ISCA Journal of Biological Sciences _____ ______________________________ ______ __ _ _ ISCA J. Biological Sci. Vol. 1 ( 1 ), 55 - 61 , May (201 2 ) International Science Congress Association 55 Decolorization of Reactive Black HFGR by Aspergillus sulphureus Salar Raj Kumar 1 , Rohilla Suresh Kumar 1 * and Rohilla Jitender Kumar 2 1 Department of Biotechnology, Chaudhary Devi Lal University, Sirsa, INDIA 2 Department of Molecular Biology and Biotechnology, CCS HAU, Hisar, INDIA Available online at: www.isca.in (Received 2 5 th APril 201 2 , revised 27 th April 201 2 , accepted , 27 th April 2 01 2 ) Abstract The strain Aspergillus sulphureus was evaluated for its ability to decolorize a textile dye, Reactive Black HFGR. The effect of physiochemical parameters (shaking vs static, pH, dye concentration and different carbon source) on the degradation of textile dye was studied in C - limited Czapek Dox broth. It was found that shaking favored to dye decolorization efficiency of fungal strain showed highest decolorization ( 93.0 4±1.86% ). Effect of different concentration of Reactive Black HFGR dyes ranging from 50 to 300 mg/L had a significant effect on decolorization ( 69.94±1.09 - 93.04±1.86% ) their maximal limits at 200 mg/L and increase in concentration of dye showed a negativ e effect on the decolorization percentage of the tested fungal strain. There was significant decolorization by the fungal strain with different pH ranging from 4 to 8. However, optimum pH was found to be pH 5. There was an influence of carbon source on dec olorization as the fungus showed considerable variation in decolorization activity in the carbon supplemented medium. The fungal strain showed maximum activity with sucrose ( 93.04±1.86% ) followed by glucose ( 85.31±0.59% ) and fructose ( 78.71±1.44% ). Therefo re, Aspergillus sulphureus is an efficient strain for the decolorization of reactive textile dyes, and it might be a practical alternative in the dyeing wastewater treatment. Keywords: Aspergillus sulphureus , decolorization, physiochemical, r eactive b lack HFGR, textile dye. Introduction Synthetic dyes are manufactured and consumed annually in large quantities in textile, food processing, paper and pulp, cosmetics and pharmaceutical industries. The textile industries account for two - third of the total dye stuff market. Dyes ha ve been used increasingly in textile and dyeing industries because of their ease and cost effectiveness in synthesis, firmness and variety in color compared to that of natural dyes. The most commonly used dye in the textile industry is r eactive b lack HFGR. Approximately, 100,000 commercial dyes are manufactured 1 and about 10,000 dyes with an annual production of over 7x10 5 metric tons are commercially available 2 . About 10 - 15% of dyes used in textile industries remain unutilized 3, 4 . Dyes are designed in su ch a way that they are resistant to light, water and oxidizing agents and, therefore, cannot be treated by conventional treatment processes such as an activated sludge 5 . Dye colors are visible in water concentration as low as 1 mg/l, whereas textile processing wastewater, normally contains more than 10 - 200 mg/l of dye concentration 6 resulting in aesthetic problem, affecting photosynthesis in aquatic plants 7 and have toxic and carcinogenic effect in mammals 8 . A major environmental problem facing the t extile dyeing and finishing industry is that the industry produces large volumes of high strength aqueous waste continuously. The discharge of wastewaters was containing recalcitrant residues into rivers and lakes lead to higher biological oxygen demand (B OD) causing a serious threat to native aquatic life 7 . The problem of environmental pollution is increasing day by day due to the release of xenobiotic substances into water, soil and air. These substances include organic compounds (pesticides, dy es, polymers etc.) and heavy metal ions. The damage caused by these pollutants to plants, animals and humans cannot be neglected and hence strategies must be developed to solve the problem of environmental pollution on the priority basis. Removal of dyes f rom the effluents or their degradation before discharge is a great environmental challenge for the industries 9 . Various physical, chemical and biological processes are usually employed to remove these dyes before discharge into the environment. Physical an d chemical methods include adsorption, sedimentation, flocculation, flotation, coagulation, osmosis, neutralization, reduction, oxidation, electrolysis and ion - exchange. However, these methods have some limitations like high cost and disposal of the large amount of sludge or some toxic byproduct produced during these processes 10 . Microbial communities are of primary importance in degradation of dye contaminated soils and water as microorganisms alter to dye chemistry and mobility through reduction, accumul ation, mobilization and immobilization. Among microorganisms, bacteria, fungi and algae are most commonly used for various bioremediation processes. Bioremediation is a cost effective and environment friendly promising alternative to replace or supplement present treatment processes. A white rot fungus, Phanerochaete chrysosporium has been used extensively for decolorization of dyes in wastewaters and correlates to the ability for the synthesis of ISCA Journal of Biological Sciences _________________________________________________________ ISCA J. Biological Sci. Vol. 1(1), 55 - 61 , May (2012) International Science Congress Association 56 lignin degrading exoenzymes such as lignin and manganese per oxidases (MnP) 11 or Laccases 12 . The use of species of the genera Pleurotus , Bjerkanera , Tremetes , Poyporus , Phelinus, Iprex Lacteus , Fungalia trogii , Ganoderma sp., and Thelephora sp., have been also investigated 3, 4, 13, 14 . The aim of the present study is to investigate the potential of an indigenous ascomycete, Aspergillus sulphureus for the removal of a dye, Reactive Black HFGR from aqueous solution. Various conditions required for decolorization have been optimized and dye decolorization/degradation has been analyzed and confirmed by UV - VIS - spectrophotometer. Material and Methods Dye and Chemicals : All media components and chemicals used in the present study were of analytical grade and purchased from Hi - Media Laboratories (Mumbai, India). The textile dyes, Reactive Black HFGR used for decolorization in the present investigation was a gift from M/s M ayur Paliwal Textiles, Panipat (Haryana). The property wise data of the dye are as follows: CAS No. 17095 - 24 - 8 , Chemical Name: Reactive Black 5 , Synonyms: Dimira Black B; Diamira Black B; Sumifix Black B; Celmazol Black B; Cavalite Black B; Remazol Black GF; Adizol Black B; Remazol Black B; Primazin Black BN; Begazol Black B; Reactive Black HFGR , Molecular formula: C 26 H 21 N 5 Na 4 O 19 S 6 , Formula weight: 991.82 , Melting point: �300 °C (lit.) , Product categories: Organics , Hazard codes: Xn , λ max = 594nm , S tructural formula for Reactive Black HFGR is shown in figure 1. Figure – 1 Chemical structure of textile dye, Reactive Black HFGR Growth and Decolorization Medium : The Czapek Dox Agar (CDA) medium was used as a growth medium. The following composition of medium was used [K 2 HPO 4 , 1.0 g L - 1 ; NaNO 3 , 3.0 g L - 1 ; KCL, 0.5 g L - 1 ; MgSO 4 .7H 2 O, 0.5 g L - 1 ; FeSO 4 .7H 2 O, 0.01 g L - 1 ; Yeast extract, 5.0 g L - 1 ; Sucrose 30.0 g L - 1 ; Rose Bengal, 0.0 3 g L - 1 ; Agar, 15.0 g L - 1 ] for growing the fungus. The decolorization medium is slightly modified C - limited Czapek Dox Broth (5 g/L) amended with 200 mg/L reactive black dyes. Decolorization study on Agar Plates : Decolorization of dye was studied in petri dishes containing C – limited CDA medium with dye 200 mg/L. All the plates were inoculated at the center with fungal disc (8 mm in diameter) cut from the periphery of the actively growing 5 days old culture grown on plates. The plates were incubated at 25±2°C in BOD incubator. The plates were monitored over a period of 5 days. Abiotic control was also maintained. The radial growth of fungal mycelia, the zone of color change and zone of decolorization on the agar plates were measured after 24 h. Decolor ization Assay : The ability of A. sulphureus to decolorize Reactive Black HFGR was carried out in C - limited Czapek Dox broth. Textile dye Reactive Black HFGR (λ max 594nm) was used at 200 mg/L concentration. Agitated cultures of fungal species were grown for 10 days in an incubator shaker at 25±2ºC and 120 rpm. Samples were withdrawn aseptically on alternate days, centrifuged at 5000 rpm for 10 min and the supernatant was scanned in a spectrophotometer at λ max of the dye. Control flask without fungus was also maintained. Percent decolorization was calculated using the formula: ( % ) = − 100 Ao a nd A t is the absorbance at different time intervals 15 . Above mentioned protocol was followed during the study of the effect of physiochemical parameters on decolorization. Effect of Physico - Chemical Parameters on Decolorization : Physiochemical parameters such as carbon sources (Glucose, Fructose, Sucrose), static vs shaking conditions, pH (4.0 - 8.0) and dye concentration (50, 100, 150, 200, 250 mg/L) in the aqueous medium was studied. The Decolorization ability of Aspergillus sulphureus at various pH studied and pH 5 was found to be the optimal pH for the decolorization activity. Based on that acidic pH 5 was selected to study the effects of various physico - chemical factors in a decolorization process. The ability of this isolated fungus to decolorize Reactive Black dye (200 mg/l) based on changes in their optical density. Statistical Analysis : All analysis was conducted in duplicate and results presented here are the mean of duplicate ± standard deviations (SD). Results and Discussion Screening of Fungus for Decolorization on solid and liquid medium : Aspergillus sulphureus used in this study was isolated from the dye disposal site and identified using microscopic analysis and cultural characteristics. Since the chances of getting microbes having the ability to decolorize the dye effluent is very high. The textile and dyeing industry are one of the industries, which contribute to the soil and water pollution. In the current research work the potential of ascomycete , Aspergillus sulphureus for the removal of a dye, Reactive Black ISCA Journal of Biological Sciences _________________________________________________________ ISCA J. Biological Sci. Vol. 1(1), 55 - 61 , May (2012) International Science Congress Association 57 HFGR was studied. Fungal strain was initially screened for their growth and decolorization with Reactive Black HFGR dyes (200 mg/L) on Czapek Dox agar media (CDA). Fungus applied (8mm disc) at the center of the plates and were incubated at 25±2°C for 5 days. The apparent dye deco lorization efficiency of native fungal strain was critically visualized and measured after 24 h based on the zone of decolorization around the fungal biomass excluding the size of the inoculum confirm the dye decolorizing activity (Figure - 2). The results are inconsistent with an earlier report of the screening of fungi and bacteria on solid media showing growth and decolorization on solid culture 16 . Further, Aspergillus sulphureus were screened for their ability to decolorize reactive black was carried out in liquid media. Effect of Physiochemical Parameters on Dye Degradation : Fungal treatment of textile dyes and effluents have been found to be influenced by temperature, pH, salts, inhibitory molecules (sulfur compounds, surfactants, heavy metals and blea ching chemicals), carbon and nitrogen sources and other nutrients 17, 18 . The effects of various parameters such as pH, carbon source, dye concentration and agitation were tested in this study. Effect of Static / Shaking on Decolorization : The Decolorization process was carried out using shaking (120 rpm ) and static culture inoculating with fungal disc (8 mm) from fungal colony growing on a CDA plate in Erlenmeyer flasks (250 ml) containing sterilized liquid dye (100 ml) containing medium and incubated in an incubator at 25±2°C for ten days. It was found that the fungus efficient in decolorization in shaking condition (93.04 ±1.86 %) than static (77.33 ±0.76 %). Figure 3 clearly indicating that the shaking condition had a significant impact on decolorization. The extent of per cent decolorization may be increased by treatment under shaking conditions because shaking facilitates the transfer and distribution of nutrients and oxygen between the medium and the microbial cells. Effect of pH on Decolorization : Effect of pH (4.0 - 8.0) was investigated, keeping other parameters constant (temp. 25±2°C, rpm 120 and dye conc. 200 mg/L). Figure - 4 showed that the highest color removal was determined at acidic pH 5 (93.04±1.86%) when compared to neutral and basic pH 8. The optimum pH was found 5.0 for maximum removal of dye. The pH has a major effect on the efficiency of dye decolorization, and the optimal pH for color removal is often between 4.5 to 11.5 for most of the dyes and reported sharp changes in decolorizati on towards both ends of the optimum pH values 16, 19 . The pH significantly influenced the dye biosorption properties of fungi 20, 21, 22 . Higher uptake obtains at lower pH value may be due to the electrostatic attraction between negatively charged dye anions and positively charged cell surface. These results provide an information that acidic pH is required for growth and decolorization . Effect of Dye Concentration on Decolorization : Generally, the concentration of color compounds found in the effluent or ri vers ranged as low as 12 to 16 mg/L. In the present study effect of different concentration of dye (50 - 300 mg/L) on decolorization was investigated. The Decolorization efficiency of the fungus was found to be highest (93.04±1.86%) with 200 mg/L under shaki ng condition (Figure - 5). In addition, the growth of fungi was strongly inhibited at dye concentration above 200 mg/L as a higher concentration of dye may be toxic to metabolic activities. Similar finding has been observed that an elevated concentration of dyes is found to be growth limiting 23 . Besides, decolorization of dyes at higher concentration may create an acidic situation, which further facilitate their better removal (enzymatic or by cell wall adsorption) by the fungi 24, 25, 26 . The desorption of the dyes from the fungal cells especially at higher dye concentrations may be due to higher molecular mass, structural complexity and the presence of inhibitory groups, SO 3 Na in the dyes 27 . Effect of carbon source on Decolorization : Three carbon sources such as glucose, fructose and sucrose were used at 5 g/L. The range of activity on decolorization of Reactive Black HFGR with sucrose was the highest (93.04±1.86%) followed by glucose (85.31±0.59%) and fructose (78.71±1.44%) after ten days of incubation in shaking condition (Figure - 6). The primary mechanism of decolorization may be due to dye adsorption/degradation by mycelium of fungi as well as the reduction of dye intensity in solution because of changes caused by them 28, 29 . Growth media enhance the adsorption/degradation capacities of fungi and on the addition of carbon and other nutrient increased decolorization reached up to 92.9% in a short time 30 . Decolorization of dye involved adsorption of the dye compound at the initial stage followed by the d ecolorization through microbial metabolism 13 . Microorganisms are used for decolorization of dyes and effluents and the fungal biomass proved to be more efficient 24 . Rapid growth of the fungus in C - limited medium with dye indicated that the fungus utilized the dye as the sole source of carbon and produced enzymes to degrade the dyes. No dye decolorization was observed in the control flask without inoculum. Conclusion Although decolorization is a challenging process to both the textile industry and the wastewater treatment, the results of this finding suggest a great potential for fungi to be used to remove color from dye wastewaters. The textile dye (Reactive Black HFGR) is degradable under aerobic conditions with a concerted effort of fungus isolated from an effluent disposal site. Physiochemical parameters (pH, carbon source, dye concentration and agitation) had a significant effect on dye decolorization. Further, it can b e suggested that the potential of ISCA Journal of Biological Sciences _________________________________________________________ ISCA J. Biological Sci. Vol. 1(1), 55 - 61 , May (2012) International Science Congress Association 58 the fungus need to be demonstrated in its application for treatment of dye bearing waste water using appropriate practice, through biotechnological approaches to color removal. Acknowledgements The authors are thankful to the Chairperson for providing necessary laboratory facilities to carry out the present research work and M/s Mayur Paliwal Textiles, Panipat (Haryana) for providing the color. References 1. Zollinger H., Color chemistry syntheses, properties and application of organic dyes and pigments , ( 1 st Edn), VCH Publishers, New York (1987) 2. 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Wataru S . , Miyashita T . , Yokoyama J . and Arail M ., Isolation of Azo dye degrading micro organisms and their application to white discharge printing on fabric , J. Biosci. Bioeng . , 5 ( 8 8), 577 - 581 (1999) Figure - 2 Solid plate decolorization of Reactive Black HFGR (5days, 25±2°C, pH 5.0, Dye 200 mg/L, Sucrose 5 g/L, error bars represents the standard deviation (±) mean of duplicate analysis) 0 2 4 6 8 10 12 14 16 18 20 1 2 3 4 5 Zone of decolorization (mm) Days ISCA Journal of Biological Sciences _________________________________________________________ ISCA J. Biological Sci. Vol. 1(1), 55 - 61 , May (2012) International Science Congress Association 60 Figure - 3 Effect of shaking/static condition on decolorization of Reactive Black HFGR (10 days, 25±2°C, pH 5.0, Dye 200 mg/L, Sucrose 5 g/L, error bars represents the standard deviation (±) mean of duplicate analysis) Figure - 4 Effect of pH on decolorization of Reactive Black HFGR (10 days, 25±2°C, 120 rpm , error bars represents the standard deviation (±) mean of duplicate analysis) Figure – 5 Effect of dye concentration on decolorization of Reactive Black HFGR (10 days, 25±2°C, pH 5.0, 120 rpm , Sucrose 5 g/L, error bars represents the standard deviation (±) mean of duplicate analysis) 0 20 40 60 80 100 0 2 4 6 8 10 Decolorization (%) Days Static Shaking 0 20 40 60 80 100 4 5 6 7 8 Decolorization (%) pH 0 20 40 60 80 100 0 50 100 150 200 250 300 Decolorization (%) Dye concentration ISCA Journal of Biological Sciences _________________________________________________________ ISCA J. Biological Sci. Vol. 1(1), 55 - 61 , May (2012) International Science Congress Association 61 Figure - 6 Effect of carbon source on decolorization of Reactive Black HFGR (10 days, 25±2°C, pH 5.0, 120 rpm, Dye 200 mg/L, error bars represents the standard deviation (±) mean of duplicate analysis) 0 10 20 30 40 50 60 70 80 90 100 2 4 6 8 10 Decolorization (%) Days Glucose Fructose Sucrose Control