Selection of bacterial strains efficient in decolorization of remazol black-B.

Azo dyes are released into wastewater streams without any pretreatment and polluted water and soil environments. To prevent contamination of our vulnerable resources, removal of these dye pollutants is of great importance. For this purpose, wastewater samples were collected from dye-contaminated sites of Ankleshwar, Gujarat, India. About 50 bacterial isolates were isolated through enrichment and then tested for their potential to remove Remazol Black-B azo dye in liquid medium. Three bacterial isolates capable of degrading Remazol Black-B azo dye efficiently were screened through experimentation on modified mineral salt medium. Isolate ETL-1 was able to completely remove the Remazol Black-B dye from the liquid medium in 18 h. Further, the isolate showed the best performance at the dye concentration of 100 mg L-1 medium (pH 7) and at temperature 35 degrees C. Similarly, yeast extract proved to be the best carbon source for decolorization purpose. The results imply that the isolate ETL-1 could be used for the removal of the reactive dyes from textile effluents.


Introduction
Duetorapidindustrialisation,alotof chemicals including dyes are manufacturedandusedindaytodaylife 1 . Synthetic dyes are extensively used in textile, dyeing, paper printing, colour photography, food, cosmetics and other industries. Approximately 10 000 different dyes and pigments are used industrially and over 0.7 million tons of synthetic dyes are produced annually, worldwide 2 . Explosion of population coupled with industrial revolution results in pollution of water, air and soil. The dischargeofpollutantsfromvarious industriesposesathreattothebiodiversityoftheearth.Thetextilefinishinggeneratesalargeamountofdyes containing wastewater from dyeing andsubsequentstepsthatformsone ofthelargestcontributionstowater pollution 3 . The traditional textile finishing industry consumes about 100 litres of water to process about 1 kg of textile material. The new closed-loop technologies such as the reuse of microbially or enzymatically treatment of dyeing effluents could help in reducing this enormous amount of water consumption 4 . It wasalreadyreportedthat10%-15% ofdyesarelostintheeffluentduring the dyeing process 5 . Azo dyes have been used increasingly in industries becauseoftheireaseandcosteffectiveness in synthesis compared to naturaldyes.However,mostazodyes are toxic, carcinogenic and mutagenic 6 .Azobondspresentinazodyes areresistanttobreakdown,withthe potential for the persistence and accumulationofhighlevelsofdyein the environment 7 . These dyes cannot be easily degraded, while some aretoxictohigheranimals 8 .Azodyes areverystableinacidicandalkaline conditions and are resistant to temperatureandlight.However,theycan bedegradedbybacteriaunderaerobic and anaerobic conditions 9 . Azo dyes are environment pollutants 10 and they contribute to the mutagenic activity of ground and surface water that are polluted by textile effluents 11,12 . The complex aromatic substituted structures make conjugated systems and are responsible for intense colour and high water solubility 13 . Their discharge in to surfacewateralsoleadstoaesthetic problems, obstructing light penetrationandoxygentransferintowater bodies 14,15 . Several physicochemical techniques have been proposed for treatment of coloured textile effluents. These include adsorption on different materials, oxidation and precipitation by Fenton's reagent, bleaching with chloride or ozone, photo degradation or membrane filtration 16 . The economic and safe removal of the polluting dyes is still an important issue. Because all these physicochemical methods are very expansive and result in the production of large amount of sludge, they create the secondary level of land pollution. In this situation bioremediation is becoming important, because it is cost-effective, environmentally friendly and produces less sludge 17 . Therefore, in such situations, biological treatment may be a real hope. These methods have the advantages of being environment friendly. Microorganisms have developed enzyme systems for the decolourisation and mineralisation of azo dyes under certain environmental conditions [18][19][20] .So,thisstudy wasdesignedtoisolateefficientazo dye decolourising bacterial strains from the textile effluents. Since the Introduction Azo dyes are released into wastewater streams without any pretreatmentandpollutewaterandsoil environments. To prevent contamination of our vulnerable resources, removalofthesedyepollutantsisof great importance. For this purpose, wastewater samples were collected from dye-contaminated sites of Ankleshwar, Gujarat, India. The aim ofthisstudyistodiscussaselection ofbacterialstrainsefficientindecol-ourisationofRemazolBlack-B.

Materials and methods
About50bacterialisolateswereisolated through enrichment and then tested for their potential to remove Remazol Black-B azo dye in liquid medium. Three bacterial isolates capable of degrading Remazol Black-B azo dye efficiently were screened through experimentation on modifiedmineralsaltmedium.

Conclusion
The results imply that the isolate ETL-Acouldbeusedfortheremovalof thereactivedyesfromtextileeffluents.

Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)
Competing interests: none declared. Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure. bacterial isolates were originated from the dye-contaminated textile wastewater of local industry, they can easily adapt to the prevailing local environment. Therefore, such bacteria can be used to develop an effectivebiologicaltreatmentsystem for the wastewaters contaminated with azo dyes. This study discusses some bacterial strains efficient in decolourisationofRemazolBlack-B.

Materials and methods
The protocol of this study has been approved by the relevant ethical committee related to our institution inwhichitwasperformed.

Sampling
Water and sludge samples were collected from Ankleshwar Industrial Estate, Ankleshwar, Gujarat, India aroundwhichmanytextileprocessing unitsaresituated.Samplesweretaken from drains at different locations and sampling sites were selected on the basis of the allocation of outlet from textile units. Electrical conductivity and pH were determined to assess the presence of total suspended solidsandacidityoralkalinityofthecollectedsamples(Table1).

Isolation of azo dye decolourising bacteria
Bacterial strains were isolated from wastewater and sludge samples of dyeindustryunits.Isolatesfromeach inoculumsourcewerefirstenriched usingMSMmediumamendedwithan azodyeRemazolBlack-Basthesole source of C and N 20 . Dye was added at a concentration of 150 mg L -1 . The cultures containing 200 mL of MSMbrothwithdyein500mLErlenmeyer flasks were inoculated with 10 mL volume of wastewater or sludgesuspensions.Theflaskswere incubated at 32°C for seven days under static conditions. After incubation, cell suspensions from each flask were plated onto MSM agar medium and incubated at 32°C for 24 hours. Microbial colonies that appeared on the agar medium were washed gently with sterile water and resuspended into the flasks containing fresh MSM broth spiked with the Remazol Black-B dye.About50activelygrowingcolonieswereselectedforpurification.

Purification of bacterial isolates
Selected isolates were purified by streaking on MSM medium containing agar at the concentration of 20gL -1 .Streakingwasdonethricein a zig-zag manner. The purified cultures were preserved in a refrigeratorforsubsequentstudy.

Screening efficient azo dye decolourising bacterial isolates
Screening was done to find out the efficient bacterial strains capable of decolourising the Remazol Black-B azodyeusingmodifiedMSM.Forthis purpose,50isolatesthathadtheability to decolourise Remazol Black-B fromallsampleswereselected.After that the decolourisation ability of each isolate was tested in the liquid medium. Media inoculated with the respectiveinoculawereincubatedat 35°Cfor24hours.After24hours,the respective cells were harvested by medium centrifugation at 10 000 rpm (REMI R-23, India) for 10 minutes. Thendecolourisationwasdetermined with the help of a spectrophotometer (SHIMADZU, Japan) at 597 nm. Uninoculated blanks were run to determine abiotic decolourisation. The three most effective bacterial isolates (ETL-A, ETL-B and ETL-C) fromthefinalscreeningwerefurther examined for their decolourisation

Statistical analysis
Data were entered in a Microsoft ® Excel2007spreadsheet.

Results
Efficiency of the bacterial isolates to decolourise Remazol Black-B was examinedbymeasuringcolourintensity in a liquid medium. Based upon therelativedecolourisationefficiency ofdifferentisolates,threeofthemost efficient isolates (ETL-A, ETL-B and ETL-C ) with more than 80% decolourising efficiency were selected for furtherexperiments(datanotshown).

Biodecolourisation of Remazol Black-B by selected bacterial isolates
BiodecolourisationofRemazolBlack-B by the selective bacterial isolates (ETL-A, ETL-B and ETL-C) was confirmed by conducting another experimentinliquidmediumatdifferenttime periods ( Figure 1). It was found that differentbacterialisolateshadvariable potentialtoremoveRemazolBlack-Bin thegrowingcultures.Themost efficient

Factors affecting biodecolourisation of Remazol Black-B in liquid medium
The potential of selected isolates (ETL-A, ETL-B and ETL-C) was furtherinvestigatedfortheoptimisation of various incubation/environmental conditions for decolourising the azo dye in liquid medium. It was evident (Figure 3) that Remazol Black-B azo dye decolourisation sharply increased up to 100 mg L -1 ofsubstrateconcentrationandmaximum decolourisation was observed at 100 mg L -1 of substrate concentration. Then, there was a gradual decrease in the azo dye decolourisation. Isolate ETL-A was the most efficientazodyedecolourisingstrain withmoreorlesscompleteremoval of the colour, that is, 100% decolourisation at 100 mg L -1 and minimum decolourisation was recorded at 50 mg L -1 while after 100 mg L -1 substrateconcentration,againETL-A showed a decreasing trend. Isolate ETL-B was the second at the rank with 90% decolourisation at 100mgL -1 .But,ETL-Cshowedadifferenttrendfromtheotherisolates, it indicated enhanced decolourisa-tionupto200mgL -1 (82%).

Types of carbon sources
Effects of different carbon sources such as maltose, mannitol, glucose andyeastextractwereevaluatedon Remazol Black-B decolourisation by bacterial isolates (Figure 4). It was found that maximum decolourisation occurred with 4% yeast extract in all selected strains (85%-95%) that was followed by glucose in which decolourisation occurred in the range of 20%-25%. However, least decolourisation was observed inthecaseofmannitol(10%-15%).

Effect of pH
For studying effect of pH value, different levels of pH ranging from 5to9wereusedandincubationofall selected isolates was done at these levels ( Figure 5). Initially with the increaseinpHvaluefrom5to7,decolourisation increased and maximum decolourisation occurred at pH 7.

Effect of incubation temperature
Five levels (25°C, 30°C, 35°C, 40°C and 45 o C) of temperature were used for assessing optimal biodecolourisation of Remazol Black-B by selected bacterial isolates. It is evident ( Figure 6) that when the temperature was raised from 25°C to 35 o Ctherewasaninconsistenttrend in decolourisation from the different isolates. The ETL-A and ETL-B isolates showed gradual increase in decolourisation, while one isolate ETL-Cdisplayedmaximumdecolourisation at 25°C. The remaining two bacterialisolates(ETL-AandETL-B) withagradualrisefrom25°Cto35°C showedmaximumdecolourisationat 35°C. As the temperature increased furtherfrom35°Cto45°C,therewas a sharp decline in decolourisation capacityinalltheisolates.Itwasalso observed that with the rise in temperature,abioticdecolourisationalso increased.Maximumdecolourisation wasobservedwiththeisolateETL-A (98%) at 35°C and it is followed by ETL-B (94%) at the same temperature. Least decolourisation was observed at 45°C in all the selected isolates.

Discussion
Industrialeffluentisnotstableandit variesofteninawiderangedepending upon the process practiced. South-Asiancountriesareexperiencing severe environmental problems due to rapid industrialisation. This phenomenonisverycommonwhere the polluting industries like textile    32 . Azo dyes generally contain one or more sulphonic acidgroupsonaromaticrings,which mightactasdetergentstoinhibitthe growthofmicroorganisms 32 .Another reason of the toxicity at higher concentration may be due to the presenceofheavymetals(metal-complex dyes)and/orthepresenceofnonhydrolysedreactivegroups,whichmay retard the bacterial growth (reactive dyes) 31 . Similarly, reduction in decolourisationatlowconcentration of the substrate might be due to the decrease in enzyme ability to recognisethesubstrateefficiently.Whereas inthecaseofdifferentcarbonsources tested on yeast extract proved to be the best amongst tested carbon sources. Our results were in agreement with the research conducted   byGuoetal. 33 inwhichthebacterial strains grew well and completely decolourised K-2BP when either yeastextractorpeptonewaspresent in the medium; however, glucose, glycerol, sucrose, lactose and starch resultedinlowerratesofgrowthand decolourisation of these dyes. Other studies also reported the maximum decolourisation of azo dyes in the presenceofyeastextractbybacteria 34 .
InthecaseofpHasavariable,decolourisation was on a higher side at pH 7. Whereas higher pH values (alkaline conditions) decreased the decolourisation efficiency of all the testedisolates.So,fromthisstudy,it could be concluded that neutral pH supportedbacterialactivitytodecolourise Remazol Black-B in liquid medium 35,36 . Temperature is another veryimportantparameterforanaerobictreatmentofwastewater.Selected isolates were mesophilic bacteria becausetheyallshowedbetterdecolourisation in the temperature range of 25°C-35°C. Similar results were alsoreportedbyGuoetal. 33 .Themesophilic range is traditionally used 37 since it is generally thought that maintaininghightemperaturewould be uneconomical, while degradation withinthepsychrophilicrangeistoo slow.Overall,oneoftheselectedisolates(ETL-A)ofbacteriawasableto completely remove the colour of the dye in 18 hours. However, these isolatesshouldbetestedatalarge-scale treatment system to examine their potential for bioremediation of dyepollutedwastewaters.

Conclusion
This study reveals that the selected three cultures can be used successfully for decolourising Reactive Black-B dye. The cultures exhibited maximum decolourisation ability at a pH of 7 for all the three isolates, and35°CforETL-A,ETL-Band25°C for ETL-C. Moreover, 4 g/L yeast extractwasfoundtobeoptimumfor decolourisation. In conclusion, bacterialspeciescanbestudiedfurther for bioremediation of dye-polluted watersincludingrateofdegradation of azo dyes other than the selected Black dye through an application of bioaugmentation.