1. Review Article
Role of Genetically Engineered Endophytic Bacteria in phytoremediation of heavy
metal contaminated soils
Saba Dilnawaz
Soil Microbiology, Department of Microbiology, Jinnah University for Women, Karachi, Pakistan
Abstract:
Industrial growth are now a days effecting the environment causing extreme destruction by disposing of
hydrocarbons very frequently. About 60 million barrels of waste water, with a salt content up to 20
times higher than sea water mixed with hazardous chemicals. These chemicals must be degraded in
order to protect the lives of plants, animals and human beings. Best technique that are now a days
exercising include phytoremediation in which phytoextraction, rhizofiltration, phytostabilization, and
phytovolatilization . Genetically engineered endophytic bacteria plays promising role in this regard
specifically for toluene degradation. This article will show how B. cepacia strain helps to degrade toluene
and also variety of different methods including phytoremediation with transgenics and some other
bacterial and fungus genera that usually used to detoxify soil from organic and inorganic contaminants.
Introduction:
Heavy metal pollution of soil and organic and inorganic pollutants is a significant environmental problem
and has its negative impact on human health, animals and agriculturei
. Phytoremediation uses different
plant processes and mechanisms normally involved in the accumulation, complexation, volatilization,
and degradation of organic and inorganic pollutantsii
. Rhizosphere1
, as an important interface of soil and
plant, plays a significant role in phytoremediation2
of contaminated soil by heavy metals, in which,
microbial populations are known to affect heavy metal mobility and availability to the plantiii
. Emerging
phytoremediation techniques are evolving to overcome the concentration of pollutants in soiliv
. Evolving
techniques such as phytoextraction, rhizofiltration, phytostabilization, and phytovolatilization are also
playing a leading role in phytoremediation processesv
. Bio degradative bacteria, plant growth-promoting
bacteria facilitate phytoremediation by other meansvi
. To deal with moderately hydrophobic pollutants,
such as benzene, toluene, ethylbenzene and xylene (BTEX) compounds, chlorinated solvents,
nitrotoluene ammunition wastes and excess nutrients, this article aimed to increase the degradation of
volatile, water soluble organic contaminants during their transport in the plant’s vascular system using
engineered endophytic bacteria.ii
Material and Methods:
Construction of a toluene-degrading endophyte is our chief task for which endophyte. B. cepacia
strain BU0072 (Nir
, Kmr
), a derivative of the endophytic bacterium B. cepacia L.S.2.4, was used. B.
cepacia strain BU0072 has nre and B. cepacia G4 carry pTOM donate its plasmid (carrying toulene
resistance) through conjugation and this pTOM served as a donor strain for toluene degradation.
1 The rhizosphere is the narrow region of soil that is directly influenced by root secretions and associated soil
microorganisms
2 Phytoremediation is a bioremediation process that uses various types of plants to remove, transfer, stabilize,
and/or destroy contaminants in the soil and groundwater.

2. Transconjugants that were resistant to nickel and kanamycin and could grow on toluene as sole
carbon source. The presence of the nre Ni resistance marker and the pTOM plasmid in the
transconjugants helps to develop resistance against toulene. Second important job is inoculation of
yellow lupine with B. cepacia VM1330 which was grown in 284 gluconate medium (250 ml culture)
at 22 °C on a rotary shaker for approximately 7 d and the inoculum was prepared . The cells were
collected by centrifugation, and suspended in 1/10 of the original volume 10 mM MgSO4 to obtain
an inoculum with a cell density of 1010 CFU/ml. Seeds of L. leutes were sterilized, rinsed and then
dried, then these seeds were planted in a sterile plastic jar (800 ml), completely filled with sterilized
perlite and saturated with 400 ml of sterile Hoagland’s nutrient solution. Subsequently, the
bacterial inoculum was added to each jar at a final concentration of 108 CFU/ml Hoagland’s
solution. The jars were covered with sterile tinfoil to facilitate bacterial colonization and prevent
contamination. After the seeds had germinated, holes were made in the tinfoil and plants were
allowed to grow through them over 21 d in a growth chamber. Same procedure was followed for
inoculation of L.luteus with the B. cepacia strains BU0072 and G4. Plants were harvested after 21 d.
Roots and shoots were treated separately. Fresh root and shoot material was vigorously washed in
distilled water After sterilization, the roots and shoots were softened in 10 ml 10 mM MgSO4
Samples (100 μl) were plated on different selective and nonselective media to test for the presence
of the endophytes and their characteristics. Three-week-old L. luteus L. plants (both controls and
those inoculated with B. cepacia VM1330, BU0072 or G4) were used to evaluate the phytotoxicity
of toluene and in its degradation. The lupine plants were carefully taken out of the jars and
their roots were vigorously rinsed in sterile water to remove bacteria from the surface, plants were
grown hydroponically, settled in a two compartment glass cuvette system such that gas exchange
between the upper and lower compartments are separated. The lower compartment was filled with
300 ml of sterile, half-strength Hoagland’s solution. Different toluene concentrations of 0, 100, 500,
and 1,000 mg/l were added to the Hoagland’s solution at the beginning of the experiment after
which they are placed in the growth chamber. After 3 weeks of growth under the above conditions,
control plants and lupine plants inoculated with B. cepacia strains VM1330, BU0072 and G4 were
transferred into half-liter pots filled with a nonsterile sandy soil, irrigated with half-strength
Hoagland’s solution. Subsequently toluene was added at concentrations of 0, 100, 250 and 500
mg/l. After two weeks plants were harvested and their biomass was determined.
Results:
We examined the effect of the different endophytic bacteria on toluene. The smallest amount of
evaporated toluene, 2,523 (±853) μg, was obtained from plants inoculated with
B. cepacia VM1330, compared to 3,378 (±987) μg, 4,362 (±733) μg and 7,367 (±298) μg for the
control plants and the plants inoculated with BU0072 or G4, respectively.
Discussion:
This article reflects phytoremediation techniques to get rid from soil with heavily contaminated
with organic and inorganic wastes in which the most preferred technique is the use of engineered
bacterium species. One of the toxic substances is toluene which can be degraded by an engineered
endophytic bacterium. Endophytic bacterium equipped with the appropriate degradation pathway
not only protects its host plant against the phytotoxic effect of an environmental contaminant, but
also improves the overall degradation of the contaminant, resulting in its decreased
evapotranspiration to the environment iv
. Hydrogen peroxide, potassium permanganate in soil and
addition of some genes in transgenic plants increase the ability of absorbance, transportation and
degradation with in the plant for e.g. Nitroaeromatics explosives are phytotoxic, when bacterial
genes involved in degradation of the nitroaromatics were expressed in plants; the plants became
more tolerant of the pollutant and could more readily remove it. Another toxic compound is arsenic

3. cause liver, lung, kidney, and bladder cancers ii
, and has therefore received a lot of attention now
adays which can be treated by growing hyper accumulating fern plant Pteris vittata can tolerate
1,500 parts per million (ppm) of arsenic in soilvii
. Plants may play a vital role in metal removal
through absorption, cation exchange, filtration, and chemical changes through the root for e.g.
Typha domingensis is highly salt-tolerant. The translocation factor indicates the efficiency of the
plant in translocating the accumulated metal from its roots to shoots viii
. Microbial populations such
as genera of bacterium and fungus play a very important role in phytoremediation because they are
involved in the petroleum degradation; Pseudomonas fluorescens, P. aeruginosa, Bacillus subtilis,
Bacillus sp., Alcaligenes sp., Acinetobacter lwoffi, Flavobacterium sp.,Micrococcus roseus, and
Corynebacterium sp. Other bacterial genera, namely, Gordonia, Brevibacterium, Aeromicrobium,
Dietzia, Burkholderia, and Mycobacterium isolated from petroleum contaminated soil proved to be
the potential organisms for hydrocarbon degradation. Fungal genera, namely, Amorphoteca,
Neosartorya, Talaromyces, and Graphium and yeast genera, namely, Candida, Yarrowia, and Pichia
were isolated from petroleum contaminated soil were involved in its degradationix
. Other important
materials include chelants which can desorb toxic metals from soil solid phases by forming strong
water-soluble complexes, which can be removed from the soil by plants through enhanced
phytoextraction or by using soil washing techniquesx
.
Conclusion:
As the industries are increasing, chemical dumps leads to the soil toxicity which can cause severe
damage to the animals, plants as well as human beings. In order to overcome these contaminants
we have variety of techniques in which the most promising technique includes phytoremediation
through genetically engineered endophytic bacteria.
Future Aspects:
Unlike organic pollutants, heavy metals cannot be degraded easily and can immobilize only which
may also cause loss of biological activity in soil, biotechnology may help us in this regard by adding
particular genes of metal eating bacteria for e.g. Geobacter metallireducens synthesizing enzymes
that may degrade metals in plant friendly environment. Another technique is the use of Shewanella
bacteria found in deep sea, is discovered of eating toxic waste and producing electricity, genes of
such bacteria can be added in the soil bacteria in order to save the plants and other organisms from
toxicity of excess metals.

4. References:
i
Cheng, S. (2003). Heavy Metal Pollution in China: Origin, Pattern and Control. Chinese Journals, 192-197.
ii
Doty, S. L. (2008). Enhancing phytoremediation through the use of transgenics and endophytes. New Phytologist,
Vol. 179, 318–333
iii
JING Yan-de†1, 2. H.-l.-e. (July 31, 2006). Role of rhizobacteria in phytoremediation of heavy metal contaminted
soils. Journal of Zhejiang University SCIENCE B, 192-202.
iv
Tanja Barac1, S. T. (MAY 2004). Engineered endophytic bacteria improve phytoremediation of water-soluble,
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v
Jose´-Miguel Barea*, M. J.-A. (July 2005). Microbial co-operation in the rhizosphere. Journal of Experimental
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vi
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vii
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viii
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