Biotechnology Letters (2005) 27: 323326 DOI 10.

1007/s10529-005-0691-7

Springer 2005

Textile dye decolorization using cyanobacteria

Amit Parikh & Datta Madamwar*
Postgraduate Department of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388 120, Gujarat, India *Author for correspondence (Fax: +91-2692-226865; E-mail: datta_madamwar@yahoo.com)
Received 26 October 2004; Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005; Accepted 10 January 2005

Key words: chlorophyll a, cyanobacteria, decolorization, dyes Abstract Cyanobacterial cultures isolated from sites polluted by industrial textile euents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC conrmed that color removal was due to degradation of the dyes.

Introduction Dye and dyestus in the aquatic environment are degraded by a range of microorganisms, but studies have concentrated exclusively on the role of bacteria and fungi (Banat et al. 1996, Wesenberg et al. 2002, Keharia & Madamwar 2003). Cyanobacteria have a ubiquitous distribution but their role in functioning of ecosystems, including degradation of recalcitrant compounds such as dye and dyestus, is scanty (Semple et al. 1999). The present study was undertaken to investigate the dye degrading potential of cyanobacteria isolated from waste contaminated sites. Materials and methods Chemicals The structures of the dyes used are given in Figure 1. Isolation, identication and screening of cyanobacterial cultures Soil and water samples collected from the vicinity of dye manufacturing units in Gujarat, India, were

used for enrichment and isolation of cyanobacteria. Isolation, purication and identication of cyanobacterial cultures were carried out using procedures described by Shah et al. (2001). All the cultures were maintained and incubated at 27 C in BG11 medium with light/dark cycle of 12/12 h under white uorescent light of 30 W m)2. Initial screening of isolated cultures for the dye decolorization was made in BG11 medium amended with 50 mg dye l)1. Cultures showing 7090% decolorization with dye at 50 mg l)1 were further investigated for decolorization of 100 mg dye l)1.

Dye decolorization study Pre-grown cultures (5 ml) were inoculated in 250-ml Erlenmeyer asks containing 45 ml BG11 medium with dye (100 mg l)1). At the same time, two other sets, one of biotic control (culture inoculated medium devoid of dye) and another of abiotic control (medium with 100 mg dye l)1 without inoculation) were also incubated. Samples were withdrawn at regular intervals and centrifuged (4000 g for 10 min.).

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Fig. 1. Chemical structure of dyes. Color index names are given within square brackets. (a) Amido Black 10B (kmax in water 618 nm) [C.I. Acid Black 1]; (b) Acid Red 97 (kmax in water 495 nm) [C.I. Acid Red 97]; and (c) FF Sky Blue (kmax in water 572 nm) [C.I. Direct Blue 1].

Residual dye content in the supernatant was measured at the dyes kmax in water using UVVisible diode array spectrophotometer. Percentage decolorization was calculated from the absorbance values obtained against the abiotic control. Growth was measured by extracting chlorophyll a from the cell mass according to Tandeau De Marsac & Houmard (1988) procedure and measured spectrophotometrically at 665 nm. Each experiment was repeated thrice to conrm the decolorization. At the end of incubation (26th day) cultures were centrifuged and the supernatant was freeze dried. Dried powder was redissolved in 2.5 ml of Milli Q water and 15 ll samples were spotted on pre-coated silica gel TLC plates. Plates were developed using n-propanol/ethyl acetate/water (12:1:6, by vol.) for FF Sky Blue, n-propanol/ethyl acetate/water (10:4:5, by vol.) for Amido Black 10B and n-propanol/25% ammonia (4:3, by vol.) for Acid Red 97 dyes. Video documentation system in conjunction with Reprostar 3 (Camag) was used for imaging the resolved compounds on TLC plates.

Results and discussion Fourteen cyanobacterial cultures were isolated and studied individually for decolorization of dyes at 50 mg l)1. Majority of isolates grew eciently in medium containing dyes. However, only 5 cultures showed 7090% color removal. Amongst these, only Phormidium ceylanicum, Gloeocapsa pleurocapsoides and Chroococcus minutus showed rapid decolorization of 100 mg dye l)1 (Table 1). These were then used for further studies.

Dye decolorization study Gloeocapsa pleurocapsoides decolorized 90% and 83% of FF Sky Blue and Acid Red 97, respectively. Decolorization of same dyes was also observed with Phormidium ceylanicum (Table 2), indicating the potential role of cyanobacteria towards dye decolorization. Amongst 14 cultures, only Chroococcus minutus decolorized Amido Black 10B, 55% after 26 days. Table 2 also shows a large decrease in chlorophyll a content

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Table 1. Screening of cyanobacterial cultures for textile dye decolorization of 100 mg dye l)1, after 30 days of incubation. Culture Dye Decolorization, (%)a 88 77 78 90

Phormidium ceylanicum Gloeocapsa pleurocapsoides

Acid Red 97 FF Sky Blue Acid Red 97 FF Sky Blue

Gloeothece sp. Acid Red 97 40 Oscillatoria splendida FF Sky Blue 58 Chroococcus minutus Amido Black 10B 49 Preliminary screened cultures for decolorization of 50 mg dye l)1 were studied further for decolorization of 100 mg dye l)1 at 27 C. a Decolorization, given as % decrease of absorbancy at the kmax of a dye was measured after 30 days of incubation. Control experiments were carried out for each dye, no decolorization was observed.

occurred in presence of the dyes. This was due to attenuation of light transmission which then reduces the rate of photosynthesis and ultimately the growth rate. A slower rate of decolorization was attributed to higher molecular weight, structural complexity and the presence of inhibitory functional groups like NO2 and SO3Na

in the dyes (Hu & Wu 2001). The absorption ratio at two distinct wavelengths changed as time progressed (Figure 2). Sequential reduction in absorbance at dyes kmax was attributed to the cleavage of color imparting chromophores (NO2, N@N, NH2) and fused aromatic rings with the simultaneous formation of UV absorbing intermediates. Transformation of dyes was further conrmed by thin layer chromatography. The dye chromatograms when observed in daylight showed decrease in intensity of dye bands and no band was observed for spots of decolorized medium. When dye chromatograms were observed under UV light, uorescent bands were observed against spots of decolorized medium with Rf values different from that of dye. Photo-oxidative changes in FF Sky Blue converted it to highly uorescent compounds, which also got degraded as the corresponding band disappeared upon cyanobacterial treatment. Certain band patterns of treated dye and of biotic control were similar, which revealed the secretion of growth-associated, soluble microbial products (Laspidou & Rittmann 2001). Jinqi & Houtian (1992) were the rst to report that microalgae could be used for dye decolorization. They found that Oscillatoria sp. and Chlorella sp. completely mineralized aniline to CO2 on extended incubation. Oxygenic pho-

Table 2. Decolorization of 100 mg dyes l-1 by cyanobacteria and their chlorophyll a contents, after 26 days of incubation. Culture/Dye studied Chlorophyll a (mg l)1) a Decolorization (%) b

FF Sky Blue Gloeocapsa pleurocapsoides controlc Gloeocapsa pleurocapsoides experimentalc Phormidium ceylanicum control Phormidium ceylanicum experimental Amido Black 10B Chroococcus minutus control Chroococcus minutus experimental Acid Red 97 Gloeocapsa pleurocapsoides Phormidium ceylanicum

9.9 3.3 7.3 2

0.3 0.4 0.4 0.4

90 80

5.7 0.3 3.1 0.5 ND ND

55

83 89

ND, Not determined. a Substraction of initial chlorophyll a concentration from nal concentration. Chlorophyll a was extracted in chilled methanol at 4 C under dark and the extract was measured at 665 nm. b Decolorization, given as % decrease of absorbancy at the kmax of a dye was measured after 26 days of incubation. No decolorization was observed with control experiments. c Control, without dye; experimental, with dye.

326 tosynthesis releases abundant amount of O2 during light reaction eliminating the possibility of ring cleavage by reduction. On the other hand, oxidative degradation of lignin and naphthalene-like compounds by cyanobacteria is well known (Cerniglia et al. 1980, Malliga et al. 1996). The present study conrms the ability of cyanobacteria to decolorize and degrade structurally dierent dyes. Further studies are needed to identify the biochemical machinery involved in the degradation.

Acknowledgement This work was supported by the University Grants Commission, New Delhi, India.

References
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Fig. 2. Spectrophotometric analysis of textile dye decolorization by cyanobacteria. (A) Decolorization of Amido Black 10B by Chroococcus minutus; (B) decolorization of Acid Red 97 by Phormidium ceylanicum; and (C) decolorization of FF Sky Blue by Gloeocapsa pleurocapsoides [a Untreated dye at 0 days, b treated dye after 26 days]. Cultures incubated in BG 11 medium amended with 100 mg dye l)1 at 27 C, samples withdrawn over 26 days.