1. LIFE12 ENV/IT/000352 BIONAD “Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle” Start: 01/01/2014 End: 30/06/2016 13/06/2014 6-month meeting Biokimica premises

2. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle The University of Florence Department of Chemistry “Ugo Schiff” Organic Chemistry

3. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle The University of Florence Laboratory No 8 Prof. Roberto Bianchini Room 161

4. B1 : traditional chemical acid dyes in leather dyeing (M1-M6) B2 : quality features of dyed leather with acid dyes (M1-M6) B3 : naturalised dyes as stable chemical substances (M1-M3) B4 : naturalised dyes in leather dyeing at laboratory level (M3-M9) C1 : environmental impact of acid dyes within leather manufacture (M1-M9) C2 : depuration of dyeing effluents using bacteria at laboratory level (M3-M9) ACTIONS OF THE FIRST SIX MONTHS LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle

5. Commercial Dye ≡ Chemical Formulation chromophore surfactans dispersants paraffins salts dyeing effluents high impact 15 - 50 % of the commercial product Unknown chemical species from the production process of the chromophore LIFE12 ENV/IT/000352 «BIONAD» Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle

6. Naturalyzed dyes are a striking evolution, to revolutionize the way of production and use of dyes Standard dyes Naturalyzed dyes 40% 60% Intermediates Surfactants Dispersants Inorganic salts Paraffins Chromophore Sugar "unknown" chemical formulation single species LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle 50% ?

7. 1 st Patent: EP1462484B1, 2004 2 nd Patent: FI2013A000093, 2013 Innovative research carried out by Prof. Bianchini and his team Innovative research carried out by Prof. Bianchini and his team LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle LactoseChromophoreChemical Bridge

8. Laboratory scale ; period: 0 - 3 months Nature of naturalized dyes Elaboration of lactose to the naturalizing unit The naturalization process Synthetic procedures for different chromophores UNIFI Synthetic strategy complying to REACH Action B.3 LIFE12 ENV/IT/352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Restrictions: Annex VIII and IX of REACH Annex I of Directive 67/548/EEC Analytical data to demonstrate the chemical stability of naturalised dyes Spectrophotometric, mass spectrometric and spectroscopic analysis

9. Analytical instrumentation Action B.3 LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Nuclear Magnetic Resonance Varian Mercury 400 MHz Mass Spectrometry ThermoScientific LCQ-Fleet UV-Visible Spectroscopy Varian Cary-4000 Chemical species identificationMolecular weight Light absorption (color observation)

10. Elaboration of lactose Action B.3 LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle 72% 1) BnNH 2, MeCN 20 h, 80 C p-TsOH · H 2 O 4 h, 60 o C DMP naturalizing units TEA, DCM ovn, 0 – 20 C p-TsCl 93% >95% piperazine MeCN 4 -6 h, 80 o C 92% 2) H 2, Pd/C, MeOH ovn, 20 C

11. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Some examples of chromophore elaboration Action B.3 THF, TEA, DMAP ovn, 20 o C DO30 92% DY42 97% 1) ClCH 2 CO 2 Et, K 2 CO 3 KI, acetone, 4 h, 58 C 2) KOH, MeOH, H 2 O ovn, 20 C DR1 60% 1) BrCH 2 CO 2 Et, NaH THF, 8 h, 0 C 2) KOH, MeOH, H 2 O ovn, 20 C 86% 1) i) SOCl 2, TEA, DCM, 4 h, 0 C ii) diethyl glutamate.HCl, TEA 2) KOH, MeOH, H 2 O ovn, 20 C succinic anhydride

12. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Coupling and deprotection Action B.3 THF, ovn, 20 o C >95% DMTMM HCl, acetone ovn, 20 o C 90% DO30 Some examples of nat-dye purification DB27DB72DO30DR202

13. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Naturalised dyes Action B.3 Sample dyes for action B.3

14. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Stability of naturalised dyes Action B.3 Stability in open airStability in acid water mimicking dyeing conditions mimicking storing conditions

15. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Synthetic strategy complying to REACH Action B.3 Azodyes which, […], may release one or more of the aromatic amines […] above 30 ppm in the finished articles or in the dyed parts thereof, […], Azodyes, which are contained in Appendix 9, ‘List of azodyes’, shall not be […] used for coloring textile and leather articles […] Appendix VIII Appendix IX Annex XVII Restrictions on the manufacture, placing on the market and use of certain dangerous substances, […]

16. metal content (atomic absorption spectrophotometry) Quantification of contamination parameters from the use of acid dyes biodegradability COD and BOD Preparation of dyed leathers on laboratory scale LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Laboratory scale ; period: 0 - 12 months Environmental impact of acid dyes within leather manufacture ICCOM-CNR, UNIFI, BIOKIMICA, INESCOP Action C.1 mass of dye: ca. 3% (w/w) of leather sample mass of leather sample: ca. 15 g Biokimica Inescop Iccom Unifi Awaiting dyeing effluents from Biokimica

17. Determination of COD and BOD before and after treatment (as for action C.1) Potential recycling of bacterial biomass Action of bacterium strain E. coli on dye effluents Considerations on the mechanism of action of E. coli Assessment of dye reduction (visible analysis) Potential reuse of dyeing effluent after depuration LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Laboratory scale ; period: 3 - 9 months Depuration of dyeing effluents using bacteria UNIFI Action C.2

18. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action of bacterium strain E. coli on dye effluents Action C.2 Tilting shaker Escherichia coli: DH5 Centrifuge Regeneration on a Petri dish Shaking at 37 C Separation of the biomass

19. LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action C.2 Preliminary tests of decoloration on simulated effluents 12-UNIFI-DO30-NAT04-UNIFI-DR202-NAT before 56 %90 % after >95 %

20. Assessment of dye reduction by visible spectrophotometry LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action C.2 12-UNIFI-DO30-NAT04-UNIFI-DR202-NAT ovn before -56%-90%

21. Assessment of dye reduction by visible spectrophotometry LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action C.2 12-UNIFI-DO30-NAT04-UNIFI-DR202-NAT ovn -40%-5% >95 % 1 week >95 % 1 week

22. Determination of COD and BOD before and after treatment (as for action C.1) Considerations on the mechanism of action of E. coli LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action of bacterium strain E. coli on dye effluents Action C.2 Analyses to be carried out at a later stage Decoloration of DR202 and DO30 indicated that E. coli may both metabolise the dye and adsorb it on the cell walls Metabolism is indicated by the visible analisys of the supernatant: novel peaks may arise in the absorption pattern Evidence of adsorption is shown by the color of the pellet biomass after centrifugation of the E. coli culture for decoloration Decoloration is carried out in aerobic conditions at 37 C

23. Potential recycling of bacterial biomass LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for environmentally friendly leather dyeing and water recycle Action C.2 First test of decoloration of DO30 simulated effluent: 0.5 L 14% 66% 44% Oxygenation of the culture may be an issue for the treatment of "large volumes" in the laboratory The nutrient Luria-Bertani extract is added to aid bacteria growth/replication in each cycle A fraction of the bacteria biomass is used in further cycles of decoloration The reuse of dyeing effluent after depuration will be assessed at a later stage -14%-30%-22% start >90% > -24%