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Efficacy of Bacterial Strains Isolated from Textile Wastewater for Degradation of Azo Dye Associated Aromatic Amines.

Byline: Farzana Kausar, Azeem Khalid, Tariq Mahmood and Nosheen Ilyas


Wastewater from textile processing and dyestuff manufacturing industries contains different kinds of dyes and their metabolites, which are mutagenic and carcinogenic in nature. Such wastewater should be treated in order to avoid ground and surface water contamination. The aim of present research was to isolate such bacterial strains which are capable of degrading dye-originated aromatic amines. Ten samples of wastewater and sludge were collected from outlets and wastewater streams of different textile industries. The analysis of wastewater showed a wide variation in the pH (8.10-12.3), total dissolved solids (500-20100 mg L-1) and chemical oxygen demand (125-556 mg L-1).

A total of 256 bacterial isolates were collected through enrichment of the mineral salt medium with an aromatic amine, 4-nitroaniline (100 umol L-1), and dyes (100 mg L-1) using 10 mL wastewater/sludge as inoculum source. Based on ability to degrade 4-nitroaniline most efficient bacterial strain was identified as Raoultella planticola (IL11) through 16S rRNA gene analysis. These findings suggest that the indigenous bacterial strains have potential for bioremediation of dye-containing textile effluents, and for complete mineralization, 4- nitroaniline degrading strains may be used in combination with dye degrading strains.

Keywords: Azo dyes; Aromatic amines; Wastewater; Biodegradation; Bacteria


Dyes are used in textile sector on large scale among which azo dyes constitute more than 50% of total dye production worldwide (Meng et al., 2012). Inefficient textile dyeing process results in the release of large amount of dyes in effluent which is directly discharged into the water bodies (Khalid et al., 2011; Mahmood et al., 2011; Kurade et al., 2016; Rawat et al., 2016). About 10-15% of dyestuff is discharged into environment during manufacturing and usage of these dyes (Verma and Madamwar, 2002; Andleeb et al., 2010). Textile finishing process requires an average amount of 100,000-150,000 L of water, as a result of which large amount of dyes residues are released in to the environment (Bezerra, 2005). Even very small quantity of the dye (<1 mg L-1 for some dyes) is clearly visible in water and does not only affect aesthetic quality but also affect oxygen solubility in water bodies.

The wastewater produced by the textile industry contains dyes and their residues, which cannot be treated by conventional treatment methods (Kangwansupamonkon et al., 2010; Aravind et al., 2016). Such wastewaters are known as carcinogenic and mutagenic and have toxic effects on aquatic organisms including fish, algae and other aquatic fauna (Annuar et al., 2009). Also the dye wastewater has variable range of BOD, COD, pH, color and salts (Peyton et al., 2002; Seesuriyachan et al., 2007). Untreated dye wastewater, which is used for different purposes such as agriculture has serious harmful impacts on human health (Pourbabaee et al., 2006). Carcinogenicity, mutagenicity and toxicity of dyes and their metabolites has increased the health concerns over the period of time. Dye degradation in most of cases take place under anaerobic conditions.

These anaerobically decolorized dye effluents are still harmful as it contains colorless aromatic amines and many of these metabolites are carcinogenic and mutagenic in nature. It has been observed that azo dyes can reduce into aromatic amines in the digestive tract of mammals (Chung et al., 1992). Study conducted by Xu et al. (2007) confirmed that biological reduction of Para Red and Sudan dyes by human intestinal microbes leads to production of various aromatic amines such as aniline, 2,4-dimethylaniline, o-toluidine and p-nitroaniline. These amines are easily absorbed by the human intestine and cause toxicity (Bomhard and Herbold, 2005; Wu et al., 2005). It is most likely that the textile wastewater could be highly toxic to the environment especially when groundwater is getting contaminated and further used for drinking purposes.

In developing countries such as Pakistan, the dye-contaminated water in urban and suburban areas are also used for growing vegetables and other crops, which represent a high health risk for people living in the vicinity of textile industry.

In view of the above, it is imperative to develop a strategy not only for the degradation of dyes but also their intermediate colorless products that are highly toxic to the environment. For this purpose, several strains of bacteria were isolated form textile wastewater and sludge and screened for efficient degradation of aromatic amine, 4- nitroaniline. The selected strains could be used for developing strategy for treating textile wastewater before being its discharge into the environment.

Materials and Methods

Chemicals and Culture Medium

The 4-nitroaniline was used for the isolation of bacteria capable of degrading dye originated aromatic compounds. Similarly three types of structurally different four azo dyes Reactive Black 5, Reactive Blue BRS, Direct Blue and Disperse Yellow were used for enrichment of the medium to isolate bacteria. Mineral salts medium (MSM) containing (g L-1) NaCl (1.0), CaCl2. 2H2O (0.1), MgSO4. 7H2O (0.5), KH2PO4 (1.0), Na2HPO4 (1.0) and yeast extract (4.0) was used for isolation of bacterial strains through enrichment technique (Khalid et al., 2008). The pH of the medium was adjusted to 7.2. All chemicals used in the experiments were of AR grade.

Collection of Wastewater/Sludge Samples

Ten samples of wastewater and/or sludge were obtained from wastewater streams of the textile industry in three districts (Rawalpindi, Faisalabad and Sheikhupura) in the province of Punjab, Pakistan. Textile processing units are widespread in the district Faisalabad (situated at latitude 31.43deg N and longitude 72.07deg E) and Sheikhupura (31.72deg N 72.98deg E), while a large processing unit is located in the district of Rawalpindi (36.60deg N 73.03deg E). Five wastewater samples were obtained from Faisalabad textile factories. Three samples were collected from the waste streams of textile units in Sheikhupura. Two samples were obtained from a textile factory in Rawalpindi. All samples were collected in sterile 50 mL Falcon tubes and stored at 4C prior to use.

Analysis of Wastewater

The wastewater and sludge samples were analyzed for color intensity, pH, total dissolved solids (TDS) and chemical oxygen demand (COD). Dye levels in colored wastewater were determined by spectrophotometer (Modified MA 02052-USA) at 600 nm. Since different types of dyes are used by the textile industry therefore, so pH and TDS of each sample was measured to find the acidic or basic nature of the dye-contaminated wastewater and overall presence of total soluble salts. The pH and TDS were analyzed by Multimeter (Crison MM-40+). COD of wastewater was measured by using the open reflux method (APHA, AWWA, WEF, 1999).

HPLC Analysis

Residues of 4-nitroaniline were analyzed using HPLC (Shimadzu CLASS-VP V6.13 SP1). HPLC analysis was performed using a C18 reverse phase column. The mobile phase was operated at rate of 1 mL min-1 with 40:60 ratio of methanol to water (v/v). The injection volume was 20 uL per sample. The oven temperature was set at 40degC. To determine the biodegradation of 4-nitroaniline, 1.5 mL liquid volume was taken from each vial and then solution was centrifuged at 10,000 rpm for 15 min. After centrifugation, the residues of 4-nitroaniline in supernatant were analyzed by HPLC. Elution of the molecules was analyzed at 254 nm by using UV detector. Standard solution of 4-nitroaniline was run at different concentrations (0-100 umol L-1) on HPLC to draw a standard curve. The samples were compared with standards.

Isolation of Bacterial Strains through Enrichment Technique

For the isolation of bacterial strains from dye- contaminated wastewater and sludge, suspension was first prepared in 250 mL conical flasks containing mineral salt medium (MSM) and 4-nitroaniline (100 umol L-1) as a source of carbon and nitrogen. Similarly, suspensions in MSM were prepared using 100 umol L-1 4-nitroaniline and different types of dyes at 100 mg L-1 concentration. The suspension in conical flasks was inoculated with 10 mL of wastewater or equivalent amount of sludge. For each sample, the suspension was prepared in a separate conical flask. Flasks containing the suspension was covered and incubated for seven days at 35C in a shaking incubator at 180 rpm. Degradation rate was measured by centrifugation of the culture of each suspension at 10,000 rpm for 15 min to remove cells. Supernatants were run on HPLC to check the biodegradation of aromatic amine (4- niroaninline).

After seven days of incubation, 1 mL culture suspension was spread on agar plate using dilute plate technique. For this purpose, ten dilutions (10-1-10-10) were prepared and last three dilutions were spread on agar medium. Bacterial colonies showing prolific growth on the medium were selected and these bacterial colonies were used to obtain pure cultures by streaking then on fresh agar medium. This process was repeated twice. A total of 256 isolates were selected: 173 isolated by using 4-nitroaniline as source of C and N, while 83 on the basis of 4-nitroaniline plus dyes.

Screening of Efficient Bacterial Strains Capable of Degrading 4-nitroaniline

A total of 256 purified bacterial isolates which were finally obtained after purification were further subjected to screening to acquire the efficient strains capable of degrading aromatic amines.

Screening on the Basis of Bacterial Cell Biomass

Cell biomass was calculated to determine the ability of bacterial isolates to grow efficiently in MSM containing 4- nitroaniline. The vial containing 2 mL MSM along with 4- nitroaniline was inoculated with 0.5 mL bacterial isolates and incubated at 35degC for 48 h. Biomass increase was calculated at different time intervals.

Screening on the Basis of Biodegradation of 4- Nitronailine in Liquid Medium

For the selection of efficient strains, all selected bacterial isolates were grown in glass vials containing 2 mL MSM broth having 100 umol L-1 of 4-nitroaniline. The solution was inoculated by selected bacterial isolates with the help of sterilized tooth picks. These vials were sealed and incubation was done at 35oC for 24 h in a shaking incubator at 180 rpm to give aerobic conditions. After HPLC analysis, 18 isolates showing highest degradation of 4-nitroanailine were selected. Based on cell biomass and 4-nitroanailine degradation rate, most efficient bacterial strain IL11 was identified.

Identification of Selected Bacterial Strains

Most efficient bacterial isolate IL11 was identified by 16S rRNA gene analysis. To identify the bacterial strain, nearly complete 16S rRNA gene was amplified by the PCR using forward and reverse primers, 9F (5'-GAGTTTGATC CTGGCTCAG-3') and 1510R (5'-GGCTACCTTGT TACGA-3'). The PCR products were purified using PCR purification kit and sequenced using the BigDyeTM Terminator Cycle Sequencing Kits following the manufacturer's protocols (ABI PRISM(r) 3730 XL Genetic Analyzer). BioEdit software package was used to obtain the consensus sequence. Sequences of closely related type strains, used for constructing the phylogenetic tree, were selected and retrieved from the National Center for Biotechnology Information and DNA Data Bank of Japan databases by BLAST searches for bacteria. The sequence of the bacterial strain IL11was deposited in GenBank under the accession member KT361501 (Fig. 1).

Statistical Analysis

All experiments were conducted in three replications per treatment in order to secure reproducibility of the results and to ensure clear accounting of all experimental results over time and space. The average size of the data values was calculated by the arithmetic means. The mean variability of the data values was determined by calculating the standard error. The statistical difference among means of wastewater collected samples was calculated through ANOVA and Fisher's LSD was used to analyze difference between the treatments using MSTAT software.


Analysis of Wastewater and Sludge

The wastewater and sludge samples collected from textile wastewater streams were analyzed for physical and chemical characteristics. The results showed a wide variation in selected physical and chemical parameters (Table 1). The pH range was between 8.10 and 12.3 in different wastewater samples obtained from different textile industries. Four samples (AP, IL, MP and RT) showed significant different pH from each other and from all other collected samples (P<0.05). Maximum pH was recorded in case of wastewater sample collected from one of the industries (MP) of Faisalabad. Similarly, a wide variation was observed in TDS (500-20100 mg L-1) of different wastewater samples. Maximum TDS were found in the same sample (MP Faisalabad) in which pH was also high. All the collected wastewater samples were significantly different for TDS (P<0.05).

Wastewater of most of textile industry was highly colored and spectrophotometer analysis showed the different range of color intensity varying between 0.12-1.92 at 600 nm. Wastewater collected from (MP, AP, FT and RT) showed significantly different absorbance range (P0.05).

Table 1: Physico-chemical analysis of dye wastewater collected from different sites

Sampling source and wastewater type###pH###TDS (mg L-1)###COD (mg L-1)###Absorbance at 600 nm

###Mean +- SE###Mean +- SE###Mean +- SE###Mean +- SE

Faisalabad###AP (effluent/wastewater)###10.2 +-0.058b###12240 +- 5.12c###512.22+-2.2b###1.22 +-0.003b

###PD (Effluent/wastewater)###8.48 +- 0.012def###2840 +- 3.2f###350.1+-2.1c###0.18 +- 0.005fg

###IL (Effluent /wastewater)###8.78 +- 0.017c###3220 +-4.0d###301.10+-1.7d###0.12 +- 0.001g

###AT (Sludge )###8.31 +-0.015fg###2140 +- 3.1h###250.12+-2.9f###0.31 +-0.002e

###MP (Effluent/wastewater)###12.3 +-0.09a###20100 +-4.2a###556.09+-3.6a###1.92 +-0.002a

Sheikhupura###FT (Sludge )###8.32 +- 0.013efg###2860+- 2.3e###245.11+-2.1f###0.52 +-0.001c

###RT (Sludge)###8.10 +-0.071g###2210 +-4.4g###265.31+-2.4e###0.45 +-0.003d

###BT (Effluent/wastewater )###8.32 +-0.023efg###500 +-1.5j###125.12+-7.5h###0.25 +-0.001e

Rawalpindi###KT (Effluent/wastewater)###8.56 +-0.012cde###1350 +- 2.3i###145.19+-1.8g###0.19 +-0.001f

###KTD(Effluent/wastewater)###8.67 +-0.012cd###13000 +-2.4b###150.28+-3.9g###0.16 +-0.001fg


Table 2: Bacterial strains isolated through enrichment of MSM with 4-nitroaniline as a source of carbon and nitrogen

Sampling source and wastewater type###No. of bacterial isolates

Faisalabad###AP (effluent)###AP1-AP18

###PD (Effluent)###PD1-PD18

###IL (Fresh effluent )###IL1-IL26

###AT (Sludge )###AT1-AT15

###MP (Effluent)###MP1-MP16

Sheikhupura###FT (Sludge )###FT1-FT15

###RT (Sludge)###RT1-RT18

###BT (Effluent )###BT1-BT17

Rawalpindi###KT (Fresh Effluent)###KT1-KT15

###KTD(Drain WW)###KTD1-KTD15

Isolation and Screening of Bacteria Capable of Degrading Aromatic Compounds

A total of 256 bacterial strains were isolated from textile wastewater/sludge through the enrichment technique. About 173 isolates were obtained by enriching MSM with 4-nitroaniline (Table 2). About half of the above (93) strains were obtained from the wastewater samples of Faisalabad, 50 strains from wastewater of Sheikhupura and 30 from the textile industry of Rawalpindi. Eighty three bacterial isolates were obtained using different dyes with 4-nitroaniline as a source of carbon and nitrogen (Table 3). These bacterial strains showed varying potential to degrade 4- nitroaniline in broth media under shaking (aerobic) conditions. Of total bacterial isolates, approximately 7% isolates were more efficient than rest of strains and capable of degrading >75% 4-nitroaniline after 24 h under aerobic conditions. The remaining isolates showed either medium (50-75%) or low ( 75%###18 (7.03 %)

Medium degradation 50-75 %###120 (46.8 %)

Low degradation < 50%###118 (46.09) %

Total isolates###256

Table 5: Bacterial isolates capable of degrading 4- nitroaniline in liquid medium after 72 h

Bacterial isolate###Degradation (%)###Biomass gain (mg L-1)

AP10###80 +-2.21###0.65 +- 0.02

AP15###81 +- 1.23###0.38 +-0.01

PD4###60 +-2.22###0.23 +- 0.004

PD9###70 +- 2.50###0.31 +- 0.01

IL5###75 +- 1.31###0.24 +- 0.08

IL7###72 +- 2.70###0.32 +- 0.12

IL11###100 +- 0.00###0.69 +- 0.06

MP10###78 +- 2.22###0.42 +- 0.03

MP37###85 +- 2.22###0.61 +- 0.04

KT1###95 +-1.11###0.75 +- 0.13

KT3###72 +- 2.22###0.55 +- 0.27

KT6###70 +- 2.01###0.42 +- 0.12

KT9###76 +- 1.21###0.50 +- 0.19

PD1###90 +- 1.29###0.71 +- 0.07

PD4###75 +- 2.11###0.51 +- 0.09

PD9###78 +- 1.22###0.45 +- 0.12

D1###75 +- 2.22###0.50 +- 0.12

D5###90 +- 1.10###0.67 +- 0.11


This study demonstrated that the pH, TDS and COD values of wastewater released from the textile industry were substantially higher than the permissible limits set by NEQS (2000). Different kinds of salts are used in huge quantities in the textile industry. As a result, a large amount of sodium and chloride ions are present in textile wastewater, which increases the toxicity of water sources (Ali et al., 2006; Paul et al., 2012; Dey and Islam, 2015). The color of the effluent measured as absorbance was highly visible, which may have environmental concerns including aesthetic problems (Sun et al., 2009; Modi et al., 2010; Lu et al., 2010; Holkar et al., 2016).

In order to obtain bacterial strains capable of degrading dye-originated aromatic compounds, several bacterial strains were isolated from the dye-contaminated wastewater/sludge through the MSM enrichment with 4- nitroaniline and different groups of dyes. Most efficient bacterial strain obtained through the enrichment of 4- nitroaniline was identified by 16S rRNA gene analysis as Raoultella planticola (IL11). This strain was very effective and complete (100%) degradation of 100 umol L-1 of 4-nitroaniline was accomplished in 72 h of incubation with stirring (aerobic) conditions. Previously, a few species of bacteria capable of degrading dye-related aromatic compounds were reported (Khalid et al., 2008; Garg et al., 2012; Agrawal et al., 2013). Microbial degradation provides most promising possibility for complete mineralization of dyes and intermediates (aromatic amines), due to greater adaptability and increased rate of bacterial growth (Saratale et al., 2011; Singh et al., 2015).

Reduction of azo bonds in anaerobic condition is the first step in the breakdown of azo dyes, forming aromatic amines (Pandey et al., 2007; Franciscon et al., 2009). The intermediate products of first step (decolorization process) are degraded by various bacterial enzymes such as hydroxylase and oxygenase (Pandey et al., 2007; Khalid et al., 2009). Furthermore, the selected bacterial strain has the ability to cleave -N=N- bond, probably with the aid of azoreductase enzyme (McMullan et al., 2001; Khalid et al., 2010).


Textile wastewater is highly polluted and its treatment is a complex process. The isolated bacterial strain, R. planticola (IL11), showed great potential to degrade dye-associated aromatic amine (4-nitroaniline). This bacterium could be used in combination with dye-degrading strains for bioremediation of textile industrial wastewater.


Authors acknowledge financial support for this study from Higher Education Commission Islamabad under Indigenous Ph.D. Fellowship Program and research grant from Pakistan Science Foundation under NSLP.


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Author:Kausar, Farzana; Khalid, Azeem; Mahmood, Tariq; Ilyas, Nosheen
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Dec 31, 2016
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