Microalgal Proteins Research Articles

Protein and lysine improvement harnessed by a signal chain of red light-emitting diode light in Chlorella pyrenoidosa

Microalgae are emerging as a novel single-cell protein source that can substitute traditional plant protein feeds. In this investigation, lysine and protein accumulation in Chlorella pyrenoidosa were significantly enhanced under red light-emitting diode light, addressing challenge of limiting amino acid in plant proteins. The study employed targeted metabolomics, HPLC, and qRT-PCR to validate the light-induced pathway triggering lysine biosynthesis. Specifically, the pathway involves Ca2+-CaM as an intermediary in signal transduction, which directly inhibits PEPC activity. This inhibition directs a significant carbon flux towards central carbon metabolism, resulting in increased pyruvate levels—a critical precursor for lysine biosynthesis via the diaminopimelate pathway. Ultimately, the content of protein and lysine under red light increased by 36.02 % and 99.56 %, respectively, compared to those under white light. These findings provide a novel orientation for the precise regulation of lysine accumulation in microalgae, and moreover lay a solid theoretical foundation for producing microalgal proteins.

Protein extracts from microalgae and cyanobacteria biomass. Techno-functional properties and bioactivity: A review

Microalgae and cyanobacteria are photosynthetic and unicellular organisms that contain considerable amounts of proteins, lipids, carbohydrates, and polyunsaturated fatty acids, among others, with applications in the cosmetic, pharmaceutical, and food industries. These microorganisms can accumulate protein up to 70 % of total biomass depending on the microalgal strain, hence they have been regarded as an alternative protein source for the future. Microalgal proteins have important applications such as emulsifying, foaming, and gelation properties, which are important for the determination of quality and texture of foods. Some microalgal peptides possess important bioactivity with many health-benefit effects. Therefore, to maximize the production of proteins from microalgae and cyanobacteria, many protein extraction procedures have been studied to increase the economic return. They have been tested towards higher protein yields at low energy cost, the preservation of protein native properties, and lower cell debris. This later is fundamental to facilitate the subsequent purification processes so that the overall cost can be reduced. The aim of this work is to review some cell disruption processes for the extraction of protein from microalgae and cyanobacteria, considering that this step is crucial for the overall process due to the high rigidness of microalgal cell covering, which can hamper the release of proteins. It also aims at reviewing the purification techniques after cellular disruption, from conventional to more recent approaches, and finally addresses the antioxidant, antidiabetic, antihypertensive, antibacterial and other bioactive properties of microalgal protein hydrolysates and peptides.

Optimization of microalgae protein extraction from Scenedesmus obliquus and investigating its functional properties

The objective of this work was to evaluate alkaline treatment with enzymatic approaches for the optimal extraction of proteins from Scenedesmus obliquus microalgae. The experiments were designed with an emphasis on the alkaline treatment period and the enzyme-to-substrate ratio utilizing response surface methodology (RSM) with central composite design (CCD). Higher enzyme concentrations led to better extraction rates and efficiency, according to the findings. Cellulase demonstrated extraction efficiencies between 16.88% and 21.42%, but the multi-enzyme complex had efficiencies ranging from 15.28% to 27.87%. With an extraction rate of 19.13%, alkaline treatment was less advantageous than enzymatic treatment due to its longer procedure. Microalgal proteins' functional characteristics were also looked at. These proteins demonstrated foaming capacity, oil absorption, and emulsification ability, making them suitable alternatives to animal proteins in food systems such as egg white and soybean. Although microalgal protein powder had lower foam capacity (20%), stability (25%), fat absorption capacity (124%), and emulsification ability compared to egg white powder, it still exhibited favorable functional properties, highlighting its potential for various food applications. Further enhancement of the functional properties can be achieved by optimizing the environmental conditions during extraction.

Microalgal Proteins and Bioactives for Food, Feed, and Other Applications

Microalgae are a known source of proteins, prebiotics, lipids, small molecules, anti-oxidants and bioactives with health benefits that can be harnessed for the development of functional foods, feeds, cosmeceuticals and pharmaceuticals. This review collates information on the supply, processing costs, target markets and value of microalgae, as well as microalgal proteins, lipids, vitamins and minerals. It discusses the potential impact that microalgae could have on global food and feed supply and highlights gaps that exist with regards to the use of microalgal proteins and ingredients as foods and supplements.

A new paradigm to search for allergenic proteins in novel foods by integrating proteomics analysis and in silico sequence homology prediction: Focus on spirulina and chlorella microalgae.

Since novel nutrient sources with high protein content, such as yeast, fungi, bacteria, algae, and insects, are increasingly introduced in the consumer market, safety evaluation studies on their potentially allergenic proteins are required. A pipeline for in silico establishing the sequence-based homology between proteins of spirulina (Arthrospira platensis) and chlorella (Chlorella vulgaris) micro-algae and those included in the AllergenOnline (AO) database (AllergenOnline.org) is described. The extracted proteins were first identified through tryptic peptides analysis by reversed-phase liquid chromatography and high resolution/accuracy Fourier-transform tandem mass spectrometry (RPLC-ESI-FTMS/MS), followed by a quest on the UniProt database. The AO database was subsequently interrogated to assess sequence similarity between identified microalgal proteins and known allergens, based on criteria established by the World Health Organization (WHO) and Food and Agriculture Organization (FAO). A direct search for microalgal proteins already included in allergen databases was also performed using the Allergome database. Six proteins exhibiting a significant homology with food allergens were identified in spirulina extracts. Five of them, i.e., two thioredoxins (D4ZSU6, K1VP15), a superoxide dismutase (C3V3P3), a glyceraldehyde-3-phosphate dehydrogenase (K1W168), and a triosephosphate isomerase (D5A635), resulted from the search on AO. The sixth protein, C-phycocyanin beta subunit (P72508), was directly obtained after examining the Allergome database. Two proteins exhibiting significant sequence homology with food allergens were retrieved in chlorella extracts, viz. calmodulin (A0A2P6TFR8), which is related to troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Specific serum screenings based on immunochemical tests should be undertaken to confirm or rule out the allergenicity of the identified proteins.

Green synthesis and characterization of Fe3O4 nanoparticles using Chlorella-K01 extract for potential enhancement of plant growth stimulating and antifungal activity

The purpose of this research was to determine the efficacy of iron oxide nanoparticles (Fe3O4-NPs) using microalgal products as a plant growth stimulant and antifungal agent. The work was conducted with the phyco-synthesis and characterization of Fe3O4-NPs using 0.1 M ferric/ferrous chloride solution (2:1 ratio; 65 °C) with aqueous extract of the green microalga Chlorella K01. Protein, carbohydrate and polyphenol contents of Chlorella K01 extract were measured. The synthesized microalgal Fe3O4-NPs made a significant contribution to the germination and vigor index of rice, maize, mustard, green grams, and watermelons. Fe3O4-NPs also exhibited antifungal activity against Fusarium oxysporum, Fusarium tricinctum, Fusarium maniliforme, Rhizoctonia solani, and Phythium sp. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) scanning electron microscopy (SEM), transmission electron microscopy (TEM), particle size analysers (PSA), and zeta potential (ZP) measurements were used to characterize these green fabricated magnetite NPs. FTIR analysis showed that the synergy of microalgal proteins, carbohydrtates and polyphenols is responsible for the biofabrication of iron nanoparticles. A spheroid dispersion of biosynthesized Fe3O4-NPs with an average diameter of 76.5 nm was produced in the synthetic process.

Identification and effect of ozone and ultrasound pretreatments on Desmodesmussp. and Tetradesmus obliquus proteins

Either ozone or ultrasound pretreatment was applied to identify and evaluate their effect over the protein fraction of Desmodesmussp. and Tetradesmus obliquus biomass cultivated in wastewater. Studies of SDS-PAGE, protein solubility, sulfhydryl and disulphide bond contents, and DSC were performed to evaluate the effect of each pretreatment on abundance, structure, and thermal properties of the protein concentrate obtained.Seven proteins were identified in Desmodesmussp. biomass, while nine proteins were identified in Tetradesmus obliquus. The proteins identified may give a high add-value to microalgae biomass because they can find important applications in agricultural, food, and biomedical areas. On the other hand, the effect of pretreatment on these microalgal proteins mainly depends on protein structure. Desmodesmussp. proteins showed a content of S-S groups an order of magnitude higher than Tetradesmus obliquus. This results in a higher glass transition temperature (Tg) for Desmodesmussp. proteins (249 °C) than for Tetradesmus obliquus proteins (188 °C). On the contrary, the Tetradesmus obliquus native structure proteins tend towards cross-linking and protein-protein aggregation, as their primary structure has a high content of Methionine, Tyrosine, Tryptophan, Histidine, and Phenylalanine. Either ozone or ultrasound pretreatment, induces a Tg reduction in both Tetradesmus obliquus and Desmodesmussp. proteins, however, this reduction is especially remarkable for Desmodesmussp. proteins (drop in Tg of about 80 °C). The results showed that proteins in Desmodesmussp. and Tetradesmus obliquus are highly resistant to pretreatment due to the nature of their biochemical structures which imply an excellent quality for protein valorisation, even after application of highly disruptive pretreatments.

Phytochemical and Biological Studies on Proteins Isolated From Different Microalgal Species

Microalgae are rich in high protein content that enhances their nutritional and therapeutic benefits in addition to their ability for high mass production. Therefore, the present study was designed to reveal the biological efficiency of the natural proteins isolated form Dunaliella salina, Scenedesmus obliquus and Spirulina platensis. During the current study, it was found that the highest protein content was noticed in S. platensis (48.44%) followed by D. salina (31.75%) then S. obliquus (28.56%). The protein content decreased upon applying some stress conditions on S. platensis and S. obliquus. The electrophoretic detection of the proteins isolated from the microalgal species showed that the vegetative and stressed S. obliquus were physiologically similar to S. platensis by 42.11 and 57.14%, respectively. D. salina was similar to S. platensis by 46.15%. As compared to the vegetative S. obliquus, the stressed S. obliquus and D. salina were similar to 30.77 and 33.33%, respectively. The D. salina was similar to the stressed S. obliquus by 57.14%. The protein pattern was electrophoretically represented by 10 denatured bands in S. platensis, 9 bands in the vegetative S. obliquus, 4 bands in the stressed S. obliquus and 3 bands in D. salina. There was one common band identified in all microalgal species at relative mobility (Rf) 0.95 (Molecular weight (Mwt) 5.27 KDa). There were 5 characteristic (unique) bands identified in the vegetative S. obliquus at Rfs 0.07, 0.17, 0.50, 0.58 and 0.86 (Mwts 214.68, 137.74, 42.37, 30.18 and 12.44 KDa, respectively). The amino acids that were analyzed in the isolated protein hydrolysate showed that 15 amino acids were noticed in S. platensis and S. obliquus. While in D. salina, 17 amino acids were identified. Glutamic acid was the most predominant amino acid and exhibited the highest percentages in D. salina, S. obliquus and S. platensis (4.96%, 3.3% and 7.58%, respectively). Additionally, the in-vitro studies on the isolated proteins showed that the D. salina protein exhibited the highest antioxidant activity at a dose of 100 μg/ml (308.78%) followed by the S. platensis and S. obliquus proteins (140.81% and 124.49%, respectively). Furthermore, D. salina and S. obliquus showed moderate anticholinesterase activity (34% and 36%, respectively). Therefore, microalgal proteins might be used as therapeutic antioxidants and might be used in combination with treatments of Alzhiemer's disease (AD).

High efficient assimilation of NO₃⁻-N with coproduction of microalgal proteins by Chlorella pyrenoidosa

The aim of this study was to establish a novel technology using microalgae for NO₃⁻ removal from high concentration wastewater and conversion to algal proteins. The effects of cultivation modes and illumination modes on the biomass yield, NO₃⁻ assimilation rate and algal protein yield were first investigated in shaking flasks for mixotrophic cultivation of Chlorella pyrenoidosa, and subsequently the scale-up verification in 5-L photo fermenter was successfully conducted. Fed-batch cultivation without medium recycling was the best cultivation mode in shaking flask system, in which the highest biomass yield (35.95 g/L), the average NO₃⁻ assimilation rate (2.06 g/(L·d)) and algal protein content (up to 42.44% of dry weight) were achieved. By using a staged increase of light intensity as illumination modes, the specific growth rate of cells could be significantly promoted to the highest (0.65 d⁻¹). After a 128-hour continuous cultivation in a 5-L photo fermenter, the highest biomass yield and the average NO₃⁻ assimilation rate were reached to 66.22 g/L and 4.38 g/(L·d) respectively, with the highest algal protein content at 47.13% of dry weight. Our study could provide a photo fermentation technology of microalgae for highly efficient treatment of waste industrial nitric acid and/or high concentration nitrate wastewater. This microalgae-based bioconversion process could coproduce protein-rich microalgal biomass, which facilitates the resource utilization of these type wastewater by trash-to-treasure conversion.

Marine Microalgae Biomolecules and Their Adhesion Capacity to Salmonella enterica sv. Typhimurium

Different molecules have been tested as analog receptors due to their capacity to bind bacteria and prevent cell adhesion. By using in vitro assays, the present study characterized the aqueous and alkaline extracts from microalgae Pavlova lutheri and Pavlova gyrans and evaluated the capacity of these extracts to adhere to enterobacteria (Salmonella Typhimurium). The aqueous and alkaline extracts of both species were fractionated via freeze-thawing, giving rise to soluble and insoluble (precipitate) fractions in cold water. The obtained fractions were studied using thermogravimetric, methylation analyses, and using 1D and 2D NMR techniques. The cold-water-soluble fractions obtained from the aqueous extracts were mainly composed of highly branched (1→3),(1→6)-β-glucans, whereas the cold-water-precipitate fractions were constituted by (1→3)-β-glucans. The alkaline extract fractions showed similar compositions with a high protein content, and the presence of glycosides (sulfoquinovosylglycerol (SQG), digalactosylglycerol (DGG)), and free fatty acids. The linear (1→3)-β-glucans and the alkaline extract fractions showed an adhesion capacity toward Salmonella. The chemical composition of the active fractions suggested that the presence of three-linked β-glucose units, as well as microalgal proteins and glycosides, could be important in the adhesion process. Therefore, these microalgal species possess a high potential to serve as a source of anti-adhesive compounds.

Extraction of proteins from the microalga Scenedesmus obliquus BR003 followed by lipid extraction of the wet deproteinized biomass using hexane and ethyl acetate

A current problem of the lipid extraction from wet biomass is the formation of emulsions during the mixing of the microalgal biomass and organic solvents. It has been suggested that microalgal proteins play an important role in the formation and stability of such emulsions. Herein, the extraction of proteins of the freshwater microalga Scenedesmus obliquus BR003 was optimized for further extraction of lipids from the wet deproteinized biomass. The optimal (pH 12 at 60 °C for 3 h) and moderate (pH 10.5 at 50 °C for 2 h) conditions of protein extraction resulted in protein yields of 20.6% and 15.4%, respectively. Wet lipid extraction of deproteinized biomass resulted in a less stable emulsion that released twice the solvent than the control biomass. However, the faster separation of phases that occurred during the wet lipid extraction of the deproteinized biomass resulted in a lipid yield twice lower than the control biomass.

The use of polyelectrolytes for simultaneous dewatering and fractionation of microalgal proteins, lipids, and pigments

For making microalgae-based bio-refinery platform sustainable and economically viable, it is necessary to simplify the harvesting process while enabling co-extraction of high-value bio-products from biomass. In this work, the ability of poly-diallyl dimethylammonium chloride (polyDADMAC) to dewater and extract cellular debris, lipids, and pigments to hexane phase while retaining protein fraction in the aqueous phase was investigated. It was observed that different components could be migrated from one phase to the other by modulating the system pH. Close to the isoelectric point, proteins can be retained in the aqueous phase while migrating algal debris to the hexane phase via targeted binding of the polyelectrolyte. Approximately 80% of total proteins were retained in the aqueous phase at pH 4, and ∼90% of cellular debris were migrated to the hexane phase at pH 4.5. Results indicate the possibility of separating multiple components from microalgae in an aqueous-organic solvent two-phase system using polyDADMAC.

Towards protein production and application by using Chlorella species as circular economy

In this study, productions of microalgal proteins were explored via a circular economy concept. First, production of proteins from Chlorella vulgaris FSP-E (CV) and Chlorella sorokiniana (CS) was optimized by using favorable cultivation conditions and strategies. The optimal CO2 concentration for the growth of both microalgae was 5% (v/v), while the optimal nitrogen source for CV and CS were 12 mM of NaNO3 and NH4Cl, respectively. Addition of 12 mg/L ammonium iron (III) citrate enhanced protein production. Next, semi-batch cultivation strategy was employed to achieve a protein production of 793.3 and 812.8 mg/L for CV and C S, representing a 4.86 and 2.77 fold increase, respectively, in protein productivity. The obtained microalgal proteins consist of 40% essential amino acids. The CV and CS proteins possess prebiotic activities as they enhanced the growth of Lactobacillus rhamnosus ZY by 48 and 74%, respectively, with a good antibacterial activity against predominant pathogens.

Extraction Methods and Functional Properties of Protein from <i>Arthospira platensis</i> for Bioavailability of Algal Proteins

Protein is one of the main nutrients that will be in short supply in the future. Alternative protein sources and production methods are required to fulfil the demand of protein requirements. Proteins from microalgae represent potential raw materials for the generation of protein based food ingredients. Arthospira platensis harbors high protein concentrations and one of the most important factors influencing successful extraction of protein is accessibility of the protein molecules. Process optimization and statistical analysis is necessary to maximize protein extraction. This study attempts to evaluate and compare various methods for their reliability in extracting microalgal proteins. Five different extraction methods namely alkali, enzymatic, thermal, microwave assisted and ultrasonic extraction were performed to obtain protein from A. platensis. Functional properties of the protein isolates were determined at various pH levels. Highest protein yield of 84% was obtained in ultrasound extraction. The lowest solubility of protein was found at pH 5.0 (0.27%) and highest solubility of protein was obtained at pH 9.0 (74.90%). Water holding capacity of protein isolates of S. platensis was in the range of 0.902 – 1.341 gwater/gprotein. The foaming capacity ranged from 19.37 to 41.28%, with the lowest and maximum values obtained at pH 5.0 and 3.0, respectively. Maximum value of foam stability at pH 5.0 was 31.24% and this subsequently decreased when the pH increased. The results revealed that both microwave assisted and ultrasound extraction methods were found suitable to make bioavailability of algal proteins from Arthospira platensis.

Effect of ammonium nitrogen on microalgal growth, biochemical composition and photosynthetic performance in mixotrophic cultivation

To enhance microalgal growth and optimize ammonium utilization, the effect of ammonium on microalgal growth, biochemical composition and photosynthetic performance were investigated by mixotrophic cultivation of microalga Spirulina platensis comparing with autotrophic cultivation. The results indicated that elevated ammonium significantly affected the microalgal growth, but the microalga in mixotrophic cultivation showed better growth and stronger tolerance to higher ammonium. The microalgal proteins were increased by increasing nitrogen concentration. The synthesis of microalgal carbohydrates was inhibited by higher ammonium, especially in mixotrophic cultivation. The addition of ammonium decreased the microalgal lipids in autotrophic cultivation but increased microalgal lipids in mixotrophic cultivation. Ammonium negatively affected the microalgal photosynthetic performance. The inhibition was intensified by elevated ammonium, inducing stronger photosystem protection mechanism, particularly in mixotrophic cultivation. The rate of ammonium inhibition to the microalgal photosystem was quick in the early stage by decreasing electron transport rate of PS II.

Effects of pulses and microalgal proteins on quality traits of beef patties.

Physicochemical parameters (pH, colour and texture), proximate composition (moisture, protein, lipid and ash content), amino acid content, and taste profile of beef patties elaborated with soy (control), pulses (pea, lentil and bean) and microalgal (Chlorella and Spirulina) proteins were assessed. The pH, colour, ash content, total, essential and non-essential amino acids and amino acid content were significantly different among the beef patties studied. In this regard, beef patties prepared with pea protein presented the highest values for pH; whereas beef patties manufactured with pea also showed the highest values for lightness and patties elaborated with bean the highest values for redness. Similar textural parameters were observed among the six batches of beef patties manufactured. Regarding ash content, a significant higher content was observed in the beef patties prepared with soy compared to the other ones. On the other hand, the beef patties elaborated with bean and seaweeds showed the highest values for the total amino acids content. The inclusion of bean and seaweed proteins increased the concentrations of all amino acids in beef patties, being glutamic acids, lysine and aspartic acid the predominant amino acids. Regarding the taste analysis, a similar profile was found among the six batches of beef patties studied. Considering all studied parameters, beef patties elaborated with bean protein could be used as an alternative protein source respect to soy protein because of it showed a similar nutritional content and taste profile and higher amino acid content.

Selective and mild fractionation of microalgal proteins and pigments using aqueous two-phase systems.

BACKGROUNDMicroalgal biomass is generally used to produce a single product instead of valorizing all of the cellular components. The biomass production and downstream processes are too expensive if only one product is valorized. A new approach was proposed for the simultaneous and selective partitioning of pigments and proteins from disrupted Neochloris oleoabundans cultivated under saline and freshwater conditions.RESULTSAn aqueous two‐phase system composed of polyethylene glycol and cholinium dihydrogen phosphate selectively separated microalgal pigments from microalgal proteins. 97.3 ± 1.0% of lutein and 51.6 ± 2.3% of chlorophyll were recovered in the polymer‐rich phase. Simultaneously, up to 92.2 ± 2.0% of proteins were recovered in a third phase (interface) in between the aqueous phases (interface). The recovered proteins, including Rubisco with a molecular weight of ∼560 kDa, seem to be intact and pigments did not suffer degradation, demonstrating the mildness of this system for fractionating microalgal biomolecules.CONCLUSIONThe ability of aqueous two‐phase systems (ATPSs) to simultaneously and efficiently fractionate different biomolecules in a mild manner from disrupted microalgae is demonstrated. This is an important step towards the development of a multiproduct microalgae biorefinery. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multiple binding modes of a moderate ice-binding protein from a polar microalga.

Ice-binding proteins (IBPs) produced by cold-tolerant organisms interact with ice and strongly control crystal growth. The molecular basis for the different magnitudes of activity displayed by various IBPs (moderate and hyperactive) has not yet been clarified. Previous studies questioned whether the moderate activity of some IBPs relies on their weaker binding modus to the ice surface, compared to hyperactive IBPs, rather than relying on binding only to selected faces of the ice crystal. We present the structure of one moderate IBP from the sea-ice diatom Fragilariopsis cylindrus (fcIBP) as determined by X-ray crystallography and investigate the protein's binding modes to the growing ice-water interface using molecular dynamics simulations. The structure of fcIBP is the IBP-1 fold, defined by a discontinuous β-solenoid delimitated by three faces (A, B and C-faces) and braced by an α-helix. The fcIBP structure shows capping loops on both N- and C-terminal parts of the solenoid. We show that the protein adsorbs on both the prism and the basal faces of ice crystals, confirming experimental results. The fcIBP binds irreversibly to the prism face using the loop between the B and the C-faces, involving also the B-face in water immobilization despite its irregular structure. The α-helix attaches the protein to the basal face with a partly reversible modus. Our results suggest that fcIBP has a looser attachment to ice and that this weaker binding modus is the basis to explain the moderate activity of fcIBP.

Investigation of in vitro digestibility of dietary microalga Chlorella vulgaris and cyanobacterium Spirulina platensis as a nutritional supplement

Microalgal proteins are promising sources for functional nutrition and a sustainable candidate for nutraceutical formulations. They also gain importance due to emerging focus on a healthy nutrition and increase in the number of chronic diseases. In this study, dried dietary species of microalga, Chlorella vulgaris, and cyanobacterium Spirulina platensis were hydrolyzed with pancreatin enzyme to obtain protein hydrolysates. The hydrolysis yield of biomass was 55.1±0.1 and 64.8±3.6% for C. vulgaris and S. platensis; respectively. Digestibility, as an indicator for dietary utilization, was also investigated. In vitro protein digestibility (IVPD) values depicted that cell wall structure due to the taxonomical differences affected both hydrolysis and digestibility yield of the crude biomass (p<0.05). Epithelial cells (Vero) maintained their viability around 70%, even in relatively higher concentrations of hydrolysates in the culture. The protein hydrolysates showed no any antimicrobial activities. This study clearly shows that the conventional protein sources in nutraceutical formulations such as soy, whey, and fish proteins can be replaced by enzymatic hydrolysates of microalgae, which shows elevated digestibility values as a sustainable and reliable source.

Single-step disruption and protein recovery from Chlorella vulgaris using ultrasonication and ionic liquid buffer aqueous solutions as extractive solvents

Microalgae are natural renewable sources providing attractive high-value products such as proteins. Due to the high costs incurred during drying processes, wet extraction from fresh microalgae biomass can be a promising approach. In this work, a single process step integrating cell disruption and protein recovery from wet Chlorella vulgaris by using ultrasonication and aqueous solutions of ionic liquids (ILs) as extractive solvents was proposed. The ILs assessed belong to the group of Good’s buffer ionic liquids (GBILs), and were prepared by a combination of Good’s buffer-based anions, namely 2-hydroxy-3-morpholinopropanesulfonate and 2-[bis(2-hydroxyethyl)amino]ethanesulfonate, and tetrabutylammonium, tetrabutylphosphonium and cholinium cations. These GBILs possess high biocompatibility and buffering capacity at neutral pH region. The protein recovery capabilities of the aqueous solutions of GBILs were compared with conventional buffers, using the same cell disruption approach. After the proper selection of the buffer solution with higher capability to extract microalgal proteins, alternative disruption techniques such as freeze-thaw, non-ionic detergent-based lysis using Triton X-100 and the combined methods were studied. The most efficient strategy, namely ultrasonication-assisted extraction, was further investigated for the effects of the process variables such as the buffer concentration, the addition of NaCl, the biomass concentration, and the ultrasonic time and power.