In situ bioremediation

EKT3113
REMEDIATION TECHNOLOGY

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Bioremediation
• A technology that encourages growth and reproduction of
indigenous microorganisms (bacteria and fungi) to enhance
biodegradation of organic constituents in the saturated
zone

• Generally requires a mechanism for stimulating and

maintaining the activity of the microorganisms, e.g.,
addition of an electron acceptor (oxygen, nitrate); nutrients
(nitrogen, phosphorus); and an energy source (carbon)
Biodegradation triangle
In Situ Bioremediation
In Situ Bio Remediation

In Place Microbial Method to Fix

Biological agents (bacteria, fungi, plants, or their enzymes) used to

clean up pollution in the environment.

Reference: Lisa Alvarez-Cohen, Civil and Environmental Engineering

University of California, Berkeley, Earth Science Division, LBNL
Microbial Metabolism
• Microorganisms use the organic contaminants for their
growth.
• Major nutrients such as C, N, P, S required for growth
• However, the organic compounds (contaminant) not
necessarily be a substrate for growth
Microbial Metabolism
• Two categories of transformation:
• Growth linked
• Biodegradation provides carbon and energy to support growth
• Not- linked to multiplication
• Carbon is obtained from respiration in order for the cells to maintain their viability
In Situ Bioremediation
• Bioventing
• One of the most common approaches in soil
• Supply air and nutrients via wells
• Takes advantage of indigenous microorganisms
• In situ biodegradation
• Supply air and nutrients by circulating aqueous solutions
through contaminated soils or groundwater
• Biosparging
• Injection of air below the water table  increases
groundwater oxygen concentrations and mixing in
saturated zone
• Bioaugmentation
• Addition of indigenous or exogenous microorganisms
• Limits to use: competition and necessity
• Biostimulation
• Natural Attenuation or Intrinsic Bioremediation
Applying In Situ Bioremediation

Natural Bioaugmentatio
Biostimulation
Attenuation n

Have Have Need

Microbes Microbes Microbes

Have food Have food

Need food or
and and
nutrients
nutrients nutrients
In Situ Bioremediation
• Biostimulation - adding nutrients (N, P, etc.) and electron acceptors
(e.g., O2) to microbial environment to stimulate the activity of
microorganisms
• Bioaugmentation - adding exogenous microbes to the subsurface
where organisms able to degrade a specific contaminant are deficient
Microbial reactions and pathways
• Degradation
• The initial substrate no longer exists
• Mineralization
• Complete conversion of the organic structure to inorganic
forms such as CO2, H2O, Cl-
• Detoxification
• Transformation of the compounds to some intermediate
form that is non-toxic or less toxic
• Activation
• The process of forming toxic end products or intermediate
products
Reactions
• Dechlorination—the chlorinated compound becomes an electron
acceptor; in this process, a chlorine atom is removed and is
replaced with a hydrogen atom.

• Hydrolysis—frequently conducted outside the microbial cell by

exoenzymes. Hydrolysis is simply a cleavage of an organic
molecule with the addition of water.

• Cleavage—cleaving of a carbon–carbon bond is another

important reaction. An organic compound is split or a terminal
carbon is cleaved off an organic chain.
• Oxidation—breakdown of organic compounds using an
electrophilic form of oxygen.

• Reduction—breakdown of organic compounds by a

nucleophilic form of hydrogen or by direct electron delivery.

• Dehydrogenation—an oxidation–reduction reaction that

results in the loss of two electrons and two protons, resulting
in the loss of two hydrogen atoms.
• Dehydrohalogenation—similar to dechlorination, results in the
loss of a hydrogen and chlorine atom from the organic
compound.

• Substitution—these reactions involve replacing one atom with

another.
IN SITU BIOREMEDIATION SYSTEM
 BIOREMEDIATION
The use of microorganism to destroy or reduce the
concentration of harzadous wastes on a contaminated site.

Applications of Bioremediation
Clean up of :
- Ground water
- Soils
- Lagoons
- Sludges
- Waste streams
Factors influence bioremediation process
 Methods of Bioremediation
In Situ Ex Situ
Bioremediation Bioremediation
Treating contamination at the site often used (less movement) Remove contamination and treat elsewhere

Intrinsic
Engineered Engineered
Bioremediation

-Bioventing
-Land Farming
-Bioaugmentation
-Composting
-Biostimulation
 Biostimulation

Consists of adding nutrients such as

- Nitrogen
to microbial environment to
- Phosphorus Stimulate the activity of
- Oxygen microorganism
(increase microbial metabolism)
- Other electron acceptors
(Nitrate, Sulfate)
 Bioaugmentation
-Addition of microorganism
-That has the capability to degrade the contaminants at high rates
-To remain active under prevailing site conditions.

Applications :

-Area that where contaminants are uncontrolled

-Site conditions are extreme
-High concentration or toxicity of contaminants
 Land Farming

• Contaminated soil is spread over a prepared bed along with

some fertilizers and occasionally rotated.
• It stimulates the activity of bacteria and enhances the
degradation of oil.
 COMPOSTING
Organic contaminants are transformed into stabilized, innocuous by-products by microorganisms
under anaerobic and aerobic conditions.
Five areas that must be “controlled”:
Feedstock
Moisture
and nutrient Particle size. Oxygen flow. Temperature.
content.
balance.

Can be done by:

Aerated (Turned) Windrow

Aerated Static Pile Composting In-Vessel Composting
Composting
 BIOREACTORS
Refers to any containers or vessels where biological degradation of contaminants is isolated and
controlled.

Commonly cylindrical, ranging in size from litres to cubic meters and are often made of stainless
steel.

Types of bioreactors:

Continuous Stirred Bubble Column

Airlift Bioreactor.
Tank Bioreactor. Bioreactor.

Fluidized Bed Packed Bed

Photo-Bioreactor.
Bioreactor. Bioreactor.
 BIOVENTING

in situ remediation technology that uses microorganisms to

biodegrade organic constituents adsorbed on soils in the
unsaturated zone by drawing oxygen
 BIOVENTING
LIMITATIONS
High soil moisture / low permeability
Low temperature
Build up of vapours in basements
Difficult to aerate saturated soil
APPLICATIONS
Remediation of soils contaminated
with fuel
Remediation of soils contaminated
with chlorinated solvents, pesticides,
wood preservatives & other organic
chemicals
 BIOFILTRATION

Biofiltration is a bioremediation technology in which vapor-

phase organic contaminants, such as fuel hydrocarbons, are passed through
a soil bed where they sorb to the soil surfaces and are broken down by
microorganisms in the soil
 BIOFILTRATION

LIMITATIONS APPLICATIONS

Low temperature
Treatment of non-chlorinated VOCs
Moisture levels, pH, temperature & and fuel hydrocarbons
filter conditions
Control odours from compost piles
Accumulation of excess bacteria
Intrinsic bioremediation
• Type of bioremediation that manages the innate
capabilities of naturally occuring microbes without taking
any engineering steps to enhance the process.

Pump and treat

• Involve installing one or more wells to extract the
contaminated groundwater.
• Groundwater is pumped from the “extraction wells” to
the ground surface.
 SCREENING CRITERIA

Biodegradability of Mineralization potential of

contaminants the compounds

Specific microbial,
substrate, and other Biogeochemical Availability of nutrients
conditions parameters

Site’s hydrogeologic Extent and distribution of

characteristics contaminants
Advantages and Limitations of Bioremediation
Advantages
• Can be done on site
• Often less expensive
• Eliminates waste permanently
• Long-term liability risk reduced
• Positive public acceptance
• Can be coupled with other treatment
techniques
Limitations
• Some chemicals cannot be bioremediated
such as heavy metals, radionuclides, and
some chlorinated compounds
• Microbial metabolism of contaminants may
produce toxic metabolites
• Extensive monitoring requirements
• Scientific intensive which needs site-specific
requirements
 Bacteria
Mycetes

Major Groups
of Fungi
Microbial

Algae
Protozoa
 Bacteria stands out the
most compare to other
four types

Rapid growth capacity Degradation of a variety

of contaminants
 Purpose of classification of
bacteria
Ability to grow in the presence or absence
of oxygen
Cell morphological structure
Type of energy and carbon sources.
 A) Ability To Grow In The
Presence or Absence Of
•
Oxygen.
It is an essential biochemical trait.
• It lead to three different categories:

 Aerobes
 Anaerobes
 Facultative Anaerobes
 Facultative
Aerobes Anaerobes
Anaerobes
 Require oxygen for  Does not require  Can grow in both
their basic survival, oxygen for growth. the presence or
growth, and the absence of oxygen. 
process of
reproduction.
 Example of
 Example of aerobes  Example of facultative
bacteria: anaerobes bacteria: anaerobes bacteria:

Bacillus

Escherichia coli
Staphylococcus
 B) Morphological Types
Bacilli or rod-shaped bacteria
Cocci or spherical-shaped bacteria
Spirilla or spirals (commonly found in
freshwater)

• Spore forming genera present among aerobic &

anaerobic bacteria
 Function of Endospores
• Some bacilli persist in unfavourable conditions
• Part of normal life cycle of bacterium
• Endure in adverse environments ( great resistance to
prolonged desiccation & high temperature )
• Persist in dormant state
• Lack of water & substrate led to death of vegetative cells
• Spore germinates & new organism emerges once the
condition conducive to vegetative growth return
C) Microorganisms

MICROORGANISMS

Autotrophic Heterotrophic
- Obtain energy from sunlight or by the - Require organic substrates to serve
oxidation of inorganic compounds and as sources of energy and carbon,
their carbon by the assimilation of CO2 dominate the soil microflora

Chemoautotrophs
Photoautotrophs
- Obtain the energy needed for growth
- Energy is derived from sunlight
from the oxidation of inorganic materials
What is acclimatization lag???

Acclimatization lag is actually initial period.

During this period, no obvious biotic changes of

contaminant levels take place or may be too slow
due to various reasons such as:
• Low concentration of microbial
population.
• Low concentration of contaminant so it
will not induce the relevant enzymes, or
their chemical structure may be so
unusual that they cannot interact with
the active enzyme sites.
 MEASUREMENT OF THE INDIGENOUS
MICROBIAL ACTIVITY
Function: Method for evaluating potential toxic or inhibitory conditions at a site

Ground water bacterial Typical soil microbial counts range:

counts range: • 10 power of 3 – 10 power of 7 CFUs
per gram of soil.
• 10 power of 2 – 10 power of
• Counts below 10 power of 3
5 colony forming units organisms per gram of soil at
(CFUs) per milliliter of contaminated sites indicates
sample stressed microbial population.
 Low bacteria counts: Potential toxicity problem or a stressed microbial population.
 Higher counts: Healthy microbial population
NUTRIENT
• Carbon makes up a large fraction of the total protoplasmic material of
a microbial cell.
• The protoplasmic material is composed of the nucleus, cell
membrane, and the cytoplasm.
 Carbohydrate
(C, H, O) &
Amino Acids Protein
(COOH, NH3)
& Pyrimidines

50%
Nucleic Acids,
Purines &
Vitamins

Hydrogen (8%), Oxygen (20%),

Nitrogen (14%)
 HETEROTROPH MICROORGANISM AUTOTROPH MICROORGANISM

• Group of microorganisms (yeast, • An organism able to make its own

moulds & bacteria) that use organic food. Autotrophic organisms take
carbon as food (as opposed to inorganic substances into their bodies
autotrophs like algae that use and transform them into organic
sunlight) and are found in every nourishment.
type of water.
• Carbon is usually supplied by
organic substrates (organic Photoautotroph Chemoautotroph
contaminants) in the case of
bioremediation.
Light Chemical
energy energy
LIEBIG’S LAW OF THE MINIMUM
• Smallest quantity will be the limiting
factor of growth.
• The essential constituent that is
present in the smallest quantity
relative to the nutritional requirement
of microorganisms will become the
limiting factor of growth.
• Biological survival and growth are limited by a variety of environmental
factors, and the least amount of supply, that is, the most important
component that limiting biological growth, therefore must increase the
supply of this important component, so that the organism can grow.

• For example, various nutrients affect plant growth, while nutrients that
are least supplied in the environment have the greatest restrictions on
plant growth. The amount of this substance will limit the growth of
plants.
A) TEMPERATURE
• As temperature increases, the rates of chemical as well as
biochemical reactions generally increase.
• This phenomenon is referred to as Arrhenius behavior
• The same phenomenon also occurs with microorganisms and the
myriad of chemical and biochemical reactions that constitute
“microbial activity,” but only to a point.
• Beyond some optimum temperature, the activity of any organism
declines precipitously.

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ARRHENIUS BEHAVIOUR

48
Three categories of microorganisms are defined, based upon
temperature optima :
1) Psychrophiles: Psychrophilic (or cryophilic) organisms have
an optimum temperature of 15 ± 5°C, and a minimum
temperature of 0°C or below. Psychrophiles usually possess
membranes rich in unsaturated fatty acids, a feature which
is alleged to provide a more fluid structure at low
temperatures.
2) Mesophiles: Mesophilic organisms have an optimum
temperature between 25°C and 40°C. Microorganisms
commonly found effective in bioremediation perform over a
temperature range of 10 to 40°C.
3) Thermophiles: Thermophilic organisms have temperature
optima above 45°C. Thermophiles have membranes rich in
saturated fatty acids.

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B) pH
1. pH will affect the microorganism's ablitiy to :

 conduct cellular function

 cell membrane transport
 equilibrium of catalyzed reactions
 protonmotive forces
responsible for energy production within the cells
2. Most bacteria tolerate pH 5 - 9 (prefer pH 6.5 - 7.5)
- Neutrophiles bacteria

E. coli
- Acidophilic bacteria (optimum near pH 2.5)

Thiobacillus thioxidans
 - Alkalophilic bacteria ( pH 10 -12)

Bacillus firmus

3. Metabolic activities of microorganisms produces organic acids and HCl.

4. concentration of organic compounds are present in the subsurface

( alkalinity) , pH control may be necessary to sustain its continuous
biodegradation.
C) Moisture Content

Why it is important?
 It will affects:

› Bioavailability of › Effective toxicity › Movement and

contaminants level of growth stage of
contaminants microorganism

› Transfer of gases
› Species
distribution
› Soil moisture frequently measured as a
gravimetric percentage/reported as field
capacity and these methods provides
information on the “water availability” or
water activity (aw) as defined by biologists
for microbial metabolism.

(formula)
Water activity is basically the ratio of the
system’s vapour pressure to that of pure
water (at the same temperature).
 
Microbial transport of water through the
bacterial plasma membrane:

› Passive process
› Governed strictly by diffusion and
gradient in aw across membrane
REDOX POTENTIAL ( )
•  • 
• 

   

   
• Make use of combination • Natural system
of platinum electrode and are seldom at
some reference electrode. equilibrium
 will cause impact on the mechanisms of microbial
transformation of contaminant during bioremediation.
 The transformation could be

 Aerobic reaction

 Denitrification

 Valence reduction

 Sulfate reduction

 Methanogenesis

 Particular [oxidants] or [reductants] will bring effect on

microbial metabolic activity no matter the but through 3D
conformation.
Scientific and Non-scientific
Factors affecting
bioremediation
 Energy Sources

Bioavailability

Scientific Bioactivity &

Biochemistry

Factor affecting
bioremediation Regulatory

Non- Scientific Research & Technical

Human Resources

Economic & Liability

Factor
 1. ENERGY
SOURCES
• One of the primary variables
affecting the activity of bacteria is
the ability and availability of reduced
organic materials to serve as energy
sources.
• In general, higher oxidation states
correspond to lower energy yields
which thus provide less energetic
incentive for microorganism
degradation.
• The outcomes of each degradation
process depends on microbial,
substrate, and a range of
environmental factor.
2. BIOAVAILABILITY
The rate of microbial cells convert contaminants during
bioremediation that depends on the rate of contaminant uptake
and metabolism and the rate of mass transfer

• Increase microbial conversion capabilities do not lead to

higher biotransformation rates when mass transfer is a
limiting factor
• Example: The contaminating explosives in soil did not
undergo biodegradation process even after 50 years
 Sorption and
The bioavailability of
desorption
contaminant is control
by number of physico-
chemical process Diffusion and
dissolution

• The bioavailability reduce in course of time is usually

due to aging or weather
• Result : Slow diffusion into very small pores and
absorption into organic matter
3. BIOACTIVITY &
vBIOCHEMISTRY
Bioactivity: To indicate the operating state of microbiological processes
v Biodegradation is attempted to be optimized when the bioactivity was enhanced.
v Biochemistry: Chemical processes within and related to living things
v The organisms have different ability to transfer contaminants to both simpler and
more complex molecules
v Favorable and unfavorable biochemical conversion were evaluated in order to
measure and control biochemical pathways in complex environments.
 1. REGULATORY

Create Markets Control Products

NON
SCIENTIFIC Toxic Substance
Control Process
FACTOR Control Act (TSCA)
 2. RESEARCH
& TECHNICAL
Bioremediation technique or
FACTOR
• Some biodegradable contaminants
technology should be given
may contain chemicals such as
specific emphasis to:
chlorinated solvents and polynuclear
aromatic hydrocarbons which are not
easily to be degraded.
• Each waste site or polluted site contain
particular contaminants that need The type of Each of the
specific bioremediation treatment. contaminant polluted site
• However, none of distinct criteria for
evaluating the success or failure of a
particular treatment have been
established yet.
3. HUMAN RESOURCES
FACTORS
• Bioremediation is a New Technology
• Lacked of Trained Human Resources
• Universities do not offer
qualifications in Bioremediation
Engineering
• A successful Bioremediation Program
requires a Multi-disciplinary
Approach and Integrating Fields.
4. ECONOMIC AND LIABILITY FACTOR

Slow venture capital to invest in the technology.

The research and development cannot be well developed.
Tighter restriction and regulatory agencies lead to greater
risk from liability.
 Credit to: All SEL3
Students (Batch 2016)