Downstream Bioprocessing

Downstream Bioprocessing encompasses the design, development and implementation of biomanufacturing schemes, with an emphasis on separation technology and microbial biotechnology. Of current interest is integrated bioprocessing where sustainable-by-design criteria apply. Moreover, the group actively seeks to implement bioprocess digitalization to alleviate process bottlenecks, increase productivity and reduce hardware and operational costs.

The group is particularly involved with the recovery, purification and advanced formulation of bioproducts, including low molecular weight compounds (“Organics”), macromolecular species (proteins, polypeptides, enzymes, others) and supra-macromolecular constructs (“Nanoplexes”). Preferred separation methods are based on the principles of adsorption or partitioning, including continuous chromatography.

The group has an interest in the exploitation of fungal strains to produce secondary metabolites, enzymes, and other products by combining high throughput metabolomic/proteomic screening of compounds with a cutting-edge bioprospecting platform for the heterologous expression of entire fungal biosynthetic gene clusters (BGCs) in genetically tractable hosts. Fungus-based bioproduction deploys both submerged fermentation and solid-state fermentation strategies. Classical mutation-and-screening and synthetic biology approaches are employed to gain process productivity.

The Downstream Bioprocessing group also develops end-to-end solutions for valorizing, characterizing, modifying, and functionalizing industrially relevant hydrocolloids (polysaccharides and proteins) sourced from food industry side-streams or underexploited food biomass resources adopting the circular bioeconomy and clean/green label principles. The group designs new product innovation concepts based on soft matter (structurally and interfacially engineered bio-templates) to enhance the quality, organoleptic, shelf-life and bio-functional aspects of food, nutraceutical, and personal care product formulations.

Main expertise fields

Adsorption and Chromatography: Adsorbent screening and ligand design; packed bed and fluidized bed chromatography; continuous chromatography, including periodic counter-current (PCC) and true moving bed (TMB) systems; fibrous adsorbents, including moving belt systems.
Non-chromatographic methods e.g. Liquid-liquid extraction and Leaching: Partitioning in aqueous two-phase systems of cells and macromolecules; deep eutectic solvents; pilot extraction of natural compounds.
Fungal Biotechnology: Genome sequence-independent methods for the heterologous expression of intact fungal biosynthetic gene clusters (BGCs) in genetically tractable hosts; genome-scale metabolic modelling coupled with multi-omics analyses, classical mutation, and synthetic biology approaches for microbial strain and bioprocess improvement; scale-up of fermentation processes [SSF and SmF] from the bench to the pilot plant (up to 300 L scale).
Microbial Metabolomics and Fluxomics: Mass spectrometry-based metabolomics to assess the metabolic state of microbial cells and characterize key extracellular metabolic processes; determination of metabolic flux distributions and metabolic pathway activity of microorganisms using intracellular metabolite profiling, quantitative metabolomics and isotope-labelling data; chemical characterization of fungal secondary metabolites.
Advanced bioproduct formulation. The key technologies the group relyies on to provide solutions for the food, nutraceuticals and personal care product sector are as follows: a) Design and optimization of clean label methods for hydrocolloid extraction, isolation and purification, based on soft-processing i.e. physical or enzymatic processes; b) Hydrocolloid modification and functionalization for imparting novel technological (i.e. thickening, gelling, texturizing, air-water and lipid-water interface stabilizing, bulking) and bio-functional (digestibility, prebiotic activity, wall materials for controlled/sustained release of bioactive compounds, muco-adhesion, colon fermentability) characteristics; c) Design and optimization of soft matter (hydrogels, dry particulates, complex emulsion templates, xerogels, aerogels, 3D printed scaffolds, food composites etc.) -based product formulations for the food nutraceutical and personal care product industry.

Research challenges

Continuous biomanufacturing
Bioprocess development and scale-up
Bioprocess efficiency and techno-economic competitivity
Sustainable design of bioprocess scenarios
Eco-green isolation and modification of bio-macromolecules
Soft-matter inspired product development
Biodiscovery

Application AREAS

Food and nutraceuticals: functional foods, alternative proteins, and clean label food biopolymers
Cosmetics and para-pharmaceuticals, particularly when challenging formulation options are needed
Green chemical processing, as materialized via bioconversion and biocatalysis
Biorefining, with emphasis on product biorefining and biomass conversion
Biopesticides and biostimulants
Biomaterials and functional molecules [biopolymers].

Main assets

PROCEED project (2019 – 2023): Exploitation of plant seed mucilage in the development of protein based xero-scaffolds embedding human gut relevant probiotic cells (FNR- CORE/2018)
ALGRPO project (2021 – 2025): Impact of microalgal proteins on the adhesion properties, release profile and biological activity of microporous scaffolds hosting probiotic living cells (FNR-Industrial fellowships/2021)
DEEPCELL project (2022 – 2026): Exploration Of The Potential Of Deep Eutectic Solvent – Physical Processing Driven Strategies In The Production Of Functional Nanocellulose Pickering Particles (FNR-CORE/2021)
ROUTAKTIV project (2021 – 2022): Characterisation of a water-soluble antifungal compound produced by an endophytic fungus (Initialist)
BIOHOLISTICS project (2023 – 2025): Developing integrated bioprocesses for a holistic chemical recycling of plastics (FNR-FORMAS-SNF-WEAVE)
SHIELD project (2022 – 2023): Radical optimisation of fluidised bed adsorption systems via polymer shielding (Initialist)
JUMPRO project (2023 – 2025): A Fluidized Bed Riser Adsorption System for Selective Protein Recovery (FBRAS) (FNR)

equipment

Oscillatory rheometer
Laser static light scattering and dynamic light particle size analysers
Light and confocal laser scattering microscopes
In-vitro digestion system based on sequential mini-reactors
High pressure and ultra-high-pressure homogenizers
3D bioprinter
Microplate reader
Turbovap® evaporator
Rotary evaporator
Bioreactors (0.5 L-300 L, stirred- and wave-tank, with and without illumination)
Bioreactor for solid state fermentation
Ultrasound devices
Freeze dryers
Vacuum oven
Controlled atmosphere/temperature incubators
Gas and liquid chromatographers coupled with high resolution mass spectrometers
Preparative and semi-preparative chromatographer
ÄKTA avant protein purification system
ÄKTA PCC continuous protein purification system

Selected publications

2022

Rathore, A.S., Fernandez-Lahore, H.M. In Focus: Continuous bioprocessing (2022) Journal of Chemical Technology and Biotechnology, 97 (9), pp. 2287-2289.
Schneider, A., Herlevi, L.-M., Guo, Y., Fernandez Lahore, H.M. Perspectives on adsorption technology as an effective strategy for continuous downstream bioprocessing (2022) Journal of Chemical Technology and Biotechnology, 97 (9), pp. 2305-2316.
Qasim, F., Diercks-Horn, S., Gerlach, D., Schneider, A., Fernandez-Lahore, H.M. Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture (2022) Journal of Food Science, 87 (10), pp. 4348-4362.
Hellebois et al. Freeze − thaw induced structuration of whey protein − alfalfa (Medicago sativa L.) galactomannan binary systems (2022) Food Hydrocolloids.
Lindsay, M. A.; Granucci, N.; Greenwood, D.R.; Villas-Boas, S.G. Fermentative production of volatile metabolites using Brettanomyces bruxellensis from fruit and vegetable by-products (2022) Fermentation 8, 457.
Berni, R.; Hausman, J.-F.; Villas-Boas, S.G.; Guerriero, G.Impact of Pseudomonas sp. SVB-B33 on stress and cell wall related genes in roots and leaves of hemp under salinity (2022) Horticulturae 8: 336
Jelley, R.E.; Lee, A. J.; Zujovic, Z.; Villas-Boas, S.G.; Barker, D.; Fedrizzi, B. First use of grape waste-derived building blocks to yield antimicrobial materials (2022) Food Chemistry 370: 131025
Lindsay, M. A.; Granucci, N.; Greenwood, D.R.; Villas-Boas, S.G. Identification of new natural sources of flavour and aroma metabolites from solid-state fermentation of agro-industrial by-products (2022) Metabolites 12: 157

2021

Hellebois et al., Cryotropic gel-forming capacity of alfalfa (Medicago sativa L.) and fenugreek (Trigonella foenum graecum) seed galactomannans (2021) Carbohydrate Polymers, 267(9) 118190
Oliviera, F.A.B.; Bang, K.W.; Zarate, E.; Kinzurik, M.; Chudakova, D; Ganley, A.R.D.; Villas-Boas, S.G. Aerial warfare: An inducible production of volatile bioactive metabolites in a novel species of Scytinostroma sp. (2021) Fungal Genetics and Biology 158: 103646
Harris, A.; Lindsay, M. A.; Ganley, A. R. D.; Jeffs, A.; Villas-Boas, S.G. Sound stimulation can affect Saccharomyces cerevisiae growth and production of volatile metabolites in liquid medium (2021) Metabolites 11, 605.

2020

Lee, A.J.; Cadelis, M.M.; Kim, S.H.; Swift, S.; Copp, B.R.; Villas-Boas, S.G.Epipyrone A, a broad-spectrum antifungal compound produced by Epicoccum nigrum ICMP 19927 (2020) Molecules 25: 5997.
Da Silveira, F…Villas-Boas, S.G.; Silveira W. Assessment of ethanol tolerance of Kluyveromyces marxianus CCT7735 selected by adaptative laboratory evolution (2020) Applied Microbiology and Biotechnology 104: 7483 – 7494

2019

Han, T.L.; Cannon, R.D.; Gallo, S.M.; Villas-Boas, S.G. A metabolomic study of the effect of Candida albicans glutamate dehydrogenase deletion on growth and morphogenesis (2019) Biofilms and Microbiomes 5: 13.
Pinu, F.R.; Tumanov, S.; Grose, C.; Raw, V.; Albright, A.; Stuart, L.; Villas-Boas, S.G.; Martin, D.; Harker, R.; Greven, M. Juice Index: an integrated Sauvignon blanc grape and wine metabolomics database shows mainly seasonal differences (2019) Metabolomics 15: 3.
Pinu, F.R.; Villas-Boas, S.G.; Martin, D. Pre-fermentative supplementation of fatty acids alters the metabolic activity of wine yeasts (2019) Food Research International 121: 835 – 844.
Teixeira, L.L.; Dörr, F.; Dias, C.T.S.; Pinto, E.; Lajolo, F.M.; Villas-Boas, S.G.; Hassimotto, N.M.A. Human urine metabolomic signature after ingestion of polyphenol-rich juice of purple grumixama (Eugenia brasiliensis Lam.) (2019) Food Research International 120: 544 – 552.
Souza, R.T.; McKenzie, E. J.; Jones, B.; de Seymour, J.V.; Thomas, M. M.; Zarate, E.; Han, T.L.; McCowan, L.; Sulek, K.; Villas-Boas, S.G.; Kenny, L.C.; Cecatti, J.G.; Baker, P.N. Trace biomarkers associated with spontaneous preterm birth from the maternal serum metabolome of asymptomatic nulliparous women – parallel case-control studies from the SCOPE cohort (2019) Scientific Reports 9: 13701.
Kang, K.; Bergdahl, B.; Machado, D.; Dato, L.; Han, T.; Li, J.; Villas-Boas, S.G.; Herrgard, M.J.; Forster, J.; Panagiotou, G. Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations (2019) GigaScience 8: 1–14.
Roager, H.M.; Vogt, J.K; Kristensen, M.; Hansen, L.B.S.; Ibrugger, S.; Markedahl, R.B.; Bahl, M.I; Lind, M.V; Nielsen, R.L.; Frokiar, H.; Gobel, R.J.; Landberg, R.; Ross, A.B.; Brix, S.H.; Jesper; M.; Anne S.; Sparholt, M.H.; Christensen, A.F.; Carvalho, V.; Holst, J.J; Rumessen, J.J.; Linneberg, A.; Sicheritz-Ponten, T.; Dalgaard, M.D.; Blennow, A.; Frandsen, H.L.; Villas-Boas, S.G.; Kristiansen, K.; Vestergaard, H.; Hansen, T.; Ekstrom, C.T.; Ritz, C.; Nielsen, H.B.; Pedersen, O.B.; Gupta, R.; Lauritzen, L.; Licht, T. R. Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial (2019) Gut 68: 83 – 93.

2018

Casu, F.; Pinu, F.R.; Stefanello, E.; Greenwood, D.R.; Villas-Boas, S.G. The fate of linoleic acid on Saccharomyces cerevisiae metabolism under aerobic and anaerobic conditions (2018) Metabolomics 14: 103.
Pinu, F.R.; Granucci, N.; Daniell, J.; Han, T-L.; Carneiro, S.; Rocha, I.; Nielsen, J.; Villas-Boas, S.G. Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test. (2018) Metabolomics 14: 43
Tumanov, S.; Pinu, F.R.; Greenwood, D.; Villas-Boas, S.G. The effect of free fatty acids and lipolysis on Sauvignon Blanc fermentation (2018) Australian Journal of Grape and Wine Research 24: 398 – 405.
Sherman, E.; Harbertson, J.F.; Greenwood, D.R,; Villas-Boas, S.G.; Fiehn, O.; Heymanne, H. Reference samples guide variable selection for correlation of wine sensory and volatile profiling data (2018) Food Chemistry 267: 344 – 354.
Hansen,L.B.S.; Roager, H.M.; Søndertoft, N.B.; Gøbel, R.J.; Kristensen, M.; Vallès-Colomer, M.; Vieira-Silva, S.; Ibrügger, S.; Lind, M.V.; Mærkedahl, R.B.; Bahl, M.I.; Madsen, M.L.; Havelund, J.; Falony, G.; Tetens, I.; Nielsen, T.; Allin, K.H.; Frandsen, H.L.; Hartmann, B.; Holst, J.J.; Sparholt, M.H.; Holck, J.; Blennow, A.; Moll, J.M.; Meyer, A.S.; Hoppe, C.; Poulsen, J.H.; Carvalho, V.; Sagnelli, D.; Dalgaard, M.D.; Christensen, A.F.; Lydolph, M.C.; Ross, A.B.; Villas-Boas, S.G.; Brix, S.; Sicheritz-Pontén, T.; Buschard, K.; Linneberg, A.; Rumessen, J.J.; Ekstrøm, C.T.; Ritz, C.; Kristiansen,K.; Nielsen, H.B.; Vestergaard, H.; Færgeman, N.J.; Raes, J.; Frøkiær, H.; Hansen, T.; Lauritzen, L.; Gupta, R.; Licht, T.R.; Pedersen, O. A low-gluten diet induces changes in the intestinal microbiome of healthy Danish adults (2018) Nature Communications 9: 4630.