LIFE12 ENV/IT/000352 BIONAD

LIFE12 ENV/IT/000352BIONAD

“Naturalised dyes replacing commercial colorants for

environmentally friendly leather dyeing and water recycle”

Start: 01/01/2014End: 30/06/2016

13/06/20146-month meeting

Biokimica premises

LIFE12 ENV/IT/000352 “BIONAD“ Naturalised dyes replacing commercial colorants for

environmentally friendly leather dyeing and water recycle

The University of FlorenceDepartment of Chemistry “Ugo Schiff”

Organic Chemistry



The University of FlorenceLaboratory No 8 Prof. Roberto Bianchini

Room 161

• 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



Commercial Dye ≡ Chemical Formulation

chromophore

surfactans

dispersants

paraffins

saltsdyeing 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


Naturalyzed dyesare 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

Chromophore

Sugar

"unknown" chemical formulation

single species



50%

50%?

• 1st Patent: EP1462484B1, 2004

• 2nd Patent: FI2013A000093, 2013

Innovative research carried out by

Prof. Bianchini and his team



Lactose ChromophoreChemical Bridge

Laboratory scale ; period: 0 - 3 monthsNature 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



Restrictions: Annex VIII and IX of REACH Annex I of Directive 67/548/EEC

• Analytical data to demonstrate the chemical stability of naturalised dyesSpectrophotometric, mass spectrometric and spectroscopic analysis

Analytical instrumentationAction B.3



Nuclear Magnetic Resonance

VarianMercury 400 MHz

Mass Spectrometry

ThermoScientificLCQ-Fleet

UV-Visible Spectroscopy

VarianCary-4000

Chemical species identification Molecular weight Light absorption(color observation)

Elaboration of lactoseAction B.3



72%

O

OH

O

HOO

HO

HO

OH

OH

OHHO

.H2O

O

OH

O

HO

O

O

O

O

OO CH(OMe)2

1) BnNH2, MeCN 20 h , 80 C

p-TsOH · H2O4 h, 60 oC

DMP

naturalizingunits

O

OH

O

TsO

O

O

O

O

OO CH(OMe)2TEA, DCM

ovn, 0 – 20 C

p-TsCl

93%

>95%

O

OH

O

N

O

O

O

O

OO CH(OMe)2HN

O

OH

O

H2N

O

O

O

O

OO CH(OMe)2

piperazineMeCN

4 -6 h, 80 oC

92%

2) H2, Pd/C, MeOH ovn, 20 C



Some examples of chromophore elaborationAction B.3

THF, TEA, DMAPovn, 20 oCO2N N

Cl

Cl

N N

CN

OH

DO30

O2N N

Cl

Cl

N N

CN

OO

CO2H

92%

DY42 97%

NH

S

NO2

NH

OO

N

S

NO2

NH

OO

CO2H

1) ClCH2CO2Et, K2CO3 KI, acetone, 4 h , 58 C2) KOH, MeOH, H2O ovn, 20 C

DR1

R

OCO2H

60%

R

OH

1) BrCH2CO2Et, NaH THF, 8 h, 0 C

2) KOH, MeOH, H2O ovn, 20 C

R

ONH

OHO2C CO2H

2

O2N NN NR =

86%

1) i) SOCl2, TEA, DCM, 4 h, 0 C ii) diethyl glutamate.HCl, TEA

2) KOH, MeOH, H2O ovn, 20 C

succinic anhydride



Coupling and deprotectionAction B.3

THF, ovn, 20 oCR3NHN

R2 OH

O

>95%

DMTMM

O2N NN NR2 =

NC

OO

Cl

Cl

R3NNR2

O

O

OH

O

O

O OO

OO

CH(OMe)2R3 =

HCl, acetone

ovn, 20 oCO

HO

O

N+ NR2

OO

OH

OH

HO

HOOH

HO

H

Cl-

90%DO30

Some examples of nat-dye purification

DB27 DB72 DO30 DR202



Naturalised dyes

Action B.3

Sample dyes for action B.3



Stability of naturalised dyes

Action B.3

Stability in open airStability in acid water

mimicking dyeing conditions

mimicking storing conditions



Synthetic strategy complying to REACHAction 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, […]

• 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



Laboratory scale ; period: 0 - 12 monthsEnvironmental impact of acid dyes within leather manufacture

ICCOM-CNR, UNIFI, BIOKIMICA, INESCOP

Action C.1

mass of dye: ca. 3% (w/w) of leather samplemass of leather sample: ca. 15 g

BiokimicaInescop

IccomUnifi

Awaiting dyeing effluents from Biokimica

• 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



Laboratory scale ; period: 3 - 9 monthsDepuration of dyeing effluents using bacteria

UNIFI

Action C.2



Action of bacterium strain E. coli on dye effluents

Action C.2

Tilting shakerEscherichia coli: DH5 Centrifuge

Regeneration on a Petri dish Shaking at 37 C Separation of the biomass



Action C.2

Preliminary tests of decoloration on simulated effluents

12-UNIFI-DO30-NAT 04-UNIFI-DR202-NAT

beforebefore56 % 90 %

after after

>95 % >95 %

Assessment of dye reduction by visible spectrophotometry



Action C.2


ovn ovn

beforebefore-56% -90%

Assessment of dye reduction by visible spectrophotometry



Action C.2


ovn ovn

-40% -5%

>95 %1 week

>95 %1 week

• Determination of COD and BOD before and after treatment (as for action C.1)

• Considerations on the mechanism of action of E. coli



Action of bacterium strain E. coli on dye effluentsAction 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

Potential recycling of bacterial biomass



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%

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