Bioremediation (Microbial)

Elective Course: MSc (Bot), Sem-4, Paper-4

(Biotechnological & Environmental Use of Plants)

Prof. AMRITESH C. Shukla

Botany Dept, Lucknow University
 Definition as per
Environmental Protection Agency (EPA)
 Bioremediation is defined as the process
whereby organic wastes are biologically
degraded under controlled conditions to an
innocuous state, or to levels below
concentration limits established by regulatory
authorities.
 It uses naturally occurring microorganisms
like bacteria and fungi or plants to degrade or
detoxify substances hazardous to human
health and/or the environment.
 Definitions
 Bioremediation is any process that uses organisms
(microorganism, algae and plant) or their enzymes to
return the polluted environment to its original condition.
 Biodegradation is the use of these organisms in the
degradation of different pollutants.
 Xenobiotic compounds are chemical compounds found in
an organism but it is not normally produced or expected
to be present in it.
 Cometabolism: in this process the microorganism
produces an enzyme to utilizes its nutrients, but by
chance this enzyme can degrade a pollutant.
 Bioremediation is a triple-corners process:
Solid
Inorganic Organic Liquid
Pollutants
Gas

Environments Organisms

Soil Microorganisms
Water Plants
Air Enzymes
 Bioremediation related topics
Environments

Organisms Pollutants
Bioremediation of
Phytoremediation metals polluted
environment
Bioremediation
Enzymes
Cells immobilization
immobilization
Bioremediation
Biosurfactants techniques
Enzymes separation
and identification
 Kinds of Bioremediation
Different kinds of bioremediation technologies are currently being
used for soil treatment and many more innovative approaches
involving bioremediation are being developed. Considering the
similarity in their cross-media transfer potential, listed below are
a few examples of bioremediation technologies and processes:
 Natural Attenuation  Biodegradation
 Aerobic/Anaerobic
 Composting
biodegradation
 Land Treatment  Bioreactors
 Bioscrubbers  Dehalogenation
 Methanotrophic Process (in  Binding of Metals
Situ)
 Plant Root Uptake  Fungi Inoculation Process
(Phytoremediation)  Slurry Phase
 Solid Phase Bioremediation bioremediation
 Bio Wall for Plume  Bioventing (Vapor
Decontamination (In Situ) Extraction)
 Key Features of Bioremediation
 Most bioremediation treatment technologies destroy
the contaminants in the soil matrix.
 These treatment technologies are generally designed to
reduce toxicity either by destruction or by transforming
toxic organic compounds into less toxic compounds.
 Indigenous micro-organisms, including bacteria and
fungi, are most commonly used. In some cases, wastes
may be inoculated with specific bacteria or fungi known
to biodegrade the contaminants in question. Plants may
also be used to enhance biodegradation & stabilize the
soil.
 The addition of nutrients or electron acceptors (such as
hydrogen peroxide or ozone) to enhance growth and
reproduction of indigenous organisms may be required.
 Key Features of Bioremediation…contd.
 Field application of bioremediation may involve:
– Excavation/ dig-out
– Soil handling
– Mixing of contaminated soils
– Aeration of contaminated soils
– Injection of fluid
– Extraction of fluid
– Introduction of nutrients and substrates
 Bioremediation - Technology description
Bioremediation involves the use of micro-organisms to
chemically degrade organic contaminants. Aerobic processes use
organisms that require oxygen to be able to degrade
contaminants. In some cases, additional nutrients such as
nitrogen and phosphorous are also needed to encourage the
growth of biodegrading organisms. A biomass of organisms –
which may include entrained constituents of the waste, partially
degraded constituents, and intermediate biodegradation
products – is formed during the treatment process (United States
Environmental Protection Agency, 1990)

Although bioremediation is applied in many different ways, the

description of typical solid phase bioremediation, composting,
bio-venting, and traditional in situ biodegradation is provided
here, besides the description of a few common bioremediation
technologies.
 Bioremediation – Concept (Contd..)
Recent studies in molecular biology and ecology offer
opportunities for more efficient biological processes to
clean-up of polluted water and land areas

 Bioremediation allows natural processes to clean up

harmful chemicals in the environment.
 Microscopic “bugs” or microbes that live in soil and
groundwater like to eat certain harmful chemicals.
 When microbes completely digest these chemicals,
they change them into water and harmless gases such
as carbon dioxide.
 Schematic

Bioremediation is an option that offers the

possibility to destroy or render harmless various
contaminants using natural biological activity.
 Fungi
Bacteria Fungi

Actinomycetes Actinomycetes Actinomycetes

 Bioremediation - Basic facts
 The microorganisms may be indigenous to a
contaminated area or they may be isolated from
elsewhere and brought to the contaminated site
 Contaminant compounds are transformed by living
organisms through reactions that take place as a part of
their metabolic processes.
 Biodegradation of a compound is often a result of the
actions of multiple organisms.
 Microorganisms must enzymatically attack the pollutants
 Bioremediation can be effective only where environ-
mental conditions permit microbial growth and activity
 Manipulation of environmental parameters needed for
microbial growth & degradation to proceed at a faster
rate.
 Stages of Microbial Purification
1- Isolation of the microorganism

2- Purification of the obtained isolates

3- Identification of the microbial isolate

4- Optimization of the biodegradation
conditions
5- Determination of the biodegradation
efficiency
6- Identification of the biodegradation products.

7- Cell or enzyme immobilization.

8- Enzyme identification.
 Factors of Bioremediation

The control and optimization of bioremediation

process is a complex system of many factors:
 existence of a microbial population
 availability of contaminants to the microbial
population
 the environment factors (type of soil, temperature,
pH, the presence of oxygen or other electron
acceptors, and nutrients).
 Microbial Populations
 Microorganisms can be isolated from almost
any environmental conditions. Microbes will
adapt and grow at subzero temperatures, as
well as extreme heat, desert conditions, in
water, with an excess of oxygen, and in
anaerobic conditions, with the presence of
hazardous compounds or on any waste stream.

 The main requirements are an energy source

and a carbon source. Because of the
adaptability of microbes and other biological
systems, these can be used to degrade or
remediate environmental hazards.
 Types of microorganisms
 Aerobic Grows in presence of oxygen, degrade pesticides and
hydrocarbons, both alkanes and polyaromatic compounds.
Many of these bacteria use the contaminant as the sole source
of carbon and energy. Examples: Pseudomonas, Alcaligenes,
Sphingomonas, Rhodococcus, and Mycobacterium.
 Anaerobic Grows in absence of oxygen. are not as frequently
as aerobic, degrade polychlorinated biphenyls (PCBs),
dechlorination of the solvent trichloroethylene (TCE), and
chloroform. Examples: Escherichia coli, Staphylococcus genus,
Clostridium genus
 Methylotrophs Aerobic bacteria that grow utilizing methane
for carbon and energy. The initial enzyme in the pathway for
aerobic degradation, methane monooxygenase, has a broad
substrate range and is active against a wide range of
compounds, including the chlorinated aliphatics
trichloroethylene. Examples: Bacillus methanicus, Pseudomonas
methanica, Methanomonas methanooxidans and M
ethylococcus capsulatus
 Composition of a microbial cell (%).

Carbon 50 Sodium 1.0

Nitrogen 14 Calcium 0.5
Oxygen 20 Magnesium 0.5
Hydrogen 8.0 Chloride 0.5
Phosphorous 3.0 Iron 0.2
Sulfur 1.0 All others 0.3
Potassium 1.0
 The Science – How Does it Work?
Microbial Metabolism refers to all the chemical reactions that happen
in a cell or organism. All living processes are based on a complex
series of chemical reactions.
Anabolism – Building complex molecules into simpler molecules.
In anabolism, chemicals taken up by the microorganism are used to
build various cell parts. Carbon and nitrogen are the basic chemicals
in the proteins, sugars and nucleic acids that make up microbial cells.
Microorganisms take up carbon and nitrogen from the soil, water, and
air around them. In order to take up nutrients and make them into cell
parts, a microorganism needs energy. This is where catabolism
comes in.
Catabolism – Breaking complex molecules into simpler molecules.
Catabolism allows microorganisms to gain energy from the chemicals
available in the environment. Although most microorganisms are
exposed to light and to chemical energy sources, most rely on
chemicals for their energy. When chemicals break down, energy is
released. Microorganisms use this energy to carry out cellular
functions, such as those involved in anabolism.
 Bio-stimulation
Although the microorganisms are present in contaminated soil,
they cannot necessarily be there in the numbers required for
bioremediation of the site. Their growth and activity must be
stimulated:
 Bio-stimulation usually involves the addition of nutrients and oxygen
to help indigenous microorganisms.
 These nutrients are the basic building blocks of life and allow
microbes to create the necessary enzymes to break down the
contaminants. All of them will need nitrogen, phosphorous & carbon.
 Carbon is the most basic element of living forms and is needed in
greater quantities than other elements. In addition to hydrogen,
oxygen, and nitrogen it constitutes about 95% of the weight of cells.
 Phosphorous and sulfur contribute with 70% of the remainders. The
nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon
to phosphorous is 30:1.
 Biostimulation (Contd..)
 For degradation it is necessary that bacteria and the
contaminants be in contact. This is not easily
achieved, as neither the microbes nor contaminants
are uniformly spread in the soil.
 Some bacteria are mobile and exhibit a chemotactic
response, sensing the contaminant and moving
toward it.
 Other microbes such as fungi grow in a filamentous
form toward the contaminant.
 It is possible to enhance the mobilization of the
contaminant utilizing some surfactants such as
sodium dodecyl sulphate
 Methods of Bioremediation:
There are two broad classes of bioremediation-

1) In-situ bioremediation – Onsite treatment for

detoxification

2) Ex-situ bioremediation- Of site treatment toxic materials

3) Sometimes bioremediation takes place by natural ways

& means called Intrinsic bioremediation or natural
attenuation.
Summary of strategies
If appropriate biodegrading microorganisms are not
present in soil or if microbial populations have been
reduced because of contaminant toxicity, specific
microorganisms can be added as “introduced organisms”
to enhance the existing populations. This process is
known as bio-augmentation. Scientist is now capable of
creating ‘super bugs’ organisms that can degrade
pollutants at extremely rapid rates. Such organisms can
be developed through successive adaptations under
laboratory condition or can be genetically engineered.
 Advantages of bioremediation
 Bioremediation is perceived by the public as an acceptable waste treatment
process. Microbes able to degrade the contaminant increase in numbers
when the contaminant is present; when the contaminant is degraded, the
biodegradative population declines.
 It is safe as the residues for the treatment are usually harmless products
and include carbon dioxide, water, and cell biomass.
 It is useful for the complete destruction of a wide variety of contaminants.
This eliminates the chance of future liability associated with treatment and
disposal of contaminated material.
 Instead of transferring contaminants from one environmental medium to
another, for example, from land to water or air, the complete destruction of
target pollutants is possible.
 It can often be carried out on site, without disruption of normal activities, no
need to transport waste off site.
 It does not require too much of sophisticated equipments.
 Bioremediation can prove less expensive than other technologies that are
used for clean-up of hazardous waste.
 Disadvantages of bioremediation
 Bioremediation is limited to those compounds that are
biodegradable. Not all compounds are susceptible to rapid and
complete degradation.
 Biological processes are often highly specific. Important site factors
required for success include the presence of metabolically capable
microbial populations, suitable environmental growth conditions,
and appropriate levels of nutrients and contaminants.
 It is difficult to extrapolate from bench and pilot-scale studies to full-
scale field operations.
 Research is needed to develop and engineer bioremediation
technologies for complex mixtures of contaminants that are not
evenly dispersed in the environment.
 Bioremediation often takes longer than other treatment options,
such as excavation and removal of soil or incineration.
 THANK YOU

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