2 type TF Wrinkled1 in Arabidopsis (AtWRI1) known as a key regulator of lipid biosynthesis in plants, and applied it to industrial microalgae, Nannochloropsis salina. RESULTS: We expressed AtWRI1 TF heterologously in N. salina, named NsAtWRI1, in an effort to re-enact its key regulatory function of lipid accumulation. Stable integration AtWRI1 was confirmed by RESDA PCR, and its expression was confirmed by Western blotting using the FLAG tag. Characterizations of transformants revealed that the neutral and total lipid contents were greater in NsAtWRI1 transformants than in WT under both normal and stress conditions from day 8. Especially, total lipid contents were 36.5 and 44.7% higher in NsAtWRI1 2-3 than in WT under normal and osmotic stress condition, respectively. FAME contents of NsAtWRI1 2-3 were also increased compared to WT. As a result, FAME yield of NsAtWRI1 2-3 was increased to 768 mg/L/day, which was 64% higher than that of WT under the normal condition. We identified candidates of AtWRI1-regulated genes by searching for the presence of the AW-box in promoter regions, among which lipid metabolic genes were further analyzed by qRT-PCR. Overall, qRT-PCR results on day 1 indicated that AtWRI1 down-regulated TAGL and DAGK, and up-regulated PPDK, LPL, LPGAT1, and PDH, resulting in enhanced lipid production in NsAtWRI1 transformants from early growth phase. CONCLUSION: AtWRI1 TF regulated several genes involved in lipid synthesis in N. salina, resulting in enhancement of neutral lipid and FAME production. These findings suggest that heterologous expression of AtWRI1 TF can be utilized for efficient biofuel production in industrial microalgae.

Reconstruction of the microalga Nannochloropsis salina genome-scale metabolic model with applications to lipid production.

BACKGROUND: Nannochloropsis salina (= Eustigmatophyceae) is a marine microalga which has become a biotechnological target because of its high capacity to produce polyunsaturated fatty acids and triacylglycerols. It has been used as a source of biofuel, pigments and food supplements, like Omega 3. Only some Nannochloropsis species have been sequenced, but none of them benefit from a genome-scale metabolic model (GSMM), able to predict its metabolic capabilities. RESULTS: We present iNS934, the first GSMM for N. salina, including 2345 reactions, 934 genes and an exhaustive description of lipid and nitrogen metabolism. iNS934 has a 90% of accuracy when making simple growth/no-growth predictions and has a 15% error rate in predicting growth rates in different experimental conditions. Moreover, iNS934 allowed us to propose 82 different knockout strategies for strain optimization of triacylglycerols. CONCLUSIONS: iNS934 provides a powerful tool for metabolic improvement, allowing predictions and simulations of N. salina metabolism under different media and genetic conditions. It also provides a systemic view of N. salina metabolism, potentially guiding research and providing context to -omics data.

Impact of inorganic contaminants on microalgae productivity and bioremediation potential.

As underdeveloped nations continue to industrialize and world population continues to increase, the need for energy, natural resources, and goods will lead to ever increasing inorganic contaminants, such as heavy metals, in various waste streams that can have damaging effects on plant life, wildlife, and human health. This work is focused on the evaluation of the potential of Nannochloropsis salina to be integrated with contaminated water sources for the concurrent production of a biofuel feedstock while providing an environmental service through bioremediation. Individual contaminants (As, Cd, Cr, Co, Cu, Pb, Ni, Hg, Se, and Zn) at various concentrations ranging from a low concentration (1X) to higher concentrations (10X, and 40X) found in contaminated systems (mine tailings, wastewater treatment plants, produced water) were introduced into growth media. Biological growth experimentation was performed in triplicate at the various contaminant concentrations and at 3 different light intensities. Results show that baseline concentrations of each contaminant slightly decreased biomass growth to between 89% and 99% of the control with the exception of Ni which dramatically reduced growth. Increased contaminant concentrations resulted in progressively lower growth rates for all contaminants tested. Lipid analysis shows most baseline contaminant concentrations slightly decrease or have minimal effects on lipid content at all light levels. Trace contaminant analysis on the biomass showed Cd, Co, Cu, Pb, and Zn were sorbed by the microalgae with minimal contaminants remaining in the growth media illustrating the effectiveness of microalgae to bioremediate these contaminants when levels are sufficiently low to not detrimentally impact productivity. The microalgae biomass was less efficient at sorption of As, Cr, Ni, and Se.

High-EPA Biomass from Nannochloropsis salina Cultivated in a Flat-Panel Photo-Bioreactor on a Process Water-Enriched Growth Medium.

Nannochloropsis salina was grown on a mixture of standard growth media and pre-gasified industrial process water representing effluent from a local biogas plant. The study aimed to investigate the effects of enriched growth media and cultivation time on nutritional composition of Nannochloropsis salina biomass, with a focus on eicosapentaenoic acid (EPA). Variations in fatty acid composition, lipids, protein, amino acids, tocopherols and pigments were studied and results compared to algae cultivated on F/2 media as reference. Mixed growth media and process water enhanced the nutritional quality of Nannochloropsis salina in laboratory scale when compared to algae cultivated in standard F/2 medium. Data from laboratory scale translated to the large scale using a 4000 L flat panel photo-bioreactor system. The algae growth rate in winter conditions in Denmark was slow, but results revealed that large-scale cultivation of Nannochloropsis salina at these conditions could improve the nutritional properties such as EPA, tocopherol, protein and carotenoids compared to laboratory-scale cultivated microalgae. EPA reached 44.2% ± 2.30% of total fatty acids, and α-tocopherol reached 431 ± 28 µg/g of biomass dry weight after 21 days of cultivation. Variations in chemical compositions of Nannochloropsis salina were studied during the course of cultivation. Nannochloropsis salina can be presented as a good candidate for winter time cultivation in Denmark. The resulting biomass is a rich source of EPA and also a good source of protein (amino acids), tocopherols and carotenoids for potential use in aquaculture feed industry.

Continuous flocculation-sedimentation for harvesting Nannochloropsis salina biomass.

A continuous flow process is developed for recovery of the biomass of the marine microalga Nannochloropsis salina. Flocculation-sedimentation is used to recover the biomass from an algal suspension with an initial dry biomass concentration of 0.5 g L(-1), as would be typical of a raceway-based biomass production system. More than 85% of the biomass initially in suspension could be settled by gravity in a flocculation-sedimentation device with a total residence time of ∼148 min. Aluminum sulfate was used as an inexpensive, readily available and safe flocculant. The optimal flocculant dosage (as Al2(SO4)3) was 229 mg L(-1). Relative to a highly effective 62-min batch flocculation-sedimentation process for the same alga and flocculant, the continuous flow operation took longer and required nearly double the flocculant dose. The design of the flocculation-sedimentation system is explained.

Heterotrophic cultivation of Nannochloropsis salina for enhancing biomass and lipid production.

Response surface methodology (RSM) was used to enhance the biomass and lipid content in Nannochloropsis salina due to its economic importance. Preliminary screening results revealed that the heterotrophically cultivated N. salina with various carbon and nitrogen sources yielded higher biomass (0.91 ± 0.0035 g/L) and lipid content (37.1 ± 0.49 mg/L) than that of the photoautotrophical cultivation (0.21 ± 0.009 g/L and 22.16 ± 0.27 mg/L). Significant sources that greatly influenced on biomass and lipid content of the alga were optimized through RSM. The medium consisting of glucose (7.959 g/L), sodium acetate (1.46 g/L), peptone (7.6 g/L) and sodium thiosulphate (1.05 g/L) was found to be the optimal concentration for heterotrophic cultivation by response optimizer. Confirmation experiment results for the RSM optimized concentration yielded the biomass of 1.85 g/L and total lipid content of 48.6 mg/L. In this study, we provide with a strategy for enhancing the biomass and lipid content in N. salina.

Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina.

BACKGROUND: Microalgae are considered promising alternative energy sources because they consume CO2 and accumulate large amounts of lipids that can be used as biofuel. Nannochloropsis is a particularly promising microalga due to its high growth rate and lipid content, and the availability of genomic information. Transcription factors (TFs) are global regulators of biological pathways by up- or down-regulation of related genes. Among these, basic helix-loop-helix (bHLH) TFs regulate growth, development, and stress responses in plants and animals, and have been identified in microalgae. We identified two bHLH TFs in the genome of N. salina CCMP1776, NsbHLH1, and NsbHLH2, and characterized functions of NsbHLH2 that may be involved in growth and nutrient uptake. RESULTS: We obtained NsbHLH2 overexpressing transformants of N. salina CCMP1776 by particle bombardment and confirmed that these were stable transformants. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting using antibodies against the FLAG tag that was attached at the end of the coding sequence confirmed the expression of the NsbHLH2 protein under various culture conditions. The qRT-PCR results also indicated that the endogenous and transgenic expression of NsbHLH2 was reduced under stressed conditions. Overexpression of NsbHLH2 led to increased growth rate in the early growth period, and concomitantly higher nutrient uptake, than wild type (WT). These enhanced growth and nutrient uptake resulted in increased productivities of biomass and FAME. For example, one of the transformants, NsbHLH2 3-6, showed increased biomass productivity by 36 % under the normal condition, and FAME productivity by 33 % under nitrogen limitation condition. Conclusively, the improved growth in the transformants can be associated with the enhanced nutrient uptake. We are currently assessing their potential for scale-up cultivation with positive outcomes. CONCLUSION: Overexpression of NsbHLH2 led to enhanced growth rate and nutrient uptake during the early growth phase, and increased biomass and FAME productivity, especially in the later period under normal and stressed conditions. Based on these results, we postulate that NsbHLH2 can be employed for the industrial production of biodiesel from N. salina.

Development of direct conversion method for microalgal biodiesel production using wet biomass of Nannochloropsis salina.

In this work, the effects of several factors, such as temperature, reaction time, and solvent and acid quantity on in situ transesterification yield of wet Nannochloropsis salina were investigated. Under equivalent total solvent volume to biomass ratio, pure alcohol showed higher yield compared to alcohol-chloroform solvent. For esterifying 200 mg of wet cells, 2 ml of methanol and 1 ml of ethanol was sufficient to complete in situ transesterification. Under temperatures of 105 °C or higher, 2.5% and 5% concentrations of sulfuric acid was able to successfully convert more than 90% of lipid within 30 min when methanol and ethanol was used as solvents respectively. Also, it was verified that the optimal condition found in small-scale experiments is applicable to larger scale using 2 L scale reactor as well.

Use of conditioned medium for efficient transformation and cost-effective cultivation of Nannochloropsis salina.

The oleaginous microalga Nannochloropsis sp. has been spotlighted as a promising candidate in genetic engineering research for biodiesel production. However, one of the major bottlenecks in the genetic manipulation against Nannochloropsis sp. is low transformation efficiency. Based on the idea that they grow rapidly in broth culture, the effect of conditioned medium on colonization and transformation efficiency of Nannochloropsis salina was investigated. Cells grown on agar plates with 20-40% conditioned medium produced colonies that were approximately 2.3-fold larger than cells grown without conditioned medium. More importantly, the transformation efficiency was about 2-fold greater on plates with 30% conditioned medium relative to those without conditioned medium. In addition, FAME productivity in liquid cultures with 100% conditioned medium increased up to 20% compared with cultures of control medium. These results suggest that conditioned medium can be applied for efficient transformation and cost-effective cultivation of N. salina for biodiesel production.