Research Article Low-Cost Biodegradation and Detoxification of Textile...

Research ArticleLow-Cost Biodegradation and Detoxification of Textile Azo DyeCI Reactive Blue 172 by Providencia rettgeri Strain HSL1

Harshad Lade1 Sanjay Govindwar2 and Diby Paul1

1Department of Environmental Engineering Konkuk University Seoul 143-701 Republic of Korea2Department of Biochemistry Shivaji University Kolhapur 416004 India

Correspondence should be addressed to Sanjay Govindwar spg biochemunishivajiacin and Diby Paul dibypaullivecom

Received 21 May 2015 Accepted 16 June 2015

Academic Editor Mohd D Rafatullah

Copyright copy 2015 Harshad Lade et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Present study focuses on exploitation of agricultural waste wheat bran (WB) as growth medium for degradation of textile azodye CI Reactive Blue 172 (RB 172) using a single bacterium P rettgeri strain HSL1 (GenBank accession number JX8537681) Thebacterium was found to completely decolorize 50mg Lminus1 of dye RB 172 within 20 h at 30 plusmn 02∘C under microaerophilic incubationconditions Additionally significant reduction in COD (85) and TOC (52) contents of dye decolorized medium was observedwhich suggested its mineralization Induction in the activities of azoreductase (159) and NADH-DCIP reductase (88) providedan evidence for reductive cleavage of dye RB 172 The HPLC FTIR and GC-MS analysis of decolorized products confirmed thedegradation of dye into various metabolites The proposed metabolic pathway for biodegradation of RB 172 has been elucidatedwhich showed the formation of 2 intermediate metabolites namely 4-(ethenylsulfonyl) aniline and 1-amino-1-(4-aminophenyl)propan-2-oneThe acute and phytotoxicity evaluation of degradedmetabolites suggests that bacterial strain favors the detoxificationof dye RB 172 Thus WB could be utilized as a low-cost growth medium for the enrichment of bacteria and their further use forbiodegradation of azo dyes and its derivatives containing wastes into nontoxic form

1 Introduction

Synthetic textile dyes are of complex aromatic structuresspecially designed for chemical stability and versatility andto resist the effect of high temperature during wet process-ing operations which makes them highly recalcitrant [1]Thousands of such synthetic dyes are extensively used inthe textile industry for dyeing and printing purposes [2]Approximately 40ndash65 L of textile wastewater is producedper kg of cloth during dyeing processes [3] Among alltextile dyestuff used the azo dyes constitute about 70 andare being used worldwide [4] The discharge of azo dyescontaining wastewaters into the environment may lead to thebioaccumulation which causes toxic effect on aquatic life andeven carcinogenic and mutagenic effect on humans becauseof the conversion of azo group into aromatic amines [5 6]Aside from the human toxicity colour of dyes interrupts theaquatic environment by reducing light penetration gas solu-bility and interference of phytoplanktonrsquos photosynthesis [7]

Therefore treatment of textile wastewater becomes essentialbefore discharging into the water streams Additionallylimited supply and increasing cost of water for industrialsector have made the treatment and reuse of dyeing effluentmandatory to avoid the environmental pollution as well asreduce the production cost

Several physicochemical methodologies such as coagula-tion and flocculation aremost commonly used worldwide fortreatment of textile effluent [8 9] But some shortcomingssuch as excessive use of chemicals secondary pollutionlarge amount of sludge generation low efficacies and highoperational cost discourage the employment of these meth-ods [10] Alternatively the modern method bioremediationwhich utilizes the ability of bacteria fungi or its combinationsystem has emerged as an effective method for the treatmentof textile wastewaters [11ndash13]However higher price ofmicro-bial growth medium makes biological treatments expensiveand beyond the use at commercial levels Thus to overcomethe problem of higher cost of microbial growth medium and

Hindawi Publishing CorporationJournal of ChemistryVolume 2015 Article ID 894109 10 pageshttpdxdoiorg1011552015894109

2 Journal of Chemistry

make the bioremediation an efficient treatment technologythe use of agricultural waste as growth medium has beensuggested

A number of agricultural wastes and its by-products suchas sugarcane bagasse wheat straw corn cob rice bran andwheat bran are cheapest and abundantly available carbonsources [14] These are normally utilized as animal fodderand domestic fuel while a large portion is being disposed ofas waste [15] For instance approximately 14520 million tonsper year of wheat straw is available inAsia [16] However onlya small portion of wheat residues is used as animal feed andthe rest is removed from the field by burning which causes airpollution and affects humanhealth [17] Recently agriculturalwaste wheat bran has been used as growth medium formicrobial consortium and their further use in biodegradationof azo dye Trypan Blue under submerged conditions [18]

In this view the easily available agricultural waste wheatbran was further evaluated as a low-cost growth medium fordegradation of model azo dye RB 172 using a single cultureof P rettgeri strain HSL1 bacterium under submerged condi-tions Initially the optimization of conditions for enhanceddye degradation efficacy was performed The activities ofdye degrading enzymes laccase azoreductase and NADH-DCIP reductase were assayed spectrophotometrically Min-eralization of dye was determined by the reduction in CODand TOC values whereas biodegradation was confirmedby HPLC FTIR and GC-MS analysis Possible metabolicpathway for degradation of dye RB 172 has been constructedFinally the environmental risk assessment was performed byacute and phytotoxicity tests

2 Materials and Methods

21 Chemicals and Textile Azo Dye RB 172 21015840-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methylred nicotinamide adenine dinucleotide (NADH) anddichlorophenolindophenol (DCIP) were purchased fromSigma-Aldrich (St Louis MO USA) Textile azo dyeRB 172 (CAS number 85782-76-9 molecular formula =C28

H26

N6

O10

S3

molecular weight = 70274) was generouslygiven byMahesh Textile Processors (Ichalkaranji MS India)

22 Preparation of WB Medium Agricultural waste wheatbran was obtained from local market (Kolhapur MS India)sieved and oven-dried at 80∘C plusmn 10∘C for several hours untilthe weight was constant and then 5 gm dry wheat bran wastaken in 100mL distilled water This content was boiled for15min and the extract was separated by filtration throughWhatman grade number 1 filter paper which then dilutedto 100mL with distilled water and called AWWB mediumThe pH of AWWB medium was adjusted to 70 autoclavedfor 15min at 121∘C and used for further degradation experi-ments

23 Preenrichment of P rettgeri Strain HSL1 Prior to decol-orization experiments the preenrichment of P rettgeri strainHSL1 was routinely carried out in AWWBmedium A loopfulof bacterial stock culture was inoculated in 250mL Erlen-meyer flask containing 100mL of AWWB medium (pH 70)

and incubated at 30plusmn02∘C for 24 h under shaking conditions(120 rpm) The overnight grown culture was then used asinoculum for further dye decolorization experiments

24 Optimization of Decolorization Conditions All the decol-orization experimentswere carried out in 250mLErlenmeyerflask containing 100mL of preenriched P rettgeri strainHSL1 culture The optimization of conditions for enhanceddecolorization of dyeRB 172was carried out by one parameterapproach at a time Initially the effect of microaerophilic andaerobic incubation (shaking at 120 rpm) preenriched culturemedium pH (3ndash12) incubation temperature (20 30 37 40and 50plusmn02∘C) and dye concentrations (50ndash250mg Lminus1) wasevaluated At defined time of intervals the aliquots of culturesupernatant (3mL) were withdrawn and suspended particleswere removed by adding equal volume of methanol followedby centrifugation (7500timesg for 15min 4 plusmn 02∘C) [13] Theresulted clear supernatant was analyzed for decolorization atmaximum absorbance wavelength of 570 nm using UV-visspectrophotometer (Hitahi U-2800 Hitachi Tokyo Japan)The control flasks which were without dye or bacterialculture were also tested under the same conditions All theexperiments were conducted at least in triplicate at 30plusmn02∘Cand average values were calculated The decolorization wasexpressed in terms of percent using the formula

Decolorization ()

=

Initial absorbance(0 h) minusObserved absorbance after incubation

(119905)

Initial absorbance(0 h)

times 100

(1)

25 Dye Mineralization Analysis The mineralization of dyeRB 172 was confirmed by chemical oxygen demand (COD)and total organic carbon (TOC) analysis For this the controland decolorized culture broths were centrifuged (7500timesg for15min 4 plusmn 02∘C) and filtered through 045120583m celluloseacetate filter (Sterlitech Corporation Kent WA USA) toremove cell biomass The reduction in COD was determinedby dichromate closed reflux titrimetricmethod [19] and TOCby using a Sievers 5310C automated analyzer (GE Water ampProcess Technologies Boulder CO USA)

26 Analysis of Metabolites after RB 172 Decolorization Theextraction of metabolites produced after degradation of RB172 by P rettgeri strainHSL1 was carried out by centrifugation(10000timesg for 20min 4 plusmn 02∘C) The resulting supernatantwas added into an equal volume of ethyl acetate and mixedvigorously to dissolve metabolites The organic layer wasseparated air-evaporated and dried over anhydrousNa

2

SO4

The remaining metabolite residues were scrapped and dis-solved in 3mL of HPLC grade methanol Finally the samplewas filtered through 045120583m cellulose acetate syringe filter(Sterlitech Corporation Kent WA USA) evaporated to250 120583L in a fume hood and subjected to HPLC FTIR andGC-MS analysis to confirm biodegradation

HPLC analysis of control dye RB 172 and its decolorizedmetabolites were performed with Waters 2690 instrument(Waters Limited Hertfordshire UK) equipped with C

18

Journal of Chemistry 3

column (symmetry 46 times 250mm) The isocratic methodusing the methanol with a flow rate of 050mLminminus1 for10min and UV detector set at 280 nm was used [13] Atotal of 10 120583L of dye RB 172 dissolved in methanol andits degradation metabolites were manually injected into thecolumn and elution profile was observed FTIR analysiswas done in the mid IR region of 600ndash4000 cmminus1 withscan speed 16 using the Shimadzu 8400S spectrophotometer(ShimadzuCorporation Kyoto Japan)The samples preparedwith spectroscopic pure KBr were fixed in the sample holderand analyzed [13] The identification of metabolites formedafter decolorization was carried out using a QP2010 gaschromatography coupled with mass spectroscopy (ShimadzuCorporation Kyoto Japan) The ionization voltage was set at70 eV and gas chromatographywas performed in temperatureprogramming mode with Restek column (025mm times 30mlong) The initial column temperature was set at 40∘C for4min then increased linearly at 10∘Cmin to 270∘C and heldfor 4min Injection port temperature was 275∘C and massinterface was maintained at 300∘C The helium with a flowrate of 1mLmin was used as carrier gas for 30min of runtime [20]

27 Extraction and Activities of Biotransformation EnzymesExtraction of enzymes after decolorization of dye RB 172by P rettgeri strain HSL1 and control medium (withoutdye) was carried out as per the procedure described earlier[21] The bacterial cells were separated by centrifugation(7500timesg for 15min 4 plusmn 02∘C) and the resulted supernatantwas considered test sample for determination of extracellu-lar enzyme activities The separated bacterial biomass wasresuspended in 50mM potassium phosphate buffer (pH 74)homogenized and sonicated by giving 7 strokes of 30 s eachfor 2min interval based on 50 amplitude output at 4 plusmn 1∘C(Sonics-Vibracell ultrasonic processor) These sonicated cellswere again centrifuged (7500timesg for 15 4 plusmn 02∘C) and thesupernatant was used as a source of intracellular enzymesSimilar protocol was followed to quantify the enzyme activ-ities of control medium Enzyme extracted from the culturemedium without adding dye was considered control

Activities of oxidoreductive enzymes such as laccaseazoreductase and NADH-DCIP reductase were assayedspectrophotometrically at room temperature (30 plusmn 1∘C)Laccase activity was determined by measuring the oxidationof ABTS at 420 nm (120576

420

nm = 36000 (M cm)minus1) [22] Deter-mination of azoreductase activity was performed as per theprocedure of Chen et al [23] while NADH-DCIP reductaseactivity was assayed as reported previously [24] All enzymeactivity assays were conducted in triplicate and average rateswere calculated The protein content was determined by themethod of Lowry et al with bovine serum albumin as thestandard [25]

28 Toxicity Studies Environmental risk assessment of dyeRB 172 and its degradation metabolites accumulation inanimals was assessed by acute toxicity test with freshwaterorganism Daphnia magna as described elsewhere [11 26]The dye treated sample with P rettgeri strain HSL1 was

centrifuged (7500timesg for 20min 4 plusmn 02∘C) supernatant-collected and sterilized by passing through 045 120583m celluloseacetate syringe filterThe clear filtrate (100mL) was taken intoa 250mL Erlenmeyer flask and five 24 h old neonates of Dmagna were added The tests were performed at 20 plusmn 02∘Cfor 48 h in the absence of light and number of immobileorganisms was counted after exposing to light for 20 seconds

Toxicity of dye RB 172 and its degradation metabolitesto plants was analyzed at room temperature on two kinds ofeconomically important agricultural crops Sorghum vulgare(monocot) and Phaseolus mungo (dicot) as described earlier[13] Briefly ten seeds of both plants were daily irrigatedwith 10mL each of RB 172 (50mg Lminus1) and its degradationmetabolites (50mg Lminus1) Length of shoot root and seedgermination () was recorded after 13 days Both the testswere conducted in triplicate with control in distilled water

29 Statistical Analysis One-way ANOVA was analyzedand Tukey-Kramer multiple comparison test was performedwith GraphPad Prism to determine the significance of theparameter studied

3 Results and Discussion

31 Decolorization of Textile Dye RB 172 in AWWB MediumThe preliminary investigation on WB as growth mediumfor decolorization of RB 172 by P rettgeri strain HSL1 wascarried out under microaerophilic conditions The result ofthe UV-vis spectral analysis (400ndash800 nm) of the dye andits decolorized medium suggested that the P rettgeri strainHSL1 treated medium (20 h) showed enhanced reduction inthe absorbance indicating dye decolorization (Figure 1(a))The removal of colour indicates that WB can be utilized asgrowthmedium for decolorization of dyes which signifies thelow-cost treatment approach It is reported that Providenciasp SRS82 could decolorize textile triazo dye Acid Black 210in nutrient medium [20] In addition degradation of textileeffluent by a developed bacterial consortium consisting ofProvidencia sp SDS and Pseudomonas aeruginosa strain BCHhas been reported in yeast extract medium [21] As perour best knowledge this is the first report showing thedecolorization of textile azo dye by Providencia sp usingWBas growth medium under submerged conditions

For the successful operation of biological wastewatertreatment systems the impact of aeration that providesoxygen for bacterial growth and stimulates its contact withmedium substrates should be properly analyzed Moni-toring the efficiency under microaerophilic condition Prettgeri strain HSL1 showed gt99 decolorization dye RB172 (50mg Lminus1) within 20 h at 30 plusmn 02∘C whereas aerobiccondition achieved only 12 performance within the sametime and even 18 in 24 h (Figure 1(b))These results indicatethat aerobic condition strongly inhibited the decolorization ofdyeRB 172 Similar findingswere reported in a previous studywherePseudomonas sp SUK1 exhibited higher decolorizationrate of reactive azo dye Red BLI under microaerophilic con-dition whereas aerobic incubation showed only the growthbut no decolorization [27] It is reported that azoreductase


0

05

1

15

2

25

3

35

400 450 500 550 600 650 700 750 800

Abso

rban

ce (A

U)

Wavelength (nm)

Reactive Blue 172Decolorized broth

(a)

0

20

40

60

80

100

0 4 8 12 16 20

Dec

olor

izat

ion

()

Incubation time (h)

MicroaerophilicAerobic (shaking at 120 rpm)

(b)

Figure 1 (a) UV-vis spectral analysis of control dye RB 172 and its decolorized broth by P rettgeri strain HSL1 (b) Percentage of dyedecolorization under microaerophilic and aerobic conditions Data point represents the mean of three independent replicates plusmnstandarderror of mean (SEM) is indicated by error bars

is the key enzyme responsible for breakdown of azo bondof azo dyes and presence of oxygen normally inhibits theazo bond reduction [28] Furthermore aerobic conditionmay dominate the use of NADH and impedes the electrontransfer from NADH to azo bonds resulting in the decreaseddecolorization performance [29]Hence in this study furtherdecolorization of azo dye RB 172 was carried out only inmicroaerophilic conditions

32 Optimization of Decolorization Conditions To scale upthe decolorization process and provide an affordable treat-ment technology for textile wastewater the optimizationof decolorization conditions such as growth medium pHincubation temperature and dyes concentration was carriedout Result of the study demonstrated that bacterial straincould decolorize the dye at broad range of pH however theoptimum pH was found to be 70 (Figure 2(a)) A significantdecrease in the decolorization performance was observed atlower pH (3ndash5) and higher pH (9ndash12) The transport of dyemolecules across cell membrane has been known to governby pH of the medium which is considered the rate limitingstep in decolorization process [30]

The enhanced and maximum decolorization activity ofdye RB 172 by bacterial culture was observed at 30 plusmn 02∘Ctemperatures within 20 h of incubation in microaerophiliccondition (Figure 2(b)) Further increase (37 40 and 50∘C) ordecrease (20∘C) in incubation temperature resulted in reduc-tion in the decolorization performance Effect of temperatureon biodegradation of dyes might be associated with themicrobial growth and enzymatic status of bacterial cultureat respective conditions which determines its degradationabilities Agrawal et al reported that Providencia sp SRS82exhibited maximum dye decolorization activity for dye AcidBlack 210 at 30∘C temperature whereas lower and highertemperature than optimum have considerably decreased itsdecolorization rates [20]

The ultimate aim of wastewater treatment is to reducethe concentration of dyes Result of the decolorizationstudy at various concentrations (50ndash250mg Lminus1) showed thatcomplete and rapid performance was observed at 50mg Lminus1within 20 h by P rettgeri strain HSL1 (Figure 2(c)) Thedecolorization efficiency of bacterial culture was found to bedecreased at dye concentration above 100mg Lminus1 It has beensuggested that the concentration of dyes can influence thedecolorization efficiency of bacteria due to the toxic effectimposed at higher concentrations [31]

33 Dye Mineralization Analysis The efficacy of textilewastewater treatment is determined by the mineralizationof dye molecules in terms of decrease in COD and TOCcontents [32] Result of the dye decolorization by P rettgeristrain HSL1 at optimum conditions that is WB mediumpH 70 incubation temperature 30 plusmn 02∘C 50mg Lminus1 ofdye concentration and microaerophilic incubation suggeststhat the complete decolorization with significant reductionin COD (85) and TOC (52) was observed within 20 h(Table 1)These decreased magnitudes of analyzed parametersuggest the applicability of WB medium for growth of Prettgeri strain HSL1 and their use in mineralization of azodye RB 172 Additionally the remained agricultural residuesafter preparation of WB medium could be used as low-cost adsorbent for dye removal and subsequent degradationby SSF [33] But the SSF based methods work better withwater soluble dyes as dye must adsorb on solid substrateprior to degradation This signifies the importance of ourwork over several studies where biodegradation of textiledye was carried out using nutrient medium [34 35] It iswell known that cost of growth medium used has stronginfluence on overall bioremediation economics The marketprice of wheat bran displayed on the worldrsquos biggest onlinecommerce company httpwwwalibabacom is US $154ndash162metric ton while the cost of mostly used defined growth


0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

()

pH

(a)

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

()

Temperature (∘C)

(b)

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

()

Dye concentration (mg Lminus1)

(c)

Figure 2 (a) Effect of culture medium pH (b) incubation temperature and (c) initial dye concentrations on the percentage of dye RB 172decolorization by P rettgeri strain HSL1 Data point represents the mean of three independent replicates plusmnSEM is indicated by error bars

Table 1 Analysis of control dye RB 172 and its decolorized brothafter treatment with P rettgeri strain HSL1

Parameters Control dye Treated (after 20 h)COD (mg Lminus1) 1020 plusmn 50 153 plusmn 30TOC (mg Lminus1) 1587 plusmn 70 762 plusmn 40Colour removal () 0 99 plusmn 10Values are mean of three experiments plusmn standard deviation (SD)

medium nutrient broth is US $5000ndash20000metric ton Thishuge difference in price of wheat bran and nutrient mediumsignifies the importance of our work for designing affordablebiological wastewater treatment processes

34 Enzyme Analysis Results of the enzyme activity analysissuggest that P rettgeri strain HSL1 possesses laccase azoreductase and NADH-DCIP reductase enzyme system incontrol cells On the other hand significant induction inthe activities of laccase (60) azo reductase (159) andNADH-DCIP reductase (88) from decolorized mediumcells indicates its active involvement in breakdown of dye RB172 (Table 2) Higher induction in the activity of azoreductaseas compared to laccase highlights the dominance of reductiveenzymes in decolorization process Lade et al reported theinvolvement of azo reductase in enzymatic cleavage of azodye Trypan Blue by bacterial consortium [18] Additionally

Table 2 Enzyme activities during decolorization of dye RB 172 byP rettgeri strain HSL1

Enzymes Control cells (0 h) After decolorization(20 h)

Laccase1 0285 plusmn 004 0456 plusmn 005lowast

Azo reductase2 0162 plusmn 002 0420 plusmn 003lowast

NADH-DCIPreductase3 17 plusmn 205 32 plusmn 212lowast

Values are mean of three experiments plusmn standard error of mean (SEM)significantly different from control cells at lowast119875 lt 0001 by one-way analysisof variance (ANOVA) with Tukey-Kramer comparison test1120583M of ABTS oxidized minminus1mL of enzymeminus1mg of proteinminus12120583M of methyl red reduced minminus1mL of enzymeminus1mg of proteinminus1

3120583M of DCIP reduced minminus1mL of enzymeminus1mg of proteinminus1

the roles of oxidoreductive enzymes in the decolorization ofreactive azo dye Red HE3B have also been characterized inProvidencia sp SDS [21]

35 Biodegradation Analysis The HPLC analysis of controldye showed the presence of one major peak at retentiontime of 2702min and three minor peaks at retention timesof 2125 2801 and 3394min (Figure 3(a)) After the dyedecolorization process the disappearance of peaks as seen incase of the control and the formation of completely differentthree major peaks at retention times of 2521 3241 and


2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)

Figure 3 (a) HPLC chromatogram of the control dye RB 172 and (b) its decolorized products obtained after treatment with P rettgeri strainHSL1

297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500

(a) RB 172(b) Decolorized products

Tran

smitt

ance

()

Wavenumbers (cmminus1)

(a)

(b)

Figure 4 FTIR spectrum of (a) control dye RB 172 and (b) itsdecolorized products obtained after treatment with P rettgeri strainHSL1

3564min and two minor peaks at retention time of 3123and 3910min were observed (Figure 3(b)) The appearanceof new minor peaks and disappearance of the major peakin the decolorized dye products elution profile support thebiodegradation of RB 172

The FTIR spectrum of control dye RB 172 comparedwith extracted products is shown in Figure 4 The FTIRspectrum of the control dye exhibits specific peaks at 162029and 159851 cmminus1 due to the presence of azo groups ndashN=Nndashstretching (Figure 4(a)) The peak at 118552 cmminus1 corre-sponds to S=O stretching of sulfonyl chlorides while thepresence of peak at 99959 cmminus1 showed PndashO stretching as inphosphorus compounds

The FTIR spectrum of extracted products after decol-orization of dye RB 172 showed variation in the positionsof peaks when compared to control dye spectrum Thedisappearance of peaks at 162029 and 159851 cmminus1 indicatesthe reductive cleavage of dye RB 172 at azo bond position(Figure 4(b)) The peak at 297486 cmminus1 indicates the CndashHstretching of alkanes while the peak at 283371 cmminus1 showsCndashH stretching of ethers The peak obtained at 144205 cmminus1

is due to CndashH deformation of alkanes In addition peak at124696 cmminus1 shows OndashNO

2

vibration of nitrates while thepeak at 106706 cmminus1 suggests CndashOH stretching of primaryalcohols These changes in the FTIR spectrum are clearevidence for the degradation of dye RB 172 into simplermolecules like aliphatic amines and carboxylic acids Addi-tionally significant induction in the activities of azoreductaseand laccase suggested initial reductive cleavage of azo bond ofdye RB 172 and further breakdown of formed metabolites

The GC-MS analysis was carried out to identify themetabolites formed during decolorization of RB 172 bybacterial strain The gas chromatogram of degraded dyemetabolites showed the presence of several peaks howeveronly two peaks were identified by mass spectrum at retentiontimes of 1954 and 2310min (Figure 6) The structure ofidentified compounds assigned from fragmentation pat-tern and 119898119911 values obtained indicates the formation of4-(ethenylsulfonyl) aniline and 1-amino-1-(4-aminophenyl)propan-2-one as low molecular weight degradation metabo-lites

The pathway for RB 172 biodegradation by P rettgeristrain HSL1 has been proposed showing the possiblemetabolites produced (Figure 5) The GC-MS analysis andenzyme activities suggested the initial reductive cleavage ofazo bond which yields 4-(ethenylsulfonyl) aniline (119898119911 183)via formation of 34-diamino-6-[4-(1-amino-2-oxo-propyl)-phenylazo]-methyl-5-hydroxy-naphthalene-27-disulfonicacid (MW 52354) as unidentified metabolite The significantinduction in the activity of azoreductase and disappearanceof azo peak in the FTIR spectrum of decolorized productsalso support the reduction of dye RB 172 It is known thatazoreductase is responsible for the reductive cleavage ofazo bond which results in dye decolorization [36] Theunidentified metabolite [I] is supposed to be further cleavedat azo position to form low molecular weight compound1-amino-1-(4-aminophenyl) propan-2-one (119898119911 165) as finalproduct via azoreductase activity

36 Toxicity Analysis The treated textile wastewaters arebeing commonly discharged into the environmental sinksHence it becomes essential to assess the risk of treated


NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H

Reactive Blue 172(MW = 70274)

Azo bond cleavage by azoreductase


NH2 NH2

CH3

CH3

HO3S

NH2 +

4-(Ethenylsulfonyl) aniline mz = 183)

NH2

H2N

CH3

1-Amino-1-(4-aminophenyl) propan-2-one mz = 165)

34-Diamino-6-[4-(1-amino-2-oxo-propyl)-phenylazo]-methyl-5-hydroxy-naphthalene-27-disulfonic acid(MW = 183

(MW = 52354)

(MW = 164

Figure 5 Proposed metabolic pathway for the biodegradation of dye RB 172 by P rettgeri strain HSL1

wastewaters for animal and plants with high accuracy andecological relevance The acute and phytotoxicity assays areadvocated as essential tools for addressing these issues [2635] Acute tests with D magna have been suggested as aprimary screeningmethod for the evaluation of lethal toxicityof chemicals to mammals and humans [37] Result of theacute test showed 100 mortality of D magna in untreateddye RB 172 (50mg Lminus1) solution suggesting the toxic natureof dye (Table 3)The acute toxicity is assumed to occur in testorganismswhen the accumulated dye content equals a criticalconcentration In contrast the treatment of dye RB 172 withP rettgeri strain HSL1 was sufficient to completely detoxifythe dye as no mortality of D magna was observed in treatedsamples

Result of the phytotoxicity analysis revealed inhibitionof germination for each seed of S vulgare and P mungo

Table 3Mortality ofDmagna exposed to dye RB 172 and its culturesupernatants obtained after degradation by P rettgeri strain HSL1

Samples Mortality ()Distilled water 0 plusmn 00RB 172 (50mg Lminus1) 45 plusmn 20Treated dye medium 0 plusmn 00Values are mean of three experiments plusmn SD

by 70 and 60 respectively treated with 50mg Lminus1 ofdye RB 172 solution (Table 4) However near about 90germination was observed in both the seeds irrigated withdye degradation metabolites Additionally good elongationof shoot (92 and 102 cm) and root (36 and 41 cm) lengthsfor S vulgare and P mungo respectively was observed in dye


50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44

Mass spectra Mass spectra

Metabolites 4-(Ethenylsulfonyl) aniline [I]

mz 183 mz 165

Metabolites 1-Amino-1-(4-aminophenyl)propan-2-one [II]

Retention time (min) 2310Retention time (min) 1954

Figure 6 GC-MS analysis of metabolites obtained after decolorization of dye RB 172 by P rettgeri strain HSL1

Table 4 Phytotoxicity of the dye RB 172 and its metabolites obtained after degradation by P rettgeri strain HSL1

SamplesS vulgare P mungo

Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)

Distilled water 100 95 plusmn 05 38 plusmn 03 100 104 plusmn 04 45 plusmn 02RB 172 (50mg Lminus1) 30 45 plusmn 02lowast 22 plusmn 01lowast 40 58 plusmn 02lowast 21 plusmn 03lowast

Degradation metabolites 90 92 plusmn 04 36 plusmn 04 90 102 plusmn 03 41 plusmn 02Values are mean of three experiments plusmn SESeeds germinated in dye are significantly different from control (distilled water) at lowast119875 lt 0001 by one-way analysis of variance (ANOVA) with Tukey-Kramercomparison test

degradation metabolites grown plants The strong influenceof physiological characteristics in untreated dye grown plantssuggests that dye RB 172 has toxic effect on plants as itinhibited germination and affected shoot and root elongationThe overall findings of the degradation study and toxicityanalysis demonstrated that P rettgeri strain HSL1 is not onlyable to decolorize the dye RB 172 but also completely detoxifyitThis suggests the future application ofP rettgeri strainHSL1for low-cost biodegradation as well as detoxification of azodye contaminated wastewaters

4 Conclusions

Wheat bran was successfully utilized as the growth mediumfor degradation of dye RB 172 by using P rettgeri strainHSL1 A real market cost analysis of WB with defined growthmedium nutrient broth suggests that WB could be used asa low-cost growth medium for bioremediation processesThe low-cost wheat bran medium rapid degradation andcomplete detoxification of model azo dye by P rettgeri strainHSL1 revealed an economical and ecofriendly approach fordesigning azo dye containingwastewater treatment technolo-gies However further studies are required to explore the useof WB medium for growth of bacteria and their use in thetreatment of real textile effluent at reactor scale which is anobjective of our future research

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Harshad Lade performed the actual work and wrote thepaper Sanjay Govindwar and Diby Paul supervised the work

Acknowledgment

The authors would like to thank all the anonymous refereesfor their constructive comments and suggestions

References

[1] P Nigam I M Banat D Singh and R Marchant ldquoMicrobialprocess for the decolorization of textile effluent containing azodiazo and reactive dyesrdquo Process Biochemistry vol 31 no 5 pp435ndash442 1996

[2] A Bafana S S Devi and T Chakrabarti ldquoAzo dyes pastpresent and the futurerdquo Environmental Reviews vol 19 pp 350ndash371 2011

[3] B Manu and S Chaudhari ldquoAnaerobic decolorisation ofsimulated textile wastewater containing azo dyesrdquo BioresourceTechnology vol 82 no 3 pp 225ndash231 2002


[4] R G Saratale G D Saratale J S Chang and S P GovindwarldquoBacterial decolorization and degradation of azo dyes a reviewrdquoJournal of the Taiwan Institute of Chemical Engineers vol 42 no1 pp 138ndash157 2011

[5] H A Modi G Rajput and C Ambasana ldquoDecolorizationof water soluble azo dyes by bacterial cultures isolated fromdye house effluentrdquo Bioresource Technology vol 101 no 16 pp6580ndash6583 2010

[6] K Lu X-L Zhang Y-L Zhao and Z-LWu ldquoRemoval of colorfrom textile dyeing wastewater by foam separationrdquo Journal ofHazardous Materials vol 182 no 1-3 pp 928ndash932 2010

[7] V K Sharma ldquoAggregation and toxicity of titanium dioxidenanoparticles in aquatic environmentmdasha reviewrdquo Journal ofEnvironmental Science and Health Part A ToxicHazardousSubstances and Environmental Engineering vol 44 no 14 pp1485ndash1495 2009

[8] S Meric H Selcuk and V Belgiorno ldquoAcute toxicity removalin textile finishing wastewater by Fentonrsquos oxidation ozone andcoagulation-flocculation processesrdquoWater Research vol 39 no6 pp 1147ndash1153 2005

[9] A K Verma R R Dash and P Bhunia ldquoA review on chemicalcoagulationflocculation technologies for removal of colourfrom textile wastewatersrdquo Journal of Environmental Manage-ment vol 93 no 1 pp 154ndash168 2012

[10] L C Davies C C Carias J M Novais and S Martins-DiasldquoPhytoremediation of textile effluents containing azo dye byusing Phragmites australis in a vertical flow intermittent feedingconstructed wetlandrdquo Ecological Engineering vol 25 no 5 pp594ndash605 2005

[11] H Lade A KadamD Paul and S Govindwar ldquoBiodegradationand detoxification of textile azo dyes by bacterial consortiumunder sequential microaerophilicaerobic processesrdquo EXCLIJournal vol 14 pp 158ndash174 2015

[12] L Ma R Zhuo H Liu et al ldquoEfficient decolorization anddetoxification of the sulfonated azo dye Reactive Orange 16and simulated textile wastewater containing Reactive Orange16 by the white-rot fungusGanoderma sp En3 isolated from theforest of Tzu-chinMountain in Chinardquo Biochemical EngineeringJournal vol 82 pp 1ndash9 2014

[13] H S Lade T R Waghmode A A Kadam and S PGovindwar ldquoEnhanced biodegradation and detoxification ofdisperse azo dye Rubine GFL and textile industry effluent bydefined fungal-bacterial consortiumrdquo International Biodeterio-ration and Biodegradation vol 72 pp 94ndash107 2012

[14] R Singh V Kapoor and V Kumar ldquoUtilization of agro-industrial wastes for the simultaneous production of amylaseand xylanase by thermophilic actinomycetesrdquo Brazilian Journalof Microbiology vol 43 no 4 pp 1545ndash1552 2012

[15] N Sarkar S K Ghosh S Bannerjee and K Aikat ldquoBioethanolproduction from agricultural wastes an overviewrdquo RenewableEnergy vol 37 no 1 pp 19ndash27 2012

[16] S Kim and B E Dale ldquoGlobal potential bioethanol productionfrom wasted crops and crop residuesrdquo Biomass and Bioenergyvol 26 no 4 pp 361ndash375 2004

[17] B Gullett and A Touati ldquoPCDDF emissions from burningwheat and rice field residuerdquo Atmospheric Environment vol 37no 35 pp 4893ndash4899 2003

[18] H Lade A Kadam D Paul and S Govindwar ldquoA Low-CostWheat branmedium for biodegradation of the benzidine-basedcarcinogenic dye Trypan Blue using a microbial consortiumrdquoInternational Journal of Environmental Research and PublicHealth vol 12 no 4 pp 3480ndash3505 2015

[19] APHA Standard Methods for the Examination of Water andWastewater American Public Health Association WashingtonDC USA 2012

[20] S Agrawal D Tipre B Patel and S Dave ldquoOptimization oftriazo Acid Black 210 dye degradation by Providencia sp SRS82and elucidation of degradation pathwayrdquo Process Biochemistryvol 49 no 1 pp 110ndash119 2014

[21] S S Phugare D C Kalyani S N Surwase and J P JadhavldquoEcofriendly degradation decolorization and detoxification oftextile effluent by a developed bacterial consortiumrdquo Ecotoxi-cology and Environmental Safety vol 74 no 5 pp 1288ndash12962011

[22] C Eggert U Temp and K-E L Eriksson ldquoThe ligninolyticsystem of the white rot fungus Pycnoporus cinnabarinuspurification and characterization of the laccaserdquo Applied andEnvironmental Microbiology vol 62 no 4 pp 1151ndash1158 1996

[23] H Chen S L Hopper and C E Cerniglia ldquoBiochemical andmolecular characterization of an azoreductase from Staphylo-coccus aureus a tetrameric NADPH-dependent flavoproteinrdquoMicrobiology vol 151 no 5 pp 1433ndash1441 2005

[24] M D Salokhe and S P Govindwar ldquoEffect of carbon source onthe biotransformation enzymes in Serratia marcescensrdquo WorldJournal ofMicrobiology andBiotechnology vol 15 no 2 pp 229ndash232 1999

[25] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[26] F Elisangela Z Andrea D G Fabio R de Menezes CristianoD L Regina and C-P Artur ldquoBiodegradation of textile azodyes by a facultative Staphylococcus arlettae strain VN-11 usinga sequential microaerophilicaerobic processrdquo InternationalBiodeterioration amp Biodegradation vol 63 no 3 pp 280ndash2882009

[27] D C Kalyani P S Patil J P Jadhav and S P GovindwarldquoBiodegradation of reactive textile dye Red BLI by an isolatedbacterium Pseudomonas sp SUK1rdquo Bioresource Technology vol99 no 11 pp 4635ndash4641 2008

[28] K-T Chung and S E Stevens Jr ldquoDegradation of azo dyes byenvironmental microorganisms and helminthsrdquo EnvironmentalToxicology and Chemistry vol 12 no 11 pp 2121ndash2132 1993

[29] J-S Chang and C-Y Lin ldquoDecolorization kinetics of a recom-binant Escherichia coli strain harboring azo-dye-decolorizingdeterminants from Rhodococcus sprdquo Biotechnology Letters vol23 no 8 pp 631ndash636 2001

[30] N D Lourenco J M Novais and H M Pinheiro ldquoReactivetextile dye colour removal in a sequencing batch reactorrdquoWaterScience and Technology vol 42 no 5-6 pp 321ndash328 2000

[31] C I Pearce J R Lloyd and J T Guthrie ldquoThe removal of colourfrom textile wastewater using whole bacterial cells a reviewrdquoDyes and Pigments vol 58 no 3 pp 179ndash196 2003

[32] R O Cristovao A P M Tavares J M Loureiro R AR Boaventura and E A Macedo ldquoTreatment and kineticmodelling of a simulated dye house effluent by enzymaticcatalysisrdquo Bioresource Technology vol 100 no 24 pp 6236ndash6242 2009

[33] A A Kadam H S Lade S M Patil and S P Govindwar ldquoLowcost CaCl

2

pretreatment of sugarcane bagasse for enhancementof textile dyes adsorption and subsequent biodegradation ofadsorbed dyes under solid state fermentationrdquo BioresourceTechnology vol 132 pp 276ndash284 2013

[34] R G Saratale G D Saratale J S Chang and S P GovindwarldquoDecolorization and biodegradation of reactive dyes and dye


wastewater by a developed bacterial consortiumrdquo Biodegrada-tion vol 21 no 6 pp 999ndash1015 2010

[35] A A Telke S M Joshi S U Jadhav D P Tamboli andS P Govindwar ldquoDecolorization and detoxification of Congored and textile industry effluent by an isolated bacteriumPseudomonas sp SU-EBTrdquo Biodegradation vol 21 no 2 pp283ndash296 2010

[36] H Chen ldquoRecent advances in azo dye degrading enzymeresearchrdquo Current Protein and Peptide Science vol 7 no 2 pp101ndash111 2006

[37] L Guilhermino T Diamantino M Carolina Silva and AM V M Soares ldquoAcute toxicity test with Daphnia magnaan alternative to mammals in the prescreening of chemicaltoxicityrdquo Ecotoxicology and Environmental Safety vol 46 no3 pp 357ndash362 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


make the bioremediation an efficient treatment technologythe use of agricultural waste as growth medium has beensuggested

A number of agricultural wastes and its by-products suchas sugarcane bagasse wheat straw corn cob rice bran andwheat bran are cheapest and abundantly available carbonsources [14] These are normally utilized as animal fodderand domestic fuel while a large portion is being disposed ofas waste [15] For instance approximately 14520 million tonsper year of wheat straw is available inAsia [16] However onlya small portion of wheat residues is used as animal feed andthe rest is removed from the field by burning which causes airpollution and affects humanhealth [17] Recently agriculturalwaste wheat bran has been used as growth medium formicrobial consortium and their further use in biodegradationof azo dye Trypan Blue under submerged conditions [18]

In this view the easily available agricultural waste wheatbran was further evaluated as a low-cost growth medium fordegradation of model azo dye RB 172 using a single cultureof P rettgeri strain HSL1 bacterium under submerged condi-tions Initially the optimization of conditions for enhanceddye degradation efficacy was performed The activities ofdye degrading enzymes laccase azoreductase and NADH-DCIP reductase were assayed spectrophotometrically Min-eralization of dye was determined by the reduction in CODand TOC values whereas biodegradation was confirmedby HPLC FTIR and GC-MS analysis Possible metabolicpathway for degradation of dye RB 172 has been constructedFinally the environmental risk assessment was performed byacute and phytotoxicity tests

2 Materials and Methods

21 Chemicals and Textile Azo Dye RB 172 21015840-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methylred nicotinamide adenine dinucleotide (NADH) anddichlorophenolindophenol (DCIP) were purchased fromSigma-Aldrich (St Louis MO USA) Textile azo dyeRB 172 (CAS number 85782-76-9 molecular formula =C28

H26

N6

O10

S3

molecular weight = 70274) was generouslygiven byMahesh Textile Processors (Ichalkaranji MS India)

22 Preparation of WB Medium Agricultural waste wheatbran was obtained from local market (Kolhapur MS India)sieved and oven-dried at 80∘C plusmn 10∘C for several hours untilthe weight was constant and then 5 gm dry wheat bran wastaken in 100mL distilled water This content was boiled for15min and the extract was separated by filtration throughWhatman grade number 1 filter paper which then dilutedto 100mL with distilled water and called AWWB mediumThe pH of AWWB medium was adjusted to 70 autoclavedfor 15min at 121∘C and used for further degradation experi-ments

23 Preenrichment of P rettgeri Strain HSL1 Prior to decol-orization experiments the preenrichment of P rettgeri strainHSL1 was routinely carried out in AWWBmedium A loopfulof bacterial stock culture was inoculated in 250mL Erlen-meyer flask containing 100mL of AWWB medium (pH 70)

and incubated at 30plusmn02∘C for 24 h under shaking conditions(120 rpm) The overnight grown culture was then used asinoculum for further dye decolorization experiments

24 Optimization of Decolorization Conditions All the decol-orization experimentswere carried out in 250mLErlenmeyerflask containing 100mL of preenriched P rettgeri strainHSL1 culture The optimization of conditions for enhanceddecolorization of dyeRB 172was carried out by one parameterapproach at a time Initially the effect of microaerophilic andaerobic incubation (shaking at 120 rpm) preenriched culturemedium pH (3ndash12) incubation temperature (20 30 37 40and 50plusmn02∘C) and dye concentrations (50ndash250mg Lminus1) wasevaluated At defined time of intervals the aliquots of culturesupernatant (3mL) were withdrawn and suspended particleswere removed by adding equal volume of methanol followedby centrifugation (7500timesg for 15min 4 plusmn 02∘C) [13] Theresulted clear supernatant was analyzed for decolorization atmaximum absorbance wavelength of 570 nm using UV-visspectrophotometer (Hitahi U-2800 Hitachi Tokyo Japan)The control flasks which were without dye or bacterialculture were also tested under the same conditions All theexperiments were conducted at least in triplicate at 30plusmn02∘Cand average values were calculated The decolorization wasexpressed in terms of percent using the formula

Decolorization ()

=

Initial absorbance(0 h) minusObserved absorbance after incubation

(119905)

Initial absorbance(0 h)

times 100

(1)

25 Dye Mineralization Analysis The mineralization of dyeRB 172 was confirmed by chemical oxygen demand (COD)and total organic carbon (TOC) analysis For this the controland decolorized culture broths were centrifuged (7500timesg for15min 4 plusmn 02∘C) and filtered through 045120583m celluloseacetate filter (Sterlitech Corporation Kent WA USA) toremove cell biomass The reduction in COD was determinedby dichromate closed reflux titrimetricmethod [19] and TOCby using a Sievers 5310C automated analyzer (GE Water ampProcess Technologies Boulder CO USA)

26 Analysis of Metabolites after RB 172 Decolorization Theextraction of metabolites produced after degradation of RB172 by P rettgeri strainHSL1 was carried out by centrifugation(10000timesg for 20min 4 plusmn 02∘C) The resulting supernatantwas added into an equal volume of ethyl acetate and mixedvigorously to dissolve metabolites The organic layer wasseparated air-evaporated and dried over anhydrousNa

2

SO4

The remaining metabolite residues were scrapped and dis-solved in 3mL of HPLC grade methanol Finally the samplewas filtered through 045120583m cellulose acetate syringe filter(Sterlitech Corporation Kent WA USA) evaporated to250 120583L in a fume hood and subjected to HPLC FTIR andGC-MS analysis to confirm biodegradation

HPLC analysis of control dye RB 172 and its decolorizedmetabolites were performed with Waters 2690 instrument(Waters Limited Hertfordshire UK) equipped with C

18





420










0

05

1

15

2

25

3

35

400 450 500 550 600 650 700 750 800

Abso

rban

ce (A

U)

Wavelength (nm)


(a)

0

20

40

60

80

100

0 4 8 12 16 20

Dec

olor

izat

ion

()

Incubation time (h)


(b)








0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

()

pH

(a)

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

()

Temperature (∘C)

(b)

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

()


(c)
















2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)


297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500


Tran

smitt

ance

()


(a)

(b)






2






NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of





420










0

05

1

15

2

25

3

35

400 450 500 550 600 650 700 750 800

Abso

rban

ce (A

U)

Wavelength (nm)


(a)

0

20

40

60

80

100

0 4 8 12 16 20

Dec

olor

izat

ion

()

Incubation time (h)


(b)








0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

()

pH

(a)

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

()

Temperature (∘C)

(b)

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

()


(c)
















2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)


297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500


Tran

smitt

ance

()


(a)

(b)






2






NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

05

1

15

2

25

3

35

400 450 500 550 600 650 700 750 800

Abso

rban

ce (A

U)

Wavelength (nm)


(a)

0

20

40

60

80

100

0 4 8 12 16 20

Dec

olor

izat

ion

()

Incubation time (h)


(b)








0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

()

pH

(a)

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

()

Temperature (∘C)

(b)

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

()


(c)
















2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)


297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500


Tran

smitt

ance

()


(a)

(b)






2






NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0

20

40

60

80

100

3 4 5 6 7 8 9 10 11 12

Dec

olor

izat

ion

()

pH

(a)

0

20

40

60

80

100

20 30 37 40 50

Dec

olor

izat

ion

()

Temperature (∘C)

(b)

0

20

40

60

80

100

50 100 150 200 250

Dec

olor

izat

ion

()


(c)
















2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)


297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500


Tran

smitt

ance

()


(a)

(b)






2






NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


2125

2702

2801

3394

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(a)

3910

3564

32412521

3123

0035

0030

0025

0020

0015

0010

0005

0000

100 200 300 400 500 600 700 800 900 1000

(min)

Abso

rban

ce u

nit

(b)


297486

283371

144205

124696

106706

79559

99959

118552

159851

162029

102

100

98

1010

1005

1000

995

4000 3500 3000 2500 2000 1500 1000 500


Tran

smitt

ance

()


(a)

(b)






2






NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


NNN

OH

NS

O

O

O

S

O

O

N

OH

N O

O

[A]

[B]

[I]

H2C

H2C

H2N

NH2

NH2

HO3S SO3H

SO3H




NH2 NH2

CH3

CH3

HO3S

NH2 +


NH2

H2N

CH3



(MW = 52354)

(MW = 164








50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


50 100 150 200 250 300 350 400 450 500

0

50

100

()

69

49148

183118

96

50 75 100 125 150 175 200 225 2500

50

100

()

41

7461

130

81101

165115

149

44



mz 183 mz 165






Germination()

Shoot length(cm)

Root length(cm)

Germination()

Shoot length(cm)

Root length(cm)




4 Conclusions






Acknowledgment


References



































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of
































2

















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

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