Rafaella Costa Bonugli-Santos * 1; Lucia Regina Durrant2; Lara Durães Sette1

e-mail: rafaellabonugli@yahoo.com.br

1Microbial Resources Division, CPQBA – University of Campinas, CEP 13081-970, Campinas, SP, Brazil – E-mail: rafaellabonugli@yahoo.com.br

2 Food Science Department, Faculty of Food Engineering, University of Campinas, Campinas, SP, Brazil

INTRODUCTION

The dyes Remazol Brilliant Blue R (RBBR) and Indigo are one of the most important dyes in

the textile industry and represents an important class of toxic organopollutants. Since the

RBBR is an anthracene derivative, it has been proposed as an efficient screening method for

fungi that are able to degrade recalcitrant pollutants, including aromatic compounds such as

polycyclic aromatic hydrocarbons (PAHs) and synthetic dyes. In addition, fungi derived

from marine environments are emerging as one of the best alternatives in the treatment of

industrial colored effluents mainly due to their salt tolerance. Therefore, the aim of the

present study was to evaluate the decolorization of RBBR and Indigo by Peniophora sp.

isolated from marine sponge Amphimedon viridis.

Keyword: marine-derived fungi, textile dyes decolorization, saline conditions, industrial

effluents.

MATERIAL AND METHODS

Screening of decolorization activity on solid media

200 mg L- of

RBBR and Indigo

Culture media:

Agar MA2

Agar MA2+ 3% NaCl

Agar MA2ASW (artificial sea water)

After 7, 14 and 21 days the fungal growth and the decolorization ability on these plates

were compared with the controls (RBBR-free).

Incubation: 21 days at 28°C

Decolorization ability on liquid medium

Incubation: 7 day

28º C, 140 rpm

Two fungal culture plugs (0.5 cm diameter) from the edge

of the colony were transferred to Erlenmeyer flasks

containing 50 mL of MA2 broth.

A fter 72 h of incubation at 140 rpm and

28°C, RBBR was added to MA2 broth (final

concentration of 500 and 1000 mg L−1).

A liquots of 1 ml from the culture were

taken right after dye addition (zero

time) and in each 24 hours during 7

days.

Samples were centrifuged

(12,074g, 10 min) and the

supernatants were

spectrophotometrically

evaluated :

Color reduction: decolorizing activity was calculated from the decrease in the maximum

absorption peak for RBBR.

Ligninolytic activities: Laccase (ABTS); Manganese peroxidase (phenol red); Lignin

peroxidade (veratryl alcohol).

Decolorization ability on crude enzymatic extract

RBBR (500 ppm) were added to 1 ml clear

supernatant samples obtained from 7-day-old

cultures (mycelia-free culture supernatants

with ligninolytic activity -RBBR-free).

The absorption spectra were read

(range of 200–800 nm) in each 2 h

from time zero and during 24 h of

incubation at 28 °C

RESULTS

After 14 days the dye RBBR was completely decolorizated by Peniophora sp. CBMAI 1063

in the medium without salt:

Control (zero time) 7-day-old 14-day-old Control (RBBR–free)

No RBBR decolorization was obtained in saline conditions;

No decoloration was observed for Indigo dye. In order to stimulate the Indigo decolorization

the fungus was also inoculated at different concentrations of malt extract: MA1 (1% malt

extract) and MA0,5 (0,5% malt extract), but even after 21 days of growth there was no

decolorization;

In generally, RRBR in the liquid medium was decolorized in the 4 th day:

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7

% D

ec

olo

riza

tio

n

Time (day)

500 mg L -1 RBBR

1000 mg L -1 RBBRControl 1 thday 2 th day 3 th day 4 th day 5 th day 6 th day 7 th day

MnP and laccase were detected during the

decolorization process; LiP was not produced;

The highest productions were proportional to the rate of decolorization;

The activity of MnP increased in the presence of RBBR (Control – Free RBBR = 1099 Ul1,

only after 21 days);

0

1000

2000

3000

4000

5000

6000

7000

8000

1 2 3 4 5 6 7

Mn

P (

U L

-1)

Time (day)

0

50

100

150

200

250

300

350

400

450

1 2 3 4 5 6 7

La

cc

as

e (

UL

-1)

Time (day)

500 mg L -1 RBBR1000 mg L -1 RBBR

After 2 h of incubation at 28°C, the crude enzymatic extract showed a decreasing of 50% in

the absorption spectrum, reaching 100% after 24 h;

0,0

2,0

4,0

6,0

350 450 550 580 590 650 750

AB

S

Wavelength (nm)

Absorption spectra during enzymatic decolorization (mycelia-free)

of RBBR

Controle

2 h

4 h

6 h

8 h

24 h

This result showed that there

was a complete removal of the

major visible light absorbance

peak, suggesting that RBBR

decolorization can be take place

in the absence of mycelia.

CONCLUSIONS

Support:

Fungi derived from marine environments have been one of the best alternatives in the

treatment of industrial colored effluents, mainly for application in bioremediation of effluents

with high alkaline and salt concentration (e.g. colored industrial pollutants), once they are

adapted to marine saline conditions;

The results obtained stimulate the development of new studies concerning decolorization and

degradation of synthetic dyes and colored effluents from the textile industries.