Green remediation of textile dyes containing wastewater by Ipomoea hederifolia L.

Article abstract of DOI:10.1039/c4ra06840h

Wild plant and tissue cultures of Ipomoea hederifolia decolorize Scarlet RR (SRR) dye at a concentration of 50 mg L^-1 up to 96% and 90% within 60 and 96 h, respectively. Significant induction in the enzyme activities of Lignin peroxidase, laccase, 2,6-dichlorophenol indophenol reductase, superoxide dismutase, catalase and tyrosinase was found in the plant roots and shoots during decolorization. I. hederifolia was also able to treat a dye mixture and a real textile effluent efficiently with a reduction in the American Dye Manufacturers Institute (ADMI) value (color removal) up to 85% and 88%, BOD up to 65% and 63% and COD up to 62% and 68%, respectively. Detailed anatomical studies of the stem and root cells of I. hederifolia during uptake and degradation were carried out, showing a stepwise and mechanistic degradation of the model dye SRR. Products formed after dye degradation were analyzed by UV-Vis spectroscopy, FTIR, HPLC and HPTLC, which confirmed the phytotransformation of SRR, dye mixture and textile effluent. A possible pathway for the phytotransformation of SRR was proposed based on GC-MS analysis, which confirmed the formation of different metabolites with lower molecular weights. The phytotoxicity study revealed the non-toxic nature of the formed products. the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra06840h

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Green remediation of textile dyes containing
wastewater by Ipomoea hederifolia L.†
Cite this: RSC Adv., 2014, 4, 36623
Niraj R. Rane,a Vishal V. Chandanshive,b Rahul V. Khandare,c Avinash R. Gholave,d
d
b
*
Shrirang R. Yadav and Sanjay P. Govindwar
Wild plant and tissue cultures of Ipomoea hederifolia decolorize Scarlet RR (SRR) dye at a concentration of
50 mg Lꢀ1 up to 96% and 90% within 60 and 96 h, respectively. Significant induction in the enzyme activities
of Lignin peroxidase, laccase, 2,6-dichlorophenol indophenol reductase, superoxide dismutase, catalase
and tyrosinase was found in the plant roots and shoots during decolorization. I. hederifolia was also able
to treat a dye mixture and a real textile effluent efficiently with a reduction in the American Dye
Manufacturers Institute (ADMI) value (color removal) up to 85% and 88%, BOD up to 65% and 63% and
COD up to 62% and 68%, respectively. Detailed anatomical studies of the stem and root cells of
I. hederifolia during uptake and degradation were carried out, showing a stepwise and mechanistic
degradation of the model dye SRR. Products formed after dye degradation were analyzed by UV-Vis
spectroscopy, FTIR, HPLC and HPTLC, which confirmed the phytotransformation of SRR, dye mixture
and textile effluent. A possible pathway for the phytotransformation of SRR was proposed based on GC-
MS analysis, which confirmed the formation of different metabolites with lower molecular weights. The
phytotoxicity study revealed the non-toxic nature of the formed products.
Received 9th July 2014
Accepted 15th July 2014
DOI: 10.1039/c4ra06840h
www.rsc.org/advances
recalcitrant to degradation in water and soil can be ascribed to
their complex chemical structure. The persistence of toxic
1. Introduction
compounds in aquatic and soil environments may lead to
accumulation in phytoplankton, sh and vegetables.4 In devel-
oping countries, the textile dyes are not only part of textile
industry effluent but also add to the municipal sewages.3
A number of physicochemical methods, such as adsorption,
coagulation, occulation, ltration, photodegradation and
chemical oxidation, are available for dealing with the pollution
created by textile dyes. These methods are associated with high
cost, low efficiency, secondary pollution problems and the
inability to treat a wide array of dyes having structural diversity.2
Although these methods are employed to remove the colour,
their capacity to reduce the toxicity is still a matter of major
concern. These facts certainly demand the development of an
efficient, cost effective and eco-friendly technology for the
removal of these dyes from environment. The use of microor-
ganisms for purifying dye contaminated water has extensively
been studied and demonstrated but is still associated with
some limitations as far as in situ administration of pollutants is
concerned.5
Industrial developments are always associated with a cost in
terms of pollution of air, water bodies and soil by a number of
toxic compounds. The fast growing textile industry is one of the
sources to release toxic chemical compounds in the form of
dyes. Note that approximately 10–15% of the synthetic dyes
having carcinogenic and mutagenic effects are released during
the dying process, ultimately threatening all life forms.1 The
valuable water bodies needed for irrigation, drinking and other
household and industrial purposes are being polluted because
of the textile dyes released through effluent. Inclusion of textile
dyes in water reservoirs alters the vital parameters of water
bodies by inuencing the levels of chemical oxygen demand
(COD), biological oxygen demand (BOD), total organic carbon
(TOC), total dissolved solids (TDS), total suspended solids (TSS),
pH and color.2 Therefore, addressing the issue of these envi-
ronmental contaminants has become very important and needs
attention from environmentalists.3 This, at the end, results in
the development of odour and deterioration of overall water
quality in the reservoir. The ability of textile dyes to remain
The use of plants to decolorize and detoxify the textile dyes in
the laboratory, as well as at the actual site of contamination,
appears as an attractive option.6 Phytoremediation appears to
be an ideal potential approach for the treatment of pollutants; it
is more admired because of its solar-driven nature, cost effec-
tiveness, aesthetic advantages and long-standing applicability
as it can be directly employed at the polluted sites compared to
aDepartment of Biotechnology, Shivaji University, Kolhapur, India
bDepartment of Biochemistry, Shivaji University, Kolhapur, India. E-mail:
spg_biochem@unishivaji.ac.in; Fax: +91-231-2691533; Tel: +91-231-2609152
cSchool of Nanoscience and Technology, Shivaji University, Kolhapur, India
dDepartment of Botany, Shivaji University, Kolhapur, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra06840h
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other expensive methods of treatment.7 Some wild plants like
Phragmites australis, Blumea malcolmii, Typhonium agelliforme,
Rheum rabarbarum (rhubarb) and R. hydrolapatum have been
proposed for textile dye removal.6,8–11 Recently, common garden
plants such as Aster amellus, Glandularia pulchella, Portulaca
grandiora, Petunia grandiora, Zinnia angustifolia and Tagetes
patula have been shown to have great potential for textile dye
degradation.12–18 Though the use of nursery-grown garden
plants appears attractive, their use for the clean-up of actually
contaminated sites has yet to be demonstrated.
This work deals with the decolorization and degradation of a
commonly used disperse textile dye Scarlet RR (SRR), a dye
mixture and a real textile effluent by Ipomea hederifolia Linn.
This plant was procured from the actual site of dye disposal,
Ichalkaranji, India. Therefore, it was thought to have the ability
to grow in high dye concentrations; thus, it was better adapted
to these dye stress conditions. The plant also possesses char-
acteristics like a dense root system, the ability to grow in marshy
places and a non-edible nature. Moreover, the seeds of I.
hederifolia can germinate in the soil contaminated with dyes,
which makes it a favourable candidate plant for phytor-
emediation of textile dyes. This work also explores the removal
and degradation pattern by the plant at an anatomical level and
reveals the histological dye degradation process by I. hederifolia.
2.3. In vitro culture of I. hederifolia and its decolorization
potential
The role of bacteria in the decolorization was doubted upon the
rst exposure to dye; therefore, in vitro grown cultures of
I. hederifolia were obtained by a seed culture method. Seeds of
I. hederifolia were collected from the plants found at the actual
site of contamination, surface sterilized and cultured on MS
basal medium as per a method previously described.5 The half-
strength MS medium was discovered suitable for 90% of seed
germination. A one week old seedling with profuse roots was
used for phytoremediation studies. The tissue culture plants of
I. hederifolia also showed degradation of SRR, mixture of textile
dyes and real textile effluent taken for the study, which is
explained below.
2.4. Decolorization studies
Decolorization experiments were initially carried out with the
wild plants in the dye SRR and the dye mixture containing
Green HE4B, SRR, Remazol Black B, Remazol Red and Navy
Blue HE2R. The structures and names of all the dyes used in
this study are shown in the appendix. The experiments were
carried out in 250 mL Erlenmeyer asks containing 100 mL of
50 mg Lꢀ1 of dye solution in plain distilled water. As the
bacterial contamination was doubted during decolorization, the
experiments were independently performed with the tissue
culturally grown plantlets of I. hederifolia exposing them to
15 mL of dye solution in test tubes. Absorbance of the dye
solution was recorded at intervals of 6 h, each by removing 1 mL
of solution. This solution was centrifuged at 4561g for 10 min to
remove any solid matter if present and the absorbance of the
clear solution was measured at a wavelength of 510 nm. The
decolorization percentage was calculated as follows:
2. Materials and methods
2.1. Chemicals
2,20-Azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS)
and riboavin were available from Sigma Aldrich (St Louis, MO,
USA). Nicotinamide adenine dinucleotide (di-sodium salt), n-
propanol, 2,6-dichlorophenol indophenol (DCIP), veratryl
alcohol and catechol were purchased from Sisco Research
Laboratories, Mumbai, Maharashtra, India. Tartaric acid was
available from BDH chemicals (Mumbai, Maharashtra, India).
The MS Basal Medium was purchased from HiMedia Labora-
tories, Pvt. Ltd., India. The textile dyes SRR, Navy Blue HE2R,
Green HE4B, Blue 2 RNL, Direct Red 5B, Reactive Red 2, Red
HE3B, Remazol Red, Remazol Black B dyes and effluent were
obtained from Mahesh, dye processors and Common Effluent
Treatment Plant, Ichalkaranji, Maharashtra, India, respectively.
All the chemicals used were of the highest purity available and
an analytical grade. The seeds of Sorghum vulgare, Phaseolus
mungo and Vigna radiata were purchased from a local grain
market, and the seeds of I. hederifolia for tissue culture were
purchased from Naamdhari Seeds Pvt. Ltd., India.
Decolorization ð%Þ ¼
initial absorbance ꢀ final absorbance
ꢁ 100
initial absorbance
Abiotic controls contained the respective dye solutions,
which were devoid of plants whereas plants in distilled water
were kept as biotic controls.
2.5. Characterization of dye mixture and textile effluent
SRR, textile effluent and the mixture of dyes were characterized
for American Dye Manufacturers Institute (ADMI) value,3
hardness, BOD, COD and TOC before and aer dye
degradation.19
2.2. Potential of I. hederifolia plants for dye decolorization
The plants of I. hederifolia were uprooted carefully and their
roots were carefully washed with owing water and exposed to
100 mL solutions of Navy Blue HE2R, Green HE4B, Blue 2 R,
2.6. Anatomical studies of stem and roots during dye
degradation
Direct Red 5B, Reactive Red 2, Red HE3B and SRR at a Transverse sections of stem and root were taken and mounted
concentration of 50 mg Lꢀ1 independently. Based on the in glycerin aer overlaying with cover slip, and the results were
decolorization performance and longevity, I. hederifolia was micro-photographed with a Zeiss Axio Imager 2 Upright Trin-
selected for further studies.
ocular Microscope with attached camera at 100ꢁ magnication.
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using Gas Chromatography-Mass Spectrometry (GC-MS), High
Performance Liquid Chromatography (HPLC), High Perfor-
mance Thin Layer Chromatography (HPTLC) and Fourier
Transform Infrared Spectroscopy (FTIR). For the extraction of
products, plants were removed from the distilled water con-
taining decolorized dye, which was then centrifuged to remove
any solid matter (e.g., root hairs), if present. The decolorized
and degraded solution was then extracted with equal volumes of
ethyl acetate in a separating funnel. The organic extracts were
combined and then evaporated in vacuum over anhydrous
Na2SO4 and dried out. The solid residue obtained was dissolved
in small volumes of HPLC grade methanol and these samples
were used for analytical study.
2.7. Photosynthetic pigments analysis
5 gm of leaves of both exposed and unexposed plants and a
pinch of MgCO3 powder were crushed using a mortar and
pestle, which contained 4 mL Lꢀ1 liquor ammonia. The extracts
were centrifuged for 10 min at 2000g. Chlorophyll a, chlorophyll
b and carotenoids were determined spectrophotometrically at
652, 645, and 663 nm.20
2.8. Preparation of cell free extract and enzyme assays
Roots and shoots of the plants of I. hederifolia were cut, weighed
equally (2 g) were nely chopped and then suspended separately
in 50 mM potassium phosphate buffer (pH 7.4). The chopped
shoot tissue was then ground using a mortar and pestle, fol-
lowed by homogenization in a glass homogenizer and then
centrifugation at 8481g for 20 min. The cell free extract thus
obtained was used as an enzyme source. The supernatant
obtained aer harvesting the plant roots and shoots was used as
a source of extracellular enzymes aer centrifugation. The cell-
free solution obtained aer harvesting cells was used as a
source of extracellular enzymes.21
Identication of the metabolites produced was carried out
using GC-MS. GC-MS analysis of the metabolites was carried out
using a Shimadzu 2010 MS Engine equipped with an integrated
gas chromatograph with an HP1 column (60 m long and
0.25 mm). Helium was used as the carrier gas at a ow rate of
1 mL minꢀ1. The injector temperature was maintained by an
oven at 80 ꢂC for 2 min. The temperature was increased to
200 ꢂC with a rate of 10 ꢂC minꢀ1 and then raised to 280 ꢂC at a
rate of 20 ꢂC minꢀ1. The compounds were identied on the
basis of mass spectra and using the database of the National
Institute of Structure and Technology (NIST) library. HPLC
analysis was carried out (Waters model no. 2690; Waters Corp.,
Milford, MA) on a C18 column (symmetry, 4.6 mm ꢁ 250 mm)
by using methanol with ow rate of 1 mL minꢀ1 for 10 min and
a UV detector at 254 nm. HPTLC analysis was carried out by
using an HPTLC system (CAMAG, Switzerland). Samples of dye
SRR, dye mixture and its biodegradation metabolites (dissolved
in HPLC-grade methanol) were loaded on precoated HPTLC
plates (Silica gel 60F 254, Merck, Germany), by using nitrogen as
a spraying gas and a TLC sample loading instrument (CAMAG
LINOMAT 5). The bands (12 mm) were applied at 10 mm from
the lower edge of the plate with the rst application position at
20 mm from the le edge of the plate and 5 mm apart from each
other. The HPTLC plate was kept in a presaturated twin-trough
chamber (10 ꢁ 20 cm) for the development of dyes and ethyl
acetate extracted metabolites (products) with 10 mL of a stan-
dardized developing solvent system of toluene to methanol
(8 : 2). Aer development, the plate was observed in a UV
chamber (CAMAG) and scanned at 254 nm with a slit dimension
of 5 ꢁ 0.45 mm by using a TLC scanner (CAMAG). The results
were generated by using HPTLC soware WinCATS 1.4.4.6337.
The phytotransformed dye SRR was characterized by FTIR
(Agilent Cary 630 FTIR spectrometer) and compared with the
control sample. The FTIR analysis was conducted in the mid-IR
region of 400–4000 cmꢀ1. The samples were mixed with spec-
troscopically pure KBr in a ratio of 5 : 95, pellets were put on the
sample analyzer, and the analyses were carried out.
Activities of the enzymes lignin peroxidase (LiP), veratryl
alcohol oxidase, laccase, tyrosinase, DCIP reductase and azo
reductase were determined spectrophotometrically at room
temperature in the case of both the control and the test plants.
LiP activity was determined by monitoring the formation of
propanaldehyde at 300 nm in a reaction mixture of 2.5 mL
containing 100 mM n-propanol, 250 mM tartaric acid, and 10
mM H2O2.22 Laccase activity was determined in a reaction
mixture of 2 mL containing 10% ABTS in 0.1 M acetate buffer
(pH 4.9), and the increase in optical density was measured at
420 nm.23 Tyrosinase activity was determined as described by an
earlier report.24 NADH-DCIP reductase was measured in cell-free
extract as reported earlier by Salokhe and Govindwar.25 Veratryl
alcohol oxidase activity was determined by using veratryl alcohol
as a substrate. The reaction mixture contained 1 mM veratryl
alcohol, in 0.05 M citrate phosphate buffer (pH 3.0) and enzyme
in a total volume of 2 mL, which was used for the determination
of oxidase activity. Oxidation of the substrate at room tempera-
ture was monitored by observing an absorbance increase at
310 nm due to the formation of veratraldehyde. One unit of
enzyme activity was measured as the amount of enzyme that
releases 1 mmol product minꢀ1 26 Antioxidant enzyme status was
.
assessed by spectrophotometric assays. Antioxidant enzymes
that were analyzed include catalase and superoxide dismutase.27
All enzyme assays were performed at 27 ꢂC with reference
blanks that contained all components except the enzyme. The
protein contents of all the samples were determined using
Lowry's method.28 All enzyme assays were run in triplicate,
average rates were calculated and one unit of enzyme activity was
dened as a change in absorbance unit minꢀ1 mg of proteinꢀ1
.
2.10. Phytotoxicity study
2.9. Analysis of the degradation products
An SRR solution at 2000 ppm concentration in distilled water
Decolorization of all the dyes was monitored using UV-Vis was prepared and applied for the toxicity testing on seeds of
spectroscopy analysis (Hitachi U-2800; Hitachi, Tokyo, Japan) Sorghum vulgare, Phaseolus mungo and Vigna radiata at room
using supernatants, whereas biotransformation was monitored temperature. Similarly, treated and untreated dye mixtures and
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effluents were used for phytotoxicity tests. The above mentioned decolorized efficiently by I. hederifolia, and it afforded ADMI
three crop plants are commonly cultivated in India. 5 mL removal of 85% and 88%, BOD removal of 65% and 63% and
distilled water was used as a control. The seeds were placed in COD removal of 62% and 68%, respectively (Table 1). Effluents
5 mL solutions of dye and degraded metabolites separately at and mixtures of dyes do not have true colors, therefore, their
room temperature. The seed germination percentage was color is measured by the tristimulus method, which measures
computed and the shoot length (plumule) and root length the color value independent of the hues.12 It is highly desired to
(radicle) were measured aer 8 days.
have a phytoremediator, which is able to treat actual textile
effluents. Earlier, Phragmites australis, Aster amellus, Glandu-
laria pulchella, Portulaca grandiora, Petunia grandiora, Zinnia
angustifolia and Tagetes patula have been shown to treat the
textile effluents and bring them down to harmless levels.11–15
I. hederifolia is a natural inhabitant of the dye effluent
contaminated soils; therefore, it can be proposed for further on
eld phytoremediation trials.
2.11. Statistical analysis
Data was analyzed by one-way analysis of variance (ANOVA) with
Tukey–Kramer multiple comparisons test. Readings were
considered signicant when P was #0.05.
3. Results and discussion
3.1. Decolorization of dyes by I. hederifolia L
3.3. Anatomical studies of stems and roots during dye
degradation
The plants of I. hederifolia from a contaminated site showed
different decolorization potentials for various dyes. I. hederifolia
decolorized Navy Blue HE2R up to 96%, Green HE4B up to 90%,
Blue 2RNL up to 89%, Direct Red 5B up to 87%, Reactive Red 2
up to 68%, Orange HE2R up to 56% and SRR up to 96% within
60 h. I. hederifolia showed versatility in decolorization of the dyes
and showed the potential to be used for textile dyes treatment.
Tissue cultures of I. hederifolia also afforded 90% decolorization
of SRR within 96 h showing the direct role of this plant without
any bacterial involvement. A UV-Vis scan (800–400 nm) of
medium supernatants withdrawn aer 96 h of exposure of
I. hederifolia cultures to the dye SRR indicated its decolorization
and decrease in concentration (Fig. 1). The tissue culture is
recognized as an efficient tool for phytoremediation studies
(Doran et al., 2009).29 Phytoremediation of textile dyes has earlier
been shown by tissue cultures of P. grandiora, Z. angustifolia,
Blumea malcolmii and Sesuvium portulacastrum.5,6,10,13,30
An anatomical study of stems for understanding the histo-
chemistry, movement and metabolism of SRR was carried out.
The control plant stem (Fig. 2a) showed no coloration of any cell
and they appeared to be normal and undisturbed. Aer 6 h of
SRR dye exposure (Fig. 2b), dye was found to be accumulated in
3.2. Characterization of dye mixture and effluent before and
aer treatment
Textile dyes containing effluents are known to have high BOD
and COD. Simulated dye mixture and textile effluent were also
Fig.
1 UV-Vis spectrophotometric analysis of Scarlet RR, root Fig. 2 Anatomy of stem of I. hederifolia (a) control plant, SRR exposed
exudates of Ipomoea hederifolia and the metabolites of the dye after plants (b) at 6 h, (c) 12 h, (d) 24 h, (e) 48 h and (f) plants exposed to
treatment.
normal tap water after 48 h of SRR exposure.
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an increase in pigment concentration was observed in the case
of C4 plants.31
3.5. Enzyme induction studies during textile dye
degradation process
Enzymatic treatments possess no risk of biological contamina-
tion and do not have any harmful impact on the ecosystems. In
order to have additional insight into the decolorization mech-
anism, the activities of lignin peroxidase, veratryl alcohol
oxidase, tyrosinase, laccase, 2,6-dichlorophenol indophenol
reductase and azo reductase were assayed. Some organic
compounds can be precisely degraded and completely miner-
Fig. 3 Meristematic cells of root tips of I. hederifolia (a) control plant
(unexposed to Scarlet RR), (b) plant exposed to Scarlet RR.
the epidermal cells. As the exposure continued the accumula- alized by plant and microbial enzymes.32 In this study, the root
tion increased, reaching up to the cortical cells at 12 h (Fig. 2c). cells of in vitro grown I. hederifolia gave signicant inductions in
Furthermore, at 24 h (Fig. 2d), the accumulation increased in the enzyme activities of LiP (628%), veratryl alcohol oxidase
the cortex along with disappearance of dye around epidermal (146%), DCIP reductase (59%), laccase (31%), tyrosinase (23%),
layer, showing degradation and metabolism of SRR. Phyto- catalase (76%) and SOD (77%). While in the case of stem cells,
transformation in epidermal, as well as cortical cells, aer 48 h signicant inductions in the enzyme activities of LiP (204%),
(Fig. 2e) clearly revealed increased dye removal. The exposed veratryl alcohol oxidase (33%), DCIP reductase (41%), laccase
plants were transferred to normal tap water and the stem (214%), tyrosinase (183%), catalase (271%) and SOD (209%)
section regained the clear cortex with some distortion in were seen (Table 2). Earlier, I. palmata and Saccharum sponta-
epidermal cells having a small amount of dye in them (Fig. 2f). neum and Phragmites australis were used for dye decolorization,
The meristematic cells of I. hederifolia were observed under a showing the involvement of peroxidase enzymes.33,34 B. malcol-
microscope, and the control plant root tip cells (Fig. 3a) showed mii, A. amellus, Zinnia angustifolia, P. grandiora and G. pulchella
normal cuboidal cells but aer dye exposure the cells were also showed an active role of veratryl alcohol oxidase, tyrosi-
observed to be elongated in shape (Fig. 3b). This distortion in nase, LiP, laccase and DCIP reductase during the decolorization
morphology could have occurred because of the dye stress.
of Direct Red 5B, Remazol Red, Brilliant Blue R and Green
HE4B.6,12–15,21 Induction in the activities of lignin peroxidase,
laccase, NADH-DCIP reductase, veratryl alcohol oxidase and
tyrosinase shows their direct or indirect role in degradation of
SRR, showing their role in degradation.
3.4. Photosynthetic pigments
Photosynthetic pigments are considered to be one of the factors
sensitive to stress and the energy requirement of cells. Aer
60 h, the chlorophyll (a, b) and carotenoid concentrations were
estimated. Surprisingly, the chlorophyll and carotenoid
3.6. Analysis of metabolites
concentrations were found to be increased in the plants The FTIR spectrum of an untreated sample of SRR (Fig. 4a)
exposed to SRR compared to those in control plants, which were showed the following peaks: peaks at 3094.8 and 2904.8 cmꢀ1
kept in normal tap water (Table 3). I. hederifolia is a C4 plant, showed C–H stretching, a peak at 3517.3 cmꢀ1 represented O–H
which under abiotic stress like the presence of dye might have stretching, a peak at 2454.9 cmꢀ1 showed NH+ stretching, a
synthesized more chlorophylls and carotenoids, leading to an peak at 2247.6 cmꢀ1 revealed C^N stretching, a peak at
increase in their concentration in the exposed plants. Similar 1598.5 cmꢀ1 showed NO2 stretching, and at 1517.0 cmꢀ1 rep-
kinds of results were reported previously where temperature resented N]O as in aromatic nitro compounds. The peak at
played a role as an abiotic stress in which a decrease in 1043.1 cmꢀ1 showed the sulfonated nature of SRR, the peaks at
photosynthetic pigments were seen in the case of C3 plants and 808.7, 969.74 and 682.82 cmꢀ1 showed C–H deformation,
Table 1 Characterization of textile effluent and mixture of dyes before and after their treatment
Parameter
Untreated effluent
Treated effluent
Untreated dye mixture
Treated dye mixture
COD (mg Lꢀ1
)
1840
1630
962
580
590
108
1430
1250
678
540
430
96
BOD (mg Lꢀ1
ADMI units
)
Turbidity (NTU)
209
3478
496
56
998
408
130
87
94
28
380
35
36
17
100
TDS (mg Lꢀ1
)
TSS (mg Lꢀ1
Hardness
pH
)
410
10.2
7.9
8.1
7.5
Conductivity (S mꢀ1
)
3.80 ꢁ 10ꢀ3
0.90 ꢁ 10ꢀ3
2.80 ꢁ 10ꢀ3
1.40 ꢁ 10ꢀ3
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Table 2 Enzyme activities in root of I. hederifolia plant control tissue at 0 h and after 60 h for SRR dyea
I. hederifolia root cells
I. hederifolia shoot cells
Control
Enzymes
Control
Test
Test
Lignin peroxidaseb
Laccaseb
0.25 ꢃ 0.01
13.86 ꢃ 0.35
256.02 ꢃ 30.41
40.24 ꢃ 0.87
NAg
1.47 ꢃ 0.13
0.046 ꢃ 0.76
2.15 ꢃ 0.38
1.82 ꢃ 0.04†††
18.14 ꢃ 0.28†
407.6 ꢃ 1.45†††
49.46 ꢃ 0.53†
NAg
3.62 ꢃ 0.13††
0.081 ꢃ 0.56†
3.81 ꢃ 0.84††
0.23 ꢃ 0.65
0.70 ꢃ 0.11††
0.91 ꢃ 0.87†
0.29 ꢃ 0.95
350.55 ꢃ 0.35
16.28 ꢃ 0.33
16.39 ꢃ 0.057
3.66 ꢃ 0.88
DCIP reductasec
Tyrosinaseb
495.44 ꢃ 0.16††
46.14 ꢃ 0.26†††
19.27 ꢃ 0.41†
4.89 ꢃ 0.16†
Azo reductased
Veratryl alcohol oxidaseb
Catalasee
0.35 ꢃ 0.49
1.3 ꢃ 0.90††
Superoxide dismutasef
1.27 ꢃ 0.66
3.92 ꢃ 0.58††
a
Values are a mean of three experiments ꢃ SEM. Signicantly different from control (0 h) at †P < 0.05, ††P < 0.01 and †††P < 0.001 by one-way
ANOVA with Tukey–Kramer comparison test. Activity in units minꢀ1 mgꢀ1
.
mg of DCIP reduced minꢀ1 mg proteinꢀ1
.
mg of Azo dye reduced
g
b
c
d
minꢀ1 mg proteinꢀ1
no activity.
.
50% inhibition of the NBT photoreduction rate (U mgꢀ1 protein). Nano moles of H2O2 Utilized (U mgꢀ1 protein). NA:
e
f
Table 3 Chlorophyll and carotenoid content of I. hederifolia leaves before and after exposure to 50 mg Lꢀ1 with SRR over a period of 60 h
Sample
Chlorophyll a (mg mLꢀ1
)
Chlorophyll b (mg mLꢀ1
)
Total chlorophyll content (mg mLꢀ1
)
Carotenoids (mg mLꢀ1
)
Control plant
Test plant
10.979 ꢃ 0.68
17.952 ꢃ 0.87
7.162 ꢃ 0.89
11.755 ꢃ 1.12
18.141 ꢃ 0.76
29.707 ꢃ 1.06
13.412 ꢃ 0.66
18.262 ꢃ 0.90
Fig. 4 FTIR analysis of (a) Scarlet RR, (b) metabolites of Scarlet RR, (c) untreated dye mixture, (d) treated dye mixture, (e) untreated effluent and (f)
effluent after treatment.
whereas 620.62 cmꢀ1 showed C–Cl stretching. The treated cmꢀ1 stood for C–H deformation, a peak at 1371.4 cmꢀ1
sample of SRR (Fig. 4b) showed different peaks: 2947.8, 2835.1, revealed S]O stretching, a peak at 1324.9 cmꢀ1 showed NO2
2798.5 cmꢀ1 showed C–H stretching and 1115.6, 1021.1 cmꢀ1 stretching, peaks at 1181.8 and 909.7 cmꢀ1 showed C–OH
indicated the presence of C–OH stretching. Similarly, the stretching, a peak at 735.3 cmꢀ1 represented the aromaticity
untreated mixture of dyes (Fig. 4c) showed peaks at 3425.7 and of the compounds, whereas a single peak at 664.8 cmꢀ1 showed
2327.6 cmꢀ1, revealing N–H stretching, a peak at 1497.7 cmꢀ1
,
C–Cl stretching. The treated sample of dye mixture (Fig. 4d)
which showed C]C and C]N stretching, a peak at 1544.2 showed a peak at 3233.2 cmꢀ1, representing N]O stretching, a
cmꢀ1, which showed N]O stretching, peaks at 1408 and 1109.4 peak at 2957.6 cmꢀ1, showing C–H stretching, peaks at 1445.2,
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Fig. 5 HPLC analysis (a) Scarlet RR, (b) metabolites of Scarlet RR, (c) untreated dye mixture, (d) treated dye mixture, (e) untreated effluent and (f)
effluent after treatment.
1303.5 and 918.2 cmꢀ1, which showed C–H deformation, a peak other hand, the FTIR spectrum of the treated effluent sample
at 1227.2 cmꢀ1, which showed S]O stretching, and a peak at (Fig. 4f) showed different peaks at 3417.8 cmꢀ1, showing N–H
695.2 cmꢀ1, which showed C–Cl stretching. The FTIR spectrum stretching, the peaks at 2922.7 and 2852.1 cmꢀ1, which showed
of the untreated effluent sample (Fig. 4e) showed a peak at C–H stretching, a peak at 1613.7 cmꢀ1, which showed C]O
3442.8 cmꢀ1 for N–H stretching, peaks at 2927.1 cmꢀ1 and stretching, and a peak at 1420.6 cmꢀ1, which showed S]O
2850.8 cmꢀ1 for C–H stretching, a peak at 1635.6 cmꢀ1, showing stretching. The differential FTIR spectra obtained before and
C]C stretching, a peak at 1591.6 cmꢀ1, showing N–H defor- aer the treatment of SSR, dye mixture and real textile effluent
mation, a peak at 1495.3 cmꢀ1, showing N]O stretching, peaks supported the transformation of the dyes to different products.
at 1340.4 and 1132 cmꢀ1, showed S]O stretching, peaks at This also reveals the phytotransformation potential of
1221.9, 844.5 and 735.6 cmꢀ1, which represented C–H defor- I. hederifolia.
mation, a peak at 1015.5 cmꢀ1, which showed C–OH stretching
and a peak at 637.9 cmꢀ1 revealed the C–Cl stretching. On the peak at a retention time of 3.12 min, whereas this peak
HPLC analysis of the untreated SRR (Fig. 5a) showed the
Fig. 6 HPTLC plate image with 3-D spectral scan at 254 nm.
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disappeared aer treatment (Fig. 5b) with the appearance of 0.38, 0.20 and 0.16 with absorbances of 138.4, 95.5, 29.5, 13.9
new peaks at retention times of 2.10, 2.51, 3.03, 3.31, 3.58 and and 97.6 AU. Lane 4 (treated dye mixture) showed a mere
4.50 min. HPLC analysis of the mixture of dyes (Fig. 5c) showed presence of dye at Rf values of 0.67 and 0.2 having absorbances
the peaks at retention times of 1.90, 2.43, 2.59, 2.90, 3.28 and of 23.1 and 16.8 AU. Untreated textile effluent (Lane 5) showed
5.69 min, while the treated dye mixture (Fig. 5d) showed peaks 5 bands at Rf values of 0.54, 0.41, 0.29, 0.21 and 0.12 with
at retention times of 2.00, 2.11, 2.57, 3.33, 3.80 and 4.50 min. absorbances of 29.8, 76.4, 494.2, 279.0 and 14.7 AU, whereas
Similarly, the HPLC spectrum of untreated effluent (Fig. 5e) treated effluent showed removal of most of the peaks and only
showed distinct peaks at retention times of 3.05, 3.39, 3.68, three peaks were seen at Rf values of 0.96, 0.42 and 0.30 with
4.28, 4.55 and 6.32 min. However, treated effluent (Fig. 5f) absorbances of 69.5, 18.3 and 82.7 AU.
showed peaks at retention times of 3.14, 3.52 and 3.62 min. The
GCMS analysis was performed to know the extracted
differential HPLC spectra obtained in untreated and treated metabolites of SRR. Six separate peaks were obtained. The
samples of SRR, the mixture of dyes and the textile effluent pathway of degradation of SRR was predicted with the help of
conrms the photodegradation of dyes.
metabolites obtained and the analysis of induction in the
HPTLC analysis (Fig. 6) of the untreated SRR (Lane 1) showed activities of the enzymes in the roots of I. hederifolia Linn. SRR
three peaks at an Rf value of 0.85 with a major band at 0.76 and a undergoes asymmetric cleavage by the action of LiP to form two
third band at 0.53 with an absorbance of 0.4, 0.2 and 0.9 AU. In intermediate compounds, quinolin-4(3H)-one and 2-(methyl-
lane 2 (treated SRR) only a diminished peak was observed at amino)-N-(4-nitro-1l3-chlorinin-2-yl) acetamide. The ring of
0.85 Rf with an absorbance of 0.2 AU, conrming the biodeg- quinolin-4(3H)-one undergoes cleavage by the action of tyrosi-
radation of SRR. Lane 3 (untreated dye mixture) showed a nase to form 2-ethenyl-2,5-dihydropyridin-4(3H)-one, which
distinguished appearance of 5 bands at Rf values of 0.87, 0.67, further undergoes reduction and de-ethenylation to afford
Fig. 7 Proposed pathway for degradation of Scarlet RR by Ipomoea hederifolia L.
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Table 4 Phytotoxicity testing of disperse dye Scarlet RR, mixture of dyes and textile effluent and their respective degraded productsa
Parameters
studied
Scarlet
RR
Scarlet RR
metabolites
Untreated dye
mixture
Treated dye
mixture
Untreated textile
effluent
Treated textile
effluent
Water
Phaseolus mungo
Germination (%)
Plumule (cm)
Radicle (cm)
100
40
100
50
80
40
70
9.71 ꢃ 0.05
3.53 ꢃ 0.06†
8.06 ꢃ 0.03‡
2.83 ꢃ 0.06†
3.53 ꢃ 0.03 ‡
2.62 ꢃ 0.02†
3.24 ꢃ 0.04 ‡
6.23 ꢃ 0.03
2.42 ꢃ 0.05†
3.60 ꢃ 0.05 ‡
3.2 ꢃ 0.05†
3.63 ꢃ 0.02 ‡
2.86 ꢃ 0.03†
3.15 ꢃ 0.02 ‡
Sorghum vulgare
Germination (%)
Plumule (cm)
Radicle (cm)
100
40
90
50
90
40
70
7.26 ꢃ 0.08
4.13 ꢃ 0.06†
5.81 ꢃ 0.10 ‡
3.63 ꢃ 0.08†
4.3 ꢃ 0.05 ‡
3.5 ꢃ 0.06†
4.33 ꢃ 0.15 ‡
5.94 ꢃ 0.12
1.46 ꢃ 0.04†
2.80 ꢃ 0.03 ‡
2.3 ꢃ 0.05†
2.8 ꢃ 0.05 ‡
1.8 ꢃ 0.01†
2.41 ꢃ 0.02 ‡
Vigna radiata
Germination (%)
Plumule (cm)
Radicle (cm)
90
50
80
40
80
40
80
5.93 ꢃ 0.08
2.73 ꢃ 0.06†
5.83 ꢃ 0.06 ‡
2.53 ꢃ 0.03†
5.73 ꢃ 0.03 ‡
2.36 ꢃ 0.08†
5.83 ꢃ 0.06 ‡
2.46 ꢃ 0.06
1.02 ꢃ 0.90†
2.36 ꢃ 0.02 ‡
0.93 ꢃ 0.90†
2.3 ꢃ 0.06 ‡
0.73 ꢃ 0.06†
1.13 ꢃ 0.03 ‡
a
Values are a mean of three experiments ꢃ SE. Root and shoot lengths of plants grown in textile dye SRR, textile effluent and mixture of dyes,
respectively, are signicantly different from that of plants grown in distilled water by †P < 0.001. Root and shoot lengths of plants grown in the
degraded treated SRR, textile effluent and mixture of dyes, respectively, are also signicantly different from that of plants grown in untreated
SRR, textile effluent and mixture of dyes by ‡P < 0.001.
2,3,4,5-tetrahydropyridine. 2-(methylamino)-N-(4-nitro-1l3-chlor- reveals the reduced toxicity of the metabolites formed aer the
inin-2-yl)acetamide undergoes oxidative cleavage by laccase to treatment by I. hederifolia (Table 4).
afford 2-amino-N-(4-hydroxy-1l3-chlorinin-2-yl) acetamide,
which further upon deamination and reduction gives N-methyl-
4. Conclusion
1l3-chlorinin-2-amine (Fig. 7). Lignin peroxidase and veratryl
alcohol oxidase are known to cleave the dye molecules asym-
metrically.12–16,30 The laccase cleaves the dyes by oxidative
cleavage.35 The differential enzymatic status also inuences the
degradation mechanism. At natural sites of dye contaminated
soils, other factors such as soil microora and environmental
conditions along with plants may act on the dye molecule
synergistically and mineralization of the dye molecules may
occur. The I. hederifolia showed great potential to degrade SRR
dye to different metabolites.
The phytoremediation of textile dyes has mainly been carried
out by using common garden plants whose potential is yet to be
tested for the in situ remediation purpose. This study explores
the phytoremediation potential of I. hederifolia, a plant inhab-
itant of the actual dye disposal site. Its seeds germinate nor-
mally in high dye concentration and can complete the life cycle
in high dye load. The plant could decolorize a real textile
effluent sample and a dye mixture to meaningful levels. This
study clearly revealed the dye removal by I. hederifolia at
anatomical level. Studies to explore this plant in a constructed
wetland system for textile effluent treatment at an industrial
scale are underway.
3.7. Phytotoxicity analysis
The textile dye SRR, mixture of dyes and effluent, showed
inhibition of germination of Phaeseolus mungo, Sorghum vulgare
and Vigna radiata seeds. The untreated textile dye SRR, mixture
of dyes and effluent, showed 60%, 50% and 60% inhibition of
P. mungo seed germination respectively, whereas S. vulgare
seeds showed 60%, 50%, 60% inhibition of germination, and
for V. radiata germination inhibition percentage was found to
be 50%, 60% and 60%, respectively. The plumule and radicle
lengths also decreased in the case of untreated textile dye SRR,
effluent and dye mixture samples. The eight days of exposure of
the untreated textile dye SRR, textile effluent and dye mixture
were highly toxic to the plantlets as the root and shoot lengths
were also inhibited when compared to treated effluent and dye
mixture samples. The treated samples were found to be less
toxic and showed lower germination inhibition percentages
when compared to their untreated forms. A signicant increase
in seed germination percentage and, shoot and root lengths
Acknowledgements
Niraj R. Rane, Avinash G. Gholave, Shrirang R. Yadav and Sanjay
P. Govindwar wish to thank Department of Biotechnology
(DBT), New Delhi, India, for the funding under Interdisciplinary
Program for Life Science (IPLS) project to Shivaji University,
Kolhapur.
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