BIOTECHNOLOGY FOR

ENVIRONMENT PROTECTION

Irfan D. Prijambada
Fac. of Agriculture,
Gadjah Mada University
 THE BEGINNINGS
 World War II brings an unprecedented
growth in the economy and business
 Breakthroughs in organic chemistry
 War effort and postwar economic boom
 Huge volumes of wastes generated by this
evolving industry
 Lack of knowledge on environmental and
health ramifications of waste disposal
 LANDFILLS
 Majority of waste is deposited in
landfills
 First “sanitary” landfills developed in
1920s
 Solid wastes are spread out in thin
layers, compacted, and covered daily
with fresh clay or plastic
Solved problem of foul smell and reduced
incineration needs
But by 1960s, evident that not capable of
containing groundwater contamination
 LANDFILLS
 Modern landfills are lined with clay and
plastic before being filled with garbage
 Bottom is covered with a second
impermeable liner, usually made of
several layers of clay, thick plastic, and
sand
 This liner collects leachate, rainwater
contaminated as it percolates through the
solid waste, which is then pumped from
bottom of the landfill, stored in tanks, and
sent to a sewage treatment plant
 LANDFILLS
 Anaerobic conditions are created
within landfill waste
 Slow stabilization of waste mass occurs,
producing methane
 Explosive and toxic over long periods of
time
 One study found that aerobic degradation of
waste within a landfill can significantly
increase the rate of waste decomposition
and settlement, decrease production of
methane gas, reduce level of toxic organics
in leachate, and decrease amount of
leachate that need treatment
RESULTS OF MISMANAGEMENT
OF WASTE
 Rachel Carson publishes Silent
Spring in 1962
 Provoked widespread public alarm with
her attack on pesticide usage,
emphasizing the unintended ecological
consequences of pesticide use
 Illustrated interconnected web of life
and how such “elixirs of death” were
stored in humans
 CONTAMINATION INCIDENTS

 Accident in Japan CONTAMINATION INCIDENTS IN JAPAN

 Accident in USA CONTAMINATION INCIDENTS IN USA

CONTAMINATION INCIDENTS IN
JAPAN
 Two cases in Japan make worldwide
headlines
 Hundreds paralyzed due to mercury
poisoning caused by eating shellfish
affected by products of a chemical
plant
 Rash of miscarriages blamed on use of
rice-cooking oil contaminated with Poly
Chlorinated Biphenyls (PCBs)

CONTAMINATION INCIDENTS
CONTAMINATION INCIDENTS IN
USA
 Love Canal, August 1977
 Black sludges bleed through basement walls in
suburban subdivision of Niagara Falls, NY
 Reports of benzene fumes in kitchens,
headaches, skin problems, respiratory
discomfort
 Shortly, dioxin detection, miscarriages and birth
defects
 Government pays for evacuation, at cost of $30
million
CONTAMINATION INCIDENTS IN
USA
 Evacuation process piecemeal over three years
amidst climate of high tension, misinformation,
broken promises
 May 19, 1980 – Love Canal activists take two
government representatives hostage overnight
 Caused by one hundred thousand drums of
chemical waste dumped into an abandoned canal
by Hooker Chemical and Plastics Corporation
 Shows that some kind of regulation is needed
MOST COMMON CONTAMINANTS

 Commercial Hydrocarbons
 gasoline
 diesel and jet fuel
 naptha: raw material used in industry
 domestic heating oil
 Chemicals called BTEX compounds
 Benzene, Toulene, Ethylene, Xylene
 MOST COMMON CONTAMINANTS 2
 Organo-halogenated compounds
(solvents)
 trichloroethylene, tetrachloroethane, etc…
 Heavy hydrocarbons
 crude oil: pipeline, tanker and rail spills
 heavy fuels from electric plants
 tars
 creosotes used in wood treatments
MOST COMMON CONTAMINANTS 3

 Heavy metals
 Explosives
 CHEMICALS WHICH ARE
DIFFICULT TO DECOMPOSE
 Trichloroethylene (TCE) - threatens
water supplies
 Perchloroethylene (PCE) - a dry-
cleaning solvent
 PCBs and Dioxin
 Arsenic, chromium, and selenium
(these have been stabilized by
bacteria in the laboratory)
 DDT
 POLLUTED SITES

 Accidental spills
 Service stations
 Old Air Force bases
 Storage tanks and pipelines
 Chemical plants and other
industrial sites
 Unauthorized dump sites
 BIOREMEDIATION
 Bioremediation is the use of living
microorganisms to degrade
environmental contaminants in the soil
and groundwater into less toxic, or
nontoxic materials.
 These microorganisms can be indigenous,
commercial bacterial mixtures (“bag of
bugs” or “bug ‘n a bag”) or may be
genetically engineered.
 BIOREMEDIATION 2
 Bacteria feed on organic waste and
derive nutrition for growth and
reproduction. This is familiar to all as
the decay of dead animals and
vegetable matter.
 Municipal wastewater treatment plants
have been using this technology for
decades. Bioremediation is an
application of the same principles in a
different setting.
 BIOREMEDIATION 3
 Over time, “Mother Nature” usually
heals herself. Adding large amounts
of certain enzymes and bacteria
hastens the decay. Utilizing
bioremediation speeds up the
process by increasing the rate of
bacterial metabolism and growth.
 USES OF BIOREMEDIATION

 Bioremediation can be used to

decompose or degrade:
 Crude oil spills
 Sewage effluent
 Chlorinated and non-chlorinated
solvents in the industrial areas
 USES OF BIOREMEDIATION 2

Coal Products: phenols and

cyanide
BTEX compounds
Agricultural chemicals and
pesticides in groundwater and
rivers
 USES OF BIOREMEDIATION 3

 Gasoline and fuel oil

contamination
 Creosote contaminants
(wood preservatives)
 Ethylene glycol (antifreeze),
methanol, methylethylketone
(MEK), ethers
 REASONS TO USE
BIOREMEDIATION
Bioremediation can be cost effective
because:
 Contamination can often be treated
in place, minimizing site
disturbance.
 Natural microbial processes can be
used at some sites.
 EFFECTIVENESS
 Biodegradation is not very
effective at sites with high
concentrations of the following
materials which are toxic to
microorganisms
 Metals-solidification/stabilization
is the usual treatment process
 Highly chlorinated organics
 Inorganic salts
 DISPOSING OF HEAVY METALS
 Heavy metals are not
biodegradable, but bacteria can
be used to concentrate them into
a more easily disposable form.
 Uranium: iron-eating bacteria
can remove low levels of
radioactive waste from water.
 Mercury: experiments with
bacteria are on-going.
 MICROORGANISM TYPES
 There are large numbers of
microorganisms that can use
many of the toxic chemicals as a
source of nutrients and energy.
Some examples include:
 Bacteria
 Yeast
 Fungi
SOME MICROORGANISMS USED
IN BIOREMEDIATION
Microorganism Characteristics Significance
Yeast aerobic/ Degrades complex
micro-aerophilic compounds
Cyanobacteria aerobic/ Self-sustaining,
micro-aerophilic/ light is primary
anaerobic energy source
Oligotrophs aerobic RemovesTRACE
concentrations of
organic substances
TYPICAL BACTERIA SPECIES INCLUDE:
IN DESCENDING ORDER OF OCCURRENCE)

• Pseudomas
• Arthobacter
• Alcaligenes
• Corynbacterium
• Flavobacterium
• Achrombacter
• Acinetobacter
• Micrococcus
• Nocardia
• Mycobacterium
EXAMPLES OF MICROBES USED FOR
SPECIFIC CHEMICALS
Compound Name Microorganisms Conditions
Aliphatics Mixed culture and Aerobic
(non-halogenated) activated sludge
Ex. Acrylonitrile
Aliphatics Marine bacteria, Aerobic +
(halogenated) sewage sludge, Anaerobic
Ex. soil bacteria,
Trichloroethane methanogens
Aromatic Pseudomonas spp., Aerobic +
compounds Bacillus spp., Anaerobic
Ex. BTEX, Rhodococcus spp.,
creosol, phenol Mycobacterium
spp.
 BENEFICIAL CHARACTERISTICS

 Beneficial characteristics of
bacteria for bioremediation must
include the following:
 Consume organic waste
 Grow and reproduce rapidly in selected
environment
 Digest the waste quickly and
completely
BENEFICIAL CHARACTERISTICS 2

 Work without causing odors or

poisonous compounds
 Non-pathogenic - (Does not cause
disease in humans or animals)
 CLASSES OF BIOREMEDIATION

 Aerobic (with oxygen) -

Microorganisms use available
atmospheric oxygen to function.
Food sources are converted to
energy by the transfer of
electrons to oxygen, which is an
electron acceptor.
 CLASSES OF BIOREMEDIATION
2
 Anaerobic (without oxygen) -
Microorganisms break down
chemical compounds to release the
energy required to function. As
electron acceptors, they utilize:
 nitrates
 sulfates
 carbon dioxide
 ferrous metals (such as iron)
 HOW BIOREMEDIATION
WORKS...

 Many naturally occurring

microorganisms can digest
organic materials such as fuels
or solvents and convert them to:
 carbon dioxide
 water
 smaller, less toxic organic compounds
Basic Metabolism Process
of Bacteria
ENERGY
CARBON
NUTRIENTS
SOURCE SOURCE

Growth NEW
and CELL
CELL MASS
Reproduction
CO2
Catalyzed by Enzymes H2O
 Schematic Diagram of
Biodegradation
1.Microbe 2. 3.
CO2+H2O
CO2+H2O
Oil

CO2+H2O
Microorganisms
Microorganisms eat release CO2 and
Microorganisms
oil and other organic H20
digest oil and
contaminants.
convert it to CO2
and H 0
 OPTIMIZATION

 To optimize and accelerate the

bioremediation of contaminants
found in water and soil,
selectively adapted microbes
are combined with:
 Food - organic waste containing
water (moisture content between
30-80%)
 added nutrients (nitrogen,
phosphorous, sulfur)
 OPTIMIZATION 2
 Oxygen if required (aerobic
types) 3-5 pounds of oxygen per
pound of hydrocarbon to be
converted
 Moderate pH - between 6-9,
neither too acidic nor too alkaline
 Moderate Temperatures - 50o
to 100o F
 OXYGEN DEMAND VALUES

 Oxygen demand values are used

to measure biological treatment
processes.
 Biological Oxygen Demand
(BOD) measures the amount of
oxygen necessary for microbes
to remove waste in wastewater
in 5 days at 20oC.
 OXYGEN DEMAND VALUES 2

 Chemical Oxygen Demand

(COD) measures a chemical’s
ability to oxidize toxic chemicals
in 3 hours.
 The difference between the two
gives the operating efficiency of
a biological process.
THE EFFECT OF pH ON THE GROWTH OF
SPECIFIC MICROORGANISMS

Microorganism Optimum pH
BACTERIA:
Pseudomonas aeruginosa 6.6 – 7.0
Bacillus alcolophilus 10.6
Nitrosomas spp. 8.0 – 8.8
Thiobacillus thiooxidans 2.0 – 3.5
ALGAE:
Cynidium caldarium 2.0
FUNGI:
Physarum polycephalum 5.0
 OPTIMIZATION 3

 Enzymes, chemical catalysts to

break waste materials into smaller
pieces
 Surfactants (detergents, for
example)
 TECHNOLOGY SELECTION
CRITERIA
 The bioremediation technology
for a site is determined by:
 Microorganisms present
 Site Condition
 Quantity and Toxicity of
Contaminants
 CATEGORIES OF
BIOREMEDIATION
Bioremediation treatment applications
fall into 2 categories:
 in situ - soil or groundwater is treated in
the location where found. This is usually the
most cost effective method, but can also be
slower and hard to manage.
 ex situ - requires the excavation of soil or
pumping of groundwater before treatment.
 EXAMPLES OF IN SITU
BIOREMEDIATION
 Bio-venting: air and nutrients are
pumped into the soil through
injection wells to flush out
contaminants.
 Air Sparging: air or oxygen is
pumped into the groundwater to
flush out contaminants - the air
increases the oxygen concentration
and enhances biological degradation.
AIR SPARGING
 EXAMPLES OF IN SITU
BIOREMEDIATION 2
 Injection of Hydrogen Peroxide:
sprinklers or a system of pipes deliver
the chemical to the soil.
 Extraction Wells: remove the
groundwater to an aboveground water
treatment system where nutrients and
oxygen are added. Injection wells return
the conditioned water to the subsurface
where microorganisms degrade the
contaminants.
TYPICAL in situ Bioremediation
System
Nutrient
Injection Recover
/ Water
Well y Well
Oxygen Treatmen
Addition t
New
Water
Table
Contaminated Zone

Old
Water
Table
 EXAMPLES OF EX SITU
BIOREMEDIATION
Slurry Phase: a large tank, or
“bio-reactor” contains the soil,
water, and added nutrients or
oxygen to keep the
microorganisms in the optimum
environment to degrade
contaminants.
 BIOREACTOR
Vapor out
Agitator
Contaminated
soil
Temperature
Contaminated control
liquid
Nutrient
Liquid outlet

Soil to
Air inlet drying
 EXAMPLES OF EX SITU
BIOREMEDIATION 2
 Solid phase: soil remains on the
site, but is placed in above-ground
treatment areas where moisture,
heat, and nutrients or oxygen are
added.
 SOLID PHASE EX SITU
BIOREMEDIATION 3
Landfarming:
Contaminated soils are excavated
and spread onto a pad. Moisture
and nutrients are controlled. This
is the most widely used
bioremediation technique.
 LANDFARMING
Contaminated
soil
Air Filter/ Tank
Pump

Gravel layer
 SOLID PHASE EX SITU
BIOREMEDIATION 3
Soil Biopile:
The contaminated soil is piled in
large heaps and air is pulled
through with vacuum pumps.
 BIOPILES

Nutrient/
Gravel moisture
layer

Contaminated soil

Impermeable Leachate
layer collection
 SOLID PHASE EX SITU
BIOREMEDIATION 3
Composting:
Biodegradable waste is mixed with a
bulking agent such as straw, hay, or
corn cobs, which facilitates the delivery
of water and nutrients.
The three types of composting are:
* Static pile
* Mechanically agitated in-vessel
* Windrow composting
 DIAGRAM OF BIOREMEDIATION

Bioremediation

in situ ex situ

Engineered Intrinisic Landfarming Bioreactor

Biostimulation Bioaugmentation

Adding Oxygen Adding Oxygen Adding

•Bioventing and Nutrients Oxygen,
•Biosparging Nutrients
and Bacteria
 REMEDIATION TIME

 in situ bioremediation time

depends on the extent, depth, and
concentration of the
contamination. It varies from 1 - 6
years.
 ex situ remediation for easily
biodegradable contaminants or
when bioreactors are used can
take as little as 1-7 months.
 CASE STUDIES IN
BIOREMEDIATION (1)
Van Nuys airport in Southern California
 High concentrations of petroleum
hydrocarbons
 Bioremediation and bioventing were used
 Pollutant levels dropped 80% in 90 days
 Site no longer considered a risk
 Passive remediation continues until microbes
exhaust their ‘food’ supply
 CASE STUDIES IN
BIOREMEDIATION (2)
Minnesota Department of Transportation
 Biomounds used to clean petroleum wastes
 Mounds utilize indigenous bacteria
 Petroleum contaminants provide the energy
 Manure provides the nutrients
 Wood chips allow the entry of oxygen
 Plastic sheeting provides warmer
temperatures for bacteria growth
 CASE STUDIES IN
BIOREMEDIATION (3)
Alaska
 Bioventing used to clean diesel fuels at
Shemya Air Force station
 Landfarming succesfully demonstrated
at Fairbanks to treat soil at the airport,
and is now being used at other sites in
the state
 CASE STUDIES IN
BIOREMEDIATION (4)
Hilo, Hawaii
 Biosparging and land treatment
used to degrade hydrocarbon
contaminated soils and
groundwater.
 CASE STUDIES IN
BIOREMEDIATION (5)
Fort Polk, Louisiana
 Landfarming used continuously for over
10 years to treat petroleum spills.
 Plants sensitive to petroleum wastes
are used to give indication of
completeness of the process.
 Cleaned soil is removed and more
contaminated soil is added.
 ISSUES FOR DISCUSSION

 Bioremediation is not always the

best form of clean-up. Specific site
analysis must be done first.
 Bioremediation is big business
for companies. Microbial mixtures
can be patented and sold for much
more than the cost of growing the
cultures.
 ISSUES FOR DISCUSSION (2)

 Not all bacteria or microbes are

good.
 Some remediation studies have
shown that there is spontaneous
mutation in some bacterial
populations after remediation
efforts. Could this be “bad” for
humans, animals, and plants?
 ISSUES FOR DISCUSSION (3)

 Some companies are using

genetically altered
microorganisms. This leads to a
discussion of genetics, natural
selection, and ethics.
 Is it OK to bring in exogenous
microbes? What if they don’t die
after the contaminat is degraded?
 METHODS FOR ANALYSIS OF DNA

Size and structure Degree of relatedness

of individual DNA between molecules by
molecules hybridization procedures

Drawbacks:
Plasmid DNA i. Lack of stability in some strains
ii. Strains not containing plasmids
iii. plasmid transfer among strains

PCR-based techniques

Chromosomal DNA  Gene sequencing

 Profiling from electrophoresed
PCR-products
 DNA FINGERPRINTING OF ORGANISM

PCR-based methods
Amplified Ribosomal DNA Restriction Analysis (ARDRA)

Principle:
Based on the specificity of inserted DNA
in transgenic organisms
The inserted DNA are amplified by PCR
and then electrophoresed to obtein
patterns which can be mathematically
analysed to establish clusters.

Level of resolution at species level,

is valuable and reliable for
phylogenetical and ecological
studies.