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BIOTECHNOLOGY FORENVIRONMENT PROTECTION
Irfan D. Prijambada
Fac. of Agriculture,Gadjah Mada University
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THE BEGINNINGS
World War II brings an unprecedentedgrowth 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 andhealth ramifications of waste disposal
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LANDFILLS
Majority of waste is deposited inlandfills
First sanitary landfills developed in
1920sSolid wastes are spread out in thin
layers, compacted, and covered dailywith fresh clay or plasticSolved problem of foul smell and reduced
incineration needs
But by 1960s, evident that not capable ofcontaining groundwater contamination
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LANDFILLS
Modern landfills are lined with clay andplastic before being filled with garbage
Bottom is covered with a second
impermeable liner, usually made ofseveral layers of clay, thick plastic, andsand This liner collects leachate, rainwater
contaminated as it percolates through thesolid waste, which is then pumped frombottom of the landfill, stored in tanks, andsent to a sewage treatment plant
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LANDFILLS
Anaerobic conditions are createdwithin landfill wasteSlow stabilization of waste mass occurs,
producing methaneExplosive 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 decompositionand settlement, decrease production ofmethane gas, reduce level of toxic organicsin leachate, and decrease amount of
leachate that need treatment
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RESULTS OF MISMANAGEMENTOF WASTE
Rachel Carson publishes SilentSpring in 1962 Provoked widespread public alarm with
her attack on pesticide usage,emphasizing the unintended ecologicalconsequences of pesticide use
Illustrated interconnected web of lifeand how such elixirs of death werestored in humans
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CONTAMINATION INCIDENTS
Accident in Japan CONTAMINATION INCIDENTS IN JAPAN
Accident in USA CONTAMINATION INCIDENTS IN USA
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CONTAMINATION INCIDENTS INJAPAN
Two cases in Japan make worldwideheadlinesHundreds paralyzed due to mercury
poisoning caused by eating shellfishaffected by products of a chemicalplant
Rash of miscarriages blamed on use of
rice-cooking oil contaminated with PolyChlorinated Biphenyls (PCBs)
CONTAMINATION INCIDENTS
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CONTAMINATION INCIDENTS INUSA
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
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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
CONTAMINATION INCIDENTS INUSA
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MOST COMMON CONTAMINANTS
Commercial Hydrocarbons
gasoline
diesel and jet fuel naptha: raw material used in industry
domestic heating oil
Chemicals called BTEXcompounds
Benzene, Toulene, Ethylene,Xylene
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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
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MOST COMMON CONTAMINANTS 3
Heavy metals
Explosives
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CHEMICALS WHICH AREDIFFICULT TO DECOMPOSE
Trichloroethylene (TCE) - threatenswater supplies
Perchloroethylene (PCE) - a dry-cleaning solvent
PCBs and Dioxin
Arsenic, chromium, and selenium(these have been stabilized bybacteria in the laboratory)
DDT
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POLLUTED SITES
Accidental spills
Service stations
Old Air Force bases Storage tanks and pipelines
Chemical plants and otherindustrial sites
Unauthorized dump sites
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BIOREMEDIATION
Bioremediation is the use of livingmicroorganisms to degradeenvironmental contaminants in the soil
and groundwater into less toxic, ornontoxic materials.
These microorganisms can be indigenous,commercial bacterial mixtures (bag ofbugs or bug n a bag) or may begenetically engineered.
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BIOREMEDIATION 2
Bacteria feed on organic waste andderive nutrition for growth andreproduction. This is familiar to all as
the decay of dead animals andvegetable matter.
Municipal wastewater treatment plants
have been using this technology fordecades. Bioremediation is anapplication of the same principles in a
different setting.
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BIOREMEDIATION 3
Over time, Mother Nature usuallyheals herself. Adding large amountsof certain enzymes and bacteria
hastens the decay. Utilizingbioremediation speeds up theprocess by increasing the rate of
bacterial metabolism and growth.
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USES OF BIOREMEDIATION
Bioremediation can be used todecompose or degrade:
Crude oil spills
Sewage effluent
Chlorinated and non-chlorinatedsolvents in the industrial areas
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USES OF BIOREMEDIATION 2
Coal Products: phenols andcyanide
BTEX compoundsAgricultural chemicals and
pesticides in groundwater and
rivers
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USES OF BIOREMEDIATION 3
Gasoline and fuel oilcontamination
Creosote contaminants(wood preservatives)
Ethylene glycol (antifreeze),
methanol, methylethylketone(MEK), ethers
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REASONS TO USEBIOREMEDIATION
Bioremediation can be cost effectivebecause:
Contamination can often be treatedin place, minimizing sitedisturbance.
Natural microbial processes can beused at some sites.
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EFFECTIVENESS
Biodegradation is not veryeffective at sites with highconcentrations of the following
materials which are toxic tomicroorganisms
Metals-solidification/stabilization
is the usual treatment processHighly chlorinated organics
Inorganic salts
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DISPOSING OF HEAVY METALS
Heavy metals are notbiodegradable, but bacteria canbe 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.
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MICROORGANISM TYPES
There are large numbers ofmicroorganisms that can use
many of the toxic chemicals as asource of nutrients and energy.Some examples include:
BacteriaYeast
Fungi
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SOME MICROORGANISMS USEDIN BIOREMEDIATION
Microorganism Characteristics Significance
Yeast aerobic/
micro-aerophilic
Degrades complex
compounds
Cyanobacteria aerobic/micro-aerophilic/
anaerobic
Self-sustaining,light is primary
energy source
Oligotrophs aerobic RemovesTRACE
concentrations oforganic substances
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TYPICAL BACTERIA SPECIES INCLUDE:IN DESCENDING ORDER OF OCCURRENCE)
PseudomasArthobacter
Alcaligenes
Corynbacterium Flavobacterium
Achrombacter
Acinetobacter Micrococcus
Nocardia
Mycobacterium
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EXAMPLES OF MICROBES USED FORSPECIFIC CHEMICALS
Compound Name Microorganisms Conditions
Aliphatics
(non-halogenated)
Ex. Acrylonitrile
Mixed culture and
activated sludge
Aerobic
Aliphatics(halogenated)
Ex.Trichloroethane
Marine bacteria,sewage sludge,
soil bacteria,methanogens
Aerobic +Anaerobic
Aromatic
compoundsEx. BTEX,
creosol, phenol
Pseudomonas spp.,
Bacillus spp.,Rhodococcus spp.,
Mycobacterium
spp.
Aerobic +
Anaerobic
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BENEFICIAL CHARACTERISTICS
Beneficial characteristics ofbacteria for bioremediation mustinclude the following:Consume organic waste
Grow and reproduce rapidly in selectedenvironment
Digest the waste quickly andcompletely
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BENEFICIAL CHARACTERISTICS 2
Work without causing odors orpoisonous compounds
Non-pathogenic - (Does not causedisease in humans or animals)
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CLASSES OF BIOREMEDIATION2
Anaerobic (without oxygen) -Microorganisms break down
chemical compounds to release theenergy required to function. Aselectron acceptors, they utilize:
nitratessulfates
carbon dioxide
ferrous metals (such as iron)
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HOW BIOREMEDIATIONWORKS...
Many naturally occurringmicroorganisms can digest
organic materials such as fuelsor solvents and convert them to: carbon dioxide
water
smaller, less toxic organic compounds
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Basic Metabolism Processof Bacteria
Growth
and
Reproduction
Catalyzed by Enzymes
CELL
ENERGY
SOURCENUTRIENTS
CARBONSOURCE
NEWCELL
MASS
H2O
CO2
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Schematic Diagram ofBiodegradation
Oil
MicrobeCO2+H2O
CO2+H2O
CO2+H2O
Microorganisms eat
oil and other organic
contaminants.
Microorganisms
digest oil and
convert it to CO2
and H 0
Microorganisms
releaseCO2 and
H20
1. 2. 3.
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OPTIMIZATION
To optimize and accelerate thebioremediation of contaminantsfound in water and soil,
selectively adapted microbesare combined with: Food - organic waste containing
water (moisture content between30-80%)
added nutrients (nitrogen,phosphorous, sulfur)
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OPTIMIZATION 2
Oxygen if required (aerobictypes) 3-5 pounds of oxygen perpound of hydrocarbon to be
convertedModerate pH - between 6-9,
neither too acidic nor too alkaline
Moderate Temperatures - 50oto 100o F
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OXYGEN DEMAND VALUES
Oxygen demand values are usedto measure biological treatmentprocesses.
Biological Oxygen Demand(BOD) measures the amount of
oxygen necessary for microbesto remove waste in wastewaterin 5 days at 20oC.
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OXYGEN DEMAND VALUES 2
Chemical Oxygen Demand(COD) measures a chemicals
ability to oxidize toxic chemicalsin 3 hours.
The difference between the two
gives the operating efficiency ofa biological process.
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THE EFFECT OF pH ON THE GROWTH OFSPECIFIC 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
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OPTIMIZATION 3
Enzymes, chemical catalysts tobreak waste materials into smallerpieces
Surfactants (detergents, forexample)
TECHNOLOGY SELECTION
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TECHNOLOGY SELECTIONCRITERIA
The bioremediation technologyfor a site is determined by:
Microorganisms present Site Condition
Quantity and Toxicity of
Contaminants
CATEGORIES OF
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CATEGORIES OFBIOREMEDIATION
Bioremediation treatment applicationsfall into 2 categories:
in situ- soil or groundwater is treated in
the location where found. This is usually themost cost effective method, but can also beslower and hard to manage.
ex situ - requires the excavation of soil orpumping of groundwaterbefore treatment.
EXAMPLES OF IN SITU
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EXAMPLES OF IN SITUBIOREMEDIATION
Bio-venting: air and nutrients arepumped into the soil throughinjection wells to flush out
contaminants. Air Sparging: air or oxygen is
pumped into the groundwater to
flush out contaminants - the airincreases the oxygen concentrationand enhances biological degradation.
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AIR SPARGING
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TYPICAL i it Bi di ti
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TYPICAL in situ BioremediationSystem
Contaminated Zone
OldWaterTable
NewWaterTable
WaterTreatmen
t
Nutrient/
OxygenAddition
Recovery Well
InjectionWell
EXAMPLES OF EX SITU
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EXAMPLES OF EX SITUBIOREMEDIATION
Slurry Phase: a large tank, orbio-reactor contains the soil,water, and added nutrients oroxygen to keep themicroorganisms in the optimum
environment to degradecontaminants.
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Contaminatedliquid
Contaminatedsoil
BIOREACTOR
Liquid outlet
Soil todrying
Temperature
control
AgitatorVapor out
Air inlet
Nutrient
EXAMPLES OF EX SITU
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EXAMPLES OF EX SITUBIOREMEDIATION 2
Solid phase: soil remains on thesite, but is placed in above-groundtreatment areas where moisture,heat, and nutrients or oxygen areadded.
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SOLID PHASE EX SITUBIOREMEDIATION 3
Soil Biopile:
The contaminated soil is piled inlarge heaps and air is pulledthrough with vacuum pumps.
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BIOPILES
Nutrient/moistureGravel
layer
Leachatecollection
Impermeablelayer
Contaminated soil
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SOLID PHASE EX SITUBIOREMEDIATION 3
Composting:
Biodegradable waste is mixed with a
bulking agent such as straw, hay, orcorn cobs, which facilitates the deliveryof water and nutrients.
The three types of composting are:
* Static pile* Mechanically agitated in-vessel
* Windrow composting
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DIAGRAM OF BIOREMEDIATION
Adding OxygenBioventingBiosparging
Adding Oxygenand Nutrients
Biostimulation
AddingOxygen,Nutrients
and Bacteria
Bioaugmentation
Engineered Intrinisic
in situ
Landfarming Bioreactor
ex situ
Bioremediation
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REMEDIATION TIME
in situ bioremediation timedepends on the extent, depth, andconcentration of the
contamination. It varies from 1 - 6years.
ex situ remediation for easilybiodegradable contaminants orwhen bioreactors are used cantake as little as 1-7 months.
CASE STUDIES IN
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CASE STUDIES INBIOREMEDIATION (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
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CASE STUDIES INBIOREMEDIATION (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
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CASE STUDIES INBIOREMEDIATION (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
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CASE STUDIES INBIOREMEDIATION (4)
Hilo, Hawaii
Biosparging and land treatment
used to degrade hydrocarbon
contaminated soils and
groundwater.
CASE STUDIES IN
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CASE STUDIES INBIOREMEDIATION (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.
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ISSUES FOR DISCUSSION (2)
Not all bacteria or microbes aregood.
Some remediation studies have
shown that there is spontaneousmutation in some bacterialpopulations after remediation
efforts. Could this be bad forhumans, animals, and plants?
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ISSUES FOR DISCUSSION (3)
Some companies are usinggenetically alteredmicroorganisms. This leads to a
discussion of genetics, naturalselection, and ethics.
Is it OK to bring in exogenous
microbes? What if they dont dieafter the contaminat is degraded?
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METHODS FOR ANALYSIS OF DNA
Size and structure
of individual DNA
molecules
Degree of relatedness
between molecules by
hybridization procedures
Plasmid DNA
Chromosomal DNA
Drawbacks:i. Lack of stability in some strains
ii. Strains not containing plasmids
iii. plasmid transfer among strains
PCR-based techniques
Gene sequencing Profiling from electrophoresed
PCR-products
DNA FINGERPRINTING OF ORGANISM
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DNA FINGERPRINTING OF ORGANISM
PCR-based methods
Principle:Based on the specificity of inserted DNA
in transgenic organismsThe inserted DNA are amplified by PCRand then electrophoresed to obteinpatterns which can be mathematicallyanalysed to establish clusters.
Level of resolution at species level,is valuable and reliable forphylogenetical and ecologicalt di
Amplified Ribosomal DNA Restriction Analysis (ARDRA)