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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.
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