Enzymatic decolorization of spent textile dyeing baths composed by mixtures of synthetic dyes and additives

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Enzymatic decolorization of spent textile dyeing baths composedby mixtures of synthetic dyes and additives

Ilaria Ciullini & Antonella Gullotto & Silvia Tilli &Giovanni Sannia & Riccardo Basosi &Andrea Scozzafava & Fabrizio Briganti

Received: 18 July 2011 /Revised: 1 December 2011 /Accepted: 5 December 2011 /Published online: 17 January 2012# Springer-Verlag 2012

Abstract The effects of different components of real dye-ing bath formulations, such as the equalizing and fixingadditives—acids, salts, and surfactants—on the decoloriza-tion catalyzed by Funalia trogii enzymatic extracts, wereinvestigated to understand their influence on the recalci-trance to biodegradation of this type of wastewater. Thedecolorization of selected dyes and dye mixtures after tissuedyeing was performed in the presence/absence of auxiliarycompounds. All spent dyeing baths were enzymaticallydecolorized to different extents, by the addition of extractscontaining laccase only or laccase plus cellobiose dehydro-genase. Whereas surfactant auxiliaries, in some instances,inhibit the decolorization of spent dyeing baths, in severaloccurrences the acid/salt additives favor the enzymatic pro-cess. In general, the complete spent dyeing formulations arebetter degraded than those containing the dyes only. Thecomparison of extracellular extracts obtained from spentstraws from the commercial growth of Pleurotus sp. mush-rooms with those from F. trogii reveals similar decoloriza-tion extents thus allowing to further reduce the costs ofbioremediation.

Keywords Trametes trogii .Funalia trogii .Pleurotus .

Laccase . Cellobiose dehydrogenase . Textile dye mixtures .

Decolorization . Bioremediation . Additives

Introduction

Synthetic dyes are extensively used in textile and leatherdyeing, pharmaceutical, food, paper printing, color photog-raphy, cosmetics, and other industries. Worldwide over10,000 different dyes and pigments are used in textile andprinting industries (Kaushik and Malik 2009). The totalworld colorant production is estimated to be 800,000 tons/year, and generally 20% of the used dyestuff enters theenvironment through wastes. Most of these dyes are toxicand potentially carcinogenic, and their removal from indus-trial effluents is a major environmental problem (Golka et al.2004). Furthermore, being highly colored, dyes are readilyapparent in wastewater, which is a further reason for theirbreakdown before discharge into the environment. The newenvironment regulations concerning textile products havebanned the discharge of colored waste in natural waterbodies.

Several traditional techniques have been used to treat dyewastewaters (Robinson et al. 2001). Adsorption, coagula-tion, and membrane processes are effective physical andchemical techniques for color removal, but they use moreenergy and chemicals consuming than biological processesand may cause secondary pollution problems in the form ofsludge.

The microbial decolorization and degradation of dyes hasbeen of considerable interest due to their inexpensive andeco-friendly nature as well as the property of producinglower amounts of sludge (Robinson et al. 2001). In particular,decolorization of azo dyes by bacteria starts by reductive

I. Ciullini :A. Gullotto : S. Tilli :A. Scozzafava : F. Briganti (*)Dipartimento di Chimica, Università degli Studi di Firenze,Via Della Lastruccia 3,50019 Florence, Italye-mail: [email protected]

G. SanniaDipartimento di Chimica Organica e Biochimica,Università degli Studi di Napoli “Federico II”,Naples, Italy

R. BasosiDipartimento di Chimica, Università degli Studi di Siena,Siena, Italy

Appl Microbiol Biotechnol (2012) 96:395–405DOI 10.1007/s00253-011-3809-y

cleavage of azo bonds under anaerobic conditions (McMullanet al. 2001). Although this step leads to decolorization of dyes,it generates amines of the dye-related structures that are notdegraded under anaerobic conditions and accumulate to toxiclevels (Gottlieb et al. 2003).

On the contrary, white rot fungi, by virtue of their abilityto degrade lignin in nature, produce enzymes like ligninperoxidases, Mn peroxidases, versatile peroxidases, andlaccases that are able to carry out oxidative decolorizationof dyes thus bypassing the danger of formation of carcino-genic amines (Fu and Viraraghavan 2001; Wesenberg et al.2003). However, due to the fact that peroxidases are depen-dent on the cosubstrate hydrogen peroxide, these have notbeen considered for large-scale applications (Wesenberg et al.2003). Laccases seem to be the most promising candidates forenzyme-mediated remediation processes because of theirbroad substrate specificity, use of molecular oxygen as finalelectron acceptor, easy production, and rapid action at milderpH and temperature (Giardina et al. 2010; Husain 2006).

In the present study, we tested the catalytic action ofextracellular extracts from fungi such as Funalia trogii andPleurotus sp. HK35 which mainly secrete laccases under theconditions utilized (Ciullini et al. 2008). Their decoloriza-tion capabilities were investigated on single dyes and on dyemixtures in the presence and in the absence of auxiliarycompounds generally added to the industrial dyeing bathssuch as salts and specific surfactants as color equalizers andfixers at high concentrations. The effects of such com-pounds on the extent of decolorization are analyzed anddiscussed.

Materials and methods

Chemicals

All the chemicals were purchased from Sigma Chemical Co.Agar and yeast extract were fromOxoid Ltd. Textile dyes, andadditives utilized were from Eurocolor S.p.A., InternationalColor S.p.A., and Ciba Specialty Chemicals S.p.A. (seeTable 1).

Organism and culturing conditions for laccase production

The white rot fungi F. trogii 201 (DSM 11919) and Pleurotussp. HK35 (a commercial strain for mushroom productionavailable at Sylvan Inc., Langeais, France) were maintainedon basidiomycete-rich medium (BRM) (Bezalel et al. 1997)agar plates at 4 °C and periodically transferred onto freshBRM agar plates and grown at 28 °C. After 4–6 days ofgrowth on agar plates, 500-ml shaken flask cultures contain-ing 150-ml liquid BRMwere prepared and inoculated with tenplugs of fungal mycelia (about 25mm2) and grown in the dark

at 28 °C under continuous stirring at 130 rpm. After 4 days,the grown mycelia were transferred in baffled 2,000-mlErlenmeyer flasks containing 1,000 ml of fresh BRM liquidmedium and grown under the same conditions. The laccaseexpression was further induced by the addition of 150 μMCuSO4. When the extracellular laccase activity reached amaximum on days 7–9 (7–8 Uml−1), the culture supernatantwas collected by filtration through Whatman No. 1 paper andconcentrated using an ultrafiltration Vivaflow 200 module(Sartorius group) with a 30,000-Da cutoff membrane.

Culturing conditions for simultaneous CDH and laccaseproduction

The fungus F. trogii 201 was cultivated in the above-described conditions, but detectable cellobiose dehydroge-nase (CDH) production occurred only in media containingcellulose powder as carbon source. Therefore, we modifiedthe BRM medium substituting the glucose, which inhibitsCDH expression, with 10 gl−1 cellulose (Stapleton andDobson 2003). When the extracellular CDH activity reacheda maximum, about on days 8–9 (up to 0.6 Uml−1), theculture supernatant was collected by filtration throughWhatman No. 1 paper and concentrated using an ultrafiltra-tion Vivaflow 200 module (Sartorius group) with a 30,000-Da cutoff membrane. Manganese peroxidase and ligninperoxidase activities were not detected in any of the differ-ent conditions utilized for fungal growth.

Enzyme assays

Because of the complexity and the heterogeneity of theextracellular enzymes in F. trogii 201 and Pleurotus sp.HK35 broth culture, the activities of three ligninolytic en-zyme families—peroxidases, laccases, and CDH—that canbe involved in the textile dyes decolorization have beendetermined by using a variety of methods. Laccase activitywas determined spectrophotometrically based on the capac-ity of this enzyme to oxidize the non-phenolic compound2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), measur-able at 420 nm (ε420036,000M

−1 cm−1), pH 3 at 25 °C; 1 U oflaccase activity was defined as the amount of enzyme oxidiz-ing 1 μmol of substrate per minute (Wolfenden and Willson1982). CDH activity was assayed by following the decrease inabsorbance of the electron acceptor, i.e., 2,6-dichlorophenol-indophenol (DCPIP), at 520 nm (ε52006.8×10

3 M−1cm−1),pH 4.0 and 37 °C. One unit of enzyme activity is defined asthe amount of enzyme reducing 1 μmol of DCPIP/min underthe above reaction conditions (Baminger et al. 1999). Thecombined determination of laccase and CDH activities wasperformed using 0.1 mM DCPIP following the method byVasil’chenko et al. (2005). To understand if the DCPIP reduc-ing activity observed was really due to CDH and not to a sugar

396 Appl Microbiol Biotechnol (2012) 96:395–405

Table 1 Textile dyes studied in the present investigation

Acid Violet 90 (Avi-90)

N

CH3

OH

N

N

OH

N

SO3Na

Reactive Red 272 (Rre-272) BrH2CHO3S

SO3HSO3H

EtPh

Br

ON

H

N

N

OHNHCl

N

N

N

Acid Yellow 241 (Aye-241)

SO3Na

N

Cl

CH3

N

OH

Cl

N

N

CH3

Reactive Yellow 39 (Rye-39)

Cl

Cl

O

CH3

N

N

N

N

CH2

SO3H

HNOCCBr

SO3H

Acid Blue 193 (Abu-193)

OH

OH

N

N

SO3Na

Acid Black 194 (Aba-194)

SO3Na

NO2

OH

N

N

OH

Acid Yellow 99 (Aye-99)

NO2-

NaO3S

OH

CH3

OH

O

N

H

N

N

Cr+

Acid Yellow 129 (Aye-129)

CH3

ON

N

N

N

HOOC

Acid Red 183 (Are-183)

SO3Na

SO3Na

Cl

OH

OH

N

CH3

N

N

N

Acid Red 42 (Are-42)

SO2

SO3Na

OH

NH2

NN

Acid Blue 158 (Abu-158)

SO3Na

SO3Na

OH

NN

OH Acid Yellow 49 (Aye-49)

SO3H

NH2

CH3

N

NNN

Cl

Cl

Acid Yellow 42 (Aye-42)

Acid Red 374 (Are-374)

SO3Na

SO3Na

SO3Na

OH

NNNN

OH

Acid Blue 40(Abu-40)

Acid Blue 80 (Abu-80)

Reactive Blue 69 (Rbu-69)

SO3H

SO3H

Br

CH2

O

NH

NH

NH2

O

O

Appl Microbiol Biotechnol (2012) 96:395–405 397

oxidase usually present in the cellulolytic systems offungi, i.e., glucose oxidase, in this assay we substitutedcellobiose with D-glucose; no glucose oxidase activitywas observed (Ander and Marzullo 1997; Henriksson etal. 2000). Manganese-oxidizing peroxidase activity wasestimated by the formation of Mn3+–tartrate complex(ε23806,500 M−1 cm−1) at pH 5, 25 °C. Lignin perox-idase activity was determined by the H2O2-dependentveratraldehyde (3,4-dimethoxybenzaldehyde) formation(ε31009,300 M−1 cm−1), pH 3 and 25 °C.

Spent dyeing bath preparation

The textile dyeing baths formulations were utilized as pro-vided by the manufacturers. The percentages of dyes andauxiliaries utilized are to be considered in w/v for solids andv/v for liquids. A bath volume of 20 l was used to treat 1 kgof wool.

The dyeing procedure for the individual dyes and for thereactive mixture was as follows: Kollasol LO-BD was addedto the dyeing bath at room temperature; once 40 °C temper-ature was reached, the other auxiliaries were added and atlast the dye was dissolved in the bath. The wool dyeingoccurred at 98 °C for 45 min, and then the bath was broughtback to room temperature.

The dyeing procedure for all the other dyeing bathsmixtures was as follows: The additives and the dyes weredissolved in the bath at room temperature in the presence ofthe wool. The dyeing occurred by heating the bath at 98 °Cand maintaining that temperature for 40 min, and the bathwas then brought back to room temperature.

Enzymatic dyes decolorization

The reaction mixtures for dyes-decolorizing tests were pre-pared in 50–500 ml flasks and consisted of an aqueoussolution of the single dye (0.2–0.5 mg/ml) or of thecorresponding spent dyeing bath up to total volumes of20–200 ml, respectively. The decolorizing reactions wereinitiated adding crude fungal extract (containing 1.5 U/mllaccase; or 1.5 U/ml laccase + 0.9 U/ml CDH) and incubatedat 30 °C with shaking (300 rpm) for the appropriate times(see “Results” and “Discussion” sections). Samples of dyesolutions were taken at regular times, centrifuged at13,000 rpm for 5 min to eventually remove suspendedparticles, and the decolorization extent was measured afterappropriate dilution (1:20–1:5) in triplicate. Control sampleswere tested under identical conditions. UV–visible measure-ments were carried out on double-beam Perkin Elmer EZ301 spectrophotometer using 1 cm path length Hellma 110quartz suprasil cells thermostated with a Lauda Ecoline low-temperature thermostat RE112. The UV–visible absorptionspectrum was recorded (400–800 nm) for each dye mixture,

and decolorization was followed monitoring the absorptionareas in the above-mentioned wavelength range utilizing theformula: (Areainitial−Areafinal) 100/Areainitial.

Results

Decolorization of individual dyes in water solution

The dyes studied in the present investigation were selectedamong the most commonly used for wool dyeing in the azoand anthraquinonic classes (see Table 1). As referencepoints, the decolorization extents were first determined forthe individual dyes in water solutions after different times ofthe enzymatic treatment with crude extracts containinglaccase only. The final results, shown in Table 2, evidenceassorted degrees of decolorization mostly depending on thedye structure. Among the mono-azo chromo-complexes(hereafter called pre-metallized dyes) Avi-90, Abu-193,Abu-158, and Aba-194 are decolorized more than 85%,Aye-129 and Aye-99 approximately 30%, whereas Are-183 and Aye-241 remain substantially unaffected. Themono-azo are generally less degraded by the action of lac-case activity; only Are-42 is decolorized more than 90%whereas Rre-272, and Aye-49 show color losses of about10% and 25%, respectively; lastly, Rye-39 is not decolor-ized at all. Both dis-azo dyes tested: Aye-42 and Are-374 aredecolorized more than 90%. The anthraquinonic dyes Abu-80 and Rbu-69 show almost complete decolorization where-as Abu-40 reaches 54% color decrease.

Decolorization of spent dyeing baths containing single dyesin the presence of different auxiliaries

Two acidic (Aba-194 and Aye-129) and two reactive (Rre-272 and Rye-39) dyes, among those investigated in watersolutions, were then tested for their enzymatic decoloriza-tion, after wool dyeing, in the presence of the auxiliariesutilized in the appropriate dyeing baths (see Table 2). Thedecolorization of the acid dye baths was analyzed in solu-tions containing each of the following auxiliaries: KollasolLO-BD, a silicone-based antifoam and deaerating agentwith wetting properties; Breviol SCN, a cationic amineethoxylate leveling agent; the third additive was acetic acid80% for the Aba-194 dye and ammonium sulfate for theAye-129 dye in the quantities reported in Table 2. The bathdyeing procedure was performed according to “Materialsand methods” section. All the spent baths containing thesedyes, independently from the added auxiliaries, were totallydecolorized by the enzymatic extract (see Table 2).

In the case of the reactive dyes Rre-272 and Rye-39,chosen because particularly recalcitrant to decolorization inwater solution, the auxiliaries Kollasol LO-BD (see above),

398 Appl Microbiol Biotechnol (2012) 96:395–405

Setavin RE (a non-ionic alkylamine ethoxylate used toincrease the absorption rate of reactive dyestuffs during theheating-up phase and to improve the dyestuff migration),and acetic acid were added (in the quantities reported inTable 2) and tested after wool dyeing. Regarding Rre-272,the addition of Kollasol LO-BD and/or Setavin RE didresult in an inhibition of the enzymatic decolorization(0%) if compared to the dye only (9.4%). Instead, the furtheraddition of the fixing auxiliary acetic acid contributed toincrease the decolorization extent to about 40% (seeTable 2). Rye-39, a peculiarly recalcitrant dye, was notdecolorized at all, also in the presence of each or all of theauxiliaries mentioned above.

Decolorization of spent dyeing baths containing dyemixtures and auxiliaries

More complex spent dyeing bath formulations composed bydye mixtures with or without the addition of the additives,

required for optimal wool dyeing, were then investigated(see Table 3). The first formulation (pre-metallized 1:2mixture) composed by Avi-90, Aye-241, and Abu-193 dyes(Fig. 1a) was tested with or without the addition of the dyesfixing ammonium sulfate and the equalizing Setavin MSN(a non-ionic ethoxylated alkylamine utilized as an equaliz-ing agent for wool dyeing with pre-metallized and aciddyes) agents in the quantities reported in Table 3. For thisspent bath, with or without additives, no color reduction wasobserved after the addition of laccase containing extractswhereas the utilization of extracts including laccase andCDH activities brought to an almost compete color disap-pearance (91%) (Fig. 1b). In both cases, we observed a shiftin pH from an initial 7.1 to a final 8.6.

The addition of laccase extract to the spent dye mixtureof the second formulation (pre-metallized 1:1 mixture) com-posed by Aye-99, Are-183, and Abu-158 dyes without addi-tives produced only a 5% color reduction (pH changed from7.2 to 8.6 final) whereas in the presence of the additives

Table 2 Single dyes decolorization with fungal extracellular extracts

Class Dye Decolorization %in water

Decolorization % in spent dyeing bath

Monoazo chromo-complex Avi-90 93

Aye-241 0.0

Abu-193 86

Aye-99 29.4

Are-183 0.0

Abu-158 87

Aba-194 89 100 (dye 2%)

100 (dye 2% + Kollasol LO-BD 0.5%)

100 (dye + Kollasol LO-BD 0.5% + Breviol SCN 0.3%)

100 (dye + Kollasol LO-BD 0.5% + Breviol SCN 0.3% + acetic acid 1%)

Aye-129 27.5 100 (dye 1%)

100 (dye 1% + Kollasol LO-BD 0.5%)

100 (dye 1% + Kollasol LO-BD 0.5% + Breviol SCN 0.5%)

100 (dye 1% + Kollasol LO-BD 0.5% + Breviol SCN 0.5% + ammonium sulfate 1%)

Monoazo Rre-272 9.4 0 dye 1.1% no auxiliaries

0 (dye + Kollasol LO-BD 0.5%)

0 (dye + Kollasol LO-BD 0.5% + Setavin RE 2%)

41 (dye + Kollasol LO-BD 0.5% + Setavin RE 2% + acetic acid 1.5%

Rye-39 0.0 0 dye 1.1% no auxiliaries

0 (dye + Kollasol LO-DB 0.5% + Setavin RE 2% + acetic acid 1.5%)

Are-42 93.4

Aye-49 24.5

Disazo Aye-42 93.9

Are-374 97

Antraquinones Abu-80 98.6

Abu-40 53.8

Rbu-69 88.7

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Setavin MSN, sulfuric acid, and sulfamic acid (in the quan-tities reported in Table 3), a 30% color decrease was ob-served (pH started at 3.9 and ended at 6.8). Looking at theindividual dyes, Are-183, under the same conditions, is notdecolorized (pH increased from an initial 7.6 to 8.4), whilethe Aye-99 color is diminished of about 30% (pH increasedfrom an initial 6.7 to 8.1) and Abu-158 is high degraded(87%) (the pH increased to a lower extent starting from 6.3to a final value of 6.7).

A third formulation (Supracen mixture) was made up bythe acidic Aye-49, Are-42, and Abu-40 dyes plus the addi-tives Setavin MSN and acetic acid. A limited color reduc-tion (5%) was noticed in the absence of auxiliaries (pH from7.8 to 9.0) with the laccase containing extract whereas theeffect was substantially larger on the complete recipe (83%,pH from 5.6 to 9.2).

With the fourth dyeing bath (Mix Follone) prepared withAye-42, Are-374, and Abu-80 (Fig. 2a) with or without theadditives Setavin MSN and ammonium sulfate, the laccaseextract effect resulted in a 22% decolorization (Fig. 2b) (pHfrom 8.0 to 8.8) in the absence of the additives whereas thecomplete bath was decolorized to 82% (Fig. 2c) (pH 7.6 to7.9) and 87% (pH 6.9 to 8.8) if treated with laccases only orlaccases plus CDH, respectively.

A reactive dye formulation (reactive mixture) with Rbu-69, Rre-272, and Rye-39 was tested only in the presence ofthe additives Kollasol LO-BD, Setavin RE, and acetic acidsince they were necessary to maintain in solution all the

dyes and to reduce hydrolytic processes which usually in-hibit dyes fixation to fibers. In the present study, weevidenced that the complete spent dye bath was totallydecolorized with the sole action of laccases.

Comparison of the decolorizing action of extracellularextracts of Pleurotus sp. HK35 and F. trogii 201

To further reduce the costs of the remediation process, theutilization of fungal extracellular extracts obtained by wash-ing and squeezing spent straws used for the commercialgrowth of Pleurotus sp. HK35 mushrooms was investigated.The screening of oxidative enzymatic activities on thePleurotus sp. extracts revealed the presence of laccases only.

The decolorizing action of the obtained extracts was thencompared to the previously evaluated extracts from F. trogii.The individual dyes Aye-42, Are-374, and Abu-80 wereexamined. As shown in Table 4, the dyes Abu-80 and Are-374, although the same amount of enzymatic activity wasutilized, were decolorized to a lower extent (70–75% insteadof 97–99%) by Pleurotus sp. than by F. trogii. On the otherhand, Aye-42 is better decolorized by Pleurotus sp. than byF. trogii (93% versus 88%).

The corresponding spent dyeing mixtures (Mix Follone)with or without additives were at last tested to compare theaction of the two different extracts. The dye mixture wasbetter decolorized by Pleurotus sp. (80% instead of 22%)whereas in the presence of the additives Setavin MSN and

Table 3 Spent dyeing bathsdecolorization with fungalextracellular extracts

aLac 1.5 U/ml laccase, Lac + CDH1.5 U/ml laccase + 0.9 U/ml CDHbAdditives 1: Setavin MSN 0.75%+ ammonium sulfate 3%cAdditives 2: SetavinMSN0.75%+sulfuric acid 1%+ sulfamic acid 3%dAdditives 3: Setavin MSN 0.75%+ acetic acid 3%eAdditives 4:Kollasol LO-BD0.5%+ Setavin RE 2% + acetic acid 80%1.5%

Composition of dyeing bath % decolorizationa

MIX pre-metallized 1:2 Dyes Mix: 0 (Lac)

Avi 90 (0.9%) Dyes Mix+Additives 1b 0 (Lac)

Aye 241 (1.2%) 91 (Lac + CDH)Abu 193 (1.0%)

MIX pre-metallized 1:1 Dyes Mix: 5 (Lac)

Aye 99 (1.2%) Dyes Mix+Additives 2c 30 (Lac)

Are 183 (1.2%) 100 (Lac + CDH)Abu 158 (0.8%)

MIX Supracen Dyes Mix: 5 (Lac)

Aye 49 (1.5%) Dyes Mix+Additives 3d 83 (Lac)Are 42 (1.5%)

Abu 40 (1.2%)

MIX Follone Dyes Mix: 22 (Lac)

Aye 42 (0.9%) Dyes Mix+Additives 3d 82 (Lac)

Are 374 (1.2%) 87 (Lac + CDH)Abu 80 (1.2%)

MIX reactive Dyes Mix+Additives 4e 100 (Lac)Rbu 69 (0.4%)

Rre 272 (0.4%)

Rye 39 (0.4%)

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acetic acid comparable results were obtained with bothextracts (82–83% decolorization).

Discussion

A real bioremediation process for textile wastewater needsto take into account all the factors involved in the textiledyeing processes. The recalcitrance to decolorization of theindividual dyes, related to their molecular structure, thepresence of dye mixtures as well as the effect of relevantconcentrations of equalizing and fixing additives, such asacids, salts, and surfactants, need to be evaluated.

Usually the majority of the biological decolorizationstudies of synthetic textile dyes have been limited to analyzethe behavior of individual dyes to the enzymatic attack.Only a few studies have been performed to date on dyemixtures simulating real dyeing baths (Abadulla et al. 2000;

Anastasi et al. 2010; Casieri et al. 2008; Di Gregorio et al.2010; Vanhulle et al. 2008).

Moreover, after textile dyeing, the exhausted baths alsocontain partially hydrolyzed dyes and traces of other sub-stances released from the tissue fibers which could influence

Fig. 2 a Visible absorption spectra of single textile dyes: Aye 42(dash-dot-dashed line), Abu 80 (dashed line, and Are 374 (solid line).Spectral changes of the decolorization of the corresponding spentdyeing bath (Follone dye mixture) without auxiliaries (b) and withauxiliaries (c): before (solid line) and after 24 h (dash-dot-dashed line)treatment with crude extracellular culture extract of F. trogii containinglaccase

Fig. 1 a Visible absorption spectra of single textile dyes: Avi 90 (solidline), Aye 241 (dash-dot-dashed line), Abu 193 (dashed line). bSpectral changes observed for the corresponding spent dyeing bathmixture (pre-metallized 1:2 dyes) before (solid line), after 4 h (dash-dot-dashed line), and after 24 h (dashed line) treatments with crudeextracellular culture extract of F. trogii containing laccase and CDHactivities

Appl Microbiol Biotechnol (2012) 96:395–405 401

the remediation process too. The present study attempts toexamine the effect of each constituent of real dyeing bathformulations on the decolorization process catalyzed byfungal extracts, containing laccases and CDH as the mainoxidizing agents, in order to understand their influence onthe recalcitrance to biodegradation of this type of wastewater.

Individual dyes decolorization: molecular structureinfluence

In order to perform such investigation, we first deter-mined the extent of enzymatic decolorization of thesingle dyes in water solutions. A variety of dyes be-longing to the azo and anthraquinonic classes, chosenamong the most utilized in textile applications for wooltissues dyeing, were tested for a comparative enzymaticdecolorization study (Table 1).

As shown in Table 2, the analyzed dyes exhibit differentdegrees of susceptibility to degradation with the yellowcolored being generally the most recalcitrant to decoloriza-tion (Aye241, Aye-99, Aye-129, and Aye-49). It can benoted that the chemical structures of the dyes largely influ-ence their enzymatic decolorization extent and rates. This ismainly due to differences in electronic distribution, chargedensity, as well as steric hindrances (Chivukula andRenganathan 1995; Ciullini et al. 2008; Pasti-Grigsby etal. 1992). Laccases have been shown to decolorize anthra-quinonic dyes more efficiently than other classes of dyes asconfirmed with Abu-80 and Rbu-69 which show almostcomplete decolorizations and Abu-40 which reaches 54%color decrease (Champagne and Ramsay 2010). Nevertheless,the most employed dyes belong to the azo class whichaccounts for the 70% of all textile dyes produced. Laccasesmodify azo dye structures by destroying their chromophoricassemblies (Chivukula and Renganathan 1995). The best bio-chemical decolorizations are achievedwith those azo dyes thatcarry hydroxyl groups in ortho and para positions to the azobond, which are known to be strongly electron-donating moi-eties such as in the mono-azo pre-metallized dyes Avi-90,Abu-193, Abu-158, and Aba-194 which are decolorized more

than 85%. On the contrary, electron-withdrawing substituentssuch as halogen or nitro groups on the aromatic rings make itdifficult for oxidases to form cation radicals thus inhibitingdyes degradation like in the mono-azo pre-metallized dyesAre-183 and Aye-241 which remain substantially unaffected.Instead, azo dyes characterized by even weakly electron-donating groups are decolorized efficiently as the mono-azodye Are-42 and the dis-azo dyes tested Aye-42 and Are-374decolorized more than 90%. Furthermore, heterocyclic azodyes, containing pyrazole or triazole rings, are not decolorizedsignificantly as in the case of the mono-azo Rye-39. Anyway,the presence of hydroxyl and other electron-donating groupson the heterocyclic and vicinal aromatic rings, in ortho posi-tion to the azo bond, results in a partial decolorization (15–30%), like in the cases of the mono-azo pre-metallized dyesAye-129 and Aye-99 and the mono-azo Rre-272 and Aye-49.Other effects, such as those caused by reaction intermediates,may contribute as well (Kandelbauer et al. 2004).

Influence of different auxiliaries on the decolorizationof spent dyeing baths containing single dyes

The effects of the presence of dyeing auxiliaries on theextent of enzymatic decolorization on the spent dyeing bathscontaining single dyes were then analyzed. The reactivedyes Rre-272 and Rye-39 were chosen because particularlyrecalcitrant to decolorization in water solution. For Rre-272,the presence of Kollasol LO-BD and Setavin RE, i.e., anti-foam and surfactant agents, resulted in a total lack of decol-orization whereas the presence of the third additive, aceticacid, increased the decolorization to about 40%. This obser-vation is in agreement with Champagne and Ramsay (2010)who showed that Reactive blue 19 decolorization wasinhibited increasing the concentration of a non-ionic surfac-tant. The enhanced decolorization observed in presence ofacetic acid (spent bath pH 5.1–5.2) can instead be associatedto the higher enzymatic activity shown by laccases at lowerpH which would contribute to overcame the surfactant in-hibition (Ciullini et al. 2008).

The Rye-39 dye, totally recalcitrant to decolorization inwater solution, remains unaffected also in the presence ofsuch auxiliaries thus showing that laccases are not able toattack this dye even at low pHs. In the case of the acidicdyes Aba-194 and Aye-129, the behavior to decolorizationwas substantially different since, after tissue dyeing, all thespent baths were completely decolorized with the additionof laccase containing extracts independently on theauxiliaries present. Therefore, whereas the acid additive(acetic acid) favored the decolorization of Rre-272 over-coming the observed surfactant inhibition, for the acid dyesthe presence of surfactant, antifoam, and acid/salt agents didnot substantially affect the decolorization process of thespent dye baths.

Table 4 Comparison of the decolorization effects of F. trogii andPleurotus sp. KH35 extracellular extracts

Dyes Funalia trogiia %decolorization

Pleurotus sp. KH35a

% decolorization

Abu-80 98.6 72.0

Aye-42 87.8 93.0

Are-374 97.0 75.0

Abu-80/Aye-42/Are-374 mix 22 80

Dyes mix + Setavin MSN0.75% + acetic acid 3%

82 83

a 1.5 U/ml laccase

402 Appl Microbiol Biotechnol (2012) 96:395–405

Influence of auxiliaries on decolorization of spent dyeingbaths containing dye mixtures

More complex spent dyeing bath formulations composed bydye mixtures with or without the addition of the additives,required for optimal wool dyeing, were then investigated.The first formulation (pre-metallized 1:2 mixture) composedby dyes Avi-90, Aye-241, and Abu-193 (Fig. 1a) was testedwith or without the addition of the dyes fixing ammoniumsulfate and the equalizing Setavin MSN (a non ionic ethoxy-lated alkylamine utilized as an equalizing agent for wooldyeing with pre-metallized and acid dyes) agents in thequantities reported in Table 3, but in both cases, no colorreduction was observed after application of laccase contain-ing extracts. This result seems to contradict the fact that inwater solutions, both Avi-90 and Abu-193 are decolorized toa large extent (93% and 86%, respectively) whereas onlyAye-241 results to be totally recalcitrant. Actually the start-ing visible spectrum of the spent bath (Fig. 1b) is mainlycharacterized by the features of Avi-90 with smaller contri-butions by the Aye-241 and Abu-193 dyes. We also notedsome differences in the pH between the spent bath mix andthe single dyes decolorization; the first was 7.1 at the be-ginning and ended up to 8.6 whereas the individual dyesshowed a starting pH between 6.4 and 7.0 and a final, afterthe enzymatic treatment of 6.2–6.8. The pH increase ob-served for the spent bath would surely result in lower or nolaccase activities thus explaining the lack of decolorizationascertained for the spent dye mixture (Fig. 1). This obser-vation resulted to be independent from the presence ofauxiliaries but due to differences between unused and spentdyes mix. In fact, after textile dyeing, the exhausted bathsalso contain partially hydrolyzed dyes and traces of othersubstances released from the tissue fibers which could in-hibit the remediation process.

Anyway, the further utilization of laccase plus CDHcontaining extracts resulted in a nearly complete decoloriza-tion (91%) (Fig. 1b). CDH, an extracellular fungal flavocy-tochrome enzyme, can, in fact, induce the production, by aFenton type reaction, of highly reactive hydroxyl radicals(Cameron and Aust 2001; Henriksson et al. 2000). Theattack of hydroxyl radicals generated by CDH can yieldthe demethoxylation and/or hydroxylation of many aromaticcompounds, possibly leading to the conversion of nonphe-nolic structures to phenolic ones, thus rendering the mole-cule easily oxidized by laccases (Hildén et al. 2000).

The addition of laccase extract to the spent dye mixtureof the second formulation (pre-metallized 1:1 mixture) com-posed by Aye-99, Are-183, and Abu-158 dyes without addi-tives produced only a 5% color reduction (pH changed from7.2 to 8.6 final) whereas in the presence of the additivesSetavin MSN, sulfuric acid, and sulfamic acid (in the quan-tities reported in Table 3), a 30% color decrease was

observed (pH started at 3.9 and ended at 6.8). Looking atthe individual dyes Are-183, under the same conditions, isnot decolorized (pH increased from an initial 7.6 to 8.4),while the Aye-99 color is diminished of about 30% (pHincreased from an initial 6.7 to 8.1) and Abu-158 is highdegraded (87%) (the pH increased to a lower extent startingfrom 6.3 to a final value of 6.7). The comparison of thevisible spectral data between the spent bath mix and theindividual dyes evidences that the larger contribution todecolorization is due to Abu-158, in particular for the spentmix including the auxiliaries since the acidic pH determinesa higher laccase activity. Also for the pre-metallized 1:1complete bath, the enzymatic extract containing laccase plusCDH induced the complete decolorization of the spent dye-ing bath thus evidencing the higher oxidizing environmentgenerated by CDH.

The third formulation (Supracen mixture) was made up bythe acidic Aye-49, Are-42, and Abu-40 dyes plus the additivesSetavin MSN and acetic acid. A limited color reduction (5%)was noticed in the absence of auxiliaries (pH changing from7.8 to 9.0) with the laccase containing extract whereas theeffect was substantially larger on the complete recipe (83%,pH from 5.6 to 9.2). Since for the individual dyes a variabledegree of discoloration was observed: Are-42 was decolorizedup to 93.4% (at pH 7.0), Abu-40 53.8% (with pH shiftingfrom 7.5 to 7.6), and Aye-49 24.5% only (with pH shiftingfrom 7.3 to 7.6), the noticeable differences between the spentbath mixture with no auxiliaries and the single dyes could beascribed to the pH which was higher for the spent dye bathwithout additives. Furthermore, from the comparison of thespectral data and the behavior of the individual dyes in watersolution, it is apparent that the Aye-49 dye, the most recalci-trant, is the only remaining in the mixture bath after theenzymatic treatment.

With the fourth dyeing bath (Follone mixture) preparedwith Aye-42, Are-374, and Abu-80 with or without theadditives Setavin MSN and ammonium sulfate, the laccaseextract effect resulted in a 22% decolorization (Fig. 2b) (pHfrom 8.0 to 8.8) in the absence of the additives whereas thecomplete bath was decolorized to 82% (Fig. 2c) (pH 7.6 to7.9) and 87% (pH 6.9 to 8.8) if treated with laccases only orlaccases plus CDH, respectively.

The individual dyes of the Follone mixture in watersolution were all degraded to a large extent: Aye-42,93.9% decolorization (pH from 6 to 7.1), Are-374 97%(pH 7), and Abu-80 98.6% (pH 7). Comparable values areobtained for the complete spent bath but not with the spentdye mixture without additives probably due to its higher pH(>8.0) (Fig. 2). Since the decolorization degree for thecomplete spent dyeing bath was very high, no trials withCDH were performed.

The reactive dye formulation (reactive mixture) withRbu-69, Rre-272, and Rye-39 was tested only in the

Appl Microbiol Biotechnol (2012) 96:395–405 403

presence of the additives Kollasol LO-BD, Setavin RE, andacetic acid since they were necessary to maintain in solutionall the dyes and to reduce hydrolytic processes which usu-ally inhibit dyes fixation to fibers. In fact hydrolysis of dyesis particularly pronounced for the reactive class, and severalstudies have investigated this aspect also showing that thehydrolyzed forms are usually more toxic (Gottlieb et al.2003; Tauber et al. 2005). In the present study, we evidencedthat the complete spent dye bath was totally decolorizedwith the sole action of laccases. This is an important resultsince, in water solution, both Rre-272 and Rye-39 weresubstantially recalcitrant to decolorization (9.4% and 0.0%,respectively), Rbu-69 being the only one degraded to a largeextent (>88% at pH 7). This could be probably related to therecent finding that hydrolyzed reactive dyes presented betterbiodegradability than the starting ones (Mezohegyi et al.2010). The degree of dyes hydrolysis for the different com-pounds and for the various dyeing procedures remains aquestion to be answered into details although some studyrecently appeared (Gottlieb et al. 2003; Mezohegyi et al.2010; Tauber et al. 2005). Furthermore, the hypothesis thatthe anthraquinonic dyes such as Rbu-69 could favor thedegradation of other dyes present in the mixture could alsohave support (Wong and Yu 1999).

Decolorizing action of different extracellular fungal extracts

To further reduce the costs of bioremediation, the employ-ment of fungal extracellular extracts obtained from spentstraws from the commercial growth of Pleurotus sp. HK35mushrooms was investigated. The screening of oxidativeenzymatic activities on the Pleurotus sp. extracts revealedthe presence of laccases only.

The decolorizing action of the obtained extracts wascompared to the previously evaluated extracts from F. trogii.The individual dyes Aye-42, Are-374, and Abu-80 as wellas the corresponding spent dyeing mixture (Mix Follone)with or without additives were finally tested to compare theaction of the two different extracts (Table 4).

Among the differences observed, the better decoloriza-tion of Aye-42 performed by Pleurotus sp. rather than by F.trogii (93% versus 88%) could be explained by the differentnature of the Pleurotus sp. extract. Pleurotus was in factgrown on natural straw. It is known that the fungal action onsuch media could generate natural mediators which shouldgenerally improve the decolorization process. Natural andchemical mediators have been studied for their ability toenhance the oxidation process catalyzed by laccases(Brogioni et al. 2008; Camarero et al. 2005; Johannes andMajcherczyk 2000; Li et al. 1999).

In summary, the response of single and mixed dyesto enzymatic decolorization in the presence of severalauxiliaries for textile dyeing resulted to be composite.

Surfactants, antifoams, and acid/salts necessary at rela-tively high concentrations (0.5–3.0% each) for optimaltextile dyeing affect the remediation process to differentdegrees.

The acidic fixing agents generally contribute to increasethe decolorization extent. This result has to be related to thehigher activity shown by the utilized laccase at lower pH.The presence of surfactants/antifoams has generally noeffect on the extent of dyes breakdown whereas in a fewinstances among those tested revealed an inhibitory out-come. The degree of dyes hydrolysis for the different com-pounds and for the various dyeing procedures remains aquestion to be answered into details although some studyrecently appeared (Gottlieb et al. 2003; Mezohegyi et al.2010; Tauber et al. 2005).

Furthermore, a previous study from our laboratoryshowed that the utilization of the combined oxidizing capa-bilities of laccases and CDH for the most recalcitrant dyesusually further aided the decolorization process (Ciullini etal. 2008). Analogous results have been obtained in thepresent investigation on spent dyeing baths where the con-tribution of CDH was fundamental for the breakdown of thedye mixtures most resistant to decolorization.

Several surveys evidenced that the azo-dyes breakdownthrough bio-oxidative processes also leads to a reducedtoxicity of the wastewaters (Abadulla et al. 2000; Anastasiet al. 2010; Casieri et al. 2008; Di Gregorio et al. 2010;Vanhulle et al. 2008). Toxicity studies will be performed onthe above-reported resulting dyeing baths in order to sub-stantiate the behavior observed in the literature. Ultimatelythis study remarks that raw fungal extracellular extracts arevalid alternatives to more expensive and less environmen-tally friendly chemical treatments of real complex textilewastewaters.

Acknowledgments We gratefully acknowledge the support of theItalian MIUR PRIN 2007 project funding and MECHOS Project,POR Ob3 Tuscany Region.

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