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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
University of South Carolina Arnold School of Public Health, 800 Sumter Street, Columbia, South Carolina 29208, USA US Army Engineer Research Development Center, Cold Regions Research and Engineering Laboratory, 72 Lyme Road, Hanover, New Hampshire 03755-1290, USA
2 1

The use of plants and bacterial to clean up environmental pollutants has gained momentum in past years. A limitation to phytoremediation of solvents has been toxicity of the compounds to plants, and the uncertainty as to the fate of many of the compounds. In a recent study, engineered endophytes have been shown to increase plant tolerance to toluene, and to decrease the transpiration of toluene to the atmosphere. This type of work has the potential to increase the use of phytoremediation by decreasing toxicity and increasing degradation of toxins.

degradation of recalcitrant compounds in the soil. Plants can be useful in many ways for this type of system; they can make contaminants in soil more bioavailable by releasing low molecular-weight organic acids [4], root release of carbon and nitrogen compounds can nourish microbes in the rhizosphere, and by releasing exudates that can be not only plant specific but also plant growthstage specific, plants can enhance degradation of soil contaminants by inducing biochemical pathways within bacteria [5].

Introduction The associations of endophytic organisms with their hosts are varied and complex and we are only starting to understand these interactions. Bacterial and fungal symbionts exist across all areas of life, from the bacteria that colonize the human intestinal tract to the ancient cells that became the plastids in plants cells, and the mitochondria that are present in almost all cells. Rhizobium and other bacteria colonize root nodules and form a symbiotic relationship with a plant, whereby the plant protects and supplies nutrients to the bacteria, and the bacterium makes nitrogen available to the plant [1]. However, we now know that plant endophytes go beyond the well-studied root nodules and exist within the leaf, root and vascular tissue of the plant [2,3]. Plant endophytes have been studied since the 1940s, with much research done on fungi. Endophytes have a variety of interactions with the plants, ranging from being active pathogens, opportunist pathogens and bacteria that exist within the plant and gain some physical protection, to bacteria that actively interact with the host plant for the benefit of both organisms. Bioremediation, the use of biological agents to remediate contaminants in the environment, has many components; however it is generally regarded as the use of microorganisms to degrade toxins. Phytoremediation, the use of plants to remediate environmentally toxic compounds, is a rapidly expanding field. Owing to its ‘green’ approach, it is gaining a significant amount of public attention. A division of phytoremediation, rhizodegradation, is the use of plants to stimulate the microbial community near the root–soil interface to enhance the
Corresponding author: Newman, L.A. (newman2@gwm.sc.edu). Available online 2 December 2004
www.sciencedirect.com

Endophytes Recently, work has been done showing that plant endophytes might be partially responsible for the degradation of environmental toxins. Work at the University of Iowa has shown that a newly discovered organism Methylobacterium populum sp. nov., strain BJ001 [6], which exists as a plant endophyte, is involved in the degradation of energetic compounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro1,3,5-triazine (HMX) and hexahydro-1,3,5- trinitro-1,3,5triazine (RDX) [7]. Other work has suggested that not only do plant endophytes have a role in toxin degradation, but also that the presence of some toxins can affect the make-up of endophytic populations. Siciliano et al. [8] showed that the genes encoding catabolic pathways increased within the root endophyte population in response to the presence of a given pollutant. This increase was pollutant-concentration-dependent. However, they also noted that the increase was dependent on the type of pollutant, the gene being looked at, the plant being screened and the soils that the plants were grown in. However, not every microbe possesses the ability to degrade every toxic compound, and not every bacterium that has a degradative capacity toward a given contaminant has the ability to successfully thrive in the sites where the contamination is present. Because of this, there has been a great deal of work done to genetically engineer microbes that can survive in a given environment, and make them capable of degrading the contaminant present [9]. Unfortunately there are many instances in which survival was decreased by giving the bacteria the degradation capability. Also, the plasmid carrying the degradative genes is not necessarily stable, and the trait can be lost under other environmental selective pressures.

whereas uninoculated plants and plants inoculated with BU0072 were dead or dieing. Thus. (1998) Regulation of symbiotic root nodule development. 32. of course. Although these organisms have been transformed using naturally occurring bacterial traits for ‘gene swapping’ it will be up to the researchers to educate the public on the usefulness of this technology. and Kondorosi. including nervous system and kidney damage. Plants inoculated with VM1330 grew in hydroponic doses of toluene up to 1000mg/L without reduction in vigor. In this project. This approach would lessen the need for expensive sampling and analysis on heterogeneous sites. Toluene is toxic to plants at moderate levels. Acknowledgements The authors gratefully acknowledge support from the US Army Environmental Quality Basic Research Program. cepacia BU 0072 and B. and the genes involved in these pathways have been identified.sciencedirect. There are. toluene. cepacia G4 were also grown as controls. the pTOM plasmid from Burkholderia cepacia G4 was naturally inserted into the lupine endophyte B. only one bacterial line would need to be created. grant P42 ES04696. The second major change in the plants was the reduction in volume of toluene transpired. Plants inoculated with either BU0072 or G4 had reductions in growth by 500 mg/L. M. in developing this article. Will it be possible to engineer bacteria that are capable of degrading these more toxic compounds. cepacia BU0072 via bacterial conjugation [11]. Toluene can cause a multitude of health problems in humans. A major concern of phytoremediation systems working on volatile solvent contamination has been the fact that the plants can transpire these compounds through the leaf stomata or stem lenticels. Previous work by this group focused on isolating. obstacles to overcome before this technology can move toward application. might then be an effective measurement tool. The resulting bacterial strains were tested for the presence of the degradative genes by growing the transformants using toluene as the sole carbon source and by PCR of the toluene degrading genes.1 January 2005 7 Engineered endophytes Daniel van der Lelie of the Brookhaven National Laboratory in Long Island and his colleagues in Belgium have developed a novel idea for making better use of the activities of plants and bacteria for environmental remediation purposes. if strains are selected that can successfully colonize multiple plants. In addition. and decrease release of the compound to the atmosphere. References 1 Schultze. Toluene is one of the four components of BTEX (benzene. First. the movement of engineered genes in the environment would be greatly reduced. M. would be reduced. and will the production of metabolites potentially harm the plants? Finally. One line of the transformed bacteria (VM1330) was introduced onto surfacesterilized lupine seeds and allowed to recolonize the vascular tissue of the plants. Plants inoculated with VM1330 showed a 50–70% reduction in toluene release from the aerial portions of the plant. persistence of the engineered organism in plants is crucial. (ii) Gene expression within endophytes might be useful as a site-monitoring tool. identifying and characterizing the bacteria that exist as bacterial endophytes in plants that might be used for phytoremediation. Genet. (iii) Bacterial endophytes might function more effectively than bacteria added to soil would because of a process known as bioaugmentation. Plants without introduced bacteria and plants with introduced but nontransformed B. this technology shows great promise for increasing the number of sites where phytoremediation can be deployed by showing that it is possible to increase plant tolerance to a given toxin. and when inhaled in high levels can cause unconsciousness or death. In summary. Rev. such as that possible with quantitative PCR. 33–57 . whereas uninoculated controls showed growth reduction with as little as 100mg/L toluene. The plant provides a ready-made environment for endophytic bacteria so competition pressure against colonization of the desired organism. the toxicity of toluene to the plants decreased significantly.23 No. Trials will be necessary to determine if this advantage remains stable in field-grown plants. the concept of releasing engineered bacteria into the environment must be addressed. and assure the public that science has not created something harmful to the environment. As a test system this has shown great promise but there are other organic contaminants that have much greater risks for human and environmental exposure. ethylbenzene and xylenes) which is a common gasoline additive.com Conclusions This work has major implications for the genetic engineering of systems for the remediation of recalcitrant organic contaminants. The root colonizing G4 imparted some increased tolerance to toluene but plant mass was w50% less than the VM1330 inoculated plants. In their paper van der Lelie’s group has developed a method for genetically endowing a plant endophyte with the ability to degrade toluene [10].Update TRENDS in Biotechnology Vol. plants inoculated with VM1330 and dosed with 500mg/L toluene for two weeks were still vigorous. as often occurs in soils. Plants grown in the presence of toluene had two important characteristics imparted by the new endophyte. this decrease in transpired toluene could prove to be significant. Using plants as soil and groundwater samplers would yield both active and passive sampling characteristics at a low cost. generally well above the regulatory limits for toluene in ground water. the Department of Energy and the US-NIEHS. Specific gene expression within endophytes. (iv) If bacterial lines are carefully selected so that the strains are at a competitive disadvantage when not living as a plant endophyte. Bacterial pathways for toluene degradation are well known. (i) Genetic engineering of endophytic bacteria is easier than the genetic engineering of plants. Because phytoremediation projects can conceivably last decades. In greenhouse studies. It is hoped that these researchers will continue this work and expand on the opportunities recognized. www. Annu.

S. 67. Hexahydro-1. et al. as the contaminant. thus potentially allowing plant growth on sites contaminated with pollution levels above the normal phytotoxicity threshold. In that case the endophytic inoculum should be considered as a starter culture that allows the initial revegetation of the contaminated site owing to its activity. volatile.1016/j. 1579–1583 6 Van Aken. Sci.). T. M. Appl. there will be a selective advantage for the endophytic population possessing the appropriate degradation characteristics.5-Trinitro-1. et al.5-Tetrazocine by a 8 9 10 11 Phytosymbiotic Methylobacterium sp. Horizontal gene-transfer has been shown to play a key role in rapidly adapting a microbial community to an environmental stress factor. and Octahydro-1. nov.1016/j. 2004. (2002) Endophytic bacteria and their potential applications.5. Environ. B.tibtech. Environ. Building 463. Appl.11.3. it is also possible that other bacteria that are better adapted to the local conditions will gradually replace the original population of engineered endophytic bacteria. organic pollutants [1]. Universitaire Campus. Gebouw D.4. facultatively methylotrophic. (vdlelied@bnl. In Biotechnology (Volume 11b) (Klein. (2000) Genetically engineered microorganisms and bioremediation. F-M. USA Limburgs Universitair Centrum. B.E. et al. et al. 583–588 Shields. Crit.11. New York 11973-5000.1 January 2005 2 Zinniel. Technol. equipped with the appropriate degradation pathway. Obstacles to applying engineered endophytic bacteria Newman and Reynolds (this issue. 441–452. Available online 26 November 2004 www. a new aromatic degradtive plasmid from Burkolderia (Pseudomonas) cepacia G4. et al. Plant Sci. Microbiol. (2002) Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants. below-ground irrigation of the plants with contaminated groundwater can be used to maintain the appropriate selection pressure until the tree roots arrive at the level of the contaminated groundwater. can assist its host plant by degrading contaminants that are readily taken up by plants which then fail to degrade them to completion.D.gov). Environ.5Triazine. 1191–1196 7 Van Aken. et al. Environ. 54. Using poplar inoculated with engineered endophytic bacteria to remediate a contaminated groundwater plume.p 0 -DDE by Cucurbita pepo. Environ. B3590 Diepenbeek. (2004) Methylobacterium populum sp. As long as selection pressure is Corresponding author: van der Lelie. (2004) Engineered endophytic bacteia improve phytormediationof water-soluble.7-Tetranitro-1. 70.8 Update TRENDS in Biotechnology Vol. Syst. Microbiol.6-Trinitrotoluene. 22. (1995) TOM. pink-pigmented.23 No.B. Another issue to address is pathway selection for the degradation of the organic contaminant. pp. Rev. Evol. (2002) Root turnover: an important source of microbial substrates in rhizosphere remediation of recalcitrant contaminants. Biotechnology.com present. Biology Department. 68. Wiley Barac. et al. In addition an active population of endophytic bacteria. D. J. 21. et al.3. J. we demonstrated that compared with controls the plants inoculated with an endophytic Burkholderia cepacia strain able to degrade toluene could grow in the presence of elevated levels of toluene.C.. 4368–4373 5 Leigh. 2469–2475 Meen. 2198–2208 3 Lodewyckx. M. methane-utilizing bacterium isolated from poplar trees (Populus deltoides x nigra DN34).a novel aerobic. D. Using yellow lupine as the model plant and toluene. Associated with Poplar Tissues (Populus deltoides!nigra DN34). Technol. S. Microbiol. Tanja Barac2. 583–606 4 White. 36. 1352–1356 0167-7799/$ . 37. Maintaining the selection pressure is no guarantee that the endophytic inoculum will become an integrated part of the endophytic community. C. This demonstrated that engineered endophytic bacteria could be used to decrease the phytotoxicity of organic contaminants. Finally. The first issue is the stability of the degradation capabilities within the endophytic community. We therefore speculate that horizontal gene-transfer could play an important role in adapting the endogenous endophytic community: rather than integrating a new bacterium in a stable community.3. 508–517 Siciliano. Environ. Appl. et al.2004. Biotechnol. Belgium Recently we published a concept for using engineered endophytic bacteria to improve phytoremediation of water soluble. All rights reserved.see front matter Q 2004 Elsevier Ltd. Safiyh Taghavi1 and Jaco Vangronsveld2 1 2 Brookhaven National Laboratory. organic pollutants. et al. 61. (2001) Selection of specific endophytic bacterial genotypes by plants in response to soil contamination. J. a common solvent as well as a gasoline additive. (2004) Biodegradation of Nitro-Substituted Explosives 2. (2003) Subspecies-level variation in the phytoextraction of weathered p.3.sciencedirect. Microbiol. ed.010 Research Focus Response Response to Newman: New uses of endophytic bacteria to improve phytoremediation Daniel van der Lelie1. Nat. The general idea . Microbiol. Sci. This implies that during the construction of the remediation site appropriate measurers should be taken to maintain the selection pressure on the trees inoculated with endophytic community to maintain its degradation potential.tibtech. the degradation pathway is transferred among the members of the community. Appl. resulting in the accumulation of intermediates or the release of these contaminants through the leaf stomata or stem lenticels. volatile. doi:10. doi:10. and that the amount of transpired toluene decreased significantly. Upton. Int.010) correctly point out several obstacles that should be overcome before engineered endophytes can be applied in field-scale phytoremediation projects.