Optimization of conditions for the biological treatment of textile dyes using isolated soil bacteria

The article titled “Optimization of conditions for the biological treatment of textile dyes using isolated soil bacteria” ([version 1; referees: peer review discontinued]. F1000Research 2018, 7:351  https://doi.org/10.12688/f1000research.13757.1 ) by Shafkat Shamim Rahman and colleagues, has been retracted by F1000Research on grounds of misconduct by the first author. Following publication of the article, the editorial team at F1000Research were notified by Romana Siddique, from BRAC University, that the data presented in this paper significantly overlaps with the data in her recently published article : Siddique and Alif; ARRB, 22(5): 1-12, 2018; Article no.ARRB.38637;  https://doi.org/10.9734/ARRB/2018/38637 .  
In response to our queries to the authors, the second and last author listed on this article, Fahim Ahmed Alif and M. Mahboob Hossain, have stated that they were not aware of the submission of this article to F1000Research, and did not agree to be authors. We have evidence which confirms their statement.  
After further investigation by the F1000Research team, and a separate investigation by BRAC University, it has become clear that Shafkat Shamim Rahman was not involved with the research presented in this paper, and that the decision to submit and publish the article was taken independently by him, and not his listed co-authors. BRAC University has confirmed that Shafkat Shamim Rahman is not currently based at their institution.

Editorial note: 23 rd May 2018 : Significant concerns have been raised about the overlap in the data presented in this paper, and that of another recently published article [Siddique and Alif; ARRB, 22(5): 1-12, 2018; Article no.ARRB.38637;https://doi.org/10.9734/ARRB/2018/38637].In addition, the second and last author listed on this article, Fahim Ahmed Alif and M. Mahboob Hossain have stated that they were not aware of the submission of this article to F1000Research, and did not agree to be authors.We are currently investigating this and have suspended all peer review activity in the meantime.

Introduction
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.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 .Inoculation in dye-containing media, isolation and screening One gram of each of the soil (effluent) samples (A, B, C, D) were taken to prepare a homogenous suspension.The suspension of each sample was individually applied to sixteen 1% dye containing (eight Orange M2R -OM2R and eight Green GS -GGS) SM broth media (glucose -10 g/L; #G8270, dipotassium phosphate -0.6 g/L; #1551128 USP, peptone -10 g/L; #P7750, monopotassium phosphate -1.9 g/L; #1551139 USP, magnesium sulfate -1 g/L; #M2643, yeast extract -1 g/L; #Y1625, pH: 6.0 -6.4; Sigma Aldrich, St Louis, MO, USA) to detect the dye degrading capability.1% solution of OM2R and GGS was made by adding 0.5g of dye into the 50ml distilled water.After 5 days, each conical flask was compared with the control and decolorization was observed.As each of the samples (A, B, C and D) contained a highly concentrated mixture of different types of microorganism, up to 10 -4 and 10 -5 dilutions were performed, and spread plate technique applied on NA plates followed by incubation for 24 hours at 37°C to obtain eight soil isolates (AO, BO, CO, DO, AG, BG, CG, DG).Selected isolates, based on morphology, were enriched in NA media and incubated for 24 hours at 37°C.The plates were sealed, refrigerated at 4°C and were frequently subcultured.

Media optimization
Optimum growth conditions for the isolates were identified applying different physiochemical state (dye concentration, pH, NaCl concentration, temperature) to the time of growth.The

R E T R A C T E D
best four selected isolates were cultured in 50 ml SM broth with three different dye concentrations of 1%, 3% and 5%; for pH (5 to 8; adjusted by adding drops of basic NaOH or acidic diluted HCl in the solution) (Model: E-201-C, China), for NaCl tolerance (added 2%, 4%, 6% and 8% concentrated Sigma brand solution to the broth and incubated), and for temperature (30°C, 37°C, 45°C and 55°C) and incubated (Model: SAARC) at 37°C (except temperature parameter) for a period of 5 days with corresponding dye OM2R and GGS.Then, O.D. was measured by adjusting the wavelength at 590nm for OM2R and 510nm for GGS (Model: UV-VIS spectrophotometer UVmini-1240; Shimadzu, Kyoto, Japan).Each experiment was repeated to validate the results.The representative data is the average of all results.
( )  26 were conducted according to individual standard protocols.

Results
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).

Dye concentration optimization
After five days of reaction, CO isolates achieved 98% decolorization in SM broth containing 1% OM2R dye.The same rates were also observed in 3% dye concentration.AO lagged in all three (1%, 3% and 5%) reactions.Degradation rate gradually decreased at higher concentration dye-containing media.Intriguingly, CG isolates resulted in 93% decolorization for both 1% and 3% dye concentration.
In GGS dye-containing media, 97%, 94% and 81% decolorization were achieved with the BG isolate in 1%, 3% and 5% dye concentration media respectively.CG isolates demonstrated a lesser rate in all three parameters (Table 2

Discussion
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][31][32] .The requirement of near neutral pH for optimum growth had been reported in several studies [33][34][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 ).
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.

Conclusion
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

R E T R A C T E D
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.
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Table 1 . Results of O.D. at fifth day after de-colorization of 1% concentration dye in SM broth at room temperature.
28Dataset 1 27 ).pHoptimizationshowed,highestrate (90%) in pH 7 by AO isolate in OM2R dye and 92% by BG isolate in the same parameter.AO isolate also showed a slightly higher response in 2% NaCl media compared to CO.Both isolates resulted in gradually reduced rates of decolorization in 4%, 6% and 8% NaCl containing media.For GGS dye, BG isolate was superior to all other isolates, achieving 89% decolorization in 2% NaCl.The optimum temperature was 37°C in all four isolates.AO achieved 93% decolorization and BG 90% in two different dye-containing media.The decolorization rate gradually decreased with temperature elevation (Table3; Dataset 228).In summary, 1% dye concentration, pH 7, 2% NaCl and 37°C appeared to be the optimum physiochemical condition for dye decolorization.
tests results AO, CO and CG isolates were gram -ve rod and AG and BG were +ve rod bacteria.BO and DO isolates were gram -ve cocci and DG was +ve coccoid.Other biochemical results (Table4) also showed all the isolates were catalase, oxidase, nitrate and aerobic growth positive.All were capable of tolerating 6.5% NaCl in media and withstand 45°C.All recorded negative results for urease, citrate, VP, starch hydrolysis and 10%, 15% NaCl tolerance.Motility, Indole, MR, Casein hydrolysis, 7% NaCl soln.tolerance,carbohydratetests produced mixed results (A mixture of positive and negative results, which indicated the disparity of a wide range of characteristics in these primarily unidentified organisms) (Table4).Finally, software analysis using ABIS (Version: July 29, 2015) based on morphology characteristics and biochemical tests postulated AO as Enterococcus termitis, BO as Enterococcus camelliae, CO as Bacillus farraginis, AG as Bacillus muralis, BG as Paenibacillus macerans, CG as Bacillus decolorationis and DG as Macrococcus brunensis.DO isolate remain unidentified.