In vitro phytoremediation potential of heavy metals by duck weed Lemna polyrrhiza L. (Lemnaceae) and its combustion process as manure value A BSTRACT

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Journal of
Environment &
Research VINANIE PUBLISHERS
In vitro phytoremediation potential of heavy metals by duck weed Lemna
polyrrhiza L. (Lemnaceae) and its combustion process as manure value
M.N. Abubacker,1,* C. Sathya2
1 PG & Research Department of Biotechnology, National College (Autonomous), Tiruchirapalli- 620001, Tamilnadu, India
2 Department of Botany, Seethalakshmi Ramaswami College, Tiruchirapalli- 620002, TamilNadu, India
ORIGINAL RESEARCH ARTICLE
ABSTRACT
In vitro experiments on chromium, copper, lead and zinc bioaccumulation using duck weed Lemna polyrrhiza L.
(Lemnaceae) was conducted with 5, 10 and 20 mg/100 mL concentrations for a period of 20 days. The SEM-EDX elemental
analysis was used to characterize the interaction between the metal and plant. The results revealed the bioaccumulation
of lead was high as 20.91%, followed by copper 9.71%, zinc 5.66% and chromium 1.86% was observed. The combustion
process of metal–loaded L. polyrrhiza biomass shows the total reduction of lead 1.72%, zinc 1.47%, chromium 0.93% and
copper 0.86%. The combusted biomass in the form of ash 10% + river sand passed through 1 mm sieve, sterilized was
supplemented to Brassica juncea pot culture, which revealed the healthy growth and ensured the manure value of metal-
loaded biomass.
KEYWORDS
combustion process; heavy metals; Lemna polyrrhiza; manure value; metal loaded; phytoremediation
1. INTRODUCTION
Accumulation of heavy metals caused due to
anthropogenic activities often results in nutrient
imbalance and productivity loss in land and aquatic
ecosystem (Pergent and Pergent-Martini, 1999).
Related researches in bioaccumulation of essential
and non-essential metals using aquatic macrophytes
(Singh and Ghosh, 2005; Vesk and Allaway, 1997)
was found useful in monitoring and ameliorating the
heavy metals in water bodies (Vajpayee et al. 1995;
Whitton and Kelley, 1995). Usually the plants have
the ability to accumulate heavy metals such as Cr,
Fe, Mn, Pb, Zn, Cu and Ni which are utilized for the
growth. Certain aquatic plants also have the tendency
to absorb and accumulate heavy metals with no
known specic biological function. However, excessive
accumulation of heavy metals will be toxic to plants.
The ability to tolerate elevated levels of heavy metals
and accumulation in high concentration has evolved
independently or in combination of both in dierent
plant species (Cheng, 2003; Ernst et al.1992) The
emphasis of most studies gradually shifted towards the
use of aquatic plants as monitors for heavy metal water
pollution. Soil and water contaminated with metals
pose a major environmental and human health hazard
that needs an eective and aordable technological
solution. Microbial bioremediation has been successful
in degradation of specic organic contaminants, but is
ineective at addressing the challenge of certain toxic
heavy metal contamination (Raskin et al. 1997). In
recent years, there has been a lot of interest in the study
of heavy metal accumulating plants which are used for
environmental remediation as well as for application,
termed as phytoremediation. Phytoextraction is one
method of phytoremediation in which the metal
accumulating plants are used to remove pollutants from
contaminated sites by concentrating in the harvestable
form from the plant (Salt et al. 1995; Zhuang et al.
2007). This is a cost-eective ‘green’ technology which
can be employed to remove toxic metals from soil and
water (Chen and Cutright, 2002; Huang et al. 2011).
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Corresponding author: M. N. Abubacker
Tel: +91 9894058524
Fax: +91 431 2458169
E. mail: abubacker_nct@yahoo.com
Received: 03-10-2016
Revised: 14-11-2016
Accepted: 30-11-2016
Available online: 01-01-2017
Journal of Environment and Biotechnology Research, Vol. 6, No. 1, Pages 82-87, 2017
82

Abubacker and Sathya, Journal of Environment and Biotechnology Research, Vol. 6, No. 1, Pages 82-87, 2017
In the present study the aquatic duck weed
L. polyrrhiza L. (Lemnaceae) was subjected to heavy
metal concentrations in in vitro conditions to examine
the bioaccumulation potential and subsequently, its
combustion process as manure value was carried out
to highlight the possibility of using accumulated heavy
metals as manure for plant growth.
2. MATERIALS AND METHODS
2.1. Collection of material
Lemna polyrrhiza, the duck weed employed in the
present study was collected from a polluted water body
in Tiruchirapalli, Tamil Nadu, India. The plant has a
small round and thick leafy structure. Flat green upper
and slightly convex purple lower structure contains
several rootlets (Gamble, 2008). The plants were
acclimatized for 5 days in tap water in 250 mL ask
and then subjected to in vitro studies.
2.2. Methods
2.2.1 Phytoremediation procedures
After acclimatization, the plants were tested in in vitro
condition for 3 dierent concentration of chromium
(potassium dichromate, Merck), copper (copper(II)
sulphate, Himedia), lead (lead acetate, Merck), and zinc
(zinc sulphate, Himedia) at 5, 10, and 20 mg/100 mL,
respectively. The respective heavy metal concentrations
were added to each of the Petridish and were exposed
to normal sun-light for detention time of 20 days. The
Petridishes were shaken at regular interval for uniform
distribution of metals in aqueous medium.
2.2.2. Anatomical studies
Control and bioaccumulated chromium, copper,
lead and zinc leaves of L. polyrrhiza was washed
with running deionised water, and were subjected to
anatomical studies. The cross sections were taken with
a thickness of 200-300 µm using a clean stainless-
steel razor. The unstained sections were mounted in
microscopic slides using a drop of glycerine covered
with a cover slip and photographed by light microscopy
with 100 X magnication (Olympus CH20i).
2.2.3. SEM EDX elemental analysis
SEM-EDX elemental analyses were carried out for
bioaccumulation of Cr, Cu, Pb and Zn. The combustion
process for manure value of all the metals was also
evaluated in this study. To study the nature of L.
polyrrhiza after bioaccumulation, the leaves were
collected on 20th day after exposure to respective heavy
metals. They were initially dried in shade, followed by
hot air oven (at 50 oC for 1 h). Using mortar and pestle,
the dried material were powdered and placed in steel
stub with carbon tape and sputter coated with gold
particle for 50 sec in high vacuum conditions for SEM-
EDX analysis. The images of L. polyrrhiza biomass
after phytoremediation was captured using scanning
electron microscope coupled with energy dispersive
X-ray consisting 3.5 nm and 2.5 nm resolution for
tungsten lament (LaB6) and EDX detector resolution
133 eV. (TESCON, Czechoslovakia) (Jamari et al.,
2014).
2.2.4. Pot culture studies
Pot culture studies were conducted using Brassica
juncea at three dierent formulations to analyze the
growth conditions. Each pot incorporated with 20
83
Figure 1. Bioaccumulation of heavy metals by L. polyrrhiza. (i) control (water), (ii) chromium, (iii) copper, (iv)
lead, and (v) zinc; (a) initial stage of bioaccumulation (day 1), (b) second stage of bioaccumulation (day 10), and
(c) third stage of bioaccumulation (day 20).

mg/100 mL of (i) heavy metals (Cr, Cu, Pb, Zn) (ii)
dried L. polyrrhiza biomass after phytoremediation
and (iii) combusted metal-loaded biomass as manure.
The controls were maintained with tap water and were
carried out for a period of 20 days.
3. RESULTS AND DISCUSSION
3.1. Bioaccumulation analysis of heavy metals
Studies on bioaccumulation of heavy metals such as Cr,
Cu, Pb and Zn were conducted on L. polyrrhiza (aquatic
weed) at 5, 10 and 20 mg/100 mL concentrations
for a period of 20 days. The results indicated that L.
polyrrhiza was able to accumulate the heavy metals
and there were no morphological changes observed
and remain healthy till 9th day of experimental
condition. Subsequently, observation on the 10th day
indicated that the plant morphology has changed due
to the accumulation of heavy metals and the survival
percentage was found dierent. Cr and Zn accumulated
plants both showed 50% survival, Cu accumulated
plants showed 40% whereas Pb accumulated plants
showed 30% survival. On the 20th day of observation,
there was further reduction in survival percentage. It
was observed that Cr, Zn accumulated plants showed
20% survival, whereas Cu and Pb accumulated plants
exhibited 10% survival (Figure 1).
Accumulation of heavy metals in plant
causes negative growth eects and also reduces their
photosynthetic process (Sandalio et al. 2001). The
metal accumulation in L. polyrrhiza and its subsequent
anatomical studies have shown that the heavy metals
were accumulated in the mesophyll tissues and more
profusely on the cell wall in accordance with study
of Thlaspi caerulescens (Wojcik et al. 2005). The
bio accumulated leaf of L. polyrrhiza was sectioned,
examined and micro-photographed as indicated in
section 2.2. The micro-photography of unstained leaf
anatomy revealed the bioaccumulation of Cr and Cu in
the mesophyll tissue (Figure. 2). Since Pb and Zn are
colorless heavy metals; leaf anatomy appears similar to
control.
3.2. SEM - EDX elemental analysis of heavy
metals
Scanning electron microscopy equipped with Energy
Dispersive X-Ray (SEM-EDX) analysis was conducted
to detect the bioaccumulation of heavy metals at cellular
and sub-cellular levels in L. polyrrhiza biomass in
single analysis revealed 1.86% for chromium, 9.71% for
copper, 20.91% for lead and 5.65% for zinc. In control
sample, these metals were not detected (Table 1, Figure
3).
SEM analysis of L. polyrrhiza biomass
samples clearly reveals the surface texture and pores
in the materials along with the morphological changes
with respect to shape and size of the materials after
accumulation of heavy metal ions. A clear dierence
in the surface of control compared to metal-loaded
biomass samples was visualized. It was also observed
that the surface of materials has changed into new
particles ensure the metal sorption as reported by Giri
and Patel (2012).
3.3. Pot culture studies
In nal stage of experiments, the metal-loaded biomass
was subjected to combustion process and the results
indicated signicant reduction in the percentage of
heavy metals 0.93% for Cr, 0.86% for Cu, 1.47% for Zn
and 1.72% for Pb than that of metal- loaded biomass
before combustion process (Figure 3). The product
after combustion (ash) was supplemented to Brassica
Abubacker and Sathya, Journal of Environment and Biotechnology Research, Vol. 6, No. 1, Pages 82-87, 2017
84
Figure 2. Leaf anatomical studies of heavy metals by L. polyrrhiza. (a) control, (b) bioaccumulation of chromium
in mesophyll tissue, and (c) bioaccumulation of copper in mesophyll tissue

Abubacker and Sathya, Journal of Environment and Biotechnology Research, Vol. 6, No. 1, Pages 82-87, 2017
85
(i)
(ii)
(iii)
(iv)
(v)
(vi)
Figure 3. Elemental analysis of L. polyrrhiza L. using SEM-EDX. (i) control (ii) bioaccumulation of chromium
(iii) copper (iv) lead (v) zinc and (vi) bioaccumulation of metals and its product after combustion
Figure 4. Pot culture Studies of Brassica juncea. C - control, Pot cultures supplemented with 20 mg/100 mL
of (a) heavy metals (Cr, Cu, Pb, Zn) (b) Dried metal-loaded L. polyrrhiza biomass (c) Combusted metal-loaded
biomass as manure.

juncia pot culture and it was observed that plants were
found to grow healthy (Figure 4), which ensures the
manure value of metal-loaded L. polyrrhiza biomass.
This result reveals the possibility of further application
of metal-loaded products in detoxication of heavy
metals as reported earlier by Lassat (2002).
4. CONCLUSIONS
Contamination of the aquatic bodies by various pollutants
like heavy metals and poly-aromatic hydrocarbons
have caused imbalance in the natural functioning of
the aquatic ecosystem. Phytoremediation works best at
sites by reducing the pollutant concentration through
bioaccumulation onto biomass. SEM-EDX analysis
conrms the bioaccumulation of heavy metals by L.
polyrrhiza biomass. Due to this special characteristic
feature, this aquatic plant can be employed easily
for cost eective and eco-friendly green technology
for heavy metal reduction from the polluted aquatic
ecosystem and also recycle these heavy metal pollutant
as manure through combustion process.
ACKNOWLEDGEMENTS
Author (MNA) wish to thank DST-FIST, Government
of India, New Delhi for providing the infrastructure
facilities to the Department of Botany, National College,
Tiruchirappalli, Tamil Nadu. Authors also expresses
thanks to Padmavibhushan Dr. V. Krishnamurthy,
President, Sri. K. Ragunathan, Secretary and Dr. K.
Anburasu, Principal, National College, Tiruchirappalli
for all the supports and encouragement given to PG
and Research Department of Biotechnology to carry
over the research work.
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Metals
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Combustion
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C 45.28 52.56 44.60 44.01 50.52 48.71
N 5.26 6.28 12.79 – – –
O28.65 31.20 30.10 31.92 38.94 23.21
Na 0.80 0.25 – – – 6.23
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P 2.25 0.32 0.38 0.45 0.42 0.34
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Cl 1.98 0.18 – 0.35 0.21 9.22
K 4.93 0.48 – – 0.53 1.61
Ca 4.59 2.23 0.39 1.00 0.58 1.92
Mn 0.29 1.11 – – – –
Fe 1.75 0.97 0.28 – 0.17 0.13
Al – 0.20 0.13 0.20 0.20 0.14
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Cu – 0.89 9.71* – – 0.86
Pb – – – 20.91* – 1.72
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Ti – – – – 0.11 –
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