Selective and energy efficient extraction of functional proteins from microalgae for food applications.

The use of a single controlled bead milling step of the microalga Tetraselmis suecica resulted in a soluble fraction, rich in functional proteins. This was achieved by fine-tuning the processing time, thereby exploiting the difference in rates of protein and carbohydrate release during milling. Soluble proteins were extracted under mild conditions -room temperature, no addition of chemicals, pH 6.5-, with a yield of 22.5% and a specific energy consumption of 0.6 kWh kgDW-1, which is within the recommended minimum energy for an extraction step in a biorefinery process. The resulting protein extract contained 50.4% (DW) of proteins and 26.4% carbohydrates, showed light green color and displayed superior surface activity and gelation behavior compared to whey protein isolate. The proposed process is simple (only one bead milling step), scalable, and allows the mild extraction of functional proteins, making it interesting for industrial applications in the food industry.

Techno-Functional Properties of Crude Extracts from the Green Microalga Tetraselmis suecica.

A mild fractionation process to extract functional biomolecules from green microalgae was implemented. The process includes bead milling, centrifugation, and filtration with several membrane cut-offs. For each fraction, the corresponding composition was measured, and the surface activity and gelation behavior were determined. A maximum protein yield of 12% was obtained in the supernatant after bead milling and between 3.2 and 11.7% after filtration. Compared to whey protein isolate, most of the algae fractions exhibited comparable or enhanced functionality. Surface activity for air-water and oil-water interfaces and gelation activities were notably superior for the retentate fractions compared to the permeates. It is proposed that such functionality in the retentates is due to the presence of hydrophobic compounds and molecular complexes exhibiting a similar behavior as Pickering particles. We demonstrated that excellent functionality can be obtained with crude fractions, requiring minimum processing and, thus, constituting an interesting option for commercial applications.

Biorefinery of the macroalgae Ulva lactuca: extraction of proteins and carbohydrates by mild disintegration.

The effect of osmotic shock, enzymatic incubation, pulsed electric field, and high shear homogenization on the release of water-soluble proteins and carbohydrates from the green alga Ulva lactuca was investigated in this screening study. For osmotic shock, both temperature and incubation time had a significant influence on the release with an optimum at 30 °C for 24 h of incubation. For enzymatic incubation, pectinase demonstrated being the most promising enzyme for both protein and carbohydrate release. Pulsed electric field treatment was most optimal at an electric field strength of 7.5 kV cm-1 with 0.05 ms pulses and a specific energy input relative to the released protein as low as 6.6 kWh kgprot-1. Regarding literature, this study reported the highest protein (~ 39%) and carbohydrate (~ 51%) yields of the four technologies using high shear homogenization. Additionally, an energy reduction up to 86% was achieved by applying a novel two-phase (macrostructure size reduction and cell disintegration) technique.

Multi-Product Microalgae Biorefineries: From Concept Towards Reality.

Although microalgae are a promising biobased feedstock, industrial scale production is still far off. To enhance the economic viability of large-scale microalgae processes, all biomass components need to be valorized, requiring a multi-product biorefinery. However, this concept is still too expensive. Typically, downstream processing of industrial biotechnological bulk products accounts for 20-40% of the total production costs, while for a microalgae multi-product biorefinery the costs are substantially higher (50-60%). These costs are high due to the lack of appropriate and mild technologies to access the different product fractions such as proteins, carbohydrates, and lipids. To reduce the costs, simplified processes need to be developed for the main unit operations including harvesting, cell disruption, extraction, and possibly fractionation.

Energy efficient bead milling of microalgae: Effect of bead size on disintegration and release of proteins and carbohydrates.

The disintegration of three industry relevant algae (Chlorella vulgaris, Neochloris oleoabundans and Tetraselmis suecica) was studied in a lab scale bead mill at different bead sizes (0.3-1mm). Cell disintegration, proteins and carbohydrates released into the water phase followed a first order kinetics. The process is selective towards proteins over carbohydrates during early stages of milling. In general, smaller beads led to higher kinetic rates, with a minimum specific energy consumption of ⩽0.47kWhkgDW-1 for 0.3mm beads. After analysis of the stress parameters (stress number and stress intensity), it appears that optimal disintegration and energy usage for all strains occurs in the 0.3-0.4mm range. During the course of bead milling, the native structure of the marker protein Rubisco was retained, confirming the mildness of the disruption process.

Understanding the salinity effect on cationic polymers in inducing flocculation of the microalga Neochloris oleoabundans.

A mechanistic study was performed to evaluate the effect of salinity on cationic polymeric flocculants, that are used for the harvesting of microalgae. The polyacrylamide Synthofloc 5080H and the polysaccharide Chitosan were employed for the flocculation of Neochloris oleoabundans. In seawater conditions, a maximum biomass recovery of 66% was obtained with a dosage of 90mg/L Chitosan. This recovery was approximately 25% lower compared to Synthofloc 5080H reaching recoveries greater than 90% with dosages of 30mg/L. Although different recoveries were obtained with both flocculants, the polymers exhibit a similar apparent polymer length, as was evaluated from viscosity measurements. While both flocculants exhibit similar polymer lengths in increasing salinity, the zeta potential differs. This indicates that polymeric charge dominates flocculation. With increased salinity, the effectivity of cationic polymeric flocculants decreases due to a reduction in cationic charge. This mechanism was confirmed through a SEM analysis and additional experiments using flocculants with various charge densities.

Selective extraction of intracellular components from the microalga Chlorella vulgaris by combined pulsed electric field-temperature treatment.

The synergistic effect of temperature (25-65 °C) and total specific energy input (0.55-1.11 kWh kgDW(-1)) by pulsed electric field (PEF) on the release of intracellular components from the microalgae Chlorella vulgaris was studied. The combination of PEF with temperatures from 25 to 55 °C resulted in a conductivity increase of 75% as a result of cell membrane permeabilization. In this range of temperatures, 25-39% carbohydrates and 3-5% proteins release occurred and only for carbohydrate release a synergistic effect was observed at 55 °C. Above 55 °C spontaneous cell lysis occurred without PEF. Combined PEF-temperature treatment does not sufficiently disintegrate the algal cells to release both carbohydrates and proteins at yields comparable to the benchmark bead milling (40-45% protein, 48-58% carbohydrates).

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 (0.49-1.37 g L(-1)) and flocculant dosages (0-150 mg L(-1)) the model simulations predicted well the optimal flocculant-to-biomass ratio between 43 and 109 mgflocculant/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.

Cell disruption for microalgae biorefineries.

Microalgae are a potential source for various valuable chemicals for commercial applications ranging from nutraceuticals to fuels. Objective in a biorefinery is to utilize biomass ingredients efficiently similarly to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. Downstream processes in microalgae biorefineries consist of different steps whereof cell disruption is the most crucial part. To maintain the functionality of algae biochemicals during cell disruption while obtaining high disruption yields is an important challenge. Despite this need, studies on mild disruption of microalgae cells are limited. This review article focuses on the evaluation of conventional and emerging cell disruption technologies, and a comparison thereof with respect to their potential for the future microalgae biorefineries. The discussed techniques are bead milling, high pressure homogenization, high speed homogenization, ultrasonication, microwave treatment, pulsed electric field treatment, non-mechanical cell disruption and some emerging technologies.

Mild disintegration of the green microalgae Chlorella vulgaris using bead milling.

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25-145 gDW kg(-1)) over a range of agitator speeds (6-12 m s(-1)). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9-10 m s(-1) regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5 kWh kgDW(-1).

Cationic polymers for successful flocculation of marine microalgae.

Flocculation of microalgae is a promising technique to reduce the costs and energy required for harvesting microalgae. Harvesting marine microalgae requires suitable flocculants to induce the flocculation under marine conditions. This study demonstrates that cationic polymeric flocculants can be used to harvest marine microalgae. Different organic flocculants were tested to flocculate Phaeodactylum tricornutum and Neochloris oleoabundans grown under marine conditions. Addition of 10 ppm of the commercial available flocculants Zetag 7557 and Synthofloc 5080H to P. tricornutum showed a recovery of, respectively, 98% ± 2.0 and 94% ± 2.9 after flocculation followed by 2h sedimentation. Using the same flocculants and dosage for harvesting N. oleoabundans resulted in a recovery of 52% ± 1.5 and 36% ± 11.3. This study shows that cationic polymeric flocculants are a viable option to pre-concentrate marine cultivated microalgae via flocculation prior to further dewatering.