Bacterial Enzymes and Multi-enzymatic Systems for Cleaning-up Dyes from the Environment - Microbial Degradation of Synthetic Dyes in Wastewaters - page 37

Fig. 8 Decolorization of
several azo dyes after 24 h of
reaction under anaerobic
conditions using PpAzoR
(Mendes et al. 2011a )

DB1

DB38

RB5

DRR

RY145

AO7

MB17

DR80

SOG

MR

RR4

AR266

AB210

AB194

AR299

MB3

MB9

AY49

0 20 40 60 80 100

Decolourisation (%)

city of PpAzoR was investigated by measuring the initial rates of
reduction of a set of structurally different azo dyes under anaerobic conditions
(Mendes et al. 2011b ). PpAzoR uses either NADPH or NADH as electron donor, but
the ef

The speci

ciency for NADPH is twice that of NADH (Gon ç alves et al. 2013 ). The enzyme

is particularly unspeci c with regard to the azo dyes used, showing only smooth

trends with methyl red and reactive black 5 representing the substrates reduced with
higher speci 10 3 M − 1 s − 1 ) and mordant black 9 and acid orange city (around 1 2.5 ×

-

10 3 M − 1 s − 1 ). The af 7 reduced at the lowest ef nity for

dyes is reduced with K m values between 0.1 and 4 mM indicating the need of adding
1

ciency (around 0.3 0.4 ×

-

the K m value) to the reaction mixtures in order to accurately
measure the maximal rates of dye degradation (Mendes et al. 2011b ). The high
concentration of dyes leads to initial absorbance values out of the Lambert-Beer law

- 40 mM of dyes (10 ×

'

s
applicability range. Therefore, the reaction assays to determine the kinetic parameters
for dye consumption need to be performed using a photometric discontinuous
method, where samples are withdrawn from reactions at time intervals, diluted and the
absorbance measured at the maximum wavelength for each substrate.
To further characterize the properties of PpAzoR, in particular the oxygen-
sensitivity of PpAzoR to oxygen, the initial rate of reactive black 5 degradation was
measured as a function of oxygen concentration (Fig. 9 a). The results show that the
rates of dye decolorization decreased with increased O 2 concentration. This is in
conformity with the low levels of dye decolorization by growing or resting cells of
P. putida MET94 cells under aerobic conditions. In order to test if oxygen is a non-
competitive, competitive inhibitor or instead is substrate for the PpAzoR enzyme,
oxygen consumption was measured in a reaction containing only enzyme, buffer
and NADPH (Fig. 9 b). The addition of catalase resulted in a 2-fold increase in the
concentration of dioxygen in the mixture, showing that peroxide is in the solution
most likely as a result of PpAzoR activity (Fig. 9 b). When catalase was added at the
beginning of the reaction, only half of the possible concentration of oxygen pro-
duced was attained (Fig. 9 b). These results clearly show that oxygen is reduced to
peroxide by PpAzoR.