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04/07/2023 1
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
04/07/2023 2
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
04/07/2023 3
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)
04/07/2023 4
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!
04/07/2023 5
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?
04/07/2023 6
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.
04/07/2023 7
Bioremediation makeseffective better approach possible.
Either by destroying or render them harmless using natural biological activity.
Use of plants
Use of Microorganisms
BIOREMEDIATION
04/07/2023 8
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)
04/07/2023 9
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)
04/07/2023 10
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)
04/07/2023 11
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.
04/07/2023 12
Fragmentation
Cl
Cl C N HOH
Cl
Cl is replaced with OH
OH
OH OH
2, 6-Dichlorobenzonitrile
NH2CH2
(Prasad MNV., 2003)
04/07/2023 13
Mineralization
NH32ClHOH
Completely converted into inorganic forms
Cl
Cl C N
2, 6-Dichlorobenzonitrile
(Prasad MNV., 2003)
04/07/2023 14
IF ANY OF THESE PROCESSES IS TRIGERED / STIMULATED TO GET A LESS CONTAMINATED
PRODUCT
THEN IT IS CALLED AS
BIOREMEDIATION
(Prasad MNV., 2003)
04/07/2023 15
Bioremediation Effectiveness• Depends on:
• Microorganisms
• Environmental factors
• Contaminant type & state
(Prasad MNV., 2003)
04/07/2023 16
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)
04/07/2023 17
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)
04/07/2023 18
Microorganisms can live at different pH conditions
(Bodishbaugh, D.F., 2006)
04/07/2023 19
MO’s can live at any temperature conditions
(Bodishbaugh, D.F., 2006)
04/07/2023 20
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)
04/07/2023 21
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
04/07/2023
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
04/07/2023 23
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)
04/07/2023 24
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.
04/07/2023 25
Bioremediation Strategies
(Barathi S and Vasudevan N, 2001)
04/07/2023 26
Bioremediation Strategies
In situ Bioremediation(at the site)
Ex situ Bioremediation(away from the site)
(Barathi S and Vasudevan N, 2001)
04/07/2023 27
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)
04/07/2023 28
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
04/07/2023 29
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
04/07/2023 30
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)
04/07/2023 31
Insitu Engineered bioremediation typesBioventing
involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria.
(Vidali,M., 2001)
04/07/2023 32
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)
04/07/2023 33
• 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)
04/07/2023 34
Ex situ engineered bioremediation Strategies
(Source: http://ndpublisher.in/ndpjournal.php?j=IJAEB)
04/07/2023 35
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)
04/07/2023 36
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)
04/07/2023 37
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)
04/07/2023 38
Bioremediation using bioreactor System
(Rittmann,B.E and McCarty,P.L.2001)
04/07/2023 39
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)
04/07/2023 41
Biodegradation of hydrocarbons and petroleum
Source: https://www.google.co.in/search?q=bioremediation+images
04/07/2023 42
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
04/07/2023 43
Percent use of different techniques for remediation in India
Source: WHO
04/07/2023 44
Review of bioremediation strategies
(Rittmann B E and McCarty P L, 2001)
04/07/2023 45
PHYTOREMEDIATION
04/07/2023 46
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)
04/07/2023 47
Phytoremediation
Phytoextraction
1
Phytovolatilization
2
Phytostabilization
3
Rhizodegradation
Rhizofiltration
4
5
5 mechanisms based on the fate of contaminants
04/07/2023 48
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.
(Source: https://www.google.co.in/search?q=bioremediation+images)
04/07/2023 49
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
(Source: https://www.google.co.in/search?q=bioremediation+images)
04/07/2023 50
Case study
04/07/2023 51
Physicochemical properties of untreated and treated effluents
04/07/2023 52
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)
04/07/2023 53
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.
04/07/2023 54
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)
04/07/2023 55
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.
04/07/2023 56
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.
04/07/2023 57
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)
04/07/2023 58
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)
04/07/2023 59
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.
04/07/2023 60
All plant mechanisms work together
(Source: https://www.google.co.in/search?q=bioremediation+images)
04/07/2023 61
Applications
(Source: https://www.google.co.in/search?q=bioremediation+images)
Hazardous waste remediation
04/07/2023 62
Applications
(Source: https://www.google.co.in/search?q=bioremediation+images)
Waste water treatment
04/07/2023 63
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
04/07/2023 64
Research trial
04/07/2023 65
Leading users of remedial technologies2008
2007
2006
2005
2004
2003
2002
2001
(Source: https://www.google.co.in/search?q=bioremediation+images)
04/07/2023 66
Case study
04/07/2023 67
Case study
04/07/2023 68
Results
04/07/2023 69
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
04/07/2023 70
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.
04/07/2023 71
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.
04/07/2023 72
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.
04/07/2023 73