Water Research 96 (2016) 1e11

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Ipomoea hederifolia rooted soil bed and Ipomoea aquatica

rhizofiltration coupled phytoreactors for efficient treatment of textile
wastewater
Niraj R. Rane a, Swapnil M. Patil a, Vishal V. Chandanshive b, Suhas K. Kadam b,
Rahul V. Khandare a, Jyoti P. Jadhav b, Sanjay P. Govindwar b, *
a
Department of Biotechnology, Shivaji University, Kolhapur, India
b
Department of Biochemistry, Shivaji University, Kolhapur, India

a r t i c l e i n f o a b s t r a c t

Article history: Ipomoea aquatica, a macrophyte was found to degrade a highly sulfonated and diazo textile dye Brown 5R
Received 16 December 2015 up to 94% within 72 h at a concentration of 200 mg L
1. Induction in the activities of enzymes such as
Received in revised form azoreductase, lignin peroxidase, laccase, DCIP reductase, tyrosinase, veratryl alcohol oxidase, catalase and
29 February 2016
superoxide dismutase was observed in leaf and root tissue in response to Brown 5R exposure. There was
Accepted 11 March 2016
Available online 19 March 2016
significant reduction in contents of chlorophyll a (25%), chlorophyll b (17%) and carotenoids (30%) in the
leaves of plants. HPLC, FTIR, UVevis spectrophotometric and HPTLC analyses confirmed the biotrans-
formation and removal of parent dye from solution. Enzymes activities and GC-MS analysis of degra-
Keywords:
Decolorization
dation products lead to the proposal of a possible pathway of phytotransformation of dye. The proposed
Phytoremediation pathway of dye metabolism revealed the formation of Napthalene-1,2-diamine and methylbenzene.
Ipomoea aquatica Toxicity study on HepG2 cell lines showed a 3 fold decrease in toxicity of Brown 5R after phytor-
Ipomoea hederifolia emediation by I. aquatica. Hydrophytic nature of I. aquatica leads to its exploration in a combinatorial
Coupled phytoreactors phytoreactor with Ipomoea hederifolia soil bed system. Rhizofiltration with I. aquatica and soil bed
Treatment lagoon treatment by I. hederifolia treated 510 L of effluent effectively within 72 h. I. aquatica along with
I. hederifolia could decolorize textile industry effluent within 72 h of treatment as evident from the
significant reductions in the values of COD, BOD, solids and ADMI. Further on field trials of treatment of
textile wastewater was successfully carried out in a constructed lagoon.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction aesthetically pleasant and practically feasible technique for dye

effluent management (Khandare and Govindwar, 2015). Common
Textile effluents carry carcinogenic, mutagenic, allergic and garden ornamentals like Glandullaria pulchella, Petunia grandiflora,
cytotoxic dyestuff and other hazardous chemicals to our valuable Aster amellus, Gailardia grandiflora, Tagetes patula and Portulaca
water bodies and soils. These hazardous dyes are traditionally grandiflora have been shown to metabolize textile dyes with their
removed by physico-chemical techniques but the treatment lead to tissue cultures as well as nursery grown plants (Patil et al., 2009;
secondary pollution in the form of sludge and leachates formation. Kabra et al., 2011a; Khandare et al., 2011a,b; Watharkar et al.,
Use of biological agents like fungi and bacteria though appear 2013; Watharkar and Jadhav, 2014). These plants however
ecofriendly, their in situ administration is extremely difficult. Off possess limitations to treat large volumes of dye wastewater
late, phytoremediation is being endorsed as an effective secondary because of their terrestrial habit and less biomass. On the other
method for treatment of textile dyes and effluents. Use of plants side, their effective enzymatic machineries are capable of degrad-
proves advantageous being a green, solar energy driven, passive, ing complex structures of textile dyes. In vitro studies nonetheless
give an insight to actual dye metabolism by ornamental plants; use
of macrophytes can definitely prove more advantageous (Rane
et al., 2015). Therefore, combinatorial systems with aquatic and
* Corresponding author. Department of Biochemistry, Shivaji University, Kolha-
pur, 416004, India. terrestrial plants need to be evaluated to achieve better treatment.
E-mail address: spg_biochem@[Link] (S.P. Govindwar). Hybrid systems exploring plant and microbial synergisms have

[Link]
0043-1354/© 2016 Elsevier Ltd. All rights reserved.
2 N.R. Rane et al. / Water Research 96 (2016) 1e11

been tried at pilot scales. Augmentation of Pseudomonas putida cells et al., 2012) to remove solid residues, and the absorbance of the
to the soil planted with Portulaca grandiflora was found to achieve clear solution was measured at the wavelength of 470 nm and
more efficacious treatment than the individual plant and bacterial decolorization percentage was calculated.
systems (Khandare et al., 2013a). A similar reactor with Glandularia Abiotic stressful environments such as drought, salinity and
pulchella augmented with Pseudomonas montelii was reported to heat are known to cause alterations in a wide range of physiological
give an enhanced performance than unaugmented reactors (Kabra as well as biochemical processes in plants. Photosynthetic pig-
et al., 2013). A static reactor with Pogonatherum crinitum and ments are linked with electron transport chain and ultimately
immobilized Bacillus pumilus cells also revealed superior perfor- responsible for the conversion of energy from sunlight to a chem-
mance in treatment of dye effluent. Plant-plant consortium on the ical form of energy necessary for the functioning of plants body.
other hand is a less explored strategy for dye treatment and has Chlorophyll a, b and carotenoids content were estimated by
mostly been shown only with in vitro grown cultures. Consortium measuring 0.2 gm leaves of both control and test plants. The
of Petunia grandiflora and Gaillardia grandiflora was proposed to samples were crushed in 20 mL of 80% acetone taking a pinch of
reveal an enhanced decolorization of Navy Blue RX than their in- MgCO3 powder. The extracts were filtered through Whattman pa-
dividual cultures (Watharkar and Jadhav, 2014). Wild plants of Aster per No. 1 and centrifuged for 10 min at 2000g. The pigments were
amellus and G. pulchella in consortium were also found to show determined spectrophotometrically at 645 and 663 nm taking
efficient removal of Remazol Orange 3R than individual plants acetone as blank (Rane et al., 2015). Carotenoid content was esti-
(Kabra et al., 2012). The mentioned works have specifically mated at the wavelength of 470 nm and Arnon's equations were
explored common garden ornamentals for dye removal. A very used to convert absorbance measurements to mg Chl gm
1 leaf
limited number of pilot scale operational systems using macro- tissue (Arnon, 1949).
phytes are on record. For instance, Phragmites australis, Typha Calculations:
domingensis, Alternenthera philoxeroides were proposed with in- For total chlorophyll
dependent constructed wetlands studies on removal of textile dyes  
from wastewater (Ong et al., 2010; Shehzadi et al., 2014; Rane et al., Chla mg gm
1 ¼ ½ð12:7  A663 Þ
ð2:6  A645 Þ
2015). Large scale treatment of textile dye effluents with combi-
natorial system of plants has seldom been reported. A 200 L volume  mL acetone=mg leaf tissue
of waste water from tie and dye industry was shown to be treated  
by cocoyam and cattail plants in independent engineered wetland Chlb mg gm
1 ¼ ½ð22:9  A645 Þ
ð4:68  A663 Þ
systems (Mbuligwe, 2005).
Use of macrophytes appears to be more logical for treatments of  mL acetone=mg leaf tissue
large volumes of effluents. Large biomasses of macrophytes are
For carotenoids
additionally advantageous as it facilitates greater accumulation and
subsequent degradation of the dyes. Macrophytes can grow nor- Cxþc ¼ 1000 A470
1:90Chla
63:14 Chlb =214; ðx
mally under aquatic environment and are able to handle fluctuating
dye loads. In the present work, an earlier reported textile dye ¼ xanthophylls and c ¼ carotenesÞ
phytoremediator Ipomoea hederifolia (Rane et al., 2014) was used to
treat textile dye build soil bed reactor facilitating the rooting in soil
surrounding. This soil bed was coupled with another reactor with
Ipomoea aquatica plantation freely floating on a grid support on 2.2. Enzyme assays and anatomical studies of stem and roots
wastewater. A total volume of 510 L was treated successfully with during dye removal
rhizofiltration by Ipomoea aquatica followed by soil bed reactor of
Ipomoea hederifolia. Looking after decolorization performance and Roots, stems and leaves of I. aquatica were cut, equally weighed
longevity, I. aquatica was further utilized in an industrial scale (2 gm), finely chopped and then separately suspended in 50 mM
lagoon of a capacity of 60,000 L and significant treatment of the real potassium phosphate buffer (pH 7.4). The chopped tissues of roots,
textile effluent was achieved in situ. stems and leaves were then ground separately in a mortar and
pestle followed by homogenization in a glass homogenizer and
2. Materials and methods centrifuged further at 8481 g for 20 min to obtain cell free extract
and further used as the enzyme source (Khandare et al., 2012).
2.1. Collection of plant material, dye decolorization studies and Activities of all the enzymes lignin peroxidase (LiP), laccase,
photosynthetic pigments analysis DCIP reductase, tyrosinase, azoreductase, veratryl alcohol oxidase
(VAO), riboflavin reductase, catalase and superoxide dismutase
I. aquatica collected from river ‘Panchganga’ at Kolhapur, India (SOD) were determined spectrophotometrically at room tempera-
were allowed to grow in water tanks. A stock of the plant was ture in the case of control and test plants. LiP activity was deter-
maintained by placing them in normal tap water for 15 d to get mined by measuring the formation of propanaldehyde at 300 nm in
acclimatized and further exposed to the effluent for treatment. The a reaction mixture of 2.5 mL containing 100 mM n-propanol,
plants showed vigorous growth and dense root system after accli- 250 mM tartaric acid, and 10 mM H2O2 (Shanmugam et al., 1999).
matization in effluent treatment reactor. Elongation in the lengths Laccase activity was determined using o-toludine as a substrate.
of roots up to 70 cm was observed. The decolorization experiments Stocks of o-toludine were prepared as 50 mM o-toluidine in 95%
were initially carried out with wild plants using the Brown 5R and a ethanol and for the final assay reaction 2 mM o-toluidine in
dye mixture containing structurally variable dyes like Direct Red 200 mM sodium acetate was used. In a reaction mixture of 2 mL
2B, Brilliant Blue, Green HE4B, Remazol Red, Brown 5R and Methyl containing 1.5 mL of 0.1 M acetate buffer (pH 4.8), 0.3 mL of o-to-
Orange. The initial experiments were carried out in 500 mL luidine and 0.2 mL of enzyme was used. Change in optical density
Erlenmayer flask containing 200 mL dye solution at a concentration was measured at 366 nm (Miller et al., 1997). Tyrosinase activity
of 200 mg L
1 in plain distilled water. Absorbance of dye solution was measured as described in earlier report (Zhang and Flurkey,
was recorded at an interval of 6 h each by removing 1 mL of solu- 1997). NADH-DCIP reductase was measured in cell-free extract as
tion. This solution was centrifuged at 4561g for 10 min (Khandare reported earlier by (Salokhe and Govindwar, 1999). Veratryl alcohol
 N.R. Rane et al. / Water Research 96 (2016) 1e11 3

was used as a substrate for measuring veratryl alcohol oxidase 2.4. Toxicity evaluation
activity. 1 mM veratryl alcohol in 0.05 M citrate phosphate buffer
(pH 3.0) and enzyme were taken in a total 2 mL reaction mixture. Its Toxicity of Brown 5R was tested by MTT assay according to
oxidation was monitored room temperature was monitored by an Ghosh et al. (2014) with some modifications. The untreated and
absorbance increase at 310 nm due to the formation of vera- treated samples of Brown 5R were exposed to human hepatocel-
traldehyde. Riboflavin reductase and azoreductase activity was lular carcinoma cell line (Hep G2). Cell lines were incubated inde-
assayed by earlier reported methods (Kurade et al., 2011). Antiox- pendently for 24 h with Brown 5R and its metabolite solution at the
idant enzyme status was assessed by spectrophotometric assays. confluency of 90. After exposure, medium was replaced by fresh
Antioxidant enzymes that were analyzed include catalase and su- medium containing MTT at a final concentration of 0.5 mg L
1 and
peroxide dismutase (Vafaei et al., 2013). All enzyme assays were incubated further for 4 h. To dissolve formed formazan crystals,
run in triplicate. Average rates were calculated. One unit of enzyme 100 ml of solubilization solution (0.1% SDS in 0.01 N HCl) was added
activity was defined as a change in absorbance unit min
1 mg of to each well followed by overnight incubation at room tempera-
protein
1. All enzyme assays were performed at 27  C with refer- ture. Absorbance was measured at 570 nm keeping background
ence blanks that contained all components except the enzyme. The absorbance at 650 nm. EC50 values were calculated by Graphpad 6
protein contents of all the samples were determined using Lowry's Prism software.
method (Lowry et al., 1951).
Anatomy of roots and shoots were observed for the accumula-
tion and removal of dye. Transverse sections of stem and roots were 2.5. Construction of the coupled phytoreactor system
mounted in glycerin overlaying with cover slip. The results were
micro-photographed with Axio-Scope A1 Trinocular phase contrast I. aquatica, a macrophyte was found to decolorize textile dyes in
Microscope with attached camera at 40 X magnification. initial screening studies. A rhizofiltration system of a capacity of
340 L was built using I. aquatica with a surface coverage area of 75%
as proposed earlier by Rane et al. (2015). This rhizofiltration phy-
2.3. Analysis of biotransformation of Brown 5R, mixture of dyes and toreactor was coupled to a soil bed reactor system with a sub-
effluent mersible water pump with the flow rate of 150 L h
1. The soil bed
reactor had the dimensions of 1.2 m  0.61 m x 0.61 m. An iron grid
Separation of metabolites was achieved using reverse phase C- was placed at a height of 0.3 m to support the soil bed. A layer of
18 column (Enable 250  4.6 mm, 3 mm) in a Shimadzu promi- 7.5 cm was piled on the iron grid using large sand gravels. On this
nence HPLC system equipped with degasser DGU-20A 5R, low layer, another layer of same height was built using smaller sized
pressure quaternary pump LC 20 AD and photo diode array de- sand gravels. Fine sand particles were mixed with ground coconut
tector SPD-M20 A. All HPLC grade solvents used for analyses were mesh (cocopeat) and used to form another layer of similar height on
purchased from Sigma Aldrich. Solvents were filtered through 0.2 the layer of smaller sand gravels. Uppermost layer of loam soil was
m sterile CN membrane filters (C-152 M Axiva) using filtration as- put on the sand-cocopeat layer. This soil bed was used as a sub-
sembly (Riviera glasses Pvt. Ltd. Mumbai, India) and degassed by stratum for plantation of I. hederifolia. The outlet of rhizofiltration
ultrasonic cleaner (Revotek). The mobile phase consisted of water reactor via the submersible pump was connected to a drip system
(A) and organic mobile phase acetonitrile (B) with 20:80%, placed on soil layer of the bed reactor. This drip system was placed
respectively with a flow rate of 0.8 mL/min. The degradation of dye at a height of 20 cm from the soil bed. A continuous flow from
was confirmed by comparing it with UVeVisible spectra and rhizofiltration reactor to the on line drip to soil bed was maintained
spiking against control sample at 470 nm. All the data acquisition throughout the experiment. Effluent percolated through the layers
and post run analysis was performed by Lab Solution software of soil bed and treated by I. hederifolia roots and dripped down
from Shimadzu, Kyoto, Japan. through the iron grid. The collected water was again supplied to the
Ethyl acetate extracted control and treated samples were rhizofiltration reactor with valved pipes. The levels therefore were
dried on separate glass plates and mixed with pure KBr (IR- maintained due to continuous circulation for 72 h (Fig. 1). The
Spectroscopy grade) purchased from Spectrochem Pvt. Ltd., treated effluent was characterized further. I. hederifolia although
Mumbai, India. Pellets were prepared using samples and then found to tolerate marshy and dye contaminated soils, it is a
fixed in sample holder. The FTIR was first calibrated for back- terrestrial plant (Rane et al., 2014). Therefore, it was planned to
ground signal scanning by keeping dried KBr sample and then the have a soil bed to grow it in the built reactor system. Root systems
treated and untreated samples were processed one by one. FTIR of both the plants were thus utilized for effective treatment.
analysis was performed in the mid-IR region of 400e4000 cm
1
with 36 scan speed and keeping Happ-Genzell method of analysis
of spectrum. 2.6. Analysis of bacterial population from the unplanted and
GC-MS analysis of metabolites formed post degradation was planted soil bed reactor of I. hederifolia, and analysis of total
done on the basis of Mass/Charge ratio to get the idea about mo- biomass production
lecular weight of the metabolites. GC-MS analysis of metabolites of
Brown 5R was carried out using a Shimadzu make 2010 MS Engine, One gram of soil was collected every 24 h from the unplanted
attached with integrated gas chromatograph with a HP1 column and planted soil bed reactor while the effluent was passing through
(60 m long, 0.25 mm i.d., nonpolar). Helium was used as carrier gas the soil bed. The soil was dissolved in 0.9% NaCl and then serially
at a flow rate of 1 mL min
1. The injector temperature was main- diluted for 6 times. Bacterial population was calculated by counting
tained at 280  C with oven conditions maintained at 80  C kept colony forming units (CFU) on nutrient agar plates of a composition
constant for 2 min-increased up to 200  C with 10  C min
1, raised of [(g L
1): peptone 5, NaCl 5, yeast extract 1.5, beef extract 1.5] at
up to 280  C with 20  C min
1 rate. Compounds were identified on 30 ± 2  C and 1.5% agar.
the basis of mass spectra using the National Institute of Structure The root and shoot length, and fresh and dry weight (biomass)
and Technology library. Chemsketch 2.0 software was used to of I. aquatica and I. hederifolia were measured to monitor the effects
elucidate the intermediate compounds formed after degradation of textile effluent on the growth of plant. Dry weight was deter-
and pathway of degradation of Brown 5R was proposed. mined by incubating plant material at 75  C for 96 h.
4 N.R. Rane et al. / Water Research 96 (2016) 1e11

Fig. 1. Coupled phytoreactor with I. aquatica and I. hederifolia for treatment of textile effluent.

2.7. Characterization of dye mixture and textile effluent the same stock of plants were used for 10 cycles keeping the
retention period of 8 d and the plants were taken for the treatment
Simulated mixture of dyes and a real textile effluent were of real dye wastewater at the actual site of dye disposal. The effluent
characterized for American Dye Manufacturers Institute (ADMI) parameters were measured during the treatment.
value (Khandare et al., 2013a) for color estimation, Biochemical
Oxygen Demand (BOD) and Chemical Oxygen Demand (COD), total 3. Results and discussion
nitrogen content, total phosphorus content, Total Dissolved Solids
(TDS), Total Suspended Solids (TSS), concentration of chloride and 3.1. Decolorization of Brown 5R, a simulated dye mixture and a real
sulfate ions present before and after treatment (APHA, 1998). textile effluent by I. aquatica, and effect on photosynthetic pigments
Organic carbon content was determined by Walkley-Black proce- in leaves
dure (Jackson, 1967). Phosphorous was determined by Olsen's
method (Olsen and Sommers, 1982). Nitrogen content was Initial decolorization studies with I. aquatica revealed its po-
measured by Kjeldahl method (Bremner and Mulvaney, 1982). tential for phytoremediation. A 200 mL solution of Brown 5R at a
Heavy metals and micronutrient were estimated using Atomic concentration of 200 mg L
1 was decolorized up to 94% by
Absorption Spectrophotometer (Thermofisher AA203) (Lindsay and I. aquatica. Similarly, a simulated dye mixture and a textile effluent
Norvell, 1978). exposed independently to I. aquatica revealed 90 and 94% of
reduction in ADMI value, respectively within 72 h. Considering the
2.8. In situ treatment of real textile wastewater in a constructed phytoremediation potential of I. aquatica, it was explored at a pilot
lagoon scale rhizofiltration reactor reported earlier by Rane et al. (2015).
The contents of chlorophyll a, chlorophyll b and carotenoids
The on-site remediation looking at the hydrophytic nature and analyzed in the dye unexposed and exposed plants revealed a
dye removal potential of I. aquatica was carried out in a rectangular significant reduction in their concentrations. Chlorophyll a, chlo-
shaped lagoon with dimensions of (10  6  1 m) and covered with rophyll b and carotenoids were decreased by 25, 17 and 30%,
plastic paper to avoid a percolation of effluent to groundwater re- respectively after the exposure of Brown 5R when compared to
sources. Pretreated textile wastewater with notable color and odor control (Data not shown). A similar reduction in photosynthetic
was taken for phytotreatment in this lagoon. The lagoon was filled pigments concentration was observed in Alternanthera philoxer-
up to a height of 1 m, so that the total volume to be treated was oides leaves upon exposure to Remazol Red (Rane et al., 2015).
60,000 L. The effluent used was sludge-less and allowed for treat-
ment for 24 h. The experiment was carried out at static condition. 3.2. Enzymatic and anatomical studies on plant tissues of
Initially the samples were collected from lagoon and subjected to I. aquatica before and after treatment of brown 5R
analysis of removal of color, change in COD, BOD, pH, TSS, TDS,
organic load and heavy metals. This lagoon was then covered Since the I. hederifolia enzymatic involvement was already pro-
approximately 75% of its total surface area by I. aquatica for effec- posed in an earlier report (Rane et al., 2014), only I. aquatica enzyme
tive treatment. The same plants were again exposed to next cycle of analysis was carried out. The activities of oxidative enzymes like
effluent treatment and the efficiency was checked. For recycling, laccase, LiP, tyrosinase and VAO were found to be induced by 75,
 N.R. Rane et al. / Water Research 96 (2016) 1e11 5

350, 311 and 396% in root tissues; 18, 35, 459 and 108% in stem sample of Brown 5R showed the appearance of new four peaks at
tissues and 99, 33, 31 and 757% in leaf tissues, respectively. Similarly, retention time of 2.89, 3.04, 3.23 and 3.82 min (Fig. 4b). The
varying inductions in activities of reductive enzymes such as azor- mixture of five dyes displayed six peaks with retention times of
eductase, NADH-DCIP reductase and riboflavin reductase by 357, 138 0.96, 1.22, 1.38, 1.56, 2.10 and 2.54 min (Fig. 4c). Dye metabolites on
and 94% in root tissues and 207, 162 and 117% in stem tissues, the other hand showed appearance of only four peaks at the
respectively was observed. The activity of azoreductase was retention time of 0.97, 1.27, 1.39 and 1.52 min (Fig. 4d). Similarly,
completely absent in leaf tissues. However, NADH-DCIP reductase HPLC analysis of textile effluent before and after treatment was
and riboflavin reductase activities were induced by 154 and 99%, carried out. The untreated effluent sample showed the presence of
respectively. The induced activities show the probable role of these five peaks at 0.79, 1.07, 1.09, 1.41 and 1.75 min (Fig. 4e). While,
enzymes in dye removal. The involvement of similar sets of enzyme treated sample of effluent revealed the presence of new five peaks
in decolorization of a number of textile dyes is a well known phe- at 1.96, 1.38, 1.42, 1.57 and 1.78 min (Fig. 4f). Thus, the variable HPLC
nomenon (Khandare and Govindwar, 2015). The oxidative stress profiles of the treated and untreated dye, textile effluent and dye
marker enzymes such as SOD and CAT also showed induction in the mixture confirmed their biotransformation to other new
activities after a 72 h dye exposure. Root, stem and leaf tissues compounds.
revealed inductions in the activities of SOD and CAT by 708 and 213; FTIR spectra of the untreated Brown 5R showed the peaks at
104 and 240; and 76 and 58%, respectively (Table 1). The textile dyes 3453.66, 2923.70, 2856.19, 2358.06, 2097.17, 1614.95, 1565.29,
and effluent have been shown to impart abiotic stresses to plants of 1490.06, 1126.64, 1417.73, 1306.33, 1185.78, 1119.23, 1037.74, 803.86,
A. philoxeroides inducing the expression of SOD and CAT enzymes 724.78 and 673.66 cm
1 represent OeH stretching, CeH stretching,
(Rane et al., 2015). NHþ stretching, NHþ 3 stretching, NH2 group, CN Stretching and NH
Anatomical study of stem and root cells for understanding the deformations, N]O stretching, CeH deformation, S]O stretching,
histochemistry, movement and metabolism of Brown 5R was car- CeOH stretching, S]O stretching, CeH stretching in benzene ring
ried out. The control plant stem (Fig. 2a) and root (Fig. 3a) showed structure and CeCl stretching (Fig. 5a). While, after treatment with
no coloration of any cell and they appeared to be normal and un- I. aquatica showed peaks at 3416.05, 3205.80, 2943.47, 2862.46,
disturbed. After 12 h of Brown 5R dye exposure with plant (Fig. 2b) 2725.51, 1732.13, 1616.40, 1454.38, 1375.29, 1161.19, 987.59, 825.56,
was observed to be accumulated in the outer epidermal cells. As the 723.33 cm
1 representing NH2 stretching, OeH stretching, CeH
exposure continued the accumulation increased and reached up to stretching, O-D stretching, CeH deformation, CH3 deformation, S]
cortical cells at 24 h (Fig. 2c). Further, at 48 h (Fig. 2d) the accu- O Stretching and CeH deformations (Fig. 5b). Untreated simulated
mulation increased in the cortex along with disappearance of dye mixture of dyes showed peaks at 2906.82, 2345.52, 1689.70,
around epidermal layer, showing degradation and metabolism of 1627.97, 1425.44, 1251.84, 1091.75, 964.44 and 497.65 cm
1 repre-
Brown 5R. Phytotransformation in epidermal as well as cortical sent CeH stretching, NHþ stretching, C]O stretching, NO2eO
cells after 72 h (Fig. 2e) clearly revealed an increased dye removal. stretching, ReOeR stretching, CeH deformation and C-halogen
Whereas, the roots showed accumulation of Brown 5R in xylem and stretching (Fig. 5c). Whereas, biotransformed product showed
phloem tissues (Fig. 3b). The exposed plants were transferred to peaks at 4346.91, 3340.82, 3147.93, 2937.68, 2860.53, 2681.14,
normal tap water and the stem section revealed a clear cortex with 1730.21, 1635.69, 1454.38, 1377.22, 1276.92, 1120.68, 1076.32 and
some distortion in epidermal cells with a small amount of dye 725.26 cm
1 representing dimeric OeH stretching, OeH stretching,
(Fig. 2f). CeH stretching, O-D stretching, C]O stretching, C]O stretching,
CeH deformation, NO2 stretching, NO2]O vibrations, CeOH
stretching and CH deformation in aromatic structure (Fig. 5d). Real
3.3. Analysis of biotransformation and fate of metabolism of Brown
textile effluent before treatment showed peaks at 3228.95, 2933.83,
5R
2343.59, 1629.90, 1340.57, 1114.89 and 624.96 cm
1 representing
N]O stretching, CeH stretching, NHþ stretching, C]C stretching,
HPLC profile of untreated Brown 5R showed three peaks at the
CeN vibrations and S]O vibrations (Fig. 5e). However, after
retention time of 1.96, 2.21 and 2.74 min (Fig. 4a). While, treated

Table 1
Enzyme analysis of I. aquatica plants tissue at 0 h and after 72 h of 200 mg L
1 Brown 5R dye exposure.

Enzymes I. aquatica root cells I. aquatica stem cells I. aquatica leaf cells

Control Test Control Test Control Test

Laccasea 16.80 ± 0.48 29.39 ± 0.61*** 11.18 ± 0.25 13.20 ± 0.26*** 13.44 ± 0.30 26.78 ± 0.12***
Lignin peroxidasea 01.75 ± 0.07 07.87 ± 0.41* 13.63 ± 0.44 18.42 ± 0.76* 03.63 ± 0.22 04.83 ± 0.87*
Tyrosinasea 02.45 ± 0.33 10.07 ± 0.40* 0.44 ± 0.08 02.46 ± 0.13* 07.25 ± 0.27 09.52 ± 0.95*
Veratryl alcohol 0.036 ± 0.02 01.786 ± 0.17* 07.24 ± 0.23 15.048 ± 0.33*** 0.21 ± 0.36 01.80 ± 0.92*
oxidasea
DCIP reductaseb 04.67 ± 0.68 11.13 ± 0.86* 15.49 ± 0.72 40.48 ± 0.76*** 18.20 ± 0.50 46.26 ± 0.13***
Azo reductasec 08.46 ± 0.62 38.67 ± 0.63*** 03.02 ± 0.12 09.28 ± 0.22** NA NA
Riboflavin reductased 22.22 ± 0.40 43.15 ± 0.53** 24.31 ± 0.08 52.81 ± 0.42*** 14.90 ± 34 29.69 ± 0.11**
Catalasee 0.067 ± 0.005 02.11 ± 0.03* 0.05 ± 0.11 0.17 ± 0.22* 01.95 ± 0.02 03.10 ± 0.32**
Superoxide dismutasef 00.24 ± 0.007 01.93 ± 0.07* 0.94 ± 0.03 01.92 ± 0.19* 01.62 ± 0.66 02.86 ± 0.80**

Values are a mean of three experiments ± SEM. Significantly different from respective control (0h) at *P< 0.05, **
P< 0.01 and ***
P< 0.001by one-way ANOVA with Tukey
Kramer comparison test.
NA: No activity.
a
Activity in units min-1 mg
1.
b
mg of DCIP reduced min
1 mg protein
1.
c
mg of Azo dye reduced min
1 mg protein
1.
d
mg of Riboflavin reduced min
1 mg protein
1.
e
50% inhibition of the NBT photoreduction rate (U mg
1 protein).
f
Nano moles of H2O2 Utilized (U mg
1 protein).
6 N.R. Rane et al. / Water Research 96 (2016) 1e11

Fig. 2. Anatomy of stem of I. aquatica a) control plant, Brown 5R exposed plants b) at 16 h, c) 32 h, d) 48 h, e) 72 h and f) plants exposed to normal tap water for 24 h after 72 h of
Brown 5R exposure.

Fig. 3. Anatomy of root of I. aquatica a) control plant b) plant exposed to Brown 5R for 72 h.

degradation, different peaks at 3246.31, 3180.72, 2928.04, 2868.24, band at Rf of 0.79 with an absorbance of 149.4 AU. Whereas, treated
2239.43, 1716.70, 1631.83, 1452.45, 1269.20, 1093.67, 896.93, 788.91 effluent after removal of color showed three smaller peaks at Rf of
and 599.88 cm
1 representing NeH stretching, OeH stretching of 0.44, 0.79 and 0.90 with reduced absorbance of 11.1, 143.3 and 176.4
chelated compounds, CeH stretching, C^C stretching, CeH defor- AU.
mation, C]O stretching, R-O-R stretching, CeOH stretching, CeH Treated sample of Brown 5R containing intermediates and its
stretching and C-halogen stretching were revealed (Fig. 5f). Thus, metabolites were analyzed with GC-MS to know the chemical na-
the differential spectra of the treated and untreated samples of dye ture of extracted metabolites. Six separate peaks with m/z values of
Brown 5R, simulated dyes mixture and textile effluent confirmed 545, 339, 266, 171, 571 and 93 were obtained. The pathway of
their transformation. degradation of Brown 5R was predicted with the help of metabo-
HPTLC analysis (Fig. 6) of the control Brown 5R (Lane 1) showed lites obtained and analysis of induction in the enzyme activities of
two peaks at Rf value 0.08 and 0.97 with an absorbance of 67.5 and the enzymes in the roots, stem and leaves of I. aquatica. Brown 5R
237.8 AU. In lane 2 (metabolites of Brown 5R), showed a single peak was cleaved at azo bond giving a 4-aminophenyl 4-
at 0.94 Rf with an absorbance of 181.9 AU. In Lane 3 (untreated methylbenzenesulfonate and intermediate I; 4-aminophenyl 4-
simulated dye mixture) showed a distinguished appearance of five methylbenzenesulfonate undergoes deamination with the action
major bands at the Rf values of 0.02, 0.24, 0.30, 0.66 and 0.78 with of lignin peroxidase forming 4-methylbenzenesulfonic acid which
an absorbance of 58.8, 12.1, 26.8, 21 and 207.5 AU. While, Lane 4 is further undergoes desulfonation by laccase and gives a metab-
(treated dye mixture) revealed the presence of dye metabolites at Rf olite methylbenzene. Whereas, Intermediate I undergoes desulfo-
of 0.80 and 0.90 having an absorbance of 161.2 and 200.6 AU. Un- nation by laccase and gives 4'-[(E)-(2-aminonaphthalen-1-yl)
treated sample of real textile effluent (Lane 5) showed single large diazenyl]biphenyl-4-amine which further acted by azoreductase
 N.R. Rane et al. / Water Research 96 (2016) 1e11 7

Fig. 4. FTIR analysis of a) Brown 5R b) metabolites Brown 5R c) untreated dye mixture d) treated dye mixture e) untreated effluent and f) effluent after treatment.

Fig. 5. HPLC analysis of a) Brown 5R b) metabolites of Brown 5R c) untreated dye mixture d) treated dye mixture e) untreated effluent and f) effluent after treatment.

yielding Napthalene-1,2-diamine (Fig. 7). Lignin peroxidase is histological levels (Khandare and Govindwar, 2015). In this work,
known to cleave the dye molecules asymmetrically (Kabra et al., MTT assay was performed on Hep G2 mammalian cell line revealed
2011b; Khandare et al., 2011a, 2013b; Patil et al., 2012; Rane a 3 fold reduction in toxicity of Brown 5R after phytoremediation by
et al., 2015). The laccase has been reported to cleave the dyes by I. aquatica. The untreated Brown 5R showed and EC50 value of 93 mg
oxidative cleavage and desulfonation (Rane et al., 2015; Kagalkar while it was observed to be increase up to 298 mg after treatment
et al., 2015). revealing the reduced toxicity.

3.4. Toxicity analysis of Brown 5R and its metabolites 3.5. Performance of coupled phytoreactors in treatment of real
textile effluent
Most of the toxicity studies on textile dyes have been generally
carried out using phytotoxicity and cytogenotoxicity on plant cells. Co-plantation with different species was reported to be more
Aquatic animals like fishes have also been used to check toxicity at efficient for the treatment of textile dyes than individual plants
8 N.R. Rane et al. / Water Research 96 (2016) 1e11

Fig. 6. HPTLC plate image with 3-D spectral scan at 470 nm.

Fig. 7. Proposed pathway for degradation of Brown 5R by I. aquatica.

 N.R. Rane et al. / Water Research 96 (2016) 1e11 9

Table 2 3.6. Analysis of bacterial population before and after I. hederifolia

Investigation of wastewater quality before and after treatment with I. aquatica and plantation in the soil bed reactor, and effect of effluent on plant
I. hederifolia plants in coupled reactors of total volume 510 L.
growth and biomass
Treatment Before After treatment
Parameters Treatment (72 h) Plant roots are known to support a number of bacterial pop-
(0 h)
ulations in their vicinity which also helps in pollutant degradation
Color (ADMI) 138 ± 1.74 26 ± 1.78 (Glick, 2010). Analysis of soil bacteria to form colonies revealed that
Odor Specific No odor
the I. hederifolia plantation had a positive effect on bacterial pop-
pH 10.5 6.8
Alkalinity (mg CaCO3 L
1) 11.7 ± 0.76 2.4 ± 0.50 ulation even when the effluent was continuously flowing through
COD (mg L
1) 990 ± 11.74 160 ± 6.22 the system. The (CFUs) of bacterial population in unplanted and
BOD (mg L
1) 740 ± 9.04 170 ± 8.33 planted soil at 0 h was 8.8 ± 0.28  103 and 12.4 ± 0.80  103 CFUs,
Total Nitrogen (mg N L
1) 5.6 ± 0.80 2.2 ± 0.28
respectively. After 72 h, the planted soil revealed comparable CFUs
Total Phosphorus (mg P L
1) 2.92 ± 0.74 1.6 ± 0.46
Total Suspended Solids (mg L
1) 5.8 ± 0.45 2.9 ± 0.10
of 8.4 ± 1.16  103 than that of unplanted soil with CFUs of
Total Dissolved Solids (mg L
1) 456 ± 5.56 179 ± 5.00 3.8 ± 2.10  103. The unplanted soil showed 57% loss in CFU at 72 h
Chlorides (mg Cl
L
1) 156 ± 6.20 40 ± 7.90 while the planted soil revealed only a loss of 33%.

1
Sulphates (mg SO2
4 L ) 188 ± 2.38 46 ± 3.40 Root and shoot length, and root and shoot weight (fresh and
Values are a mean of three experiments with SD. dry) of I. aquatica and I. hederifolia were recorded to evaluate the
influence of textile effluent on their growth and development
(Kabra et al., 2012; Watharkar and Jadhav, 2014). I. hederifolia has (Table 3). Textile effluent slightly inhibited plant growth and
earlier been reported to possess a notable potential for dye and biomass production of both the plants in respective reactors.
effluent treatment (Rane et al., 2014). It was further planned to
explore I. hederifolia at a larger scale in combination with I. aquatica. 3.7. On site application of I. aquatica for treatment of primary
However, both these plants need different surroundings for culti- treated textile effluent
vation. As a result, a coupled system was build in which I. aquatica
being hydrophytic was directly exposed to effluent and I. hederifolia I. aquatica are hydrophytic plants and therefore were decided to
was planted on a soil bed as described in Section 2.5. be explored at industrial scale reactor system. A total of 60,000 L
The effluent after a circulatory treatment in the coupled phy- effluent treatment for 8 d by I. aquatica was carried out. The effluent
toreactors revealed significant reductions in important environ- after treatment in a lagoon for 192 h revealed significant reductions
mental safety parameters. The ADMI value, pH and alkalinity of the in important environmental safety parameters. Noteworthy
effluent after treatment were reduced by 81, 35 and 80%, respec- reduction in the ADMI, BOD, COD and total solids of the effluent
tively. The effluent before treatment had a specific pungent odor after treatment were reduced by 70, 76, 87 and 34%, respectively.
which was completely disappeared after treatment in the reactor The effluent before treatment had a specific pungent odor which
system. Other important parameters like COD, BOD, TDS, TSS, total was completely disappeared after the treatment in lagoon. Other
nitrogen, total phosphorus, chlorides and sulfates were also important parameters like organic carbon, total nitrogen, total
significantly reduced by 84, 77, 61, 50, 61, 46, 75 and 76%, respec- phosphorus, ferrous, chlorides and sulfates were reduced by 46, 63,
tively. This 72 h treatment of a total volume of 510 L effluent was 56, 32, 81 and 74%, respectively after treatment. Metals from textile
achieved with a synergistic involvement of I. aquatica and effluent were also removed by I. aquatica effectively. Concentra-
I. hederifolia plants (Table 2). In an earlier report, a plastic crate tions of chromium and lead were reduced by 15 and 27%, respec-
reactors built by exploring soil free conditions using Burhead plants tively. However, concentration of mercury was found below
revealed decolorization of Reactive Red 141 achieving a TDS and detectable levels (Table 4). Cadmium and lead are considered as
conductivity removal by 42 and 50% within 2 d (Noonpui and toxic heavy metals. The results obtained in lagoon were encour-
Thiravetyan, 2011). Interconnected reactors with P. australis plan- aging as a healthy growth of the I. aquatica, increased roots length
tation and horizontal-vertical flow system showed efficient treat- forming denser root mats as well as increase in vegetative growth.
ment of dyes from a textile effluent. Significant reduction in levels A substantial reduction in the color of treated effluent was observed
of BOD, COD, TOC, TSS and color by 66, 84, 89, 93 and 90%, in comparison with the untreated effluent. A constructed wetland
respectively was observed after treatment. Ammonia, total nitro- with Typha domingensis plantation of a capacity of 1000 L was
gen, sulfate ions and surfactant concentration were reduced found to decrease pH, and Cr, Ni and Zn from a tool industry
by
331, 52, 87 and 80%, respectively (Bulc and Ojstrsek, 2008). effluent (Haddad et al., 2006). Recently, a floating treatment
Static reactor with Pogonatherum crinitum plantation was also wetland developed using a mat of diamond jumbolon board with
revealed notable performance and removal of TDS, TSS, BOD, COD Brachiaria mutica plantation along with the root associated endo-
and TOC (Watharkar et al., 2015). phytic bacteria has been reported for effective treatment of sewage

Table 3
Effect of textile effluent on growth of I. hederifolia and I. aquatica in rooted soil bed and rhizofiltration phytoreactor.

Plant sample Length (cm) Fresh weight (g) Dry weight (g)

Root Shoot Root Shoot Root Shoot

Tap water 66 ± 4.80 38 ± 3.20 27 ± 2.66 34 ± 4.56 14 ± 1.20 16 ± 1.90

(I. aquatica)
Rhizofiltration 60 ± 2.78 30 ± 3.30 24 ± 1.80 30 ± 1.22 11 ± 1.60 14 ± 0.90
Phytoreactor
(I. aquatica)
Tap water 26 ± 3.40 35 ± 4.80 12.5 ± 2.44 22 ± 1.44 07 ± 1.99 10 ± 1.33
(I. hederifolia)
Soil bed phytoreactor 24 ± 3.44 34 ± 3.80 11.5 ± 2.56 19.5 ± 2.90 6.8 ± 1.60 8.6 ± 1.44
(I. hederifolia)
10 N.R. Rane et al. / Water Research 96 (2016) 1e11

Table 4
Performance of macrophyte I. aquatica in field application in constructed lagoon of size 10.66  6.09  0.91 m having capacity of approximately 60,000 L.

Parameters for textile effluent treatment Textile effluent treatment in lagoon of 60,000 L by
I. aquatica

0h 96 h 192 h

pH 8.1 7.8 7.5

Odor Specific No odor No odor
COD (mg L
1) 2149 ± 13.40 1087 ± 11.64 290 ± 12.40
BOD (mg L
1) 1389 ± 12.74 825 ± 13.10 345 ± 11.04
ADMI 83 ± 5.14 67 ± 4.66 25 ± 2.00
TS (gm L
1) 6.32 ± 0.70 5.65 ± 0.44 4.17 ± 0.66
TDS (gm L
1) 2.69 ± 0.06 2.16 ± 0.30 1.43 ± 0.28
TSS (gm L
1) 3.63 ± 0.20 3.49 ± 0.74 2.74 ± 0.96
Chlorides (mg Cl
L
1) 204 ± 3.26 96 ± 4.40 40 ± 2.77
Sulphates 198 ± 4.88 66 ± 3.74 52 ± 4.20

1
(mg SO2

4 L )
Organic Carbon (%) 4.46 ± 0.33 3.10 ± 0.48 2.42 ± 0.56
Total Nitrogen 6.10 ± 0.66 3.80 ± 0.84 2.30 ± 0.96
(mg N L
1)
Total Phosphorus 3.12 ± 0.90 1.90 ± 0.33 1.40 ± 0.88
(mg P L
1)
Ferrous (ppm) 416.23 e 286.66
Chromium (ppm) 4.23 e 3.60
Lead (ppm) 0.30 e 0.22
Mercury (ppm) BDL e e

Values are a mean of three experiments with SD. BDL: Below Detectable Levels.

effluents, that also removed heavy metal from the wastewater in a Appendix A. Supplementary data
microcosm (Ijaz et al., 2015). Treatment of huge volumes effluent
becomes more challenging because of pollutant load and envi- Supplementary data related to this article can be found at http://
ronmental parameters (Khandare and Govindwar, 2015). However, [Link]/10.1016/[Link].2016.03.029.
the lagoon reactor system with its performance shows prospects of
future.
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