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Decolorization of Azo, Triphenylmethane and Anthraquinone Dyes by Laccase of a Newly Isolated Armillaria sp. F022

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A newly isolated white-rot fungus, Armillaria sp. strain F022, was isolated from the decayed wood in a tropical rain forest. Strain F022 was capable of decolorizing a variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes,
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  Decolorization of Azo, Triphenylmethaneand Anthraquinone Dyes by Laccase of a NewlyIsolated  Armillaria  sp. F022 Tony Hadibarata  &  Abdull Rahim Mohd Yusoff   & Azmi Aris  &  Salmiati  &  Topik Hidayat  & Risky Ayu Kristanti Received: 23 June 2011 /Accepted: 5 August 2011 # Springer Science+Business Media B.V. 2011 Abstract  A newly isolated white-rot fungus,  Armillaria sp. strain F022, was isolated from the decayed wood in a tropical rain forest. Strain F022 was capable of decolorizing a variety of synthetic dyes, including azo,triphenylmethane, and anthraquinone dyes, with anoptimal efficiency of decolorization obtained when dyesadded after 96 h of culture, with the exception of Brilliant Green. All of the tested dyes were decolorized by the purified laccase in the absence of any redoxmediators, but only a few were completely removed,while others were not completely removed even whendecolorization time was increased. The laccase, with possible contributions from unknown enzymes, played a role in the decolorization process carried out by  Armillaria  sp. F022 cultures, and this biosorptioncontributed a negligible part to the decolorization bycultures. The effect of dye to fungal growth was alsoinvestigated. When dyes were added at 0 h of culture,the maximum dry mycelium weight (DMW) values inthe medium containing Brilliant Green were 1/6 of that achieved by the control group. For other dyes, the DMWwas similar with control. The toxic tolerance of dye for the cell beads was excellent at least up to a concentrationof 500 mg/l. The optimum conditions for decolorizationof three synthetic dyes are at pH 4 and 40°C. Keywords  Armillaria  sp. F022.Brilliant Green.Laccase activity.Microbial decolorization.ReactiveBlack5.RemazolBrilliantBlueR  1 Introduction The textile industry, by far the most avid user of synthetic dyes, is in need of ecologically efficient  Water Air Soil Pollut DOI 10.1007/s11270-011-0922-6T. Hadibarata ( * ) :  A. R. M. Yusoff  :  A. Aris : SalmiatiInstitute of Environmental and Water Research Management,Universiti Teknologi Malaysia,81310 Skudai, Johor, Malaysia e-mail: hadibarata@utm.myT. Hidayat Department of Biological Science,Faculty of Bioscience and Bioengineering,Universiti Teknologi Malaysia,81310 Skudai, Johor, Malaysia T. Hidayat Department of Biology Education,Faculty of Mathematic and Natural Science,University of Education (UPI) Bandung,Jalan Dr. Setiabudhi No. 229,Bandung 40154, Indonesia R. A. KristantiDepartment of Research, Interdisciplinary Graduate Schoolof Medicine and Engineering, University of Yamanashi,4-3-11, Takeda,Kofu, Yamanashi 400-8511, Japan  solutions for its colored effluents. Effluents from textileindustries are a complex mixture of many pollutingsubstances such as heavy metals, organochlorine-based pesticides, pigments and dyes. The wastewater contain-ing dyes are highly colored which can cause water  pollution (Zouari-Mechichi et al. 2006). Based on thechemical structure of the chromophoric group, dyes areclassified as azo, triphenylmethane, anthraquinone,heterocyclic, and polymeric dyes, among which theversatile azo and triphenylmethane dyes account for most textile dyestuffs produced. Because these dyes aremutagenic, carcinogenic, and also cannot be completelyremoved by conventional wastewater treatment sys-tems, before disposal and discharge of dye-containingeffluents, they need to be treated to reduce their levels of toxicity, which will minimize their pollutionimpact. White-rot basidiomycetes are well known for their natural ability to decompose lignin, a highlycomplex non-phenolic polymer, which also givesthem the potential capacity to degrade a wide varietyof complex organopollutants. This degradative abilityhas opened up new prospects for the development of  biotechnological processes aimed at the degradationof complex polymers such as xenobiotics for effluent decolorization and biobleaching of the lignin in kraft  pulp (Lopez et al. 2002). One promising strategy is the use of microbesincluding white-rot fungal and bacterial strains that  possess the ability to decolorize synthetic dyes(Ferreira et al. 2000). Microbial decolorization and degradation are an environmentally friendly and cost-competitive alternative to physico-chemical decom- position processes for the treatment of industrialeffluents (Verma and Madamwar  2003). There is a  considerable number of recent reports on decoloriza-tion and degradation of individual synthetic dyes bywhite-rot fungi (Asgher et al. 2006; Hamedaani et al.2007; Tavaker et al. 2006). The biodegradation ability of the white-rot fungi isassumed to be associated with the production of lignolytic enzymes such as lignin peroxidase andlaccase (Couto and Herrera  2006; Ghodake et al.2008). Laccase (EC 1.10.3.2, benzenediol/oxygenoxidoreductase) is an multicopper oxidase enzymesecreted by the most of the lignin degrading white-rot  basidiomycetes, and it has been reported as anessential enzyme for lignin degradation in fungiwithout peroxidases (Kahraman and Gurdal 2002). It  catalyzes the oxidation of a broad range of organicand inorganic substrates, including diphenols, poly- phenols, diamines, aromatic amines, and ascorbate via a one-electron transfer mechanism but its substraterange has been extended to non-phenolic compoundsin the presence of low molecular mass compoundsacting as mediators (Eggert et al. 1998; Thurston1994). Most of the studies have been carried out withlaccases from eukaryotes, principally with enzymessecreted by basidiomycetes being their distribution in prokaryotes more recently reported (Claus 2003). Previously, a white-rot fungus F022 was success-fully isolated from a tropical rain forest. The fungusshowed ligninolytic activity, and the potential todegrade synthetic dyes. In this study, therefore weinvestigated the ability of strain F022 to perform biological decolorization of azo, triphenylmethane,anthraquinone. Moreover, because there is no detailed published report on the effect of the time-point of dyeaddition on decolorization by fungi, the present studywas undertaken to explore the effect of dye addition.We also investigated the importance of laccases of thestrain F022 for dye decolorization including optimumcondition of pH, temperature and laccase activity for decolorization. 2 Materials and Methods 2.1 Dye and Chemicals2,2-Azino-di-(3-ethylbenzthiazoline sulfonic acid)(ABTS) was purchased from Tokyo Chemical Indus-try (Tokyo, Japan). Azo dyes (Reactive Black 5[RB5]), triphenylmethane dyes (Brilliant Green) aswell as anthraquinonic dye (Remazol Brilliant Blue R [RBBR]) were of analytical grade. The structure of alldyes is shown in Table 1. All other chemicals were purchased from Wako Pure Chemical Industry(Osaka, Japan) at the highest purity available.2.2 Microorganism and Culture MaintenanceWhite-rot F02 was maintained on malt extracts agar (ME) containing (in g/l): glucose (20), malt extracts(20), polypeptone (2), and agar (20). The growthmedium for production of laccase and decolorizationof dyes was prepared in ME with glucose as a carbonsource. An inoculum of white-rot F022 for liquidculture was prepared as follows: three agar plugs Water Air Soil Pollut   (5 mm in diameter) punched from the periphery of a 7-day agar plate were cultivated in a 250-ml flask containing 100 ml culture solution, and the flask wasincubated for 7  –  8 days at 25°C in a shaking incubator (120 rpm).2.3 Morphologic and Molecular CharacterizationMicro- and macroscopic observations were performedon Petri dishes with PDA and malt extract agar (MEA), respectively. The optimum temperature and pH of isolates were also determined for both theisolates. Molecular identification of F022 was con-ducted on the basis of variation of 18S rRNA gene.Extracted DNA genome was amplified using univer-sal primers, NS1 and NS8. Component polymerasechain reaction (PCR) included buffer PCR, MgCl 2 , primers, enzyme  Taq  polymerase, dNTPs Mix, andDNA template. PCR was performed using thefollowing procedure: 1 cycle at 94°C for 3 min;25 cycles at 94°C for 30 s, 50°C for 30 s, and 72°Cfor 2 min; and ended with 1 cycle at 72°C for 10 min. Amplification products were cloned into pGEM-T Easy (Promega) before sending them to1st BASE Laboratory Sdn. Bhd. Malaysia for sequencing. The resulting DNA sequence was readand edited by BIOEDIT. The sequence was further compared with other 18S rRNA gene sequencesobtained from the NCBI GenBank database. Phylo-genetic analysis was conducted based upon neighbor  joining method.2.4 Dry Mycelium Weight of Strain F022Dry mycelium weight (DMW) of the fungal mass wasobtained by removing the culture solution, which isdone by filtering the contents of each flask through pre-weighed Whatman no. 1 filter paper. The paper holding the filtered fungal mass then was left to dry toa constant weight at 100°C. DMW was expressed interms of g/l biomass.2.5 Production, Preparation and Analysis of LaccaseThe time course of laccase production by F022 wasstudied in 100 ml nutrient broth at 30°C at staticcondition. Laccase activity (as discussed in thefollowing section) was measured in a crude cellextract of F022 cells which were grown at different time intervals. For higher laccase production, 10%inoculum of F022 was inoculated in 3 l nutrient medium and incubated at 30°C for 12 h. Cells were Table 1  Structures and maximum visible wavelengths of dyes Dye max  (nm) Structure Reactive Black 5 (Azo) Remazol Brilliant Blue R (Anthraquionone)Brilliant Green (Triphenylmethane) 598 495 625 Water Air Soil Pollut   collected by centrifugation at 8,000×  g   for 15 min andsuspended (150 g/l) in a 50 mM sodium phosphate buffer (pH 7.0) containing 5 g/l lysozyme. Cells werefurther incubated at 37°C for 45 min in a water bathand then disrupted by sonication. This cell-freeextract was solubilized in cholic acid on a magneticstirrer at 4°C for 30 min. The cell lysate obtained wascentrifuged twice at 15,000×  g   at 4°C for 30 min, andthe clear supernatant is used immediately or storedat   − 20°C until its use to purify laccase.The supernatant containing laccase activity 0.04 Uwas heated at 60°C for 10 min and centrifuged at 8,000×  g   for 20 min. The clear supernatant obtainedafter centrifugation was loaded onto a DEAE cellulosefast flow column (15×120 mm). The column waswashed with the same buffer by twice the columnvolume and the enzyme was eluted with a linear gradient of 0  –  1.0 M NaCl. Fractions containing laccaseactivity were pooled and dialyzed against a 1-mMsodium phosphate buffer (pH 6.0). The dialyzedsample was concentrated (1  –  2 ml) by ultrafiltrationand loaded on a Bio-gel column (10×500 mm)equilibrated with a 50 mM sodium phosphate buffer (pH 6.0). Protein elution was carried with the same buffer at 6 ml/h flow rate. Fractions containing laccaseactivity were pooled and stored at   − 20°C until use.Laccase activity was determined at 30°C bymeasuring the increase of optical density at 420 nmin a reaction mixture of 2 ml containing 0.66 mMABTS in a 0.1 M acetate buffer (pH 4.9) and 100  μ  lenzyme (Hatvani and Mecs 2001). One unit of  enzyme activity was defined as a change in absor- bance U mg protein − 1 min − 1 . The protein concentra-tion of each fraction was monitored by absorbance at 280 nm or Lowry methods with bovine serumalbumin as a standard (Lowry et al. 1951). 2.6 Decolorization by the Fungus F022The decolorization of azo, triphenylmethane andanthraquinonic dyes was also evaluated using MEliquid medium. The amounts of Reactive Black 5,Brilliant Green and RBBR in the culture were 100  –  500 mg/l, respectively. Dyes were added either initially or after 5 days of cultivation. At regular intervals, samples (1 ml) were withdrawn from theflasks and centrifuged at 8,000×  g  . The supernatantswere analyzed by measuring the decrease in absor- bance at the absorbance maxima ( λ max ) of eachdye using a UV  –  visible spectrophotometer. Dyeremoval was determined according to the followingformulation:Decolorization % ð Þ¼  1   C C  0    100where C 0  is the absorbance of the dye beforedecolorization and  C   is the absorption of the dye after decolorization at each sampling time (Sayan 2006).Cultures containing only fungi but no dye were used ascontrol groups.2.7 Treatment of Dyes by Purified LaccaseDecolorization of all dyes by purified laccase was performed using 3-ml disposable cuvettes with 2 mlfinal reaction volume. The reaction mixture wascomposedof100mMacetatebufferpH4,200mg/ldyesand 0.5 U/ml laccase. Decolorization was monitoredin 24-h intervals by scanning the spectrum between400 and 800 nm using a UV  –  Vis spectrophotometer.All experiments were performed in triplicate, andincubated with shaking (120 rpm) for an appropriatetime. Controls were performed using heat inactivatedenzymes after incubation at 100°C for 10 min.Calculation of decolorization percentage was done asdescribed as above. 3 Results and Discussion 3.1 Isolation and Identification of FungiAwhite-rot fungus, isolated from the decayed wood ina tropical rain forest, was designated F022. This strainF022, when grown on ME agar, had a white spore (7  –  9×6  –  7  μ  m), smooth, more or less elliptical, inamy-loid with a prominent apiculus, and has a clampconnection between hyphae. F022 has a sturdy,fibrous stem, with a pendent, thin, white to pale greyring and convex to umbonate, tawny to ochre capwith sparse and fibrous scale. The gills are attached or  beginning to run down the stem; nearly distant;whitish, sometimes bruising or discoloring darker (Laessoe 2002; Pace 1998). Based on these macro- scopic morphological characteristics and the phyloge-netic position of the sample, F022 is classified as belonging to the genus  Armillaria  (Fig. 1). Water Air Soil Pollut   3.2 Fungal Growth in Liquid Media Containing DyeDye toxicity for   Armillaria  sp. F022 growth wascalculated by growing fungi in liquid media contain-ing different dyes, and then we compared the DMWto a control group growing in liquid media without dyes. As shown in Fig. 2, dyes were added at different times during culture in order to evaluate dye effectson fungal growth. When dyes were added at 0 h of culture, the maximum DMW values in the mediumcontaining Brilliant Green were 1/6 of that achieved by the control group. For other dyes, the DMW wassimilar with control. When dyes were added after 24 hof cultivation, similar DMW values were obtained inexperimental and control media of   Armillaria  sp.F022. These results indicated that Brilliant Green hastoxic effects on  Armillaria  sp. F022.To determine the maximum dye concentration toler-ated by the cell in a culture, experiments with different initialdyeconcentrations,rangingfrom100to500mg/l,were performed. Even with the dye concentration ashigh as 500 mg/l, more than 60% of color removal wasobtained at 96 h incubation time except BG. A typicalMonod-type profile was observed as the specificdecolorization rate ( r  dye ) initially increased with theconcentration of RB5, RBBR and Brilliant Green(Fig. 3). Figure 3 also indicates that the toxic tolerance of dye for the cell beads was excellent at least up to a concentration of 500 mg/l. These results suggest that cell in this work was highly promising for practical Fig. 1  Phylogenetic tree of   Armillaria  species 0123 Black 5Control Reactive RBBR Brilliant Green Dyes    D   M   W    (  g   /   L   ) 0h 48h Fig. 2  Dry myceliumweights (DMW) of   Armillaria  sp. F022 in MEliquid medium containingthree synthetic dyes(incubation time: 96 h).The dyes were added intothe culture at 0 and 48 hof cultivationWater Air Soil Pollut 

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