Green Microalga Research Articles

Efficacy of Fat Supplements with Different Unsaturated/Saturated FA Ratios Undergoing First Postpartum Ovulation in Lactating Anovulatory Goats

We investigated whether microalgae or linseed supply during the early postpartum period affects ovarian restimulation and supports the first postpartum ovulation in lactating anovulatory goats. Thirty-eight An-glo-Nubian-crossbred adult goats were allocated into three groups, one with a control diet (n = 12), fed a total mixed ration (TMR) comprising chopped elephant grass and concentrate; an algal diet (n = 13), fed TMR + green microalgae (1% dry matter); and a linseed diet (n = 13), TMR + linseed (12% dry matter). Supplements were furnished from the second to fifth week (time of weaning). Goats were estrus synchronized on day 40 by insertion of an intravaginal CIDR device for 5 days, after which 0.075 mg PGF2α was applied to induce ovulation, and estrus was monitored for 72 h. From the 5th–15th day of ovulation induction, the corpus luteum (CL) area and progesterone rate were monitored. The algal and linseed groups showed lower feed intake (p < 0.001) and higher (p < 0.001) triglyceride levels/follicle numbers, respectively. After estrus induction, no differences were observed in estrus response; however, the linseed group showed more and larger growing follicles (p = 0.016 and p < 0.01), a higher ovulation rate (p < 0.05), a larger CL area (p < 0.05), and higher progesterone levels (p < 0.001). Linseed after delivery stimulates follicular growth before and after ovulation induction, favoring better CL quality during the first ovulation.

Comparative study of the synthesis of polyhydroxyalkanoates by cyanobacteria Spirulina platensis and green microalga Chlorella vulgaris

The article presents the results of the study of the growth and synthesis of degradable polyhydroxyalkanoates (PHA) of cyanobacteria Spirulina platensis (Nordst) Geitl. and green microalga Chlorella vulgaris (Beijer), in the cultures of which the production of “green” plastics was studied, including in relation to the dynamics of the fatty acid composition of lipids associated with the formation of PHA. The reorganization of constructive metabolism during the development of both cultures and the dynamics of accumulation of lipids and PHA in cells against the background of a decrease in protein were studied. On a modified BG-11 medium, with a decrease in the NaNO3 concentration to 0.5 g/L and the addition of sodium acetate (3/0 g/L), the yields of polymers for C. vulgaris and S. platensis reached 20.0 and 16.2 %, respectively. The composition and properties of the polymer, which is a homopolymer of 3-hydroxybutyric acid (P(3HB)), were studied using GC, FTIR spectroscopy, HPLC, and DTA. The average molecular weight of P(3HB) samples was determined to be 416–467 kDa; crystallinity was 54–56 % with a gap between the melting point and thermal degradation temperature of 109–114 °C.

Peculiarities of the Influence of Phenol Carboxylic Acids and Bioflavonoids on the Growth of Green Microalgae Cultures

It has been found that the addition of phenol carboxylic acids and bio-flavonoids (50-200 μg/L) to the nutrient medium of algae during their cultivation resulted in the increase in the biomass of green microalgae <i>Selenastrum gracile, Monoraphidium griffithii</i>, and <i>Acutodesmus acuminatus</i> by a factor of 1.2-3.5 depending on the stage of culture growth and active substance. The most significant influence on the specific growth rate of green microalgae cultures was exerted by salicylic and gallic acids, selectively by oxycinnamic acids and bioflavonoids. The obtained data deserve attention in further research aimed at obtaining algal biomass with an increased content of biologically valuable compounds (proteins, carbohydrates, lipids, etc.) in cultivating in biotechnological complexes.

Regulation of nucleus-encoded trans-acting factors allows orthogonal fine-tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii.

The green microalga Chlamydomonas reinhardtii is a promising host organism for the production of valuable compounds. Engineering the Chlamydomonas chloroplast genome offers several advantages over the nuclear genome, including targeted gene insertion, lack of silencing mechanisms, potentially higher protein production due to multiple genome copies and natural substrate abundance for metabolic engineering. Tuneable expression systems can be used to minimize competition between heterologous production and host cell viability. However, complex gene regulation and a lack of tight regulatory elements make this a challenge in the Chlamydomonas chloroplast. In this work, we develop two synthetic tuneable systems to control the expression of genes on the chloroplast genome, taking advantage of the properties of the vitamin B12-responsive METE promoter and a modified thiamine (vitamin B1) riboswitch, along with nucleus-encoded chloroplast-targeted regulatory proteins NAC2 and MRL1. We demonstrate the capacity of these systems for robust, fine-tuned control of several chloroplast transgenes, by addition of nanomolar levels of vitamins. The two systems have been combined in a single strain engineered to avoid effects on photosynthesis and are orthogonal to each other. They were then used to manipulate the production of an industrially relevant diterpenoid, casbene, by introducing and tuning expression of the coding sequence for casbene synthase, as well as regulating the metabolite flux towards casbene precursors, highlighting the utility of these systems for informing metabolic engineering approaches.

Advances in Bioprocess Engineering for Optimising Chlorella vulgaris Fermentation: Biotechnological Innovations and Applications.

Chlorella vulgaris, a unicellular green microalga, has obtained significant attention due to its high protein content, abundance of bioactive compounds, and broad biotechnological potential. Used in nutraceuticals, pharmaceuticals, and functional foods, it is now gaining traction in cosmetics, biopharmaceuticals, and environmental applications. Recent advancements in fermentation technology, such as the development of high-density fermentation strategies, adaptive evolution of strains, and real-time monitoring systems, have greatly improved the efficiency, scalability, and sustainability of C. vulgaris production, enhancing bioavailability and product quality. This review explores developments in C. vulgaris fermentation, highlighting advancements in strain improvement through genetic engineering, metabolic optimization, mutagenesis, and adaptive evolution, alongside bioprocess engineering and the optimization of fermentation parameters. Key considerations include bioreactor design, downstream processing, and innovative monitoring technologies aimed at maximizing biomass yield and bioactive compound production. Emerging applications of fermented C. vulgaris across industries are also highlighted, along with future perspectives on scaling up production, addressing regulatory challenges, and ensuring biosafety. These insights provide a comprehensive outlook on the future of C. vulgaris fermentation in biotechnological applications.

Accumulation of Carotenoid and Lipid in Microalgae Dunaliella bardawil DCCBC 15 Cultivated under Nitrogen Starved Conditions

Dunaliella bardawil is a unicellular green microalgae that can accumulate large amounts of carotenoids under adverse culture conditions, so it has high commercial value and is used as a functional food. Nutrient starvation, especially nitrogen starvation, induces carotenoid and lipid production in algal cells. Dunaliella bardawil cells were grown in MD4 medium after 12 cultural days reaching maximum cell density (1,4x106 cells/mL) that changed to four stress conditions—nitrogen replete, nutrient starvation, and half and fully nitrogen-depleted conditions—to investigate the carotenoid and lipid contents of D. bardawil microalgae. The results showed that the cell density D. bardawil decreased significantly under cultural conditions. High amounts of carotenoids were detected under fully nitrogen-depleted conditions (14.631 pg/cell; car/chl: 12.346). D. bardawil was able to overproduce lipids under both nitrogen-free (351.580 pg/cell; lipid/chl: 219.792) and fully nitrogen-free (373.136 pg/cell; lipid/chl 321.401) conditions. Therefore, fully nitrogen-depleted conditions of cultivation stimulate significantly D. bardawil cells accumulating a large amounts lipid and carotenoid.

Analyzing sorbitol biosynthesis using a metabolic network flux model of a lichenized strain of the green microalga Diplosphaera chodatii.

Diplosphaera chodatii, a unicellular terrestrial microalga found either free-living or in association with lichenized fungi, protects itself from desiccation by synthesizing and accumulating low-molecular-weight carbohydrates such as sorbitol. The metabolism of this algal species and the interplay of sorbitol biosynthesis with its growth, light absorption, and carbon dioxide fixation are poorly understood. Here, we used a recently available genome assembly for D. chodatii to develop a metabolic flux model and analyze the alga's metabolic capabilities, particularly, for sorbitol biosynthesis. The model contains 151 genes, 155 metabolites, and 194 unique metabolic reactions participating in 12 core metabolic pathways and five compartments. Both photoautotrophic and mixotrophic growths of D. chodatii were supported by the metabolic model. In the presence of glucose, mixotrophy led to higher biomass and sorbitol yields. Additionally, the model predicted increased starch biosynthesis at high light intensities during photoautotrophic growth, an indication that the "overflow hypothesis-stress-driven metabolic flux redistribution" could be applied to D. chodatii. Furthermore, the newly developed metabolic model of D. chodatii, iDco_core, captures both linear and cyclic electron flow schemes characterized in photosynthetic microorganisms and suggests a possible adaptation to fluctuating water availability during periods of desiccation. This work provides important new insights into the predicted metabolic capabilities of D. chodatii, including a potential biotechnological opportunity for industrial sorbitol biosynthesis.IMPORTANCELichenized green microalgae are vital components for the survival and growth of lichens in extreme environmental conditions. However, little is known about the metabolism and growth characteristics of these algae as individual microbes. This study aims to provide insights into some of the metabolic capabilities of Diplosphaera chodatii, a lichenized green microalgae, using a recently assembled and annotated genome of the alga. For that, a metabolic flux model was developed simulating the metabolism of this algal species and allowing for studying the algal growth, light absorption, and carbon dioxide fixation during both photoautotrophic and mixotrophic growth, in silico. An important capability of the new metabolic model of D. chodatii is capturing both linear and cyclic electron flow mechanisms characterized in several other microalgae. Moreover, the model predicts limits of the metabolic interplay between sorbitol biosynthesis and algal growth, which has potential applications in assisting the design of bio-based sorbitol production processes.

Metamorphosis of a unicellular green alga in the presence of acetate and a spatially structured three-dimensional environment.

Photosynthetic protists, named microalgae, are key players in global primary production. The green microalga Chlamydomonas reinhardtii is a well-studied model organism. In nature, it dwells in acetate-rich paddy rice soil, which is not mimicked by standard liquid laboratory conditions. Here, we maintained the algae in a liquid environment with spatially structured 3-D components (S3-D) and acetate recreating natural conditions. We perform transcriptome sequencing, immunoblotting, fluorescence and electron microscopy, and Raman microspectroscopy to characterize the algae in S3-D vs homogeneous conditions. The algae undergo a metamorphosis-like process when transitioned from homogeneous aquatic to a lifestyle simulating acetate-rich rice soil. These conditions result in reduced cell size and cilia length, an enlarged eyespot and many cells with double-layered cell walls. RNA-Seq reveals alterations in c. 2400 transcripts. Four key photoreceptors including CRY-DASH1 and phototropin governing plastid metabolism along with its eyespot are altered in their protein expression. Consequently, photosynthetic pigments, lipids and starch levels vary as do starch distribution patterns. Fitness against antagonistic bacteria is enhanced concurrently with the downregulation of an involved Ca2+ channel transcript. This study highlights the profound impact of S3-D initiating processes inaccessible under homogeneous laboratory conditions. Thus, overexpression lines for certain photoreceptors and starch are naturally created.

Comparative growth inhibition of 6PPD and 6PPD-Q on microalgae Selenastrum capricornutum, with insights into 6PPD-induced phototoxicity and oxidative stress

Widespread environmental detection of tire additive N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its toxic metabolite 6PPD-Q has raised great concerns for their potential impact on aquatic biota. This study investigated the effects of 6PPD and 6PPD-Q on the model green microalgae (Selenastrum capricornutum). Results showed that 6PPD at the concentrations of 1–5 mg·L−1 stimulated S. capricornutum growth, while higher concentrations (10–50 mg·L−1) inhibited growth with an IC50(96 h) of 8.78 mg·L−1. However, at concentrations up to 10 mg·L−1, no toxicity was observed for S. capricornutum exposed to 6PPD-Q. Under the stress of 6PPD, S. capricornutum exhibited increased cellular membrane permeability and cell wall rupture, indicating structural damage to the algae cell. Microalgal oxidative stress was induced through the accumulation of reactive oxygen species (ROS), reaching levels of 1.65–5.29 times higher than the non-exposure cells, which altered enzymatic activities including superoxide dismutase (SOD) and catalase. Exposure to 6PPD at concentrations of 10–50 mg·L−1 resulted in photosynthetic toxicity as evidenced by decreased Chlorophyll a (Chl a) content and adverse effects on chlorophyll fluorescence parameters, such as maximum photochemical quantum yield (Fv/fm), PSII (photosystem II) effective quantum yield [Y(II)], and photosynthetic electron transfer rate (ETR). While the concentrations employed may be higher than those typically found in the environment, this study uncovers a significant finding that 6PPD may demonstrate even greater toxicity to microalgae than its derivative, 6PPD-Q. This underscores the need for further investigation into the ecological risks of 6PPD, particularly in the context of primary producers like microalgae.

Green microalga Chromochloris zofingiensis conserves substrate uptake pattern but changes their metabolic uses across trophic transition.

The terrestrial green alga Chromochloris zofingiensis is an emerging model species with potential applications including production of triacylglycerol or astaxanthin. How C. zofingiensis interacts with the diverse substrates during trophic transitions is unknown. To characterize its substrate utilization and secretion dynamics, we cultivated the alga in a soil-based defined medium in transition between conditions with and without glucose supplementation. Then, we examined its exometabolite and endometabolite profiles. This analysis revealed that regardless of trophic modes, C. zofingiensis preferentially uptakes exogenous lysine, arginine, and purines, while secreting orotic acid. Here, we obtained metabolomic evidences that C. zofingiensis may use arginine for putrescine synthesis when in transition to heterotrophy, and for the TCA cycle during transition to photoautotrophy. We also report that glucose and fructose most effectively inhibited photosynthesis among thirteen different sugars. The utilized or secreted metabolites identified in this study provide important information to improve C. zofingiensis cultivation, and to expand its potential industrial and pharmaceutical applications.

A sulfobetaine containing-polymethylmethacrylate surface coating as an excellent antifouling agent against Chlorella sp

Biofouling represents one of the major issues in Photobioreactors (PBRs) for microalgae cultivation. At this aim, a series of zwitterionic-containing polymethylmethacrylate (PMMA) co-ter-polymers, differing in the molar percentage of sulfobetaine (SBMA)- and 2-hydroxyethylmethacrylate (HEMA) components were designed as antifouling (AF) potential coatings for inner PMMA surface of photobioreactors (PBRs). Among these, the sample named PSBM, P(HEMA-co-MMA-co-SBMA), showing the best film capabilities on PMMA slides, was selected, characterized and its AF activity investigated. High transparent and thin PSBM films, as confirmed by AFM and optical transparency investigations, were obtained by a simple drop-casting procedure using a basic isopropylalcohol/water mixture. AFM images of raw PSBM and PSBM films clearly indicate that a conformational change occurred upon treatment with electrolytes. PSBM was further characterized by ATR-FTIR Spectroscopy, Dynamic Light Scattering, X-Ray Photoelectron Spectroscopy, Thermogravimetric analysis, Differential Scanning Calorimetry and Static Water Contact Angle (WCA) investigations. Consistent with hydrophilic behavior, the PSBM surface provided a WCA of 60.9 ± 0.8° which, interestingly, decreased to 27.7 ± 1.2° after immersion in electrolyte culture media. PSBM films displayed a high satisfactory AF behavior in that a rare or no cell adhesion was observed when placed for 7 days in a suspension of the green microalga Chlorella sp. For comparison, the experiments were carried out with untreated and rough PMMA surfaces.

Biopolymer detection in Egyptian microalgae: A pathway to eco-friendly bioplastics

With the increase in pollution resulting from plastic waste, it was a necessity to reduce the danger of its waste to the environment. Hence, replacing traditional plastic with bioplastic is a must. Bioplastic is more environmentally friendly materials, such as plastic made from biological sources. Green and bluegreen microalgae have recently emerged as two of the most valuable sources. This study examines the viability of using microalgal cells to produce biopolymers, such as polyhydroxybutyrate, proteins, and carbohydrates. The study employed spectrophotometric standards to quantify the concentrations of polyhydroxybutyrate (PHB) and carbohydrates. Additionally, the Kjeldahl method was utilised to determine the total protein content. Five green microalgal strains and five blue green microalgal strains were examined. Chlorophyll a was measured to track growth. Growth rate assessments revealed that Selenastrum gracile exhibited the highest chlorophyll a concentration (6.9 μg/L) at 15 days, accompanied by the highest protein content at 41.13 %. Additionally, it displayed a PHB concentration of 325 mg/mL, while exhibiting the lowest carbohydrate levels (12 mg/ml). Polyhydroxybutrate content maximum concentration was detected in Microcystis aeruginosa 720.5 μg/mL, and 31 % concentration in proteins and carbohydrates (12.3 mg/ml). Selenastrum sp. is easily cultivated and offers potential for further research. Its application in bioplastic production, through blending with other polymers, is a promising avenue for future exploration.

Sustainable bioremediation of cassava waste effluent using Nannochloropsis salina TSD06: an eco-technological approach

This present study explored the large-scale treatment of cassava effluent using the freshwater green microalga Nannochloropsis salina TSD06. The cassava effluent treatment exposed a significant reduction of the inorganic pollutants were 98.26% nitrate, 93.94% phosphate, 97.43% sulfate, 85.86% chloride, 94.73% calcium, 93.45% potassium, 93.1% magnesium, 98.32% of sodium and 97.36% of phosphorus on 10th-day treatment using Nannochloropsis salina TSD06 microalga under optimized conditions. The analysis of variance (ANOVA) was performed utilizing response surface methodology (RSM), and experimental results showed a high regression coefficient (R2 = 0.999). The experimental microalga produced 7.25 mg/mL of biomass, 276.65 mg/g of lipids, 125.34 mg/mL of carbohydrates, and 3.75 mL/g of biodiesel. The Nannochloropsis salina TSD06 microalga produced biodiesel compounds were analyzed through gas chromatography-mass spectrometry (GCMS) and identified the major compounds viz., 13.45% of oleic acid, 11.84% of stearic acid, 10.23% of myristic acid, 9.45% of palmitic acid, 8.31% of linoleic acid, 8.04% of hexadecanoic acid, 7.62% of eicosadienoic acid, 4.86% of octadecanoic acid and 3.19% of docosahexaenoic acid. The functional groups were revealed from biodiesel using Fourier transform infrared spectroscopy (FTIR) analysis, and the functional groups were carboxyl groups, phenol molecules, alcohol molecules, alkenes, and carbonyl groups. The Nannochloropsis salina TSD06 microalga showed high removal ability and provided an effective, eco-friendly method of wastewater treatment and biofuel production.

Phycoremediation of rice bran oil processing wastewater and biodiesel production using green microalgae, Scenedesmus obliquus: a green approach to environmental protection and remediation.

The current study investigated the enhancement of biomass in S. obliquus, using rice bran oil processing (RBOP) wastewater in different RBOP wastewater concentrations, while also aiming to produce biofuel and treat the wastewater simultaneously. The strain was grown in Blue Green-11 (BG11) media as well as RBOP wastewater at different wastewater concentrations with distilled water at 10%, 25%, 50%, 75%, and 100% under controlled experimental settings. The study findings demonstrated a notable enhancement in the characteristics of RBOP wastewater during a 16-day growth period. The 75% RBOP wastewater concentration demonstrated superior efficacy as a growth medium for producing biomass as well as lipids, among other wastewater concentrations. Accordingly, physicochemical parameters and heavy metal percent of the RBOP wastewater were assessed. The collected biomass was employed in the production of biodiesel, and the fatty acid methyl esters (FAME) were measured, along with an assessment of its characteristics. Physicochemical analysis indicated that S. obliquus was able to effectively decrease levels of nitrate, phosphate, sulfur, and chemical oxygen demand (COD) in RBOP wastewater. The heavy metal reduction percentages for iron (Fe), zinc (Zn), nickel (Ni), copper (Cu), and arsenic (As) were 69.43%, 72.94%, 72.99%, 90.49%, and 91.5%, respectively, following treatment with S. obliquus. FTIR indicated the existence of various functional groups (including alcohol, carboxylic acid) on the surface of the microalgal biomass. The FAME profile of S. obliquus exhibited a moderate level of saturated and unsaturated fatty acid content. S. obliquus demonstrated significant phycoremediation capabilities and potential for lipid production. This study has indicated that S. obliquus is a potential candidate for the treatment of wastewater.

Blue Light Enhances the Antioxidant, Antimicrobial, and Antitumor Potential of the Green Microalgae Coelastrella sp. BGV.

Green algae of the genus Coelastrella have attracted the attention of scientists due to their rich biochemical composition and potential for application in phytomedicine. The present study investigated the influence of light on the bioactive capacity of extracts from the Bulgarian strain of the green microalgae Coelastrella sp. BGV. Three LED lights were examined-red/blue (C1), blue (C2), and control white light (C3). The respective ethanol extracts were analyzed for the total content of phenolic antioxidants. The antimicrobial activity was tested using the disk-diffusion method against 10 microorganisms. The antiproliferative and cytotoxic effects on cervical carcinoma HeLa and hepatocellular carcinoma HepG2 cell lines, as well as non-tumorigenic embryonal fibroblasts BALB/3T3 control, were evaluated using a cell viability assay. The overall results highlighted blue light as a factor enhancing the antioxidant, antibacterial, and cytotoxic activities of the C2 microalgal extract. Additionally, the investigated mechanism of the antitumor activity revealed a proapoptotic effect. In contrast, the C1 extract exhibited weaker activity and selectivity, while the C3 extract was the least active but demonstrated high cytotoxic selectivity. This study could contribute to expanding knowledge about the high biological potential of green microalgae and the development of biotechnological approaches for its regulation.

Microinjection System for Single-Microalgal Cells Based on Electroosmotic Flow

Introduction Microalgae are attracting attention as promising candidates for CO2 fixation and biomaterial production, due to no competition with food and feed, and high growth rates and photosynthetic ability. To expand the use of microalgae as a host for biomaterial production, it is crucial to develop efficient and versatile gene introduction technologies for microalgae, enabling diverse metabolic modifications. Several methods, such as microparticle bombardment, electroporation, and bacterial conjugation, have been developed to deliver foreign DNA to microalgae. However, these methods resulted in low transformation efficiency and limited applicable microalgae species. To overcome these problems, we have developed a novel microinjection system for single-microalgal cells based on electroosmotic flow using nano-sized pipette (nanopipette). The nanopipette enables automatic and precise control of solution delivery into cells at the femtoliter level. In this study, we investigated optimum conditions for substance introduction using the electrochemically controlled nanopipette into a green alga, Haematococcus sp.. Materials & Methods A pipette with tip outer diameter of several tens of nanometers, was used for the injection. Fluorescein isothiocyanate (FITC)-labeled dextran (MW: 70,000) was used as an injection material. Initially, FITC-dextran dissolved in PBS was filled inside the nanopipette and an electrode was set. The cell sample was also filled with PBS and a reference electrode was placed inside. Voltage was then applied to the electrode during injection and the ion current generated was monitored. The nanopipette automatically detects the cell surface by identifying changes in ionic current. Once the cell surface is detected, the nanopipette penetrates the cell to a predetermined depth and injects the reagent using electroosmotic flow caused by the application of voltage. A green microalga, Haematococcus sp. strain NKG7001, which is an astaxanthin-producer, was used to optimize the conditions for microinjection using the nanopipette. Four types of algal cells, i.e. flagellated, palmelloid, intermediates or cyst cells based on their cell cycle were investigated. Algal cells were applied and fixed on the surface of agarose gels. Subsequently, these cells on the agarose gels were immersed in PBS, and then, dextran was injected into each cell using the nanopipette under different applied voltages and injection time. The injection efficiency was calculated as the number of successfully injected cells divided by the number of trials. Furthermore, the proposed method was applied to other green algae, a polysaccharide producer, Tetraselmis sp. strain NKG400013, and a green alga, Clamydomonas reinhardtii. Results & Discussion The injection of dextran into Haematococcus cells was succeeded in flagellated and palmelloid cells, but not intermediate and cyst cells. In general, cyst cells possess thick cell wall, so the thick cell wall might prevent the nanopipette from penetrating the cells. Therefore, flagellated and palmelloid cells were used in the following experiments. Subsequently, the optimum injection voltage and injection time for Haematococcus cells were determined. The injection efficiency of this method reached 44%. Based on the above conditions, the microinjection using nanopipette was successfully carried out in other green microalgae, Tetraselmis sp. and C. reinhardtii. These results suggest that microinjection using the nanopipette can be widely utilized in green microalgae. The proposed method will be applied to efficient genome editing and subsequent metabolic engineering of various microalgae.

Optimization of Culture Conditions for High Cell Productivity and Astaxanthin Accumulation in Vietnam's Green Microalgae Haematococcus pluvialis HB and a Neuroprotective Activity of Its Astaxanthin.

Haematococcus pluvialis, a green microalga, is a rich source of natural astaxanthin and a potent antioxidant with high commercial value. This study investigates the biological characteristics and potential of H. pluvialis HB isolated from Hoa Binh, Vietnam, for growth and astaxanthin accumulation using a two-phase culture method. H. pluvialis HB was cultured in a C/RM medium at 25 °C, and morphological characteristics were examined. NMR spectroscopy was used to determine the structure of the astaxanthin, which was extracted using the Soxhlet method. After 22 days, the highest cell density (4.96 × 106 cells mL-1) was achieved under optimized light and ultraviolet conditions. Nutrient deprivation followed by bicarbonate supplementation resulted in a maximal astaxanthin accumulation of 48.8 mg g-1 dry cell weight within two days. The extracted astaxanthin demonstrated potent antioxidant activity (IC50: 3.74 mg mL-1) compared to ascorbic acid (IC50: 18.53 µg mL-1) and exhibited strong acetylcholinesterase inhibition (IC50: 297.99 µg mL-1). It also showed neuroprotective effects against H2O2 and amyloid beta-induced neurotoxicity in C6 cells. This study highlights H. pluvialis HB as a promising source for large-scale astaxanthin production with potential applications in neuroprotective health products.

Exploration of the Biotechnological Potential of Two Newly Isolated Haematococcus Strains from Reunion Island for the Production of Natural Astaxanthin.

Haematococcus lacustris is a freshwater green microalgae species able to produce and accumulate astaxanthin in response to environmental stresses such as high light and nutrient deprivation. Astaxanthin is a xanthophyll carotenoid of growing economic interest due to its numerous biological activities, notably its strong antioxidant properties, which can be valued in the fields of nutrition, health, feed and aquaculture. The present study aims at evaluating the capacity of two newly isolated Haematococcus strains from the biodiversity of Reunion Island, to be cultivated in a photobioreactor and to produce astaxanthin. The results showed that both strains were able to grow in various nutritive media and to produce and accumulate astaxanthin in response to stresses, mainly in the form of astaxanthin monoesters, which represented up to 2% of the dry biomass weight and which were mostly composed of linoleic and linolenic acids. In fed-batch cultures using 3 L benchtop photobioreactors, the concentrations of biomass enriched in astaxanthin reached up to 3 g L-1 (dry weight) with biomass productivities of 0.04 and 0.02 g L-1 d-1 based on the durations of the vegetative stage and of the entire culture, respectively. In these cultures, the astaxanthin productivities were found to reach on average around 0.25 mg L-1 d-1. Although these results were relatively low compared to the literature, the possibility of improving growth conditions in order to improve biomass and astaxanthin yields, to guarantee economic viability for cultivation at a commercial scale, was further discussed.

Enhancement of non-oleaginous green microalgae Ulothrix for bio-fixing CO2 and producing biofuels by ARTP mutagenesis

Oleaginous green microalgae are often mentioned in algae-based biodiesel industry, but most of them belong to specific genus (Chlorella, Scenedesmus, Botryococcus and Desmodesmus). Thus, the microalgal germplasm resources for biodiesel production are limited. Mutagenesis is regarded as an important technology for expanding germplasm resources. The main purpose of this study is to screen microalgae strains with high carbon dioxide tolerance and high lipid content from mutants derived from indigenous non-oleaginous green microalgae species—Ulothrix SDJZ-17. Two mutants with high CO2 tolerance and high lipid content genetic stability were obtained from the mutants by high-throughput screening, named Ulothrix SDJZ-17-A20 and Ulothrix SDJZ-17-A23. In order to evaluate the potential of CO2 fixation and biofuel production, A20 and A23 were cultured under air and 15% CO2 (v/v) conditions, and their wild-type strains (WT) were used as controls. Under the condition of high CO2 concentration, the growth performance and lipid production capacity of mutant strains A20 and A23 were not only significantly better than those of wild strains, but also better than those of their own cultured under air conditions. Among them, A23 obtained the highest LCE (light conversion efficiency) (14.79%), Fv/Fm (maximal photochemical efficiency of photosystem II) (71.04%) and biomass productivity (81.26 mg L−1 d−1), while A20 obtained the highest lipid content (22.45%). Both mutants can be used as candidate strains for CO2 fixation and biofuel production. By ARTP (atmospheric and room temperature plasma) mutagenesis with high-throughput screening, the mutants with higher CO2 tolerance, photosynthetic efficiency and lipid productivity can be obtained, even if they are derived from non-oleaginous microalgae, which is of great significance for enriching the energy microalgae germplasm bank, alleviating the global warming and energy crisis.

Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii, a model organism for unicellular green microalgae, is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including (i) unsatisfactory genome editing (GE) efficiency and (ii) uncontrolled DNA random insertion of antibiotic resistance markers. Thus, obtaining efficient gene targeting without using marker genes is instrumental in developing a pipeline for efficient engineering of strains for biotechnological applications. We developed an efficient DNA-free gene disruption strategy, relying on phenotypical identification of mutants, to (i) precisely determine its efficiency compared to marker-relying approaches and (ii) establish a new DNA-free editing tool. This study found that classical CRISPR Cas9-based GE for gene disruption in Chlamydomonas reinhardtii is mainly limited by DNA integration. With respect to previous results achieved on synchronized cell populations, we succeeded in increasing the GE efficiency of single gene targeting by about 200 times and up to 270 times by applying phosphate starvation. Moreover, we determined the efficiency of multiplex simultaneous gene disruption by using an additional gene target whose knock-out did not lead to a visible phenotype, achieving a co-targeting efficiency of 22%. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families. Furthermore, the approach provides new perspectives on how GE can be applied to (non-) model microalgae species, targeting groups of candidate genes of high interest for basic research and biotechnological applications.