Microalga Neochloris Oleoabundans Research Articles

Application of Aqueous Two-Phase Systems with Thermoseparating Polymers (EOPO) as a Method for Extractive Fermentation with Neochloris oleoabundans

Extractive fermentation is an in situ method for the production and recovery of biomolecules of interest. Aqueous two-phase systems (ATPS) allow the product to be recovered in one phase of the system, reducing unit operations in the bioprocess. Thermosensitive polymers such as EOPOs are an interesting alternative to be applied in ATPS. In this work, different EOPOs were tested in an extractive fermentation strategy with the green microalgae Neochloris oleoabundans to provide a basis for future implementations of these systems in microalgae bioprocesses. Extractive fermentations were carried out with two EOPOs of different molecular weights (3900 and 12,000 g/mol) at concentrations of 10% and 15% (w/v). The microalga was incubated axenically under two different sets of conditions for 21 and 45 days, respectively. Cell counts were performed, and cell growth curves were obtained. Additionally, a semi-continuous and batch extractive fermentation assay was performed. The extractive fermentation with EOPO showed lower cell growth and a longer adaptation time of the microalgae in the fermentation, and EPS production yields of up to 8–23 g/L were obtained. Extractive fermentation is an interesting method to be implemented in microalgae cultures; however, further conditions need to be explored to achieve an appropriate bioprocess.

Metabolic Responses of the Microalga Neochloris oleoabundans to Extracellular Self- and Nonself-DNA.

Stressed organisms identify intracellular molecules released from damaged cells due to trauma or pathogen infection as components of the innate immune response. These molecules called DAMPs (Damage-Associated Molecular Patterns) are extracellular ATP, sugars, and extracellular DNA, among others. Animals and plants can recognize their own DNA applied externally (self-exDNA) as a DAMP with a high degree of specificity. However, little is known about the microalgae responses to damage when exposed to DAMPs and specifically to self-exDNAs. Here we compared the response of the oilseed microalgae Neochloris oleoabundans to self-exDNA, with the stress responses elicited by nonself-exDNA, methyl jasmonate (MeJA) and sodium bicarbonate (NaHCO3). We analyzed the peroxidase enzyme activity related to the production of reactive oxygen species (ROS), as well as the production of polyphenols, lipids, triacylglycerols, and phytohormones. After 5 min of addition, self-exDNA induced peroxidase enzyme activity higher than the other elicitors. Polyphenols and lipids were increased by self-exDNA at 48 and 24 h, respectively. Triacylglycerols were increased with all elicitors from addition and up to 48 h, except with nonself-exDNA. Regarding phytohormones, self-exDNA and MeJA increased gibberellic acid, isopentenyladenine, and benzylaminopurine at 24 h. Results show that Neochloris oleoabundans have self-exDNA specific responses.

Characterization of Neochloris oleoabundans under Different Cultivation Modes and First Results on Bioactivity of Its Extracts against HCoV-229E Virus

Microalgae are proposed in several biotechnological fields because of their ability to produce biomass enriched in high-value compounds according to cultivation conditions. Regarding the health sector, an emerging area focuses on natural products exploitable against viruses. This work deals with the characterization of the green microalga Neochloris oleoabundans cultivated under autotrophic and mixotrophic conditions as a source of whole aqueous extracts, tested as antivirals against HCoV-229E (Coronaviridae family). Glucose was employed for mixotrophic cultures. Growth and maximum quantum yield of photosystem II were monitored for both cultivations. Algae extracts for antiviral tests were prepared using cultures harvested at the early stationary phase of growth. Biochemical and morphological analyses of algae indicated a different content of the most important classes of bioactive compounds with antiviral properties (lipids, exo-polysaccharides, and total phenolics, proteins and pigments). To clarify which phase of HCoV-229E infection on MRC-5 fibroblast cells was affected by N. oleoabundans extracts, four conditions were tested. Extracts gave excellent results, mainly against the first steps of virus infection. Notwithstanding the biochemical profile of algae/extracts deserves further investigation, the antiviral effect may have been mainly promoted by the combination of proteins/pigments/phenolics for the extract derived from autotrophic cultures and of proteins/acidic exo-polysaccharides/lipids in the case of mixotrophic ones.

Microalgae as a Sustainable, Natural-Oriented and Vegan Dermocosmetic Bioactive Ingredient: The Case of Neochloris oleoabundans

“Vegan” and “sustainable” characteristics are strong claim trends behind the development of innovative skincare, fragrances, and makeup products. This created a need in the market for compliant ingredients. To date, there have been no records evidencing the use of the microalgae Neochloris oleoabundans (NA) in dermocosmetics. Therefore, we studied the applicability of such a natural compound in this context. NA was cultivated, and the scavenging activity (SA) of the NA extracts was evaluated. The highest SA was from the aqueous extract (54.8% ± 2.1%), being higher than that of the positive control. Two hydrogels were prepared with 1.0% ammonium acryloyldimethyltaurate/VP copolymer: (1) control gel; and (2) gel with a 1.0% NA aqueous extract. In vivo experiments were performed in healthy male and female volunteers with skin phototypes of II–IV. The stratum corneum (SC) hydration and the transepidermal water loss (TEWL) were measured in the forearm of participants to determine their biocompatibility. This parameter was determined by skin bioengineering measurements, confirming that SC hydration and TEWL were not affected by the samples. The laser Doppler measurements results showed a delayed erythema onset in the sites, where the NA hydrogel was applied. The results confirmed the biocompatibility and the anti-inflammatory activity of an innovative ingredient derived from microalgae suitable for a natural and vegan lifestyle.

Nitrogen starvation-inducible promoter of microalga Neochloris oleoabundans lipogenic gene encoding diacylglycerol acyltransferase 2

Microalgal lipid triacylglycerol (TAG) is a promising source for sustainable production of biofuels and edible oils. TAG biosynthesis in microalgae can be induced by nitrogen starvation (−N); however, regulation of the genes involved in this process is poorly known. To explore the regulation of gene encoding diacylglycerol acyltransferase 2 of oleaginous microalga Neochloris oleoabundans (NeoDGAT2) responsible for TAG biosynthesis, regulatory sequence of NeoDGAT2 gene (RDG) was cloned, and its functional regions were mapped by deletion analysis using the modified cyan fluorescent protein gene (CFP) as a reporter. The efficiency of CFP gene mTurquoise2 (Tq) without any intron, Tq1 and Tq2 with one and two copies of Chlamydomonas reinhardtii rbcS2 intron 1, respectively, was evaluated; Tq2 exhibited the highest CFP fluorescence activity in N. oleoabundans was therefore used as reporter for RDG deletion analysis. Deletion analysis of RDG revealed that the −N inducible region contained the predicted binding site of MYB transcription factor (MYB-bs). Specific binding between MYB-bs of RDG and the DNA-binding domain of MYB-related transcription factor ROC40 from C. reinhardtii was observed using electrophoretic mobility shift assay. Therefore, the corresponding MYB transcription factor in N. oleoabundans is probably the transcription factor regulating NeoDGAT2. The interaction between MYB transcription factor and the MYB-bs may play a role in regulating −N induced expression of NeoDGAT2, affecting TAG accumulation. MYB transcription factors can be the potential targets for engineering to increase TAG content. Increasing TAG content is essential for products derived from microalgal TAG to achieve economic viability.

Enhanced triacylglycerol production in oleaginous microalga Neochloris oleoabundans by co-overexpression of lipogenic genes: Plastidial LPAAT1 and ER-located DGAT2

Microalgae accumulate lipid triacylglycerol (TAG), a promising feedstock for production of natural edible oils and biofuels. To make products derived from microalgal TAG economically viable, increasing TAG content and productivity are of high importance. To increase TAG content, two endogenous key enzymes of TAG biosynthesis: plastidial lysophosphatidic acid acyltransferase (NeoLPAAT1) and endoplasmic reticulum-located diacylglycerol acyltransferase 2 (NeoDGAT2) were co-overexpressed in oleaginous microalga Neochloris oleoabundans. The neutral lipid content in NeoLPAAT1-NeoDGAT2 co-overexpressing transformant detected by Nile red staining increased 2-fold without compromising cell growth. The transcriptional levels of NeoLPAAT1 and NeoDGAT2 levels were 1.9-fold higher in the transformant than wild type. Considerably higher lipid accumulation was found in the transformant than wild type: total lipid content (73.72 ± 4.17 % DCW) increased 1.6-fold, TAG content (50.63 ± 6.15 % DCW) increased 2.1-fold, total lipid productivity (16.84 ± 0.66mg/L/day) increased 1.9-fold, and TAG productivity (11.68 ± 0.90mg/L/day) increased 2.1-fold. Fatty acid composition was slightly altered in the transformant compared to wild type; saturated fatty acid C16:0 increased to 26% from 20%, whereas C18:0 was reduced to 7% from 14%. Long-term stability of NeoLPAAT1-NeoDGAT2 co-overexpression was observed in the transformant continuously maintained on solid medium in a period of 4 years. The results suggested that targeted engineering of genes in pathway located at different organelles should be possible in microalgal lipid metabolism.

Production, isolation and bioactive estimation of extracellular polysaccharides of green microalga Neochloris oleoabundans

Previous research revealed that green microalga Neochloris oleoabundans was able to excrete extracellular polysaccharides (EPS) in the media containing certain sugar, but detail researches on the production conditions and chemical and bioactive properties of the EPS are still limited. In this research, cultivation conditions, including 5 sugars (glucose, galactose, maltose, lactose and sucrose), 10–80 g/L of sugar concentrations, 9–36 mM of NaNO3 and the salinity of 0.5–1.0 g/L NaCl, were investigated their effects on the production of N. oleoabundans EPS. In order to develop a fast and convenient method for assessment of EPS concentration in algal cultivation media, an indirect quantitative technique was proposed based on the consistency coefficient of the media. An EPS (EPS1) was isolated and purified, and its immune activities were investigated in vivo and in vitro. The results showed that glucose, galactose and maltose allowed N. oleoabundans to produce substantial EPS, and glucose was the best sugar for EPS production. The results also showed that 20 g/L glucose and 12 mM NaNO3 were favorable to EPS production, whereas addition of 0.5–1.0 g/L NaCl to the medium repressed EPS production. Chemical analysis showed that the isolated EPS1 contained 0.59% (w/w) peptides and had a monosaccharide composition of glucose, mannose, galactose, xylose, ribose, arabinose and rhamnose with the molar ratio of 40.7:19.0:18.95:8.7:6.86:4.57:1.2. The bioactive tests demonstrated that EPS1 had immunomodulatory activity in vitro and in vivo.

Stable wastewater treatment with Neochloris oleoabundans in a tubular photobioreactor

The potential of a wastewater treatment and biomass production process with the green microalgae Neochloris oleoabundans in a 45-L tubular photobioreactor was tested. In a stable and reliable alga-based process, wastewaters should undergo pre-treatment to remove colored substances. Our data show that further optimization steps may be required, such as adjustment of the N:P ratio and addition of essential metals. As a final result, parameters were established for a continuous-mode photobioreactor run with optimized wastewater for five times the hydraulic retention time (157 h) at the dry biomass concentration of 0.47 ± 0.03 g L−1. During that period, 75% and 99.6% removal efficiencies for NH4+ and PO43−, respectively, were achieved.

Enhanced biomass and lipid production of Neochloris oleoabundans under high light conditions by anisotropic nature of light-splitting CaCO3 crystal

The aim of this work was to study the anisotropic effect of crystalline CaCO3 nanoparticles (CN)-driven multiple refraction/scattering from the CN-coated agglomerated cells on the rate of photosynthesis and the product yield under high light conditions in the freshwater microalgae Neochloris oleoabundans. The CN-coating via biomineralization significantly improved the biomass and lipid production of N. oleoabundans during second stage of autotrophic induction by sustaining relatively high rate of photosynthesis at high irradiance using the multiple-splitting effect of the anisotropic polymorphism. The CN were successfully produced, adsorbed and grown on the external cells under conditions of mild alkalinity (pH 7.5–8.0), mild CaCl2 concentration (0.05 M) and under nitrogen starvation with strong light (400 µE m−2 s−1). Consequently, lipid content and productivity of N. oleoabundans cells cultured with 0.05 M CaCl2 increased by 18.4% and 31.5%, respectively, compared to the cells cultured with 0.05 M CaCl2 and acetazolamide to inhibit calcification.

Increased triacylglycerol production in oleaginous microalga Neochloris oleoabundans by overexpression of plastidial lysophosphatidic acid acyltransferase

BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for sustainable production of natural edible oils and biofuels. Nevertheless, products derived from microalgal TAG are not yet economically feasible; increasing TAG content via targeted genetic engineering of genes in TAG biosynthesis pathway are important to achieve economic viability. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with the endogenous enzyme lysophosphatidic acid acyltransferase (NeoLPAAT1) responsible for plastidial TAG biosynthesisResultsNeoLPAAT1 was found to contain all canonical motifs attributed to LPAAT proteins, two hypothetical membrane-spanning domains and a putative chloroplast transit peptide, indicating as a member of plastidial LPAAT type 1 subfamily. The NeoLPAAT1-expression cassette integrated in N. oleoabundans transformant was confirmed by PCR. The neutral lipid content in the transformant detected by Nile red staining was 1.6-fold higher than in wild type. The NeoLPAAT1 transcript was twofold higher in the transformant than wild type. Considerably higher lipid quantity was found in the transformant than wild type: total lipid content increased 1.8- to 1.9-fold up to 78.99 ± 1.75% dry cell weight (DCW) and total lipid productivity increased 1.8- to 2.4-fold up to 16.06 ± 2.68 mg/L/day; while TAG content increased 2.1- to 2.2-fold up to 55.40 ± 5.56% DCW and TAG productivity increased 1.9- to 2.8-fold up to 10.67 ± 2.37 mg/L/day. A slightly altered fatty acid composition was detected in the transformant compared to wild type; polyunsaturated fatty acid (C18:2) increased to 19% from 11%. NeoLPAAT1-overexpression stability was observed in the transformant continuously maintained in solid medium over 150 generations in a period of about 6 years.ConclusionsOur results demonstrate the considerably increased TAG content and productivity in N. oleoabundans by overexpression of plastidial NeoLPAAT1 that are important for products derived from microalgal TAG to achieve economic viability. Plastidial LPAAT1 can be a candidate for target genetic manipulation to increase TAG content in other microalgal species with desired characteristics for production of natural edible oils and biofuels.

Cultivation of the microalga Neochloris oleoabundans for biofuels production and other industrial applications (a review)

Microalgae cultivation for biofuels production and other applications has gained considerable interest recently. Despite their simple structures, microalgae can accumulate significant amounts of neutral lipids per dry cell weight compared to other energy crops. Neochloris oleoabundans is a promising microalga known for its high lipid content and biomass growth rate compared to other species cultivated for biofuels synthesis; therefore, it is considered as a suitable candidate for biodiesel synthesis. This review paper covers several key aspects associated with the cultivation and applications of the microalga N. oleoabundans. Biomass composition, factors affecting the growth, and biomass and lipid productivities of this species were addressed. In addition, different growth conditions as well as alternative readily available nutrient media to support the growth of N. oleoabundans were presented in this review.

Comparison analysis on the energy efficiencies and biomass yields in microbial CO 2 fixation

Abstract CO 2 fixation by a hydrogen-oxidizing bacterium, Cupriavidus necator, was evaluated in a packed bed bioreactor under a constant flow rate of gas mixtures (H 2 , O 2 , CO 2 ). The overall energy efficiency depends on the efficiencies of CO 2 fixation into carbohydrate and the reduced carbon into biomass and bioproducts, respectively. The efficiencies varied with the limiting gas substrate. Under O 2 limitation, the efficiency (20–30%) of CO 2 fixation increased with time and was higher than the overall efficiency (12–18%). Under H 2 limitation, the efficiency of CO 2 fixation declined with time while the biomass yield was quite similar to that under O 2 limitation. A cellular metabolic model was suggested for the lithoautotrophic growth of C. necator , including CO 2 fixation into carbohydrate followed by the main metabolic pathway of reduced carbon. Under CO 2 limitation, most H 2 energy was wasted, resulting in a very low biomass yield. Under a dual limitation of O 2 and nitrogen, biosynthesis of poly(3-hydroxybutyrate) was triggered, and the energy efficiency or yield of biopolyester was lower than those of microbial cell mass. Compared with a green microalga Neochloris oleoabundans that produces lipid under nutrient limitation, C. necator exhibited a much higher (3–6 times) energy efficiency in producing biomass and bioproducts from CO 2 .

The influence of day/night cycles on biomass yield and composition of Neochloris oleoabundans

BackgroundDay/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans. Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles.ResultsUnder day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10–15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a ‘block’ (continuous light intensity during the light period) and in a ‘sine’ (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a ‘block’ or in a ‘sine’).ConclusionsThe biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

Accelerated triacylglycerol production and altered fatty acid composition in oleaginous microalga Neochloris oleoabundans by overexpression of diacylglycerol acyltransferase 2

BackgroundMicroalgae are promising sources of lipid triacylglycerol (TAG) for biodiesel production. However, to date, microalgal biodiesel is technically feasible, but not yet economically viable. Increasing TAG content and productivity are important to achieve economic viability of microalgal biodiesel. To increase TAG content, oleaginous microalga Neochloris oleoabundans was genetically engineered with an endogenous key enzyme diacylglycerol acyltransferase 2 (NeoDGAT2) responsible for TAG biosynthesis.ResultsThe integration of NeoDGAT2 expression cassettes in N. oleoabundans transformant was confirmed by PCR. The neutral lipid accumulation in the transformant detected by Nile red staining was accelerated and 1.9-fold higher than in wild type; the lipid bodies in the transformant visualized under fluorescence microscope were also larger. The NeoDGAT2 transcript was two-fold higher in the transformant than wild type. Remarkably higher lipid accumulation was found in the transformant than wild type: total lipid content increased 1.6-to 2.3-fold up to 74.5 ± 4.0% dry cell weight (DCW) and total lipid productivity increased 1.6- to 3.2-fold up to 14.6 ± 2.0 mg/L/day; while TAG content increased 1.8- to 3.2-fold up to 46.1 ± 1.6% DCW and TAG productivity increased 1.6- to 4.3-fold up to 8.9 ± 1.3 mg/L/day. A significantly altered fatty acid composition was detected in the transformant compared to wild type; the levels of saturated fatty acid C16:0 increased double to 49%, whereas C18:0 was reduced triple to 6%. Long-term stability was observed in the transformant continuously maintained in solid medium over 100 generations in a period of about 4 years.ConclusionsOur results demonstrate the increased TAG content and productivity in N. oleoabundans by NeoDGAT2 overexpression that may offer the first step towards making microalgae an economically feasible source for biodiesel production. The strategy for genetically improved microalga presented in this study can be applied to other microalgal species possessing desired characteristics for industrial biofuel production.

Biodiesel production from microalgae oil through conventional and ultrasonic methods

ABSTRACTIn the present study, the oil extracted from the microalga Neochloris oleoabundans was used for producing biodiesel. The viscosity of microalgal oil was very high so transesterification was done by an ultrasonic method and a simple basic method. Free fatty acid content in oil was found high so a two-step transesterification reaction was used. Maximum yield of algal oil methyl ester was acquired by the ultrasonic method (98%) with improved physicochemical characteristics, whereas the biodiesel yield obtained by simple transesterification process was low (91%). The maximum oil content in N. oleoabundans was found to be 42% and major fatty acids include palmitic acid, oleic acid, and linoleic acid. Finally, it was conferred that the ultrasonic-assisted transesterification method is the best technique for biodiesel production from microalgal oil.

Anaerobic digestate as a nutrient medium for the growth of the green microalga Neochloris oleoabundans

In this study, the microalga Neochloris oleoabundans was cultivated in a sustainable manner using diluted anaerobic digestate to produce biomass as a potential biofuel feedstock. Prior to microalgae cultivation, the anaerobic digestate was characterized and several pretreatment methods including hydrogen peroxide treatment, filtration, and supernatant extraction were investigated and their impact on the removal of suspended solids as well as other organic and inorganic matter was evaluated. It was found that the supernatant extraction was the most convenient pretreatment method and was used afterwards to prepare the nutrient media for microalgae cultivation. A bench-scale experiment was conducted using multiple dilutions of the supernatant and filtered anaerobic digestate in 16 mm round glass vials. The results indicated that the highest growth of the microalga N. oleoabundans was achieved with a total nitrogen concentration of 100 mg N/L in the 2.29% diluted supernatant in comparison to the filtered digestate and other dilutions.

Pressurized liquid extraction of Neochloris oleoabundans for the recovery of bioactive carotenoids with anti-proliferative activity against human colon cancer cells

In recent years, the green microalgae Neochloris oleoabundans have demonstrated to be an interesting natural source of carotenoids that could be used as potential food additive. In this work, different N. oleoabundans extracts obtained by pressurized liquid extraction (PLE) have been analyzed in depth to evaluate the influence of different culture conditions (effect of nitrogen, light intensity or carbon supplied) not only on the total carotenoid content but also on the carotenoid composition produced by these microalgae. Regardless of the cultivation conditions, lutein and carotenoid monoesters were the most abundant carotenoids representing more than 60% of the total content in all extracts. Afterwards, the effect of the different N. oleoabundans extracts and the dose-effect of the most potent algae extracts (namely, N9, PS and CO2 (−)) on the proliferation of human colon cancer cells lines (HT-29 and SW480) and a cell line established from a primary colon cancer cell culture (HGUE-C-1) were evaluated by an MTT assay whereas a stepwise multiple regression analysis was performed to get additional evidences on the relationship between carotenoid content and the antiproliferative activity. Results revealed that, as a general trend, those extracts with high total carotenoid content showed comparably antiproliferative activity being possible to establish a high correlation between the cell proliferation values and the carotenoid constituents. Monoesters showed the highest contribution to cell proliferation inhibition whereas lutein and violaxanthin showed negative correlation and diesters and zeaxanthin showed a positive significant contribution to cell proliferation.

Dosage effect of cationic polymers on the flocculation efficiency of the marine microalga Neochloris oleoabundans

A mechanistic mathematical model was developed to predict the performance of cationic polymers for flocculating salt water cultivated microalgae. The model was validated on experiments carried out with Neochloris oleoabundans and three different commercial flocculants (Zetag 7557®, Synthofloc 5080H® and SNF H536®). For a wide range of biomass concentrations (0.49–1.37gL−1) and flocculant dosages (0–150mgL−1) the model simulations predicted well the optimal flocculant-to-biomass ratio between 43 and 109mgflocculant/gbiomass. At optimum conditions biomass recoveries varied between 88% and 99%. The cost of the usage of commercial available flocculants is estimated to range between 0.15$/kgbiomass and 0.49$/kgbiomass.

Control of protozoa contamination and lipid accumulation in Neochloris oleoabundans culture: Effects of pH and dissolved inorganic carbon

Combined effects of pH (i.e., 7.5, 8.5, and 9.5) and bicarbonate (i.e., 0, 80 and 160mM NaHCO3) on lipid accumulation and on biological contaminant viability in a protozoa-contaminated culture of the freshwater microalga Neochloris oleoabundans were studied. Cultures grown in the media containing 160mM NaHCO3 at pH 9.5 obtained the highest biomass concentration (DCWmax=1.32g/L), lipid content (LC=327mg/g), which corresponded to a lipid productivity of 56mg/(L·d), and the culture was protozoa free one day after inoculation. Other cultures, 160mM NaHCO3 at pH 8.5 (DCWmax=1.32g/L, LC=223mg/g), and 80mM NaHCO3 at pH 9.5 (DCWmax=1.25g/L, LC=264mg/g) could delay protozoan growth, but not inhibit it completely. These results suggest 160mM NaHCO3 or slightly above at pH levels of 8.5–9.5 may be used in outdoor cultivation processes of freshwater N. oleoabundans to control protozoa contamination while maintain a high lipid content.

Stable nuclear transformation of the oleaginous microalga Neochloris oleoabundans by electroporation

Biodiesel from microalgae is technically feasible, but not yet economically viable. A potential approach to improve microalgae as an economically viable biodiesel feedstock is to increase microalgal lipid content via genetic engineering. Genetic manipulation of microalgae requires the accessibility to stable nuclear transformation. In this study, we describe a strategy for developing a stable nuclear transformation system of the oleaginous microalga Neochloris oleoabundans using electroporation. The hygromycin B-resistant gene Hyg3, which was used as a positively selectable marker, consisted of aph7” gene encoding aminoglycoside phosphotransferase of Streptomyces hygroscopicus and intron1 of Chlamydomonas reinhardtii rbcS2 gene, under the control of C. reinhardtii HSP70A-RBCS2 hybrid promoter. The transformation frequency was 5.2 × 10−4 transformants mg−1 DNA. The transformants showed stable hygromycin B-resistant phenotype for at least 6 months in the absence of the antibiotic selection. Co-transformation frequency of unselectable green fluorescent protein gene (Gfp) adapted to C. reinhardtii codon usage (ChGfp) and selectable Hyg3 gene was 2.6 × 10−4 transformants mg−1 DNA; up to 90 % of the transformants exhibited green fluorescent protein (GFP) activity. The ChGfp and Hyg3 gene were integrated into the nuclear genome of N. oleoabundans. The GFP fluorescence signal of the transformants under confocal laser scanning microscope was visible. The successful stable nuclear transformation system not only provides a basis for molecular genetics study, but also enables subsequent genetic engineering in the microalga to increase lipid content for biodiesel production. The strategy for developing the stable nuclear transformation system presented in this study may be applicable to other microalgal species without sequenced genome.