1. Chapter 9
Bioremediation

2. Biotechnology and the Environment
Environment – describes everything that surrounds a particular organism
Other organisms
Soil, air, water
Temperature, humidity, radiation
Abiotic and biotic factors

3. Biotechnology and the Environment
Environmental Biotechnology -
the development, use and regulation of
biological systems for remediation of
contaminated environments (land, air,
water), and for environment-friendly
processes.
Bioremediation - the use of
microorganisms to remedy
environmental problems

4. Biotechnology and the Environment
The advent of the Industrial Revolution
increase in products and waste
people moved to the city
increase in human population
Underlying cause – Industrial Revolution increased the use of fossil fuels, an increase in mining and associated pollution.
People move to the city, concentrating pollution and have less contact with nature.
Increase in world population as a result of people living longer.

5. Biotechnology and the Environment
What are the events that triggered the interest in environmental biotechnology?
Rachel Carlson’s Silent Spring (DDT)
Love Canal
Burning of a River
Exxon Valdez in 1989
1960’s – printing of Rachel Carlson’s Silent Spring. In the 1960’s rain distributed 40 tons of DDT (pesticide) each year on England alone, it got incorporated in the food chain and has a 20 year life span.
Love Canal – 1970’s – many get cancer and die from polluted land.
Also in 1970’s - Cuyahoga River in Cleveland is so polluted it catches on fire
Exxon Valdez – human carelessness caused large oil spill in the pristine Prince William Sound area of Alaska.

6. Biotechnology and the Environment
Regulations were passed:
Resource Conservation and Recovery Act (1976)
Must identify hazardous waste and establish standards for managing it properly
Requires companies that store, treat or dispose to have permits stating how the wastes are to be managed
Record of its travels: Chain of Custody
EPA initiates the Superfund Program (1980)
Counteract careless and negligent practices
Environmental Genome Project
Study and understand the impacts of environmental chemicals on human diseases
EPA was formed in 1972 to establish standards cradle to grave for handling hazardous wasted. First act passed was the resource conservation and recovery act in 1976 which mandated a chain of custody paper trail for all hazardous chemicals, also requires that companies have permits if they work with hazardous chemicals and state how waste will be managed and disposed of.
NIH funded the environmental genome project.

7. Biotechnology and the Environment
Waste
Solid: landfills, combustion-including waste-to energy plants, recovery
slurries, composting, combustion
Liquid: septic: sewage treatment, deep-well injection
Gas: fossil fuels, chlorofluorocarbons
Hazardous –anything that can explode, catch fire, release toxic fumes, and particles or cause corrosion
Solid – landfills account for over 60% of solid waste disposal, required to have an impermeable liner to prevent toxins from leaching into ground water and a monitoring well to ensure the liner is working, it must be capped with cement, without proper aeration to allow for microbial growth, materials that normally degrade in days or weeks can survive months or years. Combustion accounts for about 16% of solid waste disposal, downside is that it contributes to air pollution. Only 17% of solid waste is recycled.
Liquid – sewage is mostly cleaned by microorganisms (pro and eu), sewage treatment plants provide a good environment for bacteria, protists, blue green algae, such as good aeration and high surface areas to let them do most of the work.
Hazardous waste – air 39% / down the drain 9%

8. Biotechnology and the Environment
Garbage Test
Banana Peel
Wood Scrap/Sawdust
Wax Paper
Styrofoam Cup
Tin Can
Aluminum Soda Can
Plastic Carton
Glass Bottles
0.5 Years
4 Years
5 Years
20 Years
100 Years
500 Years
>500 Years

9. There is no waste in Nature:
From rocks and soil to plants and animals to air and water and back again:
All materials are completely recycled.
Recycled largely by
Microbes

10. Biogeochemical Cycles are a major part of the recycling process
Carbon Cycle: The primary biogeochemical cycle organic cmpds  CO2 and back
Nitrogen Cycle: proteins amino acids NH3NO2-NO3-NO2-N2ON2 NH3 etc_
Sulfur Cycle: Just like the nitrogen cycle, numerous oxidation states. Modeled in the Winogradsky column
Phosphorous Cycle: Doesn’t cycle between numerous oxidation states only soluble and insoluble form
Biogeochemical cycles rely in part on living organisms, and can be used to clean up manmade waste.

11. Carbon Cycle CO2 Organic compounds
Carbon exists in earth’s atmosphere primarily as CO2. Plants convert CO2 to carbs to O2.
Organic compounds

12. Nitrogen Cycle N2 cyanobacteria leguminous decomposition Fixation
ammonification
NH3
NO2-
nitrosomas
Nitrification
Nitrogen is needed to make all nucleic acids and amino acids. Earth’s atmosphere is 78% N2. necessary to convert N2 gas into forms usable by living organisms. Nitrogenase enzyme found in soil living bacteria combines N with H. Further oxidized to make organic compounds. Nitrates can be reduced to nitrites and combine with ammonium in the anamox process to make nitrogen gas in a single step in yeast.
All N in animals in traced back to eating plants which absorb nitrates through root hairs, reduced to nitrites, further reduced to ammonium which is used to assemble amino acids which are the building blocks of proteins.
Pseudomonas
Bacillus
Paracoccus
NO2-
Denitrification
nitrobacter
NO3-

13. Sulfur Cycle Atmosphere SO2 H2SO4 Organic sulfur S SO4 H2S
SO2 combines with H2S to make sulfuric acid, acid rain. S travels through the food chain and is released through decomposition.

14. Phosphorus Cycle Sea simple Phosphates Phosphate rocks
Phosphates too complex
for plants to absorb
from the soil
Phosphates are essential for plants and animals to make DNA molecules and ATP. Phosphorous is not in the gaseous state. Only in water, soil and sediment. Cycle is very slow and is often the limiting factor for plant growth. When animals and plants die, phosphates return to the soil/ocean during decay. Winds up in sediment and is released through weathering.
Sea simple
Phosphates
Phosphate
rocks
Microbes Breakdown
complex compounds

15. Biotechnology and the Environment
Scientists learn from nature in the 1980’s
The concept of Gaia –the total world is a living organism and what nature makes nature can degrade (bioinfalibility); only man makes xenobiotic compounds
Clean up pollution-short and long term solutions (cost, toxicity, time frame)
Use compounds that are biodegradable
Produce Energy and Materials in less destructive ways
Monitor Environmental Health
Increase Recovery of Minerals and Oil
Manmade materials are not broken down efficiently.

16. Bioremediation Basics
Naturally occurring marshes and wetlands have been doing the job!
What Needs to be Cleaned UP?
Everything!
SO How bioremediation is used depends on
what is contaminated? (locations)
on the types of chemicals that need to be cleaned up
the concentration of the contaminants (amount and duration)
Naturally occurring marshes and wetlands have excelled at bioremediation for hundreds of years. In these environments, plant life and microbes can absorb and degrade a wide variety of chemicals and convert pollutants into harmless products.
Everything needs to be cleaned up. Soil, air, water, and sediment (a combo of soil and decaying plant and animal life located at the bottom of a body of water) are all environments affected by pollution.

17. Bioremediation Basics
How do pollutants enter the environment?
Sewage (by products of medicines and food we eat such as estrogen (birth control pills) and caffeine (coffee)
Products around the house (perfumes, fertilizers, pesticides, medicines)
Industrial – leaching into groundwater
Agricultural - pesticides
Pollutants enter the environment in many different ways and affect diverse components of the environment. Tanker spill, truck accident releasing chemicals in the air which can become trapped in the clouds and contaminate surface water when it rains, ruptured chemical tank at an industrial site that leaches into ground water, landfills, illegal dumps, pesticides, mining processes.

18. Bioremediation Basics
Some are known to be potential carcinogens and mutagens

19. Bioremediation Basics
Bioremediation finds its place
Companies begin to specialize in cleaning up toxic waste spills by using a mixture of bacteria and fungi because cleaning these spills usually requires the combined efforts of several strains.
Biotechnologists begin engineering “super bugs” to clean up wastes.
However, there are many microorganisms in nature that will degrade waste products.
1970’s first patent on a living organism awarded to chakarbarty, a scientist from GE.
Superbugs – because of FDA regulations, genetically engineered organisms are reluctant to be released into the environment because of fear of their interaction with indigenous organisms. If they are used, they are designed to die immediately after cleaning up the pollution (clock genes).

20. Bioremediation Basics
Fundamentals of Cleanup Reactions
Microbes can convert many chemicals into harmless compounds HOW?
Aerobic or anaerobically
Both involve oxidation and reduction reactions

21. Bioremediation Basics
Fundamentals of Cleanup Reactions
Oxidation and Reduction Reactions
Oxidation involves the removal of one or more electrons
Reduction involves the addition of one or more electrons
Oxidizing agents gain electrons and reducing agents lose electrons
The rxns are usually coupled and the paired rxns are known are redox reactions

22. Bioremediation Basics
Aerobic and anaerobic biodegradation
Aerobic
Oxygen is reduced to water and the organic molecules (e.g. petroleum, sugar) are oxidized
Anaerobic
An inorganic compound is reduced and the organic molecules are oxidized (e.g. nitrate is reduced and sugar is oxidized)
NOTE: Many microbes can do both aerobic and anaerobic respiration; the process which produces the most ATP is used first!

23. Bioremediation Basics
The Players: Metabolizing Microbes
Site usually contains a variety of microbes
Closest to the contaminant: anaerobes
Farthest away: aerobes
The most common and effective bacteria are the indigenous microbes (e.g. Pseudomonas in soil)
Fungus and algae are also present in the environment and do a good job of “cleaning up” chemicals (fungi do it better than bacteria)

24. Bioremediation Basics
Bioremediation Genomics Programs
Stimulating Bioremediation
Add fertilizers (nutrient enrichment) to stimulate the
growth of indigenous microorganisms
Adding bacteria or fungus to assist indigenous
microbes is known as bioaugumentation or seeding

25. Bioremediation Basics
Phytoremediation
Utilizing plants to clean up chemicals
Ex: cottonwoods, poplar, juniper trees, grasses, alfalfa
Low cost, low maintenance and it adds beauty to the site
In addition, there is growing interest in using plants to clean up chemicals in the soil and in water using an approach called phytoremediation. In phytoremediation, chemical pollutants are taken in through the roots of the plant as they absorb contaminated water from the ground. After toxic chemicals enter the plant, some types of plant cells use enzymes to degrade the chemicals. In other cases, the chemical concentrates in the plant cells so that the entire plant serves as a type of plant sponge for mopping up pollutants. Poplars are commonly used for phtyoremediation and genetically engineered poplars are being tested for their potential to remediate soil and water contaminated with mercuric compounds. After scavenging the mercuric pollutants, the poplars can be harvested and the toxic compounds can be removed.

26. Cleanup Sites and Strategies
Do the chemicals pose a fire or explosive hazard?
Do the chemicals pose a threat to human health including the health of clean-up workers? (what happened at Chernobyl to the workers?)
Was the chemical released into the environment through a single incident or was there long-term leakage from a storage container?
Where did the contamination occur?
Is the contaminated area at the surface of the soil? Below ground? Does it affect water?
How large is the contaminated area?

27. Cleanup Sites and Strategies
Soil Cleanup
Either remove it (ex situ bioremediation) or in situ (in place)
In place:
If aerobic may require bioventing
Most effective in sandy soils
Removed:
Slurry-phase, solid phase, composting, landfarming, biopiles

28. Cleanup Sites and Strategies
Bioremediation of Water
Wastewater treatment

29. Cleanup Sites and Strategies
Bioremediation of Water
Groundwater Cleanup

30. Cleanup Sites and Strategies
Turning Wastes into Energy
Biogas-a gas produced by the biological breakdown of organic matter in the absence of oxygen
Biogas typically refers to a gas produced by the biological breakdown of organic matter in the absence of oxygen. One type of biogas is produced by fermentation of biodegradable materials such as biomass, manure, sewage, municipal waste, green waste, and energy crops. Methane and carbon dioxide are the most common forms of this type of biogas. Methane gas is a common energy source with many benefits, such as increasing energy security, paving the way for fuel cell vehicles, and improving public health and the environment through reduced vehicle emissions. Compared to other hydrocarbon fuels, burning methane produces less carbon dioxide for each unit of heat released. Gas-powered plants use methane for electrical generation by burning methane in a gas turbine or steam boiler. The major source of natural gas methane is extraction from geological deposits known as natural gas fields. So what makes biogas better than natural gas? It is a domestic, renewable resource and using it offsets the use of non-renewable resources such as coal, oil, and fossil fuel-derived natural gas. Landfills already produce vast amounts of methane gas as a natural consequence of the breakdown of solid wastes deposited there. Methane is a much stronger greenhouse gas than carbon dioxide, so by collecting the gases given off of these landfills and using them for power generation, we can significantly reduce green house gases while helping to meet our energy needs. Also, the use of non-landfill anaerobic digestion systems to treat wastewater from agriculture and municipal treatment systems can reduce the amount of solid waste that must be disposed of, reduce waste odors, treat waste naturally, require less land area than aerobic composting, reduce the amount of material that must be dumped in landfills, and produce sanitized compost and nutrient-rich liquid fertilizer. Click on the video link to learn about the potential for biogas production from animal manure in the United States.
Video describing potential for biogas production from animal manure

31. Cleanup Sites and Strategies
Turning Wastes into Energy
Cowpower:
Compare 3.0 kwh of “cow power” available in one cow’s daily manure contribution with the 2.4 kwh necessary to burn a 100 w light bulb for a day
Typically, dairy farms flush large amounts of manure from their barns and pens into nearby lagoons. As bacteria break down the manure, methane and carbon dioxide are released into the atmosphere. Farmers can produce biogas from cow manure anaerobic digestion. The manure coming from the barn goes directly to an inlet valve that is directly connected to the fermentation chamber. In this chamber, the digestion process begins with bacterial hydrolysis of the input materials in order to break down insoluble carbohydrates to make them available for other bacteria. In the final step of the process, methane-producing bacteria act on the manure byproducts under anaerobic conditions to give off methane gas. The captured biogas is a versatile energy source that can be used to produce heat and power generators. The amount of energy that can be harvested from one cows daily manure output is more than enough to power a 100 watt light bulb for a 24 hour period. Anaerobic digestion facilities have been recognized as one of the most useful decentralized sources of energy supply, as they are less capital intensive than large power plants. Additionally, anaerobic digestion helps farmers comply with nutrient management plans by reducing odor and water quality issues and generating fertilizer and other useful products from the degraded manure. Huckabay Ridge is a biogas facility in Stephenville, Texas that is a model for future biogas facilities, providing renewable natural gas directly to the Lower Colorado River Authority. At the Huckabay Ridge facility, manure from 10,000 cows from a local county in Texas is expected to produce enough pipeline-quality methane to power 11,000 homes.

32. Applying Genetically Engineered Strains to Clean Up the Environment
Heavy metals (bioaccumulation)
Bacteria sequester heavy and radioactive metals
Petroleum eating bacteria
Ananda Chakrabarty at General Electric
Biosensors
lux genes
D. RADIODURANS CAN ENDURE DOSES OF RADIATION OVER 3000 TIMES HIGHER THAN OTHER ORGANISMS

33. Environmental Diagnostics
A promising new area of research involves using living organisms to detect and assess harmful levels of toxic chemicals.
Frogs are sensitive to changes in UV light and air quality, lichens are sensitive to pollutants. clams close up in the presence of pollutants, clam biosensors where a laser light is trained an a piece of aluminum that is attached to the top of the clamshell. The laser detects when the clam closes as a result of pollution. The closing of the shell triggers a water sampling event.

34. Environmental Diagnostics
Daphnia magna
Transparent Thorax and Abdomen

35. Environmental Diagnostics
When healthy Daphnia are fed a sugar substrate (-galactoside attached to a fluorescent marker), they metabolize the sugar and fluoresce under UV light.
Environmental Diagnostics
                                                                                                              
When Daphnia are stressed by toxins, they do not have the enzymatic ability to digest the sugar and therefore do not fluoresce under UV light.

36. Environmental Diagnostics
Toxicity reduction involves adding chemicals to hazardous waste in order to diminish the toxicity.
For example, if the toxicity results from heavy metals, EDTA will be added to the waste and the effluent will be tested again to determine if the toxicity has been acceptably reduced.
EDTA chelates (binds to) metals, thereby making them unavailable to harm organisms in a particular body of water.
Genetically engineered bacteria to sequester heavy metals.

37. Environmental Disasters: Case Studies in Bioremediation
The Exxon Valdez Oil Spill
In the end, the indigenous microbes did the best job
Oil Fields of Kuwait
Poses a problem due to the environmental conditions
One gallon of spilled oil can spread to cover 4 acres of water. The exxon valdes released 11 million gallons of crude oil over 1000 miles of the Alaskan shoreline.
Oil fields of kuwait released 250 million gallons of oil into the deserts.

38. Future Strategies and Challenges for Bioremediation
Microbial genetics
New types of microbes (from the ocean etc)
DO A BETTER JOB OF DETERMINING RISK and ASSESSMENT OF EXISTING SITES
Greater understanding of the microorganisms involved in biodegradative processes, including the identification of novel genes and proteins involved in the metabolic process.

39. Careers in Environmental Biotech
Biodegradation
Wastewater treatment plants, organic farming
Bioremediation
Environmental clean-up companies, labs developing super bugs
Biocatalysis
Plastics, degradable and recyclable products
Other
Mining companies, oil companies
Biodegradation – companies making plant growth compounds and organic fertilizers
Bioremediation
Biocatalysis – labs to select plastic producing organisms as a renewable resource. Companies to make biodegradable products such as starch in polyethylene garbage bags so garbage bags can be recycled.
Other – biomonitoring – companies that produce sensors.