Biotechnology Advances 33 (2015) 1697–1714

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Biotechnology Advances

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Research review paper

Phytoremediation of textile dyes and effluents: Current scenario and

future prospects
Rahul V. Khandare a, 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: Phytoremediation has emerged as a green, passive, solar energy driven and cost effective approach for environ-
Received 16 April 2015 mental cleanup when compared to physico-chemical and even other biological methods. Textile dyes and efflu-
Received in revised form 31 August 2015 ents are condemned as one of the worst polluters of our precious water bodies and soils. They are well known
Accepted 15 September 2015
mutagenic, carcinogenic, allergic and cytotoxic agents posing threats to all life forms. Plant based treatment of
Available online 18 September 2015
textile dyes is relatively new and hitherto has remained an unexplored area of research. Use of macrophytes
Keywords:
like Phragmites australis and Rheum rhabarbarum have shown efficient removal of Acid Orange 7 and sulfonated
Textile dyes anthraquinones, respectively. Common garden and ornamental plants namely Aster amellus, Portulaca grandiflo-
Effluents ra, Zinnia angustifolia, Petunia grandiflora, Glandularia pulchella, many ferns and aquatic plants have also been ad-
Toxicity vocated for their dye degradation potential. Plant tissue cultures like suspension cells of Blumea malcolmii and
Phytoremediation Nopalea cochenillifera, hairy roots of Brassica juncea and Tagetes patula and whole plants of several other species
Decolorization have confirmed their role in dye degradation. Plants' oxidoreductases such as lignin peroxidase, laccase, tyrosi-
Phytoreactors nase, azo reductase, veratryl alcohol oxidase, riboflavin reductase and dichlorophenolindophenol reductase are
Constructed wetlands
known as key biodegrading enzymes which break the complex structures of dyes. Schematic metabolic pathways
Oxidoreductive enzymes
of degradation of different dyes and their environmental fates have also been proposed. Degradation products of
Metabolic fate
dyes and their fates of metabolism have been reported to be validated by UV–vis spectrophotometry, high per-
formance liquid chromatography, high performance thin layer chromatography, Fourier Transform Infrared
Spectroscopy, gas chromatograph-mass spectroscopy and several other analytical tools. Constructed wetlands
and various pilots scale reactors were developed independently using the plants of P. australis,
Portulaca grandiflora, G. pulchella, Typha domingensis, Pogonatherum crinitum and Alternanthera philoxeroides.
The developed phytoreactors gave noteworthy treatments, and significant reductions in biological oxygen de-
mand, chemical oxygen demand, American Dye Manufacturers Institute color removal value, total organic car-
bon, total dissolved solids, total suspended solids, turbidity and conductivity of the dye effluents after
phytoremediation. Metabolites of dyes and effluents have been assayed for phytotoxicity, cytotoxicity,
genotoxicity and animal toxicity and were proved to be non/less toxic than untreated compounds. Effective strat-
egies to handle fluctuating dye load and hydraulics for in situ treatment needs scientific attention. Future studies
on development of transgenic plants for efficacious phytodegradation of textile dyes should be focused.
© 2015 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698
2. Water consumption: a key issue in dying processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699
3. Toxicity of textile dye stuff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699
4. Color removal analysis using characteristic light absorbance of dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1699
5. Available methods for textile wastewater treatment and their shortcomings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700

Abbreviations: ADMI, American Dye Manufacturers Institute; BOD, biological oxygen demand; COD, chemical oxygen demand; DCIP, dichlorophenol indophenol; FTIR, Fourier
Transform Infrared Spectroscopy; GC–MS, gas chromatography–mass spectroscopy; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatogra-
phy; LC–MS, liquid chromatography–mass spectroscopy; LiP, lignin peroxidase; PPO, polyphenol oxidase; TDS, total dissolved solids; TOC, total organic carbon; TSS, total suspended solids;
UV–Vis, ultra violet–visible.
⁎ Corresponding author at: Department of Biochemistry, Shivaji University, Kolhapur 416 004, India.
E-mail addresses: spg_biochem@unishivaji.ac.in (R.V. Khandare), spgovindwar@rediffmail.com (S.P. Govindwar).

http://dx.doi.org/10.1016/j.biotechadv.2015.09.003
0734-9750/© 2015 Elsevier Inc. All rights reserved.
1698 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714

5.1. Physico-chemical methods and their limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700

5.2. Biological methods for dye removal from effluents and their limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700
6. Phytoremediation studies on removal of textile dyes and effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1700
6.1. Hydroponics for degradation of textile dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1701
6.2. Plant tissue cultures for dye degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1701
6.2.1. Whole plant in vitro cultures for dye degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702
6.2.2. Hairy root cultures for dye degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702
6.2.3. Callus and suspension cultures for dye degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702
6.3. Purified plant enzymes for degradation of textile dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1702
6.4. Synergistic strategies for degradation of textile dyes and effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1703
6.5. Reactor development and constructed wetland strategies for phytoremediation of textile dyes and effluents . . . . . . . . . . . . . . 1703
7. Plant mechanisms for treatment of textile dyes and effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1704
7.1. Adsorptive and/or accumulative removal of textile dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705
7.2. Plant enzymes involved in stress response and dye degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1705
8. Scrutiny of the products after dye phyto-removal/degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1706
8.1. Characterization of the textiles dyes and effluents after phytotreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1706
8.2. Structural elucidation of dye products and analysis of fate of metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1707
8.3. Toxicity assessment of textile dyes, effluents and their degradation metabolites after phytoremediation . . . . . . . . . . . . . . . . 1709
9. Factors affecting the phytoremediation of textile dyes and effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1709
9.1. Growth form of the plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1709
9.2. Dye availability, hydraulics and pollutant load (dye concentration) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1709
9.3. Water, oxygen and nutrient availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1710
9.4. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1710
9.5. Solar energy and radiations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1710
9.6. Weathering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1710
10. Disadvantages and advantages of phytoremediation of textile dyes and effluents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1710
11. Research needs and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711

1. Introduction possess practical complications when actual on site management of

In the past few decades, phytoremediation approaches have meticu-
lously been scrutinized as potential tools to remove and/or degrade
many hazardous environmental contaminants like heavy metals, landfill
leachates, pesticides, polyaromatic hydrocarbons, radionuclides, petro-
leum, crude oil, chlorinated solvents, polychlorinated biphenyls, explo-
sives, munitions and even the poisonous gases (Pilon-Smits, 2005;
Nwoko, 2010). Phytoextraction of heavy metals is a thoroughly studied,
engineered, proven and successful method for treatment of contaminated
sites as per the current scientific information. Efficacy of phyto-
remediation for the treatment of many other pollutants is under
examination by scientific community all over the world. Phyto-
remediation of textile dyes is still an unexplored area of research
and understanding the underlying mechanisms with subsequent
degradation of dyes by plants needs to be comprehensively worked
out.
Manufacturing and processing textile dye industries are one of the
foulest contaminators of water and soil. The recalcitrant nature of textile
effluents largely containing high concentrations of dyestuffs, salts, acids,
bases, surfactants, dispersants, humectants, oxidants and detergents
render these waters esthetically unacceptable and unusable. Textile
dyes are well known mutagens and carcinogens posing risks to various
ecosystems, animals' health and agriculture (Saratale et al., 2011),
therefore the treatment of these high volumes of wastewater becomes
crucial. Available techniques such as physical and biological adsorption,
membrane filtration, oxidation, ozonation and microbial biodegrada-
tion are generally employed for remediation of dye containing effluents.
These treatment and removal practices are not always followed as per
the governing standards and thus ultimately cause serious pollution.
These approaches are expensive and unaffordable by small scale indus-
tries and processors. This financial burden perhaps is the key reason
which slows down efforts to control pollution predominantly in under-
developed and developing countries (Khandare et al., 2013a). Bioreme-
diation approaches exploring bacteria, fungi, yeast and even their
consortial schemes have been proved effective in removal of dyes but
waste is concerned.
Phytoremediation enjoys advantages such as being a solar energy
dependent and an esthetically pleasant method of treatment. It offers
a carbon neutral and thus environmental friendly approach for removal
of toxic contaminants from the environment (Dietz and Schnoor, 2008).
Restoration of habitats and in situ cleanup of contaminants can be
achieved with significantly reduced remedial costs by this phyto-
technology. It may possibly obtain wood, flowers and even bio-energy
as per growth forms of the phytoremediator plants (Cluis, 2004). A
huge amount of literature published in past two decades has strength-
ened the notion that plants can be an interesting alternative for removal
of textile dyes. This perception is essentially grounded on the extraordi-
nary metabolic and extractive capabilities of plants which endow them
with incomparable potential for dye decontamination (Govindwar and
Kagalkar, 2010). Plants naturally provide roots, stems and leaves as hab-
itats for a wide array of microorganisms which simultaneously can
breakdown contaminants enhancing the treatment process. Species
possessing fibrous and deep roots, and rapid growth rate such as annual
herbs, creepers and grasses may possibly prove to be suitable for
phytoremediation of textile dyes (Khandare et al., 2011a, 2013a; Rane
et al., 2014). Information about the biochemical routes involved during
phytotreatments could give additional insights to develop and engineer
efficient plants for better pollution controlling practices.
Plant species having different growth forms have been proposed for
the removal of textile dyes and effluents; for instance, Typha angustifolia,
Phragmites australis, Tagetes patula, Blumea malcolmii, Typhonium
flagelliforme, Glandularia pulchella, Portulaca grandiflora, Aster amellus, Zin-
nia angustifolia, Petunia grandiflora, Ipomoea hederifolia, Typha domingensis
and Alternanthera philoxeroides (Davies et al., 2009; Patil et al., 2009;
Kagalkar et al., 2009, 2011; Govindwar and Kagalkar, 2010; Kabra et al.,
2011a, 2011b, 2012; Khandare et al., 2011a, 2011b, 2012; Watharkar
et al., 2013a; Shehzadi et al., 2014; Rane et al., 2014, 2015). Most of the
studies on phytoremediation of dyes have loitered at laboratory bench
levels and only a very few number of reports on pilot scale demonstra-
tions of dye effluent treatments are available. This review attempts to
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
provide a comprehensive catalog on the available information on
phytoremediation of textile dyes and effluents. The mechanisms of dye
metabolism by plants and further toxicological assessments of degrada-
tion products on plant and animal models are systematically discussed.
Effective and employable treatment strategies like phytoreactors and
wetland development for large scale applications have also been focused.
Future prospects and research needs for efficacious phytoremediation of
dye effluents and allied promising possibilities are further resolved.
2. Water consumption: a key issue in dying processes
Textile dye manufacturing and processing industries liberate huge
volumes of effluents to the ecosphere. A normal sized textile mill
which produces 8000 kg of fabric everyday consumes about 1.6 million
liters of water. Out of which 16% is used up in dying and around 8% is
spent in printing. Water, a finite resource is on the verge of becoming
scarce because of such indiscriminate uses (Kant, 2012). Dye processors
further need substantial amounts of water for cleaning the printed and
dyed fabric. A number of other chemicals like mordants, fastners, fixing
agents, surfactants, defoamers, acids, bases and alkalis are utilized for
achieving bright colors and fastness. These substances largely contrib-
ute to chemical oxygen demand (COD), biological oxygen demand
(BOD), total suspended solids (TSS), total dissolved solids (TDS) and
total organic carbon (TOC) of the textile wastewaters making it even
more polluted (Kurade et al., 2011; Khandare et al., 2014). As per esti-
mates of the World Bank, around 17 to 20% of total industrial water pol-
lution is solely contributed by dyeing and finishing processes. Around
72 different toxic chemicals are proposed to be present in water exclu-
sively from dyeing processes. In the same report the World Bank also
enlisted 30 different compounds which are impossible to be removed
and most recalcitrant in nature (Chen and Burns, 2006). It is a well-
known fact that the dyes even in very small quantity show notable bril-
liance making colored water esthetically unacceptable. The treatment of
water polluted by textile dyes therefore becomes indispensable
(Govindwar and Kagalkar, 2010). At the same time this water needs
to be consumed before forming toxic leachates and plants can appropri-
ately be endorsed to vitalize that function with immense competence.
3. Toxicity of textile dye stuff
Modern science-based chemical industry started to come up in the
beginning of nineteenth century. The first synthetic dye ‘Mauveine’ syn-
thesized by William Henry Perkin laid a new foundation and announced
the arrival of these xenobiotics. Thousands of synthetic dyes are pre-
pared and extensively used since then. A grave threat of pollution and
deterioration of our valuable water resources aroused after a few
years of the industrial revolution. Occurrence of textile dyes in water
bodies decrease the dissemination of daylight reducing the overall
rate of photosynthesis of algae and other aquatic vegetation. Lower con-
centrations of dissolved oxygen ultimately hamper the water quality
causing a wide array of toxic effects to the aquatic ecosystems
(Saratale et al., 2011).
Mutagenicity and carcinogenicity of azo dyes and their derivatives
have been reported earlier by Yahagi et al. (1975). It was shown that
the complex structures of aminoazo benzene dyes and their various de-
rivatives may lead to mutagenesis which is a major cause of cancer
(Garg et al., 2002). The International Agency for Research on Cancer
(IARC) has declared benzidine like dyes to be extremely powerful
carcinogen to many mammals and alarmingly human beings (IARC,
1982). Experiments on Swiss albino rats as model organisms have
shown the toxicity of textile wastewater to animals (Sharma et al.,
2007). Erythrosine, a xanthene dye impart hazards like being allergic,
carcinogenic, DNA damaging, neurotoxic and xenoestrogenic to a varie-
ty of animals including humans (Mittal et al., 2006). Disperse Orange 1
and Disperse Blue 291 caused genotoxic and mutagenic effect like in-
creased micronuclei in human hepatoma derived HepG2 cells (Tsuboy
1699
et al., 2007). Malachite Green and its metabolite leuco malachite green
was also reported to remain persistent in two human cell lines namely
epithelium resembling Caco2 and HepG2 causing significant decrease
in cell viability, total protein content and colony-forming ability
(Stammati et al., 2005). Reactive dyes are famous as most efficient
colors among all but a survey of dye manufacturing industry workers re-
vealed that these dyes are responsible to boost the rate of bladder can-
cer (Rehn, 1895). In Spain and Algeria, spraying of a textile paint
chemical was reported to cause a pulmonary disease called ‘Ardystil
syndrome’ in many spray workers leading to deaths. The term ‘Ardystil
syndrome’ was coined after the name of textile factory ‘Ardystil’ (Hoet
et al., 1999).
Oliveira et al. (2007) did a thorough toxicological characterization of
industrial dye effluents and identified mutagenic dyes like Disperse Or-
ange 37, Disperse Blue 373, Disperse Violet 93 and some components of
an unknown black color and their products to contain aromatic amine
rings. Commonly used Malachite Green was demonstrated to be respon-
sible for 20 times increase in eggs mortality rate of largemouth bass
Micropterus salmoides (Wright, 1976). Malachite Green was also found
to induce mitochondrial damages, nuclear alterations, liver focal necrosis
and sinusoidal congestions in rainbow trout fishes (Gerundo et al., 1991).
Serum calcium and protein levels were observed to be dropped signifi-
cantly leading to increased blood cholesterol levels in freshwater catfish
Heteropneustes fossilis upon exposure of Malachite Green (Srivastava
et al., 1995). Teratogenesis assay on the embryos of Xenopus laevis frog
have exposed the toxicity of Remazol Red, Remazol Turquoise, Astrazon
Blue FGRL, Astrazon Red FBL, Cibacron Blue FN-R, Cibacron Red FN-3G
and Blue G-A dyes (Birhanli and Ozmen, 2005). Deaths of cladoceran
freshwater water flea Daphnia magna was observed when exposed to tex-
tile dyes (Bae and Freeman, 2005). Various dyes and textile effluent have
shown a very strong genotoxic effects on the root cells of Allium cepa
(Phugare et al., 2011; Watharkar and Jadhav, 2014).
Disperse Orange 1 was found to impart mutagenic effects on Salmonel-
la typhimurium strains viz. TA98, YG1041, TA100 and YG1042 (Chequer
et al., 2009; Ferraz et al., 2011). Mutagenicity evaluation of Disperse
Blue 291 was reported using Salmonella assay (Umbuzeiro et al., 2005).
Congo Red and Reactive Orange 16 azo dyes were reported to cause inhi-
bition of algal growth and affected the natural bacterial luminescence
(Novotny et al., 2006). Textile wastewaters containing mixture of dyes
generally have high BOD, COD, TSS, TDS and alarming toxicities (Casieri
et al., 2008). It has therefore become imperative to work on detoxification
and degradation of textile dyes and effluents. In the view of these facts,
development of novel cost-effective and sustainable technologies to con-
trol dye pollution and enormous water consumption is highly desirable.
4. Color removal analysis using characteristic light absorbance of
dyes
Textile dyes possess characteristic chromophoric groups which
are responsible to impart colors to compounds. Each dye therefore
shows independent absorbance maxima (λmax) at particular wave-
lengths of light. This property makes it easier to monitor dye removal
over time. Decrease in absorbance clearly means that the dye is being
removed or transformed and can easily be measured using a simple
colorimeter or UV–visible spectrophotometer. Change in the absor-
bance and loss of the peak at λmax gives the first evidence of dye re-
moval and decolorization. Decolorization of Navy Blue HE2R by
in vitro cultures of Portulaca grandiflora (Fig. 1) was monitored at
it's λmax of 620 nm using a UV–vis spectrophotometer and the dis-
tinctive dye peak was observed to be disappeared after treatment
(Khandare et al., 2011a). The decolorization percentage was thus calcu-
lated using Eq. (1).
Initial absorbance−Final absorbance
Decolorization % ¼  100 ð1Þ
Initial absorbance
1700 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
Fig. 1. Experimental setup for decolorization of Navy Blue HE2R showing dye as abiotic
control, plant exposed to distilled water as biotic control, and degradation metabolites
of the dye after 40 h exposure and by Portulaca grandiflora.
It is however difficult to measure decolorization of dye mixtures
and/or real textile effluents as they do not have any particular color
and thus wavelength of maximum absorption. A tristimulus method ap-
proved by American Dye Manufacturers Institute (ADMI) called as
ADMI value is therefore employed for measuring dye removal from
mixtures and effluents. This method uses three different wavelengths
to calculate the color value independent of the hue of the samples and
color removal is measured using Eq. (2).
Initial ADMI−Final ADMI
ADMI removal % ¼  100 ð2Þ
Initial ADMI
Eqs. (1) and (2) are used for initial assessment of dye removal from
solutions after recording absorbance on a simple colorimeter/spectro-
photometer. It is apparent that the light absorbance of any dye in a so-
lution is directly proportional to its concentration. In this article, the
initial dye concentrations henceforth should be considered as 100%
wherever dye removal percentage is mentioned.
5. Available methods for textile wastewater treatment and their
shortcomings
5.1. Physico-chemical methods and their limitations
Several factors including dye type, effluent composition, costs of
chemicals, operations, handling and environmental fate determines
the methodology and financial practicability of every dye removal
practice. Many physical and chemical methods including adsorption
(Bestani et al., 2008), precipitation/coagulation/flocculation (Zhou
et al., 2008), filtration (Avlonitis et al., 2008), oxidation (Gupta and
Suhas, 2009), ozonation (Wu et al., 2007), advanced oxidation process-
es, ultra violet photolysis and sonolysis techniques (Bandala et al., 2008)
are offered for the treatment of textile dye effluents. Advanced methods
like filtration (Nano, micro and ultra) and even reverse osmosis have
also been additionally scrutinized for dye elimination (Avlonitis et al.,
2008).
These methods possess potential for removal of color from dye
wastewaters to varying extents but there are serious concerns about
toxicity of treated effluents which make them clearly inadvisable.
Mere precipitation, coagulation, flocculation, filtration and oxidation
fail to render the effluent nontoxic leading to serious consequences
such as secondary pollution from sludge and leachates. Dumping sludge
as landfills might temporarily solve the issue but threats of leachates in
future becomes unavoidable. Oxidation, ozonation, filtration, photolysis
and reverse osmosis are not reasonably priced for small scale dye
processers. Developing countries hold their own legislations for
treatment of dye effluents but industries owing to the costs and tech-
nical constraints do not follow them strictly and tend to release these
extremely toxic wastes into open environmental sinks.
5.2. Biological methods for dye removal from effluents and their limitations
Exploring various classes of living organisms for treatment of
pollutants like textile dyes is termed as ‘bioremediation’. Environ-
mental biologists have started advocating the use of microorgan-
isms by virtue of their dynamic metabolisms and abilities to
acclimatize to toxic wastes in the environment. Such adaptations
equip them with new tools to utilize the pollutants as nutrients
and transform into harmless products. Use of microbes and their
enzymatic machineries for complete degradation and removal of
dyes from textile effluent is advantageous in being cost-effective,
less sludge forming, environmental-friendly (yielding less harmful
metabolite), possessing the capability of complete mineralization
and most importantly require less water when compared to the
physicochemical methods (Jadhav and Govindwar, 2006).
Success of microbial decolorization largely depends on the compli-
ance and action of candidate microbes. A wide range of microbial spe-
cies have been tested for degradation of varied classes of textile dyes
in the last decade (Pandey et al., 2007; Reema et al., 2011). For instances,
fungi (Parshetti et al., 2006; Saratale et al., 2006, 2009a, 2009b;
Humnabadkar et al., 2008), actinomycetes (Machado et al., 2006),
algae (Daneshvar et al., 2007; Khataee et al., 2010a, 2010b, 2013a),
yeasts (Jadhav and Govindwar, 2006; Parshetti et al., 2007; Jadhav
et al., 2007, 2008a, 2008b, 2008c, 2009a; Waghmode et al., 2011) and
bacteria (Kalme et al., 2007, 2008; Dawkar et al., 2008, 2009a, 2009b;
Kalyani et al., 2008; Patil et al., 2009; Telke et al., 2008, 2010a;
Parshetti et al., 2009; Ghodake et al., 2009a; Gomare et al., 2009;
Jadhav et al., 2009b, 2012; Tamboli et al., 2010; Kurade et al., 2011;
Phugare et al., 2011) have shown the potential for degradation of textile
dyes.
6. Phytoremediation studies on removal of textile dyes and effluents
Phytoremediation of textile dyes is reasonably a new method of
textile effluent treatment. Various strategies for laboratory scale studies
have been employed. Studies on textile dye removal have mostly been
focused on the use of wild macrophytes. P. australis was explored for
the removal of Acid Orange 7 as the model dye (Davies et al., 2005;
Carias et al., 2007). Narrow leaved cattail (T. angustifolia) showed the
potential in treatment of Reactive Red 41 at concentrations of
100–300 mg L − 1 and could achieve around 60% decolorization
(Nilratnisakorn et al., 2007). Lemna minor was also proposed to re-
move Methylene Blue dye with accumulation to be the prominent
mechanism (Reema et al., 2011). Aquatic plants like Eichhornia
spp. were also found to remove Direct Dark Blue 6B, Black HY and
Congo Red (Anjana and Thanga, 2011).
A narrow range of herbaceous plant species are known to perform the
removal of harmful dyes. Z. angustifolia, Brassica juncea, B. malcolmii,
T. patula, T. flagelliforme, A. amellus, G. pulchella, Sesuvium portulacastrum,
Gaillardia grandiflora, Rosmarinus officinalis, Petunia grandiflora, L. minor,
Azolla filiculoides, Portulaca grandiflora, Thymus vulgaris, I. hederifolia and
Hydrocotyle vulgaris are some of the examples which have been proposed
for the removal of textile effluents and dyes (Zheng and Shetty, 2000;
Davies et al., 2005, 2009; Nilratnisakorn et al., 2007; Patil et al., 2009,
2012; Govindwar and Kagalkar, 2010; Kabra et al., 2011a, 2011b;
Khandare et al., 2011a, 2011b, 2012; Khataee et al., 2012, 2013b;
Vafaei et al., 2012, 2013; Watharkar et al., 2013a, 2013b;
Watharkar and Jadhav, 2014; Rane et al., 2014; Torbati et al., 2014).
Table 1 enlists the potential of different wild/indigenous plants and
their dye remediation performances.
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714 1701

Table 1
Phytoremediation performances of different wild/indigenous plants for textile dyes and effluents.

Sr. Name of the plant Dye/effluent Concentration Decolorization % Reference

no. time (h) decolorization

1 Alternanthera Remazol Red 70 mg L−1 72 100 Rane et al. (2015)

philoxeroides
2 Pogonatherum Effluent NA 288 74 Watharkar et al. (2015)
crinitum
−1
3 Nasturtium Acid Blue 92 20 mg L 96 78 Torbati et al. (2014)
officinale
4 Ipomoea hederifolia Scarlet RR 50 mg L−1 60 96 Rane et al. (2014)
5 Typha angustifolia Reactive Blue 19 75 mg L−1 144 70 Mahmood et al. (2014)
6 Bouteloua Effluent NA 24 92 Vijayalakshmidevi and
dactyloides Muthukumar (2014)
7 Petunia grandiflora Brillaint Blue G 20 mg L−1 36 86 Watharkar et al. (2013a, 2013b)
8 Azolla filiculoide Basic Red 46 and Acid Blue 92 20 mg L−1 144 and 168 90 and 80 Vafaei et al. (2012), Khataee
et al. (2013a)
9 Lemna minor Methylene Blue and Acid Blue 92 10 mg L−1 and 144 98 and 77 Reema et al. (2011), Khataee
2.5 g L−1 et al. (2012)
10 Portulaca Reactive Blue 172 20 mg L−1 40 98 Khandare et al. (2011a)
grandiflora
−1
11 Glandularia Green HE4B and Remazol Orange 3R 20 mg L 48 and 96 92 and 100 Kabra et al. (2011a, 2011b)
pulchella
−1
12 Aster amellus Remazol Red, Remazol Orange 3R 20 mg L 60 and 72 96 and 100 Kabra et al. (2011b), Khandare
et al. (2011b)
13 Typhonium Brilliant Blue R 20 mg L−1 96 65 Kagalkar et al. (2010)
flagelliforme
14 Blumea malcolmii Malachite green, Red HE4B, Methyl orange, Reactive Red 20 mg L−1 72 96, 76, 88, 80 Kagalkar et al. (2009)
2 and Direct Red 5B and 42
15 Phragmites australis Acid Orange 7 750 mg L−1 and 8 times a day and 68 and 98 Davies et al. (2009), Ong et al.
100 mg L−1 144 h (2010)

6.1. Hydroponics for degradation of textile dyes
Hydroponic systems provide another alternative for cultivation of
plants for experimental purposes. Soil-less grown plants offer advan-
tage as most of the root system can be made available for remediation.
Hydroponic systems are cost effective when compared to tissue cultures
and experimentation befits close to the field applications. Studies on hy-
droponically grown duckweed L. minor with artificial neural network
modeling has given promising results and was found to achieve a sub-
stantial decolorization of Acid Blue 92 (Khataee et al. 2012). In indepen-
dent studies using hydroponics, Basic Red 46, Acid Blue 92 and Reactive
Blue 19 dyes were found to be removed successfully by A. filiculoides,
Nasturtium officinale and T. angustifolia, respectively (Khataee et al.,
2013b; Torbati et al., 2014; Mahmood et al., 2014). Phytoremediation
performances of various plants in hydroponic systems are included in
Table 1 with initial dye concentrations, percentage removal and respec-
tive time reported for treatment.
Rumex hydrolapatum, Rumex acetosa, Rheum rhabarbarum and
Apium graveolens were screened for the removal of sulfonated anthra-
quinones in hydroponic solutions (Aubert and Schwitzguébel, 2004).
Use of hydroponic systems for phytoremediation studies also holds
some disadvantages. Water absorption capacity becomes a key factor
during studies on hydroponics. Among the tested plants, R. acetosa pos-
sessed a lower water absorption capacity and normal metabolism. The
transpiration was also observed to decline when compared to the plants
in soil (Aubert and Schwitzguébel, 2004). In another experiment with
Rumex spp. grown hydroponically; collection of leaves of same age,
growth stage and development was found to be unmanageable (Page
and Schwitzguébel, 2009). Hydroponically grown grasses namely
Cyperus javanicus, Eleocharis calva, Fimbristylis cymosa and Pennisetum
purpureum were observed to decolorize Poly R-478 up to 59, 61, 66,
and 88%, respectively within 1–4 weeks at an initial concentration of
20 mg L−1 (Paquin et al., 2006). In the same study, herbaceous plants
like Rumex crispus and Hibiscus furcellatus were also found to decolorize
Poly R-478 up to 70 and 55%, respectively.
Nutritional status, photoperiod and microbial contaminants can
affect the enzyme activities of a phytoremediating plant (Govindwar
and Kagalkar, 2010). To overcome these issue with hydroponically or
wetland grown plants; the importance of tissue culture based technolo-
gies was stressed by a few researchers. Though tissue culture requires
high costs it can prove to be an effective tool to understand the actual
role of plants in pollutant treatment. Exploitation of plants in tissue cul-
tures or hydroponics is certainly different than the actual soil and
rhizospheric environment (Zabłudowska et al., 2009). Such in vitro
built systems therefore must be accompanied or followed by actual
field trials for testing their viability.
6.2. Plant tissue cultures for dye degradation
Plant tissue cultures have proven to be useful tools for phyto-
remediation studies. Different types of cultures like whole plants,
hairy roots, cell suspensions and calli have frequently been utilized in
phytoremediation research. Tissue cultures offer a clear advantage in
understanding metabolic capabilities, toxicity tolerance and phyto-
remediation mechanisms by the model or selected plant. In spite of
availability of a large amount of literature on phytoremediation of
dyes, the lack of information on mechanistic and basic metabolism
limits our knowledge. Tissue culture technologies provide an additional
advantage of growing plants under controlled conditions and establish-
ing them with indefinite propagation. These plants can be made avail-
able throughout the year on contrary to wild plants which have a
limited natural life span. In vitro culture techniques involve a sterile en-
vironment which is free of any unwanted microbes and thus can further
be useful to differentiate the actual potential and responses of plants.
Tissue culturing also offer controlled conditions in terms of light,
humidity requirements, nutrients, hormone levels, photoperiods and
differentiation patterns etc. (Doran, 2009). Exploring tissue culture
techniques for phytoremediation on the other hand hold limitations
for the reason that the actual pollutant load and hydraulics during prac-
tical phytoremediation are entirely different and constantly fluctuating.
Use of tissue culturally grown plants for remediation purposes is
however clearly inadvisable but this technique surely is a boon in
providing basic information on mechanisms of metabolism during
phytoremediation processes.
1702 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
6.2.1. Whole plant in vitro cultures for dye degradation
Application of in vitro grown whole plantlets appears to be ideal for
understanding the actual mechanism of phytoremediation of dyes.
With a complete plant form, the fate of metabolism of a pollutant
depends upon its translocation from roots to various shoot parts.
Whole plant cultures thus offer almost factual insight to the study of
phytoremediation mechanisms. Though roots are the first tissues to
come in contact with dyes, their further metabolism occurs partially in
the shoot tissues (Nilratnisakorn et al., 2007). If the biotransformation
enzymes involved in further transformation are present in shoot parts
of plants then it may take longer time for metabolism of the compound.
Use of whole plants is also advantageous for standardization of different
parameters. Initial optimization studies using whole plant in vitro sys-
tems will be useful in obtaining vital information for further pilot scale
applications. A few in vitro grown plant species like B. malcolmii,
T. flagelliforme, Portulaca grandiflora, T. patula, S. portulacastrum,
G. pulchella, G. grandiflora, Petunia grandiflora, Z. angustifolia, Nopalea
cochenillifera and I. hederifolia have been utilized for degradation of
dyes like Brilliant Blue R, Remazol Red, Remazol Black B, Reactive Red
198, Direct Red 5B, Navy Blue HE2R, Green HE4B and Reactive Blue
160 (Kagalkar et al., 2009, 2011; Patil et al., 2009, 2012; Khandare
et al., 2011a, 2012, 2013a; Adki et al., 2012; Kabra et al., 2013;
Watharkar et al., 2013a, 2013b; Rane et al., 2014). Whole plants possess
greater pollutant tolerance making it more suitable for treatment of
dyes. Future strategies consequently will be based on the exploration
of whole plants for treatments at actual sites of dye pollution.
6.2.2. Hairy root cultures for dye degradation
Roots are first organs to have a contact with different pollutants
which come in their vicinity. Understanding primary interaction of the
roots with pollutants is indispensable as subsequent metabolism clearly
rely on initial uptake. Hairy root cultures have earlier been used for
phytoremediation of pharmaceuticals, heavy metals, radionuclides,
PCBs, TNT and phenolics (Guillon et al., 2006). A very few reports on uti-
lization of hairy roots for dye decolorization are available. Patil et al.
(2009) have shown the potential of T. patula hairy roots in degradation
of Reactive Red 198. In the same study, hairy roots of Nicotiana tabacum,
Solanum xanthocarpum and Solanum indicum decolorized Reactive Red
198 at a concentration of 20 mg L−1 up to 95, 96 and 86%, respectively
within 12–30 days. B. juncea hairy root cultures were also found to de-
colorize Methyl Orange in an independent study (Telke et al., 2011).
Practical applications of hairy roots for remediation of textile dyes at
higher concentrations and large scales nevertheless remain uncertain
(Govindwar and Kagalkar, 2010).
6.2.3. Callus and suspension cultures for dye degradation
Leaf wax, cuticles, epidermis, endodermis and bark are regulatory
plant parts which control penetration and entry of foreign substances
from the environment. Use of callus and suspension plant cell cultures
favors the overall increase in phytoremediation of contaminant as
they are devoid of barriers possessed by whole plants. Since callus and
suspension cultures are not differentiated, their translocation processes
are undefined and hence the uptake of external compounds is more uni-
form than whole plants (Doran, 2009). It is thus well-established that
uptake of pollutants is generally more in free cells than complete plants
providing a uniform exposure to the contaminant. Plant tissue culture
like suspension cells are relatively homogenous and can be easily stan-
dardized with various procedures. This improves the chances of repro-
ducibility of outcomes as compared to complete plant growth form
(Doran, 2009). Isolation of products or metabolites formed after degra-
dation of the dye molecules by cells becomes easier and requires fewer
purification steps as the chlorophyll and starch do not interfere with
measurements. Cell cultures thus form ideal systems for dye degrada-
tion studies with respect to understanding the biochemical basis of
degradation. Cell suspension culture of Rheum palmatum has been
employed for its accumulation of sulfonated anthraquinones (Duc
et al., 1999). Kagalkar et al. (2011) have demonstrated the application
of B. malcolmii suspension cells for degradation of a triphenylmethane
dye Malachite Green. Cell cultures of N. cochenillifera have also been
shown to independently transform various toxic textile dyes like Red
HE7B, Malachite Green, Toluene Blue, Green HE4BD, Navy Blue HE2R,
Golden Yellow HER, Orange M2R and Methyl Orange (Adki et al.,
2011). Performances of various plant tissue cultures in treatment of tex-
tile dyes with initial dye concentrations and percentage decolorization
has been enlisted in Table 2.
6.3. Purified plant enzymes for degradation of textile dyes
Plants mediated decolorization of textile dyes were mostly
employed relying on their competent enzymatic machineries. Enzymes
possess highly specific catalytic actions and have proven applicability
for environmental cleanup (Rao et al., 2010). Low yield and high pro-
duction costs of purified plant enzymes are some of the major draw-
backs of using them for remediation purposes. Utilizing inexpensively
available plant sources, and raising the purification fold and recovery
percentage can however meet the purpose (Shaffiqu et al., 2002). En-
zymes can easily be immobilized on appropriate carriers after purifica-
tion and could be explored for degradation of dyes. Immobilization
techniques also add several advantages like easy separation from reac-
tion products and untreated substrates. It also allows repeated usage
and can reduce cost of the process (Mohan et al., 2005). Saccharum
spontaneum and Ipomoea palmate were proposed to be fine sources of
peroxidases. The I. palmate peroxides was observed to decolorize Meth-
yl Orange, Crystal Violet, Chrysoidine, Brilliant Green and Supranol
Green at initial concentrations of 25 mg L− 1 up to 26, 36, 44, 54 and
68% within 1–4 h. Peroxidase from S. spontaneum however could de-
grade Supranol Green up to 100% with an initial concentration of
25 mg L−1 within 1 h. The S. spontaneum peroxidase was immobilized
on a modified polyethylene hydrophobic matrix. It was further used in
a batch reactor giving efficient decolorization (100%) of Green
HE-4BD, Navy Blue HER, Brilliant Blue H-7G and Supranol Green dyes
at initial concentrations of 50 mg L−1 (Shaffiqu et al., 2002).
Immobilization of horseradish peroxidase obtained by calcium algi-
nate and polyacrylamide gel entrapment method was explored for re-
moval of azo Direct Yellow-12 and was found to perform better than
free enzymes at an optimum pH of 4 (Maddhinni et al., 2006). Calcium
alginate-starch beads poured with concavalin A was used for immobili-
zation of bitter gourd peroxidase. A batch reactor exploring the
immobilized enzymes was further constructed and applied for treat-
ment of a textile effluent which gave a noteworthy decolorization per-
formance (Matto and Husain, 2009). Peroxidase obtained from
Brassica rapa has shown the potential to degrade textile dyes. Various
mixtures of acid dyes involving Black 1, Black 21, Yellow 42, Red 92
and Blue 92 were degraded by turnip peroxidase achieving 57–95% de-
colorization within 1 h at varying (40–170 mg L−1) concentrations
(Kulshrestha and Husain, 2007).
Purified laccase from hairy roots of B. juncea was found to show
complete degradation of dyes namely Congo Red, Methyl Orange,
Malachite Green, Reactive Red BLI and Remazol Brilliant Blue at
20 mg L − 1 concentrations, independently within 1 h with 2, 2′
Azino-bis 3-ethylbenzothiazoline 6-sulfonic acid as the redox medi-
ator (Telke et al., 2011). Polyphenol oxidase (PPO) obtained from po-
tato (Solanum tuberosum) and brinjal (Solanum melongena) were
explored for dye degradation. Potato PPO could achieve 52 to 85%
decolorization of reactive dyes like Blue 4, Orange 4, Red 11, Yellow
84, Orange 86, Red 120, Blue 160 and Reactive Blue 171 at 50–
100 mg L− 1 concentrations within 30 min. Brinjal PPO conversely
was found less efficient in decolorization (Khan and Husain, 2007).
Since purified enzymes appear to be efficient systems for biodegra-
dation of textile dyes further investigations should be carried out
to explore the use of various redox mediators and inducers for
achieving improved decolorization performances of respective
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714 1703

Table 2
Phytoremediation performance of various tissue cultures for removal of textile dyes.

Sr. Name of the plant Tissue culture Dye/effluent (concentration) Decolorization time % Reference
no. type (h) decolorization

1 Ipomoea hederifolia Whole plant Scarlet RR (50 mg L−1) 96 90 Rane et al. (2014)
2 Gaillardia grandiflora Whole plant Simulated dyes mixture 36 62 Watharkar and Jadhav (2014)
3 Physalis minima Hairy roots Reactive Black 8(30 mg L−1) 120 76 Jha et al. (2015)
4 Glandularia pulchella Whole plant Scarlet RR (50 mg L−1) 48 97 Kabra et al. (2013)
5 Petunia grandiflora Whole plant Brillaint Blue G and Navy Blue RX (20 mg L−1) 36 86 and 80 Watharkar et al. (2013a, 2013b)
6 Tagetes patula Whole plant Reactive Blue 160 96 90 Patil et al. (2012)
7 Zinnia angustifolia Whole plant Remazol Black B (20 mg L−1) 72 94 Khandare et al. (2012)
8 Portulaca grandiflora Whole plant Reactive Blue 172 and Direct Red 5B (20 mg L−1) 40 and 96 98 and 92 Khandare et al. (2011a, 2013a)
9 Nopalea cochenillifera cell cultures Malachite Green and Red HE7B (40 mg L−1) 91 and 65 168 Adki et al. (2012)
10 Sesuvium portulacastrum Whole plant Green HE4B (30 mg L−1) 120 70 Patil et al. (2012)
11 Brassica juncea Hairy roots Methyl Orange (20 mg L−1) 96 92 Telke et al. (2011)
12 Blumea malcolmii Cell suspension Malachite Green (20 mg L−1) 24 93 Kagalkar et al. (2011)
13 Typhonium flagelliform Whole plant Brilliant Blue R 96 80 Kagalkar et al. (2010)
14 Blumea malcolmii Whole plant Direct Red 5B (20 mg L−1) 72 60 Kagalkar et al. (2009)
15 Tagetes patula Hairy roots Reactive Red 198 (110 mg L−1) 288 95 Patil et al. (2009)

enzymes. Use of agricultural wastes could be attempted to procure
low cost redox mediators. Economic substrata and methods ought
to be tried for immobilization making the overall process cost effec-
tive. Various purified and subsequently immobilized enzymes have
shown prospective to degrade a variety of textile dyes but their ap-
plication for actual remediation is apparently costlier than the use
of wild plants (Govindwar and Kagalkar, 2010).
6.4. Synergistic strategies for degradation of textile dyes and effluents
Phytoremediation actually is the method of treatment of pollutants
by plants and their root associated micro-flora (Ma et al., 2011; Nie
et al., 2011). Plant root exudates offer a rich source of nutrients and
growth factors for proliferation of microorganisms. The plant root envi-
ronment supports around 100 to 10,000 times greater microbial popu-
lation than other soil habitats (Glick, 2010). These bacteria serve a
number of purposes like providing plants with growth promoting fac-
tors, reducing abiotic stresses, increasing metal ion uptake, inducing
systemic resistance, producing antibiotics and synthesizing fungal cell
wall degrading enzymes (Glick, 2003; Yang et al., 2009). A large number
of reports on degradation of textile dyes by individual plants and micro-
bial systems are available. Synergistic actions of plants and microbes
have been proposed to be more effective as evident by some of the
recent works. Phytoremediation of a textile effluent by Bouteloua
dactyloides was carried out with a prior treatment of sonication followed
by augmentation with three bacterial species namely Ochrobactrum sp.,
Pseudomonas aeruginosa and Providencia vermicola giving up to 95% COD
reduction after treatment (Vijayalakshmidevi and Muthukumar, 2014).
In vitro built plant-bacterial consortia of Z. angustifolia-Exiguobacterium
aestuarii and Portulaca grandiflora–Pseudomonas putida were found to
accomplish efficient removal of Remazol Black B and Direct Red 5B, re-
spectively than their independent cultures. Degradation metabolites
obtained with consortia also showed reduced phytotoxicity than the
products obtained after independent plant and bacterial treatments
(Khandare et al., 2012, 2013a). Synergism of Portulaca grandiflora and
P. putida was explored at a 20 L capacity pilot scale reactor. Bacterial
augmentation was found to significantly boost the treatment of a simu-
lated dye mixture and a real textile effluent (Khandare et al., 2013b).
Phytoremediation potential of Petunia grandiflora was found to be en-
hanced by Bacillus pumilus augmentation and decolorized Navy Blue
RX at 50 mg L−1 within 36 h. The consortial system gave 96% decolori-
zation while plant and bacterial sets individually showed up to 80 and
77% decolorization, respectively (Watharkar et al., 2013b). An in vitro
developed consortium of tissue cultures of G. pulchella and its root asso-
ciated Pseudomonas monteilii gave a complete decolorization of Scarlet
RR at a concentration of 50 mg L−1. G. pulchella and P. monteilii individ-
ually however gave 97 and 84% decolorization within 72 and 96 h,
respectively. This consortium was further explored in a vertical flow
constructed wetland of a volume of 30 L for a successful achievement
of the treatment of real textile wastewater and a mixture of dyes
(Kabra et al., 2013). Direct augmentation of bacterial cultures to
soil could be bothersome as growth of unwanted and pathogenic mi-
crobes may take place concurrently. A static phytoreactor of a vol-
ume of 25 L with Pogonatherum crinitum grass was developed and
augmented with immobilized cells of B. pumilus. This reactor could
also achieve an enhanced degradation of a real textile effluent than
the individual reactors of plant and immobilized bacteria
(Watharkar et al., 2015).
Plant-plant consortium was proposed to be an attractive alternative
for treatment of textile dyes and showed improved efficacy towards dye
removal because of synergistic enzyme actions (Kabra et al., 2011b). A
consortium of A. amellus and G. pulchella showed a better performance
of removal of Remazol Orange 3R at 20 mg L−1 concentration and de-
graded it completely within 36 h, while individual plant systems were
found to take 72 and 96 h, respectively. A differential fate of metabolism
of the Remazol Orange 3R by individual plants, bacteria and consortia
was also reported which revealed an enhanced dye removal efficacy
by the synergism (Kabra et al., 2011b). In vitro grown plants of
G. grandiflora and Petunia grandiflora gave 62 and 76% decolorization
of a simulated dye mixture at 20 mg L− 1 concentration, respectively
while their consortium could achieve 94% color removal within 36 h
(Watharkar and Jadhav, 2014). Exploring these consortia for actual
phytoremediation is still a challenge as the environmental condi-
tions at the dye disposal sites are entirely different.
6.5. Reactor development and constructed wetland strategies for phyto-
remediation of textile dyes and effluents
Pilot scale reactor design and development can be anticipated as the
pioneering step towards in situ phytoremediation. Constructed wet-
lands are large sized plotted reactors especially planned to impersonate
conditions close to real dye wastewater situations. Treatments in con-
structed wetlands can assure greater efficacies as proper monitoring
of the whole bed becomes feasible by virtue of manageable hydraulics
and pollutant loads (Barbera et al., 2009). Many studies involving
P. australis for phytoremediation of dyes have been carried out. A verti-
cal flow constructed wetland was developed with 1 m3 capacity- feed-
ing reservoir with P. australis plants. It was successively fed with 700 L
of Acid Orange 7 at 130–700 mg L−1 concentration in batches. A sub-
mersible centrifuge pump was used for mixing and feeding with a Fisher
flow meter to control the inlet flow. A gravel layer of sandy–clay soil for
supporting plant growth was maintained with drainage. An intermit-
tent feeding of the dye was monitored. The reactor was found to reveal
noteworthy treatment achieving 69 and 67% COD and TOC removal,
1704 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
respectively (Davies et al., 2005, 2009). A triple bedded interconnected
pilot scale reactor with vertical and horizontal flow filled with a layer of
sand and gravel with P. australis plantation was developed for treatment
of textile effluents in another study. It was found to remove textile dyes
from an effluent achieving reductions in COD, BOD, TOC, total nitrogen,
organic nitrogen, ammonia, sulfate ions, surfactant, TSS and color by 84,
66, 89, 52, 87, 331, 88, 80, 93 and 90%, respectively (Bulc and Ojstrsek,
2008).
An aerated and non-aerated up flow constructed wetland was built
with a bed of glass beads and sand gravel provided with an air supply.
This reactor showed 96% Acid Orange 7 removal at 50–100 mg L− 1
dye concentration in both types of reactors within 6 days. The aerated
and non-aerated reactors gave a COD removal efficiency of 86 and
75%, respectively (Ong et al., 2009, 2010). A vertical flow constructed
wetland with P. australis was independently developed and employed
for the treatment of effluent containing Direct Red 81 and showed a
COD removal of 90% and color removal of 89% (Ferreira et al., 2014).
Echinodorus cordifolius (burhead) has been explored for the treatment
of structurally different dyes in a constructed wetland system. A reactor
was assembled with 0.44 × 0.60 × 0.28 m3 plastic crates and tests were
performed with soil and soil-free surroundings. Burhead plants were in-
dependently grown in soil and water. Flat plastic mesh was used to hold
the plants. A significant removal of Reactive Red 141 was observed with
reductions in TDS and conductivity by 42 and 50% within 2 days in soil-
free conditions (Noonpui and Thiravetyan, 2011). In another work, a
microcosm planted with Canna indica enriched with sulfate reducing
bacteria was found to achieve dye removal to the tune of 99.50, 98.53,
97.05, 94.62, and 96.52% from a synthetic wastewater containing Meth-
yl Orange at 500, 1000, 1500, 2000 and 2500 mg L−1 concentrations,
respectively (Yadav et al., 2012).
Vertical subsurface flow constructed wetlands fabricated indepen-
dently using G. pulchella and Portulaca grandiflora have also been
Fig. 2. a) A phytoreactor with Portulaca grandiflora plants for treatment of textile effluent
and b) cross section view of the phytoreactor has been adapted from Khandare et al.
(2013b) to illustrate the recently developed constructed wetland which has been
reevaluated for textile effluent treatment by Shehzadi et al. (2014) using T. doningensis
plants.
explored for textile wastewater systems (Fig. 2a). Since these are arid
plants, the designed systems could retain half of the roots in soil and re-
maining in wastewater. Reactors had a continuous circulatory flow of
the wastewater enabling repeated exposure and increased retention
time of effluents which can be understood from graphic illustration of
the reactor (Fig. 2b). Efficacies of these reactors were enhanced by bac-
terial augmentation. The reactor with G. pulchella and P. monteilii bacte-
rial cells could achieve ADMI value, BOD, COD and TOC removal by 93,
70, 66 and 74%, respectively which was found to be superior than the in-
dividual reactor of plants and bacteria when monitored for 60 h (Kabra
et al., 2013). A similar reactor built utilizing Portulaca grandiflora and
P. putida bacterial augmentation also showed noteworthy reductions
in BOD, COD, TOC, TDS, TSS and turbidity by 54, 73, 52, 83, 71 and
57%, respectively after treatment of a textile effluent for 48 h, whereas
the individual plants' and bacterial sets took 72 h to give comparable
performances (Khandare et al., 2013b). The similar system was
reevaluated with T.domingensis with Microbacterium arborescens and
B. pumilus augmentation revealing significant reductions in COD, BOD,
TDS and TSS by 79, 77, 59 and 27%, respectively after 72 h (Shehzadi
et al., 2014). An open aerated system made up of polyvinyl chloride
pipes called phyto-tunnel was developed using Portulaca grandiflora
plants in another pilot scale demonstration. It was a half filled horizontal
continuous flow system providing open aeration to the roots. After
treatment in phyto-tunnel the ADMI value, COD, BOD, TOC, TSS, TDS,
conductivity and turbidity of a textile effluent and dye mixture were re-
duced up to 45, 43, 57, 77, 24 52 and 76%, within 96 h and 62, 49, 41, 71,
33, 63 and 58% within 60 h, respectively (Khandare et al., 2014). A static
reactor was developed with P. crinitum planted on a thermocol base and
the roots were exposed to a real textile wastewater. Immobilized
B. pumilus cells were further augmented directly to the wastewater in
root zone. The augmentation was found to enhance the treatment effi-
cacy and reduced BOD, COD, ADMI value, TDS, TSS, conductivity and tur-
bidity of the effluent by 70, 78, 93, 13, 70, 4 and 90%, respectively within
12 days (Watharkar et al., 2015). Advantage of using immobilized bac-
terial cells can be attributed to their reusability and reduced risk of
growth of pathogenic microorganisms. Rhizofiltration has also been
proposed to be an effective method of dye removal from effluents. A
steel reactor with dimensions of 1.2 m × 0.61 m × 0.61 m with a capac-
ity of 340 L was constructed and planted with A. philoxeroides. An iron
grid just below the inlet was placed in such a manner that it was able
to hold plants and allow the roots to float and grow in the effluent. A
very efficient treatment of textile effluents of varying pH (3.5–10.5)
was successfully carried out achieving significant reductions in ADMI
value, COD, BOD and total solids of six different effluent samples after
96 h of reactor treatment. The pH of different effluents (acidic and alka-
line) was observed to be increased or decreased accordingly to attain
near neutral pH after treatment in the reactor (Rane et al., 2015).
It should be noted that the efficacy of any system largely depends on
the basic physicochemical and biological processes stimulated by the in-
teraction of plants, microorganisms, pollutants, substrates, and many
other biotic and abiotic components. The above discussed reactor sys-
tems therefore showed varying efficiencies but they certainly provide
a window of opportunity to develop technologies for treatment at larger
scales.
7. Plant mechanisms for treatment of textile dyes and effluents
Very limited information on plants' mechanisms for metabolism of
textile dyes is available. Plants are autotrophic and are thought to take
up xenobiotics during their natural mineral and water absorption.
Over the long periods of time during evolution, plants have adapted
stress countering mechanisms along with synthesis of enzymes which
are now being explored as tools for the treatment of pollutants
(Govindwar and Kagalkar, 2010). Rhizodegradation, phytodegradation,
phytoaccumulation, phytovolatilization and phytostabilization are
classical and well studied mechanisms of phytoremediation. These
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
mechanisms are not exclusive and can occur simultaneously depending
upon nature of pollutants and the involved vegetation. Plants however
remove textile dyes predominantly by adsorption, accumulation and
subsequent enzyme mediated degradation in different parts.
7.1. Adsorptive and/or accumulative removal of textile dyes
Removal of textile dyes has been carried out using both dead and liv-
ing plant biomass. It is obvious that the dead plant parts can only per-
form adsorptive removal of dyes and therefore cannot be called as a
true phytotreatment. Giant duck weed (Spirodela polyrrhiza) dried bio-
mass was used as an adsorbent for the elimination of Methylene Blue
from its solution (Waranusantigul et al., 2003). Prosopis cineraria saw-
dust was explored for adsorptive removal of Malachite Green. Indepen-
dent activation of this adsorbent with formaldehyde and sulfuric acid
was found to enhance the adsorption capacity (Garg et al., 2004). Use
of peanut hulls, neem leaves, banana peels, orange peels and different
agricultural wastes has been proposed for the removal of colors from
effluents. Leaf sheaths of Posidonia oceanica also showed effective ad-
sorption of Reactive Red 228 (Ncibi et al., 2007). Sulfuric acid and phos-
phoric acid treated Parthenium hysterophorus adsorbents were
successfully used to remove Methylene Blue at 50–250 mg L−1 concen-
trations (Lata et al., 2007). Carbonization and activation by ZnCl2 of the
pulverized leaves of Salsola vermiculata were found to increase adsorp-
tion of Methylene Blue and iodine from aqueous solutions (Bestani et al.,
2008). Reactive Blue 172 at a concentration of 100 mg L−1 was adsorbed
up to 60% on sugar cane bagasse which was further exposed to
Providensia staurti bacterial culture and degraded under solid state fer-
mentation (Waghmare et al., 2014).
Elimination of organic pollutants like textile dyes by degradation
and transformation are supposed to be the principal mechanisms
adopted by plants. It is now well-known that binding of pollutants to
roots takes place by adsorption followed by its uptake into the plant tis-
sues (Davies et al., 2005). Adsorptive removal of dyes has also been re-
ported in living plants and cells. For instance, Malachite Green was
found to be adsorbed on the roots of B. malcolmii achieving 45% dye re-
moval (Kagalkar et al., 2009). Many dyes like Red HE7B, Red HE8B,
Direct Red 5B, Golden Yellow HER, Methyl Orange, Malachite Green,
Patent Blue, Reactive Red 2 and Brilliant Blue R have been reported to
be adsorbed on the roots of in vitro grown T. flagelliforme plantlets
(Kagalkar et al., 2010). Involvement of living tissues however is advan-
tageous as the remediation processes do not only imply adsorption but
also accumulate dyes for further process. Sulfonated anthraquinones-
raw materials for synthesis of a number of synthetic textile dyes were
removed by R. rhabarbarum with accumulation as the prime mechanism
which was evident by transpiration stream concentration factor (TSCF)
determination. If the value of TSCF exceeds unity should mean that
movement of pollutant is faster than water. Studies on R. rhabarbarum
and R. hydrolapatum on various anthraquinones showed a TSCF value
of 2.5 for anthraquinone-1-sulfonic acid which clearly indicated that
the pollutant was moving quicker than water in the plant's vascula-
ture. Other pollutants like anthraquinone-2-sulfonic acid and
anthraquinone-2,6-disulfonic acid also gave greater TSCF values
(Aubert and Schwitzguébel, 2004). Plants have been observed to be
accumulating and subsequently degrading textile dyes in different
tissues. These mechanisms are not exclusive and can occur simulta-
neous in various plant parts. Anatomical studies performed on
Eichhornia crassipes have shown severe deterioration in the cellular
structures and reduced the growth of root cells. Parenchymatous
cells in roots, stem and leaves revealed presence of raphide crystals
(Mahmood et al., 2005a). T. angustifolia commonly called as narrow
leaved cattails a well known hyper extractor of dissolved nutrients
has shown an efficient uptake of Reactive Red 141. Transmission
electron microscopic imaging of plant roots and leaves revealed
the accumulation of dye in tissues after 28 days of exposure
(Nilratnisakorn et al., 2007). Anatomical studies on I. hederifolia
1705
upon exposure to Scarlet RR showed presence of dye in the epider-
mal cells after 6 h which moved to cortex after 12 h. After 24 h, accu-
mulation was found to be increased in the cortical cells with
subsequent degradation and removal in epidermis of stem. The accu-
mulated dye started disappearing after 48 h from the stem tissues re-
vealing a specialized cellular mechanism of dye degradation (Rane
et al., 2014). A similar accumulation of Remazol Red specifically in
the stem tissues of A. philoxeroides was also revealed anatomically.
The dye was found to be accumulated in epidermal cells at 8 h of ex-
posure and subsequently moved to cortex after 32 h. Decolorization
was observed to commence from 40 h and the color disappeared
completely after 48 h (Rane et al., 2015).
7.2. Plant enzymes involved in stress response and dye degradation
Transformation abilities of plants are of immense significance as
they can completely degrade dyes rendering them into products
which are harmless to life forms and can be safely released into the en-
vironment. Plants have developed different mechanisms for protection
from abiotic stresses including the presence of xenobiotics in their vicin-
ity (Page and Schwitzguébel, 2009). They own very diverse and multi-
farious systems through which they are able to treat compounds that
are not degradable by a variety of microorganisms. Plants possess effi-
cient enzymatic machineries that can take up aromatic compounds as
substrates (Aubert and Schwitzguébel, 2004). Oxidative stress response
enzymes like catalase (CAT), superoxide dismutase (SOD) and glutathi-
one S-transferase (GST) etc. are known to be expressed as a function xe-
nobiotic exposure including textile dyes. Further detoxification of dyes
by means of complete mineralization involves a systemic interplay of
different oxidoreductive enzymes including cytochrome P450. A precise
detection of every dye metabolite throws light on the role of each of
these enzymes in degradation pathways and helps to improve our un-
derstanding. Occurrences of dye containing solutions in the root vicini-
ties offer stress and plants try to overcome it with intrinsic mechanisms.
Concentration of reactive oxygen species (ROS) increases by the en-
zymes like NADPH oxidase as a part of plant defense mechanism
resulting in activation of antioxidant scavenging enzymes. SOD is
known to catalyze the conversion of ROS into H2O2 which later is trans-
formed into oxygen and water by the action of peroxidases and CAT.
Plant detoxification mechanisms are broadly constituted by three
different phases. In phase I cytochrome P450 and peroxidases oxidize
the xenobiotics. In phase II the oxidized xenobiotics are conjugated to
glutathione by the action of GST. In phase III these conjugates are
translocated into vacuoles differentially (Carias et al., 2007, 2008). A
multigenic cytochrome P450 monooxygenases family of enzymes is im-
plicated for detoxification of many xenobiotic compounds. Cytochrome
P450 plays a vital role in metabolism of plant compounds involved in
defense mechanisms and cell signaling. Dioxygenase was reported to
add oxygen to double bond holding sulfonate group in sulfonated an-
thraquinones and eliminated it subsequently (Schwitzguébel et al.,
2002). Capillary electrophoresis was used as a tool to expose the trans-
location site on studies with rhubarb and sulfonated anthraquinones
were found to accumulate in the leaves (Aubert and Schwitzguébel,
2002). Presence of new metabolites was also detected in further studies
supporting the biotransformation of these compounds (Aubert and
Schwitzguébel, 2004). Rhubarb leaves with different anthraquinone
substrates showed a significant rise in the activities of monooxygenases.
Highest enzyme activity was observed in leaf tissues indicating that the
uptake of anthraquinones was taking place in the leaves (Page and
Schwitzguébel, 2009). Enzymes such as ascorbate oxidase and catechol
2,3-dioxygenase earlier have shown their presence in plants like cress,
alfalfa and mustard but their role in dye degradation was not studied
(Gramss and Rudeschko, 1998).
GST is another detoxifying enzyme that has been studied in plants.
This enzyme covalently link glutathione to a broad variety of reactive
hydrophobic and electrophilic substrates resulting into polar and less
1706 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
reactive moieties. Enzyme activity of GST was reported to be increased
in P. australis while degrading Acid Orange 7 in a pilot scale constructed
wetland (Carias et al., 2008). Hairy root cultures of Physalis minima ex-
posed to Reactive Black 8 was found to show induction in SOD activity
up to nine fold after 24 h. While the activities of peroxidase and ascor-
bate peroxidase were increased by 22 and 50 folds, respectively after
72 h (Jha et al., 2015). Molecular level studies performed after Acid
Orange 7 degradation by P. australis revealed the triggered gene expres-
sion for Cu/Zn and Mn SOD, CAT isoforms and glutathione peroxidase.
Over expression of these genes was proposed to be related to the sud-
den production of ROS after dye exposure (Davies et al., 2009).
Increased content of dehydroascorbate reductase which regulates
ascorbate glutathione pathway was also found in P. australis exposed
to Acid Orange 7. Significant increase in GST activity revealed the com-
partmentalization of dye in plant cells (Carias et al., 2008).
Peroxidases have been focused and studied to have a significant role
in degradation of the textile dyes by plants. Oxido-reductive enzymes
were assayed in three different plants namely mustard, alfalfa and
cress which showed that the peroxidases were most dominating en-
zymes in shoots, roots and exudates (Gramss and Rudeschko, 1998).
Lignin peroxidases (LiP) are the key enzymes which can oxidize lignin
structures of wood by yielding a cation radical as an intermediate that
undergoes spontaneous fission. Studies on lignin model like compounds
have revealed that LiP attacks the Cα and Cβ of its propyl side chain pres-
ent in aryl glycerol β-aryl ether substructure of lignin (Sarkanen et al.,
1991). LiP, a heme enclosing enzyme is versatile in utilizing a wide
range of organic chemical species as electron donor substrates and hy-
drogen peroxide for oxidation of the compound. Firstly it cleaves the hy-
drogen peroxide molecule leading to formation of a water molecule
followed by incorporation of oxygen atom into the initial intermediate
compound. In subsequent steps the enzyme is reduced in order to get
regenerated. The azo dyes can act as electron donors for these enzymes
(Davies et al., 2005; Carias et al., 2007). Many plant species have shown
the presence of peroxidases in their tissues. Addition of H2O2 in cultiva-
tion medium was found to clearly enhance the degradation of Remazol
Brilliant Blue R by Rumex crispus (Takahashi et al., 2005). Degradation of
Acid Orange 7 by P. australis was tuned rapidly after addition of H2O2 as
the mediator (Davies et al., 2005). MPH-4 clonal lines of Mentha
pulegium showed an increase in guaicol peroxidase activity while de-
crease in the phenolic content as a response to Polydye R-478 indicating
that the phenolics had been used up for lignin biosynthesis process
(Strycharz and Shetty, 2002a). Similar results with phenolic content
and guaicol peroxidase activity were demonstrated in case of oregano
(Origanum vulgare) cell lines. It was speculated that the dye might
being used as a substrate for peroxidase cross linking and then partici-
pate in lignification process though there was no evidence provided
(Strycharz and Shetty, 2002b). Enzyme activities of guaiacol peroxidase
in the roots of R. crispus and C. javanicus were also found to be significantly
induced upon initial exposure (1–2 days) to Polydye R-478 (Paquin et al.,
2006). These findings pointed out the primary involvement and action of
peroxidases on textile dyes. Significant inductions in the intracellular
LiP activities in roots of B. malcolmii, A. amellus, Portulaca grandiflora,
Z. angustifolia, G. pulchella, T. flagelliforme, Petunia grandiflora,
I. hederifolia, S. portulacastrum and N. cochenillifera was observed during
degradation of various textile dyes (Kagalkar et al., 2009, 2010; Kabra
et al., 2011a; Khandare et al., 2011a, 2011b, 2012; Adki et al., 2012; Patil
et al., 2012; Watharkar et al., 2013a; Rane et al., 2014).
Laccases form a class of polyphenol oxidases which catalyze the
oxidation of different substituted phenolic compounds using O2 as an
electron acceptor. Hydroxyl group of ortho/para-substituted mono
and polyphenolic substrates are the sites of attack for this enzyme. Re-
moval of H atom and formation of aromatic amines by one electron
transfer lead to release of free radicals which can further undergo de-
methylation, repolymerization, depolymerization and/or quinone for-
mation (Abadulla et al., 2000). Laccases have been shown to possess
immense potential in degradation of textile industry wastes. Bacterial
and fungal laccases are well known for their potential in dye decoloriza-
tion (Telke et al., 2010b). They are known to break the rings of aromatic
compounds and can also perform oxidative cleavage (Chivukula and
Renganathan, 1995; Strong and Claus, 2011). Very limited information
on involvement of plant laccases in phytoremediation of dyes is avail-
able. B. juncea root cells were found to show a significant induction in
activity of intracellular laccase while degrading Reactive Red 2 and a
textile effluent (Ghodake et al., 2009b). A similar opinion was also
made during removal of Reactive Red 198 by T. patula hairy root cul-
tures (Patil et al., 2009). In vitro studies on hairy root cultures of
B. juncea exposed to Methyl Orange showed an enhancement in laccase
activity along with involvement of various redox mediators favoring
degradation. Activity of laccase in the dye unexposed root however
was completely absent (Telke et al., 2011). Roots of wild plants of
G. pulchella also illustrated an induction in laccase activity while
degrading Green HE4B giving asymmetrical cleavage of the dye struc-
ture (Kabra et al., 2011a). Fig. 3 shows different enzymes involved in
textile dye degradation with their catalytic actions and mechanisms.
Tyrosinases are polyphenol oxidases with copper which bring about
the o-hydroxylation of some monophenols and oxidation of o-
diphenols to o-quinones using oxygen molecules (Chen and Flurkey,
2002). Tyrosinase have been extensively evaluated for their involve-
ment in microbial dye degradation, studies on plants for dye removal
conversely has yet remained poorly understood. B. malcolmii and
Portulaca grandiflora tissue cultures showed a clear induction in tyrosi-
nase activities in root cells while degrading Direct Red 5B (Kagalkar
et al., 2009; Khandare et al., 2011a). Intracellular and extracellular en-
zyme activities of tyrosinase was also found to show significant induc-
tion in T. patula hairy roots when exposed to Reactive Red 198 (Patil
et al., 2009). Inhibition studies on enzymes purified from roots of
G. pulchella upon exposure to Green HE4B was carried out and a com-
petitive inhibition of laccase and LiP was observed showing their in-
volvement in dye degradation. Enzyme activity of tyrosinase on the
other hand was not competitively inhibited confirming that mere in-
duction in the enzyme activity does not validate its role in degradation
(Kabra et al., 2011a).
Some other enzymes have also shown inductions in the activities
while degrading textile dyes. Activities of azo reductase, riboflavin re-
ductase and dichlorophenol indophenol (DCIP) reductase were found
to induce to varying extents during degradation of Direct Red 5B by
B. malcolmii (Kagalkar et al., 2009). Enzymatic status and mechanisms
of dye removal are found to be highly unpredictable with different
plant species. G. pulchella showed varying extent of activities LiP,
veratryl alcohol oxidase, tyrosinase and DCIP reductase when exposed
independently to Brilliant Blue R, Scarlet RR, Red HE3B, Rubine GFL
and Navy Blue 2R. Differential pattern of induction in enzyme activities
with respect to time was observed during decolorization of dye mixture.
Activities of LiP and veratryl alcohol oxidase were found to constantly
increase during 34 to 96 h of exposure whereas tyrosinase activity
was observed to be enhanced only after 24 h and started decreasing
later showing its role in degradation process (Kabra et al., 2012).
8. Scrutiny of the products after dye phyto-removal/degradation
Dyes, effluents and their remediation products must be critically an-
alyzed before their release to the environmental sink. Toxicity of dyes
on a variety of organisms is available in well documented formats.
Metabolites of dyes are known to remain toxic and recalcitrant after in-
appropriate or incomplete treatment. Examination of dye products for
various environmentally important parameters, toxicity and under-
standing of their fate of metabolism therefore becomes inevitable.
8.1. Characterization of the textiles dyes and effluents after phytotreatment
Textile wastewaters as earlier stated are one of the most toxic and
recalcitrant contaminants of our valuable soils and aquatic bodies.
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
Fig. 3. Mechanisms of catalysis of different oxido-reductase enzymes for dye degradation. The catalytic action of laccase and LiP have been adapted from Wesenberg et al. (2003) to sche-
matically understand their specific catalytic action and mechanism.
Textile dye processors release these waters containing complex mix-
tures of acids, bases, detergents, surfactants, humectants, mordants, fas-
teners, fixers, softening agents and textile dyes of different classes. This
makes these wastewaters acquire extremely high COD, BOD, TOC, TSS,
TDS, turbidity and conductivity. Plant based treatments have shown
these effluents to be rendered to less toxic levels. E. crassipes has been
used for textile effluent treatment at laboratory scale and could reduce
the BOD and COD by 40–70% (Mahmood et al., 2005b). P. australis in a
vertical flow constructed wetland was found to show 67–69% COD
and TOC removal (Davies et al., 2005). A combinatorial vertical and
horizontal flow lab scale reactor with P. australis plants also achieved
reductions in BOD and COD up to 45%; a similar system at pilot scale
showed an enhancement in the treatment efficacy (Bulc and Ojstrsek,
2008). A static bioreactor planted with T. angustifolia was found efficient
in treating textile effluent to unobjectionable level and reduced the TDS,
COD and color by 86, 59 and 58%, respectively (Nilratnisakorn et al.,
2009). In a vertical flow lab scale reactor with G. pulchella plants was
also found to reduce the COD, TOC and BOD by 70, 74 and 70%, respec-
tively within 60 h (Kabra et al., 2013). A similar kind of reactor with
Portulaca grandiflora was found to be effective in treating textile efflu-
ents to less toxic levels and attained the COD, BOD, TOC, turbidity,
TDS and TSS reductions by 59, 38, 37, 41, 71 and 60%, respectively
within 72 h (Khandare et al., 2013b). A phyto-tunnel constructed
with Portulaca grandiflora plantation revealed notable decrease in
COD, BOD, TOC, turbidity, conductivity, TDS and TSS of a textile efflu-
ent by 57, 45, 43, 76, 52, 24 and 77%, respectively within 96 h
(Khandare et al., 2014). A static hydroponic phytoreactor planted
with P. crinitum could also treat a textile effluent to harmless levels
achieving reductions in COD, BOD, ADMI, conductivity, turbidity,
1707
TDS and TSS by 59, 54, 74, 0.60, 44, 11 and 7%, respectively
(Watharkar et al., 2015).
8.2. Structural elucidation of dye products and analysis of fate of
metabolism
Loss of color is an obvious indicator of dye removal from solu-
tions but the assessment of products or metabolites; their chemical
nature and further evaluation of toxicity becomes essential. The
dye metabolites are generally extracted using organic solvents
like dimethylsulfoxide, ethyl acetate, chloroform, methanol etc.
and are evaporated to get dried residues which are further assayed
by a number of analytical tools like Fourier transform infra-red
spectroscopy (FTIR), thin layer chromatography (TLC), high per-
formance liquid chromatography (HPLC), high performance thin
layer chromatography (HPTLC), gas chromatography–mass spec-
troscopy (GC–MS), liquid chromatography–mass spectroscopy
(LC–MS) and so on.
FTIR essentially works on the principal of tracking slightest
changes in classical IR fingerprint region of the spectra. It clearly
depicts the changes in chemical structures viz. functional groups
and bond deviations like distortion, stretching, bending, deforma-
tion etc. FTIR spectrum provides a direct evidence of transforma-
tion of parent compounds to other metabolites. For example, FTIR
spectrum of Direct Red 5B showed different peaks at 1754.7 and
1619.3 cm − 1 for C = O stretching and N = N stretching, respec-
tively revealing azo nature of the compound. Peaks at 1546.7 and
1486 cm − 1 showed N–O stretching, while peaks at 1286.8,
1134.7, 1045.1 cm − 1 represented S = O stretching revealing
1708 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
Fig. 4. HPLC analysis of (a) Navy Blue HE2R (b) root exudates and c) products of the dye
after degradation by Portulaca grandiflora in vitro.
sulfonated nature of the dye. After treatment by Portulaca grandiflora
the products showed peaks at 1669.0 and 1107.3 cm− 1 representing
C = O which indicated the appearance of these groups. Peaks at
1443.5 and 1298.3 cm− 1 represents N–O stretching indicating the
Fig. 5. A proposed pathway of degradation of Navy Blue HE2R dye by Portulaca grandiflora tissue cultures showing involvement of enzymes supported with mass peaks.
formation of oxidized product. The only peak at 1225.7 cm− 1 for
S = O stretching supported desulfonation of Direct Red 5B after
phytotransformation. HPLC analysis of Navy Blue HE2R showed a
peak at 3.192 and 2.959 min (Fig. 4a); peaks of root exudates were
also taken as biological control (Fig. 4b). Dye metabolites after treat-
ment by Portulaca grandiflora however showed different peaks at
6.604, 5.124, 3.181 and 2.859 min signifying new products
(Fig. 4c). Presence of different peaks and loss of dye peaks clearly
supports the formation of different metabolites and degradation of
parent dye. HPTLC can also be a useful tool for the analysis of a dye
metabolism and clearly show disappearance of dye after treatment.
A mixture of dyes namely Malachite Green, Direct Red 2B, Remazol
Red, Scarlet RR and Brown 3REL treated by G. pulchella demonstrated
the disappearance of dyes and appearance of some new metabolites
confirming the phytoremoval and degradation of parent dyes (Kabra
et al., 2013).
Mass spectrometric analyses coupled with a GC and/or LC is exten-
sively used as valuable tools for identification of metabolites formed
after dye degradation. Parent dyes always have greater masses because
of complex structures, sulfonated and halogenated moieties. The me-
tabolites after degradation apparently show lower masses and peaks
at different retention times. Preceding information from FTIR spectra
and HPLC and/or HPTLC also helps in prediction of these metabolites.
Plant enzymes have specific and well studied mechanisms of dye cleav-
age and are therefore vital to propose the pathways of dye degradation.
Portulaca grandiflora root cells showed an induction in the enzyme ac-
tivities of LiP, DCIP reductase, tyrosinase and riboflavin reductase.
Based on this information and GC–MS analysis, a hypothetical pathway
of dye degradation was proposed (Khandare et al., 2011a). In vitro deg-
radation studies on Navy Blue HE2R by Portulaca grandiflora tissue cul-
tures and its GC–MS analysis was used for prediction and
understanding the fate of metabolism of dye. LiP brought about the
asymmetric cleavage of Navy Blue HE2R to form N-benzylacetamide
[Rt 14.764 min] and intermediate I which after subsequent deamination
and desulfonation gave 6-diazenyl-4-hydroxynaphthalene-2-sulfonic
acid [Rt 15.054 min] and an unidentified compound (Fig. 5). The records
were also supported by mass peaks.
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
8.3. Toxicity assessment of textile dyes, effluents and their degradation
metabolites after phytoremediation
Textile wastewater in treated and untreated form is observed to be
utilized for irrigation and other agricultural purposes in many develop-
ing countries (Kabra et al., 2013; Shehzadi et al., 2014). This was consid-
ered as the very basis behind performing phytotoxicity assessment
of the products of dye degradation on common crop plants. Textile
effluents treated independently by Portulaca grandiflora and
G. pulchella have shown reduced toxic effects on germination of Sor-
ghum vulgare and Phaseolus mungo seeds when compared to germina-
tion in untreated effluents (Khandare et al., 2013b). Earlier works on
treatment of individual dyes like Brilliant Blue R, Direct Red5B, Remazol
Red, Remazol Orange 3R, Remazol Black B, Green HE4B and Brilliant
Blue G also showed the reduced toxicity of these dyes after
phytotreatments. Healthy root and shoot developments after watering
with phytotreated dye solutions was observed after germination of
S. vulgare, Vigna radiata, Triticum aestivum and P. mungo confirming
that the products of dyes were nontoxic to these plants (Kagalkar
et al., 2011; Rane et al., 2014).
Dyes and their metabolites have been assayed for cytogenotoxic ef-
fects on Allium cepa root cells. Products of Navy Blue RX after in vitro
treatment by Petunia grandiflora was found to show less toxic effect
on A. cepa root cells and revealed superior mitotic indices, root lengths
and lesser frequencies of chromosomal aberrations when compared to
untreated dye. A comet assay (single cell gel electrophoresis) for detec-
tion of DNA damage in the root meristem cells of A. cepa exposed to un-
treated and treated Navy Blue RX solution was carried out. The treated
sample showed reduced percentages of tailing, DNA in head and tail
lengths (Watharkar et al., 2013b). An untreated dye mixture caused
the formation of binucleated cells with micronuclei, disintegrated pro-
phases, sticky anaphases, sticky metaphases and laggard chromosomes
formation in the A. cepa root cells. The same effluent after treatment by
Petunia grandiflora and G. grandiflora showed a reduced toxicity and
only a few cellular alterations were observed (Watharkar and Jadhav,
2014).
Toxicity analysis of an effluent and their phytotreated products was
carried out taking Etheostoma olmstedi fish as a model animal. A static
hydroponic reactor using P. crinitum was constructed and augmented
with immobilized B. pumilus cells. A 12-day effluent treatment in the re-
actor and subsequent histological studies on fish gills revealed that the
untreated effluent had severe toxic effects on gill cells. Presence of in-
flammation, hyperplasia, hypertrophy, abnormal cell sizes, desquama-
tion of lamellae and hemorrhage was observed in the fishes exposed
to untreated effluent. Exposure to treated effluent however was found
to keep the lamellae intact and no toxic effect was seen in gills of the
fishes in treated sample (Watharkar et al., 2015). The untreated effluent
was also found to exhibit extreme stress to fishes and enzymes like SOD
and CAT activities were highly induced with high level of lipid peroxida-
tion. On the other hand the treated effluent showed very less lipid per-
oxidation and reduced activities of SOD and CAT revealing the less toxic
nature of metabolites (Watharkar et al., 2015). Sulfonated Remazol Red
at a concentration of 70 mg L−1 was observed to exert deteriorating
effects on gills of Devario aequipinnatus fishes. Distortions in gill struc-
tures like lamellar curling, blood congestion, loss of secondary lamellae,
vasodilatation and telangiectasis after histological examination was
observed in the fishes after a 14 day of dye exposure. The fishes exposed
to treated dye on the other hand were found to posses' intact gill histol-
ogy and no deformities (Rane et al., 2015).
9. Factors affecting the phytoremediation of textile dyes and
effluents
Understanding the limiting factors and processes during phyto-
treatment of textile dyes and effluents would help in designing superior
strategies for in situ remediation. Most of the studies on
1709
phytoremediation of textile dyes have lingered at the laboratory
bench scale levels. It is therefore highly desirable to understand various
forces which can affect the phytoremediation at actual sites of dye dis-
posal. Various physico-chemical and biological factors like plants'
growth form, nature/structural diversity and concentration of dyes;
availability of dyes to plant roots; pH of dye wastewater, temperature
and humidity; organic matter content in media, water, oxygen and nu-
trients availability; rhizospheric process; biomass of the remediating
plants, growth of microbes in root zone and mass transfer limitations
of pollutant etc. directly affect the phytoremediation (Pilon-Smits,
2005). A few important factors are discussed ahead.
9.1. Growth form of the plants
A variety of plants with different habits like halophytes, garden or-
namentals, arid plants and even aquatic macrophytes have been used
for treatment of textile dyes. Plants with robust and fibrous root systems
are generally thought to be highly favorable for efficient treatment of
dyes. Small trees like R. rhabarbarum were used for the removal of
sulfonated anthraquinones which are precursors for dyes manufactur-
ing industries. S. portulacastrum which is a halophyte with very less
root biomass was found to facilitate the decolorization of Green HE4B
in higher salinity conditions (Patil et al., 2012). Commonly found garden
plants and ornamentals were proposed to be beneficial as they
yield other products like flowers and could bring wastelands under
public use. Garden ornamentals like A. amellus, G. pulchella,
Petunia grandiflora, G. grandiflora and Z. angustifolia with their
dense roots showed an excellent potential of dye degradation involving
a similar set of enzymes during degradation processes (Kabra et al.,
2011a, 2011b; Khandare et al., 2012; Watharkar et al., 2013b). Huge bio-
mass containing aquatic macrophytes like P. australis, T. flagelliforme,
T. angustifolia, T. domingensis and A. philoxeroides were found to exhibit
dye degradation potentials suggesting involvement of various oxido-
reductases and stress related enzymes (Nilratnisakorn et al., 2007;
Carias et al., 2008; Shehzadi et al., 2014; Rane et al., 2015). Plant anatom-
ical studies have pointed out that the C4 plants possess better
phytodegrading abilities than other physiological counterparts.
9.2. Dye availability, hydraulics and pollutant load (dye concentration)
Composition of effluent is a determining factor for efficient degrada-
tion of dye by plants. The pH of effluent is also a contributing factor
which can increase or decrease the availability of pollutant. Cation ex-
change capacity becomes important when pollutant availability is con-
cerned. Acidic pH means richness of H+ ions, these ions can replace
the cations on dyes making them available for plant uptake. The organic
matter content and charge present on dye are two major factors behind
dye bioavailability. Organic matter is composed of dead cells of mi-
crobes, plants parts, plants and their secretions etc. While it is known
that the organic matter has negatively charged groups (Pilon-Smits,
2005), dyes with positively charged groups can naturally bind to this or-
ganic matter. Such dye particles can easily become unavailable for the
uptake by plants. This factor was found to impact atrazine metabolism
and degradation by poplar trees (Burken and Schnoor, 1997).
Wastewater treatment by phytoremediation is essentially affected
by hydraulic shock loads, TSS and TDS of the effluents to be treated. It
has been observed that high TSS becomes a barrier for phyto-based
treatment strategies and can also obstruct the rhizofiltration. High TDS
can disturb oxygen transfer and interfere with biological metabolism
of pollutants (Pophali et al., 2003). Physico-chemical properties like hy-
drophobicity, volatility and dye concentrations can also affect the bio-
availability. Log Kow i.e. octanol: water distribution is the measure of
hydrophobicity of pollutant (Trapp and McFarlane, 1995). A Log Kow
value greater than 3 denotes the recalcitrant nature of any pollutant
while pollutants with Log Kow lesser than 3 are moderately or complete-
ly soluble in water making itself available for plant uptake. Henry's law
1710 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
constant (Hi) measures the tendency of pollutant to mobile in air or liq-
uid form (Volatility). It has earlier been shown that pollutant with Hi
greater than 10−4 has a tendency to move through the air spaces and
thus is highly volatile in nature and it can easily be released in atmo-
sphere by phytovolatilization process. While a pollutant with Hi lesser
than 10−6 are less volatile and more soluble in water and can finally
get sequestered or degraded by plants. Pollutant having Hi in between
of 10−4 and 10−6 can get dissolved in both air and water (Bromilow
and Chamberlain, 1995). Maintenance and monitoring the dye compo-
sition, pH and ultimately dye exchange capacity therefore becomes im-
portant for in situ phytoremediation strategies.
Concentration of dyes and effluents is a crucial factor when plant
based treatment is concerned. Higher dye concentrations have shown
slower removal whereas the lower concentrations favor the treatment.
In vitro removal of Direct Red 5B by B. malcolmii was found to show
efficient removal at 10–60 mg L−1 concentrations which was inhibited
at concentrations of 60–100 mg L−1 (Kagalkar et al., 2009). Potential of
L. minor to remove Brilliant Blue R was also found to be inhibited at
10 mg L−1 when monitored between 2.5–10 mg L− 1 concentrations
(Kiliç et al., 2010).
9.3. Water, oxygen and nutrient availability
Water, oxygen and nutrients are most important and key components
for general healthy state of plants (Eweis et al., 1998). Water, the major
component of all living systems serves as the transport medium for nutri-
ents and metabolites as well as all plant secretions. Greater water content
can restrict rhizospheric microbial activities and can form anoxic zones
limiting the gas exchange ultimately leading to decay and death of plants.
Oxygen, an essential gas for most of the living systems is provided by
plants in the rhizosphere. It was reported that the oxygen transfer ca-
pacity depends on habitat of plants (Vance, 1996). The wetland plants
are advocated to show greater capacity of oxygen transport. Growth of
aerobic microflora in rhizosphere plays an important role in phyto-
remediation of dyes which eventually rely on oxygen availability. For-
mation of anoxic zones deep down in unaerated areas where activities
of anaerobic bacteria can help in phytodegradation processes. Growth
of plant roots was reported to be healthier when roots of
Portulaca grandiflora were partially exposed to aeration while treating
a dye effluent in the phyto-tunnel reactor (Khandare et al., 2014).
Ample amount of nutrients in soil or other media support the
growth of plants and thus the root associated bacteria. Reactors with di-
rect exposure of dye wastewater was observed to affect the plant health
and needed to be exposed to normal water intermittently (Khandare
et al., 2014). A similar alternating exposure to freshwater was followed
while treating real textile effluents by A. philoxeroides plants in the
rhizofiltration reactor (Rane et al., 2015). In the same study, a 75%
plant surface coverage area was reported to be optimum for effective
phytoremediation than 25, 50 and 100% coverage areas which empha-
sizes the importance of proper nutrient availability to phytoremediating
plants. Portulaca grandiflora and G. pulchella roots in the phytoreactor
systems kept partially rooted in soil and partly exposed to effluent
were found to show increased longevity (Kabra et al., 2013; Khandare
et al., 2013b). Treatment of textile dyes with hydroponic media like
Hoagland's salts solutions were tried and efficient degradation of textile
dyes by plants like L. minor, A. filiculoides, H. vulgaris and N. officinale was
achieved (Khataee et al. 2012, 2013b; Vafaei et al., 2013; Torbati et al.,
2014). These findings made it clear that the use of combinatorial sys-
tems including soil, nutrient media and augmentation of rhizospheric
microbes along with appropriate plants could be advisable strategies
for applicative phytoremediation.
9.4. Temperature
Temperature is an important parameter which can affect the rate
of various processes involved in phytoremediation. The rate of
photosynthesis is clearly dependent upon the temperature of air at
plants habitat. It is a general understanding that the summer is bet-
ter suited for microbial degradation of contaminant than winter.
Increased temperature in summer can naturally help to enhance
soil microbial activities and thus boost the rhizodegradation pro-
cesses. Polyaromatic hydrocarbons degradation rate was found to
be elevated in spring and autumn when ambient temperature was
comparatively more than winter and relatively lower than summer
(Simonich and Hites, 1994). The daily water uptake of sunflower
plants exposed to benzotriazoles was found to be increased at
high-temperature water baths when compared to lower tempera-
tures. Pollutant uptake has also been observed to be more in summer
than spring (Castro et al., 2004). The fresh textile effluents when re-
leased generally have temperature up to 50–70 °C. This factor essen-
tially underlines the need of physicochemical methods for moderate
primary treatments. Phytoremediation has therefore been proposed
as secondary treatment technique as a temperature, pH and dye load
may adversely affect the phytoremediating plants (Khandare et al.,
2014).
9.5. Solar energy and radiations
Autotrophic nature of plants makes phytoremediation a highly ad-
vantageous method when it comes to energy input. It is a solar energy
driven system requiring very little monitoring and nutrient input. Sun-
light and solar radiation are known to transform parent pollutant com-
pounds altering their toxicity and availability. Textile dyes however
have not been reported to be chemically altered by solar radiations.
Higher light regime was found to enhance inorganic pollutant uptake
when compared to the lower light regime (Rofkar and Dwyer, 2011).
This seems obvious as the transpiration rates of plants are naturally
more at greater intensities of light. High rate transpiration systems are
directly influenced by the solar radiations and intensity of light.
Phytoremediation of dye mixtures and real textile effluents at pilot
scales using P. australis, G. pulchella and Portulaca grandiflora,
A. philoxeroides and P. crinitum were successfully demonstrated using
actual sunlight and without supply of any nutrients (Davies et al.,
2005; Kabra et al., 2013; Khandare et al., 2013b; Rane et al., 2015;
Watharkar et al., 2015).
9.6. Weathering
Weathering processes like hydrolysis, leaching, volatilization,
evapotranspiration, and biotransformation play a significant role in
phytoremediation. These processes are responsible to reduce degrad-
able fraction of contaminants to remain recalcitrant. Seasonal precipita-
tions can dilute the pollutant and favor availability for treatment by
plants but a risk of free discharge to nearby water bodies always perse-
vere. The contaminants left behind untreated tightly bind to organic
matter and are retained in the treatment systems. Well-weathered
sites are generally old and concentrations of pollutants are much greater
at such situations when compared to recently contaminated ones.
Weathering processes thus directly affect contaminant availability and
rate of degradation by microbes and plants (Cunningham and Ow,
1996). Operation and maintenance of the sites under phytoremediation
can however be controlled with irrigation, moderate nutrient supply
and facilitating aeration.
10. Disadvantages and advantages of phytoremediation of textile
dyes and effluents
Emergence of bioremediation processes including the use of mi-
crobes and plants have come into practice because of the problems
associated with physico-chemical methods. Phytoremediation has
been proven to be effective and environmental friendly alternative
mostly for heavy metal remediation. In situ phytoremediation efficacy
 R.V. Khandare, S.P. Govindwar / Biotechnology Advances 33 (2015) 1697–1714
of plants is yet to be tested for other pollutants including textile dyes
and effluents. A number of plants discussed earlier can be explored for
actual phytoremediation of textile dyes. Most of the proposed plants
are seasonal and therefore their availability all-round the year needs
to be monitored and maintained. Phytoremediation as a primary treat-
ment technique for removal of dyes has not yet been evaluated and it
still is considered as a secondary or tertiary method of treatment. Unlike
physicochemical and a few biological methods, phytoremediation is a
slow process hence it should be augmented or combined with other
chemical/biological agents for efficacy enhancement. Development of
effective phytoremediation strategies for in situ treatment of textile
effluents rely on monitoring of plant growth, optimum nutrient
input, water content and moderate physico-chemical pretreatment
(Govindwar and Kagalkar, 2010). Studies on phytoremediation of
textile dyes have not covered aspects like high rate transpiration sys-
tems and pollutant mass balance-transfer effects. Such parameters
are difficult to be studied on plant like complex systems at larger
scales.
On the other hand phytoremediation looks really promising being a
solar energy driven and ecofriendly method (Ma et al., 2011). It is also
advantageous because of low maintenance and negligible nutrient re-
quirements (Cluis, 2004). Phytoremediation of textile dyes can prove
to be a practical tool to bring the wastelands to productive use like de-
velopment of public gardens and thoroughfares (Khandare et al.,
2013b). Utilization of aquatic macrophytes and even weeds like water
hyacinths have shown a very high rate of heavy metals removal from
textile effluents showing multifaceted nature of phytoremediation pro-
cesses (Sanmuga and Senthamil, 2014). Treatment of textile dyes by
garden plants of economic importance to yield flowers and other prod-
ucts based on plant habit is also a possible outreach. Phytoremediation
is additionally more cost effective and ecofriendly method than any
other physico-chemical and even biological modes of treatment. It is a
process with practical feasibility and finds prospects in implementation
for treatments of actual sites of dye disposal. Menaces like leaching and
erosion can be successfully prevented by employing plant based treat-
ment systems. Improvement of phytoremediation efficiencies by micro-
bial augmentation, special nutrients, media supply and moderate prior
physico-chemical treatment needs to be thoroughly examined for effec-
tive and proficient in situ applications.
11. Research needs and conclusions
Plants from a wide array of habitats have been proven to be an ex-
tremely potential bioremediation tools for textile dyes and allied efflu-
ent. Phytoremediation for removal of textile dyes/effluents from
environment ought to be greeted as an ecofriendly, cost effective and ef-
ficacious alternative for future. Most of the plant based reactor develop-
ment studies have remained at laboratory scales, and their industrial
and/or large-scale execution still remain scarce. Studies at actual site
of dye disposal and full pilot scales therefore find more avenues of
research in the field of textile dye removal from wastewaters. In order
to look for better substitutes, combinatorial systems of plants-
microbes and plants-plants should be undertaken at larger scales and
on fields. Combined systems comprising bacteria and solid state fer-
mentation (Kadam et al., 2013) could also prove to be a promising
way for efficient treatment of dye effluents at the actual site of disposal
with concurrent phytoremediation. Phytoremediation using garden
ornamental plants needs to be motivated as this aspect adds esthetics
to treatment systems; bacterial augmentations can be used to
enhance the treatment efficacies of these plants (Khandare et al.,
2012; Watharkar and Jadhav, 2014). Moderate prior physico-
chemical treatments of textile effluents with sonication, UV photo-
lytic, photocatalytic, Fenton and Photo-Fenton oxidation processes
before actual exposure to plants is an attractive alternative which
would contribute to longevity of this phytotechnology and enhance
remediation (Vijayalakshmidevi and Muthukumar, 2014). High rate
1711
transpiration systems with tree vegetation also finds possibilities in
treatment of huge volumes textile effluents. Dried plants and/or
plant parts after remediation could be used as a source of lignocel-
lulosic waste for bio-fuel production (Jagtap et al., 2014). This
approach becomes important as disposal of plant biomass used
for phytoremediation is always criticized and subjected to serious
examination before applications.
Different oxidoreductive plant enzymes such as LiP, Malachite Green
reductase, laccase, tyrosinase, azo reductase, veratryl alcohol oxidase, ri-
boflavin reductase and DCIP reductase are key biodegrading engines
which break complex dye structures. Genomic and proteomic based
evaluations and engineering of these enzymes are still unattended
areas of research. Development of transgenic plants species carrying
genes for effective dye degrading enzymes and stress confronting abili-
ties could also be an important and exciting area of research. These
plants would prove to be more efficient tools for decolorization and de-
toxification of dye effluents. Textile dye degradation pathways by plants
and fate of metabolism proposed earlier are well in agreement with the
results obtained through UV–visible spectroscopy, HPLC, HPTLC, GC–
MS, LC–MS and NMR analytical techniques. This detailed analyses and
studies on active enzymes from respective plant sources will be useful
for advanced research in future. Transgenic Arabidopsis with an over-
expression of triphenylmethane reductase from a Citrobacter sp. was
shown to achieve efficacious phytoremediation of extensively utilized
triphenylmethane dyes when compared to non-transformed plantlets
(Fu et al., 2013). Attempts to produce more promising and adaptable
transgenic plants with extremely high prospective for dye degradation
are major targets for future. Development of methods for large volumes
of effluent treatment compatible with changing hydraulics and fluctuat-
ing pollutant loads is need of the hour.
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