Bioprocess development for the production of succinic acid from - PowerPoint PPT Presentation

Bioprocess development for the production of succinic acid from orange peel waste Maria Patsalou 1 , Chrysanthi Pateraki 2 , MarlenVasquez 1 , Chryssoula Drouza 3 and Michalis Koutinas 1 1 Department of Environmental Science & Technology, Cyprus University of Technology 2 Department of Food Science and Human Nutrition, Agricultural University of Athens 3 Department of Agricultural Sciences, Biotechnology and Food Science, Cyprus University of Technology Limassol , 201 6

Citrus fruits  88 x 10 6 tn worldwide production  Industrial OPW generation: 15 x 10 6 tn / y  Oranges: 82 % of the total production  50% of fruit is peel waste  Animal feed  Disposal in landfills

Orange peel waste  Peels  Composition of peel *  Seeds  Segment membranes Components % Dry mass Soluble sugar 16.90 Starch 3.75 Cellulose 9.21 Hemicellulose 10.50 Lignin 0.84 Pectin 42.50 Ash 3.50 Fat 1.95 Protein 6.50 Others 4.35 *Lopez et al ., Crit Rev Biotechnol. , 2010; 30(1): 63-69

Succinic acid  Di-carboxylic acid  Important biobased platform chemicals ◦ Polybutylene succinate (PBS) ◦ Polybutylene succinate-terephthalate (PBST) ◦ Polyester polyols ◦ Food industry ◦ Pharmaceutical industry ◦ Production of resins, coatings and pigments  Chemical technologies ◦ Catalytic hydrogenation ◦ Paraffin oxidation ◦ Electrolytic reduction of maleic acid or anhydride  High theoretical yield  Environmental friendly impact

Succinic acid producers ◦ Mannheimia succiniciproducens ◦ Anaerobiospirillum succiniciproducens ◦ Basfia succiniciproducens  Actinobacillus succinogenes ◦ Isolated from bovine rumen ◦ Capnophilic ◦ Mesophilic ◦ CO 2

Proposed Flow Diagram Orange peel waste Extraction of essential oils Enzymes Dryer Anaerobic ( T. reesei ) digestion solid solid Acid Enzyme hydrolysis hydrolysis liquid liquid Fermentation of Extraction of hydrolysate pectin

Aim and Objectives  Preliminary study for the development of an OPW bio-refinery to produce succinic acid Release of metal ions after acid hydrolysis and 3 1. acid/enzyme hydrolysis Dilute-acid hydrolysis conditions 2.  SA Fermentations, simple sugars  Selection of conditions  Presence of HMF in hydrolysates  SA Fermentations, dilute-acid hydrolysates Optimal cultivation time for cellulolytic enzyme 3. production by T. reesei

Elemental analysis (ICP-MS) of Hydrolysates Acid hydrolysis 1,4 160 1,2 140 1 120 C (mg /L) C (mg/L) 0,8 100 80 0,6 60 0,4 40 0,2 20 0 0 24Mg 44Ca 56Fe Elements Elements Acid and enzyme hydrolysis 1,4 160 1,2 140 1 120 C (mg /L) C (mg /L) 0,8 100 80 0,6 60 0,4 40 0,2 20 0 0 24Mg 44Ca 56Fe Elements Elements

Succinic acid production from simple sugars fermentation 7 Glucose Organic acids con. (g L -1 ) 6 5 4 Succinic acid 3 Formic acid 2 Lactic acid Acetic acid 1 0 37 o C 0 10 20 30 pH 7.5 Time (h) 30 gL -1 MgCO 3 5 gL -1 yeast Fructose 10 gL -1 initial sugar 4 Organic acids con. (g L -1 ) 0.5 vvm CO 2 3 Succinic acid 2 Lactic acid Formic acid 1 Acetic acid 0 0 10 20 30 Time (h)

Consumption of each simple sugar, Yields of fermentations 12 10 Sugar con. (g/L) 8 6 Fructose Galactose 4 Glucose 2 0 0 5 10 15 20 25 30 Time (h) Sugar SAYield (g p /g s ) OA Yield (g p /g s ) Glucose 0,57 1,26 Fructose 0,33 1,10 Galactose - - Glucose (Bioreactor) 0,66 1,10

Dilute-acid hydrolysis conditions ◦ 100-120 o C, fructose* ◦ >120 o C , arabinose and galactose*  116 o C, 10min, 5%  116 o C, 10min, 10%  116 o C, 20min, 5%  116 o C, 20min, 10%  109 o C, 10min, 5%  109 o C, 10min, 10%  109 o C, 20min, 5%  109 o C, 20min, 10% *Grohmann et al ., Bioresour. Technol., 1995; 54: 129-141

Release of sugar of dilute-acid hydrolysis NMR analysis Y (gts/grm) 0,12 109 oC, 20 min, 10% 109 oC, 10 min, 10% 0,12 116 oC, 20 min, 10% 0,12 116 oC, 10 min, 10% 0,09 109 oC, 20 min, 5% 0,18 109 oC, 10 min, 5% 0,19 116 oC, 20 min, 5% 0,19 116 oC, 10 min, 5% 0,21

Concentration of inhibitors of dilute-acid hydrolysate HMF (g/L) 109 oC, 20min, 10% 0,0173 109 oC, 10min, 10% 0,0150 116 oC, 20min, 10% 0,0189 0,0187 116 oC, 10min, 10% 0,0260 109 oC, 20min, 5% 109 oC, 10min, 5% 0,0225 116 oC, 20min, 5% 0,0383 116 oC, 10min, 5% 0,0207 0,15 ‐ 0,35 gL -1 * *Gunnarsson et al ., Bioresour. Technol., 2015; 182: 58-66

Succinic acid production and consumption of total sugars 9,57 109oC, 20min, 10% 4,05 109oC, 10min, 10% 9,11 116oC, 20min, 10% 5,95 116oC, 10min, 10% 6,17 109oC, 20min, 5% 5,95 109oC, 10min, 5% 5,48 116oC, 20min, 5% 37 o C 6,13 116oC, 10min, 5% pH 7.5 30 gL -1 MgCO 3 0 2 4 6 8 10 Succinic acid con. (g/L) 5 gL -1 yeast extract 0.5 vvm CO 2 10,89 109oC, 20min, 10% 7,35 109oC, 10min, 10% 17,75 116oC, 20min, 10% 12,75 116oC, 10min, 10% 8,09 109oC, 20min, 5% 13,64 109oC, 10min, 5% 8,18 116oC, 20min, 5% 7,99 116oC, 10min, 5% 0 5 10 15 20 Total sugars con. (g/L)

Yields of fermentations of dilute-acid hydrolysate Organic acids yield (goa/gts) Succinic acid yield (gsa/gts) 1,26 109 oC, 20min, 10% 0,88 0,94 109 oC, 10min, 10% 0,55 0,97 116 oC, 20min, 10% 0,47 0,88 116 oC, 10min, 10% 0,51 1,26 109 oC, 20min, 5% 0,76 0,77 109 oC, 10min, 5% 0,44 1,17 116 oC, 20min, 5% 0,67 1,20 116 oC, 10min, 5% 0,77 Y (g p /g ts )

Cellulase production  Fermentation T. reesei ◦ 28 o C, pH 5.5, 180 rpm ◦ 40 g L -1 wheat bran, 10 g L -1 avicel 0,5 0,4 0,3 FPU ml -1 0,2 0,1 0 0 1 2 3 4 5 6 7 Time [d]

Succinic acid bio-production Nitrogen Gas supply, Fermentation, Total Succinic acid Y Raw material Ref. source volume, Working volume (g /L) (g SA /g ts ) Anaerobic, fed-batch, Carvalho et Glycerol YE (10) 49.62 0.64 bioreactor 2 L -1.5 L al., 2014 Anaerobic, batch, Dorado et Wheat hydrolyzate YE (5) / Vit 62.1 1.02 bioreactor 1.8 L, 0.5 L al., 2009 BH (200 mg/L Anaerobic, batch, Leung et Bread hydrolyzate 47.3 nk FAN) bioreactor 2.5 L, n.d. al., 2012 Cotton stalk YE (30) / Urea Anaerobic, batch SSF, Li et al., 63 0.64 hydrolyzate (2) bottles 500 mL, n.d. 2013 Morales et Macroalgal Anaerobic, batch, YE (16.7) 33.78 0.63 al., 2015 hydrolyzate bioreactor 3L, 1.5L Anaerobic, fed-batch SSF a , Chen et al., Rapeseed meal YE (15) 23.4 0.115 bioreactor 3 L, 1.2 L 2011 Anaerobic, batch, YE (5) / Pep Wan et al., Whey 22.2 0.57 (10) bioreactor 2.5L, 1.2L 2008 Acid hydrolysis of Anaerobic, batch ( Fibrobacter Li et al., - 1.9 0.12 OPW succinogenes ), serum bottles 125ml, 25ml 2010 Dilute-acid hydrolysis Anaerobic, batch, 0.76 and Current ΥΕ (5) 6.17 and 6.13 of OPW bottles 100mL, 100mL 0.77 study

Conclusions  Elemental analysis on hydrolysates ◦ Mg 2+ , Ca 2+  Dilute-acid hydrolysis conditions ◦ 109 o C, 20 min, 5% (w/w), y=0,76 (g sa /g ts ) ◦ 116 o C, 10 min, 5% (w/w), y=0,77 (g sa /g ts )  Cellulase production ◦ 5 days incubation

Future Work  Ultrasound ◦ Frequency ◦ Duration  Enzyme hydrolysis ◦ Enzyme units ◦ Duration

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