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.

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.

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.

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.

Psychrophilic yeasts from Antarctica and European glaciers: description of Glaciozyma gen. nov., Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov.

Field campaigns in Antarctica, Greenland and the Italian glaciers aiming to explore the biodiversity of these disappearing environments identified several undescribed yeast strains unable to grow at temperature above 20°C and belonging to unknown species. Fourteen of these strains were selected and grouped based on their morphological and physiological characteristics. Sequences of the D1/D2 and ITS regions of the ribosomal RNA demonstrated that the strains belong to unknown species related to Leucosporidium antarcticum. The new genus Glaciozyma is proposed and two new species are described, namely Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp. nov. Additionally, re-classification of Leucosporidium antarcticum as Glaciozyma antarctica is proposed. Strains of Glaciozyma form a monophyletic clade and a well separated lineage within class Microbotryomycetes (Pucciniomycotina, Basidiomycota). The description of Glaciozyma genus and the re-classification of L. antarcticum reduce the polyphyletic nature of the genus Leucosporidium.

Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant.

BACKGROUND: Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. METHODS: We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. KEY RESULTS: Assembly of the 221,746 sequence reads resulted in 12,442 contigs. Over 50 % (6183) of 12,442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. CONCLUSIONS: This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

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.

Phylogenetic and molecular analysis of hydrogen-producing green algae.

A select set of microalgae are reported to be able to catalyse photobiological H(2) production from water. Based on the model organism Chlamydomonas reinhardtii, a method was developed for the screening of naturally occurring H(2)-producing microalgae. By purging algal cultures with N(2) in the dark and subsequent illumination, it is possible to rapidly induce photobiological H(2) evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. This procedure can be used to test algal species existing as axenic or mixed cultures for their ability to produce H(2). Using this system, five algal isolates capable of H(2) production were identified in various aquatic systems. A phylogenetic tree was constructed using ribosomal sequence data of green unicellular algae to determine if there were taxonomic patterns of H(2) production. H(2)-producing algal species were seen to be dispersed amongst most clades, indicating an H(2)-producing capacity preceded evolution of the phylum Chlorophyta.

Transcriptome for photobiological hydrogen production induced by sulfur deprivation in the green alga Chlamydomonas reinhardtii.

Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation.

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.

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.

Isolation and analysis of mRNA from environmental microbial communities.

The advent of metagenomics has revealed that our planet harbors millions of previously undiscovered microbial species. However, functional insights into the activities of microbial communities cannot easily be obtained using metagenomics. Using transcriptional analyses to study microbial gene functions is currently problematic due to difficulties working with unstable microbial mRNA as a small fraction of total cellular RNA. Current techniques can be expensive and time consuming, and still result in significant levels of rRNA contamination. We have adapted techniques to rapidly isolate high high-quality RNA from environmental samples and developed a simple method for specific isolation of mRNA by size separation. This new technique was evaluated by constructing cDNA libraries directly from uncultured environmental microbial communities, including agricultural soil samples, aquatic flocculants, organic composts, mammalian oral and faecal samples, and wastewater sludge. The sequencing of a fraction of these cDNA clones revealed a high degree of novelty, demonstrating the potential of this approach to capture a large number of unique transcripts directly from the environment. To our knowledge, this is the first study that uses gel electrophoresis to isolate mRNA from microbial communities. We conclude that this method could be used to provide insights into the microbial 'metatranscriptome' of entire microbial communities. Coupled with high-throughput sequencing or the construction of cDNA microarrays, this approach will provide a useful tool to study the transcriptional activities of microorganisms, including those of entire microbial communities and of non-culturable microorganisms.

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.

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.

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.

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.

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.