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1/20/2014
1
Tuzzy Talk 3:Crude Oil
BiodegradationJanuary 18, 2014
Nancy E. Kinner
University of New Hampshire
Center for Spills in the Environment1
Today’s Topics• Tuzzy Talks Series 1:What Is Crude Oil? and What Happens When It Gets in Water? (March 2013)
• Tuzzy Talks Series 2:
– Biodegradation of Crude Oil
– Dispersants and Dispersed Oil (Saturday, February 1)
• This Week:
– Review of Series 1: What Is Crude Oil and What Happens When It Gets in Water?
– Biodegradation of Crude Oil
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Crude Oil Composition
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Crude Oil Is Made of Hundreds of Chemical Compounds:
All Contain Carbon (C) (80‐90%) and Hydrogen (H) (10‐15%)
= Hydrocarbons
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5
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Simple Chains with Single Bonds
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Simple Rings with Single Bonds
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Simple Rings with Double Bonds
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Multiple Rings with Double Bonds
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Crude Oil Characteristics
(Crude oils vary from place to place and over time from the
same place)
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Density• Mass of Substance per Volume
– Water = 1 gram/mL
– (5 mL = 1 teaspoon)
– Crude Oil = 0.8 to 0.9+ g/mL
• Oil Less Dense than Water
– So….Oil Floats on Water
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Solubility• Desire of Compound to Dissolve into Water
• Though Most Compounds in Oil Do Not Dissolve Well in Water, They Do Dissolve to Some Extent
– Sugar Solubility in Water ~ 1 lb per cup water
– Crude Oil Compounds ~ 1 lb oil per 200 to 200 million gallons of water
• (1 ug/L to 1000mg/L )
(Microbes Prefer to Eat Things Dissolved in Water)
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What Happens When Crude Oil Gets Into
Water?
Natural Processes (Fate and Behavior of Oil in Water)
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Natural Processes for Surface Oil Slicks
• Function of:• Oil Type and
Characteristics• Environmental
Conditions– Temperature
• Water, Air– Wind– Waves– Currents, Tides
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Pathway Estimated Time Scale
(days)
Percent Initial Oil
Evaporation
(volatility)
1‐10 25
Dissolution
(solubility)
1‐10 5
Photochemical
Breakup (Sun)
10‐100 5
Biodegradation 50 ‐500 30
Disintegration
and Sinking
100‐1000 15
Residue More than 100 20
Total 100
(Butler et al., 1976)
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Crude Oil Biodegradation
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Key Concepts in Biodegradation of Oil
Microorganisms Can Degrade Compounds in Crude Oil
Use the Compounds for Energy
May Not Happen in Some Cases
Microorganisms Degrade Compounds at Different Rates
They Need Other Things Besides Oil Compounds to Live
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What Microbes Degrade Oil?
• Prokaryotes (chromosomes not in membrane‐bound nucleus)
– Bacteria
– Archaea
• Not Algae or Other Plants
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20Wiedemeier et.al. pB5-2
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Why Do Microbes Degrade Oil?
Mostly to Generate EnergyThey Need to Survive and
Grow!
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Energy Generating Processes
• We Generate Energy in Similar Way
– Not with Oil
– With Carbohydrates, Lipids etc.
• Energy Generated by Transfer of Electrons from One Compound to Another
• Need Electron Donor (ED)
– Loses e‐
• Need Electron Acceptor (EA)
– Gains e‐
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Example of Energy Generating Reaction
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Oil Biodegradation: By Bacteria
Electron DonorOil Compound + Oxygen → CO2 + H2O + Energy
ElectronAcceptor
H+
+e‐
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Energy Generation
3 Step Process
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Glycolysis
• Organic Carbon Molecule Broken into Small Pieces
– Preparatory steps
– Little energy generated
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Tricarboxylic Acid Cycle (TCA)
• Removes H+ and e‐ from Organic Carbon Molecule
– Preparatory step
– Some energy generated
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Electron Transport System (ETS)
• No Organic Molecule Involved
• Pass e‐ Removed from Organic Molecule Down a Chain (ETS)
– Series of reactions that generate energy
• Need Electron Acceptor at End of Chain
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Electron Transport System
Organic Ce‐
CO2
Cell Membrane
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Step 1
Electron Transport System
Organic C
CO2
Cell Membrane
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e-
Step 2
ENERGY
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Electron Transport System
Organic C
CO2
Cell Membrane
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Step 3
Electron Transport System
Organic C
CO2
Cell Membrane
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Step 4
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Electron Transport System
Organic C
CO2
Cell Membrane
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Step 5
Electron Transport System
Organic C Electron Acceptor
CO2 O2
Cell Membrane
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Step 6
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Electron Transport System
Organic C Electron Acceptor
CO2 O2
Cell Membrane
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Electron Acceptor•Brought Into Cell Across Cell Membrane
•At End of Electron Transport System
•Accepts Electron
•Leaves Cell with Electrons
How Far Down ETS Can e‐ Go?
• e‐ Can Only be Transferred from One Compound to Another if Receiver Has Higher Affinity for e‐ Than Donor
• Transfers Produce Energy
• Not All Transfers Produce the Same Energy
• Step 6 (Transfer to Oxygen) Produces Lots
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Electron Transport System
Organic CSO
4‐2
CO2
Cell Membrane
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e-
Other Electron Acceptors Produce Less Energy Than Oxygen….Microbes Are Less
Active at Biodegradation
Process Electron Acceptors Energy Generated
Aerobic Resp. • O20 → H2O High
‐2
Iron Reduction • Fe+3 → Fe+2
Denitrification • NO3‐ → NH3 or N2
+5N ‐3 0
Sulfate Reduction • SO4‐2 → S‐2 or S0
+6 ‐2 0
Methanogensis • CO2 → CH4 Little+4 ‐4
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Enzymes
• Enzymes in Cells Help All of the Steps to Occur
• Enzymes Are Specialized to Help Reactions Occur
• Cells Make the Enzymes They Need When They Are Needed– Enzymes that help in Crude Oil Degradation Are Made When Crude Oil Compounds Are Present in Microbes’ Environment
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Biodegradation of Crude Oil Compound
• Oil Compound is Electron Donor
– Will Donate e‐ and Create Energy
• Energy Generated is a Function of:
– Type of oil compound
– Electron acceptor used by cell
– Enzymes available
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Bacteria Requirements to Degrade Oil Compounds to Create Energy
• Oil Compounds– Serve as food to create energy
– Not all degraded at same rate• Simple Chains Easier Than Rings
• Electron Acceptor
– Mostly Oxygen (O2)
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Simple Chains with Single Bonds
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Simple Rings with Single Bonds
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Simple Rings with Double Bonds
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Multiple Rings with Double Bonds
Other Factors Affecting Biodegradation:Highlights
• Other Substances Cells Need
• Abiotic Factors
• Interaction Between Substrates
• Effects of Other Biological Processes
• Bioavailability
• Recalcitrance
• Acclimation
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Other Substances Cells Need
• Water – Moisture Content
– 50‐70% → 100% Optimal
• Nutrients
– Macro (large) and Micro (small) Amounts
• For example: Nitrogen, Phosphorus, Iron
– Must be in “right” form
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Abiotic Factors ‐ Temperature
• Affects Enzyme Activity
• For every 10oC ↑ in temperature, enzyme activity ↑ 2x
• Adaptation to Environmental Temperature
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Interaction Between Substances
• If Multiple Organic Substances Present:–One may be preferred
• Easier to degrade• More energy generated
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Interaction Between Substances
–First may enhance degradation of the second• Byproducts of first may be needed in degradation of second
• Synergism
Synergism
• Multiple Types of Microbes Accomplish Degradation That Neither Can Do Alone
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Bioavailability
• To Be Degraded, Oil Compound Must Be Available to Microbe
• Less Available If Not Dissolved in Water:
– Sorbed (adhered) to surfaces
– In oil slick
(This is one reason responders disperse oil…next talk)
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Recalcitrance
• Molecule Resistant to Biodegradation = Recalcitrant Molecule
• Affected by Molecular Structure and Environmental Conditions
NOT ALWAYS DEGRADED ALL THE WAY TO CO2…..MAY JUST BE SMALLER OIL COMPOUND
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Environmental Conditions Affecting Recalcitrance
• Bioavailability• Some Factor Needed for Growth is Absent or In Very Low Concentrations
‐ Nutrients
• “Right” Community of Microbes Not Present
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Bioaugmentation(Adding Microbes)
• Microbes That Can Degrade Recalcitrant Molecules Can Be Developed or May Exist in Other Environments
• Not Very Successful‐ Eaten by predators‐ Unable to compete with in situ microbesadapted to environment
• Hard to Prove that the Added (Special) Microbe Is Really the One Doing the Biodegradation
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Acclimation
• Time Between When Oil Spilled Into Environment and Biodegradation Occurs–Lag phase
• What’s Happening During This Period?–Production of enzymes for oil degradation
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Factors Affecting Acclimation Rates
• Small Initial Microbial Population
• Type of Organic
‐ Similarity of compound to natural
compounds
• Time of Exposure/Pre‐Exposure
‐May take less time with
pre‐exposure
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Acclimation Rates
• Hard to Predict In Situ Rate of Acclimation
‐ Lab studies may not be indicative of
rate in environment
• Rates Are Highly Variable
‐ Data from one site may not apply to
another
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Is Biodegradation Occurring?
Natural Weathering Processes
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Is Biodegradation Occurring?
• Look at Time Frame
• Look at Compounds Being Degraded
– Simple ones
– Complex ones
– Ratio to recalcitrant ones (hopane)
• Special Lab Tests with Tracers
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Thank You So Much for Participating in
These Talks
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