Biorefjnery development for the production of succinic acid and - PowerPoint PPT Presentation

Biorefjnery development for the production of succinic acid and value- added coproducts from sunfmower meal M.N. Efthymiou, C. Pateraki, H. Papapostolou, A. Koutinas mneuth@gmail.com Heraklion 06.2019

Sunfmower Meal (SFM) Production Crude Food Oil Sunfmow er Seeds Biofuel s De- Presser hulling / Flaking E stim ated production (M T ) Expelle Cooking r SFM Solvent Extracti on Defatted SFM Crude Oil European Union Global 25 Animal 20,7 Feed 19,3 20 15 10 4,8 5 3,7 0 Sunflower Oil Sunflower Meal Source:https://apps.fas.usda.gov/psdo nline/app/index.html#/app/advQuery

Succinic acid Solvents • C4-dicarboxylic acid Adipic acid • Conventional production via Polymer hydrogenation of maleic es e.g. 1,4 anhydride PBS Butanediol • Bio-based production is Maleic 50% of the total anhydrade Succinic acid • Glucose syrup and glycerol are the main carbon Maleic sources used in industry acid Food & beverages Formic • Basfja succiniciproducens , Detergents acid engineered Escherichia coli & and yeast are the main Surfactans strains used • Market size expected to reach 48.7 Million US $ by 2024 2

Composition of sunfmower meal Composition (g/g % db) This Study Literature Moisture a 7.6 ± 0.03 3.7-10.4 T otal Solids 92.4 ± 0.03 89.6-96.3 Protein 33.7 ± 0.50 26.6-41.8 T otal phenolics (CAE) 3.0 ± 0.04 2.4-4.7 Oil 0.1 ± 0.06 0.9-2.0 Ash 7.4 ± 0.11 4.3-9.0 T otal Hemicellul phenolic ose Moisture Pectins 5.7 ± 0.20 4.9 compound 10,83% 7,67% s Lignin 12.2 ± 0.03 5.9-11.4 Cellulose 2,99% 13,83% Protein Cellulose 13.8 ± 1.83 14.9-23.0 33,84% Lignin Hemicellulose 10.8 ± 1.92 7.5-12.9 12,20% Free Pectins Oil Ash T otal Free Sugars 5.3±0.03 - Sugars 5,70% 7,44% 0,17% 5,34% Sucrose 4.6 ± 0.02 - Glucose 0.5 ± 0.04 - Xylose 0.1 ± 0.01 - Galactose 0.2 ± 0.10 - IP b 63.6 ± 2.33 - FAN b 28.8 ± 0.12 - a wet basis b mg/100g db 3

Biorefjnery approach Total Crude Phenolic Phenolic SFM Extract content 100 g Ultrasound 8.2 g 33% ( CAE) Phenolic Protein rich Solid Extraction Residue EtOH: H 2 O 1:1 91.8 g 1/10 w/v Protein Extraction & Precipitation Cellulose rich Solid Residue Solid Residue Pretreatmen Protein 19.6 g 72.7 g t Concentrate Η2SO4 1.25% Hydrolysis w/v Recovery NaOH 2% w/v Crude enzymes 17% Aspergillus awamori Purity 80% Total Sugar Sugar rich concentration Hydrolysate 15.7 g/L Concentration 40 Fermentation Succinic acid g/L Actinobacilus succinogenes Productivity 0.48 g/L/h

Solid State Fermentation - Enzyme Activity Enzymes • Cellulases – CMC 1 % w/v, pH 5, 50 o C, 30 Aspergillus min awamori • Xylanases – Xylane 0.25 % w/v, pH 5, 50 Xylanas o C, 60 min es Cellulass • Proteases - Casein 0.75% w/v, pH 7, 50 es Phytase o C, 30 min s • Phytases - Phytic acid 0.2 % w/v, pH 5. Defjnition of enzyme activity units 50 o C, 15 min Conditions • Proteases & Phytases: • 5 g SFM amount of enzyme needed for the production of 1 μg Free Amino Nintrogen (FAN) or Inorganic • 1.0 × 10 6 spores/g Phosphorus (IP) per minute solid • 30 o C • Cellulases & Xylanases amount of enzyme needed for the production • Initial moisture of 1 mg Glucose or Xylose per minute content 50, 55, 60 & 65 %

Solid State Fermentation - Enzyme Activity E nzy m e a ctiv ity U/g SFM E nzy m e a ctiv ity U / g SF M  Proteases  Cellulases  107.2 U/g after 94 h  3.7 U/g after 94 h  Moisture content 60%  Moisture content 65% 4 120 3,5 100 3 50 80 2,5 % 2 60 55 1,5 % 40 1 20 0,5 0 0 Enzym e activity U / g SFM 0 25 50 75 100 125 150 0 25 50 75 100 125 150 Fermentation Time (h) Fermentation Time (h) E nzy m e a ctiv ity U/g SFM  Xylanases  2.7 U/g after 94 h  Moisture content 60% Phytases 512.7 U/g after 94 h Moisture content 60% 600 3 500 2,5 400 2 300 1,5 200 1 100 0,5 0 0 0 25 50 75 100 125 150 0 25 50 75 100 125 150 Fermentation Time (h) Fermentation Time (h)

Enzymatic Hydrolysis - optimization Efgect of T emperature  Hydrolysis yield: 40.5% Cellulose; 61.1% Hemicellulose  Temperature 50 °C H y d ro ly sis y ie ld % (g /g ) 70 60 50 40 30 20 10 15.7 g/L 0 45 50 55 T emperature (oC) Cellulose Hemicellulose Glucose Xylose T otal sugars

Enzymatic Hydrolysis - optimization Efgect of initial SFM concentration Increasing concentrations of SFM led to decreased hydrolysis yields : To ta l S u g a rs p ro d u c e d (g /g d b )  Cellulose: 17.4 – 3.9 % H ydrolysis yield % (g/g)  Hemicellulose: 27.7 – 5.6 % 70 60 0,35 50 0,30 0,25 40 0,20 30 0,15 20 0,10 10 0,05 0 50 75 100 0,00 0 20 40 60 80 100 SFM Concentration (g/L) Hydrolysis Time (h) Cellulose Hemicellulose 50 g/L 75 g/L 100g /L

Fed-batch fermentation for succinic acid production Conditions  Actinobacillus succinogenes  Temperature: 37 o C  Initial sugar concentration : 60 g/L  Feeding up to 30 g/L  Succinic acid concentration: 40 g/L  Yield: 0.53 g succinic acid/g consumed sugars  Productivity: 0.48 g/L/h Total Sugars Succinic

Conclusions Sunfmower meal was efgectively valorized through the development of an advanced biorefjnery concept for the production of proteins, phenolic compounds and succinic acid:  Phenolic rich extract (33% CAE)  Protein Concentrate (80 % purity)  Succinic acid production  40 g/L  Yield: 0.53 g succinic acid/g consumed sugars  Productivity: 0.48 g/L/h

Acknowledgements Thank you for your attention!!