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International Biodeterioration & Biodegradation xxx (2013) 1e8

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International Biodeterioration & Biodegradation

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Exploiting the potential of plant growth promoting bacteria indecolorization of dye Disperse Red 73 adsorbed on milled sugarcanebagasse under solid state fermentation

Avinash A. Kadama, Ashwini N. Kulkarni b, Harshad S. Lade c, Sanjay P. Govindwar d,*aDepartment of Biotechnology, Shivaji University, Kolhapur 416004, IndiabDepartment of Microbiology, Shivaji University, Kolhapur 416004, IndiacDepartment of Environmental Engineering, Konkuk University, Seoul 143-701, Republic of KoreadDepartment of Biochemistry, Shivaji University, Kolhapur 416004, India

a r t i c l e i n f o

Article history:Received 16 September 2013Received in revised form12 October 2013Accepted 14 October 2013Available online xxx

Keywords:DecolorizationSugarcane bagasseConsortium-RARBDisperse Red 73Solid state fermentation

* Corresponding author. Tel.: þ91 231 2609152; faxE-mail addresses: [email protected],

(S.P. Govindwar).

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.10.012

Please cite this article in press as: Kadam, Afermentation, International Biodeterioration

a b s t r a c t

Bioremediation of textile dyes adsorbed on agricultural solid wastes under solid state fermentation (SSF)using rhizospheric plant growth promoting microorganisms pose an ecofriendly, economically feasibleand promising treatment approach. The purpose of this study was to adsorb azo dye Disperse Red 73(DR73) on sugarcane bagasse (SCB) and its further bioremediation using consortium-RARB under SSF.The particle size of SCB 0.002 mm showed maximum adsorption (65%) for DR73. Kinetics of adsorption ofDR73 on milled SCB follows pseudo-second order kinetics. The individual cultures of Rhodobactererythropholis MTCC 4688, Azotobacter vinelandii MTCC 1241, Rhizobium meliloti NCIM 2757 and Bacillusmegaterium NCIM 2054 showed 44, 28, 50 and 61% decolorization of DR73 in 48 h respectively; while theconsortium-RARB showed complete decolorization in 48 h. Optimum moisture content, temperature andpH for decolorization of DR73 was found to be 90%, 30 �C and 6 respectively. DR73 biodegradationanalysis was carried out using HPTLC, FTIR and HPLC. Phytotoxicity and genotoxicity studies revealeddetoxification of DR73. Tray bioreactor study for decolorization of adsorbed DR73 on SCB suggests itsimplementations at large scale. Use of plant growth promoting bacteria’s consortium under SSF forbioremediation of adsorbed dyes gives a novel ecologically sustainable approach.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Textile dyeing processes are known to be most environmentalunfriendly industrial processes, because of the wastewaters pro-duced by them are of heavily polluted with different dyes, textileauxiliaries and chemicals (Sanghi et al., 2007). About 7 � 105 tonand approximately 10,000 different textile dyes are producedannually world-wide and 10% of these dyes may found in waste-water (Couto, 2009). Presence of color in textile wastewater causesdecrease in penetration of sunlight into waters, retards photosyn-thesis, inhibits aquatic biota (growth) and interferes with gas sol-ubility in water bodies which ultimately causes to the strongimpacts on aquatic ecosystem (Banat et al., 1996). In addition tothis, carcinogenic nature of dyes has been seriously considerationregarding human health (Kariminiaae-Hamedaani et al., 2007).

: þ91 231 [email protected]

All rights reserved.

.A., et al., Exploiting the poten& Biodegradation (2013), ht

Hence, it is essential to provide a textile wastewater treatmentapproach.

Textile effluent treatment using biological methods were moresuitable than chemical and physical methods (Saratale et al., 2009).Biological methods for decolorization mainly involve the use ofbacteria, fungi and plants (Lade et al., 2012; Khandare et al., 2013).Decolorization of textile dyes by using number of microorganismshas been already reported (Saratale et al., 2009). Textile wastewatertreatment using environmental friendly, non-pathogenic andecologically sustainable microorganisms put an additional insightover existing techniques of microbial bioremediation. Therhizobium-legume symbiosis offers an ability to convert atmo-spheric molecular nitrogen into forms usable by the plant, a processcalled biological nitrogen fixation (Mahadi et al., 2010). Plantgrowth-promoting rhizobacteria (PGPR) are naturally occurring soilbacteria which actively colonize plant roots and benefit plants byproviding growth promotion (Bashan et al., 2011). In view of this,use of PGRR for different pollutants bioremediation suggestsenvironmental friendly treatment approach (Bashan et al., 2011).There are few reports available which suggest the use of PGPR for

tial of plant growth promoting bacteria in bagasse under solid statetp://dx.doi.org/10.1016/j.ibiod.2013.10.012

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A.A. Kadam et al. / International Biodeterioration & Biodegradation xxx (2013) 1e82

bioremediation process (Ahmad et al., 1997; Ju et al., 2007), but thecombination of different plant growth promoting microorganismsfor degradation of textile dye was still scarce.

Sugar and alcohol industries produce sugarcane bagasse (SCB)as an agro-industrial waste. It is a largest natural fiber resourcebecause it mainly contains high cellulose content, higher regener-ation capacity and yield (Huang et al., 2012). Thousands of tons ofSCB have been produced daily by the sugarcane processing in-dustries (Darani and Zoghi, 2008). Removal of dyes using adsorp-tion on SCB and then adsorbed dye decolorization under the solidstate fermentation using PGPRs bacterial consortium ensure safe,ecofriendly and economical approach. Many agro-industrial wastesare found to have good potential to replace the standard mediumand the commonly used peat in rhizobial inoculant production(Rebah et al., 2007). Hence, growth of PGPRs on waste biomassleads to dyes removal and generated biomass may be further usedas rhizobial inoculants.

Large volume of textile wastewater was produced daily fromtextile industries, hence to use submerged culture conditions forwaste treatment is not practically applicable. However, removal ofdyes by adsorption process and SSF approach for biodegradationwas preferred for dye treatment (Murugesan et al., 2007). There-fore, use of SCB as a cheap agricultural waste for dye adsorption andmicrobial consortium PGPRs for biodegradation of dye gives envi-ronmental friendly and complete treatment approach.

This study shows the potential of rhizospheric bacterial con-sortium developed using Rhodobacter erythropholis MTCC 4688,Azotobacter vinelandii MTCC 1241, Rhizobium meliloti NCIM 2757and Bacillus megaterium NCIM 2054 for biodegradation of SCBadsorbed azo dye DR73 under SSF. FTIR, HPLC and HPTLC analysisused to study biodegradation. Phytotoxicity and genotoxicity con-firms detoxification of DR73. Tray bioreactor study suggests that toapply this treatment at large scale.

2. Methods

2.1. Dyestuff and chemicals

An analytical grade and of highest purity chemicals were usedfor study. The textile azo dye Disperse Red 73 (DR73) (I.U.P.A.CName e Disperse Rubin GFL, CAS Number e 16889-10-4) was ob-tained from Mahesh textile processing industry Ichalkaranji, India.Dimethyl sulphoxide were taken from S D Fine-Chem Ltd., India.Microbiological medium such as nutrient broth was taken fromHiMedia Laboratories Pvt. Ltd., India.

2.2. Milling of SCB

SCB was taken from Shri Chatrapati Rajaram Coperative SugarIndustry, Kolhapur, India. It waswashedwith tap water and dried insunlight. After drying it was ground in mixer and sieved to obtaindesired particle size using Micro-Mesh sieves (Industrial Netting,USA).

2.3. Microorganism and culture conditions

The microorganisms which already known for their rhizo-spheric plant growth promoting activity was collected. The mi-crobial cultures of Rhodobacter erythropholis MTCC 4688 andAzotobacter vinelandiiMTCC 1241 were received from the MicrobialType Culture Collection and Gene Bank, Institute of MicrobialTechnology, Chandigarh, India. RhizobiummelilotiNCIM 2757 and B.megaterium NCIM 2054 were received from the National Collectionof Industrial Microorganisms (NCIM), National Chemical Labora-tory, Pune, India. Nutrient medium containing (g l�1; peptone 10,

Please cite this article in press as: Kadam, A.A., et al., Exploiting the potefermentation, International Biodeterioration & Biodegradation (2013), ht

beef extract 2 and sodium chloride 10) was used for maintainingthe stock cultures at 4 �C.

2.4. Adsorption of textile dyestuff from textile effluent

Batch study experiments were carried out for adsorption ofDR73 on SCB. In 250 ml Erlenmeyer flasks 2 g of a SCB and 50 mlsolution of DR73 (300 mg l�1) was added. This mixture was kept atshaking 120 rpm for time 20 min. Then it was centrifuge for5000 rpm for 15 min to collect clear supernatant. Maximumabsorbance wavelength (520 nm) of DR73 was selected for mea-surement of color removal using UVevis spectrophotometer(Hitachi U-2800, Japan). The adsorption percentage was calculatedas reported earlier by Kadam et al., (2011, 2013a). Further, dyeadsorbed to SCB was used for the decolorization study. The effect ofparticle size of SCB on adsorption of DR73 (300 mg l�1) was studiedtaking the particle size of the SCB as 2, 0.2, 0.02 and 0.002 mm.

2.5. Adsorption kinetics study

The DR73 adsorbed on milled SCB (2 g, 0.002 mm) and itsadsorption kinetics was carried out using DR73 concentrations as100, 200, 300, 400 and 500 mg l�1 and keeping it for 5, 10, 15, 20and 25 min agitation time (Kadam et al., 2013a,b). Non-linearregression method was used to study pseudo-first order andpseudo second-order kinetics (Khambhaty et al., 2008; Lin andWang, 2009; Kadam et al., 2013a,b).

2.6. Consortium development and decolorization experiment

One loop full culture of Rhodobacter erythropholis MTCC 4688,Azotobacter vinelandii MTCC 1241, Rhizobium meliloti NCIM 2757and B. megaterium NCIM 2054 culture was inoculated into 3 mlnutrient medium separately and then it was incubated for 30 �C atstatic condition. In the 250 ml Erlenmeyer flasks DR73 adsorbed onthe SCB (2 g) were added. These flasks were sterilized after pHadjustment (7.5e8). The flasks were inoculatedwith 3ml of (0.5 ODat 530 nm) culture for decolorization study. The moisture contentwas maintained between 85 and 90%. All flasks were incubated for30 �C under static condition.

In order to develop the consortia RARB of Rhodobacter eryth-ropholis MTCC 4688, Azotobacter vinelandii MTCC 1241, Rhizobiummeliloti NCIM 2757 and B. megaterium NCIM 2054, each of thesebacterial culture were grown at 24 h in 3 ml of nutrient medium,and 0.75 ml from each bacterial culture were added aseptically indecolorization medium.

SCB adsorbed textile dye DR73 was desorbed using dimethylsulphoxide (DMSO) and used for measurement of decolorization assuggested by Kadam et al. (2011, 2013a,b). Decolorization per-centage was calculated by formula as described by Kadam et al.(2013a,b). All experiments were done in triplicates and abioticcontrols were included for each set.

2.7. Optimization of a carbon and nitrogen sources, pH andtemperature for decolorization

SCB was supplemented with 3 ml of 1% glucose, starch, beefextract, ammonium chloride (NH4Cl), urea, peptone, rice bran,yeast biomass or lactose in order to study its effect on a decolor-ization of DR73. The pH was kept to 2, 4, 6, 8 and 10 to analyze itseffect on DR73 decolorization by consortium-RARB. Differenttemperatures as 10,20, 30, 40 and 50 �C used to analyze their effecton DR73 decolorization with the pH of 6.5e7 and at static condi-tion. Moisture content of 80, 85, 90 and 95% were evaluated fordecolorization of DR73 by consortium-RARB.

ntial of plant growth promoting bacteria in bagasse under solid statetp://dx.doi.org/10.1016/j.ibiod.2013.10.012

Fig. 1. Structure of textile azo dye C.I. Disperse Red 73.

Fig. 2. Kinetics of adsorption of DR73 concentrations (100, 200, 300, 400 and500 mg l�1) on milled SCB with contact time (5, 10, 15, 20 and 25 min).

A.A. Kadam et al. / International Biodeterioration & Biodegradation xxx (2013) 1e8 3

2.8. Scale up using tray bioreactor

Five liter of the solution of DR73 (300 mg l�1) was mixed with200 g of milled SCB (0.002 mm). This mixture was stirred contin-uously. Dyes adsorbed SCB (solid slurry) was separated by passingthrough the muslin cloth and which removes the unabsorbed dye.The unautoclaved SCB slurry was taken in tray which having di-mensions 48 cm � 33 cm (length � width) and spread uniformlywith thickness of 2 cm pH (6e6.5) and moisture content (90%) wasadjusted. 24 h grown consortium-RARB (100ml) were inoculated inthe trays. These trays were incubated in non-sterile conditions andat the room temperature. After the 12 h interval of incubation 2 g ofsamples were removed from the trays and it was desorbed with10 ml DMSO for decolorization measurement.

2.9. Toxicological analysis

2.9.1. Phytotoxicity studyAfter the adsorption treatment the DR73 remains adsorbed on

SCB. Hence, phytotoxicity of DR73 adsorbed SCB is important.Plants Sorghum vulgare and Phaseolus mungo used for toxicityanalysis. The phytotoxicity evaluation of DR73 (500 ppm), SCBadsorbed DR73 and SCB biomass obtained after DR73 biodegrada-tion was carried out by the method described by Kadam et al.(2013a,b).

2.9.2. Genotoxicity assayComet assay was performed by the method reported by Acharya

et al. (2008) and Chakraborty et al. (2009). In this study, Allium cepabulbs were exposed to untreated and treated DR73 dye solution.Single cells were analyzed with 212 TriTek Comet Score version 1.5software. The parameters used to measure DNA damage were taillength (mm), % DNA damage (%T) and tail moment (TM).

2.10. Biodegradation analysis

The biodegraded metabolites were obtained by the methodmentioned earlier by Kadam et al. (2013a). DR73 Biodegradationhas been analyzed by using the FTIR, HPLC and HPTLC. Fouriertransform infrared spectroscopy (FTIR) has been performed usingthe model Perkin Elmer Spectrum one, USA, as method describedby Kadam et al. (2012). Analysis of high performance liquid chro-matography (HPLC) was carried out using a model of Waters 2690,Waters Corporation, UK, as per reported by Kadam et al. (2013a,b).High performance thin layer chromatography (HPTLC) analysis wasdone using model CAMAG, Switzerland as suggested earlier byKadam et al. (2013a,b).

2.11. Statistical analysis

One-way analysis of variance (ANOVA) with the TukeyeKramermultiple comparison tests was used for the data analysis (Hsu,1996).

3. Results and discussions

3.1. Adsorption studies

Removal of dyes using adsorption has been universal, fast andinexpensive method (Gupta et al., 2006; Kadam et al., 2013a,b). Useof activated carbons in adsorption method is an effective andcommercially applicable in the dyes treatment. Even, they arebeneficial for adsorption of textile dyes but its use is limitedbecause it requires adsorbent regeneration after every cycle and itshigher cost (Crini, 2006; Gupta and Suhas, 2009; Kadam et al.,

Please cite this article in press as: Kadam, A.A., et al., Exploiting the potenfermentation, International Biodeterioration & Biodegradation (2013), ht

2013a,b). Reactivation process for activated carbons causes sor-bent loss of 10e15% in each cycle (Kadam et al., 2013a,b). Therefore,the low cost adsorbents have been always superior to the activatedcarbon for removal of dye (Mittal et al., 2012; Kadam et al., 2013a,b).DR73 is a disperse azo dye (Fig. 1). Low cost adsorbent such as SCBwith different particle sizes viz. 2, 0.2, 0.02 and 0.002 mm showed38, 44, 41 and 65% adsorption for DR73, respectively. SCB withdifferent particle showed different adsorption capacities for dyeDR73. The finely ground 0.002 mm bagasse particles showed moreadsorption capacity. SCB aftermilling causes particle size reduction,surface area increased due to delignification and exposes morefunctional groups, which results in the enhancement of dyeadsorption (Kadam et al., 2013a,b). 0.002 mm particle size showedmore adsorption capacity and hence selected for further study.

3.2. Adsorption kinetics of DR73 on milled SCB

Fig. 2 shows the kinetic behavior of adsorbed DR73 qt (mg g�1)on SCB at given time t (min) (Kadam et al., 2013a,b). Non-linearpseudo first order equation is used to determine the rate constants(Kadam et al., 2013a,b).

qt ¼ qe�1� e�k1t

�(1)

where, qe is dye adsorbed (mg g�1) at equilibrium and qt is dyeadsorbed at t (min) and, the rate constant of adsorption is k1(min�1). For the k1 and calculated qe values calculation Originversion 9 software was used. Non-linear pseudo second orderequation is used to determine the rate constants (Kadam et al.,2013a,b)


Table 1Kinetic parameters of increasing initial DR73 concentrations (100, 200, 300, 400 and 500 mg l�1) on SCB (2 g) at shaking condition (120 rpm).

SR5B concentrations (mg L�1) qe,exp (mg g�1) Pseudo-first-order kinetic model Pseudo-second-order kinetic model

k1 (min�1) qe,cal (mg g�1) R2 k2 (g mg�1 min�1) qe,cal (mg g�1) R2

100 28.50 0.3481 26.11 0.992 0.02281 28.77 0.995200 64.0 0.3758 59.94 0.993 0.0114 64.64 0.996300 103.5 0.4706 98.25 0.995 0.0111 103.61 0.997400 122 0.5030 116.43 0.996 0.0110 121.97 0.998500 135 0.5308 130.46 0.998 0.0119 135.59 0.999

Table 2Decolorization of adsorbed dye DR73 by R. erythropholis, A. vinelandii, R. meliloti,B. megaterium and their consortium-RARB.

S.N. PGPRs used for decolorization % Decolorization

24 h 48 h 72 h

1. Bacillus megaterium NCIM 2054 35 � 1.0 46 � 1.2 61 � 0.662. Rhizobium meliloti NCIM 2757 38 � 1.4 40 � 2.0 51 � 0.913. Rhodobacter erythropholis MTCC 4688 18 � 1.3 36 � 1.0 42 � 0.994. Azotobacter vinelandii MTCC 1241 24 � 0.8 26 � 1.5 29 � 0.775. Consortium-RARB 50 � 1.3 71 � 1.2 CD

Values are mean of three experiments � SEM.CD-Complete decolorization.

0

20

40

60

80

100

2 4 6 8 10

% Decolorization

pH

% D

ecol

oriz

atio

n

60

80

100

% Decolorization

oriz

atio

n

[a]

[b]


qt ¼ k2q2e t=1þ k2qet (2)

where, second order rate constant is k2 (g mg�1 min�1) and Originversion 9 software was used for calculation of k2 and qecal. Pseudo-second-order qecal values were observed to be closed to qeexp valuesthan that of pseudo first order (Table 1). Similarly, pseudo-second-order R2 values are found to be more than pseudo first order(Table 1). These results designate that adsorption of DR73 onmilledSCB follows pseudo-second-order kinetic model. The obtainedpseudo second-order kinetics was indicates that, DR73 adsorptionon milled SCB shows sorption on to the external surface of adsor-bent and a diffusion into the interior. Similar mechanism of pseudosecond-order adsorption was reported by Gupta et al. (2012).

0

20

40

10 20 30 40 50

% D

ecol

Temperature (oC)

100

% Decolorization[c]

3.3. Decolorization of SCB adsorbed DR73

Phosphate solubilizing bacteria such as R. erythropholis andB. megaterium, symbiotic nitrogen fixing bacteria R. Meliloti, andfree living nitrogen fixing bacteria A. vinelandii ensures the goodrhizobial inoculum for plant growth promotion (Das andMukherjee, 1998; Mittal et al., 2008; Matias et al., 2009; Oliveiraet al., 2009). Bioremediation of oil by using R. erythropholis spe-cies was reported by Aoshima et al. (2006) and Huang et al. (2008).While, R. meliloti found to degrade aromatic and chloroaromaticcompounds (Ahmad et al., 1997). B. megaterium and A. vinelandii

Table 3Effect of various carbon and nitrogen sources on decolorization of adsorbeddye DR73 by consortium-RARB.

Carbon and nitrogen sources % Decolorization

Urea 78 � 1.00Peptone 53 � 1.20Ammonium chloride 53 � 0.56Yeast biomass 48 � 0.80Beef extract 47 � 0.75Glucose 47 � 0.98Lactose 49 � 1.50Rice bran 41 � 1.20Starch 35 � 0.31

Values are mean of three experiments � SEM.

0

20

40

60

80

80 85 90 95

% D

ecol

oriz

atio

n

% Moisture content

Fig. 3. Effect of pH (a), temperature (b), moisture content (c) on decolorization of DR73by consortium-RARB for DR73.

Please cite this article in press as: Kadam, A.A., et al., Exploiting the potential of plant growth promoting bacteria in bagasse under solid statefermentation, International Biodeterioration & Biodegradation (2013), http://dx.doi.org/10.1016/j.ibiod.2013.10.012

Table 4Tray bioreactor study for decolorization of adsorbed dye DR73 byconsortium-RARB.

Time in hours (h) % Decolorization

12 28 � 2.324 56 � 3.036 63 � 2.048 72 � 2.860 84 � 3.172 CD



species also have a bioremediation potential (Cheung and Gu,2005). But, still use of these PGPRs for textile dye treatment wasscarce. Use of PGPRs in combinationwith plants was highly studiedin phytoremediation technology. Earlier attempts were made to useof PGPR in microbes bioremediation using an individual microor-ganism (Cheung and Gu, 2005; Huang et al., 2008), however, noreports are available for the use of PGPRs in consortium for mi-crobial bioremediation of dyes. Complexity of microbial consortiumenables them to act on variety of pollutants (Lade et al., 2012). Theuse of PGPRs consortium for bioremediation of SCB adsorbed textiledyes not only gives an ecofriendly wastewater treatment but alsogenerate biomass which can be used as good rhizobial inoculantsfor plant growth promotion. R. erythropholis, A. vinelandii, R. melilotiand B. megaterium showed 42, 28, 50 and 30% decolorizationrespectively in 72 h (Table 2). While, the consortium-RARB wasshowed complete decolorization (decolorization to levels belowtheir detection limits) in 72 h (Table 2). The developed microbialconsortium-RARB showed enhanced decolorization whencompared with the individual microorganisms. Use of productsformed by one organism to another in a consortium has been en-hances degradation of the dye having complex structures (Kadamet al., 2011). Hence, the above given decolorization treatment us-ing PGPRs ensures sustainable and ecofriendly treatment approach.

3.4. Effect of carbon and nitrogen sources, pH, temperature andmoisture content on decolorization of SCB adsorbed DR73

In presence of nitrogen sources such as urea, peptone, ammo-nium chloride, yeast biomass and beef extract showed 78, 53, 53,48, and 47% decolorization by consortium-RARB in 24 h respec-tively (Table 3). However, the carbon sources such as glucose,lactose, rice bran and starch showed 47, 49, 41 and 35% decolor-ization of DR73 by consortium-RARB in 24 h respectively (Table 3).The decolorization was decreased in presence of carbon sources. Inpresence of organic nitrogen sources microorganisms can regen-erate NADH. This is then acts as an electron donor and causes to bereduction in the azo dye (Hu, 1994). Hence, a nitrogen sourceprobably enhances the decolorization potential. Consortium-RARBshowed 15, 34, 79 and 69% decolorization of DR73 at the pH of 2,4, 8 and 10 (Fig 3a). While, at the pH 6 Consortium-RARB was

Table 5Phytotoxicity study of the DR73 dye, DR73 adsorbed bagasse and metabolites obtained a

Parameters Water Phaseolus mungo

DR73 DR73 adsorbedSCB

SCB biomass obtaineafter DR73 biodegrad

Germination (%) 100 20 40 90Plumule (cm) 10 � 0.3 6.7 � 0.5** 6.5 � 0.7** 10.8 � 0.2*Radicle (cm) 7.9 � 0.5 4.8 � 0.1** 4.0 � 0.9** 8.6 � 0.3*

The values are significantly different from control at *P < 0.01, **P < 0.001 by one-wayValues are mean of three experiments � SEM.


decolorize the DR73 completely (Fig 3a). The pH shows a major rolein the dye decolonization rate (Saratale et al., 2009).

Consortium-RARB showed 47, 57, 97, 45 and 27% decolorizationat 10, 20, 30, 40 and 50 �C respectively (Fig 3b). Hence, 30 �C oftemperaturewas showed to be optimum for decolorization (Fig 3b).As increase in temperature above 30 �C might increases the mois-ture loss and causes to be decrease in the decolorization (Kadamet al., 2013a). Cessation in the microbial growth below the 30 �Cmay causes to be decrease in the decolorization. Decolorization ofadsorbed DR73 by consortium-RARB at themoisture content 80, 85,90 and 95 was found to be 26, 71, 98 and 63% respectively (Fig 3c).While, below the 90% of moisture content have been causesreduction in the decolorization (Fig 3c). Due to decrease inmoisturecontent microbial growth and contact of dye molecules with mi-crobial cell may reduce which leads to decrease in the decoloriza-tion (Kadam et al., 2013a,b). Moisture content of 90% was found tobe optimum for decolorization. These observations might helpduring scale up the protocol for the treatment of larger volumes oftextile effluents.

3.5. Tray bioreactor study

The SCB with particle size (0.002 mm) showed 65% adsorptionof DR73. The adsorbed dyestuff was decolorized in trays under non-sterile conditions. DR73 was decolorized 56, 72 and completedecolorization in 24, 48 and 72 h by consortium-RARB (Table 4).This study suggests that the flask experiments at batch scale weremade scale up to tray bioreactor. These results concluded scale upof the adsorption and decolorization experiment up to 5 lit. In theSSF conditions it will not only degrade dyes but also lead toenhancement in the nutritional value of the waste. Hence, it can beused as manure in agricultural fields (Robinson et al., 2001) andgenerated biomass as rhizobial inoculants (Rebah et al., 2007).

3.6. Toxicological analysis

3.6.1. Phytotoxicity analysisPlant bioassays are considered as the important tool to study

toxicity of compounds (Paul et al., 2012). Seeds of S. vulgare andPhaseolus mungo were showed germination of 20 and 20% in thepresence of DR73, respectively. SCB adsorbed DR73 has beenshowed 30% germination percentage (Table 5). However, S. vulgareand Phaseolus mungo have been showed enhancement in thegermination percentage of 90% in presence of SCB biomass ob-tained after DR73 biodegradation. Obtained results indicate sig-nificant increase in the germination percentage after the adsorbedDR73 biodegradation than control DR73 and SCB adsorbed DR73.Similarly, the plumule and radical lengths in presence of the SCBbiomass obtained after DR73 biodegradation was found to be veryclose to control distilled water. However, the lengths of plumuleand radical of plants were significantly reduced as compare to thecontrol plant in presence of DR73 and DR73 adsorbed on SCB(Table 5). Hence, it has been observed that dyes after adsorption

fter its degradation of adsorbed DR73 by consortium-RARB under SSF.

Water Sorghum vulgare

dation

DR73 DR73 adsorbedSCB

SCB biomass obtainedafter DR73 biodegradation

100 20 40 909.5 � 0.3 3.9 � 0.2** 5.9 � 0.2** 9.9 � 0.18.7 � 0.2 3.9 � 0.3** 3.8 � 0.6** 8.9 � 0.2

analysis of variance (ANOVA) with TukeyeKramer comparison test.


Table 6Detection of DNA damage in the root meristem cells of Allium cepa treated withDR73 and its degradation metabolites using Comet assay.

Analysis Samples

Control DR73 DR73 metabolites

TL (mm) 35 � 0.35 253 � 0.72 45 � 0.91Tail DNA (%) 11 � 0.50 28 � 0.88 13 � 0.42Tail moment 4 � 0.46 28 � 0.81 4.25 � 0.44



remains on adsorbents and having the toxic effect on to the plantbut its biodegradation under SSF significantly detoxify the dyes.

3.6.2. Genotoxicity analysisDifferent parameters such as percentage of tail DNA (% of DNA in

comet tail), tail length (mm), and tail moment (TM) were measured(Table 6). DR73 was showed very high values for these parameterscompare to control which suggest toxic nature of the dye DR73. Thevalues for these parameters in the control were found to be veryclose tovalues obtained inmetabolites suggests detoxificationof dye

Fig. 4. Comets observed; the sample analyzed as Control (a) dye DR73 (5

Fig. 5. FTIR analysis of control (a), and biodegraded metabolites of B. megaterium (b


(Table 6). Damage of the DNA found in the cells which exposed toDR73 (500 mg l�1) was significantly higher than that of control (Fig4). However, the DNA damage in the cells exposed to the degradedmetabolites has been showed significantly less (Fig 4). These resultssuggest significant reduction in the toxicity of DR73 after thebiodegradation by consortium-RARB. While, single cell gel electro-phoresis (comet assays) is a rapid, sensitive, inexpensive and simplemicroscopic method to study the genotoxicity analysis (McKelvey-Martin et al., 1993). Single-cell gel electrophoresis sensitivity wasallowed to be the rapid detection of the genotoxic potential ofcompounds (Phugare et al., 2011). The dyes were lead to stronggenotoxic effects on the A. cepa DNA in root meristems (Phugareet al., 2011). Comet assay for genotoxic analysis was proven to bebeneficial in the study of environmental biomonitoring and genetictoxicology (Fairbairn et al., 1995; Lee and Scott, 2003).

It is always a concern of toxicity, when one can use the dyedegraded SSF biomass as rhizobial inoculants (Kadam et al.,2013a,b). Phytotoxicity and genotoxicity revealed complete detoxi-fication of DR73. Hence, the degraded DR73 under SSF by PGPRsefficiently work as environmentally safe rhizobial inoculants.

00 mg l�1) (b) and degraded metabolites of DR73 (500 mg l�1) (c).

), A. vinelandii (c), R. meliloti (d), R. erythropholis (e) and consortium-RARB (f).


Fig. 6. HPLC analysis of control (a), and biodegraded metabolites of B. megaterium (b), A. vinelandii (c), R. meliloti (d), R. erythropholis (e) and consortium-RARB (f).


3.7. Biodegraded product analysis

Differential FTIR spectrum obtained after the decolorizationsuggests biodegradation of DR73 adsorbed SCB by an individualcultures as well as consortium-RARB (Fig 5). FTIR spectrum ofcontrol dye DR73 showed peaks at 3437, 2897, 2829, 2359, 2241,1597,1519,1411, 916 and 833 cm-1 which represents the presence ofNeH stretching, CeH stretching, CeH stretching, NeHþ stretching,C^N stretching, N]N stretching, NeH deformation, CeH defor-mation, CeH deformation and CeH deformation respectively (Fig5a). Differential spectrum obtained in individual cultures ofR. erythropholis, A. vinelandii, R. meliloti and B. megaterium and inconsortium-RARB suggests DR73 biodegradation (Fig 5). Peak1519 cm-1 in biodegraded product of R. erythropholis suggestspresence of azo bond (N]N) (Fig 5e). Hence, biodegradation ofDR73 by R. erythropholis does not remove the azo bond. However,

Fig. 7. HPTLC analysis of control (a), and biodegraded metabolites of B. megaterium


A. vinelandii, R. Meliloti, B. megaterium and consortium-RARBremove azo bond from the DR73 molecule. HPLC spectrum ofcontrol dye DR73 showed retention time of 2.891 min and changein retention time after decolorization of DR73 by R. erythropholis,A. vinelandii, R. meliloti and B. megaterium suggests DR73 biodeg-radation (Fig 6). Consortium-RARB showed retention time of 2.006,2.353, 2.580 and 3.062 after the decolorization of DR73 suggests itsbiodegradation. The HPTLC chromatogram showed the absence ofcontrol DR73 band in the metabolites lane suggested biodegrada-tion of DR73 by an individual as well as consortium-RARB (Fig 7).With respect to Rf values, control dye DR73 showed a peak of 0.81,where as an individual cultures and consortium-RARB biodegradeddye showed peaks of different Rf values than control DR73 suggestsits biodegradation (Fig 7). Disappearance of peaks in consortium-RARB (Fig 7) found to be more than individual cultures. Metabo-lite produced by one of the microorganisms might contribute in the

(b), A. vinelandii (c), R. meliloti (d), R. erythropholis (e) and consortium-RARB (f).



decolorization performance of other microorganisms which finallyleads to enhanced decolorization in consortium-RARB (Kadamet al., 2011).

4. Conclusion

Decolorization of SCB adsorbed DR73 under SSF conditionscarried out by an individual PGPRs as well as their consortium.While, the consortium-RARB has been showed enhanced decolor-ization as compared to an individual microorganisms. Being rhi-zospheric microorganism they are known to be efficient plantgrowth promoters and hence generated biomass after decoloriza-tion of textile dye may be successfully implemented as rhizobialinoculants. Phytotoxicity studies revealed that only adsorptionprocess does not detoxify the dye contaminant, but SSF of adsorbeddyes completely removes dye toxicity. FTIR, HPLC and HPTLCanalysis confirms biodegradation of DR73 by individual cultures aswell as by developed consortium-RARB. Successful implementationof lab scale tray bioreactor study suggests insights of developing thelarge scale treatment protocols.

Acknowledgments

The author Avinash A. Kadam is thankful to DST-SAP (Depart-ment of Science and Technology-Special Assisted Program) Gov-ernment of India for providing financial assistance during thisresearchwork. Ashwini N. Kulkarni is thankful to UGC for providingjunior research fellowship. Harshad S. Lade would like to thankUGC, New Delhi, India for providing Dr. D. S. Kothari PostdoctoralFellowship.

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