Transgenic plants for

phytoremediation

Shahid AliShahid Ali

Ph.D (Botany)Ph.D (Botany)

08-Arid-95408-Arid-954

Pictures depicting worldwide problems of pollution

OM pályázat, 2001

Bioremediation Basics

Bioremediation is a “treatment that uses naturally occurring organisms to break down hazardous substances into less toxic or non toxic substances”.

Naturally occurring marshes and wetlands have been doing the job! SO How bioremediation is used depends on

1) 1-what is contaminated? (locations)2) 2-on the types of chemicals that need to be cleaned up3) 3-the concentration of the contaminants (amount and duration)

Chemicals in the environment• - Sewage (by products of medicines and food we eat such as estrogen • (birth-control pills) and caffeine (coffee)• - Products around the house (perfumes, fertilizers, pesticides, medicines)• - Industrial• - Agricultural

OM pályázat, 2001

Environmental contaminants

OM pályázat, 2001

Pollutants•Naturally-occurring compounds in the environment that are present in unnaturally high concentrations

Examples:•crude oil•refined oil•phosphates•heavy metals

Xenobiotics•Chemically synthesized compounds that have never occurred in nature.

Examples:•pesticides•herbicides•plastics

Some challenges for bioremediation of pollutants and xenobiotics

Pollutants•may exist at high, toxic concentrations•degradation may depend on another nutrient that is in limiting

supply

Xenobiotics•microbes may not yet have evolved biochemical pathways to

degrade compounds•may require a consortium of microbial populations

OM pályázat, 2001

Sources of contamination

contributors to volatile organic compounds are from

• Industrial spills and leaks •Surface impoundments •Storage tanks and pipes •Landfills and dumps • Injection wells •Paint industry•Pharmaceutical industry•bakeries•printers•dry cleaners•auto body shops

OM pályázat, 2001

PhytoremediationPhytoremediation – –use of plants to remediate polluted soil and/or water. It is an alternative to engineering or processing on site.

•≈350 plant species naturally take up toxic materials

•Sunflowers used to remove radioactive cesium and strontium from Chernobyl site

•Water hyacinths used to remove arsenic from water supplies in Bangladesh, India

» Ex: cottonwoods, poplar, juniper trees, grasses, alfalfa

Water hyacinth Water hyacint uptake of heavy metal eg.,Pb,Cu,Cd,Hg from contaminated water

.

OM pályázat, 2001

Palm Oil Mill Effluent, Malaysia

1. Phytoextraction - uptake of substances from the environment, with storage in the plant (also known as phytoaccumulation).

2. Phytostabilization - reducing the movement or transfer of substances in the environment, for example, limiting the leaching of soil contaminants.

3. Phytostimulation - enhancement of microbial activity for the degradation of contaminants, typically around plant roots.

4. Phytotransformation - uptake of substances from the environment, with degradation occurring within the plant (phytodegradation).

5. Phytovolatilization - removal of substances from the soil or water with release into the air, possibly after degradation.

6. Rhizofiltration - the removal of toxic materials from groundwater through root activity.

Different forms of phytoremediation

From Pilon-Smits (2005) Annu Rev Plant Biol 56: 15-39

Mercury

Selenium

TCE

Heavy metals

Se, As

Radionuclides

TCE

Organics

(PCBs, PAHs)

Phytodegradation

Herbicides, TNT,

MTBE(methyl ter-butyl ether) TCE(Tri chloro ethylene)

Nonbiological remediation technologies and bio/phytoremediation are not mutually exclusive.

Development of transgenic plants forphytoremediation

Transgenic plants for high-efficiency phytoremediation can be produced when the genes involved in all three phases of herbicide metabolism are properly incorporated into the host plants

1. conversion (phase I),2. conjugation (phase II), and 3. compartmentalization (phase III).

Two enzyme systems play major roles in the increased degradation of herbicides:

1- cytochrome P450 monooxygenases (P450s)

2- glutathione S-transferases (GSTs)

OM pályázat, 2001

In phase I, hydrophobic chemicals are converted into less hydrophobic metabolites by cytochrome P450s.

In phase II, pesticides or their phase I metabolites are directly conjugated with glutathione, sugars, or amino acids to produce water-soluble compounds

Finally, in phase III, ATP-dependent membrane pumps recognize the conjugates and transfer them across the membranes. These conjugated metabolites are deposited in vacuoles, transferred to the apoplast, or incorporated into the cell wall in pectin, lignin, hemicellulose, and cellulose fractions

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Schematic diagram of bio-treatment ponds.

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We will use three examples to illustrate phytoremediation:

1-Biodegradation of Explosives 2-Biodegredation of Organomercury3-Bioaccumulation of Arsenic

OM pályázat, 2001

Biodegradation of explosives

The Problem:Contamination to explosives TNT, RDX and glycerol trinitrate.

“Exposure to TNT and RDX, and their degradation products causes symptoms such as anemia and liver damage. These chemicals can be lethaland are suspected carcinogens.

Hundreds of tons of these compounds are found in sediments at innumerable manufacturing sites and storage sites for unexploded ordnancearound the world. Tens of thousands of acres of land and water resources are unsafe because of RDX and TNT contamination.”

The “Solution”:Engineer plants that are able to degrade these compounds in situ.

15

These are the targets

Breakdown of TNT to ADNT (mono amino dinitro toluene) can create “sterile” pink water lagoons.

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The general strategy is to isolate catabolic genes and through standard cloning technologies convert them into plant genes expressed via a

• constitutive promoter such as the CaMV 35S promoter for expression– throughout the plant.• tissue-specific promoter for expression to leaves, stems etc.

Following transformation and plant regeneration, the properties of the plant are evaluated by standard tests.

Bacteria that can grow on these contaminants are the usual source of the genes.

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Conclusion:• TNT can be catabolized• RDX is catabolized and the nitrogen used for growth

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Biodegredation of Organomercury By Transgenic Poplar Methylmercury is a pollutant that biomagnifies in the aquatic food chain with severe consequences for humans and other animals.

The main targets include free cysteine in proteins and peptides leading to damage in the central nervous system.

Symptoms include sensory impairment (vision, hearing, speech), disturbed sensation and a lack of coordination.

Mercury occurs in deposits throughout the world and it is harmless in an insoluble form, such as mercuric sulfide, but it is poisonous as methylmercury [CH3Hg]+ due to its aqueous solubility.

Sources of Mercury include burning coal and mineral extraction. Many uses of mercury are being curtailed or eliminated.

OM pályázat, 2001

Conclusion:• methyl mercury can be reduced to

elemental mercury By Transgenic Poplar

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Bioaccumulation of Arsenic

• The 20th most common element in the earth’s crust and the 12th most common element in the human body.

• Arsenic is a major worldwide contaminant that can arise through industrial activity (pesticides, mining, combustion etc.) or from soil and ground water.

• Associated with acute poisoning and linked to liver, lung, kidney, bladder cancer; cause skin lesions; damage to the nervous system.

• most common element in the human body.

OM pályázat, 2001

In India and Bangladesh (around the Bay of Bengal) ~400 million people are at riskof arsenic poisoning, and up to40 million people drink well water containing toxic levelsof arsenic.

OM pályázat, 2001

Arabidopsis engineered to hyperaccumulate arsenic

Strategy behind cloning

•bacterial arsenate reductase (ArsC) catalyzes reduction of arsenate to arsenite.

•bacterial γ-glutamylcysteine synthetase (γ-ECS) catalyzes the formation of γ-glutamylcysteine (γ-EC) from glutamate and cys for synthesis of glutathione (GSH) and phytochelatins (PCs; three arrows)

•reduced arsenite can bind organic thiols (RS). Then transfer to vacuole.

Arsenic contamination: removed by phytoextraction

““The Chinese Ladder fern Pteris vittata, also known as the brake fern, is a highly efficient accumulator of arsenic.  P. vittata grows rapidly and can absorb up to 2% of its weight in arsenic.  . . When grown on soil with 100 ppm not only did it absorb more arsenic, but it grew 40% larger than normal.” Lena Q. Ma, 2001

P. vittata, a natural arsenic hyperaccumulator, can tolerate soil concentrations of 1,500 μg arsenate/g and can accumulate up to 23 mg arsenic/g in its shoots (fronds). The striking difference between P. vittata and arsenic non-accumulators is the enormous transport of arsenic from roots to shoots in P. vittata. In most plants, only a small fraction of the arsenic taken up from soil by roots accumulates in the above-ground tissue (<20%), whereas P. vittata accumulates up to 95% of the arsenic in above-ground tissue.

Phytoextraction for gold:

• Thio-ligands can induce the solubility and uptake of gold from waste, low-grade rock

• Discovery made in New Zealand

• Proof of concept achieved and the technology is being field tested

• Aim is a crop of 10 t/ha biomass with 100 mg/kg gold concentration dry weight

• This will yield 1000 g of gold per hectare as well as other metals made soluble

• Current focus is on mercury (Hg) removal at the same time as gold

Present strategies:•Bioaccumulation (transport and storage for harvest)

•Bioprocessing (chemical transformation to CO2, NH3, Cl- &

SO42-)

Advantages of plants:•Plants demonstrate tolerance to toxins•Photosynthesis-free energy•Extensive root systems to mine soil (440 million km/h/yr**)•Plants have evolved for transport of materials•Large biomass for harvesting•Species adapted to different ecosystems including wetlands

Disadvantages of plants:•Construction of transgenics•Transgenics & their envoronmental release

Use of Plants for Bioremediation

Alders:Pioneer species

OM pályázat, 2001

Frankia sp.:N-fixing actinomycete

Excellent candidatesExcellent candidates– rapid growthrapid growth– large biomasslarge biomass– large genetic poollarge genetic pool– root depthroot depth

Why poplar trees?Why poplar trees?

Why maize plants?Why maize plants? Excellent candidatesExcellent candidates

– rapid growthrapid growth– large biomasslarge biomass– large genetic poollarge genetic pool– tolerance to CAstolerance to CAs

Contaminants Potentially Amenable to Bioremediation

Readilydegradable

fuel oils, gasoline and alcohols

Monocyclic

aromatics bicyclic aromatics (naphthalene)

OM pályázat, 2001

Generally recalcitrant

(TCE) dioxins

Poly chlorinatedbiphenyls (PCB)

Difficult to degrade

chlorinated solvents

some pesticides and herbicides

Somewhat degradable

creosote, coaltars ketones

pentachloro-phenol (PCP)

Garbage Test

Banana Peel Wood Scrap/Sawdust Wax Paper Styrofoam Cup Tin Can Aluminum Soda Can Plastic Carton Glass Bottles

0.5 Years 4 Years 5 Years 20 Years 100 Years 500 Years 500 Years >500 Years

Biotechnology and the Environment

Petroleum eating bacteria The Exxon Valdez Oil Spill Heavy metals (bioaccumulation)

• Bacteria sequester heavy and radioactive metals Biosensors• lux genes

Drawbacks

•Only surface soil (root zone) can be treated

•Cleanup takes several years

Applying Genetically Engineered Strains to Clean Up the Enviroment

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 (StrobelG. 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

OM pályázat, 2001

Known endophytes

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2,4-D (dichloro phenoxy acetic acid)(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 methylo bacterium found in hybrid poplar

» Mineralized 60% of RDX and HMX in 2 months

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Phytoremediation: Organics

Phytoremediation: Inorganics

Brassica chinensis» Mucor isolate

Cd and Pb tolerance Cd and Pb more water

soluble; more bioavailable

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Comparison between phytoremediation and soil excavation to restore mine site

Technology Advantages Disadvantages

Soil excavation 1.Quick restoration 1.Expensive

2.Effective on limited areas 2.Inefficient on large areas

3.Less aesthetic

4.Very destructive Phytoremediation

1.Returns site to its aesthetical value 1.Slow restoration

2.Less expensive 2.Additional cost needed for

3.Less destructive biomass storing for sites

4.Ecological and sustainable method contaminated by

5.Supports biodiversity dangerous product

7.Give products with economical

value (woods)

OM pályázat, 2001

The Exxon Valdez Oil Spill

• In the end, the indigenous microbes did the best job Oil Fields of Kuwait

• Poses a problem due to the environmental conditions

Environmental Disasters: Case Studies in Bioremediation

future prospects

1The study of root symbioses in phytoremediation will help understand their sensitivity, tolerance and coadaptation capacity under stress

2.The biotechnology of root symbioses on contaminated soils will help understand and measure their impact on:

a)the microbial density and diversity in the rhizosphere, b)the microbial degradation of contaminants, c)the global soil quality (phytotoxicity, nutrients).

3. Appropriate experimental approaches are needed to assess the potential of these microorganisms in the management of disturbed soils following industrial activity.

4. Methagenomics, functional genomics (proteomics, transcriptomicsand metabololomics) approaches will revolutionize the traditional studies of soil microbial ecology and de novo bioremediation

OM pályázat, 2001

Conclusions

Many factors control biodegradability of a contaminant in the environment

•Before attempting to employ bioremediation technology, one needs to conduct a thorough characterization of the environment where the contaminant exists, including the microbiology, geochemistry, mineralogy, geophysics, and hydrology of the system

•Most organics are biodegradable, but biodegradation requires specific conditions: important to understand the physical and chemical characteristics of the contaminants of interest

•The use of genetic engineering to create organisms specifically designed for bioremediation has great potential.

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Contaminants must be bioavailable and in optimal concentrations

•Understand the environmental conditions required to: - Promote growth of desirable organisms - Provide for the expression of needed organisms - Engineer the environmental conditions needed to establish favorable conditions and contact organisms and contaminants

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The End