Phytoremediation

1. Phytoremediation Microbial Endophytes Katie Fishburn and Jared Bodecker

2. Endophytes • Microbes that live in plant tissue, that increase plant biomass and resistance to pathogens • Of the 300,000 known species of plants, there is at least one plant-associated endophyte (Strobel G. et al) • Can remediate pollutants just as good as rhizospheric microbes, in some cases better • There are clear advantages of endophytes compared to the closely associated rhizospheric cousins

3. Known endophytes in PR

4. Endophyte vs. Rhizospheric • Population control is easier than soil microbes, due to lack of competition (protected inside plant tissue) • Degradation of pollutants will be localized to inside the plant, being contained from leaking in the soil and air environment. • Plant species-specific selection of necessary bacteria for a specific pollutant • Microbial sampling is easier. (plant tissue)

5. Applications endophyte-phytoremediation

6. Phytoremediation: Organics 2,4-D (herbicide) degradation • Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D • Control: high levels of 2,4-D remain • Inoculated plants: no 2,4-D remains in test soils • PAH tolerance in willow and poplar • Used also as carbon source by microbes • TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar • Mineralized 60% of RDX and HMX in 2 months 2,4-D (herbicide) degradation • Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D • Control: high levels of 2,4-D remain • Inoculated plants: no 2,4-D remains in test soils

7. 2, 4-D Degradation Root tissue Stem tissue Psuedomonasputidamini Tn5 (GFP)

8. Phytoremediation: Organics 2,4-D (herbicide) degradation • Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D • Control: high levels of 2,4-D remain • Inoculated plants: no 2,4-D remains in test soils • PAH tolerance in willow and poplar • Used also as carbon source by microbes • TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar • Mineralized 60% of RDX and HMX in 2 months • PAH tolerance in willow and poplar • Used also as carbon source by microbes

9. Poly Aromatic Hydrocarbons Backdrop picture courtesy of NASA

10. Phytoremediation: Organics 2,4-D (herbicide) degradation • Isolates from poplar inoculated into pea plants; planted in soils with 13 mg of 2,4-D • Control: high levels of 2,4-D remain • Inoculated plants: no 2,4-D remains in test soils • PAH tolerance in willow and poplar • Used also as carbon source by microbes • TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar • Mineralized 60% of RDX and HMX in 2 months • TNT, RDX, HMX degradation by methylobacterium found in hybrid poplar • Mineralized 60% of RDX and HMX in 2 months

11. Methylobacterium spp. Strain BJ001

12. Phytoremediation: Inorganics

13. Brassicachinensis Brassicachinensis • Mucor isolate • Cd and Pb tolerance • Cd and Pb more water soluble; more bioavailable

14. Thlaspigoesingense • Thlaspigoesingense • Methylobacterium and Shpingomonas • Ni accumulation and tolerancesiderophores and ethylene relieve

15. Plant Yield and Growth Promotion • Enhanced nutrient/mineral availability and uptake • Phosphate, nitrogen • Phosphate solubilization • Siderophore production • Minerals made more bioavailable; solubilized by siderophore • Osmotic adjustment, stomatal regulation

16. Plant Health and Protection • Lessen/prevent effects of pathogens on plants • Fungal, bacterial, viral diseases less harmful after inoculation of endophytes • Endophytes trigger induced systemic resistance (ISR) • Immune-like response by plants

17. Industrial/Medical Applications • Pseudomonas, Burkholderia, Bacillus • Antibiotics, anti-cancer compounds, antifungal, antiviral, insecticidal, immunosuppressant agents

18. Making a (6) Million $ Endophyte • Engineering plant-associated microbes • Horizontal Gene Transfer • Improving already efficient phytoremediation • By-products of degradation can even more toxic when released into the air • When in planta, you have natural reduction of volatile toxins

19. Improving Toluene Degradation • Normal plant degradation of toluene creates toxic by-products that are volatized into the environment. • BurkholderiacepaciaG4 (rhizoshperic) with toluene-degrading qualities from pTOM plasmid (toluene-ortho-monooxygenase) • B. cepaciaVM1330 (endophyte), with the pMON plasmid

20. Enhanced Toluene-resistance Growth Index (Fresh Weight) Control- Yellow Lupine (no inoculate) BU0072- YL endophyte strain G4- YL rhizospheric strain (pTOM) VM1330- YL transformed endophyte strain (pTOM) Total Weight (grams)

21. Reduced Toluene-volatization Improved degradation in VM1330 and Yellow Lupine. Which lowers phytotoxicity and evapotranspiration Sub-phytotoxic levels used in experiments (100mg/L)

22. Lupine endophytes in Poplar Decreased Toluene-volatization Control- Poplar (no innoculate) VM1466- P., endophyte (pTOM) BU61- P., rhizospheric (pTOM)

23. Horizontal Gene Transfer • Opposite of vertical transfer (parental or ancestral) • Genetic material is transferred from another organism to another organism without being an offspring • Common routes of transfer • Transformation: Uptake of endogenous DNA (nature/lab) • Transduction: Delivery via vector (bacteriophage) • Conjugation: a physical connection between two organisms delivers DNA from one to the other

25. References Kieran J. Germaine, Xuemei Liu, Guiomar Garcia Cabellos, Jill P. Hogan, David Ryan & David N. Dowling. (2006). Bacterialendophyte-enhancedphytoremediationoftheorganochlorine herbicide 2,4 - dichlorophenoxyacetic acid. FEMS Benoit Van Aken,* Jong Moon Yoon, and Jerald L. Schnoor. (2003) Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a PhytosymbioticMethylobacterium sp. Associated with Poplar Tissues (Populusdeltoides􏰈nigra DN34) APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 508–517 Vol. 70, No. 1 ZujunDenga,b, LixiangCaoc, HaiweiHuanga, XinyuJianga, WenfengWangc, Yang Shia, RenduoZhanga,∗ (2010) Characterization of Cd- and Pb-resistant fungal endophyteMucor sp. CBRF59 isolated from rapes (Brassicachinensis) in a metal-contaminated soil. Journal of Hazardous Materials Rughia Idris,1 Radoslava Trifonova,1 Markus Puschenreiter,2 Walter W. Wenzel,2 and Angela Sessitsch1*. (2004) Bacterial Communities Associated with Flowering Plants of the Ni HyperaccumulatorThlaspigoesingense. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2667–2677  Robert P. Ryan1, Kieran Germaine2, Ashley Franks1, David J. Ryan2 & David N. Dowling2 (2007) Bacterial endophytes: recent developments and applications. FEMS  Germaine K, Keogh E, Borremans B et al. (2004) Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol Ecol 48: 109–118  Germaine K, Liu X, Cabellos G, Hogan J, Ryan D & Dowling DN (2006) Bacterial endophyte-enhanced phyto-remediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid. FEMS Microbiol Ecol 57: 302–310.  Miller CM, Miller RV, Garton-Kenny D, Redgrave B, Sears J, Condron MM, Teplow DB & Strobel GA (1998) Ecomycins, unique antimycotics from Pseudomonas viridiflava. J ApplMicrobiol 84: 937–944. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M & van derLelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21: 583–606. Lodewyckx C, Taghavi S, Mergeay M, Vangronsveld J, Clijsters H & van derLelie D (2001) The effect of recombinant heavy metal resistant endophytic bacteria in heavy metal uptake by their host plant. Int J Phytoremediation 3: 173–187. Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J & van derLelie D (2005) Horizontal gene transfer to endogenous endophytic bacteria from poplar improved phyto-remediation of toluene. Appl Environ Microbiol 71: 8500–8505. Strobel G, Daisy B, Castillo U & Harper J (2004) Natural products from endophytic microorganisms. J Nat Prod 67: 257–268.  Van Aken B, Peres C, Doty S, Yoon J & Schnoor J (2004) Methylobacteriumpopuli sp. nov., a novel aerobic, pink- pigmented, facultativelymethylotrophic, methane-ultilising bacterium isolated from poplar trees (Populusdeltoidesxnigra DN34). EvolMicrobiol 54: 1191–1196.  Xi C, Lambrecht M, Vanderleyden J & Michiels J (1999) Bi-functional gfp-and gusA-containing mini-Tn5 transposon derivatives for combined gene expression and bacterial localization studies. J Microbiol Methods 35: 85–92.