Isolation and Screening of Dye Degrading Micro-organisms from the Effluents of Dye and Textile Industries at Surat

Text ile dyes have been used since the Bronze Age. They also constitute a prototype 21-century speciality chemicals market. Effluent and soil samples were collected from textile industry at Surat. The pH, temperature, BOD, COD, Nitrate and Nitrite values were compared with the values given by the Bureau of Indian Standards. The culture medium was designed and standardized in the laboratory for the isolation and degradation of the dyes. Pure cultures were screened on the basis of colony morphology. Three different types of unique cultures were selected and named as isolates S1, S2 & S3. Out of 12 dyes used, isolate S1 showed degradation on the maximum number of dyes (five) in comparison to other isolates (isolates S2 and S3). Thus, isolate S1 was used for the further studies. The isolate S1 was used for the study of the amount of dye to be degraded. For this study Red BB dye was chosen. Because, isolate S1 showed maximum degradation on Red BB dye within less time of incubation in comparison with other dyes. Almost all isolates showed the positive results in some of the biochemical tests. Thus most of the isolates can have the capacity to produce the enzyme tryptophanase, indole production, citrate permease (citrate as carbon and energy source), catalase enzyme, degradation of glucose oxidatively as well as fermentatively, urease, gelatinase, production of acid and gas (allow to ferment lactose and/or sucrose) and fermentation of sugar, lactose, sucrose, mannitol and glucose. Total cellu lar fatty acids profiling has been considered to be one of the important and ideal tool for identification of microorganis ms. On the basis of fatty acid profiling of isolate S1 the similarity index indicated as Bacillus cereus GC subgroup A (similarity index 0.825), B. thuringiensis sub sp. israelensis (similarity index 0.552) and for B. thuringiensis sub sp. Kurstakii (similarity index 0.511). The isolate S1 was assumed to be B. cereus GC subgroup A. Thus this isolates can be used to degrade harmful azo dyes utilized by the dye, text ile, paper, ink industries etc.


Introduction
The colored effluents discharged fro m text ile processing and dye-manufacturing industries contain a significant amount of unreacted dyes. During dyeing processes, upto 15% o f the dyestuff does not bind to the fibers and is therefore released into the environment [1]. The world annual production of the dyestuffs amounts to more than 7×10 5 tonnes [2]. Azo dyes, being the largest group of synthetic dyes, constitute up to 70% of all the known co mmercial dyes produced [3]. Text ile processing wastewaters with dye contents in the range of 10-200 mg l -1 are h ighly colored.
The chemical structure of coloured dyes are characterized by highly substituted aromatic rings joined by one or mo re azo groups(-N=N-). These substituted ring structures make these mo lecules recalcitrant and, thus, they are not degraded by conventional wastewater treatment processes [4]. These dyes are therefore released into the environ ment and lead to the acute toxic effects on the flora and fauna of the ecosystem. In addition to being aesthetically displeasing, the release of colored effluents in water bodies reduces the photosynthesis as it impedes penetration of light in water [5,6]. Moreover, many azo dyes and their metabolites are mutagenic and carcinogenic [7]. A review of the mutagenicity of effluents showed that text ile and other dyerelated industries produce consistently more potent wastewaters when compared to other industrial d ischarges [8].
Recent studies by Rajaguru et al. [9] and Umbu zeiro et al. [10] have shown that azo dyes contribute to mutagenic activity of ground and surface waters polluted by text ile effluents. Thus, the color removal of text ile wastewater is a major environmental concern. Therefore, industrial effluents, like textile wastewater containing dyes must be treated before their discharge into the environment. The dye wastewater fro m the text ile is one of the most difficu lt wastewater to treat [11,12]. Because of their commercial importance, the impact and to xicity of dyes that are released in the environment have been extensively studied [13]. Colour can be removed fro m wastewater by chemical and physical methods including absorption, coagulationflocculation, o xidation and electrochemical methods. These methods are quite expensive, have operational problems [14], and generate huge quantities of sludge [15]. A mong low cost, viable alternatives, available for effluent treat ment and decolourizat ion, the biological systems are recognized, by their capacity to reduce biochemical o xygen demand (BOD) and chemical o xygen demand(COD) by conventional aerobic biodegradation. There is large variability in the quality of industrial effluents which varies with industrial processes. The effluents discharged by different industries contain a high range of physico-chemical parameters like temperature, pH, conductivity, hardness, alkalinity, COD, TSS, n itrates, nitrites, cations (Na + , K + , Ca 2+ and Mg 2+ ) and anions (Cl -, CO ). These effluents from different industries also contain heavy metals and trace metals including chro miu m, cad miu m, copper, lead, nickel, zinc, cobalt, magnesiu m, iron and arsenic [16].
The treatment systems based on using microorganisms capable of decolorizing/degrading these recalcitrant compounds are environment-friendly and can lead to mineralizat ion of the target compounds. The effectiveness of these treatment systems depends upon the survival and adaptability of microorganisms during the treatment processes [2,17]. Many microorganisms belonging to different taxonomic g roups of bacteria [2], fungi [18], actinomycetes [19] and algae [20] have been reported for their ability to decolorize azo dyes.
The use of pure-culture system ensures the reproduction of data and interpretation of the detailed mechanism of dye degradation. However, higher degree of biodegradation and mineralizat ion can be expected when metabolic act ivit ies of mixed cultures within a microbial co mmunity co mplement each other. The advantages of mixed cultures are apparent as some microbial consortia can collectively carry out biodegradation that cannot be achieved by pure culture [21,22]. Azo-dyes are also degraded efficiently under aerobic conditions by wood-rotting fungi (e. g. Phanerochaete chrysosporium, Trametes spp. etc.), which are in nature responsible for the degradation of lignin [23]. While fungal treatment of dye containing effluents is usually t imeconsuming and d ifficult to control [24], the potential of enzy mes for this purpose has clearly been demonstrated. Thus an effort has been made to isolate the bacteria capable of degrading the azo dyes present in the effluents of text ile industries located at Surat.

Materials and Methods
For present study effluent fro m the dye and text ile industries was used. The soil samp les were also collected fro m the same site for study of the microbial flora in the adjourning area. Nutrient media (Introduced by Robert Koch) was used with slight modification for the enrich ment of the culture fro m the effluent and the soil samples.

Collection of Effluent and Soil Sample
The effluent sample was collected fro m GIDC, Pandesara, Surat, India. The pH and the temperature of the sample were (10.3 and 24℃ respectively) measured at the time of collection. The Chemical o xygen demand (COD) was estimated by the titrat ion of the effluent samp les and found to be 7507.20 mg l -1 . The Biological o xygen demand (BOD) value could not be found for the effluent samp les. The value for the n itrates and nitrites was found to be 1893 mg l -1 and 70 mg l -1 respectively. The soil samp les were also collected fro m the near bank about 50 to 100 cm far fro m the effluent channel by digging the soil up to 5 cm.

Isolati on of Dye Degradi ng Microorganism
The effluent and combination o f distilled water & effluents (v/v) was inoculated in N-broth mediu m. To each of the flask containing 100 ml of mediu m 10 g of soil samp le was added and incubated at 28℃ for 72 hours. Serial dilutions fro m 10 0 to 10 -3 were made fro m the upper phase of the culture containing microorganisms for each of the med iu m separately. Fro m each dilution 100 μl was spread over the solid plate med iu m-1(Peptone 5 g; Yeast extract 2.46 g; NaCl 5 g; Agar 20 g and p H to 7.00), mediu m -2 (Glucose 30 g; KH 2 PO 4 6 g; Na 2 CO 3 10 g; MgSO 4 7H 2 O 0.2 g; Yeast extract 6 g; Agar 20 g and pH 7.00) containing appropriate and all different dyes separately using sterile glass spreader and incubated at 37℃ for up to 100 hours. After incubation the observations for the zone of clearance/decolorization on the respective plates were made and recorded.

Optimizati on of Conce ntrati on of Dye Degradation
Concentration of dye degraded by the microorganism was optimized for one dye and by isolate no.S1. The Red BB dye was taken in a concentration of 0.05 %, 0.

Biochemical Tests
Biochemical tests were performed for checking the presence of particular substance or enzy me produced by the bacterial isolate. Effluents of Dye and Textile Industries at Surat

Tests for Ut ilization of Carbohydrates and Organic Acids
Tests for utilizat ion of carbohydrates and organic acids were carried out by carbohydrate fermentation test, oxidation-fermentation test, methyl red test, voges-proskauer test and citrate utilizat ion test as per the method described by Patel [25].

Tests for Nitrogenous Compounds
Tests for n itrogenous compounds was studied by perform ing indole production test, H 2 S production test, deamination test, urea hydrolysis test, nitrate reduction test and ammon ia production test as per the method described by Patel [

Triple Sugar Iron Test
Co mbined test using composite media was performed using triple sugar iron agar test as per the method described by Patel [25].

Sample Processing
The pure culture was inoculated on to TSBA solid plate and incubated for 48 hours at 28℃. The culture so obtained was harvested for cellular fatty acid profiling in following steps using Gas Chro matography.

Harvesting
A loop of cultured microorganism (about 40 mg of bacterial cells, cultured on TSBA p late) was taken in 13 x 100 ml culture tube.

Saponification
In above harvested culture tube1.0 ml of Reagent 1 was added. The tube was tightly sealed with teflon lined caps, vortexed briefly and heated in a boiling water bath for 30 minutes. The tube was vigorously vortexed for 5 -10 seconds at an interval of 5 minutes of entire incubation period.

Methylation
After incubation the tube was cooled at room temperature, uncapped and 2 ml of Reagent 2 was added. The tube was capped again and briefly vortexed. After vortexing, the tube ware heated for 10 ± 1 minutes at 80 0 ± 1℃ .

Extract ion
After methylation 1.25 ml of Reagent 3 was added to the cooled tube followed by recapping and gentle tumbling on a clin ical rotator for about 10 minutes. The tube was uncapped and the aqueous (lower) phase was pipetted out and discarded.
2.6.6. Base Wash About 3ml of Reagent 4 was added to the organic phase remained in the tube. The tube was recapped and tumbled for 5 minutes. Follo wing uncapping, about 2/3 of the organic phase was pipetted into a GC vial, capped and ready for analysis GC analysis.
The RTSBA6 6.00 library method was used to found out the similarity index of the isolate on the basis of total cellular fatty acids profiling. The table, graph and result so obtained were recorded.

Results & Discussion
The effluents sample collected had the pH nearer to the permissible range. The average and permissible pH fo r the effluents of azo dye industries is 9 [26]. Hence, the effluents will not have adverse impact on aquatic ecosystem after being discharged. The permissible limit of BOD is 3000 mg l -1 and COD is 15000 mg l -1 as set by the Bureau of Indian Standards [27]. Wastes containing high BOD and COD are responsible for a heavy depletion of o xygen levels in the particular sector of the stream or soil [28]. The value for the nitrates (1893 mg l -1 ) and n itrites (70 mg l -1 ) was found to be higher than the permissible limit. The microorganisms present in the sewage reduce the nitrate into nitrite and then to ammonia, sulphates into sulphides and ferric iron into ferrous iron at very low concentrations of o xygen. Therefore, they create great nuisance for the environment [29]. The data for COD (7507.20 mg l -1 ) revealed that the effluents in present condition are fit for d ischarge to land/ water bodies, as it wou ld not be hazardous for human and aquatic life due to the lower concentration of toxicants.

Isolati on of Dye Degradi ng Microorganism
The result of the dye degradation by the isolate is shown in figure 1(a-f). An analysis of the data reveals that out of four solid med ia used three did not show the degradation of dye by the growing microorganis ms. All the media used differs in the capacity to support the growth of dye degrading microorganis ms. The degradation was seen in the Red BB, purple H3R, BHE 81, Dir Black and blue 171 dye.
In rest of the 7 dyes used were not degraded by any of the isolates obtained from the enrich ment cultures. Out of the above five, Red BB was found to be degraded more by the isolates. The change in color is due to the dye utilized by the isolates. The isolates were named on the basis of colony morphology fro m dye degradation and were named as S1, S2 and S3. Out of 3 isolates, one isolate (S1) showed degradation on all five dyes used. Thus S1 was subjected for the further study.
The growth of the culture on medium-2 could have attributed due to less concentration of glucose in the previous med iu m (mediu m-1) used. With the addition and/or substitution of the above factors the bacteria could grow on the med iu m plate which was conducive for the organis ms. Bacterial decolorizat ion of azo dyes under methanogenic conditions is non-specific [30]. The requirement fo r yeast extract or peptone making the process economically unavailable for industrial-scale application unless alternate cheaper sources are identified [2,21,31]. Glucose is known to enhance the decolorization activity of b iological systems [14,32,33]. However, there are reports that the glucose inhibits the decolorizing activ ity [34]. The variability may be due to the different microbial characteristics. Chen et al. [2] has found that 10 g l -1 concentrations of glucose led to the decolorization of RED RBN by A. hydrophila and glucose concentration of higher than 15 g l -1 inhib ited appreciably the azo reduction of azo dye by the same bacteria.

Optimizati on of Concentrati on of Dye Degradation
S1 isolate showed degradation of Red BB dye in varying concentration ( Table 1). Out of ten different concentrations used, 0.05% concentration was degraded most efficiently within 24 hours. While 0.10% -0.25 % concentration was degraded in 48 hours. Degradation was found after 72 hours in 0.30 % & 0.35 % concentration. Moderate degradation was seen in 0.40 % and 0.45 % concentration of dye. But the degradation was very less or meagre even after 96 hours of incubation in 0.50 %.
All microorganisms have ability to grow on different med iu m. Hence, different types of media have been introduced for the growth of different types of microorganis ms. An analysis of the result obtained for the degradation of dye shows that only five different dyes were degraded. This could have been due to the fact that the microorganis ms present in the isolates might have the efficiency to degrade only five dyes but not the rest (i.e. 7 dyes) used. Studies on 4-ABS degrading strains have also shown that the all different dye degrading microorganisms are highly specific, as they can utilize only 4-A BS and not other benzenesulfonates [35]. The 2-A BS degrading Alcaligenes sp. strain O-1 can utilize t wo other aromat ic sulfonates, benzene and toluene sulfonate, for growth. However, cell extracts of this strain can desulfonate at least six substrates [36]. This suggests the presence of highly specific transport systems for the uptake of aro mat ic sulfonates in these cultures. Thus the isolated bacteria may still have restricted substrate specificity.
In order to test the activity for the degradation of dye, all the isolates were tested for the Red BB dye degradation. Lower the concentration higher the degradation efficiency and vice-versa pattern were obtained. The biodegradation capability of the dyes varies from organis m to organism [37]. He found that out of 15 isolates 4 had the maximu m decolorizing capability after 72 hours of incubation. The similar finding has also been reported by Chen et al. [2].

Biochemical Test
The biochemical tests for all three isolates are depicted in Table 2   The result of biochemical tests (Table 3) for all three isolates reveals that isolate S1 can produce the enzyme tryptophanase and indole production, citrate permease (citrate as carbon and energy source), catalase enzyme, degradation of glucose oxidatively as well as fermentatively, production of acid and gas (ferment lactose and/or sucrose and fermentation of sugar glucose, sucrose and mannitol). Isolate S2 showed presences of enzy me en zy me tryptophanase and indole production and urease. Whereas Isolate S3 showed the presence of en zy me tryptophanase and indole production, gelatinase, degradation of glucose oxidatively as well as fermentatively, p roduction of acid and gas (allow to ferment lactose and/or sucrose) and fermentation of sugar lactose, sucrose, mannitol and glucose. All together isolate S1 showed positive tests for all the sugar used. Biochemical tests have been done by several workers [38] on bacterial co mmun ities.

Conclusions
The textile, dyeing and fin ishing industry use wide variety of dyestuffs due to the rapid changes in the customer's demands. Thus by the use of the above isolates sustainable biodegradation of the harmful azo dyes utilized by the dye, textile, paper in k etc. industries can be possible. These methods are not only eco-friendly but also commercially viable even for the small scale industries. A thorough investigation, taking into consideration of certain parameters such as optimization of the dye concentration for the isolates as well as for the dye to be degraded, effect of physicochemical parameters on degradation etc. at large scale is necessary to provide unequivocal evidence for the usefulness of these isolates in sustaining dye degradation capability.