we choose, to eat Cd contaminated rice
or to use GM plant-microbes?
By: Yoshikatsu Murooka*
The advancements in science and
technology in the 20th century provided us a comfortable and
convenient life. At the same time, however, the advancements provided many
unexpected problems as well. Mankind has suffered from unknown diseases for
the past several decades due to exposure to industrial wastes.
In Asia, a majority of chemical
pollutants responsible for several diseases are heavy metals. Similar
conditions must exist worldwide. Widespread pollution by heavy metals that
are generated by various industries has serious adverse effects on human
health and the environment. Decontamination of the soil and water around
industrial plants presents major challenges for a long time. Japanese
people, especially farmers and fishermen, were confronted with
unidentifiable symptoms for years.
Extensive research later
identified these symptoms as ‘Itai-itai’ disease and ‘Minamata’ disease.
Japanese scientists determined that these diseases resulted from rice grains
containing large amounts of cadmium and fishes containing methyl-mercury,
respectively. ‘Itai-itai’ and ‘Minamata’ diseases have yet to be completely
eradicated in Japan and their incidence is increasing worldwide.
Anthropogenic sources of heavy
metal deposition are on the increase as a result of the current industrial
revolution, especially in developing countries. Agriculture, mining,
smelting, electroplating, and other industrial activities are to blame for
undesirable concentrations of metals, such as As, Cd, Cr, Cu, Ni, Pb and Zn,
in the soil. Although trace metals are an important part of the soil
ecosystem, accumulation of these metals is harmful to people, animals,
plants and other organisms coming in contact with the soil and groundwater
In my laboratory, we analyzed
the cadmium content in soil samples from various rice fields in Japan. One
soil sample taken from a rice field, located downstream from an old mine
which was closed more than 30 years ago, had an extremely high concentration
of cadmium. This data is highly confidential and unpublished to date since
if I were to release this information to the press, the prefecture
governments involved would panic. Consumers won’t buy any rice produced from
this prefecture, even though most of rice had no contamination of cadmium.
It would be better to tell the farmer to stop the cultivation of rice in
this kind of field and clean up the soil as soon as possible.
Unlike many other pollutants,
heavy metals are difficult to remove from the environment. These metals
cannot be chemically or biologically degraded, and are ultimately
In the rhizosphere, plant roots
secrete various nutrients and attractants stimulating microbes to swarm
plant roots. Certain microbes have the ability to gather and carry metal
ions to plant roots. The metal ions released by these microorganisms are
taken in by plants. These phenomena gave me an idea for bioremediation using
plants and their microbes.
Let us recall plant symbiosis.
A typical plant symbiosis is a leguminous plant, the soybean, and its
nitrogen-fixing rhizobia, Bradirhizobium japonicum. 1/4th
to 1/3rd of the world’s soils are acidic and/or nutrient poor.
Since these acidic soils contain far fewer microorganisms and limited
species, we need to inoculate soils with appropriate rhizobia.
Dr. Hiep of Cantho University,
Vietnam and Dr. Nantakorn Boonkerd, Suranalee Institute of Science and
Technology, Thailand have shown the significant formation of nitrogen-fixing
nodules on the soybean roots by inoculation of the soils with
Bradirhizobium species in Mekong Delta in Vietnam and Thailand,
respectively. The yields of soybeans were increased.
The soybean plant produces
sugars by photosynthesis. The sugars are supplied to their rhizobia as an
energy source. Rhizobia present in the nodule fix atmospheric nitrogen
converting it into ammonia with energy supplied by the plant, and the
ammonia is supplied to the plant as nitrogen source. Most plant roots form
mycorrhizal association, which enhances the uptake of water and phosphate
ions from water and phasphorus deficient soils. Therefore, you do not supply
chemical fertilizer or at least you decrease the addition of chemical
fertilizer. These symbioses are beneficial to agriculture and interesting
with regard to their molecular mechanisms.
Another idea that we had for
heavy metal accumulation in symbiosis was to introduce useful gene, such as
metal-binding protein, into a Rhizobium. Genes for metal-binding
proteins introduced into a Rhizobium will be expressed in bacteroids in
We used Chinese milk vetch as
our symbiotic host plant. Known as renge-soh in Japanese and
Astragalus sinicus in Latin, Chinese milk vetch is a green manure
legume. This plant has been used as soil fertilizer for rice fields in China
for 1000 years and in Japan for two hundred years. In the rainy season,
farmers immerse renge-soh plants with their nodules into the paddy
water and plow in the fields. As a result of this, no nitrogen fertilizer is
required. Mesorhizobium huakuii subsp. rengei strain B3 is a
bacterium that establishes a symbiotic relationship with Astragalus
sinicus. It would be of considerable interest if we could use this
leguminous plant to increase the soil nitrogen content and, at the same
time, to remove heavy metals from the soil.
Inoculation of strain B3
resulted in increased accumulation of cadmium not only in nodules but in
renge-soh too. We found about 2% cadmium accumulation in this plant per
year. Given this accumulation rate for cadmium by renge-soh, it would
take about 50 years to eliminate cadmium from polluted soil using
phytoremediation. We cannot wait for 50 years. So, we began to genetically
bread the renge-soh plant to increase its ability to accumulate heavy
Genetic engineering suggests
the possible use of specially designed microbial biosorbents with suitable
selectivity and affinity for heavy metals. Normally we have a mechanism in
our bodies which produces metallothioneins, metal-binding proteins, to
protect us against toxic heavy metals. For the remediation of heavy metals,
we selected the metallothionein protein as a metal binding protein.
Overexpression of metal-binding proteins by bacterial cells results in
enhanced accumulation of cadmium and offers a promising strategy for the
development of microbe-based biosorbents for remediation of
To increase the binding of
heavy metal ions, we designed oligometric metallothioneins and succeeded in
expression of the tetrameric metallothionein in bacteroid in root nodules of
renge-soh. The tetrameric metallothionein bound 4 times more cadmium
and other heavy metals than the naturally occurring monomer metallothionein.
The presence of 106 to 108 bacterial progeny of the
rhizobia in each nodule on the roots of renge-soh is advantageous for
the expression of foreign genes that help to sequester heavy metals in
contaminated soil. Once symbiosis is established, the heavy metals should
accumulate in such nodules.
Phytochelatins are an
attractive alternative to metallothioneins since they offer the potential
for enhanced affinity and selectivity for heavy metals. The structure of
phytochelatins can be represented by (g-Glu-Cys)n-Gly,
where n ranges from 2 to 11. Phytochelatins have higher metal-binding
capacity than metallothioneins. Thus, phytochelatins are attractive as
metal-binding peptides for the development of a microbe-based biosorbents
for remediation of metal-polluted soils. We demonstrated the introduction of
the Arabidopsis gene for phytochelatin synthase (PCS); (AtPCS)
into strain B3. The gene of MTL4 or AtPCS was expressed under
the control of bacteroid-specific promoters, namely, the promoters of the
nifH gene and nolB gene. The gene for MTL4 or PCS
was expressed in free-living cells under microaerobic conditions when the
promoter was activated by nifA. The expression of the MTL4 and
AtPCS genes in strain B3 increased the ability of cells to bind
cadmium approximately 2-fold and 9- to 19-fold, respectively.
When recombinant strain B3
established the symbiotic relationship with renge-soh, the symbionts
increased cadmium accumulation in root nodules by 1.5-1.8 fold. The
expression of the both MTL4 and AtPCS genes showed additive
effect on cadmium accumulation in nodules. In paddy soil, addition of
recombinant strain B3 carrying a plasmid with the both MTL4 and
AsPCS genes significantly increased the accumulation of cadmium in roots
and nodules of A. sinicus. Our results showed about 9% cadmium
accumulation for this
engineered plant. Since we cannot decompose heavy metals, cadmium containing
plants are harvested, incinerated, and the ashes are then stocked to recycle
the heavy metals.
Thus, this system uses the
advantages of both plants and rhizobia, in particular, engineered genes can
be transformed to plants through infection with recombinant bacteria.
By our calculations, it would
take 3 years to clean up cadmium contaminated soils with the tri-annual
cultivation of the engineered plants. However, our government does not
permit to use genetically engineered plants and microbes in open fields. GM
plants and microbes have great potential for humanity. However, we must
carefully test and monitor their safety for humans and the environment. It
is not so easy to prove the safety of GM organisms. My contention is that
using GM plant-microbes is much safer than taking heavy metal-contaminated
*Professor of Osaka
University San Francisco Center for Education & Research, 120 Montgomery
St., Suite 1270, San Francisco, CA 94104, USA -