From waste-biomass to biodegradable plastics I will be present here today an overview of the projects our group is currently involved. Our main focus is the upgrade of the nutrients in waste materials for the production of value-added products. This is typically CIRCULAR ECONOMY, which aims at the recycling of resources and at a zero waste situation. M. Teresa Cesário, Štěpán Tůma, J. Kepa Izaguirre, Mafalda Marques, Pedro Fernandes and M.Manuela da Fonseca iBB, Bioengineering Department IST - University of Lisbon, PORTUGAL

Sustainable feedstocks and processes World population: 2018: 7 billion (7 E09) 2050: 10 billion (10 E09) people Pressure to increase: Food/ feed sources Fuels Chemicals/ materials Sustainable feedstocks and processes Decrease CO2 emissions (decarbonization) Circular economy zero waste strategy M. Teresa Cesário, iBB Técnico-Lisboa

Carbon platform: waste biomass Goal 12: Responsible production and consumption Terrestrial Marine Target 12.5:  By 2030, substantially reduce waste generation through prevention, reduction, recycling and reuse. Wheat straw OFMSW : Organic fraction of the Municipal Solid Waste Gelidium residues SDG: sustainable development goals Target 12.5 OFMSW : The waste feedstock was composed of domestic and garden wastes. OFMSW was collected from the local composting plant EPELE (Gipuzkoa, Spain) Explicar que parajá estamos a trabalhar com Ulva inteira, mas a ideia para o futuro é utilizar resíduos (após extraccao da proteína – U Wag) GOAL 14: Conserve and sustainably use the oceans, seas and marine resources for sustainable development Ulva lactuca residues

Product: Bioplastics (polyhydroxyalkanoates) Polyhydroxyalkanoates (PHAs) are biopolymers synthesized and accumulated intracellularly by over 200 microbial strains; Properties similar to petroleum-derived plastics; Biodegradable, even in marine environment Applications in agriculture, medicine and packaging. P (3HB) has properties similar to PP

Lignocellulosic agricultural residues Lower the PHA production costs associated with the C-source Use a residual, renewable, non- edible feedstock Wheat straw AFEX + enzymatic hydrolysis glucose (g/L) xylose arabinose  465.3  146.3 41.5 445.1 206.1 35.4 628.3 314.8 47.6 485.1 347.0 44.6 585.0 488.0 42.0 Abundant feedstock High carbon content

Results Fed-batch cultivation on wheat straw hydrolysates Burkholderia sacchari DSM 17165 (  70 % (g PHB/ g CDW)) Dr. Antonio A.Matos , CIIEM-ISCSEM, Monte de Caparica, Portugal P(3HB) and copolymer P(3HB-4HB) volumetric productivities are the highest ever achieved on agricultural waste hydrolysates. B. sacchari is able to consume xylose at a fairly good rate Production (g/L) Productivity (g.L-1.h-1) P( 3HB) 94 1.6 P(3HB-co-6% 4HB) 24 0.5

Seaweed as carbon platforms

Why seaweeds as C-platforms ? ADVANTAGES No competition for arable land High carbohydrate content (up to 80 % dw ) No fresh water consumption No lignin IMTA –Integrated Multi-Trophic Aquaculture = integration of aquaculture with seaweed farming GHG: green house gases Naturally abundant feedstock High photosynthetic rates Cesário M.T. et al. (2018), Marine algal carbohydrates as carbon sources for the production of biochemicals and biomaterials. Biotechnology Advances 36, 798-817

Biomass characterization Parameter Value Unit Method Dry-weight (DW) 93.90 ± 0.83 (%) NREL 60956 Moisture 6.10 ± 0.83 Proteins 0.68 (% dw) Kjeldahl method Lipids 3.36 Idem Soxhlet method Ash 16.39 ± 1.15 Carbohydrates 44.80 ± 1.90 NREL 60957 Agar 7.3 NREL60957 Cellulose 37.05 ± 0.02 Starch 0.80 ± 0.33 Megazyme Lyophilized Gelidium residues after agar extraction 16%

Selected microorganism Moderately halophilic bacteria that are good PHA-producers DSM 15516 Appropriate for Continuous Process Development Reduce contamination risk Allow the use of non-sterile conditions Halomonas boliviensis DSM 15516 tolerates up to 25 % (w/w) NaCl (NaCl opt = 4-5 % NaCl) grows at pH 6-11 (pH opt = 7.5-8.0) produces PHAs on glucose and galactose Halomonas boliviensis DSM 15516 (http://www.biotek.lu.se/)

Hydrolysis of Gelidium polysaccharides MILD ACID HYDROLYSIS ENZYMATIC HYDROLYSIS Milled seaweed residues Algal hydrolysate Sulfamic acid , 121°C, 30 min Cellulolytic enzymes 23 .1 g/L glucose, 0.01 g/L HMF; no galactose HMF < 0.1 g/L

PHA production from Gelidium hydrolysate Glucose (20 g/L) Gelidium hydrolysate (20 g/L glucose) CDW= 10.8 g/L PHB = 5.0 g/L % PHB= 46.7 % CDW= 8.7 g/L PHB = 3.3 g/L % PHB= 38.0 %

Conclusions Gelidium residues are sugar-rich feedstocks that can be upgraded to biomaterials: Gelidium residues still contained 44 % (dw) carbohydrates: 37 % cellulose 7 % agar Produced sugar-rich Gelidium hydrolysates with low inhibitor concentrations Successful production of PHB based on algal hydrolysates but lower values when compared to commercial sugars. Hydrolysate composition under study (C, N, P content). We aim to find the right conditions to achieve very high productivities with algae hydrolysates as we have with wheat straw: optimize hydrolysate production and composition and PHA production conditions in the bioreactor.

Acknowledgements The research leading to these results has received funding from the FCT projects (PTDC/BII-BIO/29242/2017) and (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317). We are particularly thankful to: Iberagar – Sociedade Luso-Espanhola de Colóides Marinhos SA for supplying Gelidium sesquipedale residues.

Thank you for your attention