Encontro Ciência-2017, 4th July, Lisboa INTERNATIONAL COOPERATION Indo-Portuguese Programme for Cooperation in Science & Technology Developing magnetic nanoparticle immobilized enzyme catalysts for biofuel applications Suzana Ferreira-Dias & Rajeev K. Sukumaran (2017-2019) Encontro Ciência-2017, 4th July, Lisboa

Tapada da Ajuda, Lisbon, Portugal School of Agriculture, University of Lisbon Portuguese Team: • SUZANA FERREIRA-DIAS, PhD, 1994 (PI) • HELENA PEREIRA, PhD , 1976 (Co PI) • JORGE GOMINHO, PhD, 2004 • ISABEL MIRANDA, PhD, 2000 • CARLA TECELÃO, PhD, 2011 • JOANA RODRIGUES, PhD, 2016 • DUARTE NEIVA, MSc, 2009 (PhD student) ISA Tapada da Ajuda, Lisbon, Portugal http://www.isa.ulisboa.pt

Indian Team: • RAJEEV SUKUMARAN, PhD, 2002 (PI) • K. MADHAVAN NAMPOOTHIRI, PhD, 1997 (Co-PI) • KV PRAJEESH, MSc, 2013 (PhD student)

Objectives: Valorization of residual and waste cellulosic materials to produce biofuels and added-value compounds, using magnetic nanoparticles (MNP) immobilized enzymes, under the Biorefinery concept.

Valorization of residual and waste cellulosic materials: Rice straw (India) (c.a. 35-40 % cellulose, 15-30 % hemicelluloses, 16-21 % lignin) Olive pomace (Portugal) (by-product of olive oil extraction, c.a. 370 000 t/y in PT, with 3.3 % oil and 40 % lignocellulosic material) De-oiled olive pomace

Enzyme-Catalyzed Reactions Advantages: Act under mild conditions (P = 1 atm; T < 80 ̊C) No need for co-factors High Specificity and Selectivity Selectivity sn-1,3 in TAG (some lipases) Energy saving Absence of side-reactions

Enzyme-Catalyzed Reactions Constraints: Inactivation of the biocatalyst (oxidation products; inorganic acids, phosphorus and Ni soaps; methanol, glycerine, etc.) High price Reutilization Immobilization in solid supports Enzymatic processes are still more expensive than chemical processes. Search for “low-cost” biocatalysts with high activity and operational stability.

Magnetic Nanoparticles (MNP) Immobilized Enzymes Easy recovery Easy reuse Increased stability Cost-effective Biomass Conversion Technology Ren et al. BMC Biotechnology 2011, 11:63

MNP immobilized enzymes: MNP- Betaglucosidases (BGL) MNP-Cellulases Glucose Bioethanol Cellulose hydrolysis (ligno-cellulosic substrates: rice straw or de-oiled olive pomace) Lignin Green chemicals Transesterification Olive pomace oil + 2Methanol MNP- Lipases sn-1,3 regioselective 2 FAME + Monoacylglycerols (Biodiesel) (MAG- Emulsifiers) (To avoid glycerol production) Legend: FAME = Fatty Acid Methyl Esters

Biorefinery Concept

Cost-effective Biomass Conversion Technology Specific Objectives MNP production and Enzyme immobilization: Production of ferro- fluid type magnetic nanoparticles (MNPs), surface modifications and characterization. Covalent immobilization of enzymes on MNP, optimization of immobilization process, activity testing and establishment of reusability. MNP-enzyme catalyzed reactions: BGL performance in biomass (Rice straw or de-oiled olive pomace) hydrolysis MNP-sn-1,3 regioselective lipases in transesterification of olive pomace oil (or rice bran oil) for biodiesel and monoglycerides (MAG; food and pharmaceutical emulsifiers). Operational stability of MNP-immobilized enzymes.

Cost-effective Biomass Conversion Technology By-products: To look into the byproducts of biomass hydrothermal treatment, such as xylo-oligosaccharides of nutritional or pharmaceutical importance from selected Biomass (de-oiled olive pomace or rice bran/straw) and MAG production. Scale-up of Enzymatic Processes: Formulation of enzyme cocktails with MNP BGL conjugates and their evaluation and optimization of biomass hydrolysis (at least at 0.5 kg rice straw biomass or de-oiled olive pomace) using these enzyme blends. Scale up of MNP-sn-1,3 regioselective lipase mediated reactions and demonstration of the transesterifcation process of (500-1000 ml) olive pomace oil with methanol for biodiesel.

Acknowledgements To the Organizing Committee of Ciência-2017 for the invitation to deliver this presentation. All of you for your time.