Decolorisation by Aeromonas hydrophila strain.

8/10/2019 Decolorisation by Aeromonas hydrophila strain.

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Appl Microbiol Biotechnol (2006) 72: 13161321DOI 10.1007/s00253-006-0418-2

EN V IRO N MEN TA L BIO TECH N O LO G Y

Suizhou Ren. Jun Guo. Guoqu Zeng. Guoping Sun

Decolorization of triphenylmethane, azo, and anthraquinone dyes

by a newly isolated Aeromonas hydrophilastrain

Received: 26 October 2005 / Revised: 7 March 2006 / Accepted: 9 March 2006 / Published online: 19 April 2006# Springer-Verlag 2006

Abstract A broad-spectrum dye-decolorizing bacterium,strain DN322, was isolated from activated sludge of atextile printing wastewater treatment plant. The strain was

characterized and identified as a member of Aeromonashydrophila based on Gram staining, morphology charac-ters, biochemical tests, and nearly complete sequenceanalysis of 16S rRNA gene and the gyrase subunit betagene (gyrB). Strain DN322 decolorized a variety ofsynthetic dyes, including triphenylmethane, azo, andanthraquinone dyes. For color removal, the most suitable

pH and temperature were pH 5.010.0 and 2537 C,respectively. Triphenylmethane dye, e.g., Crystal Violet,Basic Fuchsin, Brilliant Green, and Malachite Green(50 mg l1) were decolorized more than 90% within 10 hunder aerobic culture condition and Crystal Violet could beused as sole carbon source and energy source for cell

growth. The color removal of triphenylmethane dyes wasdue to a soluble cytosolic enzyme, and the enzyme was an

NADH/NADPH-dependent oxygenase; For azo and an-thraquinone dyes, e.g., Acid Amaranth, Great Red GR,Reactive Red KE-3B, and Reactive Brilliant Blue K-GR(50 mg l1) could be decolorized more than 85% within36 h under anoxic condition. This strain may be useful for

bioremediation applications.

Introduction

Synthetic dyes are extensively used in textile dyeing, paper

printing, color photography, pharmaceutical, food, cosmet-ic, and other industries (Rafii et al. 1990). Over 10,000different dyes with an annual production of over 7105

metric tones worldwide are commercially available(Padamavathy et al. 2003). Two percent of dyes that are

produced are discharged directly in aqueous effluent and10% are subsequently lost during the textile coloration

process (Pearce et al. 2003). Major classes of syntheticdyes include azo, triphenylmethane, and anthraquinonedyes, some of them are known to be very toxic andmutagenic to living organisms. With the increasing use of awide variety of dyes, pollution by dye-wastewater is

becoming increasingly alarming. Color removal, in

particular, has recently become a major scientific interest.Although several physico-chemical methods have beenused to eliminate the colored effluents in wastewater, thesemethods are rather costly and sometimes produce hazar-dous byproducts, and therefore other alternatives, such asmicrobial biodegradation, have attracted interest. Microbialdecolorization and degradation is an environmentallyfriendly and cost-competitive alternative to chemicaldecomposition processes (Verma and Madamwar 2003).To develop an efficient dye degradation biotechnology, thekey step is to obtain broad-spectrum and highly efficientdye-decolorizing bacteria. Although many dye-decoloriz-ing microorganisms have been reported (Banat et al. 1996;

Azmi et al. 1998; Xu et al. 2005), with exception of thedecolorization of dyes by Pseudomonas pseudomallei13NA and Citrobactersp. which decolorize both triphe-nylmethane and azo dyes by a single species of bacterium(Yatome et al.1981; AN et al.2002), there are no reports ondecolorization of triphenylmethane, azo, and anthraqui-none dyes by a single strain of bacterium, and no bacteriumthat has been reported was able to utilize Crystal Violet assole carbon source and energy source for growth up to now.

In the present study, we report the isolation andcharacterization of a highly efficient decolorizing bacteri-um, Aeromonas hydrophila strain DN322, which having

S. RenSouth China Botanical GardenChinese Academy of Sciences,Guangzhou 510650, China

S. Ren. J. Guo. G. Zeng. G. Sun (*)Guangdong Institute of Microbiology,Guangzhou 510070, Chinae-mail: [email protected].: +86-20-87684471Fax: +86-20-87684471

S. RenChina Graduate SchoolChinese Academy of Sciences,Beijing 100039, China


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decolorizing ability for triphenylmethane, azo, and anthra-quinone dyes.

Materials and methods

Dyes and chemicals

Crystal Violet was purchased from Sigma. Other triphe-nylmethane dyes (Basic Fuchsin, Brilliant Green, andMalachite Green), and six azo dyes (Acid Amaranth, GreatRed GR, Reactive Red KE-3B, Reactive Brilliant Red M-8B, Reactive Black KN-B, and Reactive Brilliant OrangeX-GN), as well as three anthraquinone dyes (ReactiveBrilliant Blue K-GR, Acid Blue 25, and Acid Blue 56)were of analytical grade.

Isolation and identification of dye decolorizingmicroorganism

The dye-decolorizing bacterial strain was isolated fromactivated-sludge of a textile-printing wastewater treatment

plant, Guangzhou, China. Screening of microorganisms fordye decolorization was carried out on agar plates with thefollowing medium: 200 ml of tap water, 0.2 g of yeastextract, 0.5 g of (NH4)2SO4, 2.66 g of KH2PO4, 4.32 g of

Na2HPO4, 4.0 g of agar, and 10 mg of dye. Afterpurification by successive single colony isolation onLuriaBertani (LB) plate, strain DN322 was identifiedfrom several aspects including morphology characters,

biochemical tests, and sequence analysis of nearlycomplete 16S rRNA gene and the gyrase subunit betagene (gyrB). Genomic DNA of this strain that served as

template was isolated using a method described previously(Zhou et al. 1996). For polymerase chain reaction (PCR)amplification, two universal primer sets, 27f (5-AGA GTTTGA TCC TGG CTC AG-3) and 1522r (5-AAG GAGGTG ATC CAG CCG CA-3) (Edwards et al. 1989) for16S rRNA gene, and primersgyrB3f (5-TCC GGC GGTCTG CAC GGC GT-3) andgyrB14r (5-TTG TCC GGGTTG TAC TCG TC-3) (Yez et al.2003) forgyrB genewere used. The sequences of the amplified 16S rRNA geneandgyrBgene were determined by TakaRa Biotechnology.Physiological characterization of strain DN322 was tested

by using the API 20NE System (BioMerieux French)according to the manufacturers instructions.

Decolorization of dyes by growing cells

Cells were cultured overnight in LB medium at 30 C withshaking at 150 rpm. When the absorbance at 600 nmreached 0.6 (about 108 cell ml1), precultured cells wereused to inoculate at 10% (v/v) into 2 ml M9 synthetic

medium (Sambrook et al. 1989) containing 0.1% (w/v)yeast extracts and each dye at different concentration. Forsole carbon and energy sources experiments, yeast extractsand glucose were omitted and the precultured cells wereinoculated at 2% (v/v) into 50 ml M9 synthetic mediumcontaining 0.12 mM Crystal Violet. The cultivation wascarried out on a shaker at 150 rpm at 30 C or maintained at30 C under microaerophilic condition. Under microaero-

philic conditions, oxygen concentrations vary between0.01 and 0.5 mg l1 without aeration. After incubation atshaking or microaerophilic condition to an appropriatesampling time, the cells were separated by centrifugation at10,000g for 10 min. After being washed twice with50 mM NaH2PO4buffer (pH 7.5), and the precipitated cellswere dried at 105 C for 4 h to measure dry weight of thecells. The supernatants were used as samples fordecolorization assay. Two types of control were used:uninoculated sterile control and heat-killed cells control.The former, containing only medium components, indi-cated the effect of medium components on decolorization,and the latter showed adsorption to cells. Dye removal by

these controls was supposed to be due to adsorption alone.

Crude extract preparation

Two hundred milliliter precultured cells were centrifugedand washed as described above, and then suspended in30 ml of 50 mM NaH2PO4buffer (pH 7.5). Cell disruptionwas carried out in ice bath by sonication (3 s, 40% output,80) using a sonification unit (SONICS VC-505, Amer-ica), with monitoring of the cells breaking progress underthe microscope. The cell debris and unbroken cells wereremoved by centrifugation for 30 min at 18,000g, and the

remaining cell extracts were stored at 4 C.

Decolorization assay

Decolorization activity was expressed in terms of thepercentage decolorization by the modified method de-scribed previously (Yatome et al.1993). Decolorization ofdyes was determined by monitoring the decrease inabsorbance at the maximum wavelength of each dye. Themaximum wavelength of each dye was shown in Table 1.An UVVisible scanning spectrophotometer (BECKMANDV640) was used for absorbance measurement and

recording of UV

Visible absorption spectra. Decoloriza-tion activity was calculated as the following, and all assayswere done in triplicate.

Decolorization rate % AB A

100A initial absorbanceB observed absorbance

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Results

Isolation and identification of dye-decolorizingbacteria

A broad-spectrum dye-degrading bacterial strain, namedstrain DN322, was isolated after screening and singlecolony purification. The strain is Gram-negative, rod-

shaped, motile with a single polar flagellum, and faculta-tive anaerobic, oxidase and catalase positive, and VogesProskauer positive. The strain produces gas from glucoseand produces H2S from cysteine. Colonies grown on LB

plate are light yellow to pink, smooth on the surface, andtrim on the edge. The API 20NE system identification wasto the species level, and the result of identification wascompared with that of type species A. hydrophilaATCC7966T. Biochemical and physiological profiles ofstrain DN322 is consistent with those of A. hydrophilaATCC7966T.

To further identify this strain, the nucleotide sequencesof 16S rRNA gene and gyrB gene were analyzed and

compared with those of related strains in GenBank. 16SrRNA gene and gyrB gene strain DN322 shared 99 and97% similarity with A. hydrophila ATCC7966T. Theseresults agree with that of API 20NE system identification,showing that this new isolate is a member ofA. hydrophila.The sequences of the 16S rRNA gene and the gyrB genehad been submitted to GenBank under accession numberAY686711 and AY968042, respectively. The strain has

been deposited in China Center for Type Culture Collection(CCTCC AB205001T) and the National Collection of TypeCultures (NCTC13358, London).

Decolorization of triphenylmethane dyesby strain DN322

Decolorization of various triphenylmethane, azo, andanthraquinone dyes by the growing cells of strain DN322were shown in Table 1. The optimum decolorizationactivity was observed at pH 5.010.0 and 2537 C in M9decolorization medium, which was also the optimum

condition for growth of this strain (Figs.1 and2).Compared with azo and anthraquinone dyes, this strain

had a higher decolorization rate for triphenylmethane dyes.In the case of triphenylmethane dyes tested, strain DN322showed the highest decolorization rate against CrystalViolet (Figs.3and4). Crystal Violet was nearly completelydecolorized for initial dye concentration less than 50 mgl1

after 6 h cultivation. With the dye concentration increased,the decolorization activity was inhibited obviously. Othertriphenylmethane dyes, e.g., Malachite Green, BasicFuchsin, and Brilliant Green, were also decolorizedeffectively by this strain (Table 1). The four triphenyl-methane dyes tested have similar decolorization rate, which

could be due to the similarity in structure of these dyes.Furthermore, strain DN322 could decolorize four tri-

phenylmethane dyes tested under both shaking and micro-aerophilic conditions, and the decolorizing speed is a littlefaster under shaking condition. However, when thedecolorization mixture was flashed with N2 for 10 min,no decolorization was observed, indicating that thedecolorization reaction was depended upon the presenceof molecular oxygen. Decolorization of the four triphenyl-methane dyes was also observed with cell extracts. Whenthe cell extracts treated with 1.0 mg proteinase K ml1 for30 min, more than 85% of decolorization activity was lost.After heated at 100 C for 10 min, the cell extracts lost all

of its decolorization activity. When 4 l of NADH orNADPH (25 mM) was added to the cell extracts (50 l)containing 50 mg l1 dyes, complete decolorization wasobserved within 5 min. Compared with the control inwhich neither NADH nor NADPH was added, thedecolorization rate was increased over 50 times. When

Absorbance(600nm)

pH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

3 4 5 6 7 8 9 10 11 12

Fig. 1 Effect of initial pH on the growth of strain DN322. Thestrain was cultured in M9 synthetic medium containing 0.1% (w/v)yeast extracts at various pHs at 30 C

Table 1 Decolorization (D) of triphenylmethane, azo, and anthra-quinone dyes by the growing cells of strain DN322 undermicroaerophilic condition

Dye max Conc.

(mg/l)

D (%) Time (h)

Crystal Violet 590 50 962 6



Malachite Green 617 50 945 10

Brilliant Green 545 50 933 6

Basic Fuchsin 630 50 956 8Acid Amaranth 477 50 963 6

Great Red GR 503 50 916 24

Reactive Red KE-3B 511 50 938 24

Reactive Brilliant Red M-8B 540 50 875 24

Reactive Black KN-B 595 50 825 24

Reactive Brilliant Orange X-GN 477 50 754 36

Reactive Brilliant Blue K-GR 603 50 857 36

Acid Blue 25 562 50 605 52

Acid Blue 56 637 50 216 52

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the cell extracts were flashed with N2 for 10 min, nodecolorization was observed in this phenomenon similar to

that of intact cells. All these results indicated that the colorremoval of triphenylmethane dyes was due to a solublecytosolic enzyme, and the enzyme was an NADH/

NADPH-dependent oxygenase.Strain DN322 was capable of utilizing Crystal Violet as

sole carbon and energy sources for the cell growth, asdepicted in Fig. 5. During DN322 growth on 0.12 mMCrystal Violet as a sole source of carbon and energy, cellmass increased approximately five times after 72 h culti-vation. No significant growth occurred in cultures lackingCrystal Violet, and controls lacking cells did not show anydecolorization of Crystal Violet.

Decolorization of azo and anthraquinone dyesby strain DN322

For azo dyes, strain DN322 decolorized 95% of AcidAmaranth (mono azo group) in 6 h at the dye concentration

of 50 mg l1 (Fig.6), whereas it would take 24 h for theGreat Red GR and Reactive Red KE-3B (diazo group) atthe same concentration. Other azo dyes, e.g., ReactiveBrilliant Red M-8B, Reactive Black KN-B, and ReactiveBrilliant Orange X-GN could be decolorized over 70%within 36 h at the same concentration. Unlike triphenyl-methane dyes, all these azo dyes were decolorized morerapidly under microaerophilic condition than that underaerobic condition. Shaking culture suppressed the decolor-ization of azo dyes, indicating that the decolorizationmechanism of azo dyes by strain DN322 is due to azo

Absorbance(600nm)

Temperature (o

C)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

4 10 20 25 30 35 40 45 50

Fig. 2 Effect of temperature on the growth of strain DN322. Thestrain was cultured in M9 synthetic medium containing 0.1% (w/v)yeast at various temperatures at pH 7.5

Fig. 3 UVVisible absorbance spectra of decolorization for CrystalViolet by strain DN322. Strain was cultured in M9 syntheticmedium containing 0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at30 C under aerobic condition

Fig. 4 Time course of the decolorization of Crystal Violet by strainDN322. Strain was cultured in M9 synthetic medium containing0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at 30 C under aerobiccondition

Fig. 5 Decolorization of Crystal Violet by strain DN322 withcompanying data for cell growth in M9 medium. The absorbance(abs) of Crystal violet at the maximum wavelength (590 nm) isshown for tubes containing a cell inoculum () and control tubeslacking cells (). Cell growth is illustrated for tubes containingCrystal violet () and for controls () containing a cell inoculumbut lacking Crystal violet. All data represent the mean of threetriplicate tubes, and error bars represent standard deviation.Lack of error bars for analytical data indicates that the error barswere smaller than the symbol

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bound reduction under anaerobic condition, which isdifferent from that of triphenylmethane dyes.

In the decolorization of anthraquinone dyes ReactiveBrilliant Blue K-GR, a different phenomenon of decolor-ization was observed. Firstly, a blue flocculate was formedand precipitated, and the cells of strain DN322 were stained

blue, indicating that Reactive Brilliant Blue K-GR wasadsorbed on the cell surface. Then the dye was decolorizedas the precipitated flocculate turned to white and more than80% of color was removed in 36 h at 50 mg l1 (Fig.7).Other anthraquinone dye, e.g., Acid Blue 25 and Acid Blue56 could also be decolorized as Reactive Brilliant Blue K-GR, but need a little longer time.

Discussion

Strain DN322 was identified as a member ofA. hydrophilaby biochemical analysis and nearly complete sequenceanalysis of 16S rRNA gene and gyrB gene. Strain DN322shared 99% similarity with A. hydrophila subsp. hydro-

phila type strain ATCC7966T in the 16S rRNA genesequence. The gyrB gene, a gene that encodes the -subunit of DNA gyrase, was proven to be an excellentmolecular chronometer for phylogenetic studies of thegenus Aeromonas (Yez et al. 2003). In our study, the

gyrBgene sequence similarity between strain DN322 andtype strain ATCC7966T was 97%. The gyrB gene resultagreed with 16S rRNA gene analysis, and the former

showed a higher capacity to differentiate between species.Rajesh and Uttam 1999 also found that decolorization

activity of Crystal Violet byKurthiasp. was inhibited whenthe dye concentration was increased to 20.4 mg l1, andMalachite Green was more easy decolorized than CrystalViolet. In our study, the decolorization activity of A.hydrophila DN322 was inhibited only when the dyesconcentration reached 50 mg l1, opposite to Kurthia sp.,Crystal Violet was easier to be decolorized than MalachiteGreen by strain DN322. Yatome et al. 1993reported thatthe growth of Nocardia corallina cells was completelyinhibited at concentration 2.9 mg l1 of Crystal Violet.However, strain DN322 was capable of utilizing 50 mg l1

Crystal Violet as sole carbon and energy sources for the cellgrowth. These results indicate that there are differentmechanisms of decolorization of triphenylmethane dyes indifferent bacteria.

Decolorization of dyes by bacteria could be due toadsorption to microbial cells or to biodegradation (Knappand Newby1995; Sani and Banerjee1999). In adsorption,cell mats become deeply colored because of adsorbingdyes, whereas those retaining their original colors areaccompanied by the occurrence of biodegradation. In ourresearch, the cells ofA. hydrophilastrain DN322 remainedcolorless during the process of decolorization. Similarresults were also observed in other triphenylmethane and

azo dyes. Consequently, according to the above results, thecolor removal by strain DN322 should be largely attributedto biodegradation. However, in the decolorization ofCrystal Violet byAeromonassp. B5, the cells were stainedviolet, indicating that Crystal Violet was adsorbed on thecell surface, and the decolorization of Crystal Violet wasdue to the sorption of dye on the cells (Nobuki et al.2000).

Although a lot of triphenylmethane dye-decolorizingmicroorganisms have been reported, the enzyme which isresponsible for the decolorization and degradation of thesetriphenylmethane dyes has never been purified from

bacteria, and its characterization has never been described(Azmi et al.1998). Therefore, it would be a meaningful and

helpful work to purify the decolorization enzyme andelucidate the decolorization mechanism of the triphenyl-methane dyes. Strain DN322 is the firstAeromonassp. thatis capable of decolorizing several triphenylmethane dyesand utilizing Crystal Violet as sole carbon source forgrowth. The color removal of triphenylmethane dyes instrain DN322 was due to a soluble cytosolic enzyme.Recently, the decolorization enzyme was purified tohomogeneity and identified as an NADH/NADPH-depen-dent, heme-containing oxygenase with a molecular of87 kDa in our lab. The decolorization enzyme would be agood material for further research of the enzymological

Fig. 6 UVVisible absorbance spectra of decolorization for AcidAmaranth by strain DN322. Strain was cultured in M9 syntheticmedium containing 0.1% (w/v) yeast and 0.12 mM dye at pH 7.5 at30 C under microaerophilic condition

Fig. 7 UVVisible absorbance spectra of decolorization forReactive Brilliant Blue K-GR by strain DN322. Strain was culturedin M9 synthetic medium containing 0.1% (w/v) yeast and 0.12 mMdye at pH 7.5 at 30 C under microaerophilic condition

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mechanism of triphenylmethane dyes decolorization bybacteria.

Based on these results, A. hydrophila strain DN322exhibited to be a highly promising microorganism both forthe application in the treatment of dyeing and printingindustrial wastewater and bioremediation to removerecalcitrant triphenylmethane, azo, and anthraquinone dye

pollutants from contaminated environment.

Acknowledgements This work was supported by the fund of ChineseNational Programs for High Technology Research and Development(No. 2003AA214040), Guangdong Provincial Natural Science Fund(No.2004A30404002), Guangdong Provincial Natural ScienceFund (No. 200115017) and Guangdong Provincial Natural ScienceFund (No. 032319).

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