PHAs with advanced properties, adequate to serve as food packaging materials from exploitation of food wastes and residues

Transcription

1 FORTH/ICE-HT activities towards bio-based materials for the FUTURE of Bio-Based-Industries based on Breakthrough technologies PHAs with advanced properties, adequate to serve as food packaging materials from exploitation of food wastes and residues George Voyiatzis Research Director, Biological Filters Catalysts for Green Diesel Production Metabolic Engineering, Synthetic and Systems Biology Polymers and Coatings with Specific Functionalities Safety Analysis

2 Polyhydroxyalkanoates (PHAs) Linear homo- or hetero-polyesters of hydroxyacids that are accumulated by numerous bacteria under unbalanced nutrients supply, as reserve of carbon and energy source as inclusion bodies ( μm) m = 1-13 x = 1-4 n = *10 4 short-chain-length (SCL) up to C 5 hydroxy-acids medium-chain-length (MCL) C 6 -C 16 hydroxyacids PHAs intracellular granules formed in Pseudomonas putida (Ward et al., 2005, Appl Env. Microb. 72: ) x=1 C m H 2m+1 = H poly(-3-hydroxypropionate) P3-HP CH 3 poly(-3-hydroxybutyrate) P3-HB C 2 H 5 poly(-3-hydroxyvalerate) P3-HV C 3 H 7 poly(-3-hydroxyhexanoate) P3-HHx C 5 H 9 poly(-3-hydroxyoctanoate) P3-HO x=2 C m H 2m+1 = H poly(-4-hydroxybutyrate) P4-HB x=4 C m H 2m+1 = H poly(-6-hydroxyhexanoate) P6-HHx Aldor and Keasling, 2003, Curr Op Biotechn 14: PhaA : beta ketotheolase PhaB : acetoacetylcoa reductase PhaC : PHA synthase PhaG : 3-hydroxylCoA tranferase PhaJ : (R)-EnoylCoA hydratase

3 Feedstocks for sustainable PHAs production Although the expanding interest shown the previous decades for the commercialization of bio-based biodegradable polymers, there are still different issues to be addressed in order PHAs to become antagonistic in the market, since the market price of fossils bioplastics is up to 10 times lower compared to that of PHAs. The cost of the chosen carbon source for PHAs production can be a hurdle for their commercialization Carbohydrates, fatty acids and lipids can easily be converted by various bacteria towards scl or mcl PHAs Wastes and residues of low or zero cost such as food wastes, can be used as feedstocks by PMC or MMC. Recovery of carbon sources via fractionation Acidification of carbohydrate content for SCFAs production such as butyrate, valerate, hexanoate etc., i.e. precursors for productions of heteropolymers Since the production of PHAs is actually based on the bio-transformation of carbohydrates, fatty acids and consequently lipids, substances that are quite abundant such as food wastes could be an ideal feedstock. Type Carbon source Pretreatment Domestic FW OMW, cheese whey (agroindustrial FW) Industrial FW Lignocellulosic biomass Sugars Starch cellulose, hemicellulose Lipids and oils cellulose, hemicellulose Direct accumulation Hydrolysis needed Hydrolysis needed Direct accumulation Removal of lignin and hydrolysis needed Acidification Fermentation of carbohydrate based wastes via acidogenic microorganisms Fatty acids

4 0,5 0,0-0,5-1, Temperature ( o C) Two stage PHAs production system based on MMCs H 2 + CO 2 1 st stage : anaerobic acidification Reactors Inoculum CSTR double jacket stainless steel, V w : 0,5L/20L, 35±1 o C, periodic strirring Up-flow Column Bioreactor double jacket, plexiglass,v w : 1,5L 35±1 o C, attached growth Mesophilic activated sludge from Municipal Wastewater Treatment Plan, heat treated (100 o C, 20min) Feedstock OMWW Waste glycerol Optimization by altering operational conditions SCFAs generated Hydraulic Retention Time :7.5h-60h Acet, But, Prop HRT : 12h-48 h Organic Loading Ratio :8-30g d 1 L 1 ph feed : 6-7 Acet, But, Hex 2 nd stage : aerobic PHAs production Waste Glycerol MMC SBR Reactors Borosilicate glass, V w : 1,5L Plexiglass, V w : 30L Inoculum Operational phases (OP) Feedstock Optimization 1 st Stage : Anaerobic Fermentation Type of PHAs produced Microbial Biomass SCFAs PDO Glycerol MMC 2 st Stage : Aerobic Polymerisation Enriched aerobic mixed culture from activated sludge or soil Enriched aerobic mixed culture from activated sludge phases: a) growth phase, N supply (carbon limitation), b) biomass settling phase (no aeration and stirring) c) supernatant withdrawal (2/3 of V w ), d) accumulation phase, C supply (nitrogen limitation), e) withdrawal of 2/3 of V W under agitation for PHAs extraction a. Clarified OMWW b. Acidified OMWW c. Waste glycerol d. Acidified Waste glycerol Acidified clarified OMWW Organic loading, Relative duration of OP, ph a. PHBHV, PHBHVHxx b. PHBHV, PHBHVHxx c. PHB, d. PHBHHx Microbial Biomass PDO RECOVERY PHBHO SOLVENT EXTRACTION H O R O Heat Flow (W/g) m n OH - FORTH/ICEHT - Laboratory of Environmental Biotechnology -

5 Evaluation of physicochemical properties Composition and structure Gas chromatography Nuclear Magnetic Resonance spectroscopy Molecular masses Size Exclusion Chromatography Thermal properties and stability Thermogravimetric analysis Differential scanning calorimetry Mechanical Properties Stress strain tests Dynamic Mechanical Analysis Assessment of properties Effect of aging Assessment of changes on PHAs films after artificially induced aging Controlled periodic alteration of combined UV radiation, temperature and humidity variation, simulating thus extreme environmental conditions. Periodic removal of PHA samples Chemical structure (FTIR) Transition and decomposition (TGA, DSC) molecular masses and polydispersity (SEC) mechanical properties by a stress-strain tests Biodegradability assessment Anaerobic degradation tests using mixed cultures Small scale batch experiments Mesophilic anaerobic conditions PHAs as the sole C source Urea as N source % Reidual film Relative Humidity [%] Temperature[ C] Note: Temperature and Humidity curves are estimated t 0 t 7 t 15 t 30 Preconditioning Time [hrs] Wavelength (cm -1 ) time (h) UV [nm] up to 1008 hrs PHBHV PHBHH

6 On going and Future research Exploitation of agro-industrial and industrial food wastes for PHAs production with advanced properties in a concept of circular economy 1. Production of selected PHAs precursors by : Recovery and separate exploitation of different carbon sources Fractionation of feedstock via physicochemical methods Manipulation of anaerobic metabolism during acidification process in order to maximize certain types of SCFAs Application of different fermentation strategies (types of reactors, operational conditions) 2. Production of PHAs products suitable for food packaging applications by: Manipulation of metabolism of PMCs and MMCs Application of different nutrient limitations strategies and feeding strategies (periodic alteration of carbon sources, different mixtures of precursors etc) Molding tests using PHAs based materials (co-polymers, blends, composites) recognition of behavior on demand production for specific applications - FORTH/ICEHT - Laboratory of Environmental Biotechnology -

7 Representative relevant publications of FORTH on the production of Microbial Bioplastics from Food Wastes Ntaikou I. *, Koumelis I., Tsitsilianis C., Parthenios J., Lyberatos G. Comparison of yields and properties of microbial polyhydroxyalkanoates generated from waste glycerol based substrates Int. J. Biol. Macromol., 2018, 112: Kourmentza C. *, Ntaikou I., Lyberatos G., Kornaros M. PHA production by mixed and pure cultures of Pseudomonas sp. using synthetic and olive mill wastewater under nitrogen and dual nitrogen-oxygen limitation. Int. J. Biol. Macromol., 2015, 74: Ntaikou I. *, Valencia Peroni C., Kourmentza C., Ιlieva V.I., Morelli A., Chiellini E., Lyberatos G. Microbial bio-based plastics from olive-mill wastewater: generation and properties of poly-hydroxyalkanoates from mixed cultures in a two-stage pilot scale system. J. Biotechnology. 2014, 18: Kourmentza C., Ntaikou I., Kornaros M. * Production of PHAs from mixed and pure cultures of Pseudomonas sp with short chain fatty acids as carbon source under nitrogen limitation. Desalination 2009, 248: Ntaikou I. *, Kourmentza C., Koutrouli E., Stamatelatou K., Zampraka A., Kornaros M., Lyberatos G. Exploitation of olive oil mill wastewater for combined bio-hydrogen and biopolymers production. Bioresource Technology 2009, 100: Mosquera, A., Fra, A., Palmeiro, T, Carvalho, G., Ntaikou I., Oleskowicz-Popiel P., Reis M. A. M. and Suárez-Ojeda M.E. Recovery of organic added value products from wastewater (chapter 18). In: Innovative Wastewater Treatment & Resource Recovery Technologies: Impacts on Energy, Economy and Environment, Editors: J. M. Lema, S. Suarez Martinez, IWA publishers, ISBN13: , 2017, Pages: Contact with LABORATORY OF ENVIRONMENTAL BIOTECHNOLOGY members: Gerasimos Lyberatos: Professor, Department of Chem. Engineering, NTUA, Athens Collaborating faculty member of FORTH/ICE-HT; lyberatos@chemeng.ntua.gr and Ioanna Ntaikou: Research Scientist at FORTH/ICE-HT; ntaikou@iceht.forth.gr & Georgia Antonopoulou: Research Scientist at FORTH/ICE-HT; geogant@chemeng.upatras.gr THANK YOU FOR YOUR ATTENTION