Research Journal of Chemistry and Environment____________________________________________Vol. 22 (5) May (2018) Res. J. Chem. Environ.
Detoxification of Dyes by Aspergillus niger isolated from Dye Contaminated Soil Effluent from the sites of Textile Industry Jambulingam Ranjitha1, Palani Shalini2, Mohanam Anand3 and Srinivasan Gokul Raghavendra1* 1. Vellore Institute of Technology, Vellore, INDIA 2. St. Peter’s University, Chennai, INDIA 3. Kingston Engineering College, Vellore, INDIA *
[email protected]
combinations of biological, physical and chemical decolourization methods.5 Approximately, 10-15% of unused dyes enter into the wastewater after dyeing and after the subsequent washing processes.8 Both the chemical and physical methods for treatment of dye wastewater are not widely applied to textile industries because of their cost and disposal problems. Hence there is always a need for new green technologies to deal with this problem including adsorption of dyestuffs on bacterial and fungal biomass (or) low-cost non-conventional adsorbents.6
Abstract The present study deals with the detoxification of dye sample collected from dye industry located in Tirupur district, Tamilnadu, India. Three types of dyes like malachite green, nigrosin and basic fuchsin were biodegraded using a fungal strain isolated from dye effluent soil. The selected fungal strain Aspergillus niger has shown maximum decolourization of the dye nigrosin (93.33%), basic fuchsin (92.85%) followed by malachite green (90.05%) and dye mixture (32.33%) under tube lay method.
Lately, dye removal research area is becoming very interesting area, as Government legislation concerning the release of contaminated effluent becomes more rigorous. Several treatment methods are available for the removal of dyes from industrial effluents like chemical coagulation using alum, lime, ferric chloride, ferric sulphate and electro coagulation. In past decades, research interest in the fungal bioremediation due to their biomass compared to the bacteria, has increased significantly for decolourization and degradation of synthetic dyes.7 Several scientific reports were available based on the biodegradation of dyes collected from industrial dye effluents using microbes.8-10 Keeping the above mentioned points in view, the main objective of the problem was to screen and use the selected potential textile dye effluent soil fungal strain which is capable to decolorize and detoxify the textile dyes using solid and liquid media under shaking and stationary conditions.
Aspergillus niger exhibited better results under shaking conditions than compared to the stationary method; in addition, the inoculation of fungi also brought the pH of the dye solutions to neutral from basic. As a result, Aspergillus niger was found to be a potential fungi in biodegradation of textile dyes of different chemical structures and for environmental decontamination. Keywords: Detoxification, malachite green, nigrosin, basic fuchsin, Aspergillus niger, textile industry.
Introduction Rapid industrialization and urbanization had introduced a lot of chemicals including dyes into the environment polluting the entire ecosystem. Currently, more than 100,000 commercial dyes were manufactured including numerous varieties of dyes such as Acidic, Basic, Reactive, Azo and Diazo. Anthraquinone based metal complex dyes with an annual production of over 7 x 105 metric tons are commercially available.1 Among them, 50% of the dyes were released in the industrial effluents.2 These were used on several substrates in food, cosmetics, paper, plastic and textile industries. These dyes are dangerous to living organisms due to their potential toxicity and carcinogenicity.3
Material and Methods The selection of dye sample for present study started on 19th 2016, it was made on the basis of its solubility in water. The selected dyes (malachite green, nigrosin, basic fuchsin) were fairly soluble in water and hence used to explore the potential of fungal isolates towards decolourization of textile dyes. The decolourisation of dye sample was identified using Schimadzu UV-Visible spectrophotometer. The media used for the present study are: Potato Dextrose Agar (PDA) and Potato Dextrose broth (PDB).
In the ancient period, natural pigments are used for colouring textiles, which do not fade on exposure to light, heat and water. About 15% of the dyes used for textile dying are released into processing waters.4 Frequently applied treatment methods for colour removal from coloured effluents consist of integrated processes involving various * Author for Correspondence
Source of Fungal Strain: The dye effluent soil-isolated (from Tirupur district, Tamilnadu, India) fungal strain Aspergillus niger was maintained on Potato Dextrose Agar (Himedia, Mumbai, India) and sub-cultured periodically to maintain their feasibility. The isolated fungal strain from soil was identified as Aspergillus niger based on their morphological analysis.
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Research Journal of Chemistry and Environment____________________________________________Vol. 22 (5) May (2018) Res. J. Chem. Environ. Determination of Physiochemical Properties of Soil: The soil sample was collected from site of textile industry located in Tirupur district, Tamilnadu, India. The collected soil sample was used for the determination of physico-chemical properties. The moisture content of the sample was measured in a hot air incubator at 105°C to constant weight. The pH, temperature, humidity and air pressure were determined using digital pH meter, thermometer, hygrometer and barometer etc. using standard protocol.
0.01% (w/v) individual textile dye. All culture tubes were incubated at RT (~25°C) and observed up to four weeks. Clearing of the overlaid dye indicates full decolourization (+++). Partial dye decolourization (++) was indicated by less dye intensity in comparison with the control (uninoculated PDA overlaid with PDA + 0.01% dye).
Isolation of Microorganism: The microorganisms were isolated by serial dilution technique on Potato Dextrose Agar (PDA) and Nutrient Agar Media (NAM). In this technique, a sample suspension was prepared by adding 1.0 g sample to 10 ml distilled water and mixed well for about 15 minutes. Each suspension was serially diluted 10-1 to 10-6. About 0.1 ml was pipetted into plates with PDA and NAM media, spread with an L-rod and incubated at 28°C for fungal observation. Each colony that appeared on the plate was considered as one Colony Forming Unit (CFU).
Decolourization of Dyes in Solid Medium (Tube Overlay Method): The selected fungal strain was further tested for their ability to decolorize on PDA and Sabouraud Dextrose Agar (SDA) medium. This was done to select which medium supports better growth and dye decolourization activities of selected fungal isolate. Initially, fungal strain was grown as previously described. Following incubation, fungal mycelial agar plugs (~5mm2) were cut approximately 5mm from the colony margin and inoculated on test tubes each pre-filled with 2 mL of the Potato Dextrose Agar (PDA) and SDA medium, supplemented separately with following dye 0.01% (w/v) malachite green, nigrosin and basic fuchsin respectively.
Identification of Fungi: The fungal isolate was identified by morphological examination and its characteristics. Morphological characteristics were examined under microscope.11 Aspergillus niger was one of the successful fungal that was isolated from the dye effluent soil. Figure 1 shows the colony of Aspergillus niger isolated from dye effluent soil.
The culture tubes were then incubated at room temperature (~ 25°C). The growth of the fungi and its ability to decolorize the dye was observed up to four weeks. The depth of dye decolourization (in mm) indicated by clearing of the dye was then measured. Based upon growth of fungal strain and dye decolourization, PDA medium was chosen for further studies. Assay for the Dye Decolourization Activities of Fungi in Liquid Media: The spores and mycelia of Aspergillus niger were then extracted from Petri plates using a flame-sterilized inoculating loop and mixed properly with 1 mL of sterile distilled water. To this mixture, 10 μL of the fungal spore and mycelium inoculum were added on culture vials prefilled with 25 mL Potato Dextrose Broth (PDB) supplemented with 0.01% of either one of the following dyes: malachite green, nigrosin and basic fuchsin. Three sets were prepared and were incubated either under constant agitation/shaking (or) under stationary/without shaking condition. All culture vials were incubated at room temperature (~25°C) for about 10 days and all assays were performed in triplicate. The growth and dye decolourization were observed every day. After subsequent culture for 10 days, the culture filtrates were decanted and subjected to UVVisible spectrophotometric analysis.
Figure 1: Isolated Aspergillus niger from dye effluent soil
The absorbance maxima of the tested dyes were read as malachite green-620 nm, Nigrosin-600 nm and Basic fuchsin-550 nm wavelength. The extent of dye decolourization by the soil fungal strain on liquid media was calculated using the formula below:
Screening of Soil-Derived Fungi for Dye Decolourization Activities: The dye effluent soil fungal strain was screened for their ability to degrade dyes using the tube overlay method. Initially, the fungal strain was grown on culture plates pre-filled with Potato Dextrose Agar (PDA) and incubated at room temperature for about 14 days. Further, mycelial agar plugs (~5mm2) were cut approximately 5mm from the colony margin and inoculated on test tubes containing 5 mL of PDA overlaid with 1 mL of PDA with
PDD (%) =
2
AbsorbanceC – Absorbancel AbsorbanceC
*100
Research Journal of Chemistry and Environment____________________________________________Vol. 22 (5) May (2018) Res. J. Chem. Environ. where PDD = Per cent Dye Decolourization, AbsorbanceC = Absorbance control and AbsorbanceI = Absorbance inoculated.
PPM respectively. The ability of the soil to retain water known as Water Holding Capacity (WHC %) was in a range of 41.54%. The concentration of heavy metal was found to 4.78mg/g, 6.14mg/g and 5.87mg/g for Fe, Mn and Zn respectively. The soil quality parameters of the sample soil are tabulated in table 2.
Results and Discussion The sample soil site was selected based upon the location where the effluent discharge from dye industries was high. The contamination level was assessed using the water quality parameters which would directly indicate the contamination in soil. The water quality parameters of the dye effluent water sample were analysed based on the BIS standards and are tabulated in table 1. The waste water appeared blackish blue in colour and had a pH of 9, slightly basic in nature. The total hardness of the sample exceeded the standard limit by nearly 50% which leads to scale deposition. The BOD and COD of the effluent sample were 4 folds and 8 folds greater than the permissible limit respectively, thereby making it unfit for any consumption purposes.
The effectiveness of Aspergillus niger fungi was studied by biodegrading and decolouring the lab prepared dye samples: Malachite Green, Nigrocin, Basic Fuchsin. Under tube overlay method, in presence of medium, the highest decolourization rate was noticed for nigrocin with a decolourization rate of 93.33% followed by malachite green and basic fuchsin (92.85 and 90.05%) respectively. The decolourization rate for various dyes in presence of medium is represented in figure 2. Table 2 Soil Quality Parameters of the sample soil
Total amount of dissolved solids found in water was found to be 2,43,562mg/L whereas suspended solids were found to be 7,596mg/L. the turbidity of the waste water was found to be 90 NTU which is more compared to permissible limit. The chloride content was in range of 43650mg/L because of the bleaching steps involved in pre-dying and post dying process. Based upon the water quality study, it was concluded that the waste water possessed a high risk to environment and its surrounding. The soil sample was identified based upon this quality index and was evaluated by analysing the physico-chemical properties of soil.
Parameters
Soil Sample
pH
8.46
Electrical conductivity
0.3-0.31
Nitrogen
4.32 ppm
Phosphate
65.4 ppm
Potassium
496 ppm
WHC%
41.54 %
Iron
4.78 mg/g
Manganese
6.14 mg/g
Zinc
5.87 mg/g
Table 1 Water Quality Parameters of the sample dye effluent water Parameters Colour pH Total hardness (mg/L) BOD (mg/L) COD (mg/L) TDS (mg/L) TSS (mg/L) Turbidity (NTU) Chlorides (mg/L) Oil and greases (mg/L)
Dye Effluent Water Sample Brownish black 8.66 1020
BIS Standards colourless 5.5-9 500
1120 4060 243562 7596 90 43650 12
100 250 2100 100 10 600 10
The decolourisation rate for the combination of all these dyes in any proportion was found to be 32.33% only which is because of cumulative effect of the dye together. However, during bioremediation of the dyes, under shaking conditions, highest decolourization was observed in basic fuchsin (80.60%), followed by nigrosin (75.68%), malachite green (73.45%) and least by dye mixture (25.86%) by Aspergillus niger. Although in static conditions, highest decolourization was observed in basic fuchsin (80.15%), followed by nigrosin (57.47%) and malachite green (52.34%), least by dye mixture (30.66%) as shown in the figure 3. The maximum time taken for an effective decolourization of any dyes used was 7 days. Figure 4 shows the colour of dye water before and after fungal treatment.
The pH of the soil was slightly elevated from the normal range thereby making it basic. The electrical conductivity of the soil varied between 0.3-0.31 ds/m. The amount of nitrogen fixed in soil were found to be 4.32PPM where phosphate and potassium was found to be 65.4 PPM and 496
Conclusion It was concluded that decolourization of dyes was studied under two different conditions-stationary and shaking. The maximum decolourizations of all the dyes were obtained after 7 days. In this study we have observed higher
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Research Journal of Chemistry and Environment____________________________________________Vol. 22 (5) May (2018) Res. J. Chem. Environ. decolourization under shaking conditions by Aspergillus niger which was due to the oxygenation of the fungi. It was
probable that fungal remediation will be soon a reliable and competitive dye remediation technology.
Decolourization %
Decolourization of Dyes by A.niger in medium 100 80 60 40 20 0
93.33
92.85
90.05
32.33
Malachite Green
Nigrocin
Basic Fuchsin
Mixture
Dyes
Figure 2: Dye decolourisation by soil fungi in tube overlay method
Bioremediation %
Bioremediation Rate for various Dyes 100 80 60 40
Shaking
20
Static
0 Malachite Green
Nigrocin
Basic Fuchsin
Mixture
Dyes
Figure 3: Percept bioremediation of textile dyes by A. niger
Figure 4: Dye Water (a) Before treatment (b) After Treatment
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Research Journal of Chemistry and Environment____________________________________________Vol. 22 (5) May (2018) Res. J. Chem. Environ. 7. Dhanjal N.I.K., Mittu B., Chauhan A. and Gupta S., Biodegradation of textile dyes using fungal isolates, J Env Sci Technol, 6(2), 99-105 (2013)
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(Received 25th September 2017, accepted 21st March 2018)
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