ISTC-2002
Title: Cell selection of perspective plant
species - heavy metal hyperaccumulators for phytoremediation of the environment
from anthropogenic contamination.
Project menager:
Prof. Sarsenbayev Batyrbek
Laboratory of Root Nutrition
Institute of Plant
Physiology, genetics and Bioengineering
Alamty, Kazakhstan
The aim of the project is to obtain
perspective plant species - heavy metal hyperaccumulators via cell selection,
to fix heavy metal accumulating ability, to investigate mechanisms of plant
stability to heavy metals and to discover possible mechanisms of detoxication.
Implementation of the following
tasks is necessary to achieve the aim of the project:
1. Screening of the plant species from
Kazakhstan’s flora that are stable towards heavy metals and able to accumulate
them, discovery of physiological and biochemical features of the plants in
contaminated by heavy metal media.
2. Cell selection of perspective
species concerning to heavy metal hyperaccumulation, obtaining of regenerants
and conducting of ecological tests of the regenerants in field conditions.
3. Investigation of mechanisms
of heavy metal hyperaccumulation and plant tolerance to the metals.
Duration - 3 years
Title: Treatment of
Wastewater with the Free Floating Aquatic Plants
Investigators:
M.Ines M. Soares* & Moche
SAGI** Saule D.ATABAYEVA &
*Environmental Microbiology
Laboratory Batyrbek A. SARSENBAEV
**Biostress Research Laboratory
Laboratory of Root Nutrition
The Blaustein Institute for
Desert Research Institute of Plant Physiology
Ben Gurion University of the
Negev Genetics and Bioengineering
Sede Boqer 84993, Israel
Timiryazev St.,45
480090 Almaty, Kazakhstan
Telephone: 972-7-6596834
Telephone: 7(3272)-479292
Fax: 972-7- 6596831 Fax:
7(327)-476106
E-mail: soares@bgumail.bgu.ac.il E-mail:
sauleat @yandex.ru
Overall and scientific
objectives:
The overall objective of this
project is to clean mixed effluents to an extent that they can be safely
released into the environment, without posing a threat to water sources and
agricultural soils and crops. This will be achieved by removal of contaminants
by free-floating freshwater vascular plants (FVPs) capable of taking up and
immobilizing in their biomass large amount of the polluting agents. The
specific research objectives are:
1. To select the most suitable species to
clean up Cu, Cd, Pb and Zn under the climatic conditions in Kazakhstan.
Contamination
of most natural basins in Kazakhstan by wastewater containing heavy metals and
organic and inorganic toxic substances associated with chemical, mining,
smelting industries is reaching catastrophic proportions. Pollution by heavy
metals is mostly related to related to the production of ferrous and
non-ferrous metals in East Kazakhstan, and to processing of phosphorous and
chromium deposits in South Kazakhstan. Raw or insufficiently treated effluents
from these and other industries inputs into natural systems (rivers, lakes and
groundwater) and have a detrimental impact on the future use and function of
these basins. Furthermore, the problem is amplified by the large-scale
irrigation of agricultural lands with polluted water. Municipal wastewater has
not been adequately treated either, and the situation is aggravated by the lack
of separation between domestic and industrial effluents. Eutrophication of
receiving water bodies is widespread and fishing in once pristine rivers and
lakes has all but disappeared. Although some specific contaminants may vary in
their relative importance, this overall scenario is found in the other Central
Asian Republics.
Removal
of Cu, Cd, Pb and Zn from mixed effluents will have the effect of arresting
contamination of water and soils by these metals. For their growth FVPs will
take up nitrogen, potassium, phosphor, sulfur and other elements thus improving
the overall quality of the water. This project has the potential to bring
considerable and widespread economical and ecological benefits to Kazakhstan
and other countries in the region.
Phytoremediation
- using plants to remove inorganic or organic pollutants from soil and water –
has generated much interest in recent years. Numerous plant species are known
to be hyperacumulators of heavy metals and some of the most extraordinary
hyperacumulators have been found in the aqueous environment where vascular
plants and algae can accumulate heavy metals to a level 10,000 times higher
than their concentrations in the surrounding water. Some wetland plants can
accumulate, unspecifically, appreciable amounts of various metals (Rai et al.,
1995a,b). Compared with phytoplanckon, macrophytes (freshwater vascular plants
combined with macroscopic algae) are 10-100 times less sensitive to most
elements except copper to which they are equally sensitive (Brooks and
Robinson, 1998).
Decontamination
of polluted waters with floating vascular plants (FVPs) was established
following the pioneering work of Wolverton (1975), and there are two main ways
in which the plants may be used for remediation: free floating or growing
rooted emergents in trickling bed filters. Important advantages of the former
strategy (to be used in the project) are the simplicity and economy with which
complete harvest of the biomass can be achieved.
The
water hyacinth (Eichhornia crassipes) is the most commonly cited FVPs
for phytoremediation of polluted waters; the plant has a rapid growth rate and
can hyperaccumulate nutrients and heavy metals. However, the water hyacinth has
become a noxious weed in some warm countries. Other plants include duckweed (Lemna
minor) and water velvet (Azolla pinnata), and both have shown
bioaccumulation coefficients (elemental content of plant/ elemental content
water) of about 1000 for Cu and Cr (Jain et al., 1989; Wahaab et al.,
1995).
FVPs
usually accumulate heavy metals by absorption followed by passive or active
transport across membranes (Forstner and Witman, 1981; Smies, 1983). Elemental
levels in most macrophytes exhibit a seasonal pattern of a spring maximum
followed by a steady decline during the summer. This has been ascribed to
environmentally driven physiological changes. The capacity and mechanisms of
these plants to assimilate nitrogen, sulfate and phosphate are better known.
MoCo
is a component of the molybdoenzymes nitrate reductase (NR), xanthine
dehydrogenase (XDH) and aldehyde oxidase (AO). AO has an important role in
plant development and adaptation to environmental stresses (Sagi et al.,
1998) since members of the AO multigene family catalyze the final step in the
biosynthesis of absicic acid (ABA) and indole-3-acetic acid (IAA)
(Walker-Simmons et al., 1989; Koshiba et al., 1996). NR, the
first enzyme in the nitrate assimilation path is frequently a limiting factor
in plant growth and development. Xanthine oxidoreductase catalyzes the first
oxidative step in purine catabolism. This enzyme is necessary for ureide
biosynthesis in higher plants when exposed to stress (Sagi et al.,
1998). The involvement of Molybdenum cofactor containing enzymes (Mo-enzymes)
such as NR, AO and xanthine XDH is critical on the regulation of growth rate,
biomass production and stress (Sagi et al., 1997; Sagi et al.,
1998). Although the activities of all the Mo-enzymes can be controlled at the
genetic level through the synthesis of the molybdenum cofactor (MoCo), the
synthesis of this thiol cofactor requires a commensurate uptake of sulfur and
Mo. In the active center of all Mo-enzymes, Mo binds to MoCo via two adjacent
thiol groups. Since most of the heavy metals Hg, Pb, Cd, Cu and Zn have high affinity
to thiol groups we consider the possibility that they could bind to thiol
groups of MoCo and become inactivated. Thus, one should consider the possible
involvement of Mo, sulfate and nitrate on the regulation/activation of plant
Mo-enzymes, on the uptake of heavy metals and on the synthesis of intracellular
heavy metals chelators in plants selected for phytoremediation. The importance
of Mo-enzymes for high growth rate, biomass production and stress tolerance is
obvious. Since the rate and amount of heavy metal-accumulation depend on the
plant growth rate and biomass production, physiological approaches to increase
the activities of plant Mo-enzymes of selected FVPs are a tool for effective
phytoremadiation.
In
this project we propose to carry out the following:
(1)
Species selection: Judicious selection of appropriate plant species is
the key to a successful cleanup. FVPs such as Lemna minor, Lemna
gibba, Eichhornia crassipes, Azolla filiculoides, Azolla pinnata,
Elodea densa, Elodea canadensis, Pistia stratiotes and Ceratophyllum
demersum will be tested. Selection will be based on the type of pollutants
to be removed, the geographical location and environmental conditions and the
known uptake and accumulation capacities of the species. The latter will be
first determined in the laboratory, under controlled conditions. Studies will
be carried out with artificial wastewater and with water from a field site.
Optimal mineral nutrition (NPK) requirements will be determined as well the
limits of tolerance to Cu, Cd, Pb and Zn. Rates of biomass growth and the
kinetics of accumulation during the plant life cycle will be studied. Plants
will be separated into roots and shoots and processed as described below. Water
and biomass analysis will be as under (3).
(2)
Studies on uptake, transport and accumulation mechanisms: Selected FVPs
will be grown in containers with different concentrations of Cu, Pb, Zn, or Cd
to follow their uptake and accumulation. Metal-free and metal-bound
phytochelatins will be separated and the content of metals and chelators will
be determined in all fractions. MoCo and activities of the Mo-enzymes NR, AO
and XDH will be determined.
The
results of these studies will show: (a) the types and amounts of potential
metal-chelators in a given plant species and their specificity for different
heavy metals; (b) quantities of metals accumulated; (c) the stage of plant
development at which maximum metal accumulation occurs; and (d) the stability
of different metal+chelator complexes during plant development.
(3)
Pilot experiments: The selected species will be tested under field
conditions, in small tanks/ponds. The impact of seasonal variations (e.g.,
light intensity, temperature, etc.) on the process efficiency will be assessed
as well as its capacity to withstand oscillations in effluent composition. The
systems will be operated at various retention times and nutrient loads (P, N,
BOD, metals, etc.).
The
following water quality parameters will be monitored: temperature, pH, electric
conductivity, BOD, COD, TOC, TSS, VSS, concentrations of nitrate, nitrite,
ammonia, total N, sulfate, phosphate, and grease and oil. The concentrations of
heavy metals such as Cu, Cd, Pb and Zn and cations and anions such as Na, P,
Cl, K, Fe will be determined by atomic absorption spectroscopy. The numbers of
faecal coliphorm bacteria will be monitored. Air temperature, duration of sun
light and other relevant environmental parameters will be recorded. During the
winter, experiments will also be conducted in enclosed water treatment
facilities.
Samples
of biomass will be prepared for analyses by the following steps: washing,
drying, weighing, milling and ashing.
All
biomass containing heavy metals produced during these studies will be dumped in
a protected site; burning and recory of heavy metals may be considered in the
future, when the treatment is upscaled.
References:
Brooks and Robinson
(1998) In Plants that Hyperaccumulate Heavy Metals (R.R. Brooks ed.),
pp. 203-226. CAB International, New York. Forstner and Witman (1981)
Metal Pollution in the Aquatic Environment, 2nd ed. Springer-Verlager, Berlin. Jain
S.K. et al. (1989) Biological Wastes 28:115-126. Koshiba et
al. (1996) Plant Physiol 110: 781-789. Rai et al. (1995a)
Ecol. Eng. 5:5-12. Rai et al. (1995b) J. Environ. Manage. 3:4-8. Sagi
et al. (1998) Plant Sci 135: 125-135. Sagi et al.
(1997) Physiol Plant 99: 546-553. Smies (1983) Trace Elements
Speciation in Surface Waters and its Ecological Implications, pp. 177-193.
Plenum Press, New York. Wahaab et al. (1995) Wat. Sci. Tech.
32:105-110. Walker-Simmons et al. (1989) Plant Physiol 90:
728-733. Wolverton B.C. (1975) NASA Technical Memorandum TM-X-72721.
Capacity
strengthening
The
Kazak PI and her laboratory have the scientific capabilities to carry out this
research and preliminary studies on the suitability of various FVPs are already
in progress. Even so, they would benefit from the experience of the Israeli
team on the fields of the biochemistry of mineral nutrition and stress, and on
water treatment. A significant proportion of the budget ($18,000 training and
$……..salaries) is dedicated to training of postdoctoral and doctoral students.
The equipment and materials purchased in the framework of the project would be
a considerable boost to the Kazak laboratory.
Collaboration
A
good basis exists for a successful collaboration. The Kazak PI has been in
Sede-Boqer in the framework of an International Training Course organized by
the Ministry of Foreign Affairs (Israel), and research connections already
exist between the laboratories. The two teams will be involved in all stages of
the project. E-mail communications facilitate the discussion of plans,
experiments, methods and results. Scientists and students from Almaty will work
in Sede-Boqer and the Israeli investigators will also work in Almaty. A Ph.D.
student form Kazakhstan will be trained in the Israeli laboratory.