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

The Concept of Biorg4WasteWaterVal+

Bioorganic Novel Approaches for Food Processing Waste Water Treatment and Valorisation: Lupanine Case Study Food Processing Fresh Water Raw Food Waste Water Edible Food Food Processing Fresh Water Raw Food Waste Water Edible Food Recycle Water: 80% Valuable chemicals Biomass to biogas Linear Water Economy Circular Water Economy

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The Lupin Beans Case Study

Lupin beans for human diet Hydration 3% waste Cooking 1.4% waste Sweeting 88.5% waste Salting 7.3% waste Fresh water Lupin Beans processing Wastewater

Energy Lupanine Water

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Lupin beans for human diet Hydration 3% waste Cooking 1.4% waste Sweeting 88.5% waste Salting 7.3% waste Fresh water Lupin Beans processing Wastewater Macro molecules WP2 Ultrafiltration Nanofiltration or Reverse Osmosis Re‐used water Water Qualification Treated Water Alkaloid isolation WP3.3 Anaerobic digestion for energy recover Tartaric Acid and solvent recycle (+)‐Lupanine (‐)‐Lupanine (+)‐sparteine Diastereomeric resolution

NaBH4 NaBH4

Reduction WP4.1 (−)‐sparteine WP5&6: Valorisation: Lupanine and beyond WP3.1 & 3.2 Biocatalyst screening Bioconversion and Bioreactor design WP4.2 Chemical conversion

The Lupin Beans Case Study

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Motivation

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Chemical transformation lupanine: Strains capable of using lupanine:

Figure: Decrease of the concentrations of lupanine (●) and total alkaloids (○) Figure: Reduction (‐)‐lupanine to (+)‐sparteine. during growth (□) of strains IST 20B and IST 40D at 27oC in LUP2 medium.

Lupanine removal (stationary phase): 99%

• Quinolizidine nucleus
• Useful

functionalities for fine chemicals and pharmaceutical industries

• Synthesis requires too many steps

and the overall yield is low

• Use
• f

a natural molecule as target for biotransformation

• Produce new and known alkaloids

with high added-value to

• vercome

laborious total synthesis

Strains Metabolising Alkaloids

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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)

T

• xicological Aspects of Lupanine - Aquatic

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25 50 75 100 125 Vibrio fischeri EC50 [mg L-1]

5 min 15 min

20 40 60 80 Daphnia magna EC50 [mg L-1]

24 hours 48 hours

T= 21oC, Light

Vibrio fischeri

Marine bacteria Luminescence inhibition Highly toxic

h h

Daphnia magna

Planktonic crustacean Freshwater organism Immobilisation test Highly toxic

T

• xicological Aspects of Lupanine - Plants

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Sinapis alba Sorghum sacchratum Dicotyledonous seeds Radicle growth Positive effect Non-toxic Lupinus albus is dicotyledonous Monocotyledonous seeds Radicle growth Negative effect Highly toxic

• 40,00
• 30,00
• 20,00
• 10,00

0,00 Sinapis Alba % Radish inhibition 0,00 25,00 50,00 75,00 100,00 Sorghum Saccharatum % Radish inhibition

100 50 25 12,5 6,25

T= 21oC, Dark

Isolation of Lupanine Metabolising Strains

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Environmental Samples

Granular Sludge Anaerobic Sludge Aerobic Sludge Lupinus Wastewater

Carbon Source

Lupanine

1 g L-1 lupanine, 30 oC, pH 7

8 Microbial Isolates

Aerobic Anaerobic

Granular Sl. Digested Sl. Aerobic Sl. Lupinus WW Granular Sl. Granual Sl. Digested Sl.Digested WW

1.5 g L-1 lupanine, 30 oC, pH 7

Aerobic Anaerobic

16S rRNA Sequencing (Macrogen – The Netherlands)

Aerobic Rhodococcus rhodochrous Rhodococcus sp. Rhodococcus rubber Pseudomonas putida Anaerobic Rhodobacter sp. Ochrobactrum tritici Pseudomonas citronellolis Pseudomonas sp.

TGAATCATGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATT AGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATA GGACCTCGGGATGCATGTTCCGGGGTGGAAAGGTTTTCCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGT AACGGCCCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCT TCGGGTTGTAAACCTCTTTCAGTACCGACGAAGCGCAAGTGACGGTAGGTACAGAAGAAGCACCGGCCAACTACGTGCCAGC AGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCT GTGAAAACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGT GTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGC GAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCA CGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATACACCGGAC CGCCCCAGAGATGGGGTTTCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCAGGAGACTGCCGGGGTCAA CTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGA GGGCTGCGATACCGCGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAA GTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCAT GAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTCGTGGGAA

Phylogenetic Trees of Isolates

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Rhodococcus Rhodobacter 99% 99% 99% 99%

Alkaloids (Berberine, Ergopeptines, Nicotine) Nitroaromatics (e.g. nitrophenol) Aliphatics (e.g. n-hexadecane) Aromatics (e.g. o-xylene) Polycyclic Aromatic Hydr. (PAH) Nitroaromatics Aliphatics Aromatics Polycyclic Aromatic Hydr. (PAH)

Phylogenetic Trees of Isolates

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Pseudomonas Ochrobactrum 99% 99% 99% 99%

Versatile degrader Alkaloids (Caffeine, Nicotine) Aromatics (e.g. benzene) Herbicides (e.g. atrazine) Isoprenoids (e.g. citronellol) Alkaloids (Nicotine) Aliphatics Aromatics Polycyclic Aromatic Hydr. (PAH)

Lupanine Biodegradation – Aerobic Strains

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• P. putida LPK411: 30 h

Other 3 strains: 36 h % Removal

• R. rhodochrous LPK211: 80%
• R. sp. LPK311: 70%
• R. ruber LPK111: 69%

Other studies 1 g L-1 removed (99%) in 10 h from wastewater (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.

: P. putida LPK41 : R. ruber LPK11 : R. LPK211 : Rhodococcus sp. LPK311

Final Metabolic Products – Aerobic Strains

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N HN O OH

• P. putida LPK411
• R. ruber LPK111
• R. sp. LPK311

Complete Bioconversion

• R. rhodochrous LPK211

Lupanine Multiflorine New generation sparteine analogues via alkylation on the amide bond

Resolution of Racemic Lupanine

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0,2 0,4 0,6 0,8 1 1,2 1,4 15 30 45 60

OD [600 nm]

Time [h]

(a)

25 50 75 100 15 30 45 60

e.e. of L-(-)-lupanine [%]

Time [h]

(b)

: P. putida LPK41 : R. ruber LPK11 : R. LPK211 : Rhodococcus sp. LPK311 Racemic mixture: D-(+)-lupanine, L-(-)-lupanine Conditions: 31 οC, pH 7, minimal medium (M9) All strains e.e. 95-100% at 42 h

• P. putida LPK411: e.e. 95% at 36 h, 53%

lupanine L-(-)-lupanine: synthesis of L-(-)-sparteine

Optimisation of P. putida Growth on Lupanine

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Future Opportunities

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 Microbial kinetics and metabolic products from each enantiomer  Immobilization on microbial supports  Bioreactor studies

Conclusions

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 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

Thank You!

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