OM2R - Orange M2R; GGS - Green GS; NaCl - sodium chloride; NaOCl - sodium hypochlorite; WAO - wet air oxidation; SM - salt media; NA - nutrient agar; O.D. - optical density; MR - methyl red; VP - Voges Proskauer; MIU - Motility Indole Urease Test; ml - milliliter; g/L - gram per liter.

Industrialization has expanded in every corner of the globe in the 21 ^st-century and the textile industry has emerged as a leading sector. It uses thousands of tons of synthetic dyes (azo) annually ¹ . A large portion of those goes into water bodies untreated ² . Carcinogenic and recalcitrant molecules present in the dye penetrate into the ecosystem and harm every member of the system ³ . Humans use the polluted water directly for daily necessities which may result in diseases such as cancer, new genetic mutations or changes in the DNA etc. becoming an epidemic.

The textile industry in Bangladesh accounts for 45% of all industrial employment and contributes 5% to the total national income ⁴ . The industry employs nearly 4 million people, mostly women ⁵ . Despite the significant economic contribution, it has brought with it a range of environmental problems, mostly pollution of water resources. The textile industry consumes large quantities of water for various processes and discharges equally large volumes of wastewaters containing a variety of pollutants and coloring agents such as the azo dye ^{6, 7} .

It is estimated that over 2,80,000 tons of textile dyes are discharged in industrial effluent every year, worldwide. Therefore, pollution from these discharges contaminated with dyestuff is becoming alarming ^{8, 9} . This sector is placed as a major source of water pollution in Bangladesh ¹⁰ .

Textile wastewater is highly colored, resulting in the blocking of the majority of sunlight, thereby retarding the growth of aquatic animals and plants; it also contains the dissolved toxic substance and carcinogens ^{11, 12} . The serious damage of pollution is caused mainly due to the durability of the dyes in wastewater ¹³ . Azo dyes are widely known coloring agents used in industries and hence commonly released in the environment ¹⁴ . The dye wastewaters are extremely toxic to both aquatic fauna and flora, crop plants, and human beings ¹⁵ .

At present, there are several techniques that can be employed in dye removal from effluents. However, these methods are varied in efficiency due to the variety of existent dyes and to the effluents complexity, and the combination of various methods may be considered since each method showed its limitations. There are three categories of existing methods: physical, chemical and biological. Physical methods like Coagulation/Flocculation, Adsorption, Membrane filtration, and Ion exchange are expensive. Chemical methods like Fenton’s reagent, Ozone, Photochemical, Sodium hypochlorite (NaOCl), Electrolysis and wet air oxidation are not cost effective and produce toxic byproduct.

Biological treatment, in the form of bacterial degradation, has been mainly applied in the removal of azo dyes ^{16– 17} , which generally is resistant to aerobic degradation ^{18, 19} . However, its degradation was observed in anaerobic conditions, but aromatic amines are formed as a final product, which can be toxic, mutagenic and carcinogenic ²⁰ . Under these anaerobic conditions, it is not possible to degrade the aromatic amines formed, which in turn are only degraded in an aerobic environment. Thus, to achieve a complete degradation of azo dyes a method that combines anaerobic treatment of the dyes with the mineralization of aromatic amines under aerobic conditions should be applied ^{21, 22} . This research aimed to identify effective dye degrading bacteria from effluent soil samples and optimize the physiochemical condition for their optimum growth ²³ .

Four isolates AO, BO, CO and DO were collected from effluent soil samples preliminarily inoculated for 5 days in SM broth containing 1% OM2R azo dye at room temperature to decolorize the dye. CO and AO isolates showed the highest decolorization rate (99% and 93%) and were selected for further optimization. The other two isolates performed moderately. For 1% GGS dye degradation BG (93%) and CG (94%) isolates were better than AG and DG. ( Table 1).

Most of the isolates from the different soil samples were identified as Bacillus species. Dye decolorizing ability of isolates was investigated independently. CO ( B. farraginis) showed the highest dye decolorization capacity (98%) in SM broth media containing 1% OM2R dye for 5 days at 37°C. However, as the concentration of dye increased up to 3% and 5% the decolorization rate decreased to 94% and 90% respectively, because of the intensity of azo dyes. On GGS dye degradation BG ( P. macerans) showed 97% decolorization. This was similarly effective at 3% dye concentration (94%), however, the rate was found to slump at 5% dye concentration (81%).

pH is one of the important abiotic factors that affect the growth and metabolic homeostasis. The effect was studied at different pH values (5 – 8). At pH 7.0 AO ( E. termitis) gave maximum decolorization (90%). A similar rate was observed over the pH range of 5.0 and 6.0, with a swift reduction (59%) observed at pH 8 by AO ( E. termitis). These results suggest that acidic pH values may influence the stability of the enzyme causing denaturation. Chang et al. (2001) ¹⁴ found that azo reductase performance was affected by pH, with 2.5 times better dye reduction at pH 7 - 9 than below pH 7. These findings corresponded well to the best decolorization found between pH 7 - 9.5 ²⁹ .

In the case of GGS, the maximum decolorization rate was attained at pH 7.0 by BG ( P. macerans) at 92%. The majority of the azo dye reducing bacterial species reported so far were able to reduce the dye at pH near 7 ^{30– 32} . The requirement of near neutral pH for optimum growth had been reported in several studies ^{33– 35} . Results indicate that a pH increase from 5.0 to 7.0 enhanced the decolorization of GGS dyes. At pH 5 the decolorization rate was 80% of dye by BG ( P. macerans). A small increase was observed at pH 6 (85%) with an abrupt decrease at pH 8.0 (73%). It was observed that better decolorization rates were around pH 6 - 7 bands for both of OM2R and GGS dye by the selected isolates.

Decolorization percentage of OM2R by selected isolates was found to vary with different concentration (2 - 8 g/L) of NaCl when studied for 120 hours at 37°C. Maximum decolorization of OM2R by AO ( E. termitis) was observed as 81% at 2% NaCl, but the percentage decolorization was found to decrease with increases of NaCl concentration ( Table 3; Dataset 2 ²⁸ ). The decolorization attained by AO ( E. termitis) at 37°C for 4%, 6% and 8% NaCl was 60%, 43%, and 37%. Kargi and Dincer (1996) ³⁶ mention that high salt concentrations (>1% salt) are known to cause plasmolysis and/or loss of cell activity.

Similarly, at 2% NaCl concentration the degradation percentage of GGS dye was 89% by BG ( P. macerans). The decolorization attained by BG ( P. macerans) at 37°C for 4%, 6% and 8% NaCl was 72%, 62%, and 40% ( Table 3; Dataset 2 ²⁸ ).

To determine the optimum temperature for dye decolorization a temperature range of 30°C –55°C was examined. As seen in Table 3 the optimum temperature for OM2R dye decolorization was 37°C for the AO ( E. termitis) attained a maximum decolorization of 93%. Angelova et al. (2008) ³⁷ found that the azo bond reduction rate rose with an increased temperature, a maximum rate of around 40°C, 3–5 times faster than at 20°C. At 30°C and 45°C the degradation rate for OM2R by AO E. termitis was 82% and 72% of dye. A low decolorization of 59% of the dye was detected at 55°C by AO ( E. termitis) isolate. Temperatures above 55°C were not studied since results shown that the increase from 37°C to 45°C promoted a marginal decrease in dye decolorization ( Table 3; Dataset 2 ²⁸ ).

The optimum temperature for GGS dye decolorization was 37°C for the BG isolate ( P. macerans), attaining a maximum decolorization of 90% of dye. In the case of BG, at 30°C and 45°C the decolorization percentage was 86% and 84% of dye respectively. No improvement in dye decolorization was observed at temperatures above 45°C, with low decolorization of 58% and 45% of dye detected at 55°C by BG ( P. macerans) isolate as evident from Table 3. BG ( P. macerans) has a broad range of compatibility from 30°C to 45°C. Within the optimal values of temperature, the lowest temperature was selected as the optimum temperature since this leads to lower energy costs.

Traditional wastewater treatment is inefficient and remains a threat to environment ³⁸ . Biotreatment offers an easy, cheap and effective alternative for color removal of textile dyes ³⁹ . Hence, economic and eco-friendly techniques using bacteria can be an alternative method. The present study strongly concluded that the bacterial isolates E. termitis, E. camelliae, B. decolorationis, P. macerans species were a good microbial source for textile effluent treatment, in biological degradation of textile dye. However, decolorization potential of the isolates needs to be validated by demonstration in appropriate bioreactors before its application.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2018 Rahman SS et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1: Results of optical density (O.D) & de-colorization (%) with different concentrations of dye. Average O.D. calculated from 1st and 2nd O.D. 10.5256/f1000research.13757.d198164 ²⁷

Dataset 2: Results of optical density (O.D) & de-colorization (%) with different parameters at day 5. Average O.D. calculated from 1st and 2nd O.D. 10.5256/f1000research.13757.d198165 ²⁸