Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Neochloris oleoabundans biorefinery : Integration of cell disruption and purification steps using aqueous biphasic systems-based in surface-active ionic liquids
    Suarez Ruiz, C.A. ; Martins, M. ; Coutinho, J.A.P. ; Wijffels, R.H. ; Eppink, M.H.M. ; Berg, C. van den; Ventura, S.P.M. - \ 2020
    Chemical Engineering Journal 399 (2020). - ISSN 1385-8947
    Aqueous biphasic systems - Microalgae disruption - Multi-product approach - Purification - Tensioactive compounds

    In this work, an approach to integrate the downstream processing of bioactive compounds present in the microalgae cells by combining the use of tensioactive compounds and aqueous biphasic systems (ABS) is proposed. For this purpose, several aqueous solutions using solvents with and without tensioactive nature were investigated on their capacity to disrupt the microalgae cells as well as to extract the different classes of biomolecules, namely pigments (chlorophylls a and b, and lutein), proteins and carbohydrates. Cationic tensioactive compounds were selected due to their high ability to simultaneously extract the different classes of compounds present in the Neochloris oleoabundans biomass. To fractionate pigments, proteins and carbohydrates extracted from the microalgae, ABS formed by polyethylene glycol (PEG 8000) and sodium polyacrylate (NaPA 8000) were used, with the solvent selected to disrupt the cells acting as electrolyte. This allowed to tune the biomolecule's partition reaching a selective fractionation. This approach provided the simultaneous extraction of different biomolecules (pigments, protein and carbohydrates) from the cells and, the subsequent origin of two fractions, one rich in proteins (extraction efficiencies of 100%) and carbohydrates (extraction efficiency of 80%) and the second concentrated in pigments (e.g. lutein, extraction efficiency of 98%). The further isolation of the biomolecules from the ABS forming solvents is proposed aiming at the development of an integrated downstream process, including the cell disruption/compounds extraction, the fractionation, and the isolation of the biomolecules.

    Combined bead milling and enzymatic hydrolysis for efficient fractionation of lipids, proteins, and carbohydrates of Chlorella vulgaris microalgae
    Alavijeh, Razieh Shafiei ; Karimi, Keikhosro ; Wijffels, Rene H. ; Berg, Corjan van den; Eppink, Michel - \ 2020
    Bioresource Technology 309 (2020). - ISSN 0960-8524
    Bead milling - Combined treatment - Enzymatic hydrolysis - Fractionation - Microalgae

    A combined bead milling and enzymatic hydrolysis process was developed for fractionation of the major valuable biomass components, i.e., proteins, carbohydrates, and lipids from the microalgae Chlorella vulgaris. The cells were treated by bead milling followed by hydrolysis with different hydrolytic enzymes, including lipase, phospholipase, protease, and cellulase. Without enzymatic hydrolysis, the recovery yield of lipids, carbohydrates, and proteins for bead milled biomass was 75%, 31%, and 40%, respectively, while by applying enzymatic treatments these results were improved significantly. The maximum recovery yield for all components was obtained after enzymatic hydrolysis of bead milled biomass by lipase at 37 °C and pH 7.4 for 24 h, yielding 88% lipids in the solid phase while 74% carbohydrate and 68% protein were separated in the liquid phase. The recovery yield of components after enzymatic hydrolysis of biomass without bead milling was 44% lower than that of the milled biomass.

    Multistep Fractionation of Microalgal Biomolecules Using Selective Aqueous Two-Phase Systems
    Suarez Ruiz, Catalina A. ; Kwaijtaal, Jennifer ; Peinado, Oriol Cabau ; Berg, Corjan Van Den; Wijffels, Rene H. ; Eppink, Michel H.M. - \ 2020
    ACS sustainable chemistry & engineering 8 (2020)6. - ISSN 2168-0485 - p. 2441 - 2452.
    Aqueous two-phase systems - Biomolecules - Cholinium-based ionic liquids - Downstream processes - Microalgae

    We aim to develop liquid-liquid extraction processes for the fractionation of microalgal components (proteins, pigments, lipids, and carbohydrates). The partitioning behavior of microalgal pigments and proteins in aqueous two-phase systems (ATPS) composed of the polymer polypropylene glycol with molecular weight 400 (PPG 400) + various cholinium based-ionic liquids was studied. A process for fractionation of multiple components from disrupted Neochloris oleoabundans was developed and evaluated. Results show that cholinium dihydrogen phosphate (Ch DHp) allows the fractionation of pigments in the PPG 400-rich phase and proteins in the Ch DHp-rich phase with high selectivity. It was demonstrated that a multiproduct approach can fractionate free glucose, and proteins in the ionic liquid-rich phase, pigments in the polymer-rich phase, while starch and lipids are recovered at the interface.

    Selective fractionation of free glucose and starch from microalgae using aqueous two-phase systems
    Suarez Ruiz, Catalina A. ; Zarate Baca, Santiago ; Broek, Lambertus A.M. van den; Berg, Corjan van den; Wijffels, Rene H. ; Eppink, Michel H.M. - \ 2020
    Algal Research 46 (2020). - ISSN 2211-9264
    Aqueous two-phase systems - Carbohydrates - Extraction - Glucose - Ionic liquids - Microalgae

    Microalgae are a promising source of lipids, pigments, proteins and carbohydrates, which are valuable compounds for many industries. However, optimal fractionation and valorization of all produced compounds is necessary to improve the economic viability of microalgae production. This paper aims to understand the fractionation of microalgae carbohydrates (free glucose and starch) in aqueous two-phase systems. Three aqueous two-phase systems were investigated to efficiently and mildly separate carbohydrates from disrupted Neochloris oleoabundans. This strain contains 16 w/w% of proteins, 48 w/w% total fatty acids and 27 w/w% carbohydrates when cultivated under saline water and nitrogen depletion conditions. The protein content decreases and the amount of fatty acids and carbohydrates increases notably under stress conditions and glucose becomes the main carbohydrate in this microalgae. Glucose is present in the disrupted microalgae as part of polymeric carbohydrates (starch) or in monomeric form (free glucose). With the aqueous two-phase system Polyethylene Glycol 400 - Cholinium dihydrogen phosphate (PEG400-ChDHp) microalgal free glucose is fractionated up to a recovery of 99% to the most hydrated bottom phase in a single step. Simultaneously, a recovery of 70% is reached for microalgal starch in the interface after two additional liquid-liquid extractions with PEG400-ChDHp. The final fractions obtained were free of pigments.

    Integrated Biorefineries for Algal Biomolecules
    Suarez Garcia, E. ; Olivieri, G. ; Sijtsma, L. ; Vermuë, M.H. ; Barbosa, M.J. ; Reith, J.H. ; Berg, C. van den; Eppink, M.H.M. ; Wijffels, R.H. - \ 2020
    In: Grand Challenges in Algae Biotechnology / Hallmann, A., Rampelotto, P., Springer - ISBN 9783030252328 - p. 293 - 317.
    Algae are a renewable source of biomolecules for multiple applications ranging from fuels to specialties. However, their implementation as feedstock in industrial processes has only been achieved for few high-value products. This is due to the elevated costs in cultivation and downstream processing. In order to decrease the biorefinery costs and to enhance the overall process profitability, new separation processes need to be developed. Such processes must start from the understanding of the cell architecture, as a basis to develop an optimal fractionation strategy, and must include selective and mild disentanglement processes, in order to preserve the functionality of the target molecules. In this regard, we propose novel integration concepts such as self-disintegration, simultaneous disintegration and disentanglement, and self-separating systems within the framework of process intensification, in such a way that auxiliary chemicals, solvents and numerous unit operations become redundant.
    Preliminary Evaluation of Aqueous Protein Extraction from Black Soldier Fly Larvae (Hermetia illucens L.)
    Miron, Lucian T. ; Postma, Richard P. ; Bosch, Guido ; Eppink, Michel H.M. - \ 2019
    Industrial Biotechnology 15 (2019)6. - ISSN 1550-9087 - p. 365 - 369.
    black soldier fly larvae - insects - protein extraction - SDS-PAGE - soluble protein

    This study aimed to explore the efficacy of mild water-soluble protein extraction procedures on black soldier fly larvae (BSFL), a potential alternative and sustainable protein source for food. Different conditions were tested for extraction of water-soluble proteins including phosphate buffered saline (PBS) and Tris-HCl buffer in a pH range from 7.0 to 9.0. The total protein content and the water-soluble protein content were measured by Lowry BioRad DC™ protein assay. The molecular weight distribution of water-soluble proteins was characterized using SDS-PAGE. The black soldier fly larvae contained 42.3% protein on a dry matter basis, which is comparable to other studies. After extraction, the water-soluble protein fraction ranged from 5.5 to 17.2%, dependent on the buffer conditions, and total protein content and discoloration of the supernatant was observed, which could be an indication of oxidation. Moreover, the SDS-PAGE method allowed the determination of molecular weight distribution of the protein fraction showing no protein degradation with discrete bands and the most intensive band <75 kDa was observed. The extraction of water-soluble BSFL proteins was mild, easy, fast and in agreement with other studies. Further research should focus on the characterization and techno-functional properties of the aqueous protein extracts as food ingredient alternative.

    Mild fractionation of hydrophilic and hydrophobic components from Neochloris oleoabundans using ionic liquids
    Desai, Rupali K. ; Fernandez, Maria Salvador ; Wijffels, Rene H. ; Eppink, Michel H.M. - \ 2019
    Frontiers in Bioengineering and Biotechnology 7 (2019)OCT. - ISSN 2296-4185
    Biorefinery - Hydrophilic compounds - Hydrophobic compounds - Ionic liquids - Microalgae

    Microalgae are a promising source for proteins, lipids, and carbohydrates for the food/feed and biofuel industry. To make microalgae production economically feasible, it is necessary to optimally use all produced compounds keeping full functionality. Therefore, biorefining of microalgae is the key to lower the cost of algal products using mild and effective processing techniques. In this article, we have tested the feasibility of aqueous solutions of imidazolium and phosponium ionic liquids to selectively milk the hydrophobic lipids from Neochloris oleoabundans biomass out of intact cells and recover after cell disruption the hydrophilic fraction containing proteins and carbohydrates. The results showed that the ionic liquid tributylmethylphosphonium methylsulfate (TBP SO4; Cyphos 108) is able to permeabilize fresh intact cells of N. oleoabundans for extracting 68% of total lipids out of the cells, whereas, after cell disruption, 80% of total proteins, and 77% of total carbohydrates could be obtained in aqueous buffers. This concept kept the recovered proteins in their native form without interacting with the ionic liquids that will denature the proteins. Selective biorefinery of different components from microalgae using ionic liquid TBP SO4 explains the novelty of this concept.

    A risk based surveillance programme for Toxoplasma gondii in pigs using a combination of farm auditing and serological screening
    Wisselink, H.J. ; Swanenburg, M. ; Gonzales Rojas, Jose ; Asseldonk, M.A.P.M. van; Wagenberg, Coen van; Giessen, J. van der; Meerburg, B.G. ; Krijger, Inge ; Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. - \ 2019
    Toxoplasma gondii is recognized as one of the major foodborne pathogens with a high human disease burden. In the Netherlands, pork contributes to about 11 % of the meatborne T. gondii infections. To control T. gondii infections in pigs, EFSA has advised to perform serological testing of pigs and audits of pig farms on risk factors for T. gondii infection. In the Netherlands, a program was started to translate the EFSA advice into a practical risk based surveillance system. In first instance, a large scale serological monitoring of fattening pigs was started and seroprevalence over time was determined. Next, the association between within-herd seroprevalence and risk factors for T. gondii on fattening pig farms in the Netherlands was determined. For this, a questionnaire for auditing farms for the presence of risk factors of T. gondii was developed and used on 25 case and 50 control farms. Results show that there is a significant association between seroprevalence and risk factors as cats present on farms, use of unheated feed products and feeding wet feed. Moreover, on-farm presence of rats and mice also increases Toxoplasma transmission risks. Subsequently, a study was started on farms to quantify the effectiveness of interventions on farms. A cross-over clinical trial was set up in which case farms were their own control and the cross-over moment is the implementation of interventions on risk factors to change farm management. Farms with a high within-herd seroprevalence were followed for at least during a year and monitored periodically for seroprevalence and implementation of interventions to eventually reduce the disease burden. The break-even point was calculated for which the intervention cost at fattening pig farms equal averted human disease burden and averted cost-of-illness minus cost of the surveillance program. The results shows favourable economic perspectives for interventions to control pig meat-born transmission of T. gondii.
    Identification of potential risk factors for Toxoplasma gondii in fattening pigs in the Netherlands using a Bayesian approach
    Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. ; Urlings, H.A.P. ; Asseldonk, Marcel van; Wagenberg, Coen van; Krijger, Inge ; Giessen, J.W.P. van der; Swanenburg, M. ; Wisselink, H.J. - \ 2019
    Toxoplasma gondii is a relevant foodborne pathogen,it is estimated that up to one third of the worldpopulation has been exposed to the parasite (Tenteret al. 2000). In the Netherlands toxoplasmosis rankssecond on a list of prioritized emerging zoonosis(Havelaar et al. 2010) and also second in disease burden among 14 foodborne diseases (Mangen et al.2017). Data suggest that ingesting improperly cooked meat containing T. gondii is one of the major sources of infection in Europe and North America (Crotta et al. 2017; Guo et al. 2015). The contribution of pork to meatborne T. gondii infections is estimated to be11 % in the Netherlands (Opsteegh 2011) and is seen as an important possible source of human T. gondii infections (Foroutan et al. 2019). The European Food Safety Authority (EFSA) advised to perform serological testing of pigs and on farm audits on risk factors (EFSA 2011). To that end, a serological monitoring program was developed in a slaughterhouse in the Netherlands. In this study, the objective is to determine the association between within herd seroprevalence, corrected for misclassification of samples through Bayesian analyses, and risk factors for T. gondii on fattening pig farms in TheNetherlands.
    Materials and MethodsFrom 2015 to 2018, HACCP based audits were performedon 75 fattening pig farms in The Netherlands to identify the presence of potential T. gondii risk factors. All farms were conventional pig farms, with 15 farms being farrow to finish. As overall seroprevalence of T. gondii in pigs in the Netherlands is low, estimated at 5 % (1-12 % 95 % CI) by Foroutanet al. 2019, approached farms were chosen with the knowledge of previous serology data. In this way there would be farms with positive serum samples and farms without them included in the study. The audits were based on an updated version of the questionnaire from Mul et al. (2015) and covered the following topics: outdoor access, farm biosecurity, rodent control, presence of cats, feed and watersupply. In addition, serum samples (n=6272) from fattening pigs were obtained at slaughter throughout the year before the audit on the farm was performed. These samples were used for antibody testing bya PrioCHECK™ Toxoplasma Antibody ELISA. Data were analysed using Bayesian statistics, with the within farm T. gondii prevalence as dependent variable and potential risk factors as independent variables. As always with serology, misclassification due to false-positive or false-negative results can occur. Statistical methods have been developed to account for such misclassification, based on frequentistic as well as Bayesian approaches (Hui & Walter 1980; Josephet al. 1995). First, all independent variables wereanalysed in a univariate logistic model, and variables with a probability ≤0.25 that zero is included in the 95 % interval were analysed in a multivariable model. The multivariate logistic model was fitted using backward elimination until all remaining variables showed a probability ≤0.05 that zero is included in the 95 % interval. Two-way interaction terms were evaluated similarly to the main variables regarding statistical significance.
    ResultsDescriptive results showed that 50 out of the 75 farms had 1 or more positive serum samplein the year before the audit was performed. In total 438 samples were positive out of the 6272 samples. Final Bayesian analyses are currently being conducted. However, preliminary results from data analysis using frequentistic logistic multivariate regression identified two significant risk factors: the accessibility of pig feed for cats and theprovision of well water as drinking water for the pigs (Table 1).
    Discussion and ConclusionsThe use of serological testing seems to be a valuable guide and monitoring tool for the control of T.gondii in pork production. In a preliminary analysis, a higher within-herd T. gondii seroprevalence on fattening pig farms in the Netherlands was associated with the accessibility of pig feed for cats and the provision of well water as drinking water for the pigs. Improvements in farm management on fattening pig farms will likely contribute to reduction of the human disease burden and is presently studied.
    Understanding mild cell disintegration of microalgae in bead mills for the release of biomolecules
    Suarez Garcia, E. ; Lo, C. ; Eppink, M.H.M. ; Wijffels, R.H. ; Berg, C. van den - \ 2019
    Chemical Engineering Science 203 (2019). - ISSN 0009-2509 - p. 380 - 390.
    Bead mill - Biorefinery - Disintegration - Energy efficient - Microalgae - Selective release

    Cell disintegration is, in general, the first step in the biorefinery of algae, since it allows the release of biomolecules of interest from the cells into the bulk medium. For high-value commercial applications, the disintegration process must be selective, energy efficient and mild. Developing a process with such features would demand extensive experimental effort. In the present study, we attempt to provide a tool for developing an efficient disintegration process via bead milling, by proposing a modelling strategy that allows the prediction of the kinetics of cell disintegration while having as input not only process parameters but also strain-specific parameters like cell size and cell-wall strength. The model was validated for two different algal strains (Tetraselmis suecica and Chlorella vulgaris), at various values of bead size (0.3–1 mm) and bead fillings (2.5–75%) and at two different scales of 80 and 500 mL. Since the kinetics of disintegration is proportional to the kinetics of release of biomolecules, the model can be further used for scale-up studies and to establish a window of operation to selectively target cells or metabolites of interest. Furthermore, the energy consumption in the mill was evaluated and it was found that operating at high bead fillings (>65%) is crucial to ensure an energy efficient process.

    Biorefinery of functional biomolecules from algae
    Suarez Garcia, Edgar - \ 2019
    Wageningen University. Promotor(en): R.H. Wijffels; M.H.M. Eppink, co-promotor(en): C. van den Berg. - Wageningen : Wageningen University - ISBN 9789463435956 - 255

    Algae have been regarded as a promising source of several biomolecules with industrial interest such as proteins, carbohydrates, lipids and pigments. To obtain such biomolecules, it is necessary to implement several extraction and fractionation steps which often result in poor yields, low purities and high costs. To overcome these issues, novel processes are needed, in which the concepts of minimum processing, integration of unit operations, in situ extraction and recyclability are applied. In this PhD thesis several fractionation strategies are presented in which such concepts are implemented. The main goal is to efficiently refine algal biomass into fractions containing functional biomolecules, in particular proteins. The fractionation strategies investigated here are based on mechanical disintegration, using bead milling, and chemical dissolution by means of ionic liquids as green solvents. Furthermore, it is demonstrated how minimum processing can lead, not only to low energy consumptions, but to fractions which display technical functionality comparable to animal derived protein isolates. Algae fraction are therefore envisioned as novel animal-free protein ingredients which can be used as foaming, emulsifying and gelling agents in food products.

    Disruption of microalgae with a novel continuous explosive decompression device
    Günerken, Emre ; Hondt, Els D'; Eppink, Michel H.M. ; Wijffels, Rene H. ; Elst, Kathy - \ 2019
    Algal Research 39 (2019). - ISSN 2211-9264
    Continuous process - Explosive decompression - Microalgae - Mild cell disruption - Techno-economic assessment

    Most microalgae cells are very resistant to mechanical and chemical stresses. Because target products are mainly located inside the cells, disruption of the cell wall is an essential step in downstream processing to recover the microalgae ingredients. This study is focused on the development of a mild cell disruption method based on explosive CO 2 decompression to allow the recovery of multiple sensitive products. The principle behind cell rupture via explosive decompression is the uptake of a gas under elevated pressure into the microalgae cells, where after a sudden depressurization gas expansion causes rupture of the cells. Conventional explosive decompression uses stirred tank reactors wherein, because of inefficient mixing, the gas/liquid interfacial area is minimal, resulting in long contact times, inefficient gas uptake and inefficient cell rupture. To increase the mixing yield and the formation of a gas-liquid micro-emulsion, a new continuous apparatus and a method were developed to process microalgae biomass by CO 2 based explosive decompression. The final design consisted of 2 pumps, a tubing system, a sparger, a hydrodynamic agitation zone and a heated relief valve. Experimental comparison of the conventional batch and the new continuous devices showed that process time was reduced 3.2–9.6 fold, the biomass and biochemical release efficiency increased more than 2 fold, and CO 2 consumption reduced 2–4 fold. The method can be considered as mild, since it can operate at room temperature without addition of chemicals. Moreover, the calculated shear rate on the algae cells is only 0.33% of the shear rate recorded during bead milling, while depending on the process parameters releasing a similar amount of biomass components from the cells into the supernatant. The article describes the development of the explosive decompression systems supported by computational fluid dynamics simulations and experimental data on algae cell disruption, complemented with a first techno-economic assessment.

    Integrated Product Recovery Will Boost Industrial Cyanobacterial Processes
    Berg, Corjan van den; Eppink, Michel H.M. ; Wijffels, Rene H. - \ 2019
    Trends in Biotechnology 37 (2019)5. - ISSN 0167-7799 - p. 454 - 463.
    cyanobacteria - process integration - product recovery - separation

    Cyanobacteria promise to be an important industrial platform for the production of a variety of biobased products such as fuels, plastics, and isoprenoids. Recent advances in synthetic biology have resulted in various cyanobacterial strain improvements. Nevertheless, these new strains are still hampered by product inhibition, resulting in low volumetric productivities, product concentrations, and yields on light. To circumvent these issues, continuous product recovery will need to be applied, resulting in economically viable industrial processes. Optimal product recovery strategies can be developed by considering biological and separation process constraints as well as photobioreactor design. Integrated product recovery will be indispensable to bring the cyanobacterial cell factory to industrial scale.

    Association between within-herd seroprevalence and risk factors for Toxoplasma gondii in fattening pigs in the Netherlands
    Eppink, D.M. ; Bouwknegt, M. ; Oorburg, D. ; Urlings, H.A.P. ; Asseldonk, Marcel van; Wagenberg, Coen van; Krijger, I.M. ; Giessen, J.W.P. Van der; Swanenburg, M. ; Wisselink, H.J. - \ 2018
    In: Annual General Meeting of the European College of Veterinary Public Health, "Fading of the HACCP after four decades: new trends in VPH for food safety ", Perugia, 17th-19th October 2018. - European College of Veterinary Public Health (ECVPH) - p. 40 - 40.
    PAT van buzz naar business
    Eppink, Michel - \ 2018
    Ionic Liquids as a tool for biorefinery of biomolecules from microalgae
    Eppink, Michel - \ 2018
    Microalgae: from Bio-based Curiosity Towards a Bulk Feedstock
    Lam, G.P. 't; Vermuë, M.H. ; Janssen, M. ; Barbosa, M.J. ; Wijffels, R.H. ; Eppink, M.H.M. ; Berg, C. van den - \ 2018
    In: Intensification of Biobased Processes / Górak, Andrzej, Stankiewicz, Andrzej, Royal Society of Chemistry (RSC Green Chemistry ) - ISBN 9781782628552 - p. 289 - 302.

    This chapter provides a comprehensive overview of the recent developments in microalgae cultivation and downstream processing with a focus on microalgae biorefinery. Microalgae are promising feedstocks for the production of a variety of bulk compounds in the near future. Current process designs typically consist of microalgae cultivation, harvesting, cell disruption and finally a product extraction/fractionation step. For every unit operation, the latest insights are discussed. From this overview, the current state-of-development and future directions towards large scale bulk-chemical production from microalgae are discussed.

    Selective and mild fractionation of microalgal proteins and pigments using aqueous two-phase systems
    Suarez Ruiz, Catalina A. ; Emmery, Daniel P. ; Wijffels, Rene H. ; Eppink, Michel H.M. ; Berg, Corjan van den - \ 2018
    Journal of Chemical Technology and Biotechnology 93 (2018)9. - ISSN 0268-2575 - p. 2774 - 2783.
    aqueous two-phase systems - cholinium-based ionic liquids - microalgae biorefinery - pigments - proteins

    BACKGROUND: Microalgal 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. RESULTS: An 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. CONCLUSION: The 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.

    Selective and energy efficient extraction of functional proteins from microalgae for food applications
    Suarez Garcia, E. ; Leeuwen, J. van; Safi, C. ; Sijtsma, L. ; Eppink, M.H.M. ; Wijffels, R.H. ; Berg, C. van den - \ 2018
    Bioresource Technology 268 (2018). - ISSN 0960-8524 - p. 197 - 203.
    Bead milling - Energy efficient - Functional protein - Gelation - Surface activity

    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
    SuarezGarcia, E. ; Leeuwen, J.J.A. Van; Safi, C. ; Sijtsma, L. ; Broek, L.A.M. van den; Eppink, M.H.M. ; Wijffels, R.H. ; Berg, C. van den - \ 2018
    Journal of Agricultural and Food Chemistry 66 (2018)29. - ISSN 0021-8561 - p. 7831 - 7838.
    bead milling - crude extract - filtration - gelation - surface activity

    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.

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