Bioremediation and phytoremediation

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Various strategies of pollution mitigation

By: Rachit Raghava Kashyap

Department of Environmental Science, Dr Y S Parmar UHF, Solan (H.P.)

CREDIT SEMINAR-I ENS-691

BIOREMEDIATION

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Outline of PresentationIntroduction

Bioremediation mediated biodegradation

Bioremediation effectiveness

Bioremediation strategies

Insitu and Exsitu

Case study : Oil degradation

Phytoremediation

Different mechanisms of phytoremediation and respective case studies

Applications

Case studies in support of soil and water remediation

Disadvantages

Conclusion

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INTRODUCTION• Use of different biological systems to destroy or reduce

concentrations of contaminants from polluted sites.• Manages microbes and plants to reduce, eliminate, contain or

transform contaminants present in soils, sediments, water or air.• Microbes and plants have a natural capability to attenuate or

reduce:• Mass• Toxicity• Volume• Concentration of pollutants

without human interventions.

(Rittmann, B. E, McCarty, P. L. 2001)

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Conventional methods of remediation

Dig up and remove it to a landfill

Cap and contain

Maintain it in the same land but isolate it

Is there a better approach?

Products are not converted into harmless substances. Stay as a threat!

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Better approaches

Destroy them completely, if possible

Transform them into harmless substances

• High temperature incineration.• Chemical decomposition like dechlorination.

Methods already in use

But, are they effective?

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YesBut only to some extent

Drawbacks Technological complexity.

The cost for small scale application – expensive.

Lack of public acceptance – especially in incineration.

• Incineration generates more toxic compounds.

• Materials released from imperfect incineration – cause undesirable imbalance in

the atmosphere. Ex. Ozone depletion.

• Fall back on earth and pollute some other environment.

• Dioxin production due to burning of plastics – leads to cancer.

May increase the exposure to contaminants, for both workers and

nearby residents.

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Bioremediation makeseffective better approach possible.

Either by destroying or render them harmless using natural biological activity.

Use of plants

Use of Microorganisms

BIOREMEDIATION

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Bioremediation mediated biodegradation

• in general it is “bio” mediated decomposition of paper, paint,

textiles, hydrocarbons and other pollutants.

• Superior technique over using chemicals – why?

1. Microorganisms – easy to handle.

2. Plants – easy to grow.

Biodegradation is the initial process that results to bioremediation.

(Marshall, F. M., 2009)

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Enzymatic processes in bioremediation• Major types of reactions• Oxidation.• Decarboxylation in which the -CO2H is replaced with an H atom or –OH

group.• Hydrolysis which involves the addition of H2O to a molecule accompanied

by cleavage of the molecule into two species. • Substitution in which one group of atom is replaced by another (such as OH

for Cl- ).• Elimination whereby atoms or group of atoms are removed from adjacent

carbon atoms, which remained joined by a double bond.• Reduction, dehalogenation , demethylation, deamination, condensation, in

which two smaller molecules are joined to produce a larger one: conversion of one isomer of a compound to another with a same molecular formula but different structure ; conjugation; ring cleavage.

(Marshall, F. M., 2009)

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Biodegradation has at least 3 outcomes:

1. A minor change in an organic molecule leaving the main structure

intact.

2. Fragmentation of a complex organic structure in such a way that

the fragments could be reassembled to yield the original structure.

3. Complete mineralization, which in the transformation of organic

molecules to mineral forms.

One example to describe all 3 types

2, 6-Dichlorobenzonitrile (Marshall, F. M., 2009)

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Minor change in a molecule (Dehalogenation)

Cl

Cl C N HOH

Cl

Cl is replaced with OH

OH

Cl C N

2, 6-Dichlorobenzonitrile

(Prasad MNV., 2003)

2,6-Dichlorobenzonitrile is an herbicide and is toxic for humans.

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Fragmentation

Cl

Cl C N HOH

Cl

Cl is replaced with OH

OH

OH OH


NH2CH2

(Prasad MNV., 2003)

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Mineralization

NH32ClHOH

Completely converted into inorganic forms

Cl

Cl C N


(Prasad MNV., 2003)

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IF ANY OF THESE PROCESSES IS TRIGERED / STIMULATED TO GET A LESS CONTAMINATED

PRODUCT

THEN IT IS CALLED AS

BIOREMEDIATION

(Prasad MNV., 2003)

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Bioremediation Effectiveness• Depends on:

• Microorganisms

• Environmental factors

• Contaminant type & state

(Prasad MNV., 2003)

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Microorganisms• Aerobic bacteria:

• Examples include: Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium.

• Shown to degrade pesticides and hydrocarbons; alkanes and polyaromatics.• May be able to use the contaminant as sole source of carbon and energy.

• Methanotrophs: • Aerobic bacteria that utilize methane for carbon and energy.• Methane monooxygenase has a broad substrate range.

• active against a wide range of compounds (e.g. chlorinated aliphatics such as trichloroethylene and 1,2-dichloroethane)

• Anaerobic bacteria:• Not used as frequently as aerobic bacteria. • Can often be applied to bioremediation of polychlorinated biphenyls (PCBs) in

river sediments, trichloroethylene (TCE) and chloroform.

• Fungi:• Able to degrade a diverse range of persistent or toxic environmental pollutants.

(Bodishbaugh, D.F., 2006)

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How Microbes Use the Contaminant

• Contaminants may serve as:

• Primary substrate

• enough available to be the sole energy source.

• Secondary substrate

• provides energy, not available in high enough concentration.

• Co metabolic substrate

• Utilization of a compound by a microbe relying on some other primary substrate.


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Microorganisms can live at different pH conditions


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MO’s can live at any temperature conditions


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Environmental Factors

Environmental Factor Optimum conditions Condition required for microbialActivity

Available soil moisture 25-85% water holding capacity 25-28% of water holding capacity

Oxygen >0.2 mg/L DO, >10% air-filled pore space for aerobic degradation

Aerobic, minimum air-filled pore space of 10%

Redox potential Eh > 50 milli volts

Nutrients C:N:P= 120:10:1 molar ratio N and P for microbial growth

pH 6.5-8.0 5.5 to 8.5

Temperature 20-30 ºC 15-45ºC

Contaminants Hydrocarbon 5-10% of dry weight of soil

Not too toxic

Heavy metals 700ppm Total content 2000ppm

(Vidali , 2007)

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Bio-degradable

Petroleum products (gas, diesel, fuel oil) •crude oil compounds (benzene,

toluene, xylene, naphthalene) •some pesticides (malathion) some industrial

solvents •coal compounds (phenols, cyanide in coal tars and coke waste)

Partially degradable / Persistent

• TCE (trichlorethane) threat to ground water •PCE (perchloroethane) dry

cleaning solvent •PCB’s (have been degraded in labs, but not in field work)

•Arsenic, Chromium, Selenium

Not degradable / Recalcitrant

• Uranium •Mercury •DDT

Type of contaminants

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Organic Pollutants Organisms

Phenolic - Achromobacter, Alcaligenes,

compound Acinetobacter, Arthrobacter,

Azotobacter, Flavobacterium,

Pseudomonas putida

- Candida tropicalis

Trichosporon cutaneoum

- Aspergillus, Penicillium

Benzoate & related Arthrobacter, Bacillus spp.,

compound Micrococcus, P. putida

22

Some m.o. involved in the biodegradation of organic pollutants

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Organic Pollutants Organisms

Hydrocarbon E. coli, P. putida, P. Aeruginosa

Surfactants Alcaligenes, Achromobacter,

Bacillus, Flavobacterium,

Pseudomonas, Candida

Pesticides P. Aeruginosa

DDT Arthrobacter, P. cepacia

BHC P. cepacia

Parathion Pseudomonas spp., E. coli,

P. aeruginosa

(Vidali, 2007)

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Criteria for Bioremediation Strategies

i) Organisms must have necessary catabolic activity required for degradation of contaminant at fast rate to bring down the concentration of contaminant.

ii) The target contaminant must have bioavailability.

iii) Soil conditions must be favourable for microbial/plant growth and enzymatic activity.

iv) Cost of bioremediation must be less than other technologies of removal of contaminants.

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Bioremediation Strategies

(Barathi S and Vasudevan N, 2001)

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Bioremediation Strategies

In situ Bioremediation(at the site)

Ex situ Bioremediation(away from the site)

(Barathi S and Vasudevan N, 2001)

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In Situ Bioremediation

In situ bioremediation is when the contaminated site is cleaned up

exactly where it occurred.

There is no need to excavate or remove soils or water in order to

accomplish remediation.

In situ biodegradation involves supplying oxygen and nutrients by

circulating aqueous solutions through contaminated soils to

stimulate naturally occurring bacteria to degrade organic

contaminants. It can be used for soil and groundwater.

It is the most commonly used type of bioremediation because it is

the cheapest and most efficient, so it’s generally better to use. (Wood TK , 2008)

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Types of In situ Bioremediation

Engineered Bioremediation

Intrinsic Bioremediation

2 types

Intentional changes

Simply allow biodegradation tooccur under natural conditions

(Wood TK , 2008)

Doing nothing

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Intrinsic Bioremediation

• Intrinsic bioremediation uses microorganisms already present in the environment to biodegrade harmful contaminant.

• There is no human intervention involved in this type of bioremediation, and since it is the cheapest means of bioremediation available, it is the most commonly used.

• When intrinsic bioremediation isn’t feasible, scientists turn next to engineered bioremediation.

(Barathi S and Vasudevan N., 2001)

- a bioremediation under natural conditions

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Engineered Bioremediation

The second approach involves the introduction of certain

microorganisms to the site of contamination.

When site conditions are not suitable, engineered systems have to be

introduced to that particular site.

Engineered in situ bioremediation accelerates the degradation process

by enhancing the physicochemical conditions to encourage the growth

of microorganisms.

Oxygen, electron acceptors and nutrients (nitrogen and phosphorus)

promote microbial growth.(Barathi S, Vasudevan N., 2001)

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Insitu Engineered bioremediation typesBioventing

involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria.

(Vidali,M., 2001)

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Biosparging

involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria.

(Vidali,M.2001)

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• Bioaugmentation

involves practice of adding specialized microbes or their enzyme preparation to polluted sites to accumulate transformation or stabilization of specific pollutants.

(Rittmann B.E and McCarty, P.L. 2001)

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Ex situ engineered bioremediation Strategies

(Source: http://ndpublisher.in/ndpjournal.php?j=IJAEB)

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Solid phase system Ex Situ Bioremediation

Composting is a technique that involves combining contaminated soil with organic compounds such as agricultural wastes. The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.

(Source: https://www.google.co.in/search?q=bioremediation+images)

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Land farming Operation

Land farming is a simple technique in which contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. The practice is limited to the treatment of superficial 10–35 cm of soil.

(Rittmann, B.E and McCarty, P.L, 2001)

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Biopile SystemBiopiles are a hybrid of land farming and composting. Essentially, engineered cells are constructed as aerated composted piles. Typically used for treatment of surface contamination with petroleum hydrocarbons they are a refined version of land farming that tend to control physical losses of the contaminants by leaching and volatilization. Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.

(Rittmann,B.E and McCarty,P.L.2001)

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Bioremediation using bioreactor System

(Rittmann,B.E and McCarty,P.L.2001)

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Case study: Oil degradationOil-metabolizing bacteria were known to exist, but when introduced into

an oil spill, competed with each other, limiting the amount of crude

oil that they degraded.

Prof. Chakrabarty discovered a method for genetic cross-linking that

fixed all four plasmid genes in place and produced a new, stable,

bacteria species (now called pseudomonas putida) capable of consuming

oil one or two orders of magnitude faster than the previous four strains

of oil-eating microbes.

The new microbe, which Chakrabarty called "multi-plasmid

hydrocarbon-degrading Pseudomonas," could digest about two-thirds of

the hydrocarbons that would be found in a typical oil spill.

04/07/2023 40By use of genetic engineering:a). Plasmid transfer: CAM OCT XYL NAH

Recombination Non-recombination

CAM + OCT XYL + NAH

SUPERBUG(Dowling, DN and Doty, SL. 2009)

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Biodegradation of hydrocarbons and petroleum

Source: https://www.google.co.in/search?q=bioremediation+images

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Use of bioremediation strategies over different years by developed countries ( in percent)

2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 201220

30

40

70

60

50

80

Source: http://ndpublisher.in/ndpjournal.php?j=IJAEB

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Percent use of different techniques for remediation in India

Source: WHO

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Review of bioremediation strategies

(Rittmann B E and McCarty P L, 2001)

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PHYTOREMEDIATION

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What is it ?Phytoremediation is the use of living green plants for in situ risk reduction and/or removal of contaminants from contaminated soil, water, sediments, and air.


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Phytoremediation

Phytoextraction

1

Phytovolatilization

2

Phytostabilization

3

Rhizodegradation

Rhizofiltration

4

5

5 mechanisms based on the fate of contaminants

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PhytoextractionPlant roots uptake metal contaminants from

the soil and translocate them to their above soil tissues.

Once the plants have grown and absorbed the metal pollutants they are harvested and disposed off safely.

This process is repeated several times to reduce contamination to acceptable levels.

Hyper accumulator plant species are used on many sites due to their tolerance of relatively extreme levels of pollution.

Avena sp. , Brassica sp.

Contaminants removed:Metal compounds that have been successfully

phytoextracted include zinc, copper, and nickel.


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RhizofiltrationIt is concerned with the remediation of contaminated groundwater.

The contaminants are either adsorbed onto the root surface or are absorbed by the plant roots.

1

• Plants are hydroponically grown in clean water rather than soil, until a large root system has developed

2• Water supply is substituted for a

polluted water supply to acclimatize the plant

3

• They are planted in the polluted area where the roots uptake the polluted water and the contaminants along with it

4• As the roots become saturated

they are harvested and disposed of safely


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Case study

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Physicochemical properties of untreated and treated effluents

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Phytostabilisation

To immobilize soil and water contaminants from migration.

Mechanism

Phytochemical complexation in the root zone – precipitation

Examples:

Transfer of human MT-2 gene to tobacco (Nicotiana sp.) resulted in

transgenic plant with enhanced Cd tolerance and stabilisation. (Eapen et al.

2006)

Transfer of yeast CUPl gene in cauliflower (Brassica sp.) resulted in 16-

fold higher accumulation of cadmium (Cd) in the transgenic cauliflower.

(Sriprang, 2006)

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PhytodegradationIt is the degradation or breakdown of organic contaminants by

internal and external metabolic processes driven by the plant.

Mechanisms:

Plant enzymatic activity:

oxygenases- hydrocarbons degradation.

nitroreductases- explosives degradation.

Used in breakdown of ammunition wastes, chlorinated solvents such as TCE (Trichloroethane), degradation of organic herbicides.

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Cont. 1. Transfer of pea MT gene in

Arabidopsis thaliana resulted in

enhanced copper degradation in the

transgenic A. thaliana. (Murooka,

2006).

2. Enzyme bacterial mercuric ion

reductase has been engineered into

Arabidopsis thaliana and the

resulting transformant transgenic

plant is capable of degrading and

volatalising mercuric ions.

(Cunningham and Owe, 2009)


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RhizodegradationIt is the breakdown of organic contaminants in the soil by soil dwelling

microbes which is enhanced by the rhizosphere’s presence.Rhizosphere = soil + root + microbesSymbiotic relation Also called:

Enhanced rhizosphere biodegradation

Phytostimulation

Plant assisted bioremediationSugars, alcohols and organic acids act as carbohydrate sources for the soil

microflora and enhance microbial growth and activity. Act as signals for certain microbes. The roots also loosen the soil and transport water to the rhizosphere thus

enhancing microbial activity.Digest organic pollutants such as fuels and solvents, producing harmless

products.

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Case study of symbiotic engineering

A genetically engineered rhizobium bacteria has been suggested by (Sriprang

et al., 2010).

Rhizobium grow slowly for long times in soil, but if they infect a compatible

legume they grow rapidly.

This special feature of symbiotic relationship gives clue for biotechnological

transfer and expression of MT (metallothionein) genes that sequester heavy

metals from contaminated soil.

Once symbiosis with MT genes is established with legumes, the heavy metals

starts accumulating in the nodules.

Good alternative and more cost-effective method to remove heavy metals

from soil.

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PhytovolatilizationPlants uptake contaminants which are water

soluble and release them into the atmosphere as they transpire the water.

The contaminant may become modified along the way, as the water travels along the plant's vascular system from the roots to the leaves, whereby the contaminants evaporate or volatilize into the air surrounding the plant.

Poplar trees volatilize up to 90% of the TCE they absorb.

Selenium and Mercury - Arabidopsis thaliana L. and tobacco.

(https://www.google.co.in/search?q=bioremediation+images)

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PhytohydraulicsThe use of plants to control the migration of

subsurface water through the rapid uptake of large volumes of water by the plants.

Plants - acting as natural hydraulic pumps.

A dense root network established near the water table can transpire up to 300 gallons of water per day.

This fact has been utilized to decrease the migration of contaminants from surface water into the groundwater (below the water table) and drinking water supplies.

(Rooh et al. 2007; Bizily et al., 2008)

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Wonder species of transgenic yellow poplar

(Rooh et al. 2007; Bizily et al. 2008).

Five years old popular transpire about 100 liters of water daily and act as a good clarifier.

The genes MerA and MerB were isolated from mercury resistant bacteria which synthesizes the enzymes mercuric iron reductase and incorporated into popular to make it transgenic.

The transgenic poplar with these genes released 50 times more elemental mercury (Hg) than the untransformed plantlets.

Transgenic plants were significantly more tolerant to methylmercury and other organomercurials compared to the untransformed plants.

They were released from the plants by phytovolatalization.

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All plant mechanisms work together


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Applications


Hazardous waste remediation

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Applications


Waste water treatment

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Plant species identified for phytoremediation of heavy metals

(Source: http://en.wikipedia.org/wiki/List_of_hyperaccumulators)

Plant Species Accumulation rates (in mg/kg) /d.w.

Heavy metals

A-Accumulator P-Precipitator T-Tolerant

Barley 1000Al A, P, T

Vicia faba 100 Al A, P

Indian Mustard 1000-1200 Ag P, T

Sunflower 150 Cr A, P, T

Popular 1500 Ni A, P, T, H

Tomato 550 MnT, H

Brassica napus 800 Hg P, T, H

Spanich 750 Pb P, T, H

Salix sp. 1800 Se A, P

Trifolium Red Clover 650 Zn T, H

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Research trial

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Leading users of remedial technologies2008

2007

2006

2005

2004

2003

2002

2001


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Case study

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Case study

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Results

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The process of bioremediation is slow. Time required is in day to months.

Heavy metals are not removed completely.

For in situ bioremediation site must have soil with high permeability.

It does not remove all quantities of contaminants.

Disadvantages of bioremediation

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Lab strains become food source for soil protozoa.

Inability of GEMs to contact the compounds to be degraded.

Failure of GEMs to survive/compete indigenous

microorganisms.

Contaminant solubility may be increased leading to greater

environmental damage and the possibility of leaching.

A stronger scientific base is required for rational designing of

process and success.

Disadvantages cont.

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Disadvantages cont.

Growing conditions required by the plant (i.e., Climate, geology,

altitude, temperature).

Tolerance of the plant to the pollutant.

Contaminants collected in ageing tissues may be released back into

the environment in autumn.

Contaminants may be collected in woody tissues used as fuel.

Time taken to remediate sites far exceeds that of other technologies.

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Conclusion

Bioremediation and phytoremediation are powerful tools

available to clean up contaminated sites.

Regardless of which aspect of bioremediation that is used; this

technology offers an efficient and cost effective way to treat

contaminated ground water and soil.

Its advantages generally outweigh the disadvantages, which is

evident by the number of sites that choose to use this

technology and its increasing popularity.

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