Stella Parmaki Ioannis Vyrides Marlen I. Vasquez Ioanna - PowerPoint PPT Presentation

Stella Parmaki Ioannis Vyrides Marlen I. Vasquez Ioanna Hadjiadamou Catarina B. M. Barbeitos Frederico C. Ferreira Carlos A. M. Afonso Chryssoula Drouza Michalis Koutinas NAXOS2018 14 June 2018, Naxos, Greece

2 Environmental Bioprocessing Laboratory The Concept of Biorg4WasteWaterVal+ Bioorganic Novel Approaches for Food Processing Waste Water Treatment and Valorisation: Lupanine Case Study Recycle Water: 80% Raw Food Raw Food Waste Water Fresh Water Fresh Food Waste Valuable chemicals Food Water Processing Water Processing Biomass to biogas Edible Food Edible Food Linear Water Economy Circular Water Economy

3 Environmental Bioprocessing Laboratory The Lupin Beans Case Study Lupin Beans processing Fresh water Lupin beans for Water Hydration Cooking Sweeting Salting human 3% waste 1.4% waste 88.5% waste 7.3% waste diet Wastewater Energy Lupanine

4 Environmental Bioprocessing Laboratory The Lupin Beans Case Study Lupin Beans processing Fresh water Lupin beans for Hydration Cooking Sweeting Salting human 3% waste 1.4% waste 88.5% waste 7.3% waste diet WP2 Nanofiltration or Wastewater Reverse Osmosis Diastereomeric Reduction WP4.1 Re ‐ used water Ultrafiltration resolution NaBH 4 Alkaloid (+) ‐ sparteine (+) ‐ Lupanine isolation ( ‐ ) ‐ Lupanine ( − ) ‐ sparteine NaBH 4 Tartaric Acid and solvent recycle Qualification Macro molecules WP5&6: WP3.1 & 3.2 Water WP4.2 Biocatalyst screening Valorisation: WP3.3 Anaerobic Chemical Lupanine and Bioconversion and digestion for conversion beyond Bioreactor design energy recover Treated Water

5 Environmental Bioprocessing Laboratory Motivation • Quinolizidine nucleus • Use of a natural molecule as target for biotransformation • Useful functionalities for fine chemicals and pharmaceutical • Produce new and known alkaloids industries with high added-value to overcome laborious total • Synthesis requires too many steps synthesis and the overall yield is low Chemical transformation lupanine: Strains capable of using lupanine: Figure: Decrease of the concentrations of lupanine ( ● ) and total alkaloids ( ○ ) during growth ( □ ) of strains IST 20B and IST 40D at 27 o C in LUP2 medium. Figure: Reduction ( ‐ ) ‐ lupanine to (+) ‐ sparteine. Lupanine removal (stationary phase): 99%

6 Environmental Bioprocessing Laboratory Strains Metabolising Alkaloids Nicotine: demethylation pathway in fungi pyridine pathway in Gram-positive bacteria pyrolidine pathway in Gram-negative bacteria variant of pyridine and pyrolidine pathway in Gram-negative bacteria Caffeine: Pseudomonas sp. CES (9 metabolic enzymes involved) Lupanine: Pseudomonas sp. (lupanine 17-hydroxylase)

7 Environmental Bioprocessing Laboratory T oxicological Aspects of Lupanine - Aquatic T= 21 o C, Light Vibrio fischeri 125 5 min Marine bacteria 100 15 min EC 50 [mg L -1 ] Luminescence inhibition 75 50 Highly toxic 25 0 Vibrio fischeri Daphnia magna 80 Planktonic crustacean 24 hours h 60 48 hours h EC 50 [mg L -1 ] Freshwater organism Immobilisation test 40 20 Highly toxic 0 Daphnia magna

8 Environmental Bioprocessing Laboratory T oxicological Aspects of Lupanine - Plants T= 21 o C, Dark Sinapis alba Dicotyledonous seeds Sinapis Alba 0,00 Radicle growth % Radish inhibition -10,00 Positive effect -20,00 Non-toxic -30,00 Lupinus albus is dicotyledonous -40,00 100 50 25 100,00 Sorghum sacchratum 12,5 6,25 % Radish inhibition Monocotyledonous seeds 75,00 Radicle growth 50,00 Negative effect 25,00 Highly toxic 0,00 Sorghum Saccharatum

9 Environmental Bioprocessing Laboratory Isolation of Lupanine Metabolising Strains 1 g L -1 lupanine, 30 o C, pH 7 1.5 g L -1 lupanine, 30 o C, pH 7 Environmental Samples Aerobic Aerobic Granular Sludge Granular Sl. Digested Sl. Aerobic Sl. Lupinus WW Anaerobic Sludge Aerobic Sludge Anaerobic Anaerobic Lupinus Wastewater Carbon Source Granular Sl. Granual Sl. Digested Sl.Digested WW 8 Microbial Isolates Lupanine Aerobic Rhodococcus rhodochrous 16S rRNA Sequencing (Macrogen – The Netherlands) Rhodococcus sp. TGAATCATGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATT AGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATA Rhodococcus rubber GGACCTCGGGATGCATGTTCCGGGGTGGAAAGGTTTTCCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGT AACGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCT Pseudomonas putida TCGGGTTGTAAACCTCTTTCAGTACCGACGAAGCGCAAGTGACGGTAGGTACAGAAGAAGCACCGGCCAACTACGTGCCAGC AGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCT GTGAAAACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGT Anaerobic GTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGC GAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCA CGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACG Rhodobacter sp. GGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCGGAC CGCCCCAGAGATGGGGTTTCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCAGGAGACTGCCGGGGTCAA Ochrobactrum tritici CTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGA GGGCTGCGATACCGCGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAA GTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCAT Pseudomonas citronellolis GAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTCGTGGGAA Pseudomonas sp.

10 Environmental Bioprocessing Laboratory Phylogenetic Trees of Isolates 99% Alkaloids (Berberine, Rhodococcus Ergopeptines, Nicotine) Nitroaromatics (e.g. nitrophenol) Aliphatics (e.g. n -hexadecane) Aromatics (e.g. o -xylene) Polycyclic Aromatic Hydr. 99% (PAH) 99% Rhodobacter 99% Nitroaromatics Aliphatics Aromatics Polycyclic Aromatic Hydr. (PAH)

11 Environmental Bioprocessing Laboratory Phylogenetic Trees of Isolates 99% Versatile degrader Pseudomonas Alkaloids (Caffeine, Nicotine) Aromatics (e.g. benzene) 99% Herbicides (e.g. atrazine) Isoprenoids (e.g. citronellol) 99% Ochrobactrum Alkaloids (Nicotine) 99% Aliphatics Aromatics Polycyclic Aromatic Hydr. (PAH)

12 Environmental Bioprocessing Laboratory Lupanine Biodegradation – Aerobic Strains Conditions: 31 ο C, pH 7, minimal medium (M9) Stationary phase P. putida LPK411: 30 h Other 3 strains: 36 h % Removal R. rhodochrous LPK211: 80% R. sp. LPK311: 70% R. ruber LPK111: 69% : P. putida LPK41 : R. ruber LPK11 Other studies : R. LPK211 1 g L -1 removed (99%) in 10 h from wastewater : Rhodococcus sp. LPK311 (Santana et al. 2002) 3 g L -1 removed (99%) in 30 h from wastewater (Santana et al. 1996) Santana F.M.C. et al., (2002) J. Agric. Food Chem. 50:2318-2323. Santana F.M.C. et al., (1996) J. Ind. Microbiol. 17:110-115.

13 Environmental Bioprocessing Laboratory Final Metabolic Products – Aerobic Strains Lupanine Multiflorine N P. putida LPK411 HN OH O R. ruber LPK111 New generation sparteine analogues via alkylation on the amide bond R. sp. LPK311 Complete Bioconversion R. rhodochrous LPK211

14 Environmental Bioprocessing Laboratory Resolution of Racemic Lupanine 1,4 (a) 1,2 OD [600 nm] 1 0,8 0,6 0,4 0,2 Racemic mixture: D-(+)-lupanine, L-(-)-lupanine 0 Conditions: 31 ο C, pH 7, minimal medium (M9) 0 15 30 45 60 Time [h] 100 e.e. of L-(-)-lupanine [%] (b) All strains e.e. 95-100% at 42 h 75 P. putida LPK411: e.e. 95% at 36 h, 53% lupanine 50 L-(-)-lupanine: synthesis of L-(-)-sparteine 25 0 0 15 30 45 60 Time [h] : P. putida LPK41 : R. ruber LPK11 : R. LPK211 : Rhodococcus sp. LPK311

15 Environmental Bioprocessing Laboratory Optimisation of P. putida Growth on Lupanine

16 Environmental Bioprocessing Laboratory Future Opportunities  Microbial kinetics and metabolic products from each enantiomer  Immobilization on microbial supports  Bioreactor studies

17 Environmental Bioprocessing Laboratory Conclusions  Lupanine is highly toxic for aquatic organisms  Non-toxic for dicotyledonous  Bioconversion of lupanine under aerobic conditions  Useful metabolic end-products  P. putida performs resolution of racemic lupanine

18 Environmental Bioprocessing Laboratory Thank You!