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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


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.


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.


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



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


(µg/ml )


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 - - - - - -


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


(µg/ml )


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 - - - - - -


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


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


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.


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 )


Contact time (Days)


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


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 )


Contact time (Days)


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


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.


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).


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


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.


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.

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