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- Influence of different processing techniques on microalgal protein extractionPublication . Moreira, Catarina; Ferreira-Santos, Pedro; Nunes, Rafaela; Carvalho, Bernardo; Pereira, Hugo; Teixeira, José A.; Rocha, Cristina M.R.The nutrient-rich composition of microalgae biomass positions it as a highly promising natural food ingredient. This holds the potential to not only enhance the nutritional value of various food products but also simultaneously alter their structural attributes.This work investigated the effect of five protein extraction techniques such as freeze-thawing, enzymatic-assisted extraction, high-pressure homogenization, ultrasounds-assisted extraction, and pH adjustment (pH 7, pH 10, and pH 13) in protein yield, and subsequente cell and protein structure of three microalgal suspensions, namely, Chlorella vulgaris, Nannochloropsis oceanica, and Tetraselmis chui. In Chlorella vulgaris, freeze- thawing and high-pressure homogenization exhibited a higher effect in terms of protein yield (similar to 26.60 g (protein) /100 g (protein microalgae)). The same occurred for Nannochloropsis oceanica with also ultrasounds-assisted extractions and pH 7 and 10 having a protein yield above 30 %. Tetraselmis chui was similar to Chlorella vulgaris (>20.00 g (protein) /100 g (protein microalgae)) for freeze-thawing, high-pressure homogenization and ultrasound-assisted extraction. Enzymatic-assisted had a the lower protein yield for all the three microalgae (<10.00 g (protein) /100 g (protein microalgae)). The majority of proteins extracted from Chlorella vulgaris samples had molecular weights exceeding 337 kDa, whereas proteins extracted from Nannochloropsis and Tetraselmis had molecular weights ranging from 5 to 50 kDa. alpha-helices occurred in proteins extracted from Chlorella vulgaris through freeze-thawing and enzymatic-assisted extraction, while Nannochloropsis and Tetraselmis only had beta-sheet. In conclusion, for optimal protein yield recovery, methodologies such as freeze-thawing and high-pressure homogenization are the most efficient across all studied microalgae. The method selected for extraction had a greater impact on both the protein yield and structure for spray-dried cells.
- 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.