Phytoremediation of BTEX using poplar trees

Barac, T., et al (2009) Hydraulic Containment of a BTEX Plume Using Poplars. International Journal of Phytoremediation, 11:416-424.

CFR 521gJanousek, Griffith, Tomlinson

Phytoremediation of BTEX using poplar trees Location: Genk, BelgiumSite: car factoryContamination: groundwaterContaminents: organic solvents (BTEX), fuel, heavy metals

Ford Motor Company car factory in Genk, Belgium

First steps:

1. remove source of contamination2. treat core area with-in plume3. contain migrating plume





H

CH2

CH3

H

H

H

H

H

CH3

H

H

H

H

H

CH3

CH3

H

H

H

CH3H3C

H

H

H

HH

CH3

CH3

HH

H

H

H

H

H

H

H

BenzeneC6H6

TolueneC6H5CH3

EhtylbenzeneC6H5CH2CH3

Meta-xylene Para-xylene Ortho-xylene



BenzeneWhat is it?• a colorless liquid with a sweet odor• evaporates into the air very quickly and dissolves slightly in water• highly fl ammable • formed from both natural processes and human activities• industrial processes are the main source in the environment

How is it used?• widely used in the U.S. • ranks in the top 20 chemicals for production volume • to make plastics, resins, and nylon and other synthetic fi bers• to make rubbers, lubricants, dyes, detergents, drugs, and pesticides • natural sources emitted from volcanoes and forest fi res• natural part of crude oil, gasoline, and cigarette smoke

When benzene enters the environment:• it can pass into the air from water and soil• it reacts with other chemicals in the air and breaks down within a few days • it can attach to rain or snow and be carried back down to the ground • it breaks down more slowly in water and soil• it can pass through the soil into underground water• it does not build up in plants or animals



TolueneWhat is it?• a clear, colorless liquid with a distinctive smell• occurs naturally in crude oil and in the tolu tree

How is it used?• it is produced in the process of making gasoline and other fuels from crude oil and making coke from coal• in making paints, paint thinners, fi ngernail polish, lacquers, adhesives, and rubber and in some printing and leather tanning processes

When toluene enters the environment:• it does not usually stay in the environment long• it does not concentrate or buildup to high levels in animals• in landfi lls it can enter soil or water near the waste site• can enter surface water and groundwater from spills and leaks



What is it?

Ethylbenzene

• a colorless, fl ammable liquid that smells like gasoline • naturally found in coal tar and petroleum • found in inks, pesticides, and paints

• primarily to make styrene• as a solvent, in fuels, and to make other chemicals

How is it used?

When ethylbenzene enters the environment:• it moves easily into the air from water and soil• it takes about 3 days for it to be broken down in air into other chemicals • in surface water, it breaks down by reacting with other chemicals found nat-urally in water• can move through soil into groundwater



XylenesWhat is it?

How is it used?

When xylenes enter the environment:

• 3 forms: meta-xylene, ortho-xylene, and para-xylene (m-, o-, and p-xylene)• colorless, sweet-smelling liquid, highly fl amable• occurs naturally in petroleum and coal tar • chemical industries produce xylene from petroleum • one of the top 30 chemicals produced in U.S. in terms of volume

• as a solvent and in the printing, rubber, and leather industries• cleaning agent, a thinner for paint, and in paints and varnishes• found in small amounts in airplane fuel and gasoline

• evaporates quickly from soil and surface water into the air• sunlight quickly breaks it down into other less harmful chemicals • broken down by microorganisms in soil and water• small amount builds up in fi sh, shellfi sh, plants, and other animals living in xylene-contaminated water.

BTEX - Health HazardsBenzene

• affects the nervous system and immune system • can cause aplastic anemia (damage to the bone marrow) • can cause acute myelogenous leukemia • affects the reproductive system and development

+ =

Toluene

• affects the nervous system, respiratory system, and kidneys• affects the reproductive system and development• can cause unconsciousness and death

+ =

BTEX - Health HazardsEthylbenzene

• affects the nervous system• respiratory tract and eye irritation• affects the liver and kidneys• may cause irreversible damage to the inner ear and hearing• birth defects

+ =

Xylenes + =• affects the nervous system• respiratory tract, eye, and skin irritation• affects the liver and kidneys• can cause unconsciousness and death• birth defects



EndophytesWhat are endophytes?Bacteria often live within the internal tissues of plants. Some of this bacte-ria is pathogenic, some has no effect on the host, and some benefi t the host. Endophytes are benefi cial bacteria that live in plant roots, stems, and leaves. Endophytic bacteria can increase plant growth and root mass by:

• fi xing atmospheric nitrogen • sequestering iron from the soil • synthesising phytohormones and enzymes • exhibiting strong anti-fungal activity • antagonising bacterial pathogens • controlling plant parasitic nematodes (roundworms)

Certain endophytes can also increase plant resistance to drought and even herbivores.



Methyl violet counter staining was used to examine plant tissues and the rhizosphere for endophytecolonization. The green areas high-light the endophyte colonization within plant tissues.

Expression of bacterial endophytes

HYDRAULIC CONTAINMENT OF A BTEX PLUME USING POPLARTREES , Barac et.al., 2009Site: A Ford Motor Company factory in Genk, Belgium

Problem: BTEX, fuel and heavy metal contamination in the groundwater

- 3 sources of contamination: leaking pipes and two underground storage tanks

- It is estimated that the contamination started in the 1980s based on how long the plume is.

- Upon discovering the problem the storage tanks were removed and placed above ground, there was immediate groundwater treatment (pump and treat & air stripping), and there were considerations of how to contain the plume.

Setup:- April 1999, 275 poplar trees were planted in a 75m×270 m zone perpendicularly on the migration direction of the contaminated plume.

- The groundwater depth was 4–5 m, which is within reach of the poplarroots.

- 2 poplar cultivars, Populus trichocarpa x deltoides “Hoogvorst” and “Hazen-dans” were selected due to their increased resistance to fungal disease.

- The 4-m tall cuttings were planted in 80-cm deep holes

- The cuttings were planted reciprocally (“Hazendans”—“Hoogvorst”) with a distance of 7m between each other, in 9 rows of 30 trees each. Monitoring wells were installed up- and down-gradient from the poplar planting and groundwater was sampled every six months in order to monitor the dispersion of the contaminant plume.

A>T=0 Initial Plume SamplingB> T+42 months Plume SampleC> T+50 months Plume SampleD> T+55 months Plume Sample

- Four years after planting of the trees (June 2003), both endophytic and rhizo-sphere bacteria were sampled from poplar trees growing inside and outside the BTEX polluted area at the phytoremediation site.

- For practical reasons only microbial communities associated with Populus trichocarpa × deltoides cv. “Hoogvorst” were examined.

- 5 samples were taken from three trees situated inside and three situated out-side the contamination plume.

So what? Time after Planting Activity in Plume13 months poplars haven’t reached maturity; no effect on plume42 months roots have reached depth of the plume, concentration of

BTEX hadn’t lessened but the plume is cut off by poplars50 months plume is still contained and concentration of BTEX is

decreased in the plume but the core of the contamination is not affected

55 months during the growing season the plume all of the BTEX contamination in the plume was taken up.

Other things of note:- The degredation of BTEX is dependant on endophytes, not the poplar’s cells.

- The endophytes in the poplars within the contaminated zone for the plume were degrading the BTEX and using it as an extra energy source.

- Neither endophytic nor rhizosphere bacteria with toluene degradation capacity could be isolated from trees growing either inside or outside the former BTEX plume.

- This suggests that after disappearance of the BTEX as potential carbon source, the capacity to metabolize these components was rapidly lost.

- It also seems that the endophytes were engaging in horizontal gene transfer, improving the population’s breakdown of toulene and BTEX.

- This is unusual but suggests that there is some benefit for the endophytes if they all share the burden of degrading the contaminants.

ENDOPHYTIC BACTERIAL DIVERSITYINPOPLAR TREES GROWING ON ABTEX-CONTAMINATED SITE Site: Same Ford Motor Company factory in Genk, Belgium

Problem: Looking to describe the diversity of endophytes found in poplar trees growing on a site contaminated with toulene looking to identify bacteria that can enhance the phytoremediation of toulene and BTEX.

Setup: - Leaf, stem, root and rhizosphere soil samples were taken from P. trichocarpa x deltoides “Hoogvorst” and “Hazendans”

- In both populus cultivars the greatest population of endophytes was found in the rhizosphere, then the roots, then the stem, and fewest in the leaves which suggests that endophytes cluster in the roots and rhizosphere and decrease in concentration away from the base of the plant.

- There are a number of species-specific bacteria but the overall endophyte population is comprised of both plant-specific and non-specific populations.

Results:



ENDOPHYTES, POPLARS, & CONTEXT



H

CH3

H

H

H

H

TolueneC6H5CH3

Barac (2009) - Bacteria analysis 2003:Populus trichocarpa x deltoides cv. “Hoogvorst” • 121 morphologically distict isolates; 21 genera id’d • +200% endophytic bacteria inside plume • + 50% cultivatable bacteria in rizosphere inside plume • 3x higher colony-forming units (CFU) inside plume • By 2006, “neither endophytic nor rhizosphere bacteria w/ toluene tolerance could be isolated” [after the plume receded.]

Conclusion: Out of 121 isolates, autotropic & heterotropic bacteria popu-lations increased w/in the roots and rhizosphere of poplars ex-posed to the toloune plume, then receded.

Populus trichocarpa x deltoidesBlack cottonwood x Eastern cottonwood



H

CH3

H

H

H

H

TolueneC6H5CH3

Barac (2004) - Bacteria analysis 2003:Lupinus luteus L. - Yellow Lupine • Harvested B. cepacia BU0072 (refs 15,16) derived from naturally occuring endophytic B. Cepacia L.S.2.4 • Isolated “its toluened-degrading derivitive VM1330” • Isolated soil-based B. cepacia G4. • Innoculated Yellow Lupine plants & T+21days harvested.

Growing Media: • Toluene • Toluene + Ni & Km (Kanamycin)

Conclusion: Each bacteria colonizes L. Luteus successfully.



H

CH3

H

H

H

H

TolueneC6H5CH3

Barac (2004) - Bacteria analysis 2003:Lupinus luteus L. - Yellow Lupine

HOWEVER:G4: • Hydroponically grown Lupine w/ G4 grew substantially less than other innoculates w/o the presence of toluene, sug-gesting a negative effect of G4 on Lupine.

VM1330 • Hydroponically grown Lupine w/ VM1330 showed little dif-ference between control plants and toluene-grown plants. But at 1,000 mg/l, the bacteria prevented phytotoxicity (plant death). • ALSO: Lowest evapotranspiration of toluene through aerial parts.

BU0072+G4 • comparable to non-innoculated control plants.



H

CH3

H

H

H

H

TolueneC6H5CH3

Barac (2004) - Bacteria analysis 2003:Lupinus luteus L. - Yellow Lupine

Nonsterile sandy soil growth tests:

Control plants: • Died w/in a week

BU0072: • Similar to control

G4: • Partial reduction of phytotoxicity, from degredation in the rhizosphere.

VM1330: • Protected plant up to 500 mg/l toluene dosage.



H

CH3

H

H

H

H

TolueneC6H5CH3



H

CH3

H

H

H

H

TolueneC6H5CH3

B. cepacia strain VM1330 is derived from a genetically engineered strain of the naturally

occuring B. cepacia L.S.2.4 - BU0072 !



H

CH3

H

H

H

H

TolueneC6H5CH3

Moore (2006):• Assessment of naturally occuring endophytic bac-teria to select those best adapted to toluene & TCE breakdown.

• Verifi cation of re-colonization of poplar potential.

• Because of natural gene transfer of endo. bacteria, the GMO issue is evaded.



Italian breeding strategy for Populus



Populusroot structure



Populusroot network



Wrap up:• Selected populus varieties contain a variety of endophytic bacteria.

• Some of these are better than others at breaking down BTEX.

• Enhanced break-down of BTEX can be generated in two ways: 1. • Genetically engineered endophytic bacteria (Barac 2004).

2. • Naturally occuring enophytic bacteria strains in populus can be identifi ed on BTEX contaminated sites (Barac 2009).

• Autotrophic endophytic bacteria can be selected from poplulus growing on BTEX contaminated sites, cultivated, and re-innoculated (Moore 2006).



Sources cited:Barac, T., et al (2009). Field Note: hydraulic contaminanct of a BTEX plume using poplar trees. International Journal of Phytoremediation, 11:416-424.

Barac, T., et al (2004). Enginneered endophytic bacteria improve phytoremediation of water-soluble, volatile, organic pol-lutants. Nature Biotechnology, v22n5, May.

Cohen, R.M., and Mercer, J.W., (1993). DNAPL site evaluation: C.K Smoley, p384.

Doty, S.L., (2008). Enhancing phytoremediation through the use of transgenics and endophytes. New Phytologist, 179 pps 318-333.

Germaine, K., et al (2004). Colonisation of poplar trees by gfp expressing bacterial endophytes, FEMS Microbiol. Ecol. 48 109–118.

Moore, F. P., (2006). Endophytic bacterial diversity in poplar trees growing on a BTEX-contaminated site: The characteri-sation of isolates with potential to enhance phytoremediation, Systematic and Applied Microbiology 29 539–556.

Settler, R.F., Bradshaw, H.D., et al ed (1996). Biology of Populus and its Implications for Management and Conservation.NRC Research Press, Ottowa.

USGS - http://toxics.usgs.gov/defi nitions/btex.html

Wilson, W.E., and Moore, J.E., (1998). Benzene, toluene, ethylbenzene, xylene, in Wilson, W.E., and Moore, J.E., eds., Glossary of Hydrology: Alexandria, Virginia, American Geological Institute, p20.

Date post:	09-Dec-2021
Category:	Documents
Upload:	others
View:	11 times
Download:	0 times

Phytoremediation of BTEX using poplar trees

Documents