Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp. SUK1

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  • Journal of Hazardous Materials 163 (2009) 735742

    Contents lists available at ScienceDirect

    Journal of Hazardous Materials

    journa l homepage: www.e lsev ier .com

    Ecofrie ofnewly

    D.C. KalyDepartment of

    a r t i c l

    Article history:Received 1 FebReceived in reAccepted 4 JulAvailable onlin

    Keywords:Pseudomonas sTextile dyesBiodegradatioLignin peroxidDetoxication

    xtiledustrs straa walsoe atInducctivemoniss spect by

    1. Introduction

    The numbeen steadione of the gthe high qumated thatindustrial emake up abthemthe larsyntheticdythese indusother produhumectantsrivers and ltion, thus corganisms.have toxic eplant specietion such aserosion, andto soil fertients contai

    CorresponE-mail add

    their nal discharge to the environment. Existingphysical/chemicaltechnologies for color removal are very expensive and commer-

    0304-3894/$ doi:10.1016/j.jber of studies on the biodecoloration of dyestuffs hasly increasing in recent years [1]. The textile industry isreatest generators of liquid efuent pollutants, due toantities of water used in the dyeing processes. It is esti-280,000 tonnes of textile dyes are discharged in suchfuent every year worldwide [2], out of that azo dyesout a half of all known dyestuffs in the world, makinggest groupof synthetic colorants and themost commones released into theenvironment [3]. Theefuents fromtries are complex, containing awide variety of dyes andcts, such as dispersants, acids, bases, salts, detergents,, oxidants, etc. Discharge of these colored efuents intoakes results into reduced dissolved oxygen concentra-reating anoxic conditions that are lethal to residentMany reports indicate that textile dyes and efuentsffect on the germination rates and biomass of severals, where as plant playmany important ecological func-providing the habitat for awildlife, protecting soil fromproviding bulk of organic matter that is so signicant

    lity [4,5]. Therefore, the treatment of industrial efu-ning aromatic compounds becomes necessary prior to

    ding author. Tel.: +91 231 2609365; fax: +91 231 2691533.ress: jpj (J.P. Jadhav).

    cially unattractive [6]. Biological processes provide an alternativeto existing technologies because they aremore cost-effective, envi-ronmentally friendly, and do not produce large quantities of sludge.Manymicroorganisms belonging to the different taxonomic groupsof bacteria, fungi, actinomycetes and algae have been reported fortheir ability to decolorize azo dyes [7]. Pure fungal cultures havebeen used to develop bioprocesses for the mineralization of azodyes, but the long growth cycle and moderate decolorization ratelimit the performance of fungal decolorization system [8]. In con-trast, bacterial decolorization is normally faster. Previous studiesindicated that, bacterial strains like P. mirabilis, P. luteola, Pseu-domonas sp. and K. rosea have shown very promising results fordye degradation under anoxic conditions [912]. The decoloriza-tion of sulfonated Reactive Red 2 dye were studied by number ofinvestigator, butmost of these studies have emphasized only decol-orization/degradation of dye,with no discussion of the degradationby product and their toxicity in the environment [1316]. Veryrecently fewof investigatorshave reporteddegradationand toxicityof products formed after decolorization at static (anoxic) condition[12,1719] with respect to other dyes.

    In order to develop a practical bioprocess for the treatmentof dye wastewater, it is necessary to isolate and investigate themicroorganisms capable of degrading azo dyes. In the presentinvestigation we have evaluated the decolorization, biodegrada-tion of Reactive Red 2 andmixture of various textile dyes which are

    see front matter 2008 Elsevier B.V. All rights reserved.hazmat.2008.07.020ndly biodegradation and detoxicationisolated Pseudomonas sp. SUK1

    ani, A.A. Telke, R.S. Dhanve, J.P. Jadhav

    Biochemistry, Shivaji University, Kolhapur 416004, India

    e i n f o

    ruary 2008vised form 24 April 2008y 2008e 12 July 2008

    p. SUK1


    a b s t r a c t

    The aim of this work is to evaluate tewaste disposal sites of local textile inPseudomonas species and designated asulfonated azo dye (Reactive Red 2) in6.27.5 in static condition. This isolatedyes. Measurements of COD were don52% reduction in the CODwithin 24h.observed during decolorization of Readegradation. The biodegradation wasnaphthol was characterized by GC-maof Reactive Red 2 into non-toxic produ/ locate / jhazmat

    Reactive Red 2 textile dye by

    dyes degradation by novel bacterial strain isolated from theies. Detailed taxonomic studies identied the organisms asin Pseudomonas sp. SUK1. The isolate was able to decolorizeide range (up to 5g l1), at temperature 30 C, and pH rangeshowed decolorization of the media containing a mixture ofregular intervals to have an idea of mineralization, showingtion in the activity of lignin peroxidase and azoreductase wasRed 2 in the batch culture, which represented their role in

    tored by UVvis, IR spectroscopy, HPLC. The nal product, 2-ctroscopy. The phytotoxicity study revealed the degradationPseudomonas sp. SUK1.

    2008 Elsevier B.V. All rights reserved.

  • 736 D.C. Kalyani et al. / Journal of Hazardous Materials 163 (2009) 735742

    commonly used in textile industries of Solapur, India by novel iso-lated bacterium Pseudomonas sp. SUK1 in static condition. Enzymesinvolved in the degradation were assayed and metabolites formedafter degradthe dye oncompared b

    2. Experim

    2.1. Dyes an

    TartaricPropanol frindophenolries Pvt. LtdChemical C59, ReactiveReactive Go72, ReactiveGurudutta T

    2.2. Isolatiomicroorgani

    The isolaing microo[20]. The 16(http://wwrespondingconstructedpackage [22

    2.3. Acclim

    The accdomonas spearlier repoture into frand 300mgstatic condistudies.

    2.4. Culture

    The com10, NaCl 5, y

    2.5. Decolo

    A loopfutivated in sask contaicentration 3decolorizatconcentratiof the cultutrifuged at 7Decolorizattometer (Hdyes (ReactReactive OrReactive OrGreenHE4Bgrown cultutometrically

    oxygen demand (COD) was also studied in the same sample [24].The chloride interference was removed by using HgSO4; the mix-ture of diluted sample (before and after treatment) and K2Cr2O7


    . Theed foa:

    g l

    A ised fouivaecoldec




    al grntendyentenn prein.


    bactnsidells ((50msor)f 30 sd atof ce actls (2


    ivityd eausin

    e actay mmMe. Thby tr intelcula2.25


    decois (Hidegres of4 anded wsamation were analysed. Further, the toxicity potential oftwo common plant seed S. vulgare and P. mungo wasefore and after microbial degradation.


    d chemicals

    acid was obtained from BDH Chemicals, India. n-om SRL Chemicals, India. DCIP (2,6-dichlorophenolsodium salt) obtained from Sisco Research Laborato-. Mumbai, India and NADH were obtained from Sigmaompany (USA). The dyes Reactive Red 2, Reactive BlueRed BLI, Reactive Navy Blue HE2R, Reactive Orange 4,lden yellow HER, Reactive Red HE8B, Reactive OrangeRed Brown, Reactive GreenHE4BDwere obtained fromextile Industry, Solapur, India.

    n, screening and identication of dye degradingsm

    tion, screening and identication of novel dye degrad-rganism was done as earlier reported Kalyani et al.S rDNA sequence was initially analyzed at NCBI using BLAST (blastn) tool and cor-sequences were downloaded. Phylogenetic tree wasby Neighbour-Joining method [21], using the MEGA4].


    limatization was done by gradually exposing Pseu-. SUK1 to the increasing concentrations of dye as perrt Kalme et al. [23]. The successive transfers of cul-esh nutrient medium containing 100, 150, 200, 250l1 of the Reactive Red 2 dye was done at 30 C intion. This acclimatized microorganism was used for all


    position of decolorizing medium was (g l1): peptoneeast extract 2 and beef extract 1.

    rization experiments

    l of microbial culture (Pseudomonas sp. SUK1) was cul-tatic condition for 24h at 30 C in 250ml Erlenmeyerning 100mlmedia, after 24h the dyewas added at con-00mg l1.Where as, to examine the effect of initial dyeion in static condition, the dyewas addedwith differenton 1, 2, 3, 4, and 5g l1 after 24h growth. Aliquots (3ml)remediawaswithdrawnatdifferent time intervals, cen-000 rpm for 15min to separate the bacterial cell mass.ion of the dye was analyzed using UVvis spectropho-itachi U 2800, Tokyo, Japan) at 540nm. Mixture of 10ive Blue 59, Reactive Red BLI, Reactive Navy Blue HE2R,ange 4, Reactive Golden YellowHER, Reactive RedHE8B,ange 72, Reactive Red 2, Reactive Red Brown, ReactiveD), each at concentration 100mg l1 were added in 24hre medium, decolorization was measured spectropho-at visible wavelength 470nm. Reduction in chemical

    was reask esampletitrantwas usformul

    COD (m

    wherewas usmillieq

    All dand theage de


    = [(i

    Bacteritein coadheretein cobetweeof prot

    2.6. Pr

    The24h coThese cbufferproceseach otrifugesourceenzyminterva

    2.7. En

    Actreporteminedenzymthe ass7.4), 1enzymlowedin colowas caRed 2 (

    2.8. An

    Theanalysing biovolumNa2SOobtainand thed (acidic condition and Ag2SO4 as catalyst) in a reuxed with condenser on a hot plate for 3h. The reuxed

    s titrated against ferrous ammonium sulfate (FAS) asNB medium was used as blank and similar conditionr test. Finally COD was calculated by using following

    1) = (A B) normality 1000 8volume of sample (mL)

    the ml of FAS was used for blank, B is the ml of FASr test sample, N is the normality of FAS and 8 is the

    lent weight of oxygen.orization experimentswere performed in the three setsolorization activity is expressed in terms of the percent-ization as follows:

    ion activity (%)

    l absorbance) (observed absorbance)] 100initial absorbance

    owthwasmeasured by estimating the intracellular pro-t. Cell pellet was washed in distilled water to removeparticles and then boiled in 1M NaOH for 15min, pro-t was measured by Lowry method [25]. The relationotein concentration and OD620 was 1.0 OD620 =462mg

    tion of cell free extract

    erial cells were grown in the nutrient broth at 30 C forer being control, centrifuged at 7000 rpm for 20min.75mgml1)were suspended in a potassiumphosphateM, pH 7.4) and sonicated (Sonics-vibracell ultrasonic

    keeping sonier output at 50 amp and giving 7 strokes, with 3min interval at 4 C. The homogenate was cen-8000 rpm for 20min and supernatant was used as arude enzyme. Similar procedure was used to quantifyivities during the dye decolorization at different timeh, 4h and 6h).

    e assays

    of lignin peroxidase was assayed by using procedurerlier [20]. NADH-DCIP reductase activity was deter-g a procedure reported earlier [26]. Azoreductaseivity was assayed by modifying earlier method [27];ixture (2ml) contained 50mM phosphate buffer (pHNADH, 0.25M Reactive Red 2 and 0.2ml of crudee reaction mixture was pre-incubated for 4min fol-he addition of NADH and monitored for the decreasensity at 540nm at room temperature. Enzyme activityted by using molar absorption coefcient of Reactive0mM1 cm1) at 540nm.

    cal procedure

    lorizationwasmonitoredbyusingUVvis spectroscopytachi U 2800).Where as, themetabolites produced dur-adation of Reactive Red 2, were extracted with equalethyl acetate. The extract was dried over anhydrousevaporated todryness in rotary evaporator. The crystalsere dissolved in small volumes of HPLC grade methanole samplewasused for FTIR,HPLC andGCmass spectral

  • D.C. Kalyani et al. / Journal of Hazardous Materials 163 (2009) 735742 737

    analysis. HPLC analysis was carried out (Waters model no. 2690)on RP-C18 guard column. The mobile phase was methanol withow rate was 1mlmin1. FTIR analysis of biodegraded ReactiveRed 2 was carried out using Perkin Elmer 783 Spectrophotometerby compared with control dye. The FTIR analysis was done in themid IR region of 4004000 cm1 with 16 scan speed. The sampleswere mixed with spectroscopically pure KBr, pellets formed werexed in sample holder, and the analysis carried out. The identi-cation of metabolites formed after degradation was carried usinga QP2010 gas chromatography coupled with mass spectroscopy(Shimadzu). The ionization voltage was 70eV. Gas chromatogra-phy was conducted in the temperature programming mode with aRestek column (0.25mm, 60m; XTI-5). The initial column temper-ature was 80 C for 2min, then increased linearly at 10 Cmin1 to280 C, and held for 7min. The temperature of the injection portwas 280 C and the GC/MS interface was maintained at 290 C. Theheliumcarriergasowratewas1.0mlmin1.Degradationproductswere identied by comparison of retention time and fragmentationpattern, as well as with mass spectra in the NIST spectral librarystored in the computer software (version 1.10 beta, Shimadzu) ofthe GCMS.

    2.9. Statistical analysis

    Data were analyzed by one-way analysis of variance (ANOVA)with TukeyKramer multiple comparisons test.

    2.10. Phytotoxicity study

    Phytotoxicity of the Reactive Red 2 was performed in order toassess the toxicity of textile industry efuent. The obtained prod-uct was dis5000ppm.S. vulgare ansample of thucts (5000p

    the same time. Germination (%) and length of plumule and radiclewas recorded after 8 days.

    3. Results and discussion

    3.1. Phylogenetic position of isolates

    To analyze the phylogenetic position, the 16 S rDNA sequenceof the strain SUK1 (996bp, EF541140) was determined. Fig. 1showed the phylogenetic relationship between the strain SUK1 andother related microorganisms found in the GenBank database. Thehomology assay result indicated that the strain SUK1 was in thephylogenetic branch of the Pseudomonas. SUK1 exhibited a maxi-mum identities (97%) to P. pseudoalcaligensAB276372, P. mendocinastrain 147 AY870673. The earlier report shows other species of P.mendocina have role in biodegradation of dye [28], where as therewasno literatureavailableonP. pseudoalcaligens indyedegradation,but, this microorganism has highly developed for the degradationof other toxic compounds like nitrobenzene and cyanide [29,30].

    3.2. Effect of static and shaking conditions

    The acclimatized culture of the Pseudomonas sp. SUK1,under agitation conditions demonstrated a better growth(2962.4mg l1) than that under static conditions(2142.8mg l1). As for decolorization, agitated culture showedalmost no decolorization in 24h, while the static culture decol-orized more than 96% of the initial dye concentration (300mg l1)in 6h. To conform whether this decolorization was due to themicrobial action or change in pH, the change in pH was recorded,which was in the range of 6.27.5 at static condition. Conrms

    coloric cotheTheal de

    Fig. 1. The evo ave beships of Pseudo the lesubstitutions p umbesolved in the water to form a nal concentration ofThe study was carried out (at room temperature) usingdP.mungo (10 seeds of each) by adding separately 10mle Reactive Red 2 (5000ppm) and its degradation prod-pm) per day. Control set was carried out usingwater at

    the deor statthoughdition.bacteri

    lutionary history was inferred using the Neighbor-Joiningmethod; the sequences hmonas sp. SUK1 and other species of genus Pseudomonas. Numbers at nodes showser nucleotide position and numbers in parenthesis represent GenBank accession niza...


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