Extracts from Chlorella vulgaris Protect Mesenchymal Stromal Cells from Oxidative Stress Induced by Hydrogen Peroxide.

Microalgae as unicellular eukaryotic organisms demonstrate several advantages for biotechnological and biological applications. Natural derived microalgae products demand has increased in food, cosmetic and nutraceutical applications lately. The natural antioxidants have been used for attenuation of mitochondrial cell damage caused by oxidative stress. This study evaluates the in vitro protective effect of Chlorella vulgaris bioactive extracts against oxidative stress in human mesenchymal stromal/stem cells (MSCs). The classical solid-liquid and the supercritical extraction, using biomass of commercially available and laboratory cultivated C. vulgaris, are employed. Oxidative stress induced by 300 µM H2O2 reduces cell viability of MSCs. The addition of C. vulgaris extracts, with increased protein content compared to carbohydrates, to H2O2 treated MSCs counteracted the oxidative stress, reducing reactive oxygen species levels without affecting MSC proliferation. The supercritical extraction was the most efficient extraction method for carotenoids resulting in enhanced antioxidant activity. Pre-treatment of MSCs with C. vulgaris extracts mitigates the oxidative damage ensued by H2O2. Initial proteomic analysis of secretome from licensed (TNFα-activated) MSCs treated with algal extracts reveals a signature of differentially regulated proteins that fall into clinically relevant pathways such as inflammatory signaling. The enhanced antioxidative and possibly anti-inflammatory capacity could be explored in the context of future cell therapies.

Extraction of Bioactive Compounds from C. vulgaris Biomass Using Deep Eutectic Solvents.

C. vulgaris microalgae biomass was employed for the extraction of valuable bioactive compounds with deep eutectic-based solvents (DESs). Particularly, the Choline Chloride (ChCl) based DESs, ChCl:1,2 butanediol (1:4), ChCl:ethylene glycol (1:2), and ChCl:glycerol (1:2) mixed with water at 70/30 w/w ratio were used for that purpose. The extracts' total carotenoid (TCC) and phenolic contents (TPC), as well as their antioxidant activity (IC50), were determined within the process of identification of the most efficient solvent. This screening procedure revealed ChCl:1,2 butanediol (1:4)/H2O 70/30 w/w as the most compelling solvent; thus, it was employed thereafter for the extraction process optimization. Three extraction parameters, i.e., solvent-to-biomass ratio, temperature, and time were studied regarding their impact on the extract's TCC, TPC, and IC50. For the experimental design and process optimization, the statistical tool Response Surface Methodology was used. The resulting models' predictive capacity was confirmed experimentally by carrying out two additional extractions under conditions different from the experimental design.

Green Synthesis of Silver Nanoparticles Using the Cell-Free Supernatant of Haematococcus pluvialis Culture.

The green synthesis of silver nanoparticles (AgNPs) using the cell-free supernatant of a Haematococcus pluvialis culture (CFS) was implemented in the current study, under illumination conditions. The reduction of Ag+ to AgNPs by the CFS could be described by a pseudo-first-order kinetic equation at the temperature range tested. A high reaction rate during synthesis and stable AgNPs were obtained at 45 °C, while an alkaline pH (pH = 11.0) and a AgNO3 aqueous solution to CFS ratio of 90:10 (v/v) proved to be the most effective conditions in AgNPs synthesis. A metal precursor (AgNO3) at the concentration range tested (1-5 mM) was the limited reactant in the synthesis process. The synthesis of AgNPs was accomplished under static and agitated conditions. Continuous stirring enhanced the rate of reaction but induced aggregation at prolonged incubation times. Zeta potential and polydispersity index measurements indicated stable AgNPs and the majority of AgNPs formation occurred in the monodisperse phase. The X-ray diffraction (XRD) pattern revealed the face-centered cubic structure of the formed AgNPs, while TEM analysis revealed that the AgNPs were of a quasi-spherical shape with a size from 30 to 50 nm. The long-term stability of the AgNPs could be achieved in darkness and at 4 °C. In addition, the synthesized nanoparticles showed antibacterial activity against Escherichia coli.

Optimization of Microalga Chlorella vulgaris Magnetic Harvesting.

Harvesting of microalgae is a crucial step in microalgae-based mass production of different high value-added products. In the present work, magnetic harvesting of Chlorella vulgaris was investigated using microwave-synthesized naked magnetite (Fe3O4) particles with an average crystallite diameter of 20 nm. Optimization of the most important parameters of the magnetic harvesting process, namely pH, mass ratio (mr) of magnetite particles to biomass (g/g), and agitation speed (rpm) of the C. vulgaris biomass-Fe3O4 particles mixture, was performed using the response surface methodology (RSM) statistical tool. Harvesting efficiencies higher than 99% were obtained for pH 3.0 and mixing speed greater or equal to 350 rpm. Recovery of magnetic particles via detachment was shown to be feasible and the recovery particles could be reused at least five times with high harvesting efficiency. Consequently, the described harvesting approach of C. vulgaris cells leads to an efficient, simple, and quick process, that does not impair the quality of the harvested biomass.

Magnetic Immobilization and Growth of Nannochloropsis oceanica and Scenedasmus almeriensis.

Microalgae are used in industrial and pharmaceutical applications. Their performance on biological applications may be improved by their immobilization. This study presents a way of cell immobilization using microalgae carrying magnetic properties. Nannochloropsis oceanica and Scenedasmus almeriensis cells were treated enzymatically (cellulase) and mechanically (glass beads), generating protoplasts as a means of incorporation of magnetic nanoparticles. Scanning electron microscopy images verified the successful cell wall destruction for both of the examined microalgae cells. Subsequently, protoplasts were transformed with magnetic nanoparticles by a continuous electroporation method and then cultured on a magnetic surface. Regeneration of transformed protoplasts was optimized using various organic carbon and amino acid supplements. Both protoplast preparation methods demonstrated similar efficiency. Casamino acids, as source of amino acids, were the most efficient compound for N. oceanica protoplasts regeneration in enzymatic and mechanical treatment, while for S. almeriensis protoplasts regeneration, fructose, as source of organic carbon, was the most effective. Protoplasts transformation efficiency values with magnetic nanoparticles after enzymatic or mechanical treatments for N. oceanica and S. almeriensis were 17.8% and 10.7%, and 18.6% and 15.7%, respectively. Finally, selected magnetic cells were immobilized and grown on a vertical magnetic surface exposed to light and without any supplement.

Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications.

Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different "recovery media" with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties.

A Study on the Effect of Macro- and Micro- Nutrients on Nannochloropsis oceanica Growth, Fatty Acid Composition and Magnetic Harvesting Efficiency.

The effect of iron, manganese, phosphorus and nitrogen on growth and lipid synthesis of the microalgae Nannochloropsis oceanica CCMP1779, as well as their impact on the magnetic harvesting efficiency, are examined under their depriving cell culture conditions. Herein, it is demonstrated that nitrogen and manganese depletion primarily reduced cell growth while phosphorus and iron restriction led to higher dry biomass. Subsequently, the role of those nutrients on fatty acids profile was examined. Phosphorus and nitrogen restriction resulted in lower and higher lipid content, respectively. High amounts of polyunsaturated fatty acids like eicosapentaenoic acid are produced under iron and manganese depletion. Phosphorus deprivation favors monounsaturated fatty acids such as C18:1 and C16:1, while nitrogen restriction favors saturated fatty acid production like C14:0, C16:0 and C18:0. Since the presence/absence of macro- and micro-elements may affect the overall electrostatic charges on the outmost microalgae surface, it was also analyzed how these elements affect the magnetic harvesting efficiency. Results showed that phosphorus deprivation led to the best magnetic harvesting efficiency of N. oceanica cells (93%) as compared to other nutrient starvation as well as standard medium.

A Study on Magnetic Removal of Hexavalent Chromium from Aqueous Solutions Using Magnetite/Zeolite-X Composite Particles as Adsorbing Material.

Toxic and heavy metals are considered harmful derivatives of industrial activities; they are not biodegradable and their accumulation in living organisms can become lethal. Among other heavy and toxic metals, chromium is considered hazardous, especially in the hexavalent (Cr6+) form. Numerous established studies show that exposure to Cr6+ via drinking water leads to elevated chromium levels in tissues, which may result in various forms of cancer. The purpose of this research is to synthesize magnetite/zeolite-X composite particles for the adsorption and magnetic removal of Cr6+ ions from aqueous solutions. Synthesis and characterization of such composite nanomaterials, along with an initial experimental evaluation of Cr6+ removal from water-based solution, are presented. Results show that zeolite-X is a very promising zeolite form, that when bound to magnetic nanoparticles can be used to trap and magnetically remove toxic ions from aqueous solutions.

Studies on the catalytic behavior of a membrane-bound lipolytic enzyme from the microalgae Nannochloropsis oceanica CCMP1779.

The catalytic behavior of a membrane-bound lipolytic enzyme (MBL-Enzyme) from the microalgae Nannochloropsis oceanica CCMP1779 was investigated. The biocatalyst showed maximum activity at 50 °C and pH 7.0, and was stable at pH 7.0 and temperatures from 40 to 60 °C. Half-lives at 60 °C, 70 °C and 80 °C were found 866.38, 150.67 and 85.57 min respectively. Thermal deactivation energy was 68.87 kJ mol-1. The enzyme's enthalpy (ΔΗ*), entropy (ΔS*) and Gibb's free energy (ΔG*) were in the range of 65.86-66.27 kJ mol-1, 132.38-140.64 J mol-1 K-1 and 107.80-115.81 kJ mol-1, respectively. Among p-nitrophenyl esters of fatty acids tested, MBL-Enzyme exhibited the highest hydrolytic activity against p-nitrophenyl palmitate (pNPP). The Km and Vmax values were found 0.051 mM and of 0.054 mmole pNP mg protein-1 min-1, respectively with pNPP as substrate. The presence of Mn2+ increased lipolytic activity by 68.25%, while Fe3+ and Cu2+ ions had the strongest inhibitory effect. MBL-Enzyme was stable in the presence of water miscible (66% of the initial activity in ethanol) and water immiscible (71% of the initial activity in n-octane) solvents. Myristic acid was found to be the most efficient acyl donor in esterification reactions with ethanol. Methanol was the best acyl acceptor among the primary alcohols tested.