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

which is then pumped from bottom of the landfill. thick plastic. usually made of several layers of clay. stored in tanks. rainwater contaminated as it percolates through the solid waste. and sand  This liner collects leachate. and sent to a sewage treatment plant .LANDFILLS  Modern landfills are lined with clay and plastic before being filled with garbage  Bottom is covered with a second impermeable liner.

LANDFILLS  Anaerobic conditions are created within landfill waste  Slow stabilization of waste mass occurs. reduce level of toxic organics in leachate. 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. and decrease amount of leachate that need treatment . decrease production of methane gas.

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  Accident in USA
CONTAMINATION INCIDENTS IN JAPAN

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 ricecooking 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

misinformation.CONTAMINATION INCIDENTS IN USA  Evacuation process piecemeal over three years amidst climate of high tension. 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 .

Ethylene. Toulene.MOST COMMON CONTAMINANTS  Commercial Hydrocarbons  gasoline  diesel and jet fuel  naptha: raw material used in industry  domestic heating oil  Chemicals called BTEX compounds  Benzene. 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) . and selenium (these have been stabilized by bacteria in the laboratory)  DDT . chromium.a dry-cleaning solvent  PCBs and Dioxin  Arsenic.threatens water supplies  Perchloroethylene (PCE) .

POLLUTED SITES  Accidental spills  Service stations  Old Air Force bases  Storage tanks and pipelines  Chemical plants and other industrial sites  Unauthorized dump sites .

or nontoxic materials. commercial bacterial mixtures (“bag of bugs” or “bug „n a bag”) or may be genetically engineered.BIOREMEDIATION  Bioremediation is the use of living microorganisms to degrade environmental contaminants in the soil and groundwater into less toxic. .  These microorganisms can be indigenous.

. Bioremediation is an application of the same principles in a different setting. This is familiar to all as the decay of dead animals and vegetable matter.BIOREMEDIATION 2  Bacteria feed on organic waste and derive nutrition for growth and reproduction.  Municipal wastewater treatment plants have been using this technology for decades.

“Mother Nature” usually heals herself. Utilizing bioremediation speeds up the process by increasing the rate of bacterial metabolism and growth. Adding large amounts of certain enzymes and bacteria hastens the decay.BIOREMEDIATION 3  Over time. .

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 .

methanol. ethers . methylethylketone (MEK).USES OF BIOREMEDIATION 3  Gasoline and fuel oil contamination  Creosote contaminants (wood preservatives)  Ethylene glycol (antifreeze).

.REASONS TO USE BIOREMEDIATION Bioremediation can be cost effective because:  Contamination can often be treated in place.  Natural microbial processes can be used at some sites. minimizing site disturbance.

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 .

 Mercury: experiments with bacteria are on-going.DISPOSING OF HEAVY METALS  Heavy metals are not biodegradable.  Uranium: iron-eating bacteria can remove low levels of radioactive waste from water. but bacteria can be used to concentrate them into a more easily disposable form. .

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 Yeast Cyanobacteria Characteristics aerobic/ micro-aerophilic aerobic/ micro-aerophilic/ anaerobic aerobic Significance Degrades complex compounds Self-sustaining. light is primary energy source Removes TRACE concentrations of organic substances Oligotrophs .

TYPICAL BACTERIA SPECIES INCLUDE: IN DESCENDING ORDER OF OCCURRENCE) • • • • • • • • • • Pseudomas Arthobacter Alcaligenes Corynbacterium Flavobacterium Achrombacter Acinetobacter Micrococcus Nocardia Mycobacterium .

Bacillus spp. phenol Microorganisms Mixed culture and activated sludge Marine bacteria. BTEX.EXAMPLES OF MICROBES USED FOR SPECIFIC CHEMICALS Compound Name Aliphatics (non-halogenated) Ex. Acrylonitrile Aliphatics (halogenated) Ex.. Conditions Aerobic Aerobic + Anaerobic Aerobic + Anaerobic . Trichloroethane Aromatic compounds Ex. Rhodococcus spp. soil bacteria. methanogens Pseudomonas spp.. creosol. sewage sludge.. 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.

As electron acceptors.CLASSES OF BIOREMEDIATION 2  Anaerobic (without oxygen) Microorganisms break down chemical compounds to release the energy required to function. they utilize:  nitrates  sulfates  carbon dioxide  ferrous metals (such as iron) .

.HOW BIOREMEDIATION WORKS.. less toxic organic compounds .  Many naturally occurring microorganisms can digest organic materials such as fuels or solvents and convert them to:  carbon dioxide  water  smaller.

Basic Metabolism Process of Bacteria ENERGY SOURCE CARBON SOURCE NUTRIENTS Growth CELL and Reproduction NEW CELL MASS CO2 Catalyzed by Enzymes H2O .

Microbe Oil 2. Microorganisms release CO2 and Microorganisms H 0 2 digest oil and convert it to CO2 and H20 . CO2+H2O CO2+H2O Microorganisms eat oil and other organic contaminants.Schematic Diagram of Biodegradation 1. CO2+H2O 3.

phosphorous. selectively adapted microbes are combined with:  Food .OPTIMIZATION  To optimize and accelerate the bioremediation of contaminants found in water and soil. sulfur) .organic waste containing water (moisture content between 30-80%)  added nutrients (nitrogen.

neither too acidic nor too alkaline  Moderate Temperatures .50o to 100o F .between 6-9.OPTIMIZATION 2  Oxygen if required (aerobic types) 3-5 pounds of oxygen per pound of hydrocarbon to be converted  Moderate pH .

 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  Oxygen demand values are used to measure biological treatment processes.

.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 BACTERIA: Pseudomonas aeruginosa Bacillus alcolophilus Nitrosomas spp.0 10.6 8.0 5. Thiobacillus thiooxidans ALGAE: Cynidium caldarium FUNGI: Physarum polycephalum Optimum pH 6.5 2.0 – 8.6 – 7.0 – 3.8 2.0 .

chemical catalysts to break waste materials into smaller pieces  Surfactants (detergents. for example) .OPTIMIZATION 3  Enzymes.

TECHNOLOGY SELECTION CRITERIA  The bioremediation technology for a site is determined by:  Microorganisms present  Site Condition  Quantity and Toxicity of Contaminants .

This is usually the most cost effective method.requires the excavation of soil or pumping of groundwater before treatment. . but can also be slower and hard to manage.  ex situ .soil or groundwater is treated in the location where found.CATEGORIES OF BIOREMEDIATION Bioremediation treatment applications fall into 2 categories:  in situ .

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 .

 Extraction Wells: remove the groundwater to an aboveground water treatment system where nutrients and oxygen are added.EXAMPLES OF IN SITU BIOREMEDIATION 2  Injection of Hydrogen Peroxide: sprinklers or a system of pipes deliver the chemical to the soil. . Injection wells return the conditioned water to the subsurface where microorganisms degrade the contaminants.

TYPICAL in situ Bioremediation System Injection Well Nutrient/ Oxygen Addition New Water Table Contaminated Zone Old Water Table Water Treatment Recovery Well .

or “bioreactor” contains the soil. . and added nutrients or oxygen to keep the microorganisms in the optimum environment to degrade contaminants. water.EXAMPLES OF EX SITU BIOREMEDIATION Slurry Phase: a large tank.

BIOREACTOR Vapor out Contaminated soil Agitator Contaminated liquid Nutrient Temperature control Liquid outlet Soil to drying Air inlet .

EXAMPLES OF EX SITU BIOREMEDIATION 2  Solid phase: soil remains on the site. . heat. and nutrients or oxygen are added. but is placed in above-ground treatment areas where moisture.

This is the most widely used bioremediation technique.SOLID PHASE EX SITU BIOREMEDIATION 3 Landfarming: Contaminated soils are excavated and spread onto a pad. Moisture and nutrients are controlled. .

LANDFARMING Contaminated soil Air Filter/ Pump Tank 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 Gravel layer Nutrient/ moisture Contaminated soil Impermeable layer Leachate collection .

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

DIAGRAM OF BIOREMEDIATION Bioremediation in situ ex situ Engineered Intrinisic Landfarming Bioreactor Biostimulation Bioaugmentation Adding Oxygen • Bioventing • Biosparging Adding Oxygen and Nutrients Adding Oxygen. Nutrients and Bacteria .

and concentration of the contamination. It varies from 1 .REMEDIATION TIME  in situ bioremediation time depends on the extent. .  ex situ remediation for easily biodegradable contaminants or when bioreactors are used can take as little as 1-7 months. depth.6 years.

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 exhaust their „food‟ supply until microbes .

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 .

Hawaii  Biosparging and land treatment used to degrade hydrocarbon contaminated soils and groundwater.CASE STUDIES IN BIOREMEDIATION (4) Hilo. .

 Cleaned soil is removed and more contaminated soil is added.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.

Microbial mixtures can be patented and sold for much more than the cost of growing the cultures.  Bioremediation is big business for companies.ISSUES FOR DISCUSSION  Bioremediation is not always the best form of clean-up. Specific site analysis must be done first. .

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. animals. and plants? . Could this be “bad” for humans.

 Is it OK to bring in exogenous microbes? What if they don‟t die after the contaminat is degraded? . and ethics.ISSUES FOR DISCUSSION (3)  Some companies are using genetically altered microorganisms. natural selection. This leads to a discussion of genetics.

METHODS FOR ANALYSIS OF DNA Size and structure of individual DNA molecules Degree of relatedness between molecules by hybridization procedures Drawbacks: i. plasmid transfer among strains PCR-based techniques Plasmid DNA Chromosomal DNA  Gene sequencing  Profiling from electrophoresed PCR-products . Strains not containing plasmids iii. Lack of stability in some strains ii.

is valuable and reliable for phylogenetical and ecological studies.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. .

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