Biodecolourization and biodegradation of textile azo dye Golden Yellow HER by Pseudomonas sp. GYH-1

 

Amarja H. Bhosale, Rahul M. Khobragade

Department of Microbiology, Dr. Babasaheb Ambedkar Marathwada University,

Sub-Campus, Osmanabad. 413501(M.S.) India.

 

Abstract:

Textile industry is one of the major causes of the contaminator of environment. It produces enormous amounts of diverse complex chemical substances as a part of vacant materials that includes dye in the form of wastewater throughout various stages of textile processing. These dyes produces the obvious aesthetic belongings to ecosystem by affecting on the photosynthetic ability of plants and are also toxic to the aquatic flaura and fauna when released in water system and thus ultimately get into food chain. In the present research study, Pseudomonas sp. GYH-1 was isolated and identified by 16s rRNA sequencing and phylogenetic relationship as a potent candidate to remove textile dye Golden Yellow HER from polluted soil and water samples. The physico-chemical activities of this isolate revealed maximum decolorization at 37⁰C at pH range 6.0-8.0 within 24 hours. The FT-IR and GC-MS analysis reports confirmed that complex dye was degraded into simple products. Further phytotoxicity and Microbial toxicity studies revealed that the byproducts formed after dye degradation were non-toxic to Jawar and Mung beans and other Microorganisms. Thus it can be concluded that Pseudomonas sp. GYH-1 may be used effectively for the removal of Golden Yellow HER from textile dye stuff waste.

 

 

 

1.    INTRODUCTION:

The textile industries uses large amount of dyes which are having synthetic origin and very complex aromatic and stable structure that is difficult to degrade microbiologically. Of the all dyes used in textile industries near about 80% dyes are Azo dyes (Fu et al., 2001). The Azo dyes contain N=N (azo bond) in its structure which makes the dye more stable. Therefore it makes its major contribution in textile industries (Carliell et al., 1995). Azo dyes like azodisalecylate, direct black 38 and their derivatives such as benzidineand derivatives, dimethyl amines, and anilines nitro semis are cancerous to human and animals (Puvaneswari et al., 2006).  Some azo dyes cause bladder cancer to human, hepato carcinoma, nuclear anomalies and splenic aromas to mammalian cells (Lima et al., 2007). The common processes in textile industries are scouring, desizing, bleaching, mercerizing, dyeing etc. (EPA, 1997). Overall 2-20% aqueous dyes are released in environment during dyeing processes (Cooper, 1995). The release of such toxic dyes contained textile waste water causes ecological hazards (S. Sharma et al., 2009). The textile and dyeing industry effluent is characterized by high BOD, COD, pH, colour, heavy metals and hazardous chemicals. Among these, the dyes are present significant amount in textile effluent which is high carcinogenic, toxic and mutagenic in nature. If these dyes are not treated properly they persist in environment for longer period of time (Forgacs et al., 2004; Hao et al., 2000). Moreover about 80% dye stuffs retains on cloths, lateron get drain out on the environment (Ryan J. Paterson, USA). Hence it is a difficult task to treat such effluents (Senan et al., 2004). Sometimes dyes get decomposed into mutagenic or carcinogenic compounds which cause skin irritation, changes in different tissues or allergies (Chequer et al., 2011). There are many industries which have used traditional physical and chemical methods like adsorption, membrane filtration, chemical precipitation, electrolysis and coagulation to treat the textile effluent (S. K. Solmaz et al., 2007; M. Yun et al., 2006; D. J. Joo et al., 2007; G. Crini et al., 2006; N. Daneshvar et al., 2006). But these methods have expensive and require large set up. On the other hand the certain biotechnological methods have been invented to overcome this p (Figueroa et al., 2009; Constapel et al., 2009) by using microorganisms to treat the textile waste water (Carliell et al., 2004). There are variety of microorganisms including Actinomycetes (Mane et al., 2008), bacteria (Telke et al., 2008), yeast (Meehan et al., 2000; Jadhav and Govindwar 2006), fungi (Parshetti et al., 2006; Kaushik and Malik 2009) and algae (Daeshwar et al., 2007) which are involving in removal of toxic dyes from textile waste. In recent years bacteria are widely used for degradation or mineralization of textile dyes (Kalyani et al., 2009). It was revealed earlier that the bacteria are having greater advantage than other microorganisms for removal of dye from textile effluent. Among all bacteria Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Acenetobacter sp., Proteus vulgaris, Zooglea sp. are most dominant in dye decolorization (Sudhakar et al., 2002).  The nitro and sulfonic groups of azo dyes are quite obstinate for aerobic bacterial degradation (Dos Santos et al., 2004; Robinson et al., 2001).

 

The present research work focused on isolation and identification of bacteria from dye contaminated soil area near by textile industry and having highest ability to decolorize and degrade toxic textile dyes. All isolated bacteria were screened for their ability to remove the dye Golden Yellow HER which is highly carcinogenic in nature. Among all isolates, Pseudomonas sp. GYH-1 was having highest efficiency to decolorize Golden Yellow HER. Positive isolate was identified by Morphological, Biochemical and 16s rRNA sequencing. The isolate was further examined for its dye degrading ability by optimizing various parameters.

 

2.    MATERIALS AND METHODS:

2.1 Sample collection:

The soil and water samples were collected from the area near by waste disposal site of textile industry, effluent treatment plant, dyeing units and activated sludge in Solapur.

 

2.2 Dyes and chemicals:

Textile dye Golden Yellow HER (ƛ max 490 nm) was collected from Solapur textile industry. All the required chemicals were of highest analytical grade and highest purity.

 

Preparation of dye stock solutions:

The dye solutions of 1000ppm were prepared and stored as stock solution and used for further study.

 

Table 1- Dye properties and dye structure

Dye properties	Dye structure
Formula Weight = 536.360218   ƛ max- 490

 

2.3 Acclimatization of microorganisms:

The soil and water samples were collected from the area near by waste disposal site of textile industry, dyeing unit, effluent treatment plant in Solapur. These samples were homogenized and mixed properly and added with increasing concentration of dye Golden Yellow HER at regular interval for one month. These acclimatized samples were further used for isolation of dye decolorizing bacteria.

 

2.4 Isolation and screening of dye decolorizing bacteria:

For the isolation of Golden Yellow HER decolorizing bacteria, 1% of acclimatized sample was prepared in saline. Near about 0.1 ml aliquots were spreaded on nutrient agar plates. After incubation period the isolated bacterial colonies were used for screening. The screening was carried out in tubes containing 30 ml nutrient broth and 100ppm dye concentration. Each and every isolates were tested for its ability to decolorize Golden Yellow HER dye. After inoculation of isolates, tubes were kept for incubation at ambient temperature for 24 hours. The tubes showing decolorization were used for further study. Among all promising isolates the isolate designated as GYH-1 was showing highest decolorization of dye. Hence this isolate was used for further study.

 

2.5 Identification of isolate by 16s rRNA sequencing and phylogenetic relationship:

The phylogenetic identification of isolate was constructed by determining BLAST results. The alignment of sequences was done by Clustal X2 software.  Phylogenic tree and dendrograms was constructed with Neighbour Joining Method by MEGA 4.0 software package (Saitou and Nei, 1987). Further by using 1000 replicates data sampling, Bootstrap analysis (Felsenstein, 1985) was conducted.

 

2.6 Optimization of parameters for maximum decolorization:

2.6.1 Effect of Temperature and Ph:

The activity of isolate was influenced by optimizing different culture conditions. The pH is important factor in influencing the dye decolorizing ability of microorganisms. To study the effect of pH on dye decolorizing ability, pH of medium was kept variable in the range 6.0 to 10.0 and all the other factors were kept constant. The tubes were inoculated with selected isolate having 30 ml sterile nutrient broth and 100ppm dye concentration. These tubes were kept for incubation at ambient temperature for 24 hours. After incubation period the decolorization was supervised by spectrophotometer. Temperature is also critical factor for the activity of microorganisms. Growth of any microorganisms is carried out by its enzymatic reactions. Hence the microbial growth and enzymatic reactions are directly in proportion with each other. And the activity of enzymatic reactions is increased by temperature. But still the activity of organisms is decreased by extreme higher or lower temperature. Hence to study the effect of temperature on decolorization of selected dye, the tubes containing 30 ml nutrient broth and Golden Yellow HER dye in 100ppm concentration were inoculated with selected isolate and kept for different temperatures as Room temperature, 37⁰C, 45⁰C, 55⁰C, 66⁰C. All other parameters were kept constant. The tubes were then kept for incubation for 24 hours. The decolorization was supervised by spectrophotometer.

 

2.6.2 Effect of Inoculum’s size and Dye Concentration:

To examine the effect of Inoculums’ Size on dye decolorization efficiency of selected isolate, the tubes containing 30 ml sterile nutrient broth and 100ppm of dye concentration which was under study, were inoculated with different inoculums size of isolate such as 1%, 2%, 3% and 4%. The tubes were then kept for incubation at optimum temperature and pH for 24 hours. After incubation period the decolorization was supervised by spectrophotometer. The microorganisms are capable of decolorizing the dye upto specific concentration. Above that the dye decolorizing efficiency of organisms gets decreased. Hence the decolorizing efficiency of selected isolate was studied at various dye concentration ranging from 100ppm to 100ppm dye concentration. The dye under study in its different concentrations in different tubes containing 30 ml nutrient broth was inoculated with promising isolate and kept for incubation at optimum temperature and pH for 24 hours.

 

2.6.3 Effect of different Carbon and Nitrogen sources:

In order to study the effect of Carbon and Nitrogen sources on dye decolorization, the selected isolate was inoculated in 30 ml sterile Minimal medium having 100ppm of dye concentration and 1% of different Carbon and Nitrogen sources such as Glucose, Sucrose, Starch, Peptone, Yeast extract and Meat extract. The tubes were kept for incubation at optimum temperature and pH for 24 hours. The decolorization was then supervised spectrophotometrically.

 

2.7 Percent decolorization study:

The spectrophotometer was used to monitor decolorization. To calculate the percent decolorization of Golden Yellow HER dye after its decolorization, the decolorized broth was centrifuged at 10,000rpm for 20 min in Cooling centrifuge (BIOLAB-BL-165R). The percent decolorization of dye was determined by comparing the initial absorbance of known concentration of dye and final absorbance of dye after decolorization at its maximum absorbance wavelength (ƛ max) by spectrophotometer (Systronics-106 model) and following formula.

 

                                                 Initial Absorbance – Final Absorbance

Percent decolorization = ^{________________________________________________________} × 100

                                                                  Initial Absorbance

2.8 Percent decolorization in different culture media:

2.8.1 Percent decolorization in nutrient medium:

The 30 ml of nutrient medium with composition- Peptone- 1.0 g, Beef extract- 0.3g, NaCl- 0.5g, distilled water- 100 ml was inoculated with selected isolate containing 100ppm of selected dye concentration. The tube was then incubated at optimum temperature and pH for 24 hours. The percent decolorization was observed at maximum absorbance of dye by spectrophotometer.

 

2.8.2 Percent decolorization in Half (½) Strength nutrient broth:

The efficiency of isolate for dye decolorization in Half (½) Strength nutrient medium (Peptone- 0.5g, Beef extract- 0.15g, NaCl- 0.25g, distilled water- 100 ml) was evaluated. The selected isolate was inoculated in 30 ml of Half (½) Strength nutrient broth containing 1000ppm dye concentration. The tube was further kept for incubation at maximum temperature and pH for 24 hours. The percent decolorization was observed at maximum absorbance of dye by spectrophotometer.

 

2.8.3 Percent decolorization in Cell Free Extract:

The preparation of cell free extract was carried out to study the effect of extracellular and intracellular enzymes on dye decolorizing ability of isolate. The isolate was grown in sterile nutrient broth. The cells were then separated by centrifugation at 7000 rpm for 20 min in Cooling centrifuge (BIO-LABS 165-R). The supernatant was then collected and used as source of extracellular enzyme. The pellet was then resuspended in 50 mM Phosphate buffer (pH-7.4). the buffer containing suspension was cooled and lysed properly by Ultrasonicator (Sonic- Vibra Cell System - 130) keeping sonicator output at 50 A and giving 6 strokes each of 25s at time interval 2 min at 4⁰C temperature. After this the suspension was again centrifuged for 10 min at 10000 rpm. The cell debris was separated and this was used as intracellular source of enzyme. Further both the intracellular and extracellular enzyme containing suspensions were added in nutrient broth containing 100ppm of selected dye and isolate and kept for incubation at optimum temperature and pH for 24 hours. The percent decolorization was observed at maximum absorbance of dye by spectrophotometer.

 

2.9 Extraction, Analysis and Confirmation of biodegraded metabolites:

To find out the metabolites formed after degradation of textile dye Golden Yellow HER by promising isolate, the analysis of decolorized samples was carried out. The 100 ml sterile nutrient broth containing 100ppm of Golden Yellow HER dye was inoculated with selected isolate and kept in different flasks at ambient temperature for 24 hours. Further the centrifugation of decolorized broth was carried out at 10000 rpm for 20 min in cooling centrifuge. The supernatant was separated and mixed with equal volume of Dichloromethane (DCM) in separating funnel to extract the metabolites. Then the vigorous shaking of these samples was carried for the separation of Liquid and Solvent phases. The liquid phase was separated and discarded. Partial evaporation of solvent phase was carried out. Further FT-IR and GC-MS analysis of these samples were performed. By using GC-MS technique, the degraded metabolites of Golden Yellow HER dye were detected.

 

2.10 Prediction of metabolic pathway for dye degradation:

By observing the GC-MS analysis reports, the metabolic pathway was predicted.

 

2.11 Toxicity testing of degraded products:

The degraded products of Golden Yellow HER dye were tested for toxicity. The toxicity testing was carried out by Phytotoxicity testing and microbial testing.

 

2.11.1 Phytoxicity studies:

The Dichloromethane extracted products after degradation of Golden Yellow HER dye was evaporated and mixed with 10 ml distilled water to prepare 500ppm concentration of extracted products to carry out phytotoxicity study. The phytotoxicity study was performed by observing seed germination with two types of seeds. Jawar (Sorghum bicolor) and Mung beans (Vigna radiata) were used for phytotoxicity study of treated dye products. This experiment was carried out in Petri plates containing 10 seeds of both types separately. These seeds were watered with 1 ml of original dye solution and treated dye solution separately in respective Petri plates. The distilled water was used as control for this test. These plates were kept at room temperature. All the samples were added for consecutive 7 days. The percent germination, length if root and shoot was recorded and compared with control distilled water.

2.11.2 Microbial toxicity testing

Microbial toxicity testing of degraded products of dye was performed on three test organisms which are having ecological importance viz. E. coli, Rhizobium sp. and Azotobacter sp. This test was carried out by Agar Well Bioassay method. The original dye Golden Yellow HER was used as control and degraded dye sample was used as test solution. The 24 hour culture of test organisms was spreaded on nutrient agar plates separately. Wells were prepared on these plates and these wells were poured with original dye solution and degraded dye solution separately. These plates were kept for incubation at ambient temperature for 24 hours. After incubation period these plates were observed for zone of inhibition around the wells which proved the toxicity of dye and its degraded products.

 

3.    RESULTS:

3.1 Isolation and identification of microbial isolate:

The microorganisms were isolated from acclimatized soil samples and further screened for their ability to decolorize textile dye in nutrient medium. Positive isolate showing maximum Decolourization was further identified by morphological, biochemical characterization (Table 2, Table 3(A) and (B)) and 16s rRNA sequencing. By observing analysis report the selected isolate was identified as Pseudomonas sp. GYH-1. The Neighbor Joining method was used to construct the phylogenic tree by Kimura-2-parameterin MEGA 4.0 with 1000 replicates. (Fig. 1).

 

Table 2: Morphological characteristics of GYH-1

Utilization of					Hydrolysis of
Glucose	Fructose	Sucrose	Mannitol	Lactose	Maltose	Casein	Starch	Gelatine
A	A	-	-	+	+	-	+	-

 

Table 3(A): Biochemical characteristics of GYH-1

Enzyme Activity			Indole	Methyl red	Voges Proskauer	Citrate utilization	Nitrate reduction
Catalase	Urease	Oxidase
+	+	-	-	+	-	+	-

 

Table 3(B): Biochemical characteristics of GYH-1.

Size in mm	Shape	Colour	Margin	Elevation	Consistency	Opacity	Gram nature	Motility
1mm	Circular	Off white	Regular	Flat	Moist	Translucent	Gram Negative	Highly motile

+++

 

Fig. 1: Phylogenetic analysis of the 16S rRNA sequence of Pseudomonas sp. GYH-1. The percent numbers at the nodes indicate the level of bootstrap support based on neighbor-joining analyses of 1,000 replicates. The scale bar (0.02) indicates the base pair substitutions per site i.e. the genetic distance.

3.1.1 Accession Number of Gene Bank:

The phylogenic construction was carried out by searching DDBJ BLAST. 16s rRNA sequence of Pseudomonas sp. GYH-1 is gained under Gene Bank Accession Number LT599739.

 

3.2 Optimization of parameters for maximum decolorization:

Pseudomonas sp. GYH-1 was studied for maximum decolorization of the dye Golden Yellow HER by optimizing various physic-chemical parameters such as effect of temperature, pH, inoculums size, dye concentration, different carbon and nitrogen sources.

 

3.2.1 Effect of Temperature and pH:

To study the effect of temperature the isolate was inoculated in nutrient broth having 100ppm of dye concentration and tubes were kept at different temperatures like, Room Temperature, 37⁰C, 45⁰C, 55⁰C and 65⁰C. The Pseudomonas sp. GYH-1 showed maximum/complete decolorization of Golden Yellow HER at temperature 37⁰C. In order to study the effect of pH on dye decolorization efficiency, the isolate was inoculated in nutrient broth containing 100ppm of dye concentration and tubes were kept at different pH (6.0, 7.0, 8.0, 9.0, and 10.0) at 37⁰C temperature. After incubation period of 24 hours, the isolate Pseudomonas sp. GYH-1 was showing higher decolorization of Golden Yellow HER at pH range 6.0-8.0. Specifically it was found that the pH 7.0 (Neutral) was its optimum pH which was calculated by spectrophotometer at ƛ max 490 nm. The results are shown in Fig. 2

 

 

Fig. 2: Effect of various pH and temperature on decolorization of Golden Yellow HER by Pseudomonas sp. GYH-1

 

3.2.2 Effect of inoculums size and dye concentration:

The isolate was examined for its dye decolorizing ability when inoculated in its different size. The test was carried out by inoculating different inoculums size of Pseudomonas sp. GYH-1 (1%, 2%, 3% and 4%) in nutrient broth containing 100ppm dye concentration. After 24 hr incubation periods at 37⁰C and pH, it was observed that the isolate was showing maximum decolorization when it was inoculated with 2% inoculums size. The increasing concentration of dye decreases the dye decolorizing ability of isolate. The organisms exhibit maximum decolorization ability upto specific dye concentration. The dye could be toxic to organism at higher concentration. The Pseudomonas sp. GYH-1 was able to decolorize Golden Yellow HER at 100ppm concentration. However the maximum activity of isolate was observed at 600ppm of dye concentration. Above 600ppm concentration the activity of isolate was slightly decreased. The activity of isolate was monitored by spectrophotometer. The results are shown in Fig. 3.

 

 

Fig. 3: Effect of various Inoculums’ size and Dye concentration on decolorization of Golden Yellow HER by Pseudomonas sp. GYH-1.

 

Where, I.C. = Inoculum’s size, D.C. = Dye Concentration

 

3.2.3 Effect of different Carbon and Nitrogen sources:

The isolate was inoculated in Minimal medium having dye concentration 100ppm and 1% different sources of Carbon (Glucose, Sucrose and Starch) and Nitrogen (Peptone, Yeast extract and Meat extract) to study the effect of different carbon and nitrogen sources on the dye decolorizing ability. After incubation period the tubes were observed for decolorization. It was found that Pseudomonas sp. GYH-1 was showing highest decolorization of Golden Yellow HER when it was added with 1% Starch as carbon source like wise it showed highest decolorization of dye when it was provided with 1% Peptone as Nitrogen source. The results are shown in Fig. 4.

 

 

Fig. 4: Effect of various Carbon and Nitrogen sources on decolorization of Golden Yellow HER by Pseudomonas sp. GYH-1.

 

Where, C- Carbon source, Glu- Glucose, Suc- Sucrose, Str- Starch, N- Nitrogen source, Pep- Peptone, Y.E- Yeast Extract, M.E- Meat Extract.

 

3.3 Percent decolorization study:

The percent decolorization study of the dye Golden Yellow HER by Pseudomonas sp. GYH-1 was carried out in nutrient broth containing 100ppm of dye concentration. The nutrient broth tubes were inoculated with promising isolate and kept for incubation at ambient temperature for 24 hours. After proper incubation period these tubes were observed for decolorization. The percent decolorization was calculated by given formula at its maximum absorbance using spectrophotometer. The results are shown in Fig. 5.

 

3.4 Percent Decolourization of dye in different culture media:

3.4.1 Percent Decolourization of dye in nutrient medium:

The isolate, Pseudomonas sp. GYH-1 was inoculated in nutrient broth and dye at 1000ppm concentration of Golden Yellow HER. The percent decolorization was calculated by given formula and monitored by spectrophotometer. The isolate showed 96.71% dye decolorization. Results are shown in Fig. 5.

 

3.4.2 Percent Decolourization of dye in Half (½) Strength nutrient medium:

The Nutrient broth having Half (½) strength was prepared and added with dye in 100ppm concentration. The tube was inoculated by selected isolate and kept for incubation. The results were observed after incubation period. The isolate showed 79.4% dye decolorization Results are shown in Fig. 5.

 

3.4.3 Percent Decolourization of dye in Cell Free Extract:

The activity of promising isolate for decolorization of dye Golden Yellow HER was further studied by using cell free experiment. The effect of extracellular and intracellular enzymes produced by Pseudomonas sp. GYH-1 was examined by using sterile Nutrient broth having dye concentration 100ppm. The isolate showed 88.27% dye decolorization. Results are shown in Fig. 5.

 

 

Fig. 5: Effect of different Culture media on decolorization of Golden Yellow HER by Pseudomonas sp. GYH-1.

 

Where, CFE = Cell Free Extract, NM = Nutrient Medium, HSNM = Half Strength Nutrient Medium.

 

3.5 Extraction, Analysis and Confirmation of biodegraded metabolites:

The metabolites produced after degradation of Red F2R were analyzed by FTIR analysis (Perkin Elmer 1000) in the mid range of 400-4000 cm-1 with 16 scan speed. The FTIR spectrum of standard (Original) dye was compared with spectrum of treated dye (Fig. 6(a), 6(b)). By this analysis it was observed that original complex dye was breakdown into simple metabolites. The confirmation of dye degradation was supervised by GC-MS analysis. By observing FT-IR and GC-MS reports it was concluded that the dye was completed degraded into small fragments. The results of GC-MS analysis and molecular weights of dye fragments are given in Fig. 7 and Table 2.

 

               

Fig. 6(a): FT-IR analysis spectrum of treated dye                                       Fig. 6(b): FT-IR analysis spectrum of Standard (original) dye

 

Fig. 7: Analysis report of Golden Yellow HER degraded products by GC-MS technique.

 

Table 2: Molecular weights of degraded dye products

Name of dye degraded products	Molecular weights of dye degraded products
2-diazenyl-3-methylcyclopentanol	128.17228
(1E,3Z)-penta-1,3-dien-1-amine	83.13166
2-amino-3-methylcyclopentanol	115.17352
2,5-dichloroaniline	162.0166
(3Z)-penta-1,3-diene	68.11702
3-methylcyclopentanol	100.15888
1,4-dichlorobenzene	147.00196
(2Z)-but-2-ene	56.10632
methylcyclopentane	84.15948
chlorobenzene	112.5569
2-methylbutane	72.14878
benzene	78.11184
butane	58.1222
(3Z)-penta-1,3-diene	68.11702

 

3.6 Prediction of metabolic pathway for dye degradation:

The pathway for Golden Yellow HER biodegradation was predicted by observing GC-MS report. The report suggested that the azo dye was degraded due to reductive cleavage by Pseudomonas sp. GYH-1. The probable pathway for Golden Yellow HER is shown in Fig. 8.

 

Figure 8: Probable Pathway for degradation of Golden Yellow HER by Pseudomonas sp. GYH-1.

 

The formation of 2-diazenyl-3-methylcyclopentanol and (1E,3Z)-penta-1,3-dien-1-amine intermediates indicates the asymmetric breakdown of azo bond by the enzyme Azoreductase. The formation of 2-amino-3-methylcyclopentanol indicates presence of Azoreductase enzyme which breakdown the Azo bond in the dye structure. The formation of key metabolites such as 3-methylcyclopentanol, 1,4-dichlorobenzene, benzene, butane and (3Z)-penta-1,3-diene indicates the action of different Oxidoreductase enzymes which asymmetrically break the structure of dye.

 

3.7 Toxicity testing of degraded products:

The toxicity of degraded products of the dye was carried out by phytotoxicity and microbial toxicity analysis.

 

3.7.1 Phytoxicity studies:

The toxicity of degraded products of dye on germination of seeds was tested on two types of seeds viz. Jawar (Sorghum bicolor) and Mung beans (Vigna radiata). The plant parameters such as root and shoot length, percent seed germination were measured of these two seeds. The results showed that the degraded products of dye were non-toxic as they significantly increase the root and shoot length and seed germination. The original dye was observed as toxic to plants as it does not support the seed germination. Hence it can be concluded that the isolate Pseudomonas sp. GYH-1 degraded Golden Yellow HER and resulted in detoxification of dye.

 

Type of seed	Sample	Root length (cm)	Shoot length (cm)	Percent seed germination
Jawar (Sorghum bicolor)	Distilled water (Control)	5.02	5.87	80
	Treated dye sample	4.17	4.80	70
	Golden Yellow HER 500ppm	2.2	1.90	30
Mung beans (Vigna radiata)	Distilled water (Control)	6.67	8.55	100
	Treated dye sample	6.6	3.20	80
	Golden Yellow HER 500ppm	3.44	8.34	40

 

3.7.2 Microbial toxicity:

The microbial toxicity of dye Golden Yellow HER and its degraded products formed after degradation was carried out by Agar Well Bioassay method on three ecologically important organisms viz. E. coli, Rhizobium sp. and Azotobacter sp. the wells were poured with original dye sample and treated dye sample. It was observed that the wells having original dye sample showed zone of inhibition while wells having treated dye sample showed no zone of inhibition. Hence it confirmed that the degraded products formed after degradation was non-toxic to bacteria while the original dye was toxic to bacteria.

 

4.    DISCUSSION:

The dye structure affects the decolorization process. A small variation in dye structure can affect the whole process of decolorization (Verma and Madamwar, 2003). The present research work deals with the isolation and screening of bacteria which are having highest efficiency of decolorization and degradation of toxic textile dye Golden Yellow HER from the acclimatized soil samples. All the isolates (Total 35) were screened and tested morphologically and biochemically to check the efficiency of isolates to decolorize the dye. Among all isolates, Pseudomonas sp. GYH-1 was showing highest or complete decolorization of Golden Yellow HER, which was further, identified by16s rRNA sequencing technique. Wong et al., (1998) reported Methyl red (MR) degradation by Acetobactor liquefaciens S-1 and Klebsiella pneumonie RS-13 in which maximum degradation of DMPD requires a temperature range of 30⁰C to 37⁰C and at 45⁰C decolorization was not observed. Walaa Salah EI- Din Mohmed, in 2016 was reported that the increase in pH from 3.0 to 7.0 increases the decolorization efficiency of isolate and it was maximum at pH 7.0. Above 7.0 to 9.0 the ability of decolorization was decreased. At temperature range 25⁰C to 35⁰C there was maximum decolorization and optimum temperature was 35⁰C. When temperature was increased from 35⁰C to 45⁰C there percent decolourization decreased. However the present study showed that the isolate Pseudomonas sp. GYH-1 showed maximum decolorization of Golden Yellow HER at pH 7.0 and temperature 37⁰C. This reports were strongly accordance with the reports revealed by Geetha et al., (2016) who suggested the decolorization of dye Alizarin red S by Escherichia coli and Pseudomonas sp. They found that when the medium was optimized with 1% Glucose, 1% Peptone, temperature 37⁰C, pH 7.0, dye concentration 500mg/l and combination of 50 immobilized bacterial cells, 1% Glucose, 1% Peptone in 100 ml of Minimal salt medium, these bacteria showed highest decolorization rate as 78.04% by Escherichia coli and 69.17% by Pseudomonas sp. The bacteria get carbon source by utilizing the dye. However the azo dyes lack in carbon source so it is necessary to add extra carbon source in the medium which act as co-metabolite (Shellina Khan and Nishi Mathur, 2015). Like wise Nitrogen source contain source of electron donor which helps in reduction of azo dyes (Adya Das et al., 2015). In this research work similar reports were obtained which suggested that when medium was provided with 1% carbon and nitrogen source the isolate Pseudomonas sp. GYH-1 showed highest decolorization of dye. Some researchers were successful in isolating dye decolorizing microorganisms but the increasing concentration of dye inhibits the growth of microbes. The isolate showed lower efficiency for dye decolorization in presence of high concentration of dye due to toxicity of dye Golden Yellow HER. It was previously suggested that the higher concentration of dye causes reduction of metabolic activity and inhibit of growth of organism (Junnarkar et al., 2006). The analysis of degraded products was carried out by GC-MS technique. The cleavage of azo dyes could be symmetrically or asymmetrically by an active site present on an enzyme (Pasti-Grigsby et al, 1992). The pathway for dye degradation was predicted by observing GC-MS report. Phytotoxicity reveals the toxic nature of dye. Lange et al., (1995) suggested that many sulfonated dyes after biological treatment accrue on the water surface is the major cause of water pollution. Moreover unsulfonated dye metabolites are longstanding in aquatic conditions and mildly degradable by biological water treatment (Pinheiro et al., 2004). Both of the sulfonated and unsulfonated metabolites formed after biological treatments are also major source of environmental pollution which can possibly remain after biological treatment (Tan et al., 2005). Hence it is mandatory to access phytotoxicity and microbial toxicity before and after dye degradation. The present work suggested that the degraded products of Golden Yellow HER were non toxic to Jawar (Sorghum bicolor) and Mung beans (Vigna radiata) as they support the germination of these seeds while the original dye was proved toxic to those seeds. Similar reports were revealed by Dhanve et al., 2009 in study of dye Reactive Yellow 84A and its degraded products. Like wise microbial toxicity studies revealed that metabolites of degraded products of Golden Yellow HER were nontoxic to certain organisms like E. coli, Rhizobium sp. and Azotobacter sp. similar results were observed by Shertate et al., (2016). These all studies suggest that the potent bacteria Pseudomonas sp. GYH-1 can be exploited for treatment of textile effluent.

 

5.    CONCLUSION:

The quality of environment is getting reduced due to various man-made activities by discharging inorganic and inorganic non degradable waste into natural eco-systems. Many textile, tanning and leather industries are using large amount of synthetic dyes. Most of those industries do not have accurate technology for treating and disposing these wastes into environment and hence dispose their waste by partial physical or chemical treatment or without treatment. In this case Microbiology plays an important role in not only decolorization but also degradation of toxic and harmful textile dyes from textile effluent. The microbes reduce the toxic dyes into non-toxic byproducts as well as minimize the harmful effects of these dyes. The present research work reveals that the isolate Pseudomonas sp. GYH-1 was having efficiency for bioremediation of harmful Golden Yellow HER dye. The byproducts formed after degradation was analyzed and it was found that those byproducts were non-toxic to plants and other useful microbes. The isolate was able to complete degradation of the dye at pH 7.0 and 37⁰C and in presence of Starch and Peptone which were very convenient and easy and cost effective conditions. Hence it can be concluded that the isolate Pseudomonas sp. GYH-1 could be effectively used for treatment of Golden Yellow HER dye. Still more work is needed to reveal the potential of microbes for treatment of textile effluent. From this study it can be demonstrated that native microbial flora is having highest efficiency to degrade and detoxify the harmful dyes which are originated from textile industries. Therefore these organisms could be exploited for bioremediation of textile dyes which converts the toxic dye into non-toxic products. This bioremedial approach could be applied to remove textile dyes and maintaining the sustainable clean environment and leading to Green Technology.

 

6.    ACKNOWLEDGEMENT:

I offer my great and sincere thanks to IIT, Powai, Mumbai for helping in analysis of samples by GC-MS, National Centre for Cell Science (NCCS), Pune for identification of isolates, My Guide, Head, Department of Microbiology, Dr. Babasaheb Ambedkar Marathwada University, sub-Campus, Osmanabad for providing laboratory facilities and valuable moral support.

 

7.    CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

8.    REFERENCES:

1.      Blanquez P, Casa N, Gabarell FX, Sarra M, Caminal G, Vincent T. Mechanism of textile metal dye biotrasformation by Trametes versicolor. Water. Res. 2004; 38: 2166-2172.

2.      Carliell CM, Barclay SJ, Naidoo N, Buckley CA, Mulholland DA, Senior E. Microbial decolorization of a reactive azo dye under anaerobic conditions. Water SA. 1995; 21(1): 61-69.

3.      Constapel M, Schellentrager M, Marzinkowski JM, Gab S2. Degradation of reactive dyes in wastewater from the textile industry by ozone: analysis of the products by accurate masses. Water Res. 2009; 43: 733-743.

4.      Crini G. Non-conventional low-cost adsorbents for dye removal: a review. Bioresour. Technol. 2006; 97: 1061-1085.

5.      Daeshwar N, Ayazloo M, Khataee AR, Pourhassan M. Biological decolorization of dye solution containing Malachite green by microalgae Cosmarium sp. Bioresour. Technol. 2007; 98: 1176-1182.

6.      Daneshvar N, Oladegaragoze A, Djafarzaddeh N. Decolorization of basic dye solution by electrocoagulation: an investigation of the effect of operational parameters. J. Hazard. Mater. 2006; 129: 116-122.

7.      Das A, Mishra S, Verma V. Enhanced biodecolorization of textile dye Remazol navy blue using an isolated bacterial strain Bacillus pumilus HKG 212 under improved culture conditions. J. Biochem. Tech. 6(3): 962-969,

8.      Dhanve RS, Kalyani DC, Phugare SS, Jadhav JP. Coordinate action of Exiguobacterial oxidoreductive enzymes in biodegradation of Reactive Yellow 84A dye. Biodegradation. 2009; 20: 245-255.

9.      Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985; 39: 783-791.

10.   Figueroa S, Vazqeza L, Alvarez-Gallegos A. Decolorizing textile wastewater with Fenton’s reagent electrogenerated with a solar photovoltaic cell. Water Res. 2009; 43: 283-294.

11.   Fu Y, Viraraghavan T. Fungal decolorization of dye wastewaters: a review. Bioresour. Technol. 2001; 79: 251-262.

12.   Geetha N, Illakkiam D, Subha D, Ahila V. Decolorization of Alizarin Red by Bacterial strains isolated from industrial Effluents. International Journal of Plant, Animal and Environmental Sciences. 2016; 6(1): 268-275.

13.   Jadhav JP, Govindwar SP. Biotransformation of Malachite green by Saccharomyces cerevisiae MTCC 463 Yeast. 2006; 23: 315-323.

14.   Joo DJ, Shin WS, Chori JH, Kim MC, Han MH, Ha TW, Kim YH. Decolorization of reactive dyes using inorganic coagulants and synthetic polymer. Dyes Pigments. 2007; 73: 59-64.

15.   Junnarkar N, Murthy DS, Bhatt NS, Madamwar D. Decolorization of diazo dye Direct Red 81 by a novel bacterial consortium. World Journal of Microbiology and Biotechnology. 2006; 22: 163-168.

16.   Kaushik P, Malik A. Fungal dye decolorization: recent advances and future potential. Environ. Int. 2009; 35: 127-141.

17.   Khan S, Mathur N. Biodegradation of Different Dye by Bacterial Strains Isolated from Textile Effluents of Western Rajasthan, India. Int. J. Curr. Microbiol. App. Sci. 2015; 4(2): 994-1001.

18.   Lange FT, Wenz M, Brauch HJ. Trace level determination of aromatic sulfonates in water by online ion-pair extraction/ ion-pair chromatography and their behavior in the aquatic environment. J. High Resolut. Chromotogr. 1995; 189: 243-252.

19.   Mane UV, Gurav PN, Deshmukh AM, Govindwar SP. Degradation of textile dye reactive Navy RX (Reactive blue-59) by an isolated Actinomycetes Streptomyces krainskii SUK-5. Malays. J. Microbiol. 2008; 4: 1-5.

20.   Meehan C, Banal IM, McMullan G, Nigam P, Smyth F, Marchant R. Decolorization of Ramazol Black-B using a thermotolerant Yeast, Kluyveromyces marxianus IMB3. Environ. Int. 2000; 26: 75-79.

21.   Parshetti GK, Kalme SD, Saratale GD, Govindwar SP. Biodegradation of Malachite green by Kocuria rosea MTCC 1532. Acta. Chim. Slov. 2006; 53: 492-498.

22.   Pasti-Grigsby MB, Paszczynski A, Goszczynski S, Crawford DL, Crawford RL. Influence of aromatic substitution patterns on azo dye degradation ability by Streptomyces spp. and Phanerochate chrysosporium. Appl. Environ. Microbiol. 1992; 58: 3605-3613.

23.   Pinheiro HM, Touraud E, Thomas O. Aromatic amines from azo dye reduction: Status review with emphasis on direct UV spectrophotometric detection in textile industry wastewaters. Dyes Pigments. 2004; 61: 121-139.

24.   Saitou N., Nei M., 1987. The Neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution. 4, 406-425.

25.   Senan RC, Abraham TE. Biodegradation of textile azo dyes by aerobic bacterial consortium. Biodegradation. 2004; 15: 275-280.

26.   Sharma S, Singh P, Swami R, Sharma K. Exploring fish bioassay of textile dye wastewaters and their selected constituents in terms of mortality and erythrocyte disorders. Bull. Environ. Contam. Toxicol. 2009; 83: 29-34.

27.   Shertate RAS, Thorat PR. Bioremedial Potential of Marine Bacterium. World Journal of Pharmaceutical Research. 2016; 5(5): 1130-1150.

28.   Solmaz SKA, Ustum GE, Birgul A, Tasdemir Y. Treatment and studies with chemical precipitation and ion exchange for an organized industrial district (OID) effluent in Bursa Turkey. Desalination. 2007; 217: 301-312.

29.   Sudhakar P, Palaniappan R, Gowtisankar R. Degradation of azo dye (Black-E) by an indigenous bacterium Pseudomonas sp. BSP-4. Asian J. Microbiol. Biotech. Environ. Sci. 2002; 4: 203-208.

30.   Tan NCG, Leeuwen A. van, Voorthuizen van, Slenders P, Prenafeta-Boldu FX, Temmink H, Lettinga G, Field JA. Fate and biodegradability of sulfonated aromatic amines. Biodegradation. 2005; 16: 527-537.

31.   Telke AA, Kalyani DC, Jadhav JP, Govindwar SP. Kinetics and mechanism of Reactive Red 141 degradation by a bacterial isolate Rhizobium radiobacter MTCC 8161. Acta. Chim. Slow. 2008; 55: 320-329.

32.   Verma P, Madamwar D. Decolorization of synthetic dyes by a newly isolated strain of Serratia marcescens. World Journal of Microbiology biotechnology. 2003; 19: 615-618.

33.   Walaa M. Isolation and Screening of reactive dye decolorizing bacterial Isolates from textile industry effluent. Intl. J. Microbiol. Res. 2016; 7(1): 01-08.

34.   Wong PK, Yuen PY. Decolorization and biodegradation of N, N-dimethyl-p-phenylenediamine by Klebsiella pneumoniae RS-13 and Acetobactor liquefaciens S-1. J. Appl. Microbiol. 1998; 85: 79-87.

35.   Yun M, Yeon K, Park J, Lee C, Chun J, Lim D. Characterization of biofilm structure and its effect on membrane permeability in MBR for dye wastewater treatment. Water Res. 2006; 40: 45-52.

 

 

 

 

Received on 19.09.2019       Modified on 10.10.2019 Accepted on 31.10.2019      ©A&V Publications All right reserved Research J. Science and Tech. 2019; 11(4):246-258. DOI: 10.5958/2349-2988.2019.00035.4