Development of large-scale microalgae production in the Middle East.

Microalgae in the Middle East can theoretically address food security without competing for arable land, but concerns exist around scalability and durability of production systems under the extreme heat. Large-scale Chlorella sorokiniana production was developed in outdoor raceway ponds in Oman and monitored for 2 years to gather data for commercial production. Biological and technical challenges included construction, indoor/outdoor preculturing, upscaling, relating productivity to water temperature and meteorological conditions, harvesting, drying, and quality control. Small cultivation systems required cooling for initial scale-up, but, despite maximum temperatures of 49.7 °C, water temperatures were at acceptable levels by evaporative cooling in larger raceway ponds. Contamination with Vampirovibrio chlorellavorus was identified by 16S rDNA amplicon sequencing and addressed by culture replacement. Productivities ranged from 8 to 30 g-dry weight m-2d-1, with estimated annual productivity of 16 g-dry weight m-2d-1 as functions of solar intensity and water temperature, confirming that the region is suitable for commercial microalgae production.

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 µM), 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 with 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 Chitosan: 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.

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 × 108 cells mL-1; 0.82 g L-1). 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.

Chromosome-Scale Genome Assembly of Two Australian Nannochloropsis oceanica Isolates Exhibiting Superior Lipid Characteristics.

Nannochloropsis oceanica strains BR2 and KB1 are microalgal isolates from brackish water in the Brisbane River and a coastal rock pool at the Sunshine Coast in Australia which display superior productivity at high temperatures. We used long-read sequencing to sequence their genomes and to facilitate elucidation of loci associated with these traits.

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.

Phaeodactylum tricornutum microalgae as a rich source of omega-3 oil: Progress in lipid induction techniques towards industry adoption.

Diatoms are a major group of high omega 3-fatty acid producing algae that play a key role in global climate change and ecosystem function. Phaeodactylum tricornutum is one of only two diatoms whose genomes have been completely sequenced, leading to metabolic engineering of high eicosapentaenoic acid producing strains. Based on its rapid growth, high lipid content, and especially omega-3 long chain unsaturated fatty acids, P. tricornutum exhibits a large commercial potential. However, until now, it is predominately produced as feed for the aquaculture industry, rather than food supplement. This review compares the change of P. tricornutum lipid composition under different treatments, and identifies suitable lipid induction, cultivation and harvesting methods for industry adoption. If produced in a biorefinery setting, P. tricornutum has strong potential for value generation from human health products (omega-3-rich oil and high-value protein) with cost estimates of AU$6.14 per kg dry weight and AU$20.47 for omega-3-rich oil.

Heavy metal bioremediation of coal-fired flue gas using microalgae under different CO2 concentrations.

Sustainability assessments have revealed that integration of CO2 from coal-fired flue gas with microalgae cultivation systems could reduce greenhouse gas emissions. The technical goal of this integration is to utilize exhaust from coal power plants to enhance microalgae cultivation processes by capturing and recycling of carbon dioxide from a more toxic to a less toxic form. However, heavy metals are also introduced along with CO2 to the cultivation system which could contaminate biomass and have deleterious effects on products derived from such systems. The present study aimed at shedding some light on capability of microalgae to sustain their diversity and propagate them under different CO2 concentrations from coal-fired flue gas. Mixed microalgal culture was grown in nutrient rich medium and heavy metals (Al, Cu, Fe, Mn and Zn) are expected to be introduced from flue gas. Three concentrations (1%, 3% and 5.5%) of CO2 were evaluated (reference concentrations from flue gas). Comparative studies were carried out by flue gas and control systems in photobioreactors. Under the 3% CO2 (30% flue gas), the highest fraction of B, Mn and Zn were found to be internalized by the cells (46.8 ±9.45 gL-1, 253.66 ± 40.62 gL-1 and 355.5 ±50.69 gL-1 respectively) during their cultivation period into biomass. Hence, microalgae may offer solution to two major challenges: providing potential biofuel feedstock for energy security and reducing heavy metal pollution to the air.

Biogas production coupled to repeat microalgae cultivation using a closed nutrient loop.

Anaerobic digestion is an established technology to produce renewable energy as methane-rich biogas for which microalgae are a suitable substrate. Besides biogas production, anaerobic digestion of microalgae generates an effluent rich in nutrients, so-called digestate, that can be used as a growth medium for microalgal cultures, with the potential for a closed nutrient loop and sustainable bioenergy facility. In this study, the methane potential and nutrient mobilization of the microalga Scenedemus dimorphus was evaluated under continuous conditions. The suitability of using the digestate as culture medium was also evaluated. The results show that S. dimorphus is a suitable substrate for anaerobic digestion with an average methane yield of 199 mL g-1 VS. The low level of phosphorus in digestate did not limit algae growth when used as culture medium. The potential of liquid digestate as a superior culture medium rather than inorganic medium was demonstrated.

LED power efficiency of biomass, fatty acid, and carotenoid production in Nannochloropsis microalgae.

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.

Mixed microalgae consortia growth under higher concentration of CO2 from unfiltered coal fired flue gas: Fatty acid profiling and biodiesel production.

Biodiesel is produced by transesterification of fatty acid methyl esters (FAME) from oleaginous microalgae feedstock. Biodiesel fuel properties were studied and compared with biodiesel standards. Qualitative analysis of FAME was done while cultivating mixed microalgae consortia under three concentrations of coal fired flue gas (1%, 3.0% and 5.5% CO2). Under 1% CO2 concentration (flue gas), the FAME content was 280.3 µg/mL, whereas the lipid content was 14.03 µg/mL/D (day). Both FAMEs and lipid contents were low at other CO2 concentrations (3.0 and 5.5%). However, mixed consortia in the presence of phosphate buffer and flue gas (PB + FG) showed higher saturated fatty acids (SFA) (36.28%) and unsaturated fatty acids (UFA) (63.72%) versus 5.5% CO2 concentration, which might be responsible for oxidative stability of biodiesel. Subsequently, higher cetane number (52) and low iodine value (136.3 gI2/100 g) biodiesel produced from mixed consortia (PB + FG) under 5.5% CO2 along with 50 mM phosphate buffer were found in accordance with European (EN 14214) standard. Results revealed that phosphate buffer significantly enhanced the biodiesel quality, but reduced the FAME yield. This study intended to develop an integrated approach for significant improvement in biodiesel quality under surplus phosphorus by utilizing waste flue gas (as CO2 source) using microalgae. The CO2 sequestration from industrial flue gas not only reduced greenhouse gases, but may also ensure the sustainable and eco-benign production of biodiesel.

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-2 s-1 of white LED (W-150) compared with lower light intensities, but the highest astaxanthin amounts were produced under 70 µmol m-2 s-1 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.

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism.

To map out key lipid-related pathways that lead to rapid triacylglyceride accumulation in oleaginous microalgae, RNA-Seq was performed with Tetraselmis sp. M8 at 24h after exhaustion of exogenous nitrogen to reveal molecular changes during early stationary phase. Further gene expression profiling by quantitative real-time PCR at 16-72h revealed a distinct shift in expression of the fatty acid/triacylglyceride biosynthesis and ß-oxidation pathways, when cells transitioned from log-phase into early-stationary and stationary phase. Metabolic reconstruction modeling combined with real-time PCR and RNA-Seq gene expression data indicates that the increased lipid accumulation is a result of a decrease in lipid catabolism during the early-stationary phase combined with increased metabolic fluxes in lipid biosynthesis during the stationary phase. During these two stages, Tetraselmis shifts from reduced lipid consumption to active lipid production. This process appears to be independent from DGAT expression, a key gene for lipid accumulation in microalgae.

Selection and adaptation of microalgae to growth in 100% unfiltered coal-fired flue gas.

Microalgae have been considered for biological carbon capture and sequestration to offset carbon emissions from fossil fuel combustion. This study shows that mixed biodiverse microalgal communities can be selected for and adapted to tolerate growth in 100% flue gas from an unfiltered coal-fired power plant that contained 11% CO2. The high SOx and NOx emissions required slow adaptation of microalgae over many months, with step-wise increases from 10% to 100% flue gas supplementation and phosphate buffering at higher concentrations. After a rapid decline in biodiversity over the first few months, community profiling revealed Desmodesmus spp. as the dominant microalgae. To the authors' knowledge this work is the first to demonstrate that up 100% unfiltered flue gas from coal-fired power generation can be used for algae cultivation. Implementation of serial passages over a range of photobioreactors may contribute towards the development of microalgal-mediated carbon capture and sequestration processes.

Growth and lipid accumulation of microalgae from fluctuating brackish and sea water locations in South East Queensland-Australia.

One challenge constraining the use of microalgae in the food and biofuels industry is growth and lipid accumulation. Microalgae with high growth characteristics are more likely to originate from the local environment. However, to be commercially effective, in addition to high growth microalgae must also have high lipid productivities and contain the desired fatty acids for their intended use. We isolated microalgae from intertidal locations in South East Queensland, Australia with adverse or fluctuating conditions, as these may harbor more opportunistic strains with high lipid accumulation potential. Screening was based on a standard protocol using growth rate and lipid accumulation as well as prioritizing fatty acid profiles suitable for biodiesel or nutraceuticals. Using these criteria, an initial selection of over 50 local microalgae strains from brackish and sea water was reduced to 16 strains considered suitable for further investigation. Among these 16 strains, the ones most likely to be effective for biodiesel feedstock were Nitzschia sp. CP3a, Tetraselmis sp. M8, Cymbella sp. CP2b, and Cylindrotheca closterium SI1c, reaching growth rates of up to 0.53 day(-1) and lipid productivities of 5.62 µg mL(-1)day(-1). Omega-3 fatty acids were found in some strains such as Nitzschia sp. CP2a, Nitzschia sp. CP3a and Cylindrotheca closterium SI1c. These strains have potential for further research as commercial food supplements.

High protein- and high lipid-producing microalgae from northern australia as potential feedstock for animal feed and biodiesel.

Microalgal biomass can be used for biodiesel, feed, and food production. Collection and identification of local microalgal strains in the Northern Territory, Australia was conducted to identify strains with high protein and lipid contents as potential feedstock for animal feed and biodiesel production, respectively. A total of 36 strains were isolated from 13 samples collected from a variety of freshwater locations, such as dams, ponds, and streams and subsequently classified by 18S rDNA sequencing. All of the strains were green microalgae and predominantly belong to Chlorella sp., Scenedesmus sp., Desmodesmus sp., Chlamydomonas sp., Pseudomuriella sp., Tetraedron caudatum, Graesiella emersonii, and Mychonastes timauensis. Among the fastest growing strains, Scenedesmus sp. NT1d possessed the highest content of protein; reaching up to 33% of its dry weight. In terms of lipid production, Chlorella sp. NT8a and Scenedesmus dimorphus NT8e produced the highest triglyceride contents of 116.9 and 99.13 µg mL(-1) culture, respectively, as measured by gas chromatography-mass spectroscopy of fatty acid methyl esters. These strains may present suitable candidates for biodiesel production after further optimization of culturing conditions, while their protein-rich biomass could be used for animal feed.

Effects of long chain fatty acid synthesis and associated gene expression in microalga Tetraselmis sp.

With the depletion of global fish stocks, caused by high demand and effective fishing techniques, alternative sources for long chain omega-3 fatty acids are required for human nutrition and aquaculture feeds. Recent research has focused on land-based cultivation of microalgae, the primary producers of omega-3 fatty acids in the marine food web. The effect of salinity on fatty acids and related gene expression was studied in the model marine microalga, Tetraselmis sp. M8. Correlations were found for specific fatty acid biosynthesis and gene expression according to salinity and the growth phase. Low salinity was found to increase the conversion of C18:4 stearidonic acid (SDA) to C20:4 eicosatetraenoic acid (ETA), correlating with increased transcript abundance of the Δ-6-elongase-encoding gene in salinities of 5 and 10 ppt compared to higher salinity levels. The expression of the gene encoding ß-ketoacyl-coenzyme was also found to increase at lower salinities during the nutrient deprivation phase (Day 4), but decreased with further nutrient stress. Nutrient deprivation also triggered fatty acids synthesis at all salinities, and C20:5 eicosapentaenoic acid (EPA) increased relative to total fatty acids, with nutrient starvation achieving a maximum of 7% EPA at Day 6 at a salinity of 40 ppt.

Towards sustainable sources for omega-3 fatty acids production.

Omega-3 fatty acids eicosapentaenoic acid (EPA) and docohexaenoic acid (DHA), provide significant health benefits for brain function/development and cardiovascular conditions. However, most EPA and DHA for human consumption is sourced from small fatty fish caught in coastal waters and, with depleting global fish stocks, recent research has been directed towards more sustainable sources. These include aquaculture with plant-based feeds, krill, marine microalgae, microalgae-like protists and genetically-modified plants. To meet the increasing demand for EPA and DHA, further developments are needed towards land-based sources. In particular large-scale cultivation of microalgae and plants is likely to become a reality with expected reductions in production costs, yield increasese and the adequate addressing of genetically modified food acceptance issues.

Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis.

Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic stresses as well as during normal growth and development. Although a large number of transcription factor (TF) genes are up- or down-regulated by ROS, currently very little is known about the functions of these TFs during oxidative stress. In this work, we examined the role of ERF6 (ETHYLENE RESPONSE FACTOR6), an AP2/ERF domain-containing TF, during oxidative stress responses in Arabidopsis. Mutant analyses showed that NADPH oxidase (RbohD) and calcium signaling are required for ROS-responsive expression of ERF6. erf6 insertion mutant plants showed reduced growth and increased H2O2 and anthocyanin levels. Expression analyses of selected ROS-responsive genes during oxidative stress identified several differentially expressed genes in the erf6 mutant. In particular, a number of ROS responsive genes, such as ZAT12, HSFs, WRKYs, MAPKs, RBOHs, DHAR1, APX4, and CAT1 were more strongly induced by H2O2 in erf6 plants than in wild-type. In contrast, MDAR3, CAT3, VTC2 and EX1 showed reduced expression levels in the erf6 mutant. Taken together, our results indicate that ERF6 plays an important role as a positive antioxidant regulator during plant growth and in response to biotic and abiotic stresses.

Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters.

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.