PHYTOREMEDIATION

PHYTOREMEDIATION - Bio. Tech. ROLE

G. KANTHARAJAN

ICAR-CIFE

iNTRODUCTION

• manmade activities - POLLUTION• physical removal of polluted soil - too costly &

destructive to environment • Phytoremediation Technology is an in

innovative field of science and technology • an alternative to mechanical congenital

cleaning methodologies which mostly require high capital input, labor and intensive energy.

PHYTOREMEDIATION‘’the use of plants to reduce the volume, mobility, or toxicity of contaminants in soil, groundwater, or other contaminated media’’ (USEPA, 2000)

for centuries: wetlands used for waste treatment in Europe

metal hyperaccumulator plants discovered - used as indicators for mining

1980s: - superfund act (1986 - 8.5 billion $) in USA- idea to use hyperaccumulator plants for metal

cleanup 1994: phytoremediation term coined (Ilya Raskin)

Factors of phytoremediation

site characteristics and contaminant type

plant specieslevels of contaminationcontaminated area - size and

depth site conditions (nutrient

availability, soil organic matter content, soil water, soil aeration)

Plants have the capacity to uptake, degrade, transform, sequester contaminants in addition to producing biomass

Characteristics of plants

ability to extract or degrade the contaminants of concern

ability to take up large quantities of water through the roots

generally more effective when using large, fast-growing plants (e.g) poplar trees

Possible plants should be tolerant to metal pollution and other climatic and site stresses

be efficient in translocation to the harvestable portion of the plant

Contd…able to concentrate huge amount of essentials & nonessential metals in their foliageto produce dense root system, element selectivityease of care and establishment and resistance to disease & insect problemsease of planting and maintenance

TOP 5 phytoremediators…

Indian mustard Brassica juncea

White Willow

Poplar tree Populus deltoides

Indian grass Sorghastrum nutans

Sunflower Helianthus annuus

MECHANISMUptake and transport

- Plants and metals interact in the root environment- The contaminant has to be in contact with the roots

in order to be uptaken- transporters that are used for the macronutrients and

micronutrients entrance are also used by plants for the heavy metals uptake- Metals are stored in the subcellular compartments

like vacuoles and lignocellulosic material (cell wall)- Research is being developed to discover other

mechanisms that may be active to direct the metals in cells.

Accumulation and sequestration

-Analytical techniques (X-ray emission - SEM, microanalysis) can provide information on speciation and localization of metals in plants tissue - It is important to understand the molecular bases of the capacity of some plants to hyperaccumulate and store the metals.

Plant biochemical mechanisms in the case of phytoremediation of heavy metals

1. Adsorption – root surface absorbs the elemental nutrients, binds pollutants and nutrients and favors the interaction between plant roots and soil microbes with the aim of increasing metal bioavailability2. Accumulation and transportrole of transporter proteins – proteins and peptides that increase metal binding in plants can improve metal tolerance or accumulationchelating agents (natural and synthetic chelators) – added to the soil help Increase the metal bioavailability, uptake and translocation of heavy metals3. Translocation – root cells uptake metal ions and transport them to the shoots, process in which the membrane transport systems have a major role4. Detoxification – hyperaccumulators possess a great characteristic of being very efficient in detoxification and sequestration without having phytotoxic effects due to the adsorption of huge amounts of heavy metalsvacuolar compartmentalization – vacuoles are the main storage place of heavy metals in the cells of a plant and there is a vacuolar compartmentalization to control the distribution and concentration of metal ions in order to restrict other parts of the cell to have access to the contaminantsvolatilization – metal ions are converted into volatile state5. Hyperaccumulation – the metal ion is concentrated to > 0.1 - 1 % of the dry weight of the plant.

Selection of plant species & densityIrrigation and soil

amendment

Contaminant extraction and degradation

Monitoring

Harvesting

Monitoring

Remediation Complete

Process Flow

http://www.geoengineer.org/education/web-based-class-projects/geoenvironmental-remediation-technologies/phytoremediation?showall=1&limitstart=

Techniques of Phytoremediation

Phytoextraction

Phytotransformation

Phytodegradation Phytorhizofiltration

Dendroremediation

Phytovolatilization

PHYTOREMEDIATION

Hydraulic Control

Phytostabilization

(Schwitzguebel, 2000)

(Pilon-Smith, 2005)

PHYTOEXTRACTION…

Phytoextraction (phytoaccumulation)

o largest economic opportunities obest approach for removing and isolating the

contamination from soil - keeping its structure and fertility intact

ohyper accumulating plants are seeded/ transplanted and are cultivated

oMetals present in soil are absorbed by the plants and then translocated to the above ground shoots for accumulations.

oWhen maximum plant growth and metal accumulation are achieved, plants from above ground levels are harvested - permanent removal

o recycleoNickel, Zn & Cu (accumulation often reaches to 1-

5%)

Contd…

Rehabilitation of large areas low to moderate levels of contaminated land at shallow depthsdepends on the interaction among soil, metal and plant

Strategies in Phytoextraction Chelate Assisted Phytoextraction or induced phytoextraction -Artificial chelant (EDTA, HEDTA & EDDNA) is added for increasing the mobility and uptake of metal contaminantsContinuous Phytoextraction - Naturally Plants secrete phytosideophores (chelating agents) like mugenic and aveinc acids to enhance the bioavailability of soil bond heavy metals

advantages Inexpensive, compared to conventional methodscontaminant is permanently contaminant can be recycled from the contaminated plant biomass

disadvantageshyperaccumulator - slow growth, shallow root system, and small biomass production. Harvesting & disposal complying with standardslimited to metals and other inorganic compounds in soil or sediment

Phytostabilization (Phytorestoration)

in-place inactivation used for the remediation of soil, sediment & sludge -

metal & Inorganic contaminants Plants are used to immobilize contaminants in the soil

and ground water through absorption and accumulation by roots, adsorption onto roots, or precipitation within the root zone of plants

Reduces the mobility of the contaminant and prevents migration to the ground water - bio-availability of metal into the food chain

Treatment of Pb, As, Cd, Cr, Cu & Zn presence of the plant induces in soil chemistry and

environment - induce adsorption or precipitation onto the plant roots or soil

advantages disposal of hazardous material/biomass is not

required very effective when rapid immobilization is needed

to preserve ground and surface waters The presence of plants also reduces soil erosion

disadvantages contaminant remaining in soil application of extensive fertilization or soil amendments monitoring is mandatory stabilization of the contaminants may be primarily due to the soil

amendments

Phytovolatilizationplants that absorb elemental form of metals from soil, could convert them biologically into gaseous species inside the plant, i.e., biomethylated to form volatile molecules and finally release them to the atmosphere. Se, As and Hg may exist as gaseous species in environmentControversial… whether release of these volatilized elements in

atmosphere is safe???Volatilized element could be recycled by precipitation and then

redeposit back into ecosystemMembers of the Brassicaceae are capable of releasing up to 40g Se /ha/day-as various

gaseous compounds

Phytofiltration ‘’plant roots (rhizofilteration) or seedlings (blastofilteration) are grown in aerated water from where they participate and concentrate toxic metals from contaminated effluents ‘’

UTILIZATION Treatment of groundwater (either in situ or extracted), surface water, or wastewater for

removal of metals or other inorganic compounds Used for Pb, Cd, Cu, Ni, Zn, and Cr, which are primarily retained within the roots

Characterfast growing roots with capability for removing toxic metals from solution over extended period of time

Mechanism growing plants hydroponically - transplanting into metal polluted water Root exudates and changes in rhizospheres pH cause precipitation Metal forms precipitation over root surface Whole plants or roots are harvested for disposal after the saturation point

blaslofilteration efficient than rhizofiltertion for some metals Due to the dramatic increase in surface to volume ratio after germination, seedlings

tend to ab/adsorb large quantities of toxic metal ions (seedling of Brassica juncea)

Rhizofiltration vs PhytoextractionRhizofiltration is similar to phytoextraction, but the plants are used primarily to address contaminated ground water rather than soil.

Plants used are…

Sunflower, Indian mustard, tobacco, rye, spinach, and corn have been studied for their ability to remove lead from water, with sunflower having the greatest ability

advantages ability to use both terrestrial and aquatic plants

- in situ or ex situ contaminants do not have to be translocated to

the shoots-species other than hyperaccumulators

Terrestrial plants are preferred - fibrous and much longer root system

Disadvantages need to adjust pH plants may first need to be grown in a greenhouse or

nursery there is periodic harvesting and plant disposal tank

design must be well engineered good understanding of the chemical

speciation/interactions is needed cost of remediation estimated to be $2-$6/ 1000 gallons of

water

Phytodegradation

‘’uptake and degradation of the organic compounds by the plants’’

enzymatic breakdown of organic pollutants such as TCE & herbicides, both internally and externally and through secreted plant enzymes

Plants contain Nitroreductases, Dehalogenases, LaccasesPlants containing dehalogenases – TCEPlants containing peroxidases - xenobioticsAlgal phytodegradation of petroleum naphthenic

Rhizodegradation: It is believed to be carried out by bacteria or other microorganisms

Dendro-remediation

“the use of trees to evaporate water and thus to extract pollutants from the soil”

low cost means for treating the soil and preparing it for future use

trees are planted to reduce and eliminate toxic substances in the soil over time

Process & Mechanism of contaminant removal

Aquatic plants in Phytoremediation…

Mainly heavy metal – Eichhornia crassipes, Salvinia minima, Pistia sp. Lemna minor

TNT, RDX – Elodea michx, Ceratophyllum sp., Sagittaria latifolia

Radionuclides (Cs 137, Co 60) – Potamogeton sp., Typha

Selenium (phytovolatilisation) – Sesuvium portulacastrum

Petroleum Hydrocarbon, Pb, Zn, Cd - Rhizophora mangle, Avicennia etc…

Famous projects… Chernobyl Nuclear Plant Reactor 4 in the Ukraine

caused severe radioactive contamination in 1986 - Phytoremediation by Brassica juncea & B. carinata

Phytoremediation of explosives contaminated ground water in US Army sites (Super fund sites) – by Aquatic plants

Pariyej reservoir, Gujarat - “Wetland of International Importance”

- to ascertain the degree of heavy metal contamination aquatic macrophytes used as biomonitors

Phytoremediation VS Microbial Bioremediation

• Solar driven pollutant extraction system • less expensive; it is less intrusive and more

aesthetically pleasing• By acting as soil stabilizers, plants minimize the

amount of contaminated dust that leaves the site and could enter the surrounding neighborhoods• phytoremediation is more easily monitored - tissue

can be easily collected and tested for the presence of the pollutant over time.• avoid the production of intermediates with increased

toxicity • possibility of a producing a useful product, such as

wood, pulp, or bioenergy

Biofortification

Studies on the interaction between plant tissues, heavy metals/trace elements have led to the concept of biofortification

Plants enriched in micronutrient content are seen as an aid against malnutrition.

Differently from phytoaccumulation of metals, which is considered as a risk

for the food chain, biofortification of crops with specific elements may become advantageous

Knowledge of mechanisms controlling metal accumulation is a prerequisite

Information is also requested for those antinutrients that decrease element availability: (Phytic acid, fibres, and polyphenols)

phytotechnologyphytoremediation has been recently supplanted by the term “phytotechnologies”, used to indicate all applications in which plants are used to manage and control pollutants, even without removing or destroying it.

PROS… Amendable to a broad range of organic and inorganic contaminants

including many metals with limited alternative options. Cost effective and ecologically friendly in which plant utilizes its

natural abilities to restore environment Reduces the amount of waste to be landfilled (up to 95%), can be

further utilized as bio-ore of heavy metals. Does not require expensive equipment or highly specialized personnel Alternative or complimentary to mechanical congenital cleaning

methodologies which mostly require high capital input, labor and intensive energy.

Easy to maintain and accepted by public Fewer spread of contaminant via air and water

CONS…Compared to engineering methods… too slow or only

seasonally effective. hyper accumulators are shallow root system, slow

growth, small biomass production – limited to shallow sediment, soil and waterbody

Regulatory agencies often require significant progress in remediation to be made in only a few years, making most phytoremediation applications unsuitable.

For some pollutants such as TCE, the concentration of the pollutant is not reduced sufficiently to meet regulatory requirements.

the pollutants can be at phytotoxic concentrations or recalcitrant - plants are not effective

Disposal of contaminants harvested in the plant biomass- again pollution

wildlife and people may consume the plants - IAS

NEED of Bt. IN PHYTOREMEDIATION…

‘’Conventional plants fail to meet the requirements‘’

Ideal phytoremediator: tolerance to the pollutant; degrade or concentrate the contaminant; extensive root systems; absorb large amounts of water from the soil; and fast growth rates and high levels of biomass

trees- which have extensive root systems, high biomass, and low agricultural inputs requirements- tolerate pollutants poorly, and do not accumulate

The remedial capacity improvement - by genetic manipulation and plant transformation technologies

The introduction of novel traits for the uptake and accumulation of pollutants into high biomass plants

Identification of genes and proteins• the search for genes and proteins is being carried on with genomics

and proteomics approaches• results are showing how different genes are induced by metals in

these plants • Ectopic expression of Thlaspi genes in yeast led to the isolation of a

new gene function involved in Cd transport and probably also hyperaccumulation.• Genetic mapping - building maps of Quantitative trait Loci (QTLs) for

hyperaccumulation and tolerance in model plants.• Due to phylogenetic relationships with known hyperaccumulators in

the family Brassicaceae, Arabidopsis thaliana is the best model plant available, due to the complete knowledge of the genomic sequence and to the genetic knowledge. • The group led by Martin Broadley has recently mapped QTL involved

in Cs accumulation of A. thaliana, Several accessions were analysed for Cs accumulation, leading to the description of a 2-fold variation in Cs concentration. • Mapping of candidate genes in these regions will lead to new

hypotheses about the structure and function of these QTLs.

Development of GE plants

http://envfor.nic.in/divisions/csurv/biosafety/SAU/Presentations/Junagadh/Jeetendra_Solanki.pdf

GE of plants for enhanced metabolism of pollutants

- for enhancing the effectiveness of phytoremediation - overexpress in genes involved in metabolism, uptake, or transport of specific pollutants. - using Agrobacterium tumefaciens-mediated plant transformation To increase the phytoremediation potential of the common

pollutant TCE, genetically engineered plants with mammalian cytochrome P450 enzyme known to metabolize it.

The P450 2E1 enzyme controls the rate-limiting step in the metabolism of multiple environmental pollutants, including TCE, carbon tetrachloride, chloroform, benzene, vinyl chloride, and ethylene dibromide.

When the cytochrome P450 2E1 gene (hCYP2E1) was overexpressed in tobacco plants, metabolism of TCE was substantially increased.

Transgenic tobacco removed 98% of the ethylene dibromide, compared with 63% removal by the null vector control plants.

elemental mercury methylmercury (bacteria in the water and soil convert)

food chain – bio-magnificationTerrestrial plants are generally insensitive to the harmful

effects of mercury compounds but it affects photosynthesis and oxidative metabolism reduces plant water uptake.

Plants have no requirement for Hg and typically play a relatively passive role in the biogeochemistry of Hg compounds.

no naturally occurring plant species with significant capabilities for accumulation, degradation, or removal of Hg

Several plant species convert modest amounts of Hg (II) (TOXIC – BIOMAGNIFICATION) to Hg (0) by the activities of several redox enzymes such as catalase and peroxidase.

Hg (0) (VOLATILE) is released into the soil from roots or into the atmosphere from shoots.

Chloroplast engineering in Hg phytoremediation

the genes encoding bacterial mercury transformations have been well characterized

plants with improved abilities for Hg removal and detoxification was initiated in the early 1990s by Richard et al.

They made use of the two bacterial genes from the well-characterized mer operon, merA, and merB, to engineer an Hg transformation and remediation system in plants

Diverse plant species such as A. thaliana, Tobacco, yellow poplar, cottonwood, and rice constitutively expressing modified merA were resistant to at least ten times greater concentrations of Hg (II) than those that kill non-transgenic controls.

These transgenic plants showed significant levels of Hg (0) volatilization relative to controls.

CONTD…

CONTD…

The chloroplast and endoplasmic reticulum (ER) have been shown to be significant targets for Hg poisoning.

Engineering Hg detoxification systems in chloroplasts or ER may offer high levels of Hg tolerance and

Transgenic tobacco plants exhibited high levels of tolerance to the phenylmercuric acetate and accumulated 100- and 4-fold more Hg in the shoot in the presence of PMA or HgCl2 than untransformed plants.

Pesticide & Oil spill To increase phytoremediation of BTEX chemicals,

the genes for degrading the BTEX component, toluene, were transferred to an endophytic strain and inoculated onto lupine .

- The inoculated plants were able to tolerate levels of toluene ten times the normally phytotoxic levels.

atzA gene encoding the first enzyme, atrazine chlorohydrolase - expressed in transgenic tobacco, Arabidopsis, and alfalfa actively expressed atzA, resulting in increased tolerance to a wide range of atrazine concentrations.

- The pesticide was dechlorinated to hydroxyatrazine in all of the plant organs.

Safety concerns• Due to the strict regulations governing the release of transgenic

trees, it is likely that genetically engineered trees for phytoremediation will be used only on closely monitored sites.

• To prevent transgene flow, the trees would be cut down before they became sexually mature, after several years in the field.

• Careful selection of the species to be transformed can avoid routes of transgene release by using trees that will not resprout from wind-blown branches.

• If the transgene did “escape” into native populations, a gene involved in pollutant degradation would be unlikely to confer any selective advantage or negative environmental impact.

Future prospects… - biotechnology - for efficient, clean, cheap, and sustainable bioremediation technologies is very promising

A better understanding of the molecular basis of the pathways involved in the degradation of pollutants is needed. Further analysis and discovery of genes suitable for phytoremediation is essential.

Phytoremediation technology is still at an early development stage, and field testing of transgenic plants for phytoremediation is very limited.

Biosafety concerns need to be properly addressed - prevent gene flow into wild species

Phytoremediation technologies are currently available for only a small subset of pollutants, and many sites are contaminated with several chemicals - need to be engineered with multiple stacked genes

Doty, and Strand., 2008. Phytoremediation of Volatile Pollutants through Genetic Engineering. ISB News Report.

REFERENCES

Pilon-Smith, E. 2005. Phytoremediation: Annual Rev. Plant Biol. 56:15-39.

Ismail, S. 2012. 'Phytoremediation: a green technology'. Iranian Journal of Plant Physiology 3 (1), 567 - 576.

Nelson Marmiroli., Marta Marmiroli, and Elena Maestri., 2006. PHYTOREMEDIATION AND PHYTOTECHNOLOGIES: A REVIEW FOR THE PRESENT AND THE FUTUREParma, Italy

Etim, 2012. Phytoremediation and Its Mechanisms: A Review. International Journal of Environment and Bioenergy, 2012, 2(3): 120-136

Buhari Muhammad, L., Sulaiman Babura, R., Vyas, NL., Sulaiman, B, and Harisu Umar, Y,. 2016. Role of Biotechnology in Phytoremediation. J Bioremed Biodeg 7: 330. doi: 10.4172/2155-6199.1000330