A biorefinery for Nannochloropsis: induction, harvesting, and extraction of EPA-rich oil and high-value protein

Microalgae have been studied as biofactories for almost four decades. Yet, even until today, many aspects of microalgae farming and processing are still considered exploratory because of the uniqueness of each microalgal species. Thus, it is important to develop the entire process of microalgae farming: from culturing to harvesting, and down to extracting the desired high-value products. Based on its rapid growth and high oil productivities, Nannochloropsis sp. is of particular interest to many industries for the production of high-value oil containing omega-3 fatty acids, specifically eicosapentaenoic acid (EPA), but also several other products. This review compares the various techniques for induction, harvesting, and extraction of EPA-rich oil and high-value protein explored by academia and industry to develop a multi-product Nannochloropsis biorefinery. Knowledge gaps and opportunities are discussed for culturing and inducing fatty acid biosynthesis, biomass harvesting, and extracting EPA-rich oil and high-value protein from the biomass of Nannochloropsis sp

Development of high‐level omega‐3 eicosapentaenoic acid (EPA) production from Phaeodactylum tricornutum

Phaeodactylum tricornutum is a lipid-rich marine diatom that contains a high level of omega-3 polyunsaturated fatty acids, especially eicosapentaenoic acid (EPA). In an effort to reduce costs for large-scale cultivation of this microalga, this study first established a New BBM medium (0.3 x strength BBM with only 3% of the initial phosphate level) to replace the traditional F/2 medium. Phaeodactylum tricornutum could grow in extremely low phosphate concentrations (25\ua0µM), without compromising the EPA content. In the presence of sea salts, silicate addition was not necessary for high rate growth, high EPA content or lipid accumulation in this species. Using urea as the sole nitrogen source tended to increase EPA contents per dry biomass (by 24.7%) while not affecting growth performance. The use of sea salts, rather than just sodium chloride led to significantly improved biomass yields (20% increase) and EPA contents of total fatty acid (46-52% increase), most likely because it supplied sufficient essential elements such as magnesium. A salinity level of 35 led to significantly higher biomass yields compared to 20, but salinity had no significant influence on EPA content. EPA became the dominant fatty acid with average levels of 51.8% of total fatty acids during the exponential growth phase at 20 ppt in New BBM medium with sea salts

Effective harvesting of Nannochloropsis microalgae using mushroom ahitosan: a pilot-scale study

For efficient downstream processing, harvesting remains as one of the challenges in producing Nannochloropsis biomass, a microalga with high-value omega-3 oils. Flocculation is an effective, low-energy, low-cost method to harvest microalgae. Chitosan has been shown to be an effective food-grade flocculant; however, commercial chitosan is sourced from crustaceans, which has disadvantages including concerns over heavy-metal contamination. Thus, this study tests the flocculation potential of mushroom chitosan. Our results indicate a 13% yield of chitosan from mushroom. The identity of the prepared chitosan was confirmed by Fourier-transform infrared (FTIR) spectroscopy. Furthermore, results show that mushroom chitosan can be an alternative flocculant with >95% flocculation efficiency when tested in 100-mL jar and 200-L vertical column photobioreactor. Applications in a 2000-L raceway pond demonstrated that thorough mixing of mushroom chitosan with the algal culture is required to achieve efficient flocculation. With proper mixing, mushroom chitosan can be used to produce food-grade Nannochloropsis biomass suitable for the production of vegan omega-3 oils as a fish oil alternative

Cold and dark treatments induce omega-3 fatty acid and carotenoid production in Nannochloropsis oceanica

Nannochloropsis is one of the most-studied microalgae because of its high eicosapentaenoic acid (EPA) content and high-value carotenoids. However, during large-scale farming, decreased EPA productivity of Nannochloropsis oceanica BR2 was observed when cultured during the summer season (≥30 °C). We hypothesized that short low temperature treatments and membrane biosynthesis during reduced light exposure may increase EPA yields. Thus, we investigated the effect of 3-day cold or dark treatments on N. oceanica BR2 nutritional composition by measuring fatty acid and carotenoid profiles. Indeed, treated samples contained increased levels of polyunsaturated fatty acids including EPA and carotenoids violaxanthin and zeaxanthin. Tunnelling electron microscopy showed increased an chloroplast size and an increased number of thylakoid membranes in treated samples, visualizing how cells adapt to these environmental changes. The present study provides a practical strategy on how heat-stressed Nannochloropsis cultures can be subjected to short 3-day treatments before harvesting to enable high-value biomass production

Efficient harvesting of Nannochloropsis microalgae via optimized chitosan-mediated flocculation

Food-grade rather than synthetic or chemical flocculants are needed for microalgae harvesting by settling, if used for food products. Chitosan is effective in harvesting freshwater microalgae, but it is expensive and typically not suitable for marine microalgae like Nannochloropsis. To minimize costs for food-grade flocculation, a number of potentially important parameters are considered, including chitosan solubility and optimized chitosan-mediated flocculation of Nannochloropsis sp. BR2 by a five-factor central composite design experiment. Results show that an optical density (440 nm) of 2 (0.23 g dry weight L-1), initial pH of 6, final pH of 10, and 22 ppm chitosan with a viscosity of 1808 cP provide optimum flocculation efficiency, which is predicted to be in the range of 97.01% to 99.93%. These predictions are verified on 4.5 and 8 L Nannochloropsis sp. BR2 cultures

Blue light enhances astaxanthin biosynthesis metabolism and extraction efficiency in Haematococcus pluvialis by inducing haematocyst germination

Haematococcus pluvialis accumulates large amounts of astaxanthin during its haematocyst stage. Cyst germination is a biological pretreatment method for improved astaxanthin extraction with potential to replace energy-intensive homogenizer-based mechanical cell cracking methods. The present study demonstrates effects of different LED light wavelengths on haematocyst germination and on its astaxanthin biosynthesis pathway. Blue light enhanced the germination efficiency and slowed the nitrogen consumption rate, resulting in significantly higher astaxanthin content and improved extractability compared with white and red lights, that also had lower germination rates. After 5 days, the total astaxanthin extractability under blue light was 4.0 and 6.7 times higher than for white and red lights, respectively. The blue light receptor gene phot was significantly induced which upregulated the biosynthesis pathway genes psy and pds, as well as dgat1 and dgat2d. Hence, blue light triggers germination and astaxanthin biosynthesis, providing a strategy for improved extraction while modulating higher biosynthesis during germination

Growth-promoting bacteria double eicosapentaenoic acid yield in microalgae

High-yielding microalgae present an important commodity to sustainably satisfy burgeoning food, feed and biofuel demands. Because algae-associated bacteria can significantly enhance or reduce yields, we isolated, identified and selected highly-effective “probiotic” bacterial strains associated with Nannochloropsis oceanica, a high-yielding microalga rich in eicosapentaenoic acid (EPA). Xenic algae growth was significantly enhanced by co-cultivation with ten isolated bacteria that improved culture density and biomass by 2.2- and 1.56-fold, respectively (1.39 × 10 cells mL; 0.82 g L). EPA contents increased up to 2.25-fold (to 39.68% of total fatty acids). Added probiotic bacteria possessed multiple growth-stimulating characteristics, including atmospheric nitrogen fixation, growth hormone production and phosphorous solubilization. Core N. oceanica-dominant bacterial microbiomes at different cultivation scales included Sphingobacteria, Flavobacteria (Bacteroidetes), and α, γ-Proteobacteria, and added probiotic bacteria could be maintained. We conclude that the supplementation with probiotic algae-associated bacteria can significantly enhance biomass and EPA production of N. oceanica

Gene expression profiling of astaxanthin and fatty acid pathways in Haematococcus pluvialis in response to different LED lighting conditions

Haematococcus pluvialis is a green microalga of major interest to industry based on its ability to produce large amounts of astaxanthin. Biosynthesis of astaxanthin and its mono- and di-esters was significantly stimulated under 150 μmol m s of white LED (W-150) compared with lower light intensities, but the highest astaxanthin amounts were produced under 70 μmol m s of blue LED (B-70). Transcripts of astaxanthin biosynthesis genes psy, crtO, and bkt2 were upregulated under W-150, while psy, lcy, crtO, and crtR-B were upregulated by B-70. Total fatty acid content and biosynthesis genes fata and all dgat genes were induced under W-150, while C18:3n6 biosynthesis and dgat2a expression were specifically stimulated by B-70 which was correlated to astaxanthin ester biosynthesis. Nitrogen starvation, various LEDs and the identified upregulated genes may provide useful tools for future metabolic engineering to significantly increase free astaxanthin, its esters and fatty acid precursors in H. pluvialis

Comprehensive utilization of marine microalgae for enhanced co-production of multiple compounds

Marine microalgae are regarded as potential feedstock because of their multiple valuable compounds, including lipids, pigments, carbohydrates, and proteins. Some of these compounds exhibit attractive bioactivities, such as carotenoids, ω-3 polyunsaturated fatty acids, polysaccharides, and peptides. However, the production cost of bioactive compounds is quite high, due to the low contents in marine microalgae. Comprehensive utilization of marine microalgae for multiple compounds production instead of the sole product can be an efficient way to increase the economic feasibility of bioactive compounds production and improve the production efficiency. This paper discusses the metabolic network of marine microalgal compounds, and indicates their interaction in biosynthesis pathways. Furthermore, potential applications of co-production of multiple compounds under various cultivation conditions by shifting metabolic flux are discussed, and cultivation strategies based on environmental and/or nutrient conditions are proposed to improve the co-production. Moreover, biorefinery techniques for the integral use of microalgal biomass are summarized. These techniques include the co-extraction of multiple bioactive compounds from marine microalgae by conventional methods, super/subcritical fluids, and ionic liquids, as well as direct utilization and biochemical or thermochemical conversion of microalgal residues. Overall, this review sheds light on the potential of the comprehensive utilization of marine microalgae for improving bioeconomy in practical industrial application