Production of specialties for food, aquaculture and non-food applications via multi-product biorefinery of microalgae

Highlights of the EU FP7 project MIRACLES

Multi-product Integrated bioRefinery of Algae: from Carbon dioxide and Light Energy to high-value Specialties (2013-2017)

Hans Reith, Leen Bastiaens, Hans Kleivdal, Dorinde Kleinegris, Lolke Sijtsma, Philippe Willems, Carlos Unamunzaga, Elke Breitmayer, Macarena Sanz, René Wijffels

AlgaeBiorefineries for Europe, Brussels, 16-17 October 2017

Successful scale-up of the algae industry requires reduction of production costs and enhancement of the (economic) output.

MIRACLES addresses these challenges by:

● improving cost-effectiveness of algae production andprocessing through technology development

● development of multiple-product biorefinery for specialtiesfrom algae

● development of new products for food, aquaculture and non-food

Aims

The MIRACLES consortium: 26 partners

• 6 Universities, 5 Research Organisations, • 12 SME’s, 3 MNI’s incl. 11 end user companies in target sectors• In 6 EU countries + Norway + Chile.

● Production of biomass for biorefinery and application research using established production strains:● Nannochloropsis ● Isochrysis● Phaeodactylum● Scenedesmus

● Development molecular monitoring tools / product optimization strategies

● Pilot scale production

Algae production

Closed photobioreactors. 100% automated and Scada controlled. Induced accumulation of bioactive compounds.

FITOPLANCTON MARINO FACILITIES (2015)

CO2 concentration from the atmosphere

Aim: to become independent of CO2 point source (e.g. power plant, flue gas) >>more remote locations Based on Supported amine adsorption

Activities√ Sorbent screening (method & sorbent)

√ Sorbent characterisation√ CO2 adsorption capacity√ H2O co-adsorption√ Kinetics√ Sorbent stability√ Regeneration (Pure CO2 vs. enriched

air)√ Process & Contactor design√ Construction of Prototype √ Proof of Concept

Screening sorbents

Adsorption kinetics (H2O >CO2)

Contactor design and testing

Proof of concept ongoing

Installation fully operational

Evaluation of (semi) continuous operation (>1% CO2; > 1kg/day) coupled to algae cultivation ongoing

Update in lecture Wim Brilman + poster

Molecular tools to monitor and control production of target products during cultivation

• Molecular tools available for monitoring production inNannochloropsis gaditana:

• TriAcylGlycerides (TAG)

• Eicosa Pentaenoic Acid (EPA) ω3-essential fatty acid

• Envisioned use of the tools to optimize production:

• Real-time monitoring for appropriate timing of biomassharvesting to reach optimal productivity of TAG / EPA

• Potential use in selection programs for clones with higher capacityand/or efficiency to accumulate TAG and/or EPA

Lecture Carlos Infante

• Based on establishing correlationsbetween relative gene expressionlevels and productivity of a selectedproduct.

Concept: growth of algae in liquid foams

Foam generation controlled by foaming agents and gas distributors

Development Liquid Foam Bed PhotoBioReactor

Feature ConsequenceVery short light pathLimited amount of water in reactor

Low weight, low pressure drop,

Large interface surface areaIncreased gas residence time

High biomass concentration Reduced harvesting costs

Reduced construction costs, energy costs

Enhanced mass transfer CO2, O2

• Validation on lab scale achieved • Substantial reduction of energy use and costs projected

Membrane technology for harvesting and growth medium recycling

Harvesting and dewatering: major impact on energy use and costs

Water & nutrients: significant cost factor in large scale cultivation.

Aim: develop novel membrane technology for combined harvesting and medium recycling capable of saving water, nutrients, energy and costs

Submerged Membrane Algae Filtration tests

AlgaeCentrifugation

160-200 g/L0,2-2 g/L

Submerged membranereactor

10-20 g/L

Nannochloropsis gaditana

MAF1 (lab scale – 0,3 m²)

10 tests performed

MAF2 (semi-pilot – 1 m²)

>15 tests performed (100-600L)

MAF1e – continuous(semi-pilot– 1,3 m²)

2-stages

Outlook

Submerged membrane filtration technology is a promising approach for integrated algae pre-concentration & medium recirculation

Medium recycling (up to > 90%) is possible; no negative impact on algae growth observed

Technology positively evaluated at lab and semi-pilot scale both in batch & continuous mode

• Substantial reduction of operational costs is expected

Lecture Leen Bastiaens, + posters

Bioprospecting, screening, selection of strains

Environmentalsamples collected

Enrichmentscultures in lab

Clonal culturesplated (FACS)

Clonal isolatesestablished for

cultivationScreenedstrains

Candidatestrains

Sub-tropics,Gran Canarias, Spain 213 639 - 70 24 9

Altiplanic lagoon Antofagasta, Chile 69 360 19200 66 20 13

Arctic/Nordic, Bergen, Norway 58 110 10560 149 43 5

Total 337 1 109 38 400 285 87 27

Demonstration

Downstream processing

Application testing

SelectionIndustrial screening criteria

SamplingIsolation Cultivation Screening

BEA CODE STRAIN % BEA 0024 Rhodomonas sp 0,1%

TYPE Phaeodactylum tricornutum 0,2% BEA 0650 Tetraselmis striata 0,3% BEA 0664 Pyrocystis lunula 0,4% BEA 0136 Chlorococcum sp. 0,4% BEA 0620 Erythotrichia cf carnea 0,4% BEA 0097 Tetraselmis striata 0,8% BEA 0304 Dunaliella sp 1,3% BEA 0078 Tetraselmis convolutae 1,4% BEA 0163 Dunaliella sp 1,4% BEA 0808 Closteriun sp. 1,7% BEA 0124 Rhodomonas sp 1,9% BEA 0398 Picochlorum oklahomense 2,0% BEA 0615 Graesiella vacuolata 2,0% BEA 0788 Coelastrella saipanensis 2,1%

TYPE Scenedesmus obliquus 2,2% BEA 0274 Crucigeniella sp 2,3% BEA 0317 Desmodesmus sp. 2,4% BEA 0544 Coccomyxa sp 2,4% BEA 0406 Cosmarium sp 2,8% BEA 0316 Desmodesmus sp. 3,0% BEA 0364 Protodesmus globulifer 3,4% BEA 0069 Halochlorella rubescens 4,5%

TYPE Isochrysis galbana 7,1% BEA 0313 Sarcinochrysis marina 8,4%

TYPE Nannochloropsis gaditana 10,5% BEA 1048 Euglena sp. 22,0% BEA 0937 Euglena cantabrica 57,5%CONTROL Yeast 46% ß-Glucan 44,6%

Enrichment

Bioprospecting in extreme climatic conditions to identify novel algal strains with suitable product profiles.

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Characterisation of selected strains

• Characterisation on lab scale

• Best strains evaluated for pilot scale outdoor productivity in identical photobioreactors

Outlook

• New, validated production strains available

• Process strategies to maximize yields of oils and EPA in Nannochloropsis (based on gene studies and metabolic modeling)

Lecture Dorinde Kleinegris + posters

Complete, mild cell disruption via bead milling or homogenization effective. Operation bead mill optimized..

Suitable enzymes for processing identified

Novel extraction routes using Supercritical fluids, Gas Expanded Liquids, Pressurized Liquids

Membrane filtration (UF/DF) for protein refinery

Results Integrated in Multiproduct flowsheets

Algae oil produced and extracted on pilot scale

Lectures Lolke Sijtsma, Elena Ibanez + several posters

Achievements, innovations in biorefinery development

Soluble, functional proteins as potentialsubstitute for dairy proteins in food products

● Protein characteristics differ from otherprotein sources>> processing challenging

● Promising emulsifying properties shownin product tests using crude soluble proteinfractions

Proteins for food applications

Aquaculture will be main contributor to global seafood supply: 140 MT by 2050 (more than double in the next 15 years)

Aquaculture sector is constantly seeking novel, sustainable raw materials of high nutritional value

In the framework of MIRACLES partners EWOS and Sparos assessed the potential of microalgae as ingredients in aquafeeds

Application of microalgae in aquafeeds

• Diets for trials evaluating digestibility for seawater salmon and growth of small freshwater salmon prepared containing Nannochloropsis (disrupted*) and Phaeodactylum produced at Fitoplancton Marino.

• Reduced nutrient digestibility observed for the algae diets (esp. disrupted Nannochloropsis) compared to reference diet needs to be considered regarding nutritional value of these algae in salmon feed formulations.

• Disrupted Nannochloropsis and Phaeodactylum added at 8% of diet fed to smallfreshwater salmon (2.5g initial weight) over 8 weeks (flowchart below). Algae feeds were non-inferior for growth compared to reference diet.

Salmon trials evaluating digestibility in seawaterand growth in freshwater

*Wet algae paste went through two passes of high pressure liquid homogeniser at ILVO-Technology & Food, BENatural antioxidant used to provide extra protection to algae in algae diets only so combined algae/natural antioxidant effect compared against reference diet. Note that oxidation parameters (TBARS on all diets and PV on small fish diets) were not lower in algae diets compared to reference diet.

Demonstration of functional role of microalgae in gilthead seabream(major farmed species in the Mediterranean)

Diet with residual biomass of

N. gaditana and algal oil allowed the

successful replacement of

fishmeal and fish oil by 80% and 30%,

respectively

Diet with 1% P. tricornutum resulted in a short-term benefical modulation of several immune-related pathways

A consumer panel characterised algae-fed seabream as having a more vivid and typical external pigmentation

Demonstration of functional role of microalgae in Senegalese sole(an emergent high value species in Europe)

Diet with 1% of fucoxanthin-rich algae (P. tricornutum) improved immune response to stressful events in sole juveniles

Diets with 5% of a broken cells extract of P. tricornutum

did not affect growth, but significantly enhanced the

survival of sole larvae

Microalgae show great potential as functional ingredients in aquaculture feeds

Demonstrated benefits on:

• Animal welfare (stress resistance, immunity)• Consumer quality traits (pigmentation)• Reduce reliance on finite marine-harvested resources (e.g.

replacement of fish meal and fish oil)

Poster #40 M.Sardinha et al.

Protein hydrolysates (prepared by cell disruption + enzymatic hydrolysis) tested for bioactive properties: antioxidant, antimicrobial, ....

Potent inhibitors of food spoilage-related micro-organisms identified (longer lag phase, lower growth rate)

Different concentrations of extracts were added to a bacterial culture and the growth was monitored at 30 C directly (left) or after pre-incubation at 4 C (right), to mimick food storage conditions.

Harry Laan, Poster #30

Antimicrobial peptides

Development of a cosmetic product: facial cream

MICROENCAPSULATION

IGE: Carotenoids fraction ofIsochrysis galbana extract(fucoxanthin)

ACTIVE INGREDIENT 1 FUNCTIONALITY TESTS 2Human dermal fibroblasts (ATCC® PCS-201-012™)

CYTOTOXICITY ASSAY

PROOXIDANT TOXICITY Neutralization of ROS overproductioninduced by external agents (oxidantsand UV radiation) in human fibroblasts.Antioxidant effect up to 100 timesgreater than that of pure fucoxanthin

Selection of the optimal dose

Phospholipid complex formation

3

Stabilized active ingredient

ANTIOXIDANT PROTECTION

COSMETIC FORMULATION4

Esther de la Fuente, Natac

Development of microalgae-based resins for the wood-based panel industry

Range of algae species, materials tested: a) Untreated biomass b) fractions incl. proteins, residues after extraction of lipids.

Development of effective PF type resins with 30% phenol replacement by microalgae-derived materials.

Produce plywood panels that meet the criteria of the European standards for exterior applications (EN314.1 and EN314.2)

Development of bio-based, formaldehyde free covering material suitable for lamination of wood-based panels.

Electra Papadopoulou c.s., Poster #37

Produced batches bioplastic compound suitable for:

● Injection moulding

● Sheet extrusion

Up to 15% in compound replaced by algae

Acceptable mechanical and processing properties

Product development ongoing

Contact: Philippe Willems, exploitation officer Miracles

Biopolymers

Use of whole algae or processing residues Application in flooring, table tops, light covers, ...

Contact: Philippe Willems, VFT/Orineo

Decorative biomaterials VFT

Specialty fertilisers/plant growth promotors

• Isochrysis extract has rejuvenating and stress reduction effects in bioassays using wheat, corn seedlings

• stimulated growth rate • delayed leaf senescence• higher leaf expansion rate

• Phaeodactylum extract shows positive effect on Nitrogen-use efficiency of plants. Wheat and corn seedlings show increased growth rate at low nitrogen nutrition level.

• Relevant for improving plant quality, durability in logistics phase, and customer satisfaction.

• Product development underway

Poster #36, Daan Kuiper, Philippe Willems

Conclusions

The MIRACLES consortium has successfully developed and demonstrated:

● technological innovations that contribute to improving cost-effectiveness of algae production, harvesting and processing

● new value-added applications and product leads for food, aquaculture and non-food

We are ready for further development and commercialisation

For publications, final report, contacts see Project Website: http://miraclesproject.eu/

• The project is supported by the European Commission through the 7th Framework Program under Grant Agreement No. 613588.

• Program Officer DG RTD: Andrea Grisorio andrea.grisorio@ec.europa.eu

• More information: Hans Reith, hans.reith@wur.nl

• www.miraclesproject.eu

Acknowledgements

Thanks!

For more information visit our website and follow us on social networks:

www.miraclesproject.eu@miraclesproject

Project Website: http://miraclesproject.eu/

Coordinator: Hans Reith, Bioprocess Engineering, WU Agrotechnology& Food Sciences. Tel. +31 (0)317 480 100 , e-mail hans.reith@wur.nl

Exploitation Officer: Philippe Willems, Value for Technology bvba. Tel.: +32 (0)2 759 33 10. e-mail phw@value-for-technology.be

Dissemination Officer: Macarena Sanz, IDAction and IDConsortium, Tel. +34 911 25 03 11. e-mail msanz@idaction.es

EC Project Officer: Andrea GRISORIO, European Commission, DG Research & Innovation. F2. Bio-based products and processing., Tel.: +32 229-90538. e-mail: Andrea.GRISORIO@ec.europa.eu

Contacts