INTRODUCTION:

Rapid urbanization, industrialization and technological innovations in various walks of life have lead to the problem of environmental pollution. Although the growth impact of the environmental protection on industrial development promoted the development of ecofriendly technologies, which reduce the consumption of fresh water and lower the output of waste water, the release of important amounts of synthetic dyes to the environment causes public concern, legislation problems and is a serious challenge to environmental scientists^1-4. Effluents of several production facilities such as textiles, paper and cosmetics contain synthetic dye components which contaminate the water resources. The environmental impact assessments indicated that the contaminations affected photosynthetic activity of aquatic organisms by reducing light penetration patterns and might also include some toxic components in them⁵.

Dyes that have synthetic origin and complex aromatic molecular structures are classified as follows: anionic-direct, acid and reactive dyes; cationic –basic dyes; non-ionic-disperse dyes. Reactive dyes are typically azo-based chromophores combined with different type of reactive groups e.g., vinyl sulfone, chlorotriazine, trichloropyrimidine, difuorochloropyrimidine^6-8.

Decolourization of these dyes are possible by using physical and chemical methods such as adsorption, oxidation, coagulation-flocculation, chemical degradation and photo degradation^9-10. These methods are very expensive and have operational problems. Because synthetic textile dyes are resistant to biological degradation, colour removal by bioprocessing is also difficult. As an alternative, biological treatments are relatively inexpensive way to remove dyes from wastewater¹¹. Many fungi and bacteria are used for the development of biologic process for the treatment of textile effluents^12-14. Dead / pre-treated biomass is also used for sorption of dye and many other pollutants (biosorption). However the non-viable biomaterials and isolated potent microorganisms are considered to be limited for real application due to economics, non-availability, low binding capacity and efficiency and system control difficulty^15-20.

The present investigation was aimed to find out the decolorization efficiency of the Reactive Red dye and the effect of substrate and dye concentration on decolorization. The effect of incubation time on decolorization was also evaluated and in addition, phytotoxicity assays were to be performed.

MATERIALS AND METHODS:

Dyestuff, chemicals and instruments:

The textile azo dye Reactive Red C2G29 was obtained from Sigma Chemical Co., St Louis, USA. Double distilled water was used for preparing solutions throughout the study. Stock solutions of the dyes were stored in the dark at room temperature. The nutrient broth and agar were purchased from Navakar Bio Chemicals, Chennai. All chemicals were of the highest purity and of an analytical grade. All pH measurements were carried out with a microprocessor based pH meter model number HI 98107, Hanna Equipments Private Limited, Mumbai, India. Concentration of the dye solutions were estimated using absorbance recorded on Shimadzu UV-1800 spectrophotometer model number (Tokyo, Japan).

Isolation of dye decolorizing bacteria:

The dye decolorizing mixed microbial cultures obtained from the domestic sewage was enriched by periodic sub culturing of samples in NB medium; 1.0 g/L of KH₂PO₄, 1.0 g/L of (NH₄)₂SO₄containing 50 mg/L dye. An aliquot of 0.1 mL of the growing enriched sewage sample was spread on nutrient agar (2.5%, w/v) plate containing 100 mg/L dye adjusted to pH 6.0 and incubated at 37˚C. After an incubation period of 2 days, four distinct colony morphotypes were observed and each were selected and isolated by repeatedly transferring to agar slants. The pure cultures were mixed into 100 mL of nutrient medium containing 100 mg/Ldye in a 250 mL Erlen Meyer flasks and incubated at 37˚C in an incubator for 1-5 days. This bacterial consortium was kept at 4˚C and transferred to nutrient media including dye every three months. The initial pH was adjusted using 1N HCl and 1N NaOH.

Acclimatization

In order to produce more resistant and efficient strain, adaptation of the cells to the progressively higher concentrations of the dye was performed. Adapted bacterial strains was obtained during the serial subcultures in growth medium supplemented with different concentrations of dye changing between 50 and 500 mg/L at a constant sucrose concentration varied for each experimental set. The culture grown in the medium containing the dye at the lowest level was transferred to the next medium supplemented with a higher concentrations of dye and thus, acclimatized to higher concentrations of dye at the same NB concentration. When the adapted culture reached to its exponential growth phase, 1.5 mL of the culture medium was transferred to the next culture medium. The adaptation studies were repeated two times for each dye concentration and were carried out at 37˚C containing 100 mL of the growth medium.

PCR and Sequencing

The genomic DNA isolation was followed by 16S rRNA gene amplification. 2 µL hereof were added directly to the PCR reaction using the bacterial prime set 27F and 1390R. PCR was carried out by initial denaturation step at 94˚C for 5 min, followed by 32 cycles at 94˚C for 1 min, annealing at 45˚C for a min, and elongation at 72˚C for 1 min 20 sec cycling was completed by a final elongation at 72˚C for 7 min. Sequence and phylogenetic analysis was carried out using BLAST search.

Decolorization assay

Decolorization of the Reactive Red C2G29 of varying concentrations of 100 mg/L to 300mg/L was analyzed using UV-Vis-spectrophotometer SL 150 at 540 nm. The decolorization activity was expressed in terms of percentage decolorization.

Analysis of biodegraded product

After 48 h of decolorization, the metabolites produced during biodegradation of Reactive Red C2G29 were monitored by UV-vis spectrophotometer. The HPLC analysis was carried out (Sesimazhu AP model no. 2010) on RP-C18 column. The mobile phase used was (acetonitrile: methanol) in the ratio 70:30 by which the presence of metabolites were determined. In GC-MS analysis, the identification of metabolites formed after degradation was carried out using (Waters 2695 model) gas chromatography coupled with mass spectroscopy. The ionization voltage was 75eV using helium carrier gas. Degradation products were identified by retention time and fragmentation pattern. The identification of products after degradation was identified using LC-MS analysis (Sybyl Triton-7.2 version) using methonitrile as mobile phase.

Toxicity study

Phytotoxicity test were performed in order to assess the toxicity of reactive red and its degraded metabolites. The obtained product was dissolved in the water to form a final concentration of 1000mg/L.The study was carried out (at room temperature) using Vigna radiata. (10 seeds) by adding separately 10ml sample of the Reactive Red C2G29 and its degraded products per day [16]. Length of plumule and radicle was recorded after 8 days.

RESULTS AND DISCUSSION:

Phylogenetic position of isolate:

To analyze the phylogenetic position, the 16SrDNA sequence of the strain Acinetobacter sp.(NBCS09) was determined. The length of the sequence was 1469. Fig.1 showed the phylogenetic relationship between this strain and other related microorganisms found in the Gen Bank database. The result indicated that the strain Acinetobacter sp.was in the phylogenetic branch of the Acinetobacter sp. exhibited a maximum identity (82%) to other Acinetobacter sp. The sequence is given below

AACGCAAAGCTCGCACGGCACTGCCCTCTTTGAGTTGAGTACGCGCTTCTGGTGCACAAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATTCTGATCCGGGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTAGGATCGGGTTTTTGAGATTAGGATTACCTCCCTGTGTAGGAACCCTTTGTACCCACCATTGGAGCACGGGGGGAGCCCTGGGCGGAAGGGGCATGAAGACTTGGGGTCCTCCCCCCCCTCCCCCCGTTTGTGACTGGGGGGATCCTTAAAATTCCCCCCCCAAATGGGGGGGAAAAAAGAAAAAGGGTGCGCTCTTTGTGGGGGTTTAACCCACATCTCCCCACACCAGGGGAGGACAGCCCTGTGGAACCCGTGTTTAAAATTCCCAAAGGACCAATCCCTCTCTGGGAAGATTCTACGATGTCCAAGGCCGGGGAGGGTTTTTTCCCCTTTTATAAAAATAAAACACATGCACCACCGCGTGTGGGGGGCCCCGCCAAATTTTTTGGGATTTATTTTTCCGAAAAGCCCTCCCCAAAAACAGGCTTTGACTCAATTAGTCGTAAGCATTTACTTTCCATGGGCGGTTGGGGGTAGGGTGTTTTGTTGTTGGGGTTTGGTTTTTTGGGGGGGTTTTTTTTGGTGTTTGTGCTTTGTGGTGTTTGGGTGGTATGGGGGGTGGGGTTTTTTGGTGTTGGTGGGTGTGTTTTGTTTTTTTTTGCTTGTTTTTTCTTGTTTTGCGTTTGGTTTTTGCATCGTTGTGTGTTGGGGGGTGGTGTTGGGTGGTGGTGGTTTTTTTGTGCTGTGTTTTGTCGTTTTTGTGTTGTGTTGTCTTTGTTTTTGTTGGTGGTTGTGTGGCTTTTTTGTTTGGTAGGTATGGGTGGGTTTGTTGTTTTTGTGTTGTTGTGTCTGTGATTGTGTTGG

Fig.1. Phylogenetic tree showing position of isolates

Effect of pH on decolorization:

The influence of pH on reactive red dye on percentage decolorization was studied by varying between 4 and 6. There was no growth observed at pH 4 initially. Fig.2 indicate that the decolorization was maximum at pH 5.This indicates that the increase in pH further sharply decreased the decolorization rate.

Fig.2. Effect of pH on decolorization

Effect of substrate concentration on decolorization:

The effect of nutrient supplementation on decolorization activity varied from enhancement [3] to inhibition [5]. In this present investigation effect of substrate concentration on the decolorization of the reactive red dye was studied by varying the substrate concentration from 100 % to 20%. The maximum percentage of decolorization obtained was 96.2 is shown in (Fig.3).

Fig.3. Effect of substrate concentration on decolorization

Effect of dye concentration on decolorization:

The decolorization of reactive red dye was studied at various increasing concentration of dye from 100mg/L to 300mg/L. It was found that the rate of decolorization was decreased with increasing concentration of dye. The maximum decolorization was obtained for 200mg/L of dye concentration and the time required for decolorization was 48 hours is shown in (Fig.4). Reduction in color removal may result from the toxicity of dyes to bacteria through the incubation and metabolic activities [22].

Fig.4. Effect of dye concentration on decolorization

Effect of incubation time on decolorization:

Using the optimized parameters such as temperature of 30˚C, of pH 5 and dye concentration of 200mg/L the present investigation shows that maximum decolorization percentage obtained was 96.2 at 48 hours of incubation period is shown in Fig.5.

Fig.5. Effect of incubation time on decolorization

Analysis of biodegraded product:

The analysis of the degraded products was done using HPLC, GC-MS and LC-MS. The HPLC analysis of the dye sample collected at the beginning showed one major peak at retention time 10.023 and one minor peak at retention time 10.001.As degradation is progressed it showed two peaks major and minor with retention time 10.038 and 10.015.

Fig.6. HPLC analysis of sample before degradation

Fig.6. HPLC analysis of sample after degradation

GC-MS:

In order to verify degradation products formed during dye decolorization by Acinetobacter sp. the GC-MS analysis was carried out, which reveals presence of several peaks. The low molecular weight aromatic compound was produced from the degradation of Reactive Red C2G29 by Acinetobacter sp. is shown in (Fig.7).

Fig.7. GC-MS analysis of the dye degraded sample

LC-MS analysis

The LC-MS analysis carried out indicates the presence of degraded product Benzo (1,4) anizo sulphonic acid is shown in (Fig.8).

Fig.10. LC-MS analysis of dye degraded sample

Toxicity studies:

Dye metabolites such as unsulphonated aromatic amines are relatively stable in aquatic conditions and are poorly degraded under anaerobic or aerobic waste water treatment conditions [24]. Thus both sulphonated and unsulphonated aromatic amines are important group of pollutants formed during reduction of (sulphonated) azo dyes, that can be potentially pass through biological treatment system [28]. There fore, it was of concern to assess the toxicity of the dye before and after degradation, results in Table 5.8.1 indicates that the length of plumule and radicle of Vigna radiata was less with reactive red treatments as compared to its extracted degradation product and water. This study reveals that the metabolites generated after the biodegradation of Reactive Red C2G29 less toxic compared to original dye.

Table 1 Phytotoxicity study of Reactive Red C2G29 before treatment

Parameter studied	Length (cm)	Volume of water (mL)	Reactive Red (mg/L)
Length of plumule	10	50	250
Length of radicle	1.5	50	250

CONCLUSIONS:

The acclimated bacteria Acinetobacter sp. was capable of decolorization and detoxification of the toxic Reactive Red C2G29.The degradation metabolites formed were found to be Benzo-(1, 4) anizo sulphonic acid.which indicated the cleavage of the azodye by the azoreductase enzyme. The toxicity study carried out revealed that this dye degraded water can be used for irrigational purposes.

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Received on 30.08.2013 Accepted on 01.09.2013

Research J. Engineering and Tech. 4(4): Oct.-Dec., 2013 page 246-250