StudyStudy ofof thethe effecteffect ofof redox redox mediatorsmediators onon thethe

decolorizationdecolorization ofof azo azo dyesdyes bybyascomyceteascomycete yeastsyeasts

BiologyBiology DeptDept., ., ChemistryChemistry DeptDept. . andand TextileTextileEngeneeringEngeneering DeptDept., ., UniversityUniversity ofof MinhoMinho

P.A.RamalhoP.A.Ramalho, , M.H.CardosoM.H.Cardoso, R. , R. MillwardMillward, , A.CavacoA.Cavaco--PauloPaulo, , M.T.RamalhoM.T.Ramalho

IntroductionIntroductionSome yeasts can reductively cleave azo Some yeasts can reductively cleave azo bonds in dyes, producing colourless bonds in dyes, producing colourless amines.amines.

Reduction is growthReduction is growth-- and pHand pH--dependent.dependent.

The available evidence suggests the The available evidence suggests the involvement of an enzymatic activity in involvement of an enzymatic activity in azoreductionazoreduction..

AzoreductaseAzoreductase activity is constitutive.activity is constitutive.

The The decolorizationdecolorization process by UM41process by UM41

0 h 10 h 18 h

0,01

0,1

1

10

0 10 20 30 40

Time (h)

OD

640

nm

0

1

2

3

4

5

6

pH /

Abs

470

nm

Biomass pH Dye

The strain:Isolated from

contaminated soilkc = 0,31 h-1

Ybiomass = 16,8%td ≈ 15 h

Strain UM41:Strain UM41:

IssatchenkiaIssatchenkiaoccidentalisoccidentalis

Coloured solution containing azo dye

Colourless solution containing amines

Carbon complexes

Oxidation products

NADH NAD+

Azoreductase

Redox mediatorox

Redox mediatorred

X

NN

X

X

NH2Chromophore

NH2

X

Yeast cellYeast cell

The azo reduction by yeastsThe azo reduction by yeasts

Structures of the tested dyesStructures of the tested dyes

N N NCH3

CH3

NaO3S

N N NCH3

CH3

NaO3S

N N

NaO3S

OH

Dye A Dye B (Methyl Orange)

Dye Amaranth

Dye C Dye D (Orange II)

N N

OH

NaO3S

N

OH SO3Na

SO3Na

NaO3S N

The redox mediators: structuresThe redox mediators: structures

O

OSO3Na

AQS AQS (9,10(9,10--AnthraquinoneAnthraquinone--22--

sulfonate)sulfonate)

O

OCH3

MenadioneMenadione(2(2--MethylMethyl--1,41,4--

naphtoquinone)naphtoquinone)

ResultsResults1.1. DecolorizationDecolorization with growing with growing

cultures + AQScultures + AQS2.2. Resting cells assays: effect of Resting cells assays: effect of

diferentdiferent mediatorsmediators3.3. Effect of Effect of menadionemenadione4.4. Cyclic Cyclic voltammetryvoltammetry

DecolorizationDecolorization with growing with growing cultures + AQScultures + AQS

0,1

1

10

0 5 10 15 20 25 30

Time (h)

OD

640

nm

0

1

2

3

4

5

6

pH /

Dye

Abs

461

nm

OD W/o OD W/ pH W/opH W/ Dye Abs W/o Dye Abs W/

Dye ADye A

0,1

1

10

0 10 20 30

Time (h)O

D 6

40 n

m

0

1

2

3

4

5

6

pH /

Dye

Abs

470

nm

Dye BDye B

Resting cells assaysResting cells assaysHigh cell density (c.a. 4 gDW.LHigh cell density (c.a. 4 gDW.L--11););Low glucose concentration (0.011 M);Low glucose concentration (0.011 M);

Measurement of the Measurement of the azoreductaseazoreductaseactivityactivity in different conditions in different conditions

((μμmol/(hmol/(h**gDWgDW))))

Effect of different mediators: Effect of different mediators: resting cells assaysresting cells assays

-100%

-50%

0%

50%

100%

150%

200%

A B C D

Amaranth

Dye

Varia

tion

(%) o

f the

spe

cific

azor

educ

tion

activ

ity

FADAQSRiboflavin

Oxygen is present in the assays?Oxygen is present in the assays?

Oxygen can compete with the dye for the electrons.Oxygen can compete with the dye for the electrons.

Why Why MenadioneMenadione??MenadioneMenadione enhances the reduction of enhances the reduction of

extracellularextracellular electron acceptors by electron acceptors by yeast cells.yeast cells.

BaronianBaronian et al. et al. ApplAppl MicrobiolMicrobiol BiotechnolBiotechnol (2002) 60: 108(2002) 60: 108--113113

MethyleneMethylene BlueBlue

Modification of the Modification of the ““resting resting cellscells”” assaysassays

Decrease of the glucose concentration Decrease of the glucose concentration (0.056mM) to minimize growth during the (0.056mM) to minimize growth during the assay.assay.

MenadioneMenadione –– standard standard conditionsconditions

I I –– BeforeBefore dyedye additionaddition II II –– AfterAfter dyedye additionaddition III III –– EndEnd ofof thethe assayassay (2h)(2h)

1 1 –– MethyleneMethylene Blue + CellsBlue + Cells2 2 –– Dye D + CellsDye D + Cells3 3 –– Dye D + Cells + AQSDye D + Cells + AQS

4 4 –– Dye D + Cells + AQS + Dye D + Cells + AQS + MenadioneMenadione5 5 –– Dye D + Cells + FADDye D + Cells + FAD6 6 –– Dye D + Cells + FAD + Dye D + Cells + FAD + MenadioneMenadione

No significant effect !No significant effect !

MenadioneMenadione –– without Owithout O22I I –– BeforeBefore NN2 2 spargingsparging II II –– BeforeBefore dyedye additionaddition III III –– AfterAfter dyedye additionaddition

IV IV –– EndEnd ofof thethe assayassay (2h)(2h)

1 1 –– MethyleneMethylene Blue + CellsBlue + Cells2 2 –– Dye D + CellsDye D + Cells3 3 –– Dye D + Cells + AQSDye D + Cells + AQS

4 4 –– Dye D + Cells + AQS + Dye D + Cells + AQS + MenadioneMenadione5 5 –– Dye D + Cells + FADDye D + Cells + FAD6 6 –– Dye D + Cells + FAD + Dye D + Cells + FAD + MenadioneMenadione

MenadioneMenadione: results: results

-60-40-20

020406080

100

AQS AQS+Menad FAD FAD+Menad

Incr

ease

in th

e %

of

deco

loriz

atio

n

W/o oxygen W/ oxygen

Cyclic Cyclic VoltammetryVoltammetryA A –– Reduced formReduced formB B –– OxidationOxidationC C –– Oxidized formOxidized formD D –– ReductionReduction

EEpapa –– anodic peak potential anodic peak potential EEpcpc –– cathodiccathodic peak potential peak potential

Formal potential:Formal potential:

EE00’’ = (= (EEpapa + + EEpcpc) / 2) / 2

Sample Sample voltammogramsvoltammogramsDye B

AQS

--0.398 V0.398 V

Dye D

--0.560 V0.560 V--0.337 V0.337 V

Cyclic Cyclic VoltammetryVoltammetry: results: resultsDyeDye Reduction Reduction

peak (V)peak (V)DecolorizationDecolorization

time (h)time (h)

AA --0.3970.397 15 15

1212

2424

3030

1717

BB --0.3370.337

CC --0.5210.521

DD --0.5600.560

AmaranthAmaranth --0.4760.476

Cathodic peak potentials (literature):

NAD+ = -0.530 to -0.590 V

FAD / Riboflavin ≈ -0.46V

MediatorMediator Reduction Reduction peak (V)peak (V)

AQSAQS --0.3980.398

ConclusionsConclusions

-100%

-50%

0%

50%

100%

150%

A B C D

Amaranth

Dye

% o

f inc

reas

e in

the

spec

ific

azor

educ

tion

activ

ity

AQS

1.1. Thermodynamics is crucial. Thermodynamics is crucial. EEpcpcmediatormediator

< < EEpcpcdyedye

SubstanceSubstance EEpcpc (V)(V)

AA --0.3970.397

BB --0.3370.337

AQSAQS --0.3980.398

AmaranthAmaranth --0.4760.476

CC --0.5210.521

DD --0.5600.560

ConclusionsConclusions2. The amines N,N2. The amines N,N--dimethyldimethyl--pp--phenylenediaminephenylenediamine

and 1and 1--aminoamino--22--naphthol can not act as naphthol can not act as mediators because in this casemediators because in this case

EEpcpcaminesamines> > EEpcpcdyedye

N,NN,N--dimethyldimethyl--pp--phenylenediaminephenylenediamine 11--aminoamino--22--naphtholnaphthol

ConclusionsConclusions3. The reduction capacity should be dependent on 3. The reduction capacity should be dependent on

intracelularintracelular NAD(P)H concentration, since a NAD(P)H concentration, since a decrease in glucose concentration in the media decrease in glucose concentration in the media causes an inversion on the effect of the mediator causes an inversion on the effect of the mediator FAD (thermodynamic effect)FAD (thermodynamic effect)

0%

20%

40%

60%

80%

100%

120%

140%

160%

180%

A B C D

Dye

% v

aria

tion

in t

he d

ecol

oriz

atio

nac

tivity

-40

-20

0

20

40

60

80

100

FAD

% v

aria

tion

in th

e de

colo

rizat

ion

activ

ity

W/o oxygen W/ oxygen

[glucose] = 0.06 [glucose] = 0.06 mMmM[glucose] = 11 [glucose] = 11 mMmM

ConclusionsConclusions4. 4. MenadioneMenadione has a positive effect mainly in the has a positive effect mainly in the

absence of oxygen. This reinforces our idea of the absence of oxygen. This reinforces our idea of the involvement of a membrane system in involvement of a membrane system in azoreductionazoreductionby yeast cells.by yeast cells.

-40

-20

0

20

40

60

80

100

AQS AQS+Menad

Incr

ease

in th

e %

of d

ecol

oriz

atio

n

W/o oxygen W/ oxygen

Final conclusionsFinal conclusionsCyclic Cyclic voltammetryvoltammetry is a powerful is a powerful

instrument that can help us to predict:instrument that can help us to predict:

the effect of redox mediators on the the effect of redox mediators on the biological reduction of azo dyes;biological reduction of azo dyes;

the possibility of biological reduction the possibility of biological reduction of this class of compounds not only by of this class of compounds not only by yeasts but also by other yeasts but also by other microorganisms.microorganisms.