Biotreatment of simulated textile dye effluent containing malachite green by an

up-flow immobilized cell bioreactor

Deepak Kumar Sharma1, Harvinder Singh Saini1,*, Manjinder Singh1, Swapandeep Singh Chimni2 and BhupinderSingh Chadha11Department of Microbiology, Guru Nanak Dev University, Amritsar 143005, India2Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India*Author for correspondence: Tel.: þ91-183-2258802, ext. 3318, Fax þ91-183-2258820, E-mail: sainihs@yahoo.com

Received 6 August 2003; accepted 6 December 2003

Keywords: Biodegradation, bioreactor, dye-decolorization, malachite green and triphenylmethane dye

Summary

An up-flow immobilized cell bioreactor was developed using a microbial consortium, consisting of Bacillus sp.,Alcaligenes sp. and Aeromonas sp., immobilized on refractory brick pieces as immobilization support. malachitegreen, a model triphenylmethane dye was decolourized by more than 93% within 48 h (operating conditions: initialdye concentration 30 mg l)1; flow rate 6 ml h)1). The analytical studies based on TLC and 1H NMR showeddegradation of the aromatic rings of the malachite green into simpler metabolic intermediates.

Introduction

Wastewater originating from the textile-processingindustries (TPI) is a complex mixture of potentiallypolluting substances consisting of textile dyes, heavymetals associated with dyes and the other auxiliariesused during dyeing process (Correia et al. 1994). Duringprocessing, up to 15% of the total textile dye remainsunreacted and is lost in the effluents (O’Neill et al. 1999).Apart from the aesthetic deterioration and obscuring thepenetration of light into natural water bodies, some ofthe dyes, dye precursors and dye degradation productsare reported to be carcinogenic and mutagenic in nature(Michaels & Lewis 1985; Littlefield et al. 1985; Hender-son et al. 1997). Although different physico-chemicaltreatments viz., adsorption on activated charcoal, pre-cipitation by alum, ozonation, electrolysis, etc. are beingused for colour removal from wastewater, these pro-cesses have high operational costs and limited applica-bility (Slokar et al. 1998). Additionally, the dyes remainunaltered in most cases and the sludge so produced is ahazardous waste that requires special disposal tech-niques as per law. Thus, the focus has shifted to thedevelopment of environmentally friendly biologicaltreatment systems for such industrial effluents, so as toachieve complete mineralization of toxic compounds.Wastewater treatment systems based on biofilms ofmicroorganisms on support particles have been reportedto be successful due to their ability to work underdifferent dilution rates and to sustain shock loading(Oxspring et al. 1996; Feng et al. 1997).

The present study reports development of a bacterialconsortium by selective enrichment of microbial popu-lations, in the samples collected from waste disposalsites of TPI, using triphenylmethane (TPM) group dyescommonly used in TPI located around city of Amritsar,Punjab (India). The efficiency of the consortium wasevaluated, in an up-flow immobilized bioreactor, todecolourize and degrade malachite green, a model TPMgroup dye.

Materials and methods

Chemicals

Malachite green and the medium components werepurchased from Hi-Media, Mumbai (India). All otherchemicals used were of analytical grade.

Medium used

Mineral salts medium (MSM) of following composition(g l)1) was used for all the studies: Na2HPO4 (3.6),KH2PO4 (1.0), (NH4)2SO4 (1.0), MgSO4 (1.0), Fe(NH4)citrate (0.01), CaCl2 Æ 2H2O (0.10) and 10.0 ml of traceelement solution per litre. The trace element solution hasthe following composition (mg l)1) ZnSO4 Æ 7H2O(10.0), MnCl2 Æ 4H2O (3.0), CoCl2 Æ 6H2O (1.0), Ni-Cl2 Æ 6H2O (2.0), Na2MoO4 Æ 2H2O (3.0), H3BO3 (30.0),CuCl2 Æ 2H2O (1.0). The final pH of the medium wasadjusted to 7.0. The stock solutions of glucose (50% w/v)

World Journal of Microbiology & Biotechnology 20: 431–434, 2004. 431� 2004 Kluwer Academic Publishers. Printed in the Netherlands.

and yeast extract (25% w/v) were sterilized separatelyand were supplemented to the MSM to give a finalconcentration of 5.6 mM of glucose and 0.25% (w/v) ofyeast extract, respectively. The MSM was used in all thestudies unless stated otherwise.

Enrichment and isolation of dye-decolourizingmicroorganisms

The microbial populations present in soil and sludgesamples collected from the waste disposal sites of textile-dyeing industries were enriched on MSM. The sterilizedMSM containing 10 mg of Acid blue-15 (AB-15) (CI42645) dye/l was inoculated with soil/sludge samples(10% w/v) and incubated at 30 ± 1 �C at 100 re-v min)1. A 10% v/v sample from the flask was furthertransferred to fresh MSM containing 10 mg of dye/lafter every 15 days for a period of 6 months. Theplating of enriched populations was carried out onMSM-agar plates containing 10 mg of dye/l. Fivemorphologically distinct isolates showing clear zonesaround their colonies was selected and used for furtherstudies. The bacterial isolates were identified by Micro-bial Type Culture Collection and Gene Bank (MTCC),Institute of Microbial Technology, Chandigarh, India.The consortium constituted by mixing equal propor-tions of selected bacterial isolates was used as seedinoculum for the up-flow immobilized cell bioreactor.

Immobilization support

The pieces (7–10 mm) of refractory brick obtained froma local brick-kiln were used as immobilization supportbecause of their high porosity and inertness. The brickpieces were incubated overnight in 0.1 M HCl followedby thorough washing with tap water to remove salts/soildeposited in the cavities prior to use.

Designing of bioreactor

The bioreactor was built from a glass column (20-cmlength and 3.34-cm diameter) having a 9-cm bed ofrefractory brick pieces. The total volume of the reactorwas 148 ml with a void volume of 45 ml. To initiatebiofilm development, 500 ml of nutrient broth-growncell suspension of the consortium (initial cell concentra-tion was 6.3 · 108 c.f.u. ml)1) was fed to the bioreactorin a loop for 7 days. This was followed by a feed ofMSM supplemented with glucose (5.6 mM), yeastextract (0.25% w/v) and 10 mg of malachite greendye/l at for a period of 1 month to allow development ofbiofilm on support particles. The dye concentration wasgradually increased up to a final concentration of30 mg l)1 within a period of 1 month. The bioreactorwas fed at a flow rate of 6 ml h)1, in batch mode, from a1.5-l reservoir of MSM supplemented with 30 mg ofmalachite green dye/l. Another bioreactor was operatedunder the same conditions as abiotic control to checkthe abiotic loss of dye. The support pieces were removed

from the bioreactor after 3 months of operation andwere processed for scanning electron microscopy(SEM), as described previously by Deya et al. (1995).Scanning electron micrographs were taken using JSM-6100, JEOL (Tokyo, Japan) at Regional SophisticatedInstruments Centre (RSIC), Punjab University, Chandi-garh.

Analytical studies

Decolourization assayAliquots of cell free supernatants (5 ml) of output of thebioreactor collected after every 24 h, during the oper-ation of bioreactor, were scanned in the range of 200–800 nm (UV–VIS spectrophotometer, Shimadzu, Ja-pan). The decolourizing activity expressed in terms ofpercentage decolourization was determined by monitor-ing the decrease in absorbance at 618 nm (kmax of thedye) using MSM as reference. Decolourization activity(%) was calculated according to the formula:

Decolourization activityð%Þ¼ ½ðinitial absorbanceÞ � ðobserved absorbanceÞ�� 100=initial absorbance

Thin layer chromatography (TLC)The feed of the bioreactor containing 30 mg malachitegreen dye/l was extracted with n-butanol. The cell freesupernatant of the bioreactor output collected every24 h was extracted first with ethyl acetate to extract thebiotransformed products/intermediates and subse-quently with n-butanol to extract the residual dye. Thedeveloping solvent system used was ethyl acetate:hexane(1:9, v/v). The chromatograms were observed under u.v.light (254 nm) and after exposure to iodine vapours inan iodine chamber.

Nuclear magnetic resonance spectroscopy (NMR)The 1H NMR spectra of the n-butanol-extracted inputfeed of the bioreactor containing malachite green(30 mg l)1), ethyl acetate-extracted output and n-buta-nol-extracted output of the bioreactor were determinedusing a 200 MHz, Brucker AMX 300 NMR spectro-meter.

Results and discussion

Development of immobilized cell bioreactor

The selective enrichment of soil and sludge samples ledto the isolation of five bacterial isolates, which wereidentified by the Microbial Type Culture Collection andGene Bank (MTCC), Institute of Microbial Technology,Chandigarh, India as belonging to Bacillus sp., Alcalig-enes sp. and Aeromonas sp. The bacterial consortium,having equal proportion of the activated cell of fiveisolates, was used as seed inoculum for the development

432 D.K. Sharma et al.

of biofilm on refractory brick pieces. The bacterialconsortium in the bioreactor was initially fed with10 mg of malachite green dye/l, to allow the develop-ment and acclimatization of microbial populations inthe biofilm and to avoid initial toxic shock of the dye tothe microbial population in the biofilm. The scanningelectron micrograph (SEM) of uninoculated supportmaterial showed the highly porous surface of thesupport material that provided a large surface area forbacterial growth and attachment (Figure 1A). The SEMof the support material removed from the bioreactorafter 3 months of operation showed formation ofbiofilm by bacterial cells (Figure 1B). After the forma-tion of a stable biofilm the bioreactor was fed with feedcontaining 30 mg malachite green dye/l as the rate ofdecolourization decreased with further increase in dyeconcentration (data not shown) in the feed.

Decolorization of malachite green

The activity of the microbial populations in the biore-actor led to 97% decolourization of dye (30 mg l)1) upto day 4, which latter stabilized at 80% after day 7(Figure 2). The comparison of u.v./vis. spectra of the

input and output of the reactor indicates to decrease incolour intensity together with a spectral shift towardsthe u.v. region (Figure 3). The peaks appearing at 618and 248 nm in the input of the bioreactor disappeared inthe output of the bioreactor, suggesting that malachitegreen may have been reduced to its leuco-form. Aspectral shift in the peaks at 425 and 317 nm withsubsequent appearance of two new peaks at 357 and309 nm suggested that malachite green might be under-going N-demethylation, since the N-demethylated prod-ucts have absorption maxima at wavelengths lower thanthat of malachite green (Cha et al. 2001). Additionally,no dye was found adsorbed on the support material ofthe abiotic control bioreactor after the extraction with n-butanol, suggesting that the observed dye decolouriza-tion is due to the biological activity of the microbialconsortium. Similar microbial process has been reported

Figure 1. Scanning electron micrographs showing the refractory brick

piece support (A) before, (B) after bacterial cell immobilization.

0

20

40

60

80

100

0 1 2 3 4 5 6 7 8 9 10Time (Days)

Dec

olor

izat

ion

(%)

Figure 2. Percentage decolorization of Malachite Green by bioreactor.

Figure 3. Absorption spectra of Malachite Green before (- - -) and

after (—) treatment of bioreactor. (1) Input feed of the bioreactor (2)

618 nm (kmax of the dye) (3) 425 nm (4) 317 nm (5) 248 nm (6) output

of the bioreactor (7) 357 nm (8) 309 nm.

Up-flow immobilized cell bioreactor 433

by Kanekar et al. (1995, 1996) for treatment of effluentfrom a methyl violet-manufacturing unit using rock/brick immobilized cells of Pseudomonas mendocina andPseudomonas alcaligenes capable of decolourizingmethyl violet and phenol, respectively. Up to 60%methyl violet was removed from the effluent by recyclingit for 24 h in the form of a shower. Sani & Banerjee(1999) reported 96% decolourization of malachite green(9.27 mg l)1) by harvested cells of Kurthia sp.(0.40 g l)1) within 30 min of incubation, but the rateof decolourization decreased with increase in dyeconcentration as only 36% decolourization wasachieved at 97.33 mg of malachite green/l. Similarly,An et al. (2002) reported decolourization of malachitegreen using Citrobacter sp. (1.2–1.3 g l)1 dry wt),whereby they had reported 88% decolourization of46.35 mg of malachite green/l after 1 h of incubation atshake flask level.

Biodegradation of malachite green

During thin layer chromatographic studies, it wasobserved that the bands appearing in the chromatogramsof the ethyl acetate extracted output of the bioreactor(with Rf value 0.18, 0.22, 0.25, 0.43, 0.47, 0.51 and 0.64)were completely different from the chromatograms ofinput feed of the bioreactor (Rf value 0.25, 0.28). Yatomeet al. (1981) studied the degradation of crystal violet withPseudomonas pseudomallei 13 NA and demonstrated theformation of unknown products by TLC. The compar-ison of the 1H NMR spectra of the input feed with ethylacetate-extracted output of the bioreactor indicated thebiotransformation of malachite green into metabolicintermediates. Additionally, the 1H NMR spectrum ofthe n-butanol-extracted output of the bioreactor had nosignals corresponding to the parent dye.The formation of metabolic intermediates and more

than 90% decolourization observed, conclusively indi-cated to concerted decolourization and degradationactivities of the components of bacterial consortiumimmobilized on refractory brick pieces. Further work onidentification of the intermediates formed is beingcarried out.

Acknowledgements

Harvinder Singh Saini gratefully acknowledges theCouncil of Scientific and Industrial Research (CSIR),New Delhi (India) for funding this study in which

Deepak Kumar Sharma worked as Senior ResearchFellow (SRF).

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