Bioremediation and phytoremediation

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
6/23/2014 1

Outline of Presentation
Introduction
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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Yes
But 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 makes
effective 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)
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2,6-Dichlorobenzonitrile is an herbicide and is
toxic for humans.

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
(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
microbial
Activity
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
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Some m.o. involved in the biodegradation of organic pollutants

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)
6/23/2014 25

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 to
occur under natural conditions
(Wood TK , 2008)
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Doing nothing

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
6/23/2014 29

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 types
Bioventing
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)
6/23/2014 35

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 System
Biopiles 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)
6/23/2014 38

Case study: Oil degradation
Oil-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.
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By 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
6/23/2014 41

Use of bioremediation strategies over differentyears by developed
countries ( in percent)
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
20
30
40
70
60
50
80
Source: http://ndpublisher.in/ndpjournal.php?j=IJAEB6/23/2014 42

Percent use of different techniques for remediation in India
6/23/2014 43
Source: WHO

Review of bioremediation strategies
(Rittmann B E and McCarty P L, 2001)
6/23/2014 44

PHYTOREMEDIAT
ION
6/23/2014 45

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.
6/23/2014 46

Phytoextraction
1
Phytovolatilization
2
Phytostabilization
3
Rhizodegradation
Rhizofiltration
4
5
5 mechanisms based on the fate of contaminants
6/23/2014 47

Phytoextraction
Plant 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.
6/23/2014 48

Rhizofiltration
It 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
6/23/2014 49

Physicochemical properties of untreated and treated
effluents
6/23/2014 51

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)
6/23/2014 52

Phytodegradation
It 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.
6/23/2014 53

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)
6/23/2014 54

Rhizodegradation
It is the breakdown of organic contaminants in the soil by soil dwelling
microbes which is enhanced by the rhizosphere’s presence.
Rhizosphere = soil + root + microbes
Symbiotic relation
Also called:
Enhanced rhizosphere biodegradation
Phytostimulation
Plant assisted bioremediation
Sugars, 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.
6/23/2014 55

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.6/23/2014 56

Phytovolatilization
Plants 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)
6/23/2014 57

Phytohydraulics
The 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.
6/23/2014 58
(Rooh et al. 2007; Bizily et al., 2008)

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.
6/23/2014 59

All plant mechanisms work together
6/23/2014 60

Applications
Hazardous waste remediation
6/23/2014 61

Applications
Waste water treatment
6/23/2014 62

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 1000
Al 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 Mn
T, 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
6/23/2014 63

Leading users of remedial technologies
2008
2007
2006
2005
2004
2003
2002
2001
6/23/2014 65

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.
6/23/2014 71

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.
6/23/2014 72

Bioremediation and phytoremediation

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