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6 Update TRENDS in Biotechnology Vol.23 No.

1 January 2005

Bacteria and phytoremediation: new uses for


endophytic bacteria in plants
Lee A. Newman1 and Charles M. Reynolds2
1
University of South Carolina Arnold School of Public Health, 800 Sumter Street, Columbia, South Carolina 29208, USA
2
US Army Engineer Research Development Center, Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover,
New Hampshire 03755-1290, USA

The use of plants and bacterial to clean up environmen- degradation of recalcitrant compounds in the soil. Plants
tal pollutants has gained momentum in past years. A can be useful in many ways for this type of system; they
limitation to phytoremediation of solvents has been can make contaminants in soil more bioavailable by
toxicity of the compounds to plants, and the uncertainty releasing low molecular-weight organic acids [4], root
as to the fate of many of the compounds. In a recent release of carbon and nitrogen compounds can nourish
study, engineered endophytes have been shown to microbes in the rhizosphere, and by releasing exudates
increase plant tolerance to toluene, and to decrease that can be not only plant specific but also plant growth-
the transpiration of toluene to the atmosphere. This stage specific, plants can enhance degradation of soil
type of work has the potential to increase the use of contaminants by inducing biochemical pathways within
phytoremediation by decreasing toxicity and increasing bacteria [5].
degradation of toxins.

Endophytes
Introduction Recently, work has been done showing that plant
The associations of endophytic organisms with their hosts endophytes might be partially responsible for the
are varied and complex and we are only starting to degradation of environmental toxins. Work at the Uni-
understand these interactions. Bacterial and fungal versity of Iowa has shown that a newly discovered
symbionts exist across all areas of life, from the bacteria organism Methylobacterium populum sp. nov., strain
that colonize the human intestinal tract to the ancient BJ001 [6], which exists as a plant endophyte, is
cells that became the plastids in plants cells, and the involved in the degradation of energetic compounds such
mitochondria that are present in almost all cells. as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-
Rhizobium and other bacteria colonize root nodules and 1,3,5-triazine (HMX) and hexahydro-1,3,5- trinitro-1,3,5-
form a symbiotic relationship with a plant, whereby the triazine (RDX) [7].
plant protects and supplies nutrients to the bacteria, and Other work has suggested that not only do plant
the bacterium makes nitrogen available to the plant [1]. endophytes have a role in toxin degradation, but also
However, we now know that plant endophytes go beyond that the presence of some toxins can affect the make-up of
the well-studied root nodules and exist within the leaf, endophytic populations. Siciliano et al. [8] showed that the
root and vascular tissue of the plant [2,3]. Plant genes encoding catabolic pathways increased within the
endophytes have been studied since the 1940s, with root endophyte population in response to the presence of a
much research done on fungi. Endophytes have a variety given pollutant. This increase was pollutant-concen-
of interactions with the plants, ranging from being active tration-dependent. However, they also noted that the
pathogens, opportunist pathogens and bacteria that exist increase was dependent on the type of pollutant, the gene
within the plant and gain some physical protection, to being looked at, the plant being screened and the soils that
bacteria that actively interact with the host plant for the the plants were grown in.
benefit of both organisms. However, not every microbe possesses the ability to
Bioremediation, the use of biological agents to remedi- degrade every toxic compound, and not every bacterium
ate contaminants in the environment, has many com- that has a degradative capacity toward a given contami-
ponents; however it is generally regarded as the use of nant has the ability to successfully thrive in the sites
microorganisms to degrade toxins. Phytoremediation, the where the contamination is present. Because of this, there
use of plants to remediate environmentally toxic com- has been a great deal of work done to genetically engineer
pounds, is a rapidly expanding field. Owing to its ‘green’ microbes that can survive in a given environment, and
approach, it is gaining a significant amount of public make them capable of degrading the contaminant present
attention. A division of phytoremediation, rhizodegrada- [9]. Unfortunately there are many instances in which
tion, is the use of plants to stimulate the microbial survival was decreased by giving the bacteria the
community near the root–soil interface to enhance the degradation capability. Also, the plasmid carrying the
Corresponding author: Newman, L.A. (newman2@gwm.sc.edu).
degradative genes is not necessarily stable, and the trait
Available online 2 December 2004 can be lost under other environmental selective pressures.
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Update TRENDS in Biotechnology Vol.23 No.1 January 2005 7

Engineered endophytes Conclusions


Daniel van der Lelie of the Brookhaven National Labora- This work has major implications for the genetic engin-
tory in Long Island and his colleagues in Belgium have eering of systems for the remediation of recalcitrant
developed a novel idea for making better use of the organic contaminants.
activities of plants and bacteria for environmental (i) Genetic engineering of endophytic bacteria is easier
remediation purposes. Previous work by this group than the genetic engineering of plants. In addition, if
focused on isolating, identifying and characterizing the strains are selected that can successfully colonize multiple
bacteria that exist as bacterial endophytes in plants that plants, only one bacterial line would need to be created.
might be used for phytoremediation. In their paper van (ii) Gene expression within endophytes might be useful
der Lelie’s group has developed a method for genetically as a site-monitoring tool. Using plants as soil and
endowing a plant endophyte with the ability to degrade groundwater samplers would yield both active and passive
toluene [10]. sampling characteristics at a low cost. Specific gene
Toluene is one of the four components of BTEX expression within endophytes, such as that possible with
(benzene, toluene, ethylbenzene and xylenes) which is a quantitative PCR, might then be an effective measure-
common gasoline additive. Toluene can cause a multitude ment tool. This approach would lessen the need for
of health problems in humans, including nervous system expensive sampling and analysis on heterogeneous sites.
and kidney damage, and when inhaled in high levels can (iii) Bacterial endophytes might function more effec-
cause unconsciousness or death. Toluene is toxic to plants tively than bacteria added to soil would because of a
at moderate levels; generally well above the regulatory process known as bioaugmentation. The plant provides a
limits for toluene in ground water. Bacterial pathways for ready-made environment for endophytic bacteria so
toluene degradation are well known, and the genes competition pressure against colonization of the desired
involved in these pathways have been identified. In this organism, as often occurs in soils, would be reduced.
project, the pTOM plasmid from Burkholderia cepacia G4 (iv) If bacterial lines are carefully selected so that the
was naturally inserted into the lupine endophyte strains are at a competitive disadvantage when not living
B. cepacia BU0072 via bacterial conjugation [11]. The as a plant endophyte, the movement of engineered genes
in the environment would be greatly reduced.
resulting bacterial strains were tested for the presence of
There are, of course, obstacles to overcome before this
the degradative genes by growing the transformants
technology can move toward application. Trials will be
using toluene as the sole carbon source and by PCR of
necessary to determine if this advantage remains stable in
the toluene degrading genes. One line of the trans-
field-grown plants. Because phytoremediation projects
formed bacteria (VM1330) was introduced onto surface-
can conceivably last decades, persistence of the engin-
sterilized lupine seeds and allowed to recolonize the
eered organism in plants is crucial. As a test system this
vascular tissue of the plants. Plants without intro-
has shown great promise but there are other organic
duced bacteria and plants with introduced but non-
contaminants that have much greater risks for human
transformed B. cepacia BU 0072 and B. cepacia G4
and environmental exposure. Will it be possible to
were also grown as controls.
engineer bacteria that are capable of degrading these
Plants grown in the presence of toluene had two more toxic compounds, and will the production of
important characteristics imparted by the new endophyte. metabolites potentially harm the plants?
First, the toxicity of toluene to the plants decreased Finally, the concept of releasing engineered bacteria
significantly. Plants inoculated with VM1330 grew in into the environment must be addressed. Although these
hydroponic doses of toluene up to 1000mg/L without organisms have been transformed using naturally occur-
reduction in vigor, whereas uninoculated controls showed ring bacterial traits for ‘gene swapping’ it will be up to the
growth reduction with as little as 100mg/L toluene. Plants researchers to educate the public on the usefulness of this
inoculated with either BU0072 or G4 had reductions in technology, and assure the public that science has not
growth by 500 mg/L. In greenhouse studies, plants created something harmful to the environment.
inoculated with VM1330 and dosed with 500mg/L toluene In summary, this technology shows great promise for
for two weeks were still vigorous, whereas uninoculated increasing the number of sites where phytoremediation
plants and plants inoculated with BU0072 were dead or can be deployed by showing that it is possible to increase
dieing. The root colonizing G4 imparted some increased plant tolerance to a given toxin, and decrease release of
tolerance to toluene but plant mass was w50% less than the compound to the atmosphere. It is hoped that these
the VM1330 inoculated plants. researchers will continue this work and expand on the
The second major change in the plants was the opportunities recognized.
reduction in volume of toluene transpired. Plants
inoculated with VM1330 showed a 50–70% reduction Acknowledgements
in toluene release from the aerial portions of the The authors gratefully acknowledge support from the US Army
plant. A major concern of phytoremediation systems Environmental Quality Basic Research Program, the Department of
Energy and the US-NIEHS, grant P42 ES04696, in developing this
working on volatile solvent contamination has been article.
the fact that the plants can transpire these com-
pounds through the leaf stomata or stem lenticels. References
Thus, this decrease in transpired toluene could prove 1 Schultze, M. and Kondorosi, M. (1998) Regulation of symbiotic root
to be significant. nodule development. Annu. Rev. Genet. 32, 33–57
www.sciencedirect.com
8 Update TRENDS in Biotechnology Vol.23 No.1 January 2005

2 Zinniel, D.E. et al. (2002) Isolation and characterization of endophytic Phytosymbiotic Methylobacterium sp. Associated with Poplar
colonizing bacteria from agronomic crops and prairie plants. Appl. Tissues (Populus deltoides!nigra DN34). Appl. Environ. Micro-
Environ. Microbiol. 68, 2198–2208 biol. 70, 508–517
3 Lodewyckx, C. et al. (2002) Endophytic bacteria and their potential 8 Siciliano, S.D. et al. (2001) Selection of specific endophytic bacterial
applications. Crit. Rev. Plant Sci. 21, 583–606 genotypes by plants in response to soil contamination. Appl. Environ.
4 White, J.C. et al. (2003) Subspecies-level variation in the phytoextrac- Microbiol. 67, 2469–2475
tion of weathered p,p 0 -DDE by Cucurbita pepo. Environ. Sci. Technol. 9 Meen, F-M. et al. (2000) Genetically engineered microorganisms and
37, 4368–4373 bioremediation. In Biotechnology (Volume 11b) (Klein, J. ed.),
5 Leigh, M.B. et al. (2002) Root turnover: an important source of Biotechnology, pp. 441–452, Wiley
microbial substrates in rhizosphere remediation of recalcitrant 10 Barac, T. et al. (2004) Engineered endophytic bacteia improve
contaminants. Environ. Sci. Technol. 36, 1579–1583 phytormediationof water-soluble, volatile, organic pollutants. Nat.
6 Van Aken, B. et al. (2004) Methylobacterium populum sp. nov.,a novel Biotechnol. 22, 583–588
aerobic, pink-pigmented, facultatively methylotrophic, methane-uti- 11 Shields, M.S. et al. (1995) TOM, a new aromatic degradtive plasmid
lizing bacterium isolated from poplar trees (Populus deltoides x nigra from Burkolderia (Pseudomonas) cepacia G4. Appl. Environ.
DN34). Int. J. Syst. Evol. Microbiol. 54, 1191–1196 Microbiol. 61, 1352–1356
7 Van Aken, B. et al. (2004) Biodegradation of Nitro-Substituted
Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5- 0167-7799/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a doi:10.1016/j.tibtech.2004.11.010

Research Focus Response

Response to Newman: New uses of endophytic


bacteria to improve phytoremediation
Daniel van der Lelie1, Tanja Barac2, Safiyh Taghavi1 and Jaco Vangronsveld2
1
Brookhaven National Laboratory, Biology Department, Building 463, Upton, New York 11973-5000, USA
2
Limburgs Universitair Centrum, Universitaire Campus, Gebouw D, B3590 Diepenbeek, Belgium

Recently we published a concept for using engineered present, there will be a selective advantage for the
endophytic bacteria to improve phytoremediation of water endophytic population possessing the appropriate degra-
soluble, volatile, organic pollutants [1]. Using yellow dation characteristics. This implies that during the
lupine as the model plant and toluene, a common solvent construction of the remediation site appropriate mea-
as well as a gasoline additive, as the contaminant, we surers should be taken to maintain the selection pressure
demonstrated that compared with controls the plants on the trees inoculated with endophytic community to
inoculated with an endophytic Burkholderia cepacia maintain its degradation potential. Using poplar inocu-
strain able to degrade toluene could grow in the presence lated with engineered endophytic bacteria to remediate a
of elevated levels of toluene, and that the amount of contaminated groundwater plume, below-ground irriga-
transpired toluene decreased significantly. This demon- tion of the plants with contaminated groundwater can be
strated that engineered endophytic bacteria could be used used to maintain the appropriate selection pressure until
to decrease the phytotoxicity of organic contaminants, the tree roots arrive at the level of the contaminated
thus potentially allowing plant growth on sites contamin- groundwater.
ated with pollution levels above the normal phytotoxicity Maintaining the selection pressure is no guarantee that
threshold. In addition an active population of endophytic the endophytic inoculum will become an integrated part of
bacteria, equipped with the appropriate degradation the endophytic community. Horizontal gene-transfer has
pathway, can assist its host plant by degrading contamin-
been shown to play a key role in rapidly adapting a
ants that are readily taken up by plants which then fail to
microbial community to an environmental stress factor.
degrade them to completion, resulting in the accumulation
We therefore speculate that horizontal gene-transfer could
of intermediates or the release of these contaminants
play an important role in adapting the endogenous endo-
through the leaf stomata or stem lenticels.
phytic community: rather than integrating a new bacter-
ium in a stable community, the degradation pathway is
Obstacles to applying engineered endophytic bacteria transferred among the members of the community.
Newman and Reynolds (this issue, doi:10.1016/j.tibtech. Finally, it is also possible that other bacteria that are
2004.11.010) correctly point out several obstacles that better adapted to the local conditions will gradually
should be overcome before engineered endophytes can be replace the original population of engineered endophytic
applied in field-scale phytoremediation projects. The first bacteria. In that case the endophytic inoculum should be
issue is the stability of the degradation capabilities within considered as a starter culture that allows the initial
the endophytic community. As long as selection pressure is revegetation of the contaminated site owing to its activity.
Corresponding author: van der Lelie, D. (vdlelied@bnl.gov).
Another issue to address is pathway selection for the
Available online 26 November 2004 degradation of the organic contaminant. The general idea
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