International Journal of Biology and Nanobiomaterials1 (2021) 97-106

Original Article

Isolation and characterization of dyeeffluent discoloration bacteria from industrial wastewater

Karpagam, S *
Department of Biotechnology, Sri Ganesh College of Arts and Science, Salem, Tamil Nadu, India

ARTICLE INFO

Article history:

Received 28 May 2021
Revised 15 July 2021
Accepted 19 July 2021
Available online 23 July 2021

Keywords:

Bacterial consortiumDecolorization
Textile effluents
Vigna radiate
Hazardous

ABSTRACT

The worldwide rapidly developing in industrialization and urbanization have been leads to discharge of effluents and wastewater that causing of environmental pollution and hazardous to the aquatic animals. In moreover, major industrial effluents of different dye concentration hugely affected to agriculture soil and ground water. Bioremediation is the major key to the treatment of wastewater and ecofriendly achievement. In this method effectively removal of dye effluents that are involved heterologous biological compounds. In the present study focused on the industrial effluents decolorization by used potential bacterial consortiumof two bacterial strains such as Bacillussp and Pseudomonas sp. The bacterial strains were isolated from industrial wastewater. Suitable decolorization was observed at 37°   C for 15 days incubation to be conformed UV–Vis analysis. In furthermore, the decolorized end product was performed phytotoxicity assessment using of Vignaradiata seeds which evaluated toxicity of decolorized end product of wastewater.

  1. Introduction

Raising globalization, urbanization and rapid development of industrialization have been causes different kinds of environmental pollutions.Among various industries, the textile dying industries discharge large volume of waste water after dyeing process (Zollinger, 1987). It is estimated that around 10 -15% of the dyes are lost in the effluent during the dyeing processes (Kadiret al., 2009).  Dyes are classified to the basis of chemical structure of chromophore there are 20 -30 different groups of dyes like azo (monoazo, diazo, triazo, polyazo),anthraquinone, phthalocyanine and triarylmethane dyes are the most important groups (Chen et al., 2011). The majority of industrial important azo dyes belong to the following classes: acid dyes, basic dyes, direct dyes, Disperse dyes, mordant dyes, reactive dyes and solvent dyes (Wanyonyi et al., 2017).Dyes make up an abundant class of organic compounds characterized by the presence of unsaturated groups (chromophores) such as –C=C-, ─N═N─ and ─CH═, which are responsible for colors (Molinariet al., 2004).

Around 10,000 different dyes with an annual production of more than 7 ×105 metric tons worldwide are commercially available and are extensively used in textile processing, paper, printing, pharmaceutical, food and other industries (McMullan et al., 2001; Siddeeg et al., 2020). The textile effluents containing of various toxic substances namely suspended detergents, surfactants, carcinogenic amines, formaldehyde and organochloride pesticides associated with dyes (Jadhavet al.,2010). During the fabrication process approximately 10% of the dyes have not fixed with fibers, it released into the environment (Asadet al.,2007).The release of untreated effluents from the dyeing industries causes a major threat to the environment and reduces sun light penetration of the stream. These effluents are toxic to fish and mammalian life and to inhibit the activity and growth of microorganisms. It alters the pH, increases the biological oxygen demand (BOD) and chemical oxygen demand (COD) and gives the rivers intense colorations. It also affects the soil fertility and plant growth (Afrad et al., 2020). In humans the azo dyes are capable of producing intestinal cancers, cerebral and skeletal abnormalities in foetuses(Saha and Rao, 2020). Moreover, disposal of textile waste water on an open land contaminates the subsoil water, so that drinking water gives color as well as bad taste. Dissolved substances in industrial effluents alter the chemical and biological status of the soil and water, which may affect growth and productivity of plants and alters the color and quality of the water bodies has been proved to be hazardous to aquatic organisms (Lade et al., 2015). Toxic compounds from dye effluent get into aquatic organisms and ultimately reach man through food chain to cause various physiological disorders like hypertension, skin cancer, sporadic fever, renal damage and cramps etc. They possess toxicity like lethal effect, genotoxicity, mutagenicity, and carcinogenicity to plants and (Puvaneswariet al.,2006).

There are several physical and chemical treatment methods are used for dyes like adsorption, sedimentation, floatation, coagulation, polymerization, flocculation, reverse osmosis, ultra-filtration, ionization, radiation, reduction, oxidation, electrolysis and ion exchange are extensively used. These methods are not environmentally friendly and produces enormous amount of secondary pollutants and are cost-effective (Roy et al., 2018).Biological treatment is often the most economical alternative compared to physical and chemical processes. Biological methods like stabilization, aerated lagoons, trickling filter, activated sludge, anaerobic digestion and bio-augmentation etc. The biological methods have lower cost but it having large land spaceandlesseffective(Sheam et al., 2020).The removal of textile carcinogenic pollutants is the major task of the bioremediation. The ability of microorganisms to decolorize and metabolize dyes has long been known and the use of bioremediation based technologies for treating textile waste water has attracted interest (McMullanet al., 2001). Several microorganisms have been found to decolorize and mineralize azo dyes. Many microorganisms belonging to different taxonomic group of bacteria, fungi, actinomycetes and algae have been reported for their ability to decolourizeazo dyes (parmar, 2014; Hassan etal.,2013;Zabinet al., 2011,). Biodegradation systems of color removal through the use of bacteria have been shown to be highly effective.

The bacterial metabolism of azo dyes is initiated by a reductive cleavage of the azo bond in most cases, which results in the formation of amines. Azoreductase enzymes responsible for decolourization of azo dyes are purified from several bacterial strains. These reductive processes have been studied in some aerobic bacteria, which grow on azo compounds. Bioreactor is a engineered device, usually a vessel, used to direct the activity of a biological catalyst to achieve a desired chemical transformation. Basic principles of process control, fermentation monitoring, dissolved oxygen, PH,temperature, monitoring, subtract (Glucose).  In current research aimed to focus on isolation and characterization of effluent decoloring bacteria from the textile effluent wastewater. Two bacterial strains were screened for potential degradation of effluents industrial dye, after that 15 days incubation. The decolorized by-product was performed phytotoxic study that revealed toxicity assessment of the end by-product.

  1. Materials and methods

2.1. Sample collection

Textile dye effluent waste water was collected from in Edapadi, Salem district, Tamil Nadu, India. Effluent was collected using sterile container and aseptically transported to the laboratory and stored at 4ºC for further use.

2.2. Isolation of dye degrading bacteria

Collected effluent was mixed with sterile 100mL Bushnell Hass broth and incubated at 37ºC for 7 days. 1 mL of the above broth sample was serially diluted and spread on nutrient agar plates for the isolation of dye degrading bacterial strains and incubated at 37ºC for 24 h. Afterthatincubation period, fifteen bacterial strains were isolated and stored at 4ºC. The selected bacterial strains were noted as KADB01 to KADB15.

2.3. Identification of bacteria strain

The isolated bacterial strains were identified by following staining and biochemical methods of Gram staining, spore staining, motility, oxidase, indole, methyl red voges-prosjauer, citrate utilization, urease and carbohydrate fermentation test. The results further were compared with manual of systematic bacteriology.

2.4. Screening of dye degrading bacterial strain

The isolated bacterial strains were screening for the selection of efficient dye degradation activity. In briefly described, LB agar medium was prepared and directly added into the different concentration of azo dye solution under the ambient temperature and properly mixed. After that process immediately poured into the petri plates and allowed to solidification. Then respective isolated bacterial strains were streaked on the plates and incubated at 37ºC for 24 h. The efficiency of bacterial dye degradations were observed based on the zone of inhibition (Kabeer et al., 2019).

2.5. Bioreactor study

            Bioreactor is an engineered device, usually a vessel, used to direct the activity of a biological catalyst to achieve a desired chemical transformation. Bioreactor of 4L capacity was designed for lab scale study for the treatment of dye contaminated water. The bioreactor was filled with 4L of the dye contaminate water to which 1% of bacterial consortium was added. Temperature was maintained at room temperature and continuously aerated with constant agitation using stirrer at 250 rpm. The samples were collected fromvarious parameters such as pH, CFU and Optical density was measured by UV spectrophotometer. The percentage of dye degradation was calculated by following formula.

% degradation = Initial absorbance value-final absorbance value/Initial absorbance valuex100

2.6. Phytotoxicity assay

The soil was taken in a 250ml of sterile plastic cups and 4 Vignaradiata seeds were placed into each cup at 1.5 cm depth. All the cups were irrigated regularly with 5 ml of untreated as negative control and bioreactor treated dye effluent. The seeds were allowed to germinate and seed germination was assessed on every day of the experiment. At the end of 10th day shoot length and size of the leave was measured. Control set was carried out using distilled water to replace the textile dye effluent at same time.

  1. Results and discussion

Industrial dye contaminated water sample was collectedand physicochemical parameters analyzed such as color, oder, PH,

Table 1 Physicochemical parameters of industrial dye

S.No Parameters Dye contaminated water sample
1 Color Red
2 Oder Dyeing
3 PH 8
4 Temperature 36
5 OD Value 0.052

Table 2 Biochemical characterization of isolated bacterial strains

S.No Tests Gram’s Staining Spore Motility Test Indole Test Methyl red Test VP Test Citrate Test Urease Test Catalase Test Oxidase Test Sugar fermentation Tentative Genus
1 KDDB01 + + + + Bacillus sp
2 KDDB02 + + + + + Micrococcus sp
3 KDDB03 + + + + + + Bacillussp
4 KDDB04 + + + + Pseudomonas sp
5 KDDB05 + + + + + Bacillus sp
6 KDDB06 + + + + + + + + Bacillussp
7 KDDB07 + + + + + + Staphylococcus sp
8 KDDB08 + + + + + Staphylococcus sp
9 KDDB09 + + + + + + Bacillus sp
10 KDDB010 + + + + + + Pseudomonas sp
11 KDDB011 + + + Micrococcussp
12 KDDB012 + + Enterococcus sp
13 KDDB013 + + + + Klebsiellasp
14 KDDB014 + + + + Micrococcus sp
15 KDDB015 + + + + + + Bacillus sp

temperature, OD value were observed and shown in Table 1. The dye degrading bacterial strains were isolated from dye effluent sample by using spread plate technique. The totally fifteen bacterial strains were isolated and biochemical characterized, the results were revealed in the Table 2. Similar report revealed,

textilewastewater showed abnormal coloration of black turquoise blue colored. The pH determined the collected test sample was slightly acidic to neutral (Bhatia et al., 2018).

3.1. Screening of dye degradation bacteria

Luria Bertani Agar medium incorporated with azo dye and the bacterial isolates were inoculated. The maximum zone of clearance of the isolatedstrains were taken as an indicator of the dye degradation bacteria its shows in Table 3, the colony size and  zone of clearing size were measured in millimeters. Several researchers have been reported, similar results as related to zone of clearing on the agar plate, its indication of the dye degradation ability of the selected bacteria (Shazia et al., 2017).

Table 3 Zone of clearance dye degradation bacteria

S.No Strain No Zone of clearance (mm)
1 KDDB01 0
2 KDDB02 16
3 KDDB03 12
4 KDDB04 25
5 KDDB05 27
6 KDDB06 0
7 KDDB07 12
8 KDDB08 23
9 KDDB09 13
10 KDDB010 0
11 KDDB011 21
12 KDDB012 11
13 KDDB013 0
14 KDDB014 12
15 KDDB015 0

3.2. Construction of consortium
The Bacillussp and Pseudomonassp were used for the construction of consortium due to the synergistic effects with each other.

3.3. Dye decolourization
The dye decolourization by the bacterial consortium was detected by the following optical density value of the bioreactor

Table 4 Dye effluent decolourization measurement by UV spectrophotometer

UV Spectroscopic OD values of the treated sample
Days Treated sample Untreated sample
1 0.052 0.052
2 0.050 0.052
3 0.048 0.052
4 0.047 0.052
5 0.046 0.052
6 0.045 0.052
7 0.044 0.052
8 0.040 0.052
9 0.039 0.052
10 0.038 0.052
11 0.037 0.052
12 0.035 0.052
13 0.034 0.052
14 0.033 0.052
15 0.032 0.052

treated dye effluent shows in Table4.Our bacterial consortium degraded 38% percentages of dye within fifteen days of treatment. Similar research was conducted by Lalnunhlimi et al.,(2016) for the decolorization of mixture dyes using of  Bacillus cereus has showed 93.37% of highest decolorization within 5 days. Another related study was reported byKarim et al. (2018), the decolorization experiment using of five frequently employed textile azo dyesby different bacterial isolates strains of EK13 (41%), EK7 (41%) and EK6 (42%) for incubation after 6 days in Bushnell-Haas (BH) broth culture medium (Lalnunhlimi et al., 2016).

3.4. Phytotoxicity

The microbial treated dye effluents was applied to study the phytotoxicity of green gram (Vigna radiate) plant such as germination capability, shoot length and root length. The result was compared with control tape water it’s shown in Table 5.In comparison of similar research reported by Nouren et al., 2017, using of yellow 4 dye by-product for phytotoxicity experiment. Zea mays seeds treated with degraded by-product of test samples and untreated DY4 dye samples. The germination percentage was calculated based on the root and shoot lengths of germinated seeds were compared with control samples. The result suggested 50% of negatively affected then compared to treated samples, which is confirmed exhibit degradation revealed in the test samples (Karim et al., 2018).

Table 5 Phytotoxicity assessment of treated dye effluent

Shoot length measurement (cm)
Sl.No. Days Treated water Tape water Untreated water
1 1 0 0 0
2 2 0 0 0
3 3 0.5cm 0.5cm 0
4 4 0.7cm 0.9cm 0
5 5 1.0cm 1.2cm 0
6 6 1.5cm 1.8cm 0
7 7 2.0cm 2.5cm 0
8 8 2.3cm 3.0cm 0
9 9 2.6cm 3.4cm 0.5cm
10 10 3.0cm 3.7cm 0.7cm
11 11 3.4cm 4.1cm 0.8cm
12 12 3.8cm 4.6cm 0.9cm
13 13 4.2cm 5.0cm 1.0cm
14 14 4.6cm 5.5cm 1.5cm
15 15 5.0cm 5.9cm 1.7cm
  1. Conclusions

The textile industries are produced large amount of wastewater effluents which hazardous to ecosystem, because of their higher concentration of dyes mixtures. Although bacterial decolorization or degradation of textile effluent is a challenging process, but nowadays many researchers have been reported microbial mediated degradation of textile effluent. In the current research study, consortium of two bacterial strains such as Bacillussp and Pseudomonassp were potential effluent decolorization was screened through the plate assay method. The consortium of two bacterial strains was observed maximum growth at 37°C and pH 7.0. The construction of consortium with two bacterial isolates is able to decolor of industrial effluent dye up to 38% for 15 days incubation. The seeds germination was revealed less toxic effect in treated samples and also enhancing of plant growth. These results suggested the potential applicable of these two bacterial strains for the treatment of wastewater in the future.

        Funding: The authors received no specific funding for this work.

        Conflicts of Interest: None

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