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.
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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