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CREDIT SEMINAR-I ENS-691

BIOREMEDIATION
Various strategies of pollution mitigation

By: Rachit Raghava Kashyap

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

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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

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

Is there a better approach?

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Better approaches

Destroy them completely, if possible

Transform them into harmless substances

Methods already in use

• High temperature incineration.
• Chemical decomposition like dechlorination.

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.

BIOREMEDIATION

Use of Microorganisms

Use of plants
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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)

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

Cl C N HOH

2, 6-Dichlorobenzonitrile
OH

Cl C N Cl

Cl is replaced with OH
(Prasad MNV., 2003)
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Fragmentation

Cl

Cl C N HOH

2, 6-Dichlorobenzonitrile
OH

OH OH Cl

NH2CH2
Cl is replaced with OH
(Prasad MNV., 2003)
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Mineralization

Cl

Cl C N

2, 6-Dichlorobenzonitrile

HOH 2Cl NH3

Completely converted into inorganic forms

(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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• Aerobic bacteria:
Microorganisms
• 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.

(Bodishbaugh, D.F., 2006)

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Microorganisms can live at different pH conditions

(Bodishbaugh, D.F., 2006)

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MO’s can live at any temperature conditions

(Bodishbaugh, D.F., 2006)

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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 Aerobic, minimum air-filled pore
space for aerobic degradation 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 Not too toxic

of soil
Heavy metals 700ppm Total content 2000ppm

(Vidali , 2007)
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Type of contaminants
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
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Some m.o. involved in the biodegradation of organic pollutants

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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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

2 types

Engineered Bioremediation
 Intentional changes Doing nothing

Intrinsic Bioremediation
 Simply allow biodegradation to
occur under natural conditions

(Wood TK , 2008)
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Intrinsic Bioremediation
- a bioremediation under natural conditions

• 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)
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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 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)
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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 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)

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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.
By use of genetic engineering:
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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
Use of bioremediation strategies over different years by developed
countries ( in percent)

80

70

60

50

40

30

20
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
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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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PHYTOREMEDIAT
ION
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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.

(Source: https://www.google.co.in/search?q=bioremediation+images)
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5 mechanisms based on the fate of contaminants

5
1
4
2 3

Rhizofiltration
Phytoextraction

Phytovolatilization Rhizodegradation

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

• Plants are hydroponically grown in clean water

rather than soil, until a large root system has
1 developed

• Water supply is substituted for a polluted water

supply to acclimatize the plant
2

• They are planted in the polluted area where the roots

uptake the polluted water and the contaminants along
3 with it

• As the roots become saturated they are harvested and

disposed of safely
4

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

(Source: https://www.google.co.in/search?q=bioremediation+images)
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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.
 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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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)
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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.
(Rooh et al. 2007; Bizily et al., 2008)
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Wonder species of transgenic yellow poplar

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.

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

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All plant mechanisms work together

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

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

(Source: https://www.google.co.in/search?q=bioremediation+images)
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Plant species identified for phytoremediation of heavy metals

Plant Species Accumulation rates (in Heavy A-Accumulator P-
mg/kg) /d.w. metals 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
T, H
Tomato 550 Mn

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
(Source: http://en.wikipedia.org/wiki/List_of_hyperaccumulators)
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Research trial
Leading users of remedial technologies
2008

2007

2006

2005

2004

2003

2002

2001

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(Source: https://www.google.co.in/search?q=bioremediation+images)
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Case study
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Case study
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Results
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Disadvantages of bioremediation

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.

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Disadvantages cont.
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.
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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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