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- Microalgae protein: a comparison between spray-dryed and frozen paste cellsPublication . Moreira, Catarina; Nunes, Rafaela; Kholany, Mariam; Pereira, Hugo; Teixeira, José A.; Ferreira-Santos, Pedro; Rocha, Cristina M.R.The need for sustainable protein substitutes is being driven by the growing global population. Because of their high protein content and environmental sustainability, microalgae are a viable source. In this study, proteins extracted from frozen paste and spray-dried biomass from Nannochloropsis oceanica and Tetraselmis chui were studied. High-pressure homogenization, followed by ultrafiltration (non-purified, NPS) and ammonium sulfate precipitation (purified, PS), was used to process the protein extracts. PS extracts showed higher protein concentrations, reaching approximately three-fold higher levels than NPS in spray-dried N. oceanica and about 2.7-fold higher in frozen paste samples, while frozen paste T. chui exhibited a 2.6-fold increase. Spectroscopic and chromatographic analyses revealed that frozen paste extracts preserved a greater proportion of native protein structures and displayed higher hydrophobic site exposure, whereas spraydrying and purification reduced α-helix content and promoted protein aggregation. Despite their lower protein concentration, frozen paste NPS extracts exhibited more favorable structural characteristics that may support improved techno-functional performance. These results highlight the importance of selecting processing conditions based on intended applications. Future food formulations could benefit greatly from the use of microalgal proteins, especially those derived from frozen biomass, as adaptable and sustainable ingredients.
- Fractioning macrocomponents of nannochloropsis oceanica by high-pressure homogenization, membrane processing, and ethanolic extractionPublication . Cunha, Pedro; Carvalho, Bernardo; Kholany, Mariam; Cardoso, Helena; Pereira, Hugo; Varela, JoãoMulti-product biorefineries, which transform biomass feedstocks into multiple valuable bio-based products, are pivotal for transitioning from a fossil-based economy to a sustainable circular bioeconomy. This work proposes a processing pipeline for fractionating the macrocomponents of Nannochloropsis oceanica, which can serve as a basis for multi-product microalgae biorefineries. It consists of high-pressure homogenization (1200 bar, 1 cycle) to permeabilize the cells, and sequential membrane processing (0.2 µm dia-microfiltration followed by 100 kDa ultrafiltration) and ethanolic extraction (60 mL ethanol/g dry weight, 1 h) to fractionate the disrupted biomass. This biorefinery resulted in four final fractions: (1) enriched in water-soluble proteins (39.0 ± 2.8% w/w proteins; 10.7 ± 0.8% w/w carbohydrates); (2) remaining soluble components (5.7 ± 0.4% w/w proteins; 4.3 ± 0.9% w/w carbohydrates); (3) lipid-rich extract (62.4 ± 5.8% w/w lipids); and (4) non-extracted components (11.8 ± 4.5% w/w lipids), with mass recovery yields of 23.2 ± 2.1%, 6.9 ± 1.0%, 10.6 ± 1.9%, and 60.4 ± 4.1%, respectively. The ultrafiltration protein selectivity was not optimal, despite yielding a 2.6 times more concentrated fraction. Lipid extraction yield (35–60%) and purity (56–68%) were highly affected by the water content of the microfiltration retentate. Overall, 10.0 ± 0.9% of the proteins, 9.7 ± 1.8% of the carbohydrates, and 42.4 ± 13.4% of the lipids of N. oceanica were recovered in fractions 1, 2, and 3, respectively.
- Assessment of cell disruption methods in an integrated multi-product biorefinery for Nannochloropsis Oceanica: from process design to economic analysis.Publication . Cunha, Pedro; Carvalho, Bernardo; Kholany, Mariam; Pereira, Hugo; Varela, JoãoMicroalgae are bioresources with significant potential within a sustainable, circular, bio-economy. However, high production costs have limited the widespread use of algae biomass. This study aimed to develop a multi-product biorefinery for Nannochloropsis oceanica that generates multiple revenue streams from the biomass, thereby enhancing the economic viability of algal production. The effectiveness of cell wall disruption using high-pressure homogenization and enzymatic hydrolysis was evaluated. Enzymatic hydrolysis solubilized nearly half (48.2 ± 1.5%) of the dry cell weight, compared to only 27.3 ± 3.2% with high-pressure homogenization, resulting in more concentrated water-soluble fractions and significantly higher protein extraction yields. Lipid extracts obtained after enzymatic hydrolysis had higher lipid (72.0 ± 5.3% w/w) and eicosapentaenoic acid (28.1 ± 6.9% w/w) contents than those from high-pressure homogenization (38.8 ± 6.1% w/w lipids; 9.1 ± 0.6% w/w eicosapentaenoic acid), despite similar lipid extraction yields (around 30%). Increasing the ethanol volumetric ratio from 58% to 75% v/v significantly improved lipid extraction yields (57.4 ± 3.1%) in the enzymatic hydrolysis-based biorefinery, with even higher yields observed upon scaling up (70.1%). All fractions, including lipid extracts, exhibited a balanced essential amino acid profile that exceeded the WHO/FAO/UNU-recommended values. A preliminary economic analysis indicated that lipid production was more cost-effective when cells were permeabilized by enzymatic hydrolysis than by high-pressure homogenization.