Fucoxanthin from Algae to Human, an Extraordinary Bioresource: Insights and Advances in up and Downstream Processes.

Fucoxanthin is a brown-colored pigment from algae, with great potential as a bioactive molecule due to its numerous properties. This review aims to present current knowledge on this high added-value pigment. An accurate analysis of the biological function of fucoxanthin explains its wide photon absorption capacities in golden-brown algae. The specific chemical structure of this pigment also leads to many functional activities in human health. They are outlined in this work and are supported by the latest studies in the literature. The scientific and industrial interest in fucoxanthin is correlated with great improvements in the development of algae cultures and downstream processes. The best fucoxanthin producing algae and their associated culture parameters are described. The light intensity is a major influencing factor, as it has to enable both a high biomass growth and a high fucoxanthin content. This review also insists on the most eco-friendly and innovative extraction methods and their perspective within the next years. The use of bio-based solvents, aqueous two-phase systems and the centrifugal partition chromatography are the most promising processes. The analysis of the global market and multiple applications of fucoxanthin revealed that Asian companies are major actors in the market with macroalgae. In addition, fucoxanthin from microalgae are currently produced in Israel and France, and are mostly authorized in the USA.

A review of paramylon processing routes from microalga biomass to non-derivatized and chemically modified products.

Paramylon is a linear ß-1,3-glucan, similar to curdlan, produced as intracellular granules by the microalga Euglena gracilis, a highly versatile and robust strain, able to grow under various trophic conditions, with valorization of CO2, wastewaters, or food byproducts as nutrients. This review focuses in particular on the various processing routes leading to new potential paramylon based products. Due to its crystalline structure, involving triple helices stabilized by internal intermolecular hydrogen bonds, paramylon is neither water-soluble nor thermoplastic. The few solvents able to disrupt the triple helices, and to fully solubilize the polymer as random coils, allow non derivatizing shaping into films, fibers, and even nanofibers by a specific self-assembly mechanism. Chemical modification in homogeneous or heterogeneous conditions is also possible. The non-selective or regioselective substitution of the hydroxyl groups of glucosidic units leads to water-soluble ionic derivatives and thermoplastic paramylon esters with foreseen applications ranging from health to bioplastics.

Optimization of continuous TAG production by Nannochloropsis gaditana in solar-nitrogen-limited culture.

Nitrogen limitation and changing solar conditions are both known to affect triacylglycerol (TAG) production in microalgae. This study investigates the optimization of TAG production with a continuous nitrogen-limited culture of Nannochloropsis gaditana in simulated day-night cycles (DNc). The effect of DNc was first investigated in nitrogen-deprived condition (i.e., batch culture), emphasizing a significant change in mechanical resistance of the strain during the night. The concept of released TAG, which shows how much of the TAG produced is actually recovered in the downstream stages, that is, after cell disruption, was shown here of interest. For a maximum released TAG, the optimum harvesting time was suggested as being 4 h into the night period, which minimizes the losses due to a too great cell mechanical resistance. The protocol for continuous nitrogen-limited culture was then optimized, and a continuous nitrogen addition was compared to a pulsed-addition. For the latter, nitrogen was supplied in a single pulse at the beginning of the light periods, while the bulk medium was supplied separately at a slow but constant dilution rate of 0.005h-1$0.005\,{{\rm{h}}}^{-1}$ . The pulse dose was calculated after the study of nitrogen consumption and TAG production/consumption during the DNc. The estimated released TAG for the pulsed-addition of 1.4 × 10-3 kg/m2 d found significantly higher than the one achieved in batch culture (0.3 × 10-3 kg/m2 d) but lower than for continuous nitrogen addition which obtained the highest released TAG of 3×10-3kg∕m2 d$3\times 1{0}^{-3}\,\text{kg}\unicode{x02215}{{\rm{m}}}^{2}\unicode{x0200A}{\rm{d}}$ .

The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis.

We observed differences in lhc classification in Chromista. We proposed a classification of the lhcf family with two groups specific to haptophytes, one specific to diatoms, and one specific to seaweeds. Identification and characterization of the Fucoxanthin and Chlorophyll a/c-binding Protein (FCP) of the haptophyte microalgae Tisochrysis lutea were performed by similarity analysis. The FCP family contains 52 lhc genes in T. lutea. FCP pigment binding site candidates were characterized on Lhcf protein monomers of T. lutea, which possesses at least nine chlorophylls and five fucoxanthin molecules, on average, per monomer. The expression of T. lutea lhc genes was assessed during turbidostat and chemostat experiments, one with constant light (CL) and changing nitrogen phases, the second with a 12 h:12 h sinusoidal photoperiod and changing nitrogen phases. RNA-seq analysis revealed a dynamic decrease in the expression of lhc genes with nitrogen depletion. We observed that T. lutea lhcx2 was only expressed at night, suggesting that its role is to protect \cells from return of light after prolonged darkness exposure.

Selection of Culture Conditions and Cell Morphology for Biocompatible Extraction of ß-Carotene from Dunaliella salina.

Biocompatible extraction emerges recently as a means to reduce costs of biotechnology processing of microalgae. In this frame, this study aimed at determining how specific culture conditions and the associated cell morphology impact the biocompatibility and the extraction yield of ß-carotene from the green microalga Dunaliella salina using n-decane. The results highlight the relationship between the cell disruption yield and cell volume, the circularity and the relative abundance of naturally permeabilized cells. The disruption rate increased with both the cell volume and circularity. This was particularly obvious for volume and circularity exceeding 1500 µm3 and 0.7, respectively. The extraction of ß-carotene was the most biocompatible with small (600 µm3) and circular cells (0.7) stressed in photobioreactor (30% of carotenoids recovery with 15% cell disruption). The naturally permeabilized cells were disrupted first; the remaining cells seems to follow a gradual permeabilization process: reversibility (up to 20 s) then irreversibility and cell disruption. This opens new carotenoid production schemes based on growing robust ß-carotene enriched cells to ensure biocompatible extraction.

Two-step procedure for selective recovery of bio-molecules from microalga Nannochloropsis oculata assisted by high voltage electrical discharges.

Two-step procedure with the initial aqueous extraction from raw microalgae Nannochloropsis oculata and secondary organic solvent extraction from vacuum dried (VD) microalgae were applied for selective recovery of bio-molecules. The effects of preliminary aqueous washing and high voltage electrical discharges (HVED, 40 kV/cm, 4 ms pulses) were tested. The positive effects of HVED treatment and washing on selectivity of aqueous extraction of ionics and other water-soluble compounds (carbohydrates, proteins and pigments) were observed. Moreover, the HVED treatment allowed improving the kinetic of vacuum drying, and significant effects of HVED treatment on organic solvent extraction of chlorophylls, carotenoids and lipids were determined. The proposed two-step procedure combining the preliminary washing, HVED treatment and aqueous/organic solvents extraction steps are useful for selective extraction of different bio-molecules from microalgae biomass.

Application of high-voltage electrical discharges and high-pressure homogenization for recovery of intracellular compounds from microalgae Parachlorella kessleri.

Treatments with high-voltage electrical discharges (HVED) and high-pressure homogenization (HPH) were studied and compared for the release of ionic components, carbohydrates, proteins, and pigments from microalgae Parachlorella kessleri (P. kessleri). Suspensions (1% w/w) of microalgae were treated by HVED (40 kV/cm, 1-8 ms) or by HPH (400-1200 bar, 1-10 passes). Particle-size distribution (PSD) and microscopic analyses were used to detect the disruption and damage of cells. HVED were very effective for the extraction of ionic cell components and carbohydrates (421 mg/L after 8 ms of the treatment). However, HVED were ineffective for pigments and protein extraction. The concentration of proteins extracted by HVED was just 750 mg/L and did not exceed 15% of the total quantity of proteins. HPH permitted an effective release overall of intracellular compounds from P. kessleri microalgae including a large quantity of proteins, whose release (at 1200 bar) was 4.9 times higher than that obtained by HVED. Consequently, HVED can be used at the first step of the overall extraction process for the selective recovery of low-molecular-weight components. HPH can be then used at the second step for the recovery of remaining cell compounds.

Emerging techniques for cell disruption and extraction of valuable bio-molecules of microalgae Nannochloropsis sp.

Microalgae of Nannochloropsis sp. present valuable source of bio-molecules (pigments, lipids, proteins) that have nutritional potential for the prevention and treatment of human diseases. Moreover, some species of Nannochloropsis are the promising sources of biofuels and excellent candidates for the replacement of classical biofuel crops. This review describes and compares the efficiency of different conventional and novel techniques that can be used for cell disruption and recovery of bio-molecules from Nannochloropsis sp. Classification of different extraction techniques includes chemical, enzymatic, mechanical and other physical methods. The detailed analysis of extraction efficiency assisted by pressure and temperature (subcritical and supercritical fluids, hydrothermal liquefaction), ultrasound, microwaves, and pulsed electric energy (pulsed electric fields and high voltage electrical discharges) is presented. The general discussion includes comparison between techniques, their effectiveness for cell disruption and selectivity of bio-molecules extraction from Nannochloropsis sp. The cost-effectiveness, benefits and limitations of different techniques are also analyzed.

Bead milling disruption kinetics of microalgae: Process modeling, optimization and application to biomolecules recovery from Chlorella sorokiniana.

Industrial development of microalgae biomass valorization relies on process optimization and controlled scale-up. Both need robust modeling: (i) for biomass production and (ii) for integrated processes in the downstream processing (DSP). Cell disruption and primary fractionation are key steps in DSP. In this study, a kinetic model, including microalgal cell size distribution, was developed for Chlorella sorokiniana disruption in continuous bead milling. Glass beads of 0.4 mm size at impeller tip velocity of 14 m.s-1 were used as optimal conditions for efficient cell disruption. These conditions allowed faster disruption of big cells than small ones. A modified expression of the Stress Number, including cell size effect, was then proposed and validated. Separation of starch, proteins and chlorophyll by mild centrifugation was studied as function of the disruption parameters. Low energy consumption conditions led to extreme comminution. An intermediate zone drew attention for allowing moderate energy consumption and efficient metabolites separation by centrifugation.

Industrial case study on alkaloids purification by pH-zone refining centrifugal partition chromatography.

The industrial potential of pH-zone refining centrifugal partition chromatography has been evaluated by studying the purification of pharmaceutical ingredients at the pilot scale. For the first time, a scale up methodology based on both column capacity and mass transfer efficiency as invariants was developed. The purification of catharanthine and vindoline from an industrial crude extract of aerial parts of Catharanthus roseus, was used as a case of study. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system, triethylamine as retainer in the organic stationary phase and sulphuric acid as displacer in the aqueous mobile phase. The separation intensification was performed on a 36mL CPC column equipped with 832 partition twin-cells. The combined effects of four parameters (displacer and retainer concentrations for intensive parameters, flow rate and rotational speed for extensive parameters) were studied by design of experiment in order to maximize both recoveries and productivities. Then, scale change was done on two larger columns (305mL and 1950mL of capacity) equipped with only 231 and 238 partition cells. For this step, it has been shown that the global mass transfer coefficient k0a (the efficiency of a column design) and the stationary phase retention (the capacity of the column) were relevant and useful scale up invariants. A CPC model based on acid-base equilibriums and interfacial mass transfer in continuously stirred tank reactors in series was used to predict fully separations on larger CPC column at the optimized operating conditions and to guide the CPC user in its scale-up strategy. The experimental validation on pilot CPC column, by injecting up to 150g of Catharanthus roseus crude extract on the 1950mL column highlighted the preservation of the separation quality, the non-linear character of the scale up in centrifugal partition chromatography and that a productivity of about 4kg of processed crude extract per day can be reached by implementing developed methodology.

Modeling pH-zone refining countercurrent chromatography: a dynamic approach.

A model based on mass transfer resistances and acid-base equilibriums at the liquid-liquid interface was developed for the pH-zone refining mode when it is used in countercurrent chromatography (CCC). The binary separation of catharanthine and vindoline, two alkaloids used as starting material for the semi-synthesis of chemotherapy drugs, was chosen for the model validation. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system. First, hydrodynamics and mass transfer were studied by using chemical tracers. Trypan blue only present in the aqueous phase allowed the determination of the parameters τextra and Pe for hydrodynamic characterization whereas acetone, which partitioned between the two phases, allowed the determination of the transfer parameter k0a. It was shown that mass transfer was improved by increasing both flow rate and rotational speed, which is consistent with the observed mobile phase dispersion. Then, the different transfer parameters of the model (i.e. the local transfer coefficient for the different species involved in the process) were determined by fitting experimental concentration profiles. The model accurately predicted both equilibrium and dynamics factors (i.e. local mass transfer coefficients and acid-base equilibrium constant) variation with the CCC operating conditions (cell number, flow rate, rotational speed and thus stationary phase retention). The initial hypotheses (the acid-base reactions occurs instantaneously at the interface and the process is mainly governed by mass transfer) are thus validated. Finally, the model was used as a tool for catharanthine and vindoline separation prediction in the whole experimental domain that corresponded to a flow rate between 20 and 60 mL/min and rotational speeds from 900 and 2100 rotation per minutes.

Methodology for optimally sized centrifugal partition chromatography columns.

Centrifugal Partition Chromatography (CPC) is a separation process based on the partitioning of solutes between two partially miscible liquid phases. There is no solid support for the stationary phase. The centrifugal acceleration is responsible for both stationary phase retention and mobile phase dispersion. CPC is thus a process based on liquid-liquid mass transfer. The separation efficiency is mainly influenced by the hydrodynamics of the phases in each cell of the column. Thanks to a visualization system, called "Visual CPC", it was observed that the mobile phase can flow through the stationary phase as a sheet, or a spray. Hydrodynamics, which directly governs the instrument efficiency, is directly affected during scale changes, and non-linear phenomena prevent the successful achievement of mastered geometrical scale changes. In this work, a methodology for CPC column sizing is proposed, based on the characterization of the efficiency of advanced cell shapes, taking into account the hydrodynamics. Knowledge about relationship between stationary phase volume, cell efficiency and separation resolution in CPC allowed calculating the optimum cell number for laboratory and industrial scale CPC application. The methodology is highlighted with results on five different geometries from 25 to 5000 mL, for two applications: the separation of alkylbenzene by partitioning with heptane/methanol/water biphasic system; and the separation of peptides by partitioning with n-butanol/acetic acid/water (4/1/5) biphasic system. With this approach, it is possible to predict the optimal CPC column length leading to highest productivity.

Physicochemical factors affecting the stability of two pigments: R-phycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum.

Phycoerythrin is a major light-harvesting pigment of red algae, which could be used as a natural dye in foods. The stability of R-phycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum in relation to different light exposure times, pHs, and temperatures was studied. Regarding the light exposure time, after 48h, the reduction in concentrations of B-phycoerythrin and R-phycoerythrin were 30±2.4% and 70±1%, respectively. Phycoerythrins presented good stability from pH 4 to 10. At pH 2, the reduction in concentration was 90±4% for B-phycoerythrin and 40±2.5% for R-phycoerythrin while, at pH 12, the phycoerythrins were degraded. Phycoerythrins showed good stability toward temperature, up to 40°C. At 60°C, the reduction in concentrations of B-phycoerythrin and R-phycoerythrin were 50±3.4% and 70±0.18%, respectively. Moreover, the best conditions of storage (-20°C) were determined.

Purification of a modified cyclosporine A by co-current centrifugal partition chromatography: process development and intensification.

Synthetic hydrophobic non-ionizable peptides are not soluble in most common solvents and are thus difficult to purify by preparative reversed-phase HPLC, normally used for industrial production. The challenge exists to develop alternative purification chromatographic processes using suitable solvents and providing good yields, high purity and sufficient productivity. A 11mer hydrophobic synthetic modified cyclosporine, showing an anti-HIV activity, was successfully purified by centrifugal partition chromatography using the biphasic solvent system heptane/ethyl acetate/acetone/methanol/water (1:2:2:1:2, v/v). A 5% co-current elution - made possible by the liquid nature of the two phases - has been used in order to avoid hydrodynamic instabilities mainly due to the physico-chemical properties of the target peptide. This original solution was developed after the study of the effect of the peptide on the hydrodynamic behavior of the two phases during the separation, and the visualization of the flow patterns using the Visual-CPC device. Critical impurities were efficiently eliminated and the peptide was recovered in high yield and high productivity achieving the specifications requirements.

Centrifugal partition extraction of ß-carotene from Dunaliella salina for efficient and biocompatible recovery of metabolites.

A biocompatible extraction method for ß-carotene recovery from the microalga Dunaliella salina was studied. The centrifugal partition extraction was used for liquid-liquid mass transfer intensification during continuous extraction. Different solvents and process parameters were compared. Ethyl oleate extraction with 5% dichloromethane achieved a 65% ß-carotene recovery with the least amount of cell damage as more than 65% of the cells remained viable as demonstrated by photosynthesis activity measurements.

Separation and fractionation of exopolysaccharides from Porphyridium cruentum.

In this work the extraction of EPSs from culture media of Porphyridium cruentum, by dialysis, solvent-precipitation with 3 polar alcohols (methanol, ethanol and isopropanol) and membrane separation techniques has been studied. Diafiltration (DF) using a membrane with a 300 kDa molecular weight cut off was the most efficient technique compared to solvent-extraction and dialysis methods. After extraction, EPS fraction was characterized in terms of rheological properties and biochemical content. The product exhibited shear thinning behavior and a critical overlap concentration equal to 0.6 g/L. The monosaccharide composition was investigated after acidic hydrolysis. Xylose, galactose, glucose and glucuronic acid were identified as the main constitutive monomers.

Centrifugal partition extraction, a new method for direct metabolites recovery from culture broth: case study of torularhodin recovery from Rhodotorula rubra.

Centrifugal partition extraction (CPE), close to centrifugal partition chromatography, put in contact in a continuous way two immiscible liquid phases. This work presents early experiments on CPE use for solid-liquid-liquid extraction. It was applied to the direct treatment of culture broth for metabolites recovery. Torularhodin is one of the carotenoid pigments produced by the yeast Rhodotorula sp., with a terminal carboxylic group considered nowadays as a powerful antioxidant to be included in food and drugs formulations. Torularhodin was extracted from Rhodotorula rubra ICCF 209 cells by CPE. The recovery of torularhodin reaches 74 µg/g of biomass i.e. 294 µg/L of culture medium. The efficiency of the extraction step increased with the operating flow rate. The extraction yield could reach 91% with a contact time lower than 2 min. A 300 mL apparatus allowed a feed at 90 mL/min. The technique is proposed for extraction or sample preparation before analysis.

Strong ion exchange in centrifugal partition extraction (SIX-CPE): effect of partition cell design and dimensions on purification process efficiency.

The aim of this article was to evaluate the influence of the column design of a hydrostatic support-free liquid-liquid chromatography device on the process efficiency when the strong ion-exchange (SIX) development mode is used. The purification of p-hydroxybenzylglucosinolate (sinalbin) from a crude aqueous extract of white mustard seeds (Sinapis alba L.) was achieved on two types of devices: a centrifugal partition chromatograph (CPC) and a centrifugal partition extractor (CPE). They differ in the number, volume and geometry of their partition cells. The SIX-CPE process was evaluated in terms of productivity and sinalbin purification capability as compared to previously optimized SIX-CPC protocols that were carried out on columns of 200 mL and 5700 mL inner volume, respectively. The objective was to determine whether the decrease in partition cell number, the increase in their volume and the use of a "twin cell" design would induce a significant increase in productivity by applying higher mobile phase flow rate while maintaining a constant separation quality. 4.6g of sinalbin (92% recovery) were isolated from 25 g of a crude white mustard seed extract, in only 32 min and with a purity of 94.7%, thus corresponding to a productivity of 28 g per hour and per liter of column volume (g/h/LV(c)). Therefore, the SIX-CPE process demonstrates promising industrial technology transfer perspectives for the large-scale isolation of ionized natural products.

Intensified extraction of ionized natural products by ion pair centrifugal partition extraction.

The potential of centrifugal partition extraction (CPE) combined with the ion-pair (IP) extraction mode to simultaneously extract and purify natural ionized saponins from licorice is presented in this work. The design of the instrument, a new laboratory-scale Fast Centrifugal Partition Extractor (FCPE300(®)), has evolved from centrifugal partition chromatography (CPC) columns, but with less cells of larger volume. Some hydrodynamic characteristics of the FCPE300(®) were highlighted by investigating the retention of the stationary phase under different flow rate conditions and for different biphasic solvent systems. A method based on the ion-pair extraction mode was developed to extract glycyrrhizin (GL), a biologically active ionic saponin naturally present in licorice (Glycyrrhiza glabra L., Fabaceae) roots. The extraction of GL was performed at a flow rate of 20 mL/min in the descending mode by using the biphasic solvent system ethyl acetate/n-butanol/water in the proportions 3/2/5 (v/v/v). Trioctylmethylammonium with chloride as a counter-ion (Al336(®)) was used as the anion extractant in the organic stationary phase and iodide, with potassium as counter-ion, was used as the displacer in the aqueous mobile phase. From 20 g of a crude extract of licorice roots, 2.2g of GL were recovered after 70 min, for a total process duration of 90 min. The combination of the centrifugal partition extractor with the ion-pair extraction mode (IP-CPE) offers promising perspectives for industrial applications in the field of natural product isolation or for the fractionation of natural complex mixtures.