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DOI 10.1007/s00425-011-1556-z

ORIGINAL ARTICLE

Sesuvium portulacastrum (L.) L.: a potential halophyte

for the degradation of toxic textile dye, Green HE4B
Asmita V. Patil • Vinayak H. Lokhande •
Penna Suprasanna • Vishwas A. Bapat •
Jyoti P. Jadhav

Received: 14 August 2011 / Accepted: 14 November 2011

Ó Springer-Verlag 2011

Abstract Sesuvium portulacastrum is a common halo- 200 mM NaCl. Gas Chromatography–Mass Spectroscopy
phyte growing well in adverse surroundings and is (GC–MS) analysis of the products revealed the formation
exploited mainly for the environmental protection includ- of three metabolites such as p-amino benzene, p-amino
ing phytoremediation, desalination and stabilization of toluene and 1, 2, 7-amino naphthalene after phytotrans-
contaminated soil. In the present investigation, attempts formation of GHE4B. Based on the FTIR and GC–MS
have been made on the decolorization of a toxic textile dye results, the possible pathway for the biodegradation of
Green HE4B (GHE4B) using in vitro grown Sesuvium GHE4B in the presence of 200 mM NaCl has been pro-
plantlets. The plantlets exhibited significant (70%) decol- posed. The phytotoxicity experiments confirmed the non-
orization of GHE4B (50 mg l-1) that sustain 200 mM toxicity of the degraded products. The present study
sodium chloride (NaCl) within 5 days of incubation. The demonstrates for the first time the potential of Sesuvium for
enzymatic analysis performed on the root and shoot tissues the efficient degradation of textile dyes and its efficacy on
of the in vitro plantlets subjected to GHE4B decolorization saline soils contaminated with toxic compounds.
in the presence of 200 mM NaCl showed a noteworthy
induction of tyrosinase, lignin peroxidase and NADH- Keywords Biotransformation  Green HE4B 
DCIP reductase activities, indicating the involvement of Phytoremediation  Phytotoxicity  Sesuvium
these enzymes in the metabolism of the dye GHE4B. The
UV–visible spectrophotometer, HPLC and Fourier Trans- Abbreviations
form Infrared Spectroscopy (FTIR) analyses of the samples BAP Benzyl aminopurine
before and after decolorization of the dye confirmed the FTIR Fourier Transform Infrared Spectroscopy
efficient phytotransformation of GHE4B in the presence of GC–MS Gas chromatography mass spectroscopy
GHE4B Green HE4B
HPLC High-performance liquid chromatography
The authors A. V. Patil and V. H. Lokhande contributed equally for LiP Lignin peroxidase
the manuscript. MS Medium Murashige and Skoog medium
NAA a-Napthaleneacetic acid
A. V. Patil
Department of Biochemistry, Shivaji University, NaCl Sodium chloride
Kolhapur 416 004, India NADH-DCIP Dichlorophenol indophenol

V. H. Lokhande  P. Suprasanna
Functional Plant Biology Section, Nuclear Agriculture
and Biotechnology Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085, India Introduction

V. A. Bapat  J. P. Jadhav (&)

Fulfilling the demand of ever-growing human population
Department of Biotechnology, Shivaji University,
Kolhapur 416 004, India for food and shelter is becoming a serious problem for
e-mail: jpj_biochem@unishivaji.ac.in; jpjbiochem@gmail.com increased urbanization and industrialization. This is leading

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to the accumulation of xenobiotic compounds in the water
sources, food crops and lands under cultivation causing
contamination of non-renewable environment and natural
resources. Toxic heavy metals, hazardous chemicals and
effluents from electrochemicals, fertilizers and textile
industries are the main causative agents responsible for the
environmental contamination. Dyes constitutes one such
category of compounds released in the effluents of textile
and dyeing industries which even at very small concen-
trations, has mutagenic and carcinogenic effects on living
organisms (Moorthi et al. 2007; Hu et al. 2009).
The dye-contaminated effluents have adverse physico-
chemical properties with high chemical oxygen demand
(COD), biological oxygen demand (BOD), suspended
solids and other toxic chemical compounds (Casieri et al.
2008; Shedbalkar et al. 2008; Shedbalkar and Jadhav
2011). The textile industries utilize costly physical and
chemical treatments which are not highly effective in
reducing the effect of contaminated water bodies. These
methods have several disadvantages such as formation of
toxic by-products leading to disposal problems of con-
taminated wastes (Robinson et al. 2001; Aubert and
Schwitzguebel 2004). As an alternative option, bio-
remediation is becoming an efficient technology com-
pared with physico-chemical treatments. Applications of
algal, bacterial and fungal organisms for dye degradation
have been studied extensively (Daneshvar et al. 2007;
Shedbalkar et al. 2008; Kalme et al. 2009; Shedbalkar
and Jadhav 2011). The use of plants as phytoremediators
is considered as a safe, easy to operate and a less dis-
ruptive technique (Cunningham and Berti 2000). This
technology is eco-friendly, cost effective, less sludge-
producing and has been widely employed for the envi-
ronmental cleanup strategies (Singh et al. 2008). In
recent years, use of plant systems for detoxification has
generated an increasing interest because of its auto-
trophic nature, large biomass production, and its ability
to survive in the limited nutrient resources, protection
against water and wind erosions and prevention of con-
taminant products from spreading (Cluis 2004). Plant
metabolism of xenobiotics may lead to a total or partial
degradation or transformation by reductive, oxidative or
hydrolytic enzymes (Ghodake et al. 2009). Additionally,
plants have a remarkable potential to concentrate and
accumulate mineral elements and compounds as well as
organic contaminants from the environment and use
them in their metabolic pathways as well as store the
various molecules in their organs and tissues. The plants
have also an efficient genetic machinery of mechanisms
to coordinate metabolic activities (Salt et al. 1998).
Several plants such as Salsola vermiculata, Typhonium
flagelliforme and Blumea malcolmii have already been
studied for their ability to degrade sulfonated and non-
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sulfonated azo dyes (Bestani et al. 2008; Kagalkar et al.
2009, 2010). However, these plant species are glyco-
phytes, which may not be able to grow in water or soils
contaminated with a variety of toxic dyes, saline ions,
heavy metals and other xenobiotic compounds. In con-
trast, halophytes, the most amenable category of plants
adapted to various adverse environmental conditions
like salinity, heavy metal toxicity, high temperature and
drought are considered most appropriate for phytoreme-
diation of toxic dyes. Since halophytes have the capa-
bility to overcome the salt stress and other adverse
factors, it is logically expected that halophytes will act in
an efficient way to degrade the hazardous chemicals. In
this context, the removal of heavy metals by halophytes
has already been reported (Ghnaya et al. 2005; Lokhande
et al. 2010a). However, meager information is available
on the use of halophytes for dye degradation studies and
only Phragmites australis has been used for the degra-
dation of textile azo dyes (Davies et al. 2005; Carias et al.
2008).
Sesuvium portulacastrum (L.), a member of the family
Aizoaceae, is an important halophyte in the category of
‘‘salt accumulator’’ plants which accumulates high salt
concentration in their cells and tissues and overcomes
salt toxicity by developing succulence. This plant is used
as a fodder for animals and has an ornamental value
since it blooms throughout the year in the barren areas
(Lokhande et al. 2009a). Due to its survival in adverse
environmental conditions, the plant is recognized as a
promising candidate for the environmental protection
(Ghnaya et al. 2005, 2007; Lokhande et al. 2009a,
2010a; Rabhi et al. 2009, 2010; Moseki and Buru 2010;
Zaier et al. 2010a, b). The experiments have shown the
optimum growth potential of the plant in the presence of
salt concentrations of NaCl (200 mM) (Lokhande et al.
2010b; Moseki and Buru 2010). Recently, preliminary
experiments have also shown that the pre-plantation of
S. portulacastrum on saline soil has capacity to remove
the excess salts from the soil and helps to regain the
fertility of arable lands for the production of crop plants
(Rabhi et al. 2010).
Earlier phytoremediation studies have been carried out
using in vitro cell and tissue culture techniques and genetic
engineering (Mackova et al. 2001; Eapen and D’Souza
2005; Padmavathiamma and Loretta 2007; Guillon et al.
2008) which offer unique opportunities that complement
and extend the existing options. Against this background,
attempts have been made in the present work on the
potential of in vitro grown Sesuvium plantlets to degrade
the sulfonated dye, Green HE4B (GHE4B), with an insight
into the involvement of different enzymes and prediction of
metabolic pathway responsible for the biotransformation of
these recalcitrant compounds.
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Materials and methods
Chemicals and dyes
The chemicals and reagents used in the study were of
highest purity and of an analytical grade. ABTS (2,20 -
azino-bis, 3-ethylbenzothiazoline-6-sulfonic acid) was
procured from Sigma-Aldrich (St. Louis, MO, USA);
NADH-disodium salt, n-propanol, 2,6-dichlorophenol-
indophenol (DCIP) and catechol were purchased from Si-
sco Research Laboratories (Mumbai, Maharashtra, India).
The pulverized powder of MS (Murashige and Skoog
1962) basal medium was obtained from Himedia Chemi-
cals Ltd. (Mumbai) and tartaric acid from BDH chemicals
(Mumbai). The colored dyes namely, Red HE8B, Remazol
Orange, Golden yellow-HER, Red violet FBL and Green
HE4B (Table 1, see CI names and CAS numbers) were
procured from Manpasand textile industry (Ichalkaranji,
Maharashtra, India).
Source of plant material and culture establishment
The nodal sectors (*3.0 cm) of S. portulacastrum, col-
lected from the coastal region of Maharashtra state, India,
were maintained at the Botanic Garden, Department of
Botany, University of Pune, Pune, India (Lokhande et al.
2009b) and were used as a source for the explants. The
multiplication of axillary shoots from the nodal explants
was achieved on MS (Murashige and Skoog 1962) solid
medium supplemented with 20 lM BAP (benzyl aminopu-
rine) and rooting of shoots was obtained on MS liquid
medium containing 10 lM NAA (a-napthaleneacetic acid)
as described earlier by Lokhande et al. (2010c). The culture
establishment was carried out under controlled conditions of
temperature at 25 ± 2°C, 16 h photoperiod under cool white
fluorescent light (Philips India, Ltd., 40 lmol m-2 s-1)
and 70% relative humidity. These in vitro grown rooted
plantlets (5–7 cm height) were used for our experimental
studies.
Table 1 Influence of S. portulacastrum plantlets on percent decolorization of various textile dyes (20 mg/l) in different culture medium for
5 days
CAS numbera CI name Common name
71872-80-5 Reactive red 152 Red HE8B
71838-95-4 Reactive orange 82 Remazol orange
61951-85-7 Reactive yellow 84 Golden yellow-HER
61931-49-5 Reactive green 19 Green HE4B
6408-72-6 Disperse violet 26 Red violet FBL
a
(http://www.chemicalbook.com, http://www.guidechem.com)
Dyes-decolorization experiments
Initially, the dyes (Red HE8B, Remazol Orange, Golden
yellow-HE2R, Red 2GX and Green HE4B) procured from
the industry were screened for their ability to degrade in the
presence of Sesuvium plantlets. The plantlets with their
roots and part of the basal portion of the stem submerged
(Fig. 3) were cultured in glass test tubes (150 9 25 mm,
Borosil, Mumbai, India). The tubes contained distilled
water, 100 mM NaCl and MS basal liquid medium (10 ml/
test tube), each supplemented separately with the above-
mentioned dyes at the concentration of 20 mg l-1. The
culture tubes containing medium and respective dyes
without plantlets served as controls. The pH of the medium
was adjusted to 5.8 ± 0.05 prior to autoclaving. The cul-
tures were maintained in the culture room for 5 days under
controlled conditions as mentioned earlier. At the end of
the incubation period, the plantlets were harvested from the
culture medium of the treatment. The medium of the
control and treatment was centrifuged at 10,000g for
10 min at room temperature to remove the suspended
particles. The decolorization of the dyes by Sesuvium
plants was monitored by measuring the change in absor-
bance of the treatment supernatant against controls at the
respective absorption maxima of the dyes screened using
UV–visible spectrophotometer (Hitachi U-2800; Hitachi,
Tokyo, Japan). Decolorization percentage of the respective
dyes was calculated using the following equation:
Decolorization ð%Þ ¼ ðAinitial  Afinal =Ainitial Þ  100
Based on the maximum percent decolorization of the
dyes by in vitro grown Sesuvium plantlets, further
experiments of decolorization were carried out with the
selected dye Green HE4B (GHE4B).
The ability of the Sesuvium plantlets to decolorize
GHE4B dye was screened at variable concentrations of the
dye in the range of 25–100 mg l-1. Similarly, considering
the facultative halophytic nature of the plant and its opti-
mum growth parameters in the presence of NaCl under in
kmax % Decolorization
Distilled water NaCl (100 mM) MS Basal
548 11.5 59.75 69.3
494 15.2 43.3 23.9
370 11.4 10.0 31.7
636 47.2 71.5 61.7
535 8.4 34.4 20.0
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vitro conditions (Lokhande et al. 2010b), the effect of
variable concentrations of NaCl was studied. Further, the
GHE4B dye degradation potential of Sesuvium plantlets
was studied under optimum culture conditions such as
distilled water containing GHE4B (50 mg l-1) in the
presence or absence of 200 mM NaCl. The plantlets cul-
tured in the test tubes containing distilled water without
addition of dye and in the presence or absence of 200 mM
NaCl served as a control. The cultures were incubated for
7 days under controlled conditions as mentioned earlier.
Aliquots of the solution were aseptically removed at dif-
ferent time intervals (1, 2, 3, 5 and 7 day), centrifuged at
10,000g for 10 min at room temperature to remove the
unwanted material and absorbance of the clear solution was
measured at the absorption maxima of the GHE4B dye
for the determination of percent decolorization by the
plantlets.
Enzyme assays
The plantlets harvested from control and treatment were
separated into root and shoot and used for enzymatic
assays involved in decolorization of GHE4B dye. The
length of the selected plantlets that were robust in growth,
including roots and shoots, was approximately 10–12 cm.
The roots and shoots were ground separately in the mortar
with a pestle in the presence of ice-chilled potassium
phosphate buffer (50 mM, pH 7.4). The homogenate
was centrifuged at 8,000g for 20 min at 4°C and the
supernatant thus obtained was used for enzyme assays.
The biotransformation enzymes viz. lignin peroxidase,
tyrosinase and NADH-DCIP reductase were assayed
spectrophotometrically at the room temperature. Lignin
peroxidase activity was determined by monitoring the
formation of propanaldehyde at 300 nm in a reaction
mixture of 2.5 ml (pH 3.5) containing 100 mM n-propa-
nol, 250 mM tartaric acid and 10 mM H2O2 (Patil et al.
2009). Tyrosinase activity was determined by monitoring
the formation of catechol quinone at 495 nm in a reaction
mixture (2.0 ml) containing 0.01% catechol in 100 mM
potassium phosphate buffer (pH 6.8) (Zhang and Flurkey
1997). NADH-DCIP reductase activity was measured
according to the protocol described earlier (Salokhe and
Govindwar 1999). The assay mixture contained 50 lM
DCIP, 50 lM NADH in 50 mM potassium phosphate
buffer (pH 7.4) and 0.1 ml of enzyme solution in a total
volume of 5.0 ml. The DCIP reduction was monitored at
595 nm.
Decolorization and biodegradation analysis
The decolorization and biodegradation analysis were per-
formed at the end of 5th day of culture incubation. The
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supernatant samples obtained from control and treatments
were subjected to UV–visible spectral analysis (UV 2800
spectrophotometer, Hitachi) using absorption spectrum in
the range of 400–800 nm. The metabolites produced after
the biodegradation of GHE4B dye by Sesuvium plantlets
were extracted from the supernatant samples of control and
treated with an equal volume of ethyl acetate. The extract
was dried over anhydrous Na2SO4 and the solvent was
evaporated on a rotary evaporator. The residues obtained
after evaporation were dissolved in a small volume of high-
performance liquid chromatography (HPLC) grade meth-
anol and used for the analytical studies. HPLC analysis was
performed in an isocratic Waters 2690 system equipped
with dual absorbance detector, using C8 column in pres-
ence of mobile phase comprising methanol:acetonitrile
(75:25) at a flow rate of 1 ml min-1. The Fourier Trans-
form Infrared Spectroscopy (FTIR) analysis of the degra-
dation metabolites was done in the mid-IR region of
450–4,000 cm-1 with 16 scan speed using Perkin Elmer
783 spectrophotometer and compared with the control dye.
The samples were mixed with spectroscopically pure
potassium bromide (KBr) powder in the ratio of 5:95 prior
to injection. Pellets were kept in the sample holder for the
analysis. The metabolites formed after degradation of
GHE4B were identified using a QP2010 gas chromatog-
raphy coupled with mass spectroscopy (GC–MS, Shimadzu)
equipped with Restek column (0.25 mm, 60 m; XTI-5) at an
ionization voltage of 70 eV. Gas chromatography was
conducted in the temperature programming mode with the
initial column temperature of 80°C for 2 min, which was
increased linearly at 10°C min-1 to 200°C then at 20°C
min-1 to 280°C and held for 7 min. The temperature of the
injection port was 280°C and the GC/MS interface was
maintained at 290°C. Helium was used as a carrier gas with
flow rate at 1.0 ml min-1. Based on the fragmentation
pattern in the National Institute of Structure and Technol-
ogy (NIST) spectral library stored in the computer software
(version 1.10 beta, Shimadzu) of the GC–MS, the retention
time and mass spectra of degradation metabolites were
compared.
Phytotoxicity study
The toxicity of the dye Green HE4B and its ethyl acetate-
extracted metabolites (2,000 ppm) was tested on seeds of
Sorghum vulgare and Phaseolus mungo at room tempera-
ture. The experiments were carried out by placing 10 seeds
in a separate 10 ml solution containing either distilled
water, dye or metabolites of the degraded dye. Distilled
water was used as a control. Percent germination and the
lengths of shoot plumule (shoot) and radicle (root) were
measured and the seed germination percentage was calcu-
lated after 8 days.
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Statistical analysis 75
(a) a
a
The experiments were carried out in a randomized block 70

design with minimum three biological replicates per b

% Decolorization
treatment and repeated thrice. Data were analyzed by one- 65
b
way analysis of variance (ANOVA) with Tukey–Kramer
60
multiple comparisons test. Readings were considered sig-
nificant when P < 0.05.
55

50
Results 25 50 75 100
-1
GHE4B concentration (mg l )
Screening of dyes for decolorization
80 (b)
GHE4B GHE4B + 200 mM NaCl a
The in vitro grown Sesuvium plantlets incubated with dif-
b
ferent dyes for 5 days in the presence of different culture c
60
media (distilled water, 100 mM NaCl and MS basal),

showed variable degree of decolorization (Table 1). The
potential of plantlets for maximum decolorization was
observed for Green HE4B dye (71.5%) in the presence of
100 mM NaCl followed by Red HE8B (69.3%) in MS
basal liquid medium, whereas the least decolorization was
recorded for Red violet FBL (8.4%) in distilled water
(Table 1). Considering the halophytic nature of the plant
and on the basis of maximum decolorization of GHE4B by
the plantlets in the presence of 100 mM NaCl, this dye was
selected for further experiments.
Optimization of culture conditions
For the determination of maximum decolorization of
GHE4B by the plantlets, various culture conditions were
optimized such as concentration of the dye, NaCl and
incubation period. The plantlets exposed to different con-
centrations (25, 50, 75 and 100 mg l-1) of the GHE4B
showed variable decolorization efficiency ranging from
60–75%, and the maximum decolorization was observed
with no significant differences at 25 and 50 mg l-1
(Fig. 1a) within 5 days of culture incubation. The plantlets
exposed to GHE4B (50 mg l-1) and supplemented with
various concentrations of NaCl (0, 50, 100 and 200 mM)
revealed 75% decolorization efficiency at 200 mM NaCl
compared with control (59.16%), 50 mM NaCl (63.26%)
and 100 mM NaCl (69.3%) within 5 days of culture
incubation. To check the accuracy of the incubation time
on percent decolorization, the plantlets were incubated in
the medium containing dye (50 mg l-1) and dye plus NaCl
(200 mM) for various durations (1, 2, 3, 5 and 7 days) and
they showed a higher degree of decolorization compared
with plants incubated in GHE4B without addition of
200 mM NaCl (Fig. 1b). Significantly, the highest degree
of decolorization was recorded on the 5th and 7th days
after incubation (Fig. 1b) but there was no drastic
% Decolorization
A
d
B
40
e C
D
E
20
0
1 2 3 5 7
Culture incubation period (days)
Fig. 1 Optimization of culture conditions for the degradation of
GHE4B by S. portulacastrum plantlets. Influence of the plantlets on
percent decolorization of variable GHE4B concentrations (a), influ-
ence of incubation period on percent decolorization of GHE4B
(50 mg l-1) in the absence of 200 mM NaCl (b, white columns) and
in the presence of 200 mM NaCl (b, dark columns). Error bars
indicate SE (n = 3). Within each set of experiments, means with
different letters are significantly different at P B 0.05
difference. Therefore, the optimum culture conditions such
as GHE4B (50 mg l-1), NaCl (200 mM) and an incubation
period of 5 days were selected and used in further
experiments.
Enzymatic analysis
Activities of the biotransformation enzymes viz., lignin
peroxidase, tyrosinase and NADH-DCIP reductase were
assessed in root and shoot tissue of Sesuvium plantlets
cultured in distilled water and 200 mM NaCl in the pres-
ence or absence of GHE4B dye after 5 days of incubation
period (Tables 2, 3). The tyrosinase activity was compar-
atively higher in shoot than in root tissues of the plant-
lets exposed to distilled water and 200 mM NaCl in the
presence or absence of GHE4B dye. Increased lignin
peroxidase activity in the root tissue of plantlets expo-
sed to GHE4B in 200 mM NaCl showed significant

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Table 2 Enzyme activities in shoot tissue of S. portulacastrum plantlets exposed to GHE4B (50 mg l-1) dye decolorization in absence and
presence of 200 mM NaCl
Enzymes Enzyme activities of shoot tissue in distilled water Enzyme activities of shoot tissue in NaCl
Before decolorization After decolorization Before decolorization After decolorization

Lignin peroxidasea 0.271 ± 0.092 0.124 ± 0.012 0.156 ± 0.011 0.100 ± 0.007
Tyrosinasea 0.653 ± 0.049 0.574 ± 0.017 0.936 ± 0.143* 0.580 ± 0.095
NADH-DCIP reductaseb 0.834 ± 0.011 0.781 ± 0.028 0.736 ± 0.069 1.120 ± 0.017***
Mean values of three independent experiments ± SE, significantly different from control (0 h) at *P \ 0.05, **P \ 0.01 and ***P \ 0.001 by
one-way ANOVA with Tukey–Kramer comparison test
a
Enzyme activity unit min-1 mg protein-1
b
Micrograms of DCIP reduced min-1 mg protein-1

Table 3 Enzyme activities in root tissue of S. portulacastrum plantlets exposed to GHE4B (50 mg l-1) dye decolorization in absence and
presence of 200 mM NaCl
Enzymes Enzyme activities of root tissue in distilled water Enzyme activities of root tissue in NaCl
Before decolorization After decolorization Before decolorization After decolorization

Lignin peroxidasea 0.126 ± 0.011 0.117 ± 0.022 0.286 ± 0.013 0.156 ± 0.016
a
Tyrosinase 0.207 ± 0.020 0.158 ± 0.017 0.060 ± 0.015 0.333 ± 0.107
NADH-DCIP reductaseb 0.435 ± 0.020 0.462 ± 0.027 0.435 ± 0.047 0.590 ± 0.019
Mean values of three independent experiments ± SE, significantly different from control (0 h) at *P \ 0.05, **P \ 0.01 and ***P \ 0.001 by
one-way ANOVA with Tukey–Kramer comparison test
a
Enzyme activity unit min-1 mg protein-1
b
Micrograms of DCIP reduced min-1 mg protein-1

decolorization of GHE4B compared with plantlets exposed 1.8

to GHE4B without 200 mM NaCl. A similar trend was GHE4B (0 d)

observed in root tissues of the plantlets incubated in GHE4B (5 d)

200 mM NaCl control experiments. In general, the lignin 200 mM NaCl + GHE4B (0 d)
200 mM NaCl + GHE4B (5 d)
peroxidase activity in the shoot and root tissues was found 1.2
Optical Density

lower in the plantlets exposed to different treatments,

except in distilled water control in which the activity in the
shoot tissue was significantly higher than in roots. NADH-
DCIP reductase activity was significantly higher in the root 0.6
and shoot tissues of the plantlets exposed to GHE4B in
200 mM NaCl compared with plantlets incubated in dis-
tilled water, 200 mM NaCl or GHE4B in distilled water.
Overall, the enzyme activities of biotransformation were 0
500 540 580 620 660 700 740 780
efficiently induced in the presence of 200 mM NaCl for the
Wavelength (nm)
decolorization of GHE4B.
Fig. 2 UV-visible spectral analysis of the culture supernatants of the
Decolorization and biodegradation analysis S. portulacastrum plantlets incubated in GHE4B (50 mg l-1) dye in
absence or presence of 200 mM NaCl

UV–visible spectral analysis of GHE4B in presence or
absence of 200 mM NaCl demonstrated a maximum
absorbance at 636 nm (Fig. 2); however, when the solu-
tions were incubated with Sesuvium plantlets for 5 days,
significant decrease in absorbance in the presence of
200 mM NaCl compared with GHE4B in distilled water
alone was observed visually (Fig. 3). HPLC chromato-
grams of GHE4B produced major and minor peaks at 2.449
and 2.856 retention times, respectively (Fig. 4a). The
metabolites analyzed after degradation of GHE4B in
presence of 200 mM NaCl by Sesuvium plantlets revealed
five additional peaks at retention times 1.978, 2.866, 3.130,
3.552 and 5.732 min (Fig. 4b). The HPLC chromatogram
of plant exudates produced in the presence of 200 mM
NaCl showed peaks at retention times 2.537, 3.094, 3.499
and 5.707 (Fig. 4c).

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Fig. 3 Phenotypes of
S. portulacastrum plantlets
incubated with GHE4B in
200 mM NaCl at the initiation
of experiment (i.e. 0 day, a).
GHE4B decolorization in
distilled water (b) and in the
presence of 200 mM NaCl
(c) after 5 days
The FTIR spectral analysis of GHE4B dye, its metab-
olites formed after the biodegradation in the presence of
200 mM NaCl and exudates produced by Sesuvium plant-
lets, in the presence of 200 mM NaCl showed significant
differences between each other and confirmed the phyto-
transformation of toxic GHE4B into less-toxic metabolites
(Fig. 5a–c). The spectra for GHE4B (Fig. 5a) had different
peaks such as peak at 3430.81 cm-1 for secondary amides
(free NH) and at 1587 cm-1 for azo compounds (N=N).
Peaks at 1488.85, 1194.53, 1020.52 and 642.29 cm-1
represented aromatic homocyclic compound, S=O stretch-
ing, primary alcohols and sulphonic acid (S=O), respec-
tively (Fig. 5a). In contrast, spectra of the metabolites
formed after the biodegradation of GHE4B plus 200 mM
NaCl (Fig. 5b) revealed peak at 3444.02 cm-1 representing
N–H stretching for bonded NH groups; peaks at 2914.74
and 2845.96 represent alkanes (–CH2–), and a peak at
2364.47 corresponds to charged amines (NH2?). The peak
at 1378.57 cm-1 denoted the C–H deformation of alkane,
whereas the peak at 1111.08 cm-1 represents di-substituted
aromatic compound. Compared with the spectra of GHE4B
before and after phytotransformation, the absence of peak
at 1587 cm-1 in the GHE4B dye and present in the bio-
degraded GHE4B metabolites indicated the breaking of azo
bond, whereas the presence of the peak at 642.29 cm-1 in
the former and absent in the latter sample demonstrated the
desulfonation of GHE4B dye. The spectral analysis of
distilled water control samples (i.e. exudates produced by
Sesuvium plantlets in the presence of 200 mM NaCl)
revealed peaks at 3789.25, 2922.38, 1634.74, 1370.93 and
1072.87 representing hydroxyl group containing com-
pounds, alkanes (–CH2–), free or bonded –NH, alkanes
(–CH3) and for primary alcohols, respectively (Fig. 5c).
The peaks, namely 2914.74, 1636.79 and 1378.57 cm-1,
recorded in the sample of bio-transformed metabolites of
GHE4B in the presence of 200 mM NaCl (Fig. 5b) were
found to be more or less similar to the peaks such as
2922.38, 1634.74 and 1370.93 cm-1 observed, respec-
tively, in the distilled water control sample (Fig. 5c).
The results of FTIR spectral analyses for GHE4B
decolorization experiments were further checked by GC–
MS analyses to find out the probable metabolites produced
after phytotransformation of GHE4B by Sesuvium in the
presence of 200 mM NaCl. GC–MS analysis performed on
the metabolites formed after decolorization of GHE4B in
the presence of 200 mM NaCl resulted in the formation of
three metabolites such as p-amino benzene, p-amino tolu-
ene and 1,2,7-amino naphthalene (Table 4). Based on the
FTIR and GC–MS results, the pathway proposed in the
Fig. 6 indicated that the enzymatic actions of tyrosinase,
lignin peroxidase and NADH-DCIP reductase of Sesuvium
plantlets caused symmetric cleavage of sulfonated azo dye
GHE4B in the presence of 200 mM NaCl. The further
desulfonation leads to the formation of three metabolites as
predicted by GC–MS (Table 4).
Toxicity testing
As the textile effluent after the treatment can be utilized for
irrigation purposes, the seeds of agricultural crops mainly
S. vulgare and P. mungo were selected to study the toxicity
of the dye and the metabolites formed after degradation
of the dye. The phytotoxicity study revealed that there was
an inhibition of germination in solutions containing
2,000 ppm of the dye Green HE4B for both S. vulgare and
P. mungo (Table 5). The seeds of S. vulgare and P. mungo
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Fig. 4 HPLC chromatogram of

GHE4B (a), metabolites formed 0.08 (a) 2.449
after degradation of GHE4B by
S. portulacastrum plantlets
(b) and S. portulacastrum 0.06
immersed in 200 mM NaCl (c)

AU

2.856
0.04

0.02

0.00

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

0.018
(b) 1.978 3.130 3.552
0.016

0.014

0.012

0.010
2.866
AU

0.008

0.006

0.004 5.732

0.002

0.000

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes
3.094

(c)
3.499

0.005
2.537

0.004

0.003
AU

0.002
5.707

0.001

0.000
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes

showed a higher percentage of germination in both distilled Discussion

water and the extracted metabolites (Table 5) as compared
with that in the Green HE4B dye. The length of the plu-
mule and radicle was found to be lower in seeds germi-
nated in the dye than in those germinated in water and in
metabolites of the dye (Table 5). These observations con-
firmed that the dye was toxic to these food crops while the
metabolites formed after the degradation of the dye by
Sesuvium portulacastrum were almost non toxic.
Exploitation of plant tissue culture technique is a most
resourceful tool to understand the basic mechanisms of
plant tolerance to various toxic compounds and to check its
efficiency to cope up with the adverse environmental
conditions. The parameters related to the medium compo-
sition, nutritional requirements, phytohormone levels and
the ability to manipulate cell activities can precisely be

123
Planta
Fig. 5 FTIR spectral analysis
of GHE4B (a), degradation
product of GHE4B by
S. portulacastrum plantlets
(b) and S. portulacastrum
plantlets immersed in 200 mM
NaCl (c)
controlled in contrast to plants growing in soil, thereby
assuring the reproducibility and accuracy of the results
(Doran 2009). The present investigation confirmed the
utility of in vitro grown tissue cultured Sesuvium plantlets
for the decolorization of various toxic textile azo dyes to
varying extents. The differences observed in decolorization
of dyes might be due to the structural differences (Pas-
zcezynski et al. 1992), higher molecular weight and the
presence of inhibitory groups such as –NO2 and –SO3Na in
the dyes (Mohandass et al. 2007). To mimic the natural
habitat of Sesuvium in the screening experiment, the dye
decolorization ability of the plants by different culture
media showed significantly higher decolorization of
GHE4B in the presence of 100 mM NaCl compared with
incubations in distilled water and MS basal culture med-
ium. Taking into consideration the earlier work on GHE4B
dye degradation by Pseudomonas desmolyticum bacteria
(Kalme et al. 2009), our present study is of importance to
understand the mechanisms underlying the phyto-decolor-
ization of dyes and has proven the role of the plant in itself
in the process through maintaining the conditions similar to
its natural habitat.
Experiments carried out with optimization of culture
conditions demonstrated dye decolorization efficiency of
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Table 4 GC mass spectral data of metabolites formed after degradation of GHE4B by S. portulacastrum
No. Molecular weight of Retention time Name of Mass peaks
metabolite (m/z) (min) metabolite

1 104 20.183 p-Amino benzene 10.0

149

5.0

41 56 76 93 104 115 132 160 178 192

0.0
40 60 80 100 120 140 160 180 200

2 115 20.549 p-Amino toluene %

100 43 73
60

50
129
69
83 97 157 171 185
44 115
98 116 143 199
162
0
50.0 75.0 100.0 125.0 150.0 175.0 200.0

3 200 25.267 1,2,7-Amino %

naphthalene
100 193
63
90
50
114 166
52 75 102
62 83 138
41 124 200
0 150 173 187
50.0 75.0 100.0 125.0 150.0 175.0 200.0

more than 60% even at higher concentrations (75–100
mg l-1) of GHE4B. Moreover, considering the (50 mg l-1)
concentration of GHE4B an optimum condition for
decolorization purposes, the efficiency checked in the
presence of different NaCl concentrations revealed a sig-
nificantly higher decolorization ability of the plants in the
presence of 200 mM NaCl. The decolorization potential
increased with increase in the culture incubation period up
to 7th day in comparison with decolorization of GHE4B in
the absence of 200 mM NaCl. The reason might be that at
200 mM NaCl plants have maintained the optimum growth
potential through sequestration of more saline (Na?) ions
for the maintenance of osmotic balance between cytoplasm
and vacuole which helped in keeping the enzymes in their
more active form for detoxification of GHE4B.
The decolorization of textile dyes by the plants has been
correlated with the induction of various enzyme activities.
Davies et al. 2005 suggested the role of peroxidases from
the halophyte Phragmites australis in constructed flow
wetlands for the degradation of effluent containing azo
dyes while Carias et al. 2008 demonstrated the peroxidases
activities from P. australis in the degradation of the dye,
Acid orange 7. Recently, the use of tissue culture technique
for the identification and understanding of the mechanism
of dye decolorization potential by various other
glycophytes has also been studied, and peroxidase, tyrosi-
nase and DCIP reductase have been reported to be induced
during the decolorization of Reactive red 198 in Tagetes
patula hairy roots (Patil et al. 2009), Direct red 5B in
tissue cultured raised plants of Blumea malcolmii (Kagal-
kar et al. 2009) and Brilliant blue R in Typhonium flagel-
liforme (Kagalkar et al. 2010). Similarly, in the present
investigation, significant induction in the tyrosinase, lignin
peroxidase and NADH-DCIP reductase activities in the
shoot and root parts of Sesuvium suggested their active role
in detoxification of GHE4B and hence significant decrease
in intense color of GHE4B.
The UV–visible spectral analysis of GHE4B in the
presence of 200 mM NaCl revealed a significant decrease
in color intensity of GHE4B within 5 days of culture
incubation which further suggested and confirmed the
involvement of various enzymes in decolorization of
GHE4B. Additionally, the activity has been improved in
the presence of NaCl compared with GHE4B decoloriza-
tion in the absence of NaCl. The biodegradation of GHE4B
in the presence of 200 mM NaCl into different metabolites
due to activation of azo reductase enzymes and decrease in
UV-absorbance has been supported by the occurrence of
additional peaks in HPLC analyses. The FTIR spectra of
the metabolites confirmed the formation of the proposed

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SO3 SO3

SO3Na SO3Na
OH NH2 OH NH2

N N C C N N N N
N N
H H
Green HE4B SO3Na SO3Na
SO3Na SO3Na

Lignin peroxidase/
Symmetric
Tyrosinase/
cleavage
SO3 DCIP-reductase SO3

SO3Na SO3Na
OH NH2 OH NH2

N
(c)
N N N CH3 H3C N N N N

SO3Na SO3Na
SO3Na SO3Na

SO3 OH NH2 SO3Na SO3 OH NH2 SO3Na

H2 N NH2 H2 N NH2

H2 N CH3 H2 N CH3

SO3Na SO3Na SO3Na SO3Na

NH2 NH2
Desulfonation
Desulfonation

OH NH2 OH NH2
H2 N NH2 H2 N NH2
H2 N CH3 H 2N CH3

NH2 MW-115 NH2 MW-115

OH - OH -
MW-102 MW-102
NH2 NH2
H2 N NH2 H2 N NH2

MW-200 MW-200

Fig. 6 Proposed pathway for the phytotransformation of GHE4B

Table 5 Phytotoxicity studies of Green HE4B dye and its metabolites

Parameters Sorghum vulgare Phaseolus mungo
Control Green HE4B Metabolites Control Green HE4B Metabolites

Germination (%) 100 60 90 90 50 80

Plumule (cm) 3.10 ± 0.03 2.50 ± 0.02** 3.01 ± 0.05$$ 11.1 ± 0.10 6.84 ± 0.16** 10.44 ± 0.12$$
Radicle (cm) 10.26 ± 0.01 2.43 ± 0.04** 6.18 ± 0.05$$ 4.48 ± 0.14 1.77 ± 0.07** 3.48 ± 0.05$$
All the values are a mean of three experiments ± SE. Root and shoot lengths of plants grown in Green HE4B is significantly different from that
of plants grown in water by *P \ 0.05 and **P \ 0.001. Root and shoot lengths of plants grown in the extracted metabolites of Green HE4B is
significantly different from that of plants grown in Green HE4B by $P \ 0.05 and $$P \ 0.001

123

compounds (p-amino benzene, p-amino toluene, and 1,2,7-
amino naphthalene), produced due to symmetric cleavage
mediated by tyrosinase, NADH-DCIP reductase and lig-
nin peroxidase and desulfonation process as detected by
GC–MS analysis. Therefore, the UV-spectral, HPLC, FTIR
and GC–MS analyses have indicated that GHE4B has been
converted successfully to less-toxic metabolites by the
halophyte Sesuvium portulacastrum. Based on these anal-
yses, in the present investigation the possible degradation
pathway has been proposed for the degradation of GHE4B
in the presence of 200 mM NaCl by Sesuvium portulaca-
strum plantlets.
Conclusion
Sesuvium portulacastrum, a facultative halophyte, has
multiple advantages for the protection of the environment
due to its salt accumulating, drought and heavy metal stress
tolerant nature. Results of the present investigation on in
vitro detoxification of GHE4B suggest its eco-friendly
implication in the areas of textile industries for the removal
of toxic azo dyes into less toxic metabolites which is
otherwise released in the form of effluents and contami-
nated the natural resources such as water bodies and arable
lands. The lower percentage of germination of Sorghum
vulgare and Phaseolous mungo indicated that the dye was
toxic to these plants, while the metabolites formed after the
degradation of the dye by Sesuvium portulacastrum were
almost non-toxic. The data generated in the present work
have helped to elucidate the levels of toxic chemicals
degraded by Sesuvium and give information on the nature
of the degraded products. These findings have confirmed
the degrading potential of Sesuvium that will help remove
hazardous chemicals from the environment. The findings
may contribute to the development of a reliable option for
removal of toxins from the environment.
Acknowledgment AVP and JPJ wish to thank University Grant
Commission, New Delhi for SAP-DRS-1 support.VAB wishes to
thank Council of Scientific and Industrial Research, New Delhi for
Emeritus Scientist Fellowship.
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