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Biodegradation and detoxification potential ofrotating biological contactor (RBC) with Irpex lacteusfor remediation of dye-containing wastewater

Katerina Malachova a,*, Zuzana Rybkova a, Hana Sezimova a, Jiri Cerven a,Cenek Novotny a,b

a Faculty of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska, Ostrava, Czech Republicb Laboratory of Environmental Biotechnology, Institute of Microbiology ASCR, Vıde�nska 1083, 142 20 Prague 4, Czech

Republic

a r t i c l e i n f o

Article history:

Received 23 March 2013

Received in revised form

26 June 2013

Accepted 5 July 2013

Available online 20 October 2013

Keywords:

Liquid textile wastes

Dye decolorization

Genetic toxicity

Biological toxicity

Rotating biological contactor

Irpex lacteus

* Corresponding author. Department of BioloOstrava, Czech Republic. Tel.: þ420 2 597092

E-mail addresses: Katerina.Malachova@oSezimova), jiri.cerven@osu.cz (J. Cerven), no

0043-1354/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.watres.2013.07.050

a b s t r a c t

Use of fungal organisms in rotating biological contactors (RBC) for bioremediation of liquid

industrial wastes has so far been limited in spite of their significant biodegradation po-

tential. The purpose was to investigate the power of RBC using Irpex lacteus for decolor-

ization and detoxification of industrial dyes and dyeing textile liquors. Recalcitrant dye

Methylene Blue (150 mg L�1) was decolorized within 70 days, its mutagenicity removed,

and the biological toxicity decreased more than 10-fold. I. lacteus biofilm in the RBC

completely decolorized within 26 and 47 days dyeing liquors containing disperse or reac-

tive dyes adjusted to pH4.5 and 5-fold diluted with the growth medium, respectively. Their

respective biological toxicity values were reduced 10- to 104-fold in dependence of the test

used. A battery of toxicity tests comprising Vibrio fisheri, Lemna minor and Sinapis alba was

efficient to monitor the toxicity of textile dyes and wastewaters. Strong decolorization and

detoxification power of RBC using I. lacteus biofilms was demonstrated.

ª 2013 Elsevier Ltd. All rights reserved.

1. Introduction various dyes, desizing and scouring agents, detergents, fin-

Textile industry produces large volumes of dye-containing

effluents that are ineffectively remediated in wastewater

treatment plants and are responsible for coloration of streams

that negatively affects water life. Biological and genetic

toxicity of dyes for bacteria, protozoa, aquatic animals, plants

and mammals has been widely documented (Gottlieb et al.,

2003; Soni et al., 2006; etc.). Textile wastewaters are

extremely variable in composition due to the presence of

gy and Ecology, Faculty315; fax: þ420 2 59709238su.cz (K. Malachova), Zvotny@biomed.cas.cz (C.

ier Ltd. All rights reserved

ishing agents and inorganic salts that all can contribute to

their toxicity (Dubrow et al., 1996). Consequently, efficient

remediation must result in both decolorization and detoxifi-

cation of the wastewater.

Decolorization of dyes with ligninolytic fungi has been

proven to be an efficient, cheap and environment-friendly

process but their detoxification power has been studied less

frequently (e.g. Knapp et al., 2008). A number of chemically-

different types of persistent dyes have been shown to be

of Science, University of Ostrava, Chittussiho 10, 710 00 Slezska,2.uzana.Rybkova@osu.cz (Z. Rybkova), Hana.Sezimova@osu.cz (H.Novotny).

.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3e7 1 4 87144

effectively degraded by various fungal organisms (e.g. Singh,

2006). Methylene Blue (CAS No. 61-73-4, C.I. 52015, MB), a

heterocyclic phenothiazine dye, is widely used for dyeing

leather and textile materials. Its decolorization strongly de-

pends on the conditions of fungal culture and the efficiency of

various ligninolytic fungi is quite variable (Tychanowicz et al.,

2004; etc.). Partial to weak decolorizations were reported by

various ligninolytic fungi in liquid- and solid-state cultures, by

bacteria and the aerobic activated sludge (e.g. Ma et al., 2011).

Anaerobic sludge is able to remove the dye but due to re-

oxidation by air only a low color removal effect was ach-

ieved (Ong et al., 2005).

Broader adverse effects of MB include eye injury, breathing

problems, methemoglobinemia, bacteriostatic and fungicidal

activities, and a significant toxicity to aquatic plants, crusta-

ceans and fish (Wainwright et al., 1999 etc.). Because of the

persistence and toxicity, MB was chosen in this study as a

model industrial dye for testing the efficiency of RBC reactor

using Irpex lacteus to decolorize and detoxify dyes and textile

effluents. RBC reactors operable in repeated-batch or contin-

uous mode offer advantages in bioremediation of industrial

wastewaters due to great surface per unit volume, low power

requirement and limited flow clogging (Anderson, 1983). Lig-

ninolytic fungi behave well in RBC but so far few studies have

been conducted, comprising only a limited number of fungal

strains (e.g. Guimaraes et al., 2005; Axelsson et al., 2006).

Biodegradation and detoxification power of I. lacteus in this

type of reactor has not been thoroughly investigated.

Ecotoxicity of pollutants is usually measured with stan-

dard toxicity tests, i.e. bacterial, crustacean, algal and seed

germination tests. For instance, Vibrio fischeri bioluminiscence

test was used to measure both a decrease and increase of

toxicity resulting from degradation of various textile dyes by

Trametes versicolor (Ramsay and Nguyen, 2002) or formation of

toxic products in the course of anaerobic decolorization of

Reactive Black 5 by Enterococcus faecalis and Clostridium butyr-

icum (Gottlieb et al., 2003). Tests with Daphnia spp. were used

to monitor decolorization-linked detoxification of phthalocy-

anine- and azo dyes obtainedwith Penicillium simplicissimum or

of a raw textile effluent treated with horseradish peroxidase

(Bergsten-Torralba et al., 2009). A reduction of mutagenicity of

Reactive Orange 16 and Disperse Blue 3 dyes in a two-step

treatment with activated sludge and a static culture of I. lac-

teus was monitored by the Ames test (Malachova et al., 2006).

Our study was undertaken to investigate the dye decolor-

ization and detoxification capacity of I. lacteus under the

conditions of RBC reactor using MB and two different textile

dyeing liquors containing mixtures of reactive or disperse

dyes to test the decolorization and detoxification efficiency.

Biological toxicity changes during the treatment were

measured with a battery of standard bacterial and plant tests

and the change of genetic toxicity with Ames test.

2. Material and methods

2.1. Chemicals

The dyeing liquors were obtained from INOTEX a.s., Czech

Republic. Wastewater I contained Sumifix Black B 150% (C.I.

Reactive Black 5) (9.82 g L�1), Inosin Yellow V-GR 160% (C.I.

Reactive Yellow 15) (2.47 g L�1), NaCl (75 g L�1) and the fixation

agent Texalkon MS (7.87 g L�1). Wastewater II contained Ito-

sperse Yellow RAP dye mix (5.47 g L�1), Itosperse Red RAP dye

mix (3.75 g L�1), Itosperse Blue RAP dye mix (2.47 g L�1), the

disperging agent Nicca Sunsolt� RF-557 (1 g L�1) and acetic

acid (0.3 ml L�1).

Malt extract and agar were purchased from Oxoid, UK,

Disperse Blue 3 (DB3, anthraquinone) andMethylene Blue (MB,

phenothiazine) dyes from SigmaeAldrich, Czech Republic.

Other chemicals were of analytical grade.

2.2. Microorganism

Irpex lacteus 931 was provided by the Culture Collection of

Basidiomycetes, Institute of Microbiology ASCR, Prague and

maintained on malt extract-glucose (MEG) medium contain-

ing 2% (w/w) agar at 4 �C.

2.3. Biodegradation in RBC reactor

The rotating biological contactor (RBC) reactor consisted of a

glass vessel and a horizontal driving axis with six 1-cm thick

polyurethane foam (PUF) discs (diameter 8 cm, rotation speed

2 rpm, 40% of disc volume immersed). The experiments were

carried out aseptically inMEGmedium (per litre: 5 gmalt extract,

10gglucose,pH4.5)at22 �Candforcedaerationwithair (50Lh�1).

Sterile PUF discs were put horizontally in MEG and inocu-

lated with a homogenate (Ultra-Turrax T25 mixer, IKA Werk,

Germany, 20 s) of a 7-d-old, static MEG culture grown at 28 �C(10% v/v inoculum). The discs were colonized with the fungus

(7 d, 28 �C) and then mounted aseptically in the reactor con-

taining one litre ofMEGmediumwithDB3 orMBdyes dissolved

at a concentration of 150 mg L�1 representing the respective

dye concentrations of 0.56 and 0.47 mM. Their decolorization

was measured spectrophotometrically at 645 nm and 505/

580 nm, respectively. Wastewaters I and II were adjusted to pH

4.5 and used 5-fold diluted with MEG. Their decolorization was

measured at respective maxima of 575 and 425 nm. The fungal

biomass on the discs was estimated gravimetrically at the end

of the experiment as dry biomass.

2.4. Biological toxicity tests

The acute biological toxicity was estimated using bacterial

luminiscence, aquatic plant growth and seed germination as

the endpoints. V. fischeri test (ISO 11348-3, 2007) measured

bioluminiscence inhibition after a 30-min exposition using a

LUMIStox300 luminometer (Hach-Lange, Dusseldorf, Ger-

many). Lemna minor test (ISO CD, 20079, 2005) determined

growth inhibition of fronds, the exposition time was 7 days.

The Phytotoxkit Sinapis alba test (ISO 11269-1, 1993) deter-

mined the inhibition of root growth after a 3-d exposure. The

test was considered to be valid if the germination of the con-

trol was �90%. The stimulation effect of endproducts was

evaluated by using a linear model.

A positive toxic effect was evaluated in the tests against

negative controls containing only the culturemedium. Positive

controls using toxicants recommended in the corresponding

ISO standardwere alsomeasured to check the sensitivity of the

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 10 20 30 40 50 60 70

Days

Ab

so

rb

an

ce

Fig. 1 e Time course of decolorization of Methylene Blue

(oeo) and textile dyeing liquors containing reactive dyes

(CeC, Wastewater I) and disperse dyes (:e:,

Wastewater II) in RBC reactor with Irpex lacteus.

Absorbance was measured at the corresponding

absorption maxima: Methylene Blue 580 nm, Wastewater I

575 nm, Wastewater II 475 nm. The absorbance values

represent the means of three samples.

0

5

10

15

20

25

30

35

40

45

50

Mu

tatio

n p

oten

tia

l

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3e7 1 4 8 7145

individual tests. In thetests, EC50or IC50valueswerecalculated

and expressed as a logarithm of the dilution factor determined

as a volumepercentage (v/v%) of the diluted sampleused in the

test relative to the original undiluted sample removed from the

reactor. This enabled us to compare the changes of EC50 and

IC50measured before and after degradation.

2.5. Mutagenicity (Ames) test

Mutagenicity was detected using a plate-incorporation version

of the Salmonella typhimuriumHise reversion assay usedwith or

without the in vitro metabolic activation with the rsat liver S9

microsomal fraction and cofactor mixture (OECD Test No. 471,

1997). The auxotrophic strains TA100 and TA98 were used for

detection of the base substitution mutations and frameshift

mutations, respectively. Themutagenic activity was expressed

as a number of revertant colonies (Rt) obtainedwith the treated

sample compared to thenumber of revertant colonies obtained

with the control sample (Rc). The index of mutagenicity was

calculated as a ratio of Rt/Rc and a twofold increase of the index

was considered to be significant. Mutation potential repre-

sented the indexofmutagenicity related to the concentrationof

the tested compound expressed in micrograms. Each test was

repeated at least three times using two replicate plates for each

sample and the results were calculated using SALM software

(Broekhoven and Nestmann, 1991).

1 2 3 4 1 2 3 4 1 2 3 4

Day 0 Day 20 Day 70

Fig. 2 e Mutagenicity of Methylene Blue during

biodegradation in RBC reactor measured with Salmonella

typhimurium His- test with and without S9 activation.

Samples were withdrawn on Day 0, Day 20 (intermediates)

and Day 70 (endproducts): TA100-S9 (1); TA98-S9 (2);

TA100 D S9 (3); TA98 D S9 (4).

3. Results and discussion

3.1. Decolorization and detoxification of model dyes

Decolorization of DB3 was used as a forerunner test of the

ability of the fresh-grown I. lacteus biofilms mounted in the

RBC reactor to decolorize recalcitrant dye compounds as the

funguswas previously reported to decolorize and detoxify DB3

(Malachova et al., 2006). The dye was completely decolorized

within 25 days (data not shown). Then the reactorwaswashed

with MEG medium and, subsequently, a batch of MEG con-

taining MB was added. A complete decolorization was ach-

ieved within 70 days (Fig. 1). The total amount of fungal

biofilms on the surface of PUF discs in RBC after decolorization

of MB was 7.53 g dry biomass and the average decolorization

rate was calculated to be 0.34 mg MB d�1 g�1 dry biomass.

These data well compare with other fungi, namely, the

decolorization of 5e20 mg MB L�1 by Phanerochaete chrys-

osporium and T. versicolor (Mazmanci et al., 2002; Radha et al.,

2005) or a partial decolorization of 200 mg MB L�1 by Lenti-

nula edodes (Boer et al., 2004).

Sampling at Day 0, 20 and 70 (cf. Figs. 2 and 3) intended to

measure the toxicity of intact MB, degradation intermediates

and the endproduct in keeping with the time course of MB

decolorization shown in Fig. 1. OnDay 0, themutagenic effects

of the dye were observed in all variants of the Ames test. MB

induced frameshift and substitution, direct and indirect mu-

tations both with and without metabolic activation. However

in the tests without metabolic activation MBwas concluded to

be only potentially mutagenic since a significant, twofold in-

crease of the revertant number was not accomplished due to

the toxicity of high sample concentrations for the indicator

strains. The respectivemaximalmutagenic activities obtained

with TA100 and TA98 strains expressed as the index of

mutagenicity were 1.54 (25 mg MB per plate) and 1.85 (2.5 mg MB

per plate). In the tests with metabolic activation, the values of

the index of mutagenicity were even higher: TA100 1.78 (25 mg

MB per plate), TA98 3.85 (2.5 mg MB per plate). The results thus

confirmed the genotoxic effects of the intact dye reported by

other authors (NTP TR 540, 2008). The degradation resulted in

a complete detoxification: the sample removed at Day 70,

when the absorbance decreased to zero, exhibited no

0

1

2

3

1 2 3 1 2 3 1 2 3

Day 0 Day 20 Day 70

Lo

g E

C50 o

r IC

50

Fig. 3 e Toxicity of Methylene Blue expressed as Log EC50

or IC50 during biodegradation in RBC reactor measured

with Vibrio fischeri (1), Sinapis alba (2), and Lemna minor (3).

Samples were withdrawn on Day 0, Day 20 and Day 70 to

measure the toxicity of intact dye, intermediates and

endproducts of biodegradation, respectively.

-4

-3

-2

-1

0

1

2

3

1 2 3 1 2 3 1 2 3

Day 0 Day 10 Day 26

Lo

g E

C50 o

r IC

50

-1

0

1

2

3

1 2 3 1 2 3 1 2 3

Day 0 Day 40 Day 47

Lo

g E

C50 o

r IC

50

b

a

Fig. 4 e Toxicity of Wastewater I (a) and Wastewater II (b)

during biodegradation in RBC reactor measured with Vibrio

fischeri (1), Sinapis alba (2) and Lemna minor (3). The samples

withdrawn on Day 40 and Day 47 (Wastewater I) and Day

10 and Day 26 (Wastewater II) represented 50 and 100%

decolorization to measure the toxicity of biodegradation

intermediates and endproducts, respectively.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3e7 1 4 87146

mutagenic effect (Fig. 2). A similar removal of mutagenicity by

I. lacteus growing in static liquid cultures was demonstrated

for Reactive Orange 16 azo dye (Malachova et al., 2006).

Tests of acute biological toxicity demonstrated a significant

toxicity of the sample containing intact MB removed on Day 0.

The V. fischeri test and the plant tests with L. minor and S. alba

showed the following respective toxicity values:

EC50 ¼ 18.07 � 0.31 mg L�1, EC50 ¼ 12.22 � 0.55 mg L�1 and

IC50 ¼ 100 � 0.1 mg L�1 (Fig. 3). The germination of seeds of S.

alba was 5- and 8-fold less sensitive to the toxic effect of the

dye than the bacterial luminiscence and growth of L. minor,

respectively. These results showed that MB was toxic for or-

ganisms of various trophic levels and confirmed previous

findings of the adverse side-effects (e.g. NTP TR 540, 2008;

Wainwright et al., 1999). The EC50 values demonstrated a

good sensitivity of the test battery and were comparable to

those obtained for various azo dyes with crustaceans Daphnia

magna and Desmocaris trispinosa (Ogugbue and Oranusi, 2006;

Verma, 2008). The decrease of biological toxicity of MB dur-

ing biodegradation, expressed as EC50 or IC50, exceeded one

order of magnitude. In the tests with L. minor up to a 10%

stimulation of growth of fronds, compared to the control, was

observed with the sample withdrawn at Day 70; probably, the

degradation endproducts were usable as nutrients by the

plants. The stimulation effect along the gradient of end-

product concentration was found to be significant (F ¼ 15.57,

p ¼ 0.017). The fitted model: y (rate of growth) ¼ 0.0008

conc þ 0.137.

Similar studies reported both decrease and increase of

biological toxicity after the treatment of various dyes with T.

versicolor and Penicillium simplicissimus, when monitored with

V. fischeri and Daphnia pulex tests (Bergsten-Torralba et al.,

2009; Ramsay and Nguyen, 2002).

3.2. Decolorization and detoxification of textile dyeingliquors

Textile wastewaters containing reactive (Wastewater I) or

disperse (Wastewater II) dyes were completely decolorized in

the RBC reactorwithin 47 and 26 days, respectively (Fig. 1). The

decolorization ofWastewater I containing two reactive dyes, a

high concentration of NaCl and Texalcon MS fixation agent

was slower than that of Wastewater II containing three

disperse dyes and low concentrations of disperging agent and

acetic acid. The difference of decolorization ratesmay reflect a

high NaCl concentration in Wastewater I and the presence of

dispersant in Wastewater II (cf. Novotny et al., 2003). It is not

easy to compare the decolorization of wastewaters with other

studies due to different compositions of wastewaters and

various fungi and reactor types used. The rate of decoloriza-

tion of Wastewater II was comparable to the data obtained for

decolorization of an untreated textile wastewater by Pleurotus

flabellatus (60e70% decolorization within 10 d; Nilsson et al.,

2006) or a crude effluent from a dye manufacture by Pleuro-

tus sanguineus (70% decolorization within 14 d; Vanhulle et al.,

2008) but lower than the decolorization rate of a pigment plant

effluent by Pycnoporus cinnabarinus (100% decolorization

within 3 d; Schliephake et al., 1993). I. lacteus was able to

completely remove the color of both Wastewater I and II and

demonstrated a strong potential for decolorization of true

industrial effluents when used under the conditions of RBC

reactor.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 7 1 4 3e7 1 4 8 7147

Neither of the wastewaters tested exhibited a genetic

toxicitywhenmeasuredwith the Ames testwith orwithout S9

activation before the treatment. In order to check the possi-

bility of formation of genotoxic degradation products, samples

were removed when the absorbance decreased below 50% of

the initial value (intermediates) and at the end of the experi-

ment (endproducts), i.e. on Days 40 and 47 for Wastewater I

and on Days 10 and 26 for Wastewater II, respectively (cf.

Fig. 1). The results indicated that no genotoxic intermediates

or end-products were formed (data not shown).

The biological detoxification of Wastewater I and II was

again measured with V. fischeri, L. minor and S. alba. A signifi-

cant toxicity ofWastewater I was detected with all three tests,

S. alba being the least sensitive of the tests used (Fig. 4A). The

degradation led to a 10-fold decrease of toxicity measured

with V. fischeri and L. minor. The severe inhibition of growth of

L. minor by Wastewater I was probably caused by a high con-

centration of NaCl in the dyeing liquor as salinities exceeding

1.66% had been reported toxic for the plant (Haller et al., 1974).

Interestingly, the high toxicity was removed by I. lacteus sug-

gesting an important desalting capacity of fungal biofilms

(Fig. 4A). The respective initial toxicity values ofWastewater II

were 5e10-fold inferior to those of Wastewater I when

measured with V. fischeri and L. minor. On the other hand, the

toxicity of Wastewater II measured by germination of S. alba

seeds exceeded that of Wastewater I more than 103-fold

(Fig. 4A, B). The degradation decreased the toxicity of Waste-

water II measured with Vibrio fisheri and S. alba 10- and 104-

fold, respectively, but the toxicity for L. minor remained the

same, suggesting that the latter toxic effect was probably not

caused by the dyes but by other compounds that were not

degraded by the fungus (Fig. 4B). The results demonstrated a

strong detoxification power of I. lacteus comparable, for

instance, to T. versicolor capable to decrease the toxicity of

Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Bril-

liant Red 3G-P dyes (Ramsay and Nguyen, 2002) or of a textile

effluent from dyeing with cochineal extracts (Arroyo-Figueroa

et al., 2011).

The battery of the toxicity tests was applied to monitor the

decrease of genetic and biological toxicity during biodegra-

dation of dyes. The sensitivity of the individual tests to various

toxicants differed: V. fischeri test was themost sensitive to MB,

L. minor test to Wastewater I, and S. alba test to Wastewater II.

These differences evidently reflected the involvement of

various biological processes targeted by the toxicants in the

individual tests: bioluminiscence, growth, and seed germina-

tion. This fact stressed the importance of using batteries of

tests that include various endpoints and organisms of

different trophic levels for the evaluation of environmental

toxicity.

4. Conclusions

� I. lacteus biofilms used in the RBC-type reactor with PUF

discs demonstrated a high efficiency of decolorization and

detoxification of recalcitrant, medium-toxic dyes and textile

dye mixtures in industrial dyeing liquors and confirmed a

potential of this fungal technology for remediation of textile

wastewaters. No production of genetically or biologically

toxic compounds was observed during the dye removal. The

endproducts of MB degradation were usable as nutrients for

growth of L. minor and S. alba.

� A battery of acute toxicity tests comprising the bio-

luminiscence test and plant growth- and seed germination

tests showed good sensitivity for monitoring the toxicity of

textile dyes, comparable to often used water crustacean

tests.

� Different sensitivities of the individual tests towards

particular dyes and dyemixtures stressed the importance of

using various biological targets for the assessment of danger

represented by textile waste effluents.

Acknowledgments

The provision of textile wastewaters by INOTEX s.r.o., Czech

Republic is gratefully acknowledged. We thank R. Po�rızka, M.

Hole�sova, L. Va�sutova and I. Falgentragerova for help in real-

ization of the experiments. We acknowledge the financial

support from the following projects: IAAX00200901 (Grant

Agency of the ASCR), Institutional Research Concept No.

AV0Z50200510, SGS 19/PrF/2012, Inst. Environ. Technol. proj-

ect CZ.1.05/2.1.00/03.0100 realized within Research and

Development for Innovations Operational Programme co-

financed by Structural Funds of EU and the Czech Republic.

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