Conventional alginate extraction from brown seaweed typically relies on harsh, non-recyclable chemicals, limiting process sustainability. This study presents temperature-responsive deep eutectic solvents (TRDES) as circular, recyclable extractants for alginate recovery. Using computational screening with COSMO-RS and experimental validation of TRDES affinity and alginate partitioning, TRDES1 (o-cresol: ethanolamine) was identified as the most promising combination, and was optimised and reused over eight cycles, yielding up to 55.6 +/- 14.4 mg/g DW. COSMO-RS modelling validated the observed increase in extraction efficiency over successive cycles, showing enhanced partition coefficients and reduced Gibbs free energy of transfer with reuse. The process enabled mild extraction of functional alginate with increasing efficiency over the cycles. The main solvent parameters for TRDES design found to govern extraction and recyclability were capacity (C), partition coefficient (K), and Gibbs free energy (Delta G). Optimal performance was achieved with moderate TRDES-water capacity (1.27 x 10(1) to 3.15 x 10(1)), low TRDES capacity (<1.2), and K > 1. This work establishes a theoretical framework with design rules for future TRDES development based on computational and experimental analysis and highlights the need for novel, biocompatible TRDES systems. As demonstrated, combining computational screening with these design principles enables the use of recyclable solvents. Incorporating natural compounds into TRDES design enhances both process efficiency and sustainability, facilitating the integration of DES technologies into circular biorefineries and supporting environmentally responsible biomass valorisation.

Microalgae are a promising source for proteins, lipids, and carbohydrates for the cosmetic, nutraceutical, chemical, food/feed, and biofuel industry. In comparison with soy and palm oil, microalgae can be produced in a more sustainable way. To make microalgae production economically feasible, all biomass ingredients need to be efficiently utilized, similar to petroleum refineries in which oil is fractionated in fuels and a variety of products with higher value. However severe conditions can affect the properties of some components in the biomass. To overcome this, focus needs to be put on biorefinery techniques which are mild and effective. Microalgal biorefinery is a linear process consisting of harvesting, cell disintegration, sequential extraction, and further fractionation. Among these steps, the cell disintegration often represents a bottleneck for the extraction of hydrophilic or hydrophobic components, due to the presence of a tough cell wall in many strains. State of the art knowledge on both novel and classical techniques for product extraction within cell disintegration is presented. Comparison is made on the basis of two main criteria: yield of disintegration and energy consumption. The current work gives also a comprehensive outlook on business cases for microalgae biorefinery.

Protein extraction from Palmaria palmata remains challenging due to structural features that restrict protein release. This study evaluated alkaline solvents, xylanase enzymes, and deep eutectic solvents (DES) to assess protein recovery and structural changes using chemical analysis and microscopy. Protein yield, sugar solubilization, tissue density, protein localization, and cell wall integrity were examined. Microscopy revealed that proteins are concentrated in densely packed cortical cells with minimal intercellular space, limiting solvent penetration. Alkaline extraction achieved the highest protein recovery (∼60%) with moderate structural disruption. Xylanase-assisted extraction caused greater cell wall breakdown but resulted in lower protein recovery (∼27%), while DES treatment caused minimal disruption and yielded the lowest recovery (∼14%). Carbohydrate solubilization did not correlate with protein recovery, indicating that cell wall degradation alone is insufficient. These findings identify dense cortical organization as a key constraint and highlight the need for combined mechanical and targeted biochemical strategies to improve extraction efficiency.

Conventional alginate extraction from brown seaweed relies on alkaline treatments that limit sustainability and solvent reuse. In this study, a novel deep eutectic solvent (DES)-salt aqueous two-phase system (ATPS) was developed for the integrated extraction and separation of alginate as a more sustainable alternative. By incorporating the precipitation principle of three-phase partitioning at elevated salt concentrations in an ATPS, alginate was selectively recovered in an intermediate phase between the DES and aqueous salt solution, enabling direct isolation while preserving the solvent for reuse. Salt selection significantly influenced phase formation, with K3PO4 exhibiting stronger phase-forming ability than K2HPO4 due to its enhanced salting-out effect, whereas alginate recovery was primarily governed by salt concentration. The highest recovery (76.4 +/- 0.1%) was achieved using betaine: urea (1,2) with 50 wt% K3PO4, with most alginate accumulating in the intermediate phase. The DES maintained stable recovery over six cycles (63-86%), while preserving alginate integrity. These results demonstrate the potential of DES-based ATPS as an efficient platform for simultaneous extraction, separation, and solvent recycling, providing a promising route toward more sustainable and integrated seaweed biorefinery processes.

Microalgae are considered a potential sustainable feedstock for producing ingredients used in food, feed and cosmetics. However, the rigid cell wall structure presents a significant challenge for the efficient extraction intracellular products. Enzymatic hydrolysis of cell wall polymers could offer a mild and environmentally sustainable cell disruption approach. Nevertheless, incomplete knowledge of the structural and architectural properties of the cell wall limits the commercial application of this method. In this study, a combination biochemical and microscopy techniques was used to decipher the composition, topography, ultrastructure and the architecture of the cell wall of Nannochloropsis oceanica IMET1. Acid hydrolysis of the extracted cell wall released neutral sugars, constituting 80 % of its total weight (w/w). Of these sugars, 64 % was glucose, which primarily constitutes the cellulose layer. This was further confirmed through calcofluor white fluorescence imaging using a scanning confocal microscope. Additionally, the presence of small amounts of arabinose, fucose, galactose, and mannose suggests the presence of polysaccharide networks connecting the outer layer, the cellulose-based layer and the plasma membrane. Scanning electron microscopy revealed a rugged cell surface covered with N-acetyl-glucosamine units as confirmed by fluorescent-lectin staining. Transmission electron microscopy revealed a double-layered cell wall structure. Hydrolysis of the extracted cell wall with lichenases and cellulases generated mostly glucose monomers alongside traces of other neutral sugars. Overall, this study provides insights into the complex structure of the cell wall of N. oceanica IMET1, which can enhance enzyme assisted extraction strategies in microalgae.

Semi-hydrophobic eutectic solvents (ES) possess a great potential as lipid extraction solvent from untreated microalgae. However, the low vapor pressure of these solvents and the unknown effects on other biomolecules (e.g., proteins, carbohydrates) limit their application in microalgae biorefinery. In this work, recovery of the extracted lipids was performed by addition of antisolvents and the affecting parameters (i.e., antisolvent type, amount, temperature, ES imidazole content) were studied. The highest recovery was obtained with methanol addition to ES with 15 mol% imidazole at -20 °C, where lipid crystals were formed consisting mainly of saturated fatty acids. The remaining soluble lipids under the same condition were found to be fractions with mono- and poly-unsaturated fatty acids. Furthermore, based on the iterative extractions, the regenerated solvents could create sufficient driving force for lipid extraction despite the lipid accumulation. In addition, a scale-up study of lipid extraction and solvent recycling was performed (2 mL vs 500 mL), whereby the larger scale also showed a good performance. Finally, protein and carbohydrate isolation from the defatted biomass was feasible, but the proposed ES process was not sufficiently mild to maintain native proteins. On the other hand, opportunities are discussed to create new functionalities for proteins and carbohydrates so that a multiproduct biorefinery is feasible for this ES.

Conventional alginate extraction methods from brown seaweed typically rely on harsh chemicals that are not reused, and valuable pigments are lost during this process. This study applied a novel approach utilising reusable natural deep eutectic solvents (DES) in three-phase partitioning (TPP) to simultaneously extract alginate and pigments from Saccharina latissima. The hydrophobic DES effectively released alginate from the algal cell wall in the aqueous phase and served as a solvent for pigment extraction. Computational screening confirmed that all selected DES had an affinity for pigments chlorophyll a and fucoxanthin, while alginate extraction confirmed their role in disrupting the algal cell wall. Extraction conditions were optimised, resulting in an alginate yield of 101.8 +/- 3.1 mg/g DW compared to 55.3 +/- 14.1 mg/g DW for conventional alkaline extraction. According to physicochemical characterisation through FT-IR and M/G ratio (mannuronic to guluronic) analysis, the extracted alginate was comparable to that obtained via alkaline extraction, exhibiting similar functional groups and M/G ratios. The DES was reused successfully, showing that it could be reused for up to seven extraction cycles, during which pigments accumulated. After the seventh cycle, alginate yield declined, likely due to partial transfer into the DES phase, possibly driven by reverse micelle formation in the system. This study highlights a novel, mild multiproduct approach of a DES-based TPP system, enhancing economic feasibility by employing gentler and quicker extraction conditions. It facilitates the concurrent recovery of alginate and pigments while allowing for the repeated reuse of the DES.

Microalgae can provide a more sustainable alternative to traditional food systems which are dominated by terrestrial crops. The main economic challenges, however, relate to the downstream processing of microalgae and the valorization of their side streams. The present work explores the scientific principles and data required to develop an integrated biorefinery-on-a-chip, which replaces many of the common downstream processing unit operations by employing acoustic fields. The acoustic parameters of Tisochrysis lutea microalgal cells and their cell components are determined using the neutrally buoyant state method. Culture conditions which result in a high carbohydrate or high protein to lipid ratio led to a higher acoustic contrast factor than culture conditions favoring a high composition of lipids. The collected acoustic data is used as input in a numerical model which studies the harvesting of microalgal cells and the fractionation of microalgal cell components. High separation levels are achieved based on the size and composition of microalgal cells and the type of cell component. Subsequent studies are envisioned to determine the practical feasibility of applying these concepts and even scaling them out. Nevertheless, this study represents a steppingstone towards a novel, label-free approach to processing microalgal cells of different biomass compositions.

Deep eutectic solvents (DES) have emerged as green alternative extraction solvents. However, challenges in DES recovery and recycling limit their broader application. In this study, a novel thermo-separating aqueous twophase system (ATPS) was developed for the continuous, cyclic extraction and separation of alginate from Laminaria digitata using DES and temperature-responsive copolymers. The system enables a novel approach by repeatedly reusing both the DES phase and the EOPO copolymer phase five times, thereby reducing waste generation and enhancing process sustainability. This research demonstrated that DES can be efficiently recycled for ten cycles using temperature-responsive ethylene oxide-propylene oxide (EOPO) copolymers, remaining stable yields. For the extraction and subsequent separation of alginate, three different DESs were evaluated, all of which demonstrated to extract and recover alginate. DESs ChCl:Ethylene glycol and ChCl:Urea exhibited a preference for EOPO1000 (66 and 75 % recovery, respectively), whereas Bet:Urea achieved the highest recovery with EOPO3900 (66 % recovery). Subsequent recycling of the recovered DES showed that DES could be recycled for ten cycles, maintaining stable extraction yields between 74 and 86 mg alginate/g DW and alginate recovery yields of 55-65 %. Furthermore, combined DES and EOPO recycling could be performed for up to five cycles while maintaining an alginate recovery yield between 50 and 65 mg/g DW. This thermo-separating ATPS presents a novel, circular and sustainable approach for DES recycling compared to the non-circular conventional alkaline extraction. This proposed method can be applied in a simple and effective manner to both recover and recycle DES.

Photosynthetic microorganisms such as Arthrospira platensis are considered a more sustainable source of bioactive compounds. Moreover, their biomass composition can be altered by subjecting these cultures to physical or chemical stresses, at the expense of reduced growth rates. Alternative approaches such as external fields stimuli could enhance biomass composition while minimizing the negative impact on its growth. This study explores the impact of mild acoustic radiation on the compositions of A. platensis. Acoustic treatment variables such as power (ranging from 25% to 75%), frequency (in the range of 578-1148 kHz), duration (from 5 to 15 min) and culture age (from 4 to 12 days) were investigated using a custom multifactorial Design of Experiments approach. As a result, maximum increase in protein, lipids and carbohydrate concentrations of 10%, 96% and 88% respectively were observed with negligible impact on growth rates. This increase was achieved under different optimized acoustic treatment conditions. These findings could specifically maximize the production of certain compounds without sacrificing growth rates, fine-tunning the A. platensis biomass for various applications.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal microscopy, but also as a tracer for determination of residence time distribution in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. The site occupancy of two labeled antibodies, subclass 1 and 2 conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the IgG1 and IgG2 molecules, in positions 188 and 189, respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling despite the secondary region responsible for binding to the protein A was partly labeled. Fluorescent-labeled antibodies are a good compromise as an inert tracer in protein A affinity chromatography because they are much cheaper than isotope-labeled antibodies.

Extraction of seaweed compounds using Deep Eutectic Solvents (DES) has shown high interest. Quantification, however, is challenging due to interactions with DES components. In this research work, three chemical separation techniques were investigated to isolate and quantify alginate from a set of choline chloride-based DES. While choline chloride served as the hydrogen bond acceptor (HBA); Urea, Ethylene Glycol, Propylene Glycol, Glycerol, Sorbitol, Xylitol and Glucose were used as hydrogen bond donors (HBD). DES containing sodium alginate were subjected to precipitation with sulfuric acid 0.2 M (pH 1.6), ethanol-water mixture (80 % v/v) and calcium chloride (1 % w/v CaCl2·2H2O). Alginate in precipitates was quantified and used to evaluate the performance of each separation technique. The highest recovery yields (51.2 ± 1.3 %) were obtained using the ethanol-water mixture followed by calcium chloride (45.7 ± 1.2 %), except for polyols (e.g. sorbitol). The lowest recovery yields were obtained with acid, with a particularly low recovery yield when urea was used as HBD (9.6 ± 1.3 %). Estimations of ManA/GulA ratios showed lower values for precipitates from DES compared to the ones obtained from water. This research shows ethanolic precipitation as a suitable method for alginate separation from the studied set of choline chloride-based DES.

Deep eutectic solvents (DES) are green alternatives for conventional solvents. They have gained attention for their potential to extract valuable compounds from biomass, such as seaweed. In this framework, a case study was developed to assess the feasibility of pressure-driven membrane processes as an efficient tool for the recovery of deep eutectic solvents and targeted biomolecules. For this purpose, a mixture composed of the DES choline chloride - ethylene glycol (ChCl-EG) 1:2, water and alginate was made to mimic a DES extraction from seaweed. An integrated separation process design was proposed where ultrafiltration-diafiltration-nanofiltration (UF-DF-NF) was coupled. UF and DF were found to be effective for the separation of alginate with an 85 % yield. DES was likewise recovered by 93 %, proving the membrane filtrations' technical feasibility. The NF performance to separate the DES from the water, for its recycling, laid by a 45 %-50 % retention and a final concentrated DES solution of 18 %(v/v).

An integrated biorefinery was developed that utilizes microalgal biomass, Chlorella vulgaris, to sustainably produce proteins, fatty acids, bioethanol, and biogas. The microalgal soluble proteins and fatty acids were initially extracted through a cascading extraction process, including bead milling and solvent extraction. Subsequently, the investigation focused on utilizing the biomass residues for bioethanol and biogas production, ultimately improving energy recovery. Implementing the cascading process resulted in a 25 % enhancement in bioethanol yield and a 22.4 % increase in biomethane yield compared to untreated biomass. This approach resulted in 78.0 g of protein, 50.9 g of lipid, 20.8 ml of ethanol, and 136.5 L of methane from one kilogram of dry C. vulgaris biomass. Considering the potential of 8,640 k tons of annual microalgae production in Iran, an estimated 4.1 million tons of CO2 emissions could be averted. This reduction could result in saving approximately 1394.8 million USD in associated social costs of carbon. These improvements in fully valorizing biomass through practical cascading methods significantly advance microalgal biorefinery.

This study demonstrated an effective method for extracting alginate from the brown seaweed Ascophyllum nodosum using deep eutectic solvents (DES), achieving a higher yield than traditional alkaline extraction methods. Eight choline chloride-based DESs were tested, with DES ChCl:Urea (1:2) yielding the most alginate. The extraction conditions were optimized using response surface methodology (RSM), which identified the biomass to liquid ratio as the most significant factor influencing extraction efficiency. The optimized parameters resulted in a 74% extraction efficiency of alginate. Characterisation through FT-IR and size exclusion chromatography showed that the extracted alginate was comparable to that obtained via the conventional alkaline extraction method. Overall, DES extraction offers a more efficient and sustainable alternative to conventional methods, which typically extract only 10%-24.5% of total alginate.

Fluorescently labeled antibodies are widely used to visualize the adsorption process in protein chromatography using confocal laser scanning microscopy (CLSM), but also as a tracer for determination of residence time distribution (RTD) in continuous chromatography. It is assumed that the labeled protein is inert and representative of the unlabeled antibody, ignoring the fact that labeling with a fluorescent dye can change the characteristics of the original molecule. It became evident that the fluorescently labeled antibody has a higher affinity toward protein A resins such as MabSelect Sure. This can be due to slight differences in hydrophobicity and net charge, which are caused by the addition of the fluorescent dye. However, this difference is eliminated when using high salt concentrations in the adsorption studies. In this work, the site occupancy of two labeled antibodies, MAb1 (IgG1 subclass) and MAb2 (IgG2 subclass) conjugated with the fluorescent dye Alexa Fluor™ 488 was elucidated by intact mass spectrometry (MS) and peptide mapping LC-MS/MS, employing a sequential cleavage with Endoproteinase Lys-C and trypsin and in parallel with chymotrypsin alone. It was shown that the main binding site for the dye was a specific lysine in the heavy chains of the MAb1 and MAb2 molecules, in positions 188 and 189 respectively. Other lysine residues distributed throughout the protein sequence were labeled to a lot lesser extent. The labeled antibody had a slightly different affinity to MabSelect Sure although its primary binding site (to Protein A) was not affected by labeling, despite the secondary region responsible for binding to the protein A was partly labeled. Overall, the fluorescent-labeled antibodies are a good compromise as an inert tracer in residence time distribution and chromatography studies because they are much cheaper than isotope-labeled antibodies; However, the differences between the labeled and unlabeled antibodies should be considered.

Deep eutectic solvents (DES) can be a green alternative extraction technology for microalgae lipids, acting simultaneously as a pre-treatment agent and solvent. However, due to the low vapor pressure of both DES and lipid solute, the recovery of lipids and solvent regeneration remains difficult. In this study, we developed a novel strategy to separate the dissolved sunflower and model algae oil from imidazole/hexanoic acid DES by using polar antisolvents (water, methanol, and ethanol). The polarity and the amount of antisolvent influenced the lipid solubility in DES. While the water was the strongest antisolvent, the alcohols were easier to evaporate, ensuring easy DES regeneration. By adding small amounts of water and methanol, more than 90% of the lipids were recovered in the form of high-purity oils (>90%). In the case of ethanol, a large amount of ethanol was required, which diluted the solvent-rich phase and solubilized more lipids in it. Based on three repeated cycles with the selected antisolvent methanol, > 90% of the eutectic solvent could be regenerated.

The monoclonal antibody (mAb) industry is becoming increasingly digitalized. Digital twins are becoming increasingly important to test or validate processes before manufacturing. High-Throughput Process Development (HTPD) has been progressively used as a tool for process development and innovation. The combination of High-Throughput Screening with fast computational methods allows to study processes in-silico in a fast and efficient manner. This paper presents a hybrid approach for HTPD where equal importance is given to experimental, computational and decision-making stages. Equilibrium adsorption isotherms of 13 protein A and 16 Cation-Exchange resins were determined with pure mAb. The influence of other components in the clarified cell culture supernatant (harvest) has been under-investigated. This work contributes with a methodology for the study of equilibrium adsorption of mAb in harvest to different protein A resins and compares the adsorption behavior with the pure sample experiments. Column chromatography was modelled using a Lumped Kinetic Model, with an overall mass transfer coefficient parameter (kov). The screening results showed that the harvest solution had virtually no influence on the adsorption behavior of mAb to the different protein A resins tested. kov was found to have a linear correlation with the sample feed concentration, which is in line with mass transfer theory. The hybrid approach for HTPD presented highlights the roles of the computational, experimental, and decision-making stages in process development, and how it can be implemented to develop a chromatographic process. The proposed white-box digital twin helps to accelerate chromatographic process development.

Supplementary tables describe a comparison of half-lives of SYD983 in the different species (Table S1), PK details in mice (Table S2), PK details in tumor-bearing mice (Table S3), PK details in cynomolgus monkeys (Tables S4 and S5), Flow rates of SYD983 in RP-HPLC, HIC profile of SYD983 and SYD985 (Figure S1), Cytotoxicity of SYD983 versus SYD985 (Figure S2), and control staining for IHC of the tumors used in PDX (Figure S3).