NOTES BY Nimisha J Chennakadan : environmental biotechnology

Unit 3: PART 1 : Bioremediation & Waste management GEMs in environment; Role of environmental
biotechnology in management of environmental problems, Bioremediation, advantages and disadvantages; In
situ and ex situ bioremediation; slurry bioremediation; Bioremediation of contaminated ground water and
phytoremediation of soil metals; microbiology of degradation of xenobiotics.
Waste management - Sewage and waste water treatment and solid waste management, chemical measure of
water pollution, conventional biological treatment, role of microphyte and macrophytes in water treatment;
Recent approaches to biological waste water treatment, composting process and techniques, use of composted
materials. Microorganisms & Agriculture – Microorganisms in Agricultural waste-water treatment,
Vermiculture, Microbial pesticides.

BIOREMEDIATION
Bioremediation is the process of reduction, elimination, alteration, and transformation of contaminants
present in the natural environment like soil, sediments, air, and water through the application of
microorganisms, fungi, green plants, or their enzymes.
• It is a waste management technique that uses naturally occurring biological organisms to break down
hazardous substances into less toxic or non-toxic forms.
• Bioremediation includes a series of redox reactions for the production of energy within microbial cells for
cell maintenance and reproduction.
• Bioremediation is a global, regional and local application for removing pollutants from the environment
restoring the contaminated sites.
• Bioremediation is the process of reduction, elimination, alteration, and transformation of contaminants
present in the natural environment like soil, sediments, air, and water through the application of
microorganisms, fungi, green plants, or their enzymes.
• It is a waste management technique that uses naturally occurring biological organisms to break down
hazardous substances into less toxic or non-toxic forms.
• Bioremediation includes a series of redox reactions for the production of energy within microbial cells for
cell maintenance and reproduction. Bioremediation is a global, regional and local application for removing
pollutants from the Environment restoring the contaminated sites.
Factors Affecting Bioremediation
1. Concentration of the contaminant
The concentration of the contaminants directly affects microbial activity. Lower the concentration of the
contaminants there will be decreasing rate of degrading enzymes produced by bacteria in the soil. Toxic
effects are observed in presence of higher concentrations of contaminants.
2. Nutrient availability
Carbon, nitrogen, phosphorus, potassium, and calcium are the basic requirement for the growth of
microorganisms, the concentration of the nutrient availability directly affects the degradation of the
contaminants. The excessive presence of nitrogen, potassium, and phosphorus shows a negative impact on
the degradation of hydrocarbons..
3. Characteristics of the contaminated soil
The bioremediation process is significantly affected by the different parameters of the contaminated soil
such as pH, texture, permeability, water holding capacity, temperature, and oxygen availability.

• pH: Optimum pH is required for the bioremediation process which ranges from 6-8. Neutral pH is suitable
for the degradation of petroleum hydrocarbons whereas some fungi and acidophilic microbes degrade
contaminants in an acidic environment.
• Temperature: The degradation of the contaminants is also affected by temperature especially in the case
of hydrocarbons under both in situ and ex-situ conditions. It has been found that a higher temperature of
30°C-40°c increases the bioremediation in the soil as well as in the marine environment.
• Oxygen availability: Oxygen is a very important factor to determine the extent and rate of biodegradation
of contaminants. Aerobic biodegradation is much faster than anaerobic biodegradation. In the majority of
cases, the addition of hydrogen peroxide is used to introduce oxygen. Hydrogen peroxide is about seven
times more soluble in water than oxygen.

Types of Bioremediation

On the basis of removal and transportation of waste

for treatment, bioremediation is of two different
types.

➢ In Situ Bioremediation
➢ Ex Situ Bioremediation

The main difference between in situ and ex situ is that

the in situ refers to the original location whereas the
ex situ refers to the off-site. Furthermore, in situ
methods are less expensive and less manageable
while the ex situ methods are expensive and
manageable.

In situ and ex situ are two methods used to describe

different biological processes such as bioremediation,
and conservation of organisms.
In Situ Bioremediation
In situ remediation is the in-site treatment of contaminants using biological agents. It is a cleanup approach
between microbes and the contaminants directly for biotransformation.
In Situ Bioremediation Techniques:
Bioaugmentation,Biostimulation,Bioslurping,Bioventing,Phytoremediation
1. Bioaugmentation

Bio augmentation is the process of addition of culture microbial population which have the ability to degrade
specific soil and groundwater contaminants.
A technique of bioremediation in which strains of natural or genetically engineered bacteria with unique
metabolic profiles are added to the contaminated site in order to supplement indigenous microflora and speed
up biodegradation. Common application involves bio augmentation for chlorinated contaminants, petroleum
hydrocarbon etc. Microorganisms are isolated either from contaminated sites, historical sites or genetically
modified to support remediation process of contaminated sites.

2. Bio-stimulation

Microbes cannot use pollutants as the only source of energy thus they need to be accessed with supplied
nutrients. Bio-stimulation is the process of environmental modification via addition of limiting nutrients and
electron acceptors like phosphorous, nitrogen, oxygen or carbon in order to stimulate the existing microbial
population which are involved in bioremediation. It is most common remediation approach against
petroleum pollutants in soil.
 3. Bio-sparging

Biosparging the injection of a gas and gas-phase

nutrients pressure into the saturated zone applying
pressure to promote aerobic biodegradation.
It is the most recommended approach for aerobic
degredation of sites affected with lighter to heavier
petroleum contaminants such as oils, diesel, gasoline,
jetfuels etc.
Lighter ones are removed easily but heavier ones due
to minimum level of microbial bioavaibility requires
longer process of treatment.
In it the cost can be reduce by reducing the diameter
of injection point

4. Bioventing

The most common in situ treatment and involves supplying air and nutrients through wells to contaminated
soil to stimulate the indigenous microorganism.
Bioventing is applied for remediation of petroleum hydrocarbons contaminants in soil through air supply to
an unsaturated soil zone using a combination of pumps and blowers for continuous injection of low volumes
of air.
It can be categorized as either aerobic, anaerobic or co-metabolic depending on the amendments used. The
slow removal of air and maintaining 5% oxygen in subsurface is generally practice for bioventing.
 5. Phytoremediation

Phytoremediation is the use of plant and its products

for the decontamination or stabilization of
contaminants and metals from soil. There are
certain varieties of plants which have the ability to
vacuum heavy metals the soil via root and
concentrate them in the stems, shoots, and leaves.
These plants possess genes that regulate the amount
of metals taken up from the soil by roots and
deposited at other locations within the plant.
Depending on the underlying processes,
applicability and types of contaminant,
phytoremediation can be broadly categorized as:
➢ Phytodegradation
➢ Phytostimulation/rhizodegradation
➢ Phytovolatilization
➢ Phytoextraction
➢ Rhizofiltration
➢ Phytostabilization

Ex-Situ Bioremediation
Ex-situ bioremediation or off-site bioremediation is the removal/excavation of contaminants and pollutants by
subsequent transportation of contaminants from one site to another. Similar to in situ techniques, remediation
occurs with the role of microorganisms. These techniques are based on the type of contaminants, site of
pollution, degree of pollution, and cost of treatment.
Techniques in ex situ bioremediation are:
1. Biopile
Biopile is type of remediation process that involves enhancement techniques via above-ground piling of
excavated polluted soil, nutrient amendment, and sometimes aeration to increase the microbial population and
their activity. This technique involves aeration, irrigation, nutrient and leachate collection systems, and a
treatment bed.Biopile can reduce and limit volatilization of low molecular weight (LMW) pollutants, also help
in effective remediation of extreme polluted environments. It is a cost effective approach which ensures
effective biodegradation.
 2. Bioreactor

Bioreactor is an engineered system involving series of biological reactions in which pollutants are fed into the
bioreactor vessel for their degradation that facilitate the growth of biological mass.
Bioreactors maintains suitable controlled environment for the optimum growth conditions that leads to the
proliferation of microbial populations.

3. Composting

Composting is the process of degradation and decaying or organic waste under favorable controlled conditions
with the action waste degrading microorganisms.
Composting is a self-heating, substrate-dense and solid phase treatment process. Microbial population
metabolize the organic waste and degrade it to volume by 50% reduction forming the end product called
compost or humus. Compost is a nutrient rich soil which is very useful in application to the crops and plants
for their effective growth.
The steps involved in the process include sorting and separating, size reduction, and digestion of the refuse.
 4. Land Farming

One of the simplest bioremediation process of

excavation of polluted soil transported to above the
ground surface allowing aerobic biodegradation of
pollutant by autochthonous microorganisms.
The autochthonous microorganisms are stimulated by
tilling process which involves nutrients amendments
(nitrogen, phosphorous etc.), aeration process and
irrigation.
It is also a cost effective approach which requires
minimal environment and energy for treatment of large
volume of polluted soils.

Advantages of Bioremediation
• Complete remediation of harmful contaminants presents in the environment instead to transferring
contaminants from one site to another.
• Cost effective method with minimal requirements of complex tools and equipment
• Environment friendly approach with use of microorganism instead of harmful chemicals
• In majority cases, can be carried out on site reducing transportation cost
• Minimum site destruction and disruption.
Disadvantages of Bioremediation
• Only limited to biodegradable waste and contaminants
• Requires extensive monitoring
• Being a biological process, specificity is a major drawback in terms factors like type of environmental
growth conditions, types of microorganisms, type of nutrient requirements and type of contaminants.
• Possibility of production unknown and potentially toxic byproducts
• Comparatively a time consuming process

Waste management GEMs in environment

GEMs (Genetically Engineered Microorganisms) in Waste Management

Genetically Engineered Microorganisms (GEMs) offer promising solutions for effective waste management
and environmental remediation. Here are some key applications and benefits of GEMs in waste management:

1. Biodegradation of Pollutants
• Enhanced Breakdown: GEMs can be designed to degrade hazardous pollutants more efficiently than
naturally occurring microorganisms.
• Specificity: These microorganisms can target specific contaminants, such as petroleum hydrocarbons,
pesticides, and heavy metals.
2. Bioaugmentation

• Boosting Degradation: Introducing GEMs into contaminated environments can enhance the natural
degradation processes, especially in areas with insufficient native microbial activity.

• Rapid Action: GEMs can significantly speed up the degradation of waste materials.

3. Bioremediation

• Soil and Water Treatment: GEMs are used to clean up contaminated soil and water bodies by breaking
down toxic substances into less harmful or harmless compounds.

• Phytoremediation Support: GEMs can support plant-based remediation techniques by enhancing the
degradation of pollutants in the rhizosphere (root zone).

4. Wastewater Treatment

• Efficient Waste Breakdown: GEMs can be used in wastewater treatment plants to more effectively break
down organic waste and reduce the levels of harmful substances.

• Nutrient Removal: They can be engineered to remove excess nutrients, such as nitrogen and phosphorus,
from wastewater, preventing eutrophication in water bodies.

5. Composting and Organic Waste Management

• Accelerated Composting: GEMs can speed up the composting process of organic waste by enhancing the
breakdown of complex organic materials.

• Improved Quality: The resulting compost is often of higher quality, with better nutrient profiles and
reduced pathogen levels.

6. Industrial Waste Management

• Toxin Degradation: GEMs can be tailored to degrade industrial toxins and byproducts, reducing
environmental pollution from industrial activities.

• Resource Recovery: They can help in recovering valuable resources from industrial waste, making the
waste management process more sustainable and economically viable.

Benefits of Using GEMs in Waste Management

1. Efficiency: GEMs can degrade pollutants more efficiently and faster than natural microorganisms.
2. Specificity: They can be engineered to target specific contaminants, ensuring thorough cleanup.

3. Sustainability: GEMs offer a more sustainable approach to waste management by reducing the reliance
on chemical treatments and physical remediation methods.
4. Cost-Effectiveness: Long-term cost savings due to more efficient waste breakdown and reduced need for
extensive remediation efforts.
5. Environmental Protection: Reduced environmental impact by minimizing the release of harmful
substances and promoting cleaner ecosystems.
Examples of Genetically Engineered Microorganisms (GEMs) in Waste Management
1. Pseudomonas putida

• Application: Biodegradation of organic pollutants such as toluene, xylene, and naphthalene.

 • Modification: Engineered to express specific enzymes that break down aromatic hydrocarbons,
making it effective in cleaning up oil spills and other petroleum-based contaminants.

2. Escherichia coli

• Application: Heavy metal bioremediation.

• Modification: Engineered to express metal-binding proteins that can sequester and precipitate heavy
metals like mercury, cadmium, and lead from contaminated water and soil.

3. Deinococcus radiodurans

• Application: Radiation and heavy metal remediation.

• Modification: Modified to tolerate and remediate radioactive waste and heavy metals, making it
suitable for cleaning up nuclear waste sites.

4. Bacillus subtilis

• Application: Phosphate removal in wastewater.

• Modification: Engineered to enhance the uptake and storage of phosphate, reducing eutrophication
risks in water bodies.
5. Ralstonia eutropha

• Application: Plastic waste biodegradation.

• Modification: Engineered to produce enzymes that break down polyhydroxyalkanoates (PHAs), a

type of biodegradable plastic, aiding in plastic waste management.

6. Saccharomyces cerevisiae (Baker's yeast)

• Application: Bioremediation of industrial effluents.

• Modification: Engineered to degrade and detoxify industrial pollutants such as phenols and heavy
metals, making it useful for treating industrial wastewater.
7. Alcaligenes eutrophus

• Application: Degradation of polychlorinated biphenyls (PCBs).

• Modification: Engineered to express enzymes capable of breaking down PCBs, which are highly toxic
environmental pollutants.

Challenges and Considerations

1. Safety Concerns: Ensuring that GEMs do not pose a risk to human health or the environment.
2. Regulatory Approval: Gaining approval from regulatory bodies for the use of GEMs in waste
management.
3. Public Acceptance: Addressing public concerns and misconceptions about the use of genetically modified
organisms in the environment.

4. Containment: Preventing the unintended spread of GEMs beyond the target area to avoid ecological
imbalance
slurry bioremediation

Slurry bioremediation is a treatment process that involves creating a slurry—a mixture of contaminated soil
or sediment with water and sometimes other additives—and treating it in a controlled environment to enhance
microbial degradation of pollutants. This method is particularly effective for treating soils and sediments with
high concentrations of contaminants.

Steps in Slurry Bioremediation

1. Site Assessment Analyze the contamination levels and types of contaminants present in the soil or
sediment. This step includes Soil sampling, laboratory analysis, and environmental impact assessment.

2. Slurry Preparation Create a homogenous mixture of contaminated soil or sediment with water and
additives. Mixing soil/sediment with water to form a slurry, sometimes adding nutrients or surfactants to
enhance microbial activity.

3. Bioreactor Setup Provide a controlled environment for microbial degradation. The slurry in a bioreactor
that maintains optimal conditions such as aeration, temperature, and pH.

4. Microbial Inoculation (if needed) Enhance the degradation process by introducing specific strains of
microorganisms. Adding selected microbial cultures to the slurry.
5. Monitoring and Control Ensure optimal conditions for microbial activity and degradation. Regular
monitoring of bioreactor conditions, such as oxygen levels, temperature, pH, and adjusting them as needed.
6. Post-Treatment Processing Separate treated slurry into solid and liquid components for further handling.
Dewatering the treated slurry, managing residual solids (e.g., disposal, further treatment), and treating
effluent water if necessary.
Microorganism used :
a) Pseudomonas putida
b) Escherichia coli
c) Deinococcus radiodurans
d) Bacillus subtilis
e) Alcaligenes eutrophus
f) Arthrobacter sp.
g) Sphingomonas sp.
h) Mycobacterium smegmatis
i) Acinetobacter sp.
j) Rhodococcus sp.

Applications of Slurry Bioremediation

1. Oil Spill Cleanup Treatment of soil contaminated by oil spills, utilizing hydrocarbon-degrading
bacteria.
2. Industrial Waste Remediation Cleaning up sediments contaminated by industrial activities, such as
chemical manufacturing or metal plating.
3. Agricultural Runoff Remediation of soil contaminated with pesticides and herbicides from
agricultural runoff.

4. Mixed Contaminant Sites Treating sites with complex mixtures of organic and inorganic pollutants.

Advantages of Slurry Bioremediation

1. Enhanced Degradation Rates

o Controlled conditions in the bioreactor can significantly accelerate the microbial degradation process.

2. Uniform Treatment

o Ensures even distribution of microbes, nutrients, and oxygen, leading to more consistent treatment of
the contaminated material.

3. Flexibility

o Can be tailored to specific contaminants and site conditions by adjusting bioreactor parameters and
microbial communities.

4. Effective for High Concentrations

o Particularly suitable for treating soils and sediments with high concentrations of contaminants that are
challenging to remediate using other methods.

Disadvantages of Slurry Bioremediation

1. Cost Higher operational costs compared to some other bioremediation techniques due to the need for
specialized equipment and controlled conditions.
2. Complexity Requires careful monitoring and management of bioreactor conditions to maintain
optimal microbial activity.
 3. Post-Treatment Handling The treated slurry needs to be dewatered, and the residual solids must be
disposed of or further treated, adding to the complexity and cost.

4. Potential for Secondary Waste The process may generate secondary waste streams (e.g.,
contaminated water) that require additional treatment.

MICROBIAL DEGRADATION OF XENOBIOTICS

Biodegradation or biological degradation is the phenomenon of biological transformation of organic

compounds by living organisms, particularly the microorganisms. Biodegradation basically involves the
conversion of complex organic molecules to simpler (and mostly non-toxic) ones. The term
biotransformation is used for incomplete biodegradation of organic compounds involving one or a few
reactions. Biotransformation is employed for the synthesis of commercially important products by
microorganisms.

Bioremediation refers to the process of using microorganisms to remove the environmental pollutants
i.e. the toxic wastes found in soil, water, air etc. The microbes serve as scavengers in bioremediation.
The removal of organic wastes by microbes for environmental clean-up is the essence of bioremediation.
The other names used (by some authors) for bioremediation are bio-treatment, bio-reclamation and bio-
restoration. It is rather difficult to show any distinction between biodegradation and bioremediation.
Further, in biotechnology, most of the reactions of biodegradation/bioremediation involve xenobiotic.

Xenobiotics are chemicals found in organisms that are not naturally produced or expected to be present in
those organisms' environments. They include pollutants, drugs, and other compounds introduced into the
environment by human activity.

Microorganisms Used in the Degradation of Xenobiotics

1. Pseudomonas putida
2. Escherichia coli
3. Rhodococcus erythropolis
4. Sphingomonas sp.
5. Bacillus subtilis
6. Mycobacterium sp.
7. Arthrobacter sp.
8. Acinetobacter sp.
9. Streptomyces sp.

Examples of xenobiotics include:

1. Pesticides and Herbicides: Synthetic chemicals used to control pests (e.g., insecticides like
chlorpyrifos, herbicides like glyphosate).
2. Pharmaceuticals: Drugs and medications that are intentionally introduced into the body (e.g.,
antibiotics, painkillers, antidepressants).
3. Industrial Chemicals: Synthetic compounds used in manufacturing processes (e.g., solvents like
trichloroethylene, plasticizers like bisphenol A).
4. Personal Care Products: Chemicals found in cosmetics, soaps, and detergents (e.g., parabens,
phthalates).
5. Polycyclic Aromatic Hydrocarbons (PAHs): Found in petroleum products and combustion byproducts
(e.g., benzo[a]pyrene).
6. Heavy Metals: Inorganic compounds that can be toxic at low concentrations (e.g., lead, mercury,
cadmium).
7. Chlorinated Compounds: Chemicals containing chlorine, often persistent in the environment (e.g.,
polychlorinated biphenyls (PCBs), chlorinated solvents).
8. Plastic Additives: Chemicals added to plastics for various purposes (e.g., plasticizers like diethylhexyl
phthalate (DEHP), flame retardants).

Process of Microbial Degradation of Xenobiotics

Microbial degradation of xenobiotics involves several biochemical steps where microorganisms metabolize
and transform synthetic compounds into simpler, less harmful substances.

1. Recognition and Uptake

• Recognition: Microorganisms possess enzymes or transport systems that recognize xenobiotics as
potential substrates.

• Uptake: Xenobiotics are transported across the cell membrane into microbial cells.
2. Phase I Reactions (Initial Metabolism)
• Oxidation: Microorganisms use oxygenases or monooxygenases to introduce oxygen atoms into the
xenobiotic structure, increasing its reactivity.

• Example: Cyanide can be oxidized to less toxic forms like cyanate (OCN⁻) or thiocyanate (SCN⁻) by
microbial enzymes such as cyanide oxygenase.
3. Phase II Reactions (Conjugation)

• Conjugation: Microorganisms attach polar groups (e.g., sulfate, glutathione) to Phase I metabolites,
increasing their solubility.

• Example: Cyanate or thiocyanate produced in Phase I reactions can be conjugated with glutathione to
form less toxic mercapturic acid derivatives.

4. Cleavage and Mineralization

• Cleavage: Enzymatic cleavage breaks down complex xenobiotics into smaller, simpler fragments.

• Mineralization: Further degradation converts these fragments into basic compounds like carbon
dioxide (CO₂), ammonia (NH₃), and water (H₂O).

• Example: Cyanate or thiocyanate derivatives are further degraded to carbon dioxide and nitrogen
compounds through microbial metabolic pathways.

5. Energy and Carbon Utilization

• Energy Production: Microorganisms utilize the carbon and energy derived from xenobiotics for
growth and metabolic processes.

• Example: Microorganisms involved in cyanide detoxification can use the carbon from cyanide
derivatives as a carbon source for energy production and biomass synthesis.

6. Regulation and Optimization

• Gene Regulation: Microorganisms regulate the expression of genes encoding xenobiotic-degrading

enzymes in response to substrate availability.

• Example: Microbial communities in cyanide-contaminated environments upregulate genes encoding

cyanide-degrading enzymes under cyanide stress.

7. Environmental Impact

• Biodegradation Potential: Microbial communities vary in their ability to degrade specific xenobiotics
based on genetic diversity and environmental conditions.

• Example: Certain bacteria and fungi are specialized for cyanide detoxification in contaminated soil
and water environments, contributing to ecosystem resilience.

Example: Cyanide Detoxification

Cyanide (CN⁻) is a toxic compound found in various industrial processes, mining effluents, and certain natural
sources. Microbial degradation of cyanide involves enzymatic pathways that convert cyanide into less toxic
forms, which can be further mineralized.

• Microbial Enzyme: Cyanide oxygenase enzymes in bacteria such as Pseudomonas spp. catalyze the
oxidation of cyanide to cyanate or thiocyanate.

• Phase II Reaction: Conjugation with glutathione forms mercapturic acid derivatives, increasing
solubility and reducing toxicity.
• Mineralization: Further microbial processes degrade cyanate or thiocyanate into carbon dioxide and
nitrogen compounds.

----------------------------------------------------------------------------------------------------------------------------------