d'Haene, S.

Dowling, D.N.

Karlson, U.

Moore, E.R.B.

Trapp, Stefan

van der Lelie, D.

Vangronsveld, J.

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 20, 2017EndegradeEndophytic degrader bacteria for improving phytoremediation of organic xenobioticsKarlson, U.; Trapp, Stefan; Moore, E.R.B.; Vangronsveld, J.; Dowling, D.N.; d'Haene, S.; van der Lelie, D.Publication date:2001Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Karlson, U., Trapp, S., Moore, E. R. B., Vangronsveld, J., Dowling, D. N., d'Haene, S., & van der Lelie, D.(2001). Endegrade: Endophytic degrader bacteria for improving phytoremediation of organic xenobiotics. Postersession presented at Wastewater treatment and plants as a 'green liver': The Eyropean approach, experienceand trends", Larnaca, .F. Porteous1, L. Oeyen2, T. Barac2, S. Trapp3, B. Borremans4, D. van der Lelie5, J.Vangronsveld2, D. Ryan6, K. Germain6, D. Dowling6, S. D’Haene7, G. de Becker7,C.D. Campbell1, E.R.B. Moore1 and U. Karlson8.1. The Macaulay Institute, Craigiebuckler, Aberdeen, Scotland, UK. (E-mail: f.porteous@macaulay.ac.uk) 2. Limburgs Universitaire Centrum, Diepenbeek, Belgium.3.Institute of Science & Engineering, Technical  University of Denemark, Lyngby, Denmark. 4. Vlaamse Instelling voor Technologisch Onderzooek (Vito), Boeretang, Belgium. 5.Brookhaven National Laboratory, Upton, USA. 6. Institute of Technology Carlow, Carlow, Ireland.  7. DEC NV, Zwijndrecht, Belgium.  8. National Environmental Research Institute, Roskilde, Denmark.IntroductionPhytoremediation is the use of plants to remediate contaminated soils. Ideally, plants should have deep roots, be perennial, and use large quantities of water. Poplar and Willow trees are the most commonly used. Some pollutants, however,are not metabolised by plants and, thus, accumulate and cause phytotoxicity. Phytoremediation, alone, may not be a suitable technology for all contaminated soils.Bacterial soil bioremediation can be reduced significantly in time by the ability of the indigenous bacterial population to overcome any introduced species.“Endegrade” is an EU funded project which is attempting to utilise endophytes(bacteria living within plants) to breakdown pollutants as they are translocated through the plant, thereby reducing phytotoxicity and volatilisation(Patent :van der Lelie et al, 2001).“ENDEGRADE”Endophytic bacteria for improving  phytoremediationAims :Little is known about the bacteria that live within plants, and until recently it was thought that all were pathogenic (Lodewyckx et al, 2002). In order to successfully identify and re-introduce bacteria that can survive in plants, we need to understand the community dynamics of endophytes.This study aimed to isolate bacteria from within the xylem sap of Willow and Poplar trees, culture and identify them by16SrDNA sequencing and ascertain the community structure of the culturable microbial population.Dominant, culturable bacteria are being screened to avoid pathogens, identify species that can degrade problem pollutants, or to be used to introducedegradation genes, such as the pTOM plasmid.Hypothesis:Different tree species will have different diversity of bacterial endophytes, and these will be specific to the plant compartment sampled.Endophytic bacteria will be able to naturally degrade pollutants as they pass through the plant or can be engineered to enhance their ability to do so.Figure 1. Installation of a phytoremediation barrier forthe treatment of a BTEX-TCE plume. Phytoremediationwill be done using hybrid poplar. Phytoremediation willbe improved using modified endophytic bacteria.Compound Fate in plant Reference(toxic, build up or volatile)Phenols Toxic Pfleeger et al., 1991Chloro-phenols Toxic Pfleeger et al., 1991TNT Toxic, degraded to amino-dinitrotoluene Thomson et al., 1998Amino-dinitrotoluene Rather persistent, toxic Thomson et al., 1998MTBE Volatile Trapp et al., 1994BTEX Volatile Trapp et al., 1994TCE Volatile, build up of trichloroacetate Trapp et al., 1994PER Volatile Trapp et al., 1994Table 1. List of pollutants not metabolised sufficiently in plantae.Figure 2- Endophytic bacteria isolated from Poplarand Willow trees at Figure 1 phytoremediation site,classified by 16S rDNA sequencing to genus level.Figure 3- Classification of the Pseudomonas speciesfound within the endophytic bacteria isolated fromPoplar and Willow.Figure 4 - Number of isolates from Willow, in relationto the location within tree.Figure 5 - Number of isolates from Poplar  var.Hazendans,  in relation to the location within tree.Figure 6 - Number of isolates from Poplar  var.Hoogvorst,  in relation to the location within tree.Discussion:A wide range of  Eubacteria were found in all tree types, including a few pathogenic  strains, with more diversity than was expected.  The majority ofthe isolates were  Pseudomonas species, which are known degraders of many potential target pollutants (Table 1). Importantly, this genus was presentthroughout all compartments of the plants studies.  There were however distinct differences  in the diversity of endophytes between different planttissues (Fig 4-6).Conclusions & future work:A similar study on the non-culturable endophytic community is on-going to establish whether the information gained from studies on isolates is representative of the total bacterial population within the plant.A number of isolates have been found to have the natural ability to degrade toluene, and to have a natural resistance to a range of antibiotics. Burkholderia cepacia has also had the pTOM plasmid introduced successfully, which encodes for toluene degradation, and re-inoculation studies are continuing. At present, studies are ongoing to introduce GFP, rfp, lux and heavy metal resistance markers into strains possessing natural degradationabilities.We are currently assessing the seasonal changes in the endophytic communities of Willow and Poplar, at the inter- and intra-species levels. As Figures4-6 above shows, a considerable amount of compartmentalisation occurs within the plants, with different bacterial species being found within the same compartment of the different tree variety or species.Endegrade provides a real possibility to enable phytoremediation to tackle a greater range of polluted environments, while enhancing its socialReferences:1. Pfleeger, T. (1991) A short-term bioassay for whole plant toxicity. In: Plants for Toxicity assessment, 2nd Vol, American Society for Testing & Materials STP 1115.2.Thompson, P.L., Ramer,L.A., Guffey, A.P., and Schnoor, J.L. (1998) Decreased transpiration of poplar trees exposed to 2, 4-dinitrotoluene, Env. Science & Technology17, p902-906.2. Trapp, S., McFarlane, J.C., and Matthias, M. (1994) Model for uptake of xenobiotics into plants-validation with bromacil experiments, Env. Science & Technology  13,p413-422,4. Lodewyckx, C., Vangronsveld, J., Porteous, F., Moore, E.R.B., Taghavi, S., and van der Lelie, D. (2002) Endophytic bacteria and their potential applications. Critical reviewsin Plant Sciences (in press).Patent registered:D. Van der Lelie, S. D'Haene, D.N. Dowling, U. Karlson, E.R.B. Moore, S. Taghavi,S.A.J. Trapp, J. Vangronsveld. "METHOD FOR IMPROVING PHYTOREMEDIATIONTREATMENT OF A CONTAMINATED MEDIUM". Application nr. 60/291,344.U.S. Patent office, 16 May, 2001.Acknowledgements:Funding was obtained from the EU, contract no : QLK3-2000-0164EnterobacterBacillus (7%)AcinetobacterFlavimonasMycetocola Sphingomonas (6%)XylophilusHerbaspirillumDuganellaBurkholderia (6%)Delftia (6%)KlebsiellaPantoeaPseudomonas(42%)Xanthomonas (7%)MoraxellaArthrobacter (5%)AgreiaAerococcusPaenibacillusCurtobacterium MicrococcusP.azotoformans2%P. fulva13%P. beteli2% P. jessenii7%P.plecoglossida19%P. rhodesiae2%P. tolaasii13%P. frederiks13%P. veronii11%P. migulae2%P. mandelii4%P. fragi2%P. putida2%P. graminis4%P. stutzeri4%BacillusPseudomonasAerococcusRhizobiumSphingomonasMicrococcusPantoeaPaenibacillusXanthomonasMycetocolaXanthomonasAgreiaPseudomonas0 2 4 6 8 10 12No of isolates  SHOOT  LEAFAEROCOCCUSBACILLUSPSEUDOMONASMYCETOCOLASPHINGOMONAS MICROCOCCUSXANTHOMONASPAENIBACILLUSRHIZOBIUMPANTOEAAGREIABacillusAcinetobacterArthrobacterEnterobacterFlavimonasHerbaspirillumKlebsiellaBacillusArthrobacterPaenibacillusXanthomonasXylophilusBlastomonasPseudomonasSphingomonasPseudomonasPseudomonas0 2 4 6 8 10 12 14No of isolatesPseudomonasPseudomonasBacillusSphingomonasMoraxellaDuganellaXanthomonasAgreiaPseudomonasEnterobacterCurtobacteriumBurkholderia0 2 4 6 8 10No of isolatesROOT SHOOTLEAFBURKHOLDERIA BACILLUSPSEUDOMONASCURTOBACTERIUMSPHINGOMONASMORAXELLADUGANELLAXANTHOMONAS AGREIAENTEROBACTERROOT SHOOTLEAFHERBASPIRILLUMBACILLUSPSEUDOMONASENTEROBACTERSPHINGOMONASACINETOBACTERKLEBSIELLAXANTHOMONASARTHROBACTERFLAVIMONASPAENIBACILLUSXYLOPHILUS BLASTOMONAS

Endegrade:Endophytic degrader bacteria for improving phytoremediation of organic xenobiotics

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Endegrade:Endophytic degrader bacteria for improving phytoremediation of organic xenobiotics

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