Oleaginous Microalga Nannochloropsis Research Articles

Green integrative large scale treatment of tannery effluent, CO2 sequestration, and biofuel production using oleaginous green microalga Nannochloropsis oculata TSD05: An ecotechnological approach

In this present investigation, the freshwater microalga Nannochloropsis oculata TSD05 was used to demonstrate multiforius environmental applications viz., bioremediation of tannery effluent, carbon sequestration, and green renewable biodiesel production from treated microalgal biomass. The microalga Nannochloropsis oculata TSD05 was exhibited unique heavy metal reduction capabilities and reduced significant amount of heavy metals viz., 96.62% of copper (Cu2+), 99.9% of chromium (Cr3+), 98.36% of zinc (Zn2+), 98.71% of cadmium (Cd2+), 97.96% of lead (Pb2+), 98.88% of Cobalt (Co2+), and 98.76% of nickel (Ni2+). The biosorption capacity rate (qmax) of heavy metals reduction and highest R2 of 97.84% exhibited at 12 days of effluent treatment by Langmuir and Freundlich isotherm models. The phytotoxicity analysis was tested againt treated tannery effluent using Vigna radiate, Sapindus muukorossi seeds and 98.45%, 99.05% seeds were germinated. The Nannochloropsis oculata TSD05 microalga was utilized 98.45% of CO2 by biofixation kinetics, 372 mg g−1 of lipid, 3.65 mg mL−1 of biomass and 4.64 mLg−1 of biodiesel were produced. The produced biodiesel was confirmed and identification of 18 fatty acid methyl ester (biodiesel) compounds using GCMS. The recognition of 17 functional groups was identified using FTIR analysis and the microalga Nannochloropsis oculata TSD05 potential for sustainable and renewable green energy production. The potential future application of Nannochloropsis oculata TSD05 is to increase lipid production and biodiesel yield. Scaling up the bioremediation process can also validate these microalga's feasibility for use in wastewater treament. Enhancing heavy metal biosorption and carbon sequestration efficiency through genetic engineering could also maximize environmental benefits.

Growth and fatty acid profile of Nannochloropsis oceanica cultivated on nano-filtered whey permeate

Nano-filtered whey permeate (WP), a major by-product of dairy industry, is produced by membrane filtration of whey. The oleaginous microalga Nannochloropsis oceanica was successfully cultivated on WP without salinity and nutrient amendments. Growth, cell characteristics, and fatty acid profile of the cultures were analyzed using microscopy, flow cytometry, and GC analysis. WP was nitrogen limited, comprising primarily protein as a nitrogen source and only small amounts of free inorganic nitrogen (in the form of nitrate). Nannochloropsis oceanica (and associated bacteria) efficiently removed nitrate (100%), protein (87%), and phosphate (74%) from the whey permeate. Microscopic and flow cytometric analysis revealed diverse size distributions in whey permeate cultures, with significant cell aggregation attributed to low-salinity acclimatization and nitrogen limitation. Autofluorescence analysis revealed reduced photosynthetic activity in whey permeate-grown cells, possibly as a consequence of heightened mixotrophic activities on carbon source in the medium. Low nitrogen availability in whey permeate resulted in biomass with a fatty acid profile enriched in saturated fatty acids. Despite this, a considerable level of the omega-3 polyunsaturated fatty acid (in the form of eicosapentanoic acid or EPA) was detected at ca. 16% of total fatty acids. Whey permeate proved beneficial for the growth of N. oceanica and yielded high concentrations of eicosapentaenoic acid in the extracted lipids for potential applications in the feed/food industries.

Genome-wide adenine N6-methylation map reveals epigenomic regulation of lipid accumulation in Nannochloropsis

Epigenetic marks on histones and DNA, such as DNA methylation at N6-adenine (6mA), play crucial roles in gene expression and genome maintenance, but their deposition and function in microalgae remain largely uncharacterized. Here, we report a genome-wide 6mA map for the model industrial oleaginous microalga Nannochloropsis oceanica produced by single-molecule real-time sequencing. Found in 0.1% of adenines, 6mA sites are mostly enriched at the AGGYV motif, more abundant in transposons and 3′ untranslated regions, and associated with active transcription. Moreover, 6mA gradually increases in abundance along the direction of gene transcription and shows special positional enrichment near splicing donor and transcription termination sites. Highly expressed genes tend to show greater 6mA abundance in the gene body than do poorly expressed genes, indicating a positive interaction between 6mA and general transcription factors. Furthermore, knockout of the putative 6mA methylase NO08G00280 by genome editing leads to changes in methylation patterns that are correlated with changes in the expression of molybdenum cofactor, sulfate transporter, glycosyl transferase, and lipase genes that underlie reductions in biomass and oil productivity. By contrast, knockout of the candidate demethylase NO06G02500 results in increased 6mA levels and reduced growth. Unraveling the epigenomic players and their roles in biomass productivity and lipid metabolism lays a foundation for epigenetic engineering of industrial microalgae.

Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica.

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Biochemistry and Biotechnology of Lipid Accumulation in the Microalga Nannochloropsis oceanica.

Oils are among the most important agricultural commodities and have wide applications in food/nutrition, biofuels, and oleochemicals. The oleaginous microalga Nannochloropsis oceanica can produce large amounts of oils and the high-value ω-3 eicosapentaenoic acid, which represents a promising resource for oil production targeting biodiesel, nutraceutical, and aquaculture industries. In recent years, with the availability of omics databases and the development of genetic tools, N. oceanica has been extensively investigated as a model photosynthetic organism for studying lipid metabolism and as a green cellular factory to produce lipids for industrial applications. This review summarizes the current knowledge on the lipid composition and biosynthetic pathways of N. oceanica and reviews the recent advances in metabolic engineering of lipid production in this microalga.

Medium-chain triglyceride production in Nannochloropsis via a fatty acid chain length discriminating mechanism.

Depending on their fatty acid (FA) chain length, triacylglycerols (TAGs) have distinct applications; thus, a feedstock with a genetically designed chain length is desirable to maximize process efficiency and product versatility. Here, ex vivo, in vitro, and in vivo profiling of the large set of type-2 diacylglycerol acyltransferases (NoDGAT2s) in the industrial oleaginous microalga Nannochloropsis oceanica revealed two endoplasmic reticulum-localized enzymes that can assemble medium-chain FAs (MCFAs) with 8-12 carbons into TAGs. Specifically, NoDGAT2D serves as a generalist that assembles C8-C18 FAs into TAG, whereas NoDGAT2H is a specialist that incorporates only MCFAs into TAG. Based on such specialization, stacking of NoDGAT2D with MCFA- or diacylglycerol-supplying enzymes or regulators, including rationally engineering Cuphea palustris acyl carrier protein thioesterase, Cocos nucifera lysophosphatidic acid acyltransferase, and Arabidopsis thaliana WRINKLED1, elevated the medium-chain triacylglycerol (MCT) share in total TAG 66-fold and MCT productivity 64.8-fold at the peak phase of oil production. Such functional specialization of NoDGAT2s in the chain length of substrates and products reveals a dimension of control in the cellular TAG profile, which can be exploited for producing designer oils in microalgae.

Transcriptome analysis of lipid metabolism in response to cerium stress in the oleaginous microalga Nannochloropsis oculata

Nannochloropsis oculata can accumulate large amounts of lipids under rare earth element (REE) conditions. However, the lipid accumulation mechanism responsible for REE stress has not been elucidated. In this study, the effects of cerium (the most abundant REE) on the growth and lipid accumulation of N. oculata were investigated. The de novo transcriptome data of N. oculata under cerium conditions were subsequently collected and analyzed. The results showed that N. oculata exhibited good cerium-resistance ability, showed slightly decrease in biomass but significantly increase in lipid content (55.8 % dry cell weight) under 6.0 mg/L cerium condition. Meanwhile, about 83.4 % cerium was biological fixated. Through transcriptome analysis, we found that the inhibited photosynthesis and carbon fixation pathways coupled with the stress-sensitive expression of ribosome biogenesis genes acclimatized the cells to REE stress. The active glycolysis pathway accelerated carbon flux to pyruvate and acetyl-CoA, and the upregulation of glycerol kinase and phosphatidate cytidylyltransferase genes further induced lipid accumulation. In addition, cerium downregulated the acyl-CoA oxidase and triacylglycerol lipase genes, which inhibited the degradation of lipids. Therefore, different responses to cerium demonstrate how N. oculata cells adapt to REE stress, and this knowledge may be used to extend our understanding of triacylglycerol (TAG) and the synthesis of other important metabolites.

The nucleolus as a genomic safe harbor for strong gene expression in Nannochloropsis oceanica

Microalgae are used in food and feed, and they are considered a potential feedstock for sustainably produced chemicals and biofuel. However, production of microalgal-derived chemicals is not yet economically feasible. Genetic engineering could bridge the gap to industrial application and facilitate the production of novel products from microalgae. Here, we report the discovery of a novel gene expression system in the oleaginous microalga Nannochloropsis that exploits the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation. We identified the nucleolus as a genomic safe harbor for Pol I transcription and used it to construct transformant strains with consistently strong transgene expression. The new expression system provides an outstanding tool for genetic and metabolic engineering of microalgae and thus will probably make substantial contributions to microalgal research.

Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages.

Industrial microalgae are promising photosynthetic cell factories, yet tools for large-scale targeted genome engineering are limited. Here for the model industrial oleaginous microalga Nannochloropsis oceanica, we established a method to precisely and serially delete large genome fragments of ~100kb from its 30.01 Mb nuclear genome. We started by identifying the 'non-essential' chromosomal regions (i.e. low expression region or LER) based on minimal gene expression under N-replete and N-depleted conditions. The largest such LER (LER1) is ~98kb in size, located near the telomere of the 502.09-kb-long Chromosome 30 (Chr 30). We deleted 81kb and further distal and proximal deletions of up to 110kb (21.9% of Chr 30) in LER1 by dual targeting the boundaries with the episome-based CRISPR/Cas9 system. The telomere-deletion mutants showed normal telomeres consisting of CCCTAA repeats, revealing telomere regeneration capability after losing the distal part of Chr 30. Interestingly, the deletions caused no significant alteration in growth, lipid production or photosynthesis (transcript-abundance change for < 3% genes under N depletion). We also achieved double-deletion of both LER1 and LER2 (from Chr 9) that total ~214kb at maximum, which can result in slightly higher growth rate and biomass productivity than the wild-type. Therefore, loss of the large, yet 'non-essential' regions does not necessarily sacrifice important traits. Such serial targeted deletions of large genomic regions had not been previously reported in microalgae, and will accelerate crafting minimal genomes as chassis for photosynthetic production.

Operation Regimes: A Comparison Based on Nannochloropsis oceanica Biomass and Lipid Productivity

Microalgae are currently considered to be a promising feedstock for biodiesel production. However, significant research efforts are crucial to improve the current biomass and lipid productivities under real outdoor production conditions. In this context, batch, continuous and semi-continuous operation regimes were compared during the Spring/Summer seasons in 2.6 m3 tubular photobioreactors to select the most suitable one for the production of the oleaginous microalga Nannochloropsis oceanica. Results obtained revealed that N. oceanica grown using the semi-continuous and continuous operation regimes enabled a 1.5-fold increase in biomass volumetric productivity compared to that cultivated in batch. The lipid productivity was 1.7-fold higher under semi-continuous cultivation than that under a batch operation regime. On the other hand, the semi-continuous and continuous operation regimes spent nearly the double amount of water compared to that of the batch regime. Interestingly, the biochemical profile of produced biomass using the different operation regimes was not affected regarding the contents of proteins, lipids and fatty acids. Overall, these results show that the semi-continuous operation regime is more suitable for the outdoor production of N. oceanica, significantly improving the biomass and lipid productivities at large-scale, which is a crucial factor for biodiesel production.

Substrate degradation, biodiesel production, and microbial community of two electro-fermentation systems on treating oleaginous microalgae Nannochloropsis sp

Electro-fermentation system (EFS) emerges its effectiveness on treating microalgae for biodiesel production, but much is unknown about biodegradation behaviors, biodiesel characteristics, and microbial community. Compared with conventional fermentation system (CFS), microbial electrolysis cell-based EFS (MEC-EFS) and microbial fuel cell-based EFS (MFC-EFS) were investigated for the performance while treating microalgae Nannochloropsis sp. Results indicated that MEC-EFS presented much higher first-order decomposition rate coefficients of carbohydrates and proteins (1.212/d and 0.951/d) than those of CFS (0.615/d and 0.794/d) and MFC-EFS (0.518/d and 0.415/d). Compared with MFC-EFS, MEC-EFS showed better electrochemical performance (2.17 A/m3vs. 0.95 A/m3). Moreover, MEC-EFS reached the highest extracted lipid to biomass ratio (43.3%), followed by MFC-EFS (32.3%) and CFS (27.7%). By strengthened microbial biohydrogenation, MEC-EFS and MFC-EFS had higher saturated fatty acids ratio (78.8% and 70.6%) than that of CFS (56.1%). For MEC-EFS, enriched Ruminococcus and Geobacter in anodic biofilm might contribute to favorable biohydrogenation and electrochemical performance.

Proteomic and Transcriptomic Patterns during Lipid Remodeling in Nannochloropsis gaditana.

Nutrient limited conditions are common in natural phytoplankton communities and are often used to increase the yield of lipids from industrial microalgae cultivations. Here we studied the effects of bioavailable nitrogen (N) and phosphorus (P) deprivation on the proteome and transcriptome of the oleaginous marine microalga Nannochloropsis gaditana. Turbidostat cultures were used to selectively apply either N or P deprivation, controlling for variables including the light intensity. Global (cell-wide) changes in the proteome were measured using Tandem Mass Tag (TMT) and LC-MS/MS, whilst gene transcript expression of the same samples was quantified by Illumina RNA-sequencing. We detected 3423 proteins, where 1543 and 113 proteins showed significant changes in abundance in N and P treatments, respectively. The analysis includes the global correlation between proteomic and transcriptomic data, the regulation of subcellular proteomes in different compartments, gene/protein functional groups, and metabolic pathways. The results show that triacylglycerol (TAG) accumulation under nitrogen deprivation was associated with substantial downregulation of protein synthesis and photosynthetic activity. Oil accumulation was also accompanied by a diverse set of responses including the upregulation of diacylglycerol acyltransferase (DGAT), lipase, and lipid body associated proteins. Deprivation of phosphorus had comparatively fewer, weaker effects, some of which were linked to the remodeling of respiratory metabolism.

Improving lipid productivity by engineering a control-knob gene in the oleaginous microalga Nannochloropsis oceanica

Nannochloropsis spp. are promising industrial microalgae for scalable oil production and the lipid production can be boosted by nutrient starvation and high irradiance. However, these stimuli halt growth, thereby decreasing overall productivity. In this study, we created transgenic N. oceanica where AtDXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) derived from Arabidopsis thaliana was overexpressed in vivo. Compared with the wild type (WT), engineered Nannochloropsis showed a higher CO2 absorption capacity and produced more biomass, lipids, and carbohydrates with more robust growth in either preferred conditions or various stressed conditions (low light, high light, nitrogen starvation, and trace element depletion). Specifically, relative to the WT, lipid production increased by ~68.6% in nitrogen depletion (~1.08 ​g ​L−1) and ~110.6% in high light (~1.15 ​g ​L−1) in the transgenic strains. As for neutral lipid (triacylglycerol, TAG), the engineered strains produced ~93.2% more in nitrogen depletion (~0.77 ​g ​L−1) and ~148.6% more in high light (~0.80 ​g ​L−1) than the WT. These values exceed available records in engineered industrial microalgae. Therefore, engineering control-knob genes could modify multiple biological processes simultaneously and enable efficient carbon partitioning to lipid biosynthesis with elevated biomass productivity. It could be further exploited for simultaneous enhancement of growth property and oil productivity in more industrial microalgae.

Distinct microalgae species for food\u2014part 2: comparative life cycle assessment of microalgae and fish for eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and protein

The production of food for a growing world population is a great challenge. In particular, protein and the long-chain n-3 fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), which exert a series of potential health effects, are scarce resources in the context of global food security. Fish from wild capture and aquaculture production cannot meet the current demand for EPA and DHA; therefore, a supplementation with alternative sources is crucial. Specific microalgae species have been shown to be a lucrative source of EPA, DHA, and protein, in particular, the oleaginous microalgae Nannochloropsis sp. and Phaeodactylum tricornutum. This study aimed to compare different cultivation scenarios of Nannochloropsis sp. and P. tricornutum with the production of aquaculture and capture fish as traditional sources of EPA and DHA in terms of environmental impacts. Scenarios included borosilicate glass and acrylic glass as photobioreactor (PBR) materials, two different tube diameters, and three different cultivation seasons. In these scenarios, carbon dioxide was modeled as an avoided burden. Additionally, all scenarios were modeled with the burdens resulting from carbon dioxide production. Environmental impacts of selected fish species were obtained from systematic literature research. Life cycle assessment following ISO 14040/44 was used to analyze the global warming potential, acidification, eutrophication, cumulative energy demand, water footprint, and land use. The system boundaries were set from “cradle-to-store,” where the target store is located in Germany. Microalgae biomass as a source of EPA, DHA, and protein was found to have similar or lower environmental impacts than fish fillet from wild capture and aquaculture production when carbon dioxide was modeled as an avoided burden. Microalgae production that included the full burden of carbon dioxide production still caused similar or lower environmental impacts than aquaculture fish. It was found that the distinct microalgae species can significantly influence the results if the comparison is conducted based on nutritional values. Regarding the recommended daily intake of 250–500 mg EPA+DHA, microalgae are an advisable source of nutrients to lessen the environmental pressure on marine ecosystems.

Glucose-6-Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis.

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose-6-phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19-fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09-fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose-6-phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.

Phosphorus-Induced Lipid Class Alteration Revealed by Lipidomic and Transcriptomic Profiling in Oleaginous Microalga Nannochloropsis sp. PJ12.

Phytoplankton are primary producers in the marine ecosystem, where phosphorus is often a limiting factor of their growth. Hence, they have evolved strategies to recycle phosphorus by replacing membrane phospholipids with phosphorus-free lipids. However, mechanisms for replacement of lipid classes remain poorly understood. To improve our understanding, we performed the lipidomic and transcriptomic profiling analyses of an oleaginous marine microalga Nannochloropsis sp. PJ12 in response to phosphorus depletion (PD) and replenishing. In this study, by using (liquid chromatography couple with tandem mass spectrometry) LC-MS/MS-based lipidomic analysis, we show that membrane phospholipid levels are significantly reduced upon PD, while phosphorus-free betaine lipid levels are increased. However, levels of phosphorus-free photosynthetic galactolipid and sulfolipid are not increased upon PD, consistent with the reduced photosynthetic activity. RNA-seq-based transcriptomic analysis indicates that enzymes involved in phospholipid recycling and phosphorus-free lipid synthesis are upregulated, supporting the lipidomic analysis. Furthermore, enzymes involved in FASII (type II fatty acid synthesis) elongation cycle upon PD are transcriptionally downregulated. EPA (eicosapentaenoic acid) level decrease upon PD is revealed by both GC-MS (gas chromatography coupled with mass spectrometry) and LC-MS/MS-based lipidomic analyses. PD-induced alteration is reversed after phosphorus replenishing. Taken together, our results suggest that the alteration of lipid classes upon environmental change of phosphorus is a result of remodeling rather than de novo synthesis in Nannochloropsis sp. PJ12.

Knockdown of carbonate anhydrase elevates Nannochloropsis productivity at high CO2 level

Improving acid tolerance is pivotal to the development of microalgal feedstock for converting flue gas to biomass or oils. In the industrial oleaginous microalga Nannochloropsis oceanica, transcript knockdown of a cytosolic carbonic anhydrase (CA2), which is a key Carbon Concentrating Mechanism (CCM) component induced under 100 ppm CO2 (very low carbon, or VLC), results in ∼45%, ∼30% and ∼40% elevation of photosynthetic oxygen evolution rate, growth rate and biomass accumulation rate respectively under 5% CO2 (high carbon, or HC), as compared to the wild type. Such high-CO2-level activated biomass over-production is reproducible across photobioreactor types and cultivation scales. Transcriptomic, proteomic and physiological changes of the mutant under high CO2 (HC; 5% CO2) suggest a mechanism where the higher pH tolerance is coupled to reduced biophysical CCM, sustained pH hemostasis, stimulated energy intake and enhanced photosynthesis. Thus “inactivation of CCM” can generate hyper-CO2-assimilating and autonomously containable industrial microalgae for flue gas-based oil production.

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

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

Effective use of tannin based natural biopolymer, AFlok-BP1 to harvest marine microalgae Nannochloropsis sp.

Harvesting, a challenging step of microalgae processing appears to be promising using flocculation method by the addition of tannin based natural biopolymer, AFlok-BP1. In this study, marine microalgae Nannochloropsis sp. was flocculated using AFlok-BP1 to evaluate its effectiveness in terms of flocculation efficiency, pH and dosages used, zeta potential, floc morphology characterization with comparison to the uses of aluminium sulphate and alkaline-induced pH. Oleaginous marine microalgae Nannochloropsis sp. was found to work effectively using AFlok-BP1 at an acidic pH 5 with more than 90% efficiency obtained at 160 mg/L. The finding was further supported with great instability of microalgae surface charge of -0.33 ± 0.27 mV, hence maximum destabilization of microalgae was believed to occur. The use of alum also effective to harvest Nannochloropsis sp. at neutral pH 7 with comparable efficiency as AFlok-BP1, but high dosage needed for more than 300 mg/L was at major concerns. Similarly, improved efficiency was observed for alkaline-induced pH for almost 100% efficiency. This would provide a better alternative for marine microalgae flocculation however, high concentration of minerals in the harvested biomass due to pH increase in need of further evaluation. To summarize, AFlok-BP1 would be a viable option to harvest marine microalgae Nannochloropsis sp. for the production of valuable compounds as it is an environmental friendly flocculant and provide no potential toxicity to the harvested biomass.

Engineering the Chloroplast Genome of Oleaginous Marine Microalga Nannochloropsis oceanica

Plastid engineering offers an important tool to fill the gap between the technical and the enormous potential of microalgal photosynthetic cell factory. However, to date, few reports on plastid engineering in industrial microalgae have been documented. This is largely due to the small cell sizes and complex cell-wall structures which make these species intractable to current plastid transformation methods (i.e., biolistic transformation and polyethylene glycol-mediated transformation). Here, employing the industrial oleaginous microalga Nannochloropsis oceanica as a model, an electroporation-mediated chloroplast transformation approach was established. Fluorescent microscopy and laser confocal scanning microscopy confirmed the expression of the green fluorescence protein, driven by the endogenous plastid promoter and terminator. Zeocin-resistance selection led to an acquisition of homoplasmic strains of which a stable and site-specific recombination within the chloroplast genome was revealed by sequencing and DNA gel blotting. This demonstration of electroporation-mediated chloroplast transformation opens many doors for plastid genome editing in industrial microalgae, particularly species of which the chloroplasts are recalcitrant to chemical and microparticle bombardment transformation.