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Chapter 3
BIOREMEDIATION OF HEAVY METALS BY LOWER PLANTS
3.1 INTRODUCTION
Most of the engineering technologies have failed in effluent clean
up process; an alternative, eco friendly biological tool is substituted here
in pollution abatement. Phytoremediation is the most applicable among
the bioremedial measures and is an emerging technology. The capacity of
aquatic plants to remove potentially toxic heavy metals is well
documented. Lower plants like aquatic mosses and liverworts have the
ability to concentrate high amount of metals. The role of ferns like
Salvinia has already been established in this regard. Higher aquatic plants
like Eichornia crassipes, Pistia stratioles, Taylus latifolia, Hydrilla,
Vallisneria and members of duck weed family Lemnaceae have shown
their unique sorption potential of metals like Cd, Pb, Cu and Hg and act as
natural bioscavenger of metal effluents. Generally hydrophytes showed
varying degree of accumulation capacities. So they are screened for
selecting a suitable metal scavenger. The ease of harvesting and handling
the biomass is also taken into account during screening.
168 Chapter 3
3.2. MATERIALS AND METHODS
3.2.1 Screening for a Metal Tolerant Hydrophyte
3.2.1.1 Test plants and their culturing Young plants of Azolla pinnata, were collected from Malanadu
Development Corporation, Kanjirapally free from metal contamination.
Lemna major, Lemna minor and Hydrilla were collected from fresh water
ponds around Mannanam, Kottayam and used for the study.
They are then washed with 0.1M EDTA solution followed by
distilled water to remove the metallic elements. They were then
transferred to the culture chamber containing the Hoagland and Arnold
nutrient solution (Appendix). The culture chambers were kept for a week
at 28± 2°C in approximately 1200lux/m2 light intensity for acclimatization
and growth of the test plant.
3.2.1.2 Metal stock preparation 1000µg/ml stock solutions were prepared by dissolving analytical
grade salts of CdCl2, Pb (NO3)2 in 1000 ml distilled water. From stock
solution different volumes were added to the culture medium separately in
order to maintain the required concentration of metal (25-200µg/ml).
3.2.1.3 Screening for metal removing capacity
a) Experimental set up The fist phase of the study involved transferring a definite weight
of laboratory cultured plant species individually into aquarium
compartments each having a size of (20x20x15cm) dimensions,
containing culture medium loaded with different concentrations of metals
so as to make a total volume of 1L. The plants could easily float with the
root lying above the bottom of the chamber. A photo period of 11 hours and
Bioremediation of Heavy Metals by Lower Plants 169
a light source of 1200 lux were maintained during the treatment. pH was
adjusted to 6. Samples were transferred from each of the compartment after
5 days of interval and the residual metal concentrations were determined by
AAS. The percentage of metal removed was calculated from the residual
metal concentration (Banerjee and Sarker, 1997). The experiment was
performed to study the percentage of metal uptake and to determine the
tolerant strain under various concentrations of different metal stress.
Control experiments were performed simultaneously with the experimental
ones. Triplicate batch test for each concentration were conducted.
After the preliminary screening, Azolla pinnata was selected for
further studies since it showed more metal tolerance.
3.2.2. MECHANISM OF UPTAKE
3.2.2.1 Adsorption experiments For the determination of mode of uptake – whether it is an
absorption or adsorption, the following experiments were done. 5gm of
laboratory cultured Azolla pinnata plants were inoculated into culture
chambers containing culture medium loaded with different concentrations
of metal (25-200µg/ml) and made up to one liter. The residual
concentration of metal in culture medium was estimated after 12hrs of
contact time with different initial concentrations of each metal by AAS.
Two widely accepted adsorption isotherm models describing
adsorption/biosorption phenomenon are Freundlich (1906) and Langmuir
(1916) models. These models were fitted to the above experimental data
for the determination of mode of uptake. Neither Freundlich nor Langmuir
adsorption models was obeyed by the experimental data. This necessitates
conducting the bioaccumulation studies.
170 Chapter 3
3.2.2.2 Bioaccumulation studies (Sen and Bhattacharyya, 1993).
The metal tolerant laboratory cultured Azolla pinnata plants were
inoculated into culture chambers containing culture medium loaded with
different concentrations of metal and 5gm of Azolla was added and made
up to one liter. The pH was adjusted to 6. Samples were taken from each
of the compartment periodically at every 24hours of interval for the
determination of the residual metal concentration by AAS. The
percentage of metal removed was calculated from the residual metal
concentration.
The treated plants were analysed for the metal by ‘Wet
digestion technique’ outlined by (Chigbo et al., 1982). The oven dried
materials were thoroughly grinded with mortar and pestle and the
powder was taken in a beaker. It is digested with concentrated HNO3
and perchloric acid in the ratio 5:2 and kept in a water bath till a paste
is formed. It is diluted with 5ml of 1N HNO3 and filtered. The metal
concentration in the supernatant was estimated by AAS and expressed
in mg/gm of fresh weight (APHA et al., 1989).
3.2.2.3 Biochemical investigations The plants were taken from the culture tank and exposed to 25, 50,
100, 150 and 200µg/ml of CdCl2 separately for about 72 hrs in small
aquarium tanks. After exposure the plants were taken out, washed with
tap water, then with distilled water, dried with blotting paper and 500mg
was used for bio-chemical analysis.
The tissue was homogenized with 10 ml of phosphate buffer of
pH–7.4. The mixture was sonicated 5 to 10 cycles of 20 seconds at
110mv at 4°C and centrifuged at 10,000 rpm in a refrigerated
Bioremediation of Heavy Metals by Lower Plants 171
centrifuge kept at 4ºC for15 min. The centrifugate was taken for
analysis of total protein and total thiol.
3.2.3 PURIFICATION OF PHYTOCHELATIN The metal removal capacity of Azolla pinnata for cadmium was
assessed from the previous studies. Many of the reports on phylochelatin
induction are cadmium induced ones either in vivo or in vitro. Hence this
metal is selected for further studies. The heavy metal concentration at
which the maximum concentration of total thiol obtained was selected for
further purification studies. The extract taken from the plant material
under 100µg/ml stress of CdCl2 was found to have the maximum total
thiol content and was taken as standard for further studies including
purification. The plants were exposed to 100µg/ml of CdCl2 for 72 hours
(Inouhe et al., 1994). It was then taken out, washed thoroughly with
distilled water many times and suspended in cold Tris-HCl buffer.
Isolation and purification was done according to the method by Grill et al,
(1991) with slight modifications. All purification steps were carried out at
4°C. Purification was done in two steps viz. gel filtration chromatography and ion exchange chromatography.
3.2.3.1 Extraction Frozen plant tissue (25gm) is thawed and homogenised with 15 ml
of 20mM Tris-HCl buffer pH-7.8, containing 10mM 2-mercaptoethanol.
The homogenate was sonicated and pressed through four layers of
cheesecloth and the extract was cleared by centrifugation at 12,000 rpm
for 30min. set at 4oC.
172 Chapter 3
3.2.3.2 Ion-Exchange Chromatography This step serves primarily to concentrate the protein present in the
extract. The extract was subjected to ion exchange chromatography using
DEAE Sephadex A-50 (Pharmacia, Sweden). 4gm of Sephadex A-50 was
suspended in 10mM Tris-HCl buffer pH-7.8 and kept at 4oC overnight.
Swollen DEAE SephadexA-50 was loaded into a chromatographic column
(1.5 x 25 cm) and allowed to settle. Care was taken to avoid trapping of air
bubbles in the column. Before loading the column, it was well equilibrated
with 10 mM Tris-HCl buffer, pH-7.8 and 1mM 2-mercaptoethanol. The
extract was loaded to the top of the column. This buffer was also used to
wash the column after sample application and the bound proteins were
subsequently eluted with a linear gradient of NaCl (0-1M) in the same buffer.
Flow rate was adjusted to 60ml/hr and fractions of 5ml were collected. The
elute was tested for metal concentration, protein absorbance at 280nm and
total thiol by treating with Ellman’s reagent and absorbance was taken at
412nm. The SH positive fractions with high metal content were pooled and
collected for the next purification step.
3.2.3.4 Salting out and Concentration The protein solution was dialysed in a 0.5 KDa cut off dialysis bag.
This step was carried out by placing the dialysis bag with the protein
solution in the tank containing 10 mM Tris-HCl buffer pH-7.8, about 100
times the volume of protein inside the bag. The process was done three
times by changing of the buffer. After desalting the NaCl in the protein it
was concentrated. The final concentrate was used for further gel filtration.
Bioremediation of Heavy Metals by Lower Plants 173
3.2.3.5 Gel Filtration 4gm of Sephadex G-50 was suspended in 10mM Tris-HCl buffer
pH-7.8 and kept at 4oC overnight. Swollen Sephadex G-50 was loaded
into a chromatographic column (2.5 x 50 cm) and allowed to settle under
gravity while maintaining a slow flow rate through the column. Care was
taken to avoid trapping of air bubbles in the column. Before loading the
column, it was well equilibrated with 10mM Tris-HCl buffer, pH-7.8.
The final concentrate was loaded to the top of the column and was
eluted using Tris-HCl buffer at a flow rate of 60 ml/hr and fractions of
4ml were collected. Fractions containing Cd-PC complexes were
identified by its absorption at 254 nm, 280nm and by assay for sulphydryl
groups using Ellman’s reagent (Jocelyn, 1991). The fraction, which
showed maximum thiol content, was taken for further studies.
3.2.4 HPLC ANALYSIS
3.2.4.1 Instrumentation A basic HPLC system is used with the following capabilities: (1) a
single pump equipped with a proportionate value for gradient elution (2)
sample injector (3) a wavelength detector for monitoring UV absorbance
at 200-220 nm (4) a data–handling system capable of collecting and
integrating data from the detector (5) a fraction collector through which
fractions are collected to the peak height.
3.2.4.2 Sample Preparation
Purification of PC prior to RP-HPLC was attained through two
steps, ion exchange chromatography and gel filtration chromatography.
All purification steps were carried out at 4ºC. The purification procedure
used in the present study included the following steps.
174 Chapter 3
The homogenate was subjected to ion-exchange chromatography
with DEAE-Sephadex A-50 and 0.5 M NaCl fractions were collected and
pooled since these fractions showed high thiol and metal contents. This
pooled fractions were used for further analysis.
Gel-permeation chromatography was performed on a Sephadex G-
50 column. 4ml fractions were collected and noted absorption at 254 and
280 nm. Total thiol was also estimated.
Fractions containing thiol peaks were taken and stored at 0oC and
used for further analysis.
3.2.4.3 RP-HPLC Procedure
Test method The type of column used for isolation is Waters C18 column -
4.6×250mm Nucleosil, Waters 717 plus auto sampler and Waters 2487
UV detector.
Gradient Elution The mobile phase used to elute PCs from RP-column consists of
an equilibration buffer (buffer-A) such as water in 0.1% TFA and an
elution buffer (buffer-B) that contains an organic modifier such as 20%
acetonitrile in water with 0.1% TFA. Both the equilibration and elution
buffer was filtered and degassed by vacuum filtration prior to use in RP-
HPLC. The flow rate was 1.0ml/min. and the volume injected was 50µl.
Bioremediation of Heavy Metals by Lower Plants 175
3.3 RESULTS
3.3.1 Screening for Metal Removing Capacity The percentage of Cd and Pb removal by Azolla pinnata after 5
days of contact is presented in the Table.B3.1. The results clearly suggested
that at lower concentration the plant showed greater removal efficiency and
decreased at higher concentration. But the metal uptake increased with
increase in concentration. Growth was normal at lower concentration as
compared to control. The plant started to show morphological changes at
higher concentration (200µg/ml) after 5 days of contact. The maximum
percentage of Cd removal recorded was 90.24 and was observed at an
initial concentration of 25µg/ml.With regard to Pb it was found to be 82.2%.
The results clearly indicated that Azolla pinnata showed more tolerance and
removal efficiency when compared to other plants. Hyper tolerance which
made accumulation possible may be due to some inherent mechanisms.
Plate 8: Experimental setup for metal accumulation studies in
Azolla pinnata
176 Chapter 3
Table: B3.1 Cadmium and Lead removal by Azolla pinnata after 5 days of contact
Initial concentration of
metal (µg/ml)
Metal in culture medium after absorption by plants
(µg/ml )
Metal uptake by the whole
plant(µg/ml)
Removal of metals by plants
(%)
Cadmium Lead Cadmium Lead Cadmium Lead
25 2.44±0.68 4.45±0.58 22.56 20.55 90.24 82.2
50 12.75±1.27 15.65±0.87 37.25 34.35 74.50 68.7
100 42.61±1.68 45.61±1.61 57.39 54.39 57.39 54.39
150 76.35±2.65 79.80±2.85 73.65 70.20 49.10 46.80
200 122.42±2.56 126.45±2.63 77.58 73.5 38.79 36.77
Mean of six values ± SD
Table: B3.2 Cadmium and Lead removal by Lemna major after 5 days of contact
Metal in culture medium after absorption by
plants (µg/ml ) Metal uptake by the whole plant (µg/ml)
Removal of metal by plants (%)
Initial concentratio
n of metal (µg/ml) Cadmium Lead Cadmium Lead Cadmium Lead
25 4.5±0.12 5.10±0.62 20.47 19.90 81.88 79.60
50 17.76±0.91 19.78±0.86 32.24 30.22 64.68 60.44
100 47.15±1.16 51.6±1.72 52.85 48.35 52.85 48.35
150 77.35±1.87 80.15±2.18 72.65 69.85 48.43 46.56
200 132.65±2.75 140.25±2.56 67.35 59.75 33.62 29.88
Mean of six values ± SD
Bioremediation of Heavy Metals by Lower Plants 177
The uptake of metal by Lemna major was found to increase with
increase of metal concentration. The percentage removal of Cd and Pb
recorded were 81.88 and 79.60 respectively and observed at an initial
concentration of 25µg/ml. The results suggested that the plant showed less
removal efficiency and tolerance than Azolla pinnata. But the plant
showed any inhibition of growth at any concentration tested. Metal
tolerance is found to be high in Lemna major (Table.B3.2).
Table: B3.3 Cadmium and Lead removal by Hydrilla after 5 days of contact
Metal in culture medium after absorption by plants
(µg/ml )
Metal uptake by the whole
plant(µg/ml ) Removal of metal
by plants (%) Initial
concentration of metal (µg/ml Cadmium Lead Cadmium Lead Cadmium Lead
25 9.56±1.25 7.45±0.54 15.44 17.55 61.76 70.20
50 24.32±1.45 19.25±0.85 25.68 30.75 51.3 61.50
100 - 69.35±2.35 - 30.65 - 30.65
150 - - - - - -
200 - - - - - -
Mean of six values ± SD
Hydrilla was not tolerant even at a concentration of 100µg/ml and
died after 3 days of contact and was not at all tolerant to higher
concentrations and could not survive a day. Cd affected cellular
178 Chapter 3
parameters and was toxic to the plant. The results showed that the plant
was not at all tolerant to Cd and Pb (Table.B3.3).
Table:B3.4 Cadmium and Lead removal by Lemna minor after 5 days of contact
Metal in culture medium after absorption by plants
(µg/ml )
Metal uptake by the whole
plant(µg/ml )
Removal of metal by plants
(%)
Initial concentration
of metal (µg/ml)
Cadmium Lead Cadmium Lead Cadmium Lead
25 8.50±1.32 6.45±1.42 16.5 18.55 66.00 74.20
50 20.21±1.67 18.65±1.78 29.79 31.35 59.58 62.7
100 72.25±2.32 68.50±2.13 27.75 31.50 27.75 31.5
150 - 112.25±2.22 - 37.75 - 25.17
200 - - - - - -
Mean of six values ± SD
The percentage removal of metals by the plant decreased but
uptake of metals increased gradually with increase in concentration of the
metal in the culture medium. It is evident from the result that the plant
removed maximum percentage of Cd and Pb at a concentration of
25µg/ml. Lemna minor died at 150µg/ml of Cd after 4 days of contact
while at 200µg/ml they died within a day. The plant could not withstand
high concentration of Pb also (Table.B3.4).
The preliminary screening results clearly indicated that Azolla
pinnata showed more tolerance and metal removal efficiency compared to
Bioremediation of Heavy Metals by Lower Plants 179
other plants and was selected for further studies. The objective of our
study is to suggest an organism which could remove metal very
effectively so as to use it in bioremediation at industrial belts.
3.3.2 MECHANISM OF UPTAKE
3.3.2.1Adsorption experiments
The experimental data for fitting the Freundlich and Langmuir
adsorption models are given in Tables B3.5 and B3.6. From the
Freundlich and Langmuir adsorption studies it could be suggested that the
mode of uptake was not a physical one. The plot of log Ceq versus log qeq
is not a straight line which confirms that surface adsorption is not the
mechanism involved in the mode of uptake of metals. (Sen and
Bhattacharyya, 1993). The plot of 1/ Ceq versus 1/qeq is also not a straight
line suggesting the inapplicability of both models.
Table: B3.5Cadmium adsorption by Azolla pinnata
Initial Conc. Residual Conc. Metal adsorbed
Co(mg/L) Ceq(mg/L) qeq(mg/g) Adsorption%
25 20.70±2.23 4.30 17.20
50 42.84±1.78 7.16 14.32
100 89.31±2.45 10.69 10..69
150 134.73±2.85 15.27 10.10
200 184.89±1.43 15.11 7.5
The values are averages of six values ± SD
180 Chapter 3
Table: B3.6 Lead adsorption by Azolla pinnata
Initial Conc. Residual Conc. Metal adsorbed
Co(mg/L) Ceq(mg/L) qeq(mg/g)
Adsorption%
25 21.28±1.32 3.72 14.88
50 43.14±2.45 6.86 13.72
100 89.50±2.65 10.50 10.50
150 139.24±2.31 10.76 7.16
200 189.09±2.43 10.91 5.45
The values are averages of six values ± SD
3.3.2.2 Bioaccumulation studies The metal contact of the plant at different days with different
initial concentrations of Cd and Pb were studied and the results are shown
in the Tables. B3.7 and B3.8. It could be seen that the metal removal not
only depends on metal contact time but also the initial metal concentration
in the medium. 84.96% of Cd and 75.4% of Pb were removed after 3 days
of contact at 25µg/ml of metal concentration. With increase in initial
metal concentration in the culture medium, the percentage removal of
metals by the plant decreased. Accumulation is a time dependant process.
The metal absorbed/uptake by the plant (µg/gm) increased with increase
in time and concentration but an equilibrium is reached at a concentration
beyond which no uptake was observed.
Bioremediation of Heavy Metals by Lower Plants 181
Table: B3.7 Absorption of Cd by Azolla pinnata after 1,2 and 3 days of contact
Metal in culture medium after absorption by plants(µg/ml )
Removal of metal by plants (%)
Contact time (Days)
Initial concentration
of metal (µg/ml)Cd 1 2 3 1 2 3
Metal absorbed by plants
(µg/g) after 3days
25 9.52±0.65 7.12±0.65 3.76±1.15 61.92 71.52 84.96 3.96
50 22.35±1.13 17.73±2.15 15.45±1.65 55.3 64.54 69.10 8.29
100 68.45±2.51 57.47±2.25 48.54±2.61 31.55 42.53 51.46 9.69
150 106.75±3.12 94.15±2.15 75.65±2.34 28.83 37.23 49.57 14.55
200 148.25±2.12 135.21±2.73 127.35±2.56 25.88 32.40 36.33 14.12
The values are averages of six values ± SD
Table: B3.8 Absorption of Pb by Azolla pinnata after 1, 2 and 3 days of contact
Metal in culture medium after absorption by plants(µg/ml )
Removal of metal by plants (%)
Contact time (Days)
Initial concentration
of metal (µg/ml)Pb
1 2 3 1 2 3
Metal absorbed by plants
(µg/g) after
3days
11.50±1.20 9.24±2.2 6.15±0.15 54.00 63.04 75.40 3.65
50 26.25±1.45 20.45±1.32 15.75±1.58 47.50 59.10 68.50 6.65
100 68.25±2.45 59.23±1.89 49.35±2.76 31.75 40.77 50.65 9.32
150 119.87±1.29 110.62±1.38 95.86±2.47 20.08 26.25 36.09 10.46
200 163.24±1.43 156.40±2.65 145.18±2.45 18.38 21.80 27.41 10.50
The values are averages of six values ± SD
182 Chapter 3
3.3.2.3 Biochemical investigations
Table: B3.9 Total Protein and Thiol content of Azolla pinnata grown under Cadmium stress
Cadmium concentration(µg/ml)
Total protein (mg/g tissue weight)
Total Thiol (mM/mg protein)
Control 1.525± 0.142 0.051± 0.005
25 1.814± 0.067 0.155±0.004
50 1.734± 0.042 0.161± 0.008
100 1.632±0.115 0.277 ±010
150 0.842±0.025 0.091±0.007
200 0.706± 0.055 0.072±0.004
The values are averages of six values in each case ± SD
The total protein and thiol content of the plant, grown in presence
of Cd was studied and the results are shown in Table.B3.9. The total
protein content was not affected significantly whereas the total thiol
content increased significantly from 25µg/ml to 100µg/ml. But the thiol
content decreased with 150µg/ml to 200µg/ml of Cd. The protein content
reached maximum at 25µg/mlCd concentration.
Bioremediation of Heavy Metals by Lower Plants 183
Table: B3.10 Total Protein and Thiol content of Azolla pinnata grown under Lead stress
Lead concentration(µg/ml)
Total protein (mg/g tissue weight)
Total Thiol (mM/mg protein)
Control 1.612± 0.092 0.051± 0.008
25 1.624± 0.027 0.071± 0.004
50 1.704± 0.042 0.095± 0.005
100 1.632±0.115 0.067 ±010
150 1.042±0.125 0.081±0.007
200 0.916± 0.050 0.072±0.004
The values are averages of six values in each case ± SD
Azolla pinnata plants grown under Pb stress showed maximum
synthesis of total protein and thiol content at 50µg/ml. But the protein and
thiol contents were not so significant when compared to the control. The
protein content was lower than control in samples treated with 150 and
200µg/ml Pb. An increase in thiol content was observed but it was not
significant as compared to the control and hence was not considered for
further studies.
Azolla pinnata plants, grown under CdCl2 showed maximum total
thiol synthesis at 100µg/ml and highly significant when compared to the
control. Increase in thiol content was observed in all metal stressed
samples when compared to the control. Since maximum synthesis of thiol
was obtained with plants treated with 100µg/ml of Cd, this concentration
was selected for the further studies.
184 Chapter 3
3.3.3 PURIFICATION OF PHYTOCHELATIN The ion exchange chromatography profile of the Cd stressed
plants (Figure. B3.1) showed a greater absorption between fractions 40
and 55 at 412 nm when treated with DTNB (Ellman’s reagent) which is
specific for sulfahydryl groups. Increased thiol production was detected in
fractions between 40 and 55 which showed a peak. These fractions also
showed high metal content. Thus the sulfhydryl containing material from
the metal treated extract was isolated by ion exchange chromatography.
These fractions were pooled and subjected to gel chromatography.
0 20 40 60 80 1000
2
Protein absorbance at 280 nm Thiol absorbance at 412 nm M etal concentration (µg/m l)
N um ber of Fractions
Opt
ical
Den
city
-1
0
1
2
3
Metal C
oncentration µg/ml)
Figure: B3.1 Elution profile of ion exchange chromatographed extract
of Azolla pinnata treated with Cd showing absorbance at 280nm, 412nm & metal concentration (µg/ml).
Bioremediation of Heavy Metals by Lower Plants 185
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 5 9 13 17 21 25 29 33 37 41 45 49
Fractions
Opt
ical
den
sity
254280412
Figure: B3.2 Elution profile of Gel chromatographed extract of
Azolla pinnata treated with Cd showing absorbance at 254nm, 280nm & 412nm.
The gel filtration profile of the Cd stressed plants (Figure. B3.2)
showed a greater absorption between fractions 21 and 30 at 412 nm when
treated with DTNB (Ellman’s reagent) which is specific for sulfahydryl
groups. Increased thiol production was detected in fractions 21- 30 which
showed a peak. The chromatogram of metal treated sample clearly
displayed a U-V absorbing peak at 412nm specific for sulfhydryl
containing groups. Thus the sulfhydryl containing material from the metal
treated extract was isolated by ion exchange and gel filtration
chromatography. These fractions were pooled and subjected to HPLC.
3.3.4 HPLC ANALYSIS The figure.B3.2 shows peptides which differ in the number of γ-Glu-Cys
units they contain. PC1, PC2 and PC3 are the different peaks obtained which
depend on the number of γ-Glu-Cys units. Hirata et al, (2001) reported
186 Chapter 3
the synthesis of phytochelatin in the marine algae, Dunaliella tertiolecta
under various conditions of exposure to Cd. They found that among the
PC subtypes in the Cd-treated Dunaliella tertiolecta cells, PC4 was the
predominant PC subtype after 48 hrs of metal treatment and PC2 was the
least prevalent. During PC synthesis, it is assumed that PC synthase add
more γ-Glu-Cys units which result in the formation of different PC
subtypes like PC2, PC3 and PC4.
Figure. B3.3: RP-HPLC of Phytochelatin isolated from Azolla
Bioremediation of Heavy Metals by Lower Plants 187
3.4 DISCUSSION Heavy metals are increasingly found in aquatic habitats due to natural
and industrial processes. Aquatic plants have evolved several mechanisms to
tolerate the presence of heavy metals. Many of them have immense capability
to accumulate metals, and there is considerable potential for using them to treat
wastewaters. Plant tolerance/survival largely depends on intrinsic biochemical
and structural properties and genetic adaptations and hence seems to be
dependant on the plant species and the metal involved. Some available
hydrophytes in our area for which no earlier reports available were therefore
screened for their ability to tolerate Cd and Pb. Hyper tolerance which makes
accumulation possible depends upon the mechanism to withstand metal stress.
Screening resulted in the selection of Azolla pinnata as the best species to
remove Cd and Pb. These metals are of profound concern as highly toxic
contaminant of surface waters posing serious health hazard to humans.
In the present study, screening was used to select a metal tolerant
organism. Selection was done by enrichment of the culture medium with
graded levels of above metals( 25-200µg/ml).The culture medium used was
only a mineral salt medium and there was no other carbon source supply. The
use of Hoagland and Arnold medium for screening was reported by several
workers. The number of tolerant organisms reduced as the concentration of
metals in the culture medium increased and resulted in the selection of an
efficient tolerant one with high accumulation capacity.
The method employed for determining the metal accumulation
capacity is Atomic Absorption Spectroscopy which is most predominant and
employed by many researchers (Wilde et al., 2001). Compared to other
analytical methods this is the most accurate since micro and nanogram
concentrations of the metal pollutant can be analysed. In the present study
188 Chapter 3
AAS was used to determine the percentage of metal uptake among the
screened hydrophytes.
Many researchers had reported that a variety of mechanisms had been
employed for metal removal (Volesky and Holan, 1995). They are broadly of
three types viz, Biosorption bioaccumulation and biotransformation.
Biosorption is metabolism independent and involves rapid physical adsorption
while bioaccumulation is metabolism dependant and metal is transported into
cells by active metabolism (Gadd, 1990). In the present study adsorption
experiments were conducted to know the mode of uptake. Freundlich (1906)
and Langmuir (1916) models are two widely accepted isotherm models
describing adsorption. When the experimental data obtained were fitted to
these models, neither Freundlich nor Langmuir adsorption model was obeyed
by the plot since no straight line was obtained. This made it clear that the
mechanism involved is not adsorption and some intracellular metal binding
complexes might be involved. After three days of contact the plants treated
with metals were shaken with 1N solution of HCl, H2SO4 separately but no Cd
or Pb was found in the solution. Again 1gm of the treated plant was
homogenised with the above solution separately and after centrifugation the
solution didn’t contain any Cd or Pb.
The metal absorption by the plant was found to be biphasic, being
considerably high initially, but subsequently slowed down and gradually
reached an equilibrium which could be attributed to the gradual saturation that
can be attained by a definite biomass. The increase in concentration might have
caused increased toxic effects to the plant metabolism beyond a threshold level.
The concentration and duration dependant uptake of Hg2+ had been reported in
Hydrilla by Chatterji and Nag (1991). The higher uptake in the beginning and
its later decline is indicative of misbalance in tissue permeability.
Bioremediation of Heavy Metals by Lower Plants 189
Biochemical investigations revealed that the total thiol content of the
metal -treated plant was found to be elevated compared to the control.
According to Grill et al., (1985) cysteine rich tripeptides (GSH) are capable of
binding to cadmium and other metal ions through cysteine thiolate coordination.
This might be the reason for the increase of thiol content in metal treated plants.
The total thiol content was found to be high at a Cd concentration of 100µg/ml.
Different workers adopted different strategies for the purification of
PCs from different organisms. Some researchers used only a single step for the
purification of PCs. ( Grill et al., 1985) purified PCs by gel filtration on
Sephadex G-50 from plants and cell cultures. Zenk et al., (1987) used ion
exchange chromatography for the purification of PCs from Silene cucubalus.
Some researchers used two steps for the purification of PCs. Gekeler et al.,
(1988) employed two steps ie. ion-exchange chromatography and Sephadex
G-50 for the purification of PCs. In the present study a two step purification
strategy had been adopted ie. ion-exchange chromatography on DEAE
Sephadex A-50 followed by gel filteration on Sephadex G-50.
Phytochelatin has similarity with SH containing GSH (γ- glu- cys-gly),
and act as the substrate for the synthesis of Phytochelatin mediated by enzymes
under metals stress. So for the isolation of this metal complexing protein, the
SH containing fractions of the elution profile obtained by gel filteration is
detected by treating with DTNB and absorbance was taken at 412nm.These
fractions are pooled and used for HPLC analysis.
Many biochemical and genetic studies have confirmed that glutathione
(GSH) is the substrate for PCs synthesis (Reese et al., 1988 and Mendum et al.,
1990). In the presence of metal ions, especially Cd2+, the constitutive enzyme
named PC synthase (EC 2.3.2.15) using GSH as a substrate produces
phytochelatins with the general structure (γ-Glu-Cys)n-gly where n 2–11
190 Chapter 3
(Grill et al., 1989; Rauser,1995). As a result nontoxic complexes PC-Cd appear
where cadmium is bound by the cysteine thiols. In many plant species heavy
metals detoxification (particularly Cd ions) is associated with the synthesis of
cysteine-rich peptides called phytochelatins (PCs) (Grill et al., 1985; Cobbett,
2000; Hall, 2002).This lead to the search for such a compound which resulted
in its purification.
RP-HPLC analysis The result obtained is in accordance with Grill et al., (1985), Rauser
et al., (1995); Steffens e t al., (1986), Robinson et al., (1988) as they resolved
metal-binding complexes with RP-HPLC with solvents containing either 0.05%
phosphoric acid or 0.1% TFA. The Peaks obtained in the figure B3.1 represent
the Phytochelatin peptides which differ in the number of γ-Glu-Cys units they
contain. Such Cd-binding complexes were isolated from members of Phycophyta
by RP-HPLC and had been shown to be composed of phytochelatin peptides
which contain different number of γ-Glu-Cys units (Grill et al., 1988). In the
present study also PCs splits into smaller peptides of different units. RP-HPLC
done on Hydrilla verticillata grown in various Pb2+ concentrations indicated
involvement of PCs in Pb2+ detoxification (Gupta et al., 1995). However, the
formation of complexes and detoxification of cadmium in vivo is more complex.
Phytochelatins probably play a central role in the homeostatic control
of metal ions in plants. They may also be involved in the physiological
mechanism of metal tolerance of selected plants. Plants have been reported to
fix such metals intracellularly by the synthesis of a buffering molecule,
phytochelatin which is believed to be ubiquitous in all groups of plants. From
the above findings it could be established that the physical adsorption is not
taking place in plants even though physical adsorption is the major mode of
metal sequestration in cyanobacteria and bacteria.