Bioremediation of Petroleum and
Polyaromatic Hydrocarbons:
Theory and Applications
Dr. Carol Litchfield
Dept. Environmental Science & Policy
George Mason University
Manassas, Virginia, U.S. A.
October 2005
Outline
Brief introduction to hydrogeology and the subsurface
Overview of the theory of bioremediation
What is Bioremediation?
In the most general terms, this is the process by
which organisms (from any domain) transform
chemicals.
For our purposes it is the transformation of
chemicals considered to be contaminants or
pollutants.
When aerobic microorganisms are involved, it
means the production of CO2, biomass, water,
and perhaps other ions such as Cl-
Some Terms and Understanding
of the Subsurface
 Groundwater: subsurface water
 Water Table: water level in the subsurface where the
pressure of the water equals the atmospheric pressure
and soil pores are completely saturated with water
 Hydraulic Conductivity: water flow rate per cross
sectional area; units of cm2/m/day or meters/day
 Vadose Zone: zone of unsaturation above the water
table. It may or may not include the interface.
 Saturated Zone: soils where water is always present
 NAPL: nonaqueous phase liquid
 DNPL: dense nonaqueous phase liquid
Soil Texture & Porosity
Aerobic Heterotrophic Metabolism
Molecular oxygen provides the electron
acceptor for the oxidation of organic
compounds
Organic carbon is used for energy and
growth and this includes methane but not
carbon dioxide
Examples: mammals, birds, fish, bacteria
Anaerobic Metabolism
Anaerobic bacteria live and grow in the
presence of reduced compounds and with either
the total absence of molecular oxygen or its
greatly reduced concentration
Compounds which can serve as terminal
electron acceptors for anaerobes are: carbon
dioxide, nitrate, sulfate, iron, humates and
glucose (producing lactic acid and ethanol)
Co-metabolism
The transformation of a nongrowth
compound when an organism is growing
on another substrate and deriving its
carbon and energy from the second
substrate. That is, the first compound is
“accidentally” transformed and provides no
benefit to the organism
Some Terms and Understanding of the
Subsurface
Retardation: the extent to which substances or
chemicals are prevented from moving in soils or
in the subsurface:
sorption
entrapment
cation exchange capacity
Kow (octanol:water partition coefficient)
chemical reactions
porosity
composition
other organic matter
gases
microbial action
Retardation of Bacteria
Cross Section of the Subsurface
Hydraulic Conductivities of Different Soil
Types
MEDIUM
K (METERS/DAY)
Surface clay soils
0.01 to 0.2
Deep clay beds
10 -8 to 10 -12
Surface loam soils
0.1 to 1.0
Fine sand
1.0 to 5.0
Medium sand
5.0 to 20.0
Coarse sand
100 to 1000
Sand and gravel
5.0 to 100
Glacial till
0.001 to 0.1
Sandstone
0.001 to 0
Shale
10 -7
Dense solid rock
10 -5
Fractured or weathered rock
0.001 to 1
Traditional Technologies
Pump and Treat if a Liquid
Excavate and treat on site
 with soil washing
 incineration
 landfill
Remediation Options
 Excavate and remove to landfill or incinerate
 Containment
 Slurry wall
 Vitrification
 Stabilize
 Pump and Treat
 Free-phase removal
 Pump groundwater and air strip or carbon filter
 Chemical/physical oxidation
 Hydrogen peroxide
 UV
 Reverse Osmosis
 Thermal treatment
 Use In Situ treatments
 Soil venting
 Bioremediation
Advantages of Bioremediation
 Can be highly specific
 Less expensive than excavation or incineration
processes
 If mineralization occurs get complete
degradation and clean up
 Does not transfer contaminants from one
environment to another
 Uses a natural process
 Good public acceptance
 Process is simple
 If using ISB you will treat the groundwater and
soil at the same time
Disadvantages to Bioremediation
Not instantaneous
Often need to develop a system
Always need to test and optimize conditions
empirically – not with computer models
May have inhibitors present
Compounds may not be in a biodegradable form
– polymers, plastics
Compounds may be recalcitrant – higher
congeners of PCBs
How Do We Begin Bioremediation?
Determine there is a problem
Monitor the surface/subsurface soils and
water
Types of Organisms
Natural
Pure cultures
Flavobacterium spp.
Pseudomonas spp.
Mixed cultures

Consortia

Methane-utilizing bacteria (anaerobes)
Genetically engineered microorganisms
What Types of Organisms Have
Been Used?
• Fungi
• Plants (Phytoremediation)
• Bacteria
– The natural community
– Bioaugmentation
What Types of Compounds Can Be
Treated Biologically?
 Petroleum Hydrocarbons
 Gasoline
 Diesel Fuel
 Gasoline Additives such as MTBE
 Polyaromatic Hydrocarbons
 Creosote
 Chlorinated Hydrocarbons
 Chlorinated Aliphatics: trichlorethylene
 Chlorinated Aromatics : PCB’s, Pentachlorophenol
 Explosives
 RDX, TNT
 Inorganics via Reduction to a Lower Valence Causing
Precipitation
 Uranium, Technicium
 Sulfur and Sulfuric Acid
 Ammonia or Nitrate/Nitrite

Environmental Conditions for
Bioremediation
• Aerobic – where oxygen in some form is
added to the treatment environment
• Anaerobic – where nitrate, iron, or other
electron acceptor is added to the
treatment environment
• Combinations - where a combination of
the above is used, often in pulses
What Biological Technologies Are
Available?
In situ Bioremediation (ISB) or Enhanced
Bioremediation
Natural Bioremediation
Biopiles
Bioreactors
Bioventing/ Biosparging
Engineered Treatment Cells
Bioreactor Design Considerations
Type of contaminant: soil, sludge, water
Hydraulic residence time
Bacterial residence time
Mixing
Oxygen transfer
Contaminant levels and likelihood of interactions
Discharge standards and air emission controls
Sludge disposal
Operational costs: capital equipment, personnel,
chemicals, duration
Bioaugmentation
Definition: The addition of microorganisms
to the reaction chamber whether in situ or
above ground
Considerations before bioaugmenting:
Ability to survive
Ability to function
Assurances that they are nonpathogenic to
higher life forms
Types of Bioremediation Using Indigenous
Microorganisms
On Site
Above ground bioreactor for soils and/or
water
Solid phase treatment cells for soils
Landfarming for soils but could use for water
if not much water is contaminated
Composting of soils
In situ bioremediation
Composting
A process for treating contaminated soils
using microorganisms in a heat generating
process so that the contaminants are
degraded at an accelerated rate.
Definition of In Situ Bioremediation (ISB)
The treatment in place without excavation
of contaminated soils or sediments. This
may require construction in the
soils/sediments of barriers to contain the
movement of the groundwater, nutrients,
or contaminants
Factors to Consider in Any In Situ
Process
Geochemistry – the interaction of any
treatments with the soil mineralogy
Hydrogeology – how does the subsurface
water move
Biodegradability – rate, extent, and
pathways of degradation
Redox condition – is this an oxidizing or
reduced environment
Principles of ISB
 Bacteria occur naturally in subsurface soils and water.
 These bacteria have adapted to their environment
 Microbial activity is limited by environmental or nutrient
factors
 When these limiting factors are corrected, the microbial
community can degrade the contaminants to biomass,
carbon dioxide, water, and salts if appropriate
 Growth stimulation can be controlled by controlling the
addition of the nutrients.
 Aerobic biodegradation is preferred because it is faster
and tends to result in more complete degradation
 Microbial transformations can lead to more toxic
materials or complete degradation
Information Required for any ISB Process
Is there an acclimated population?
How many are there?
Is there a reducing zone indicating that bacteria
are already active there?
What is the hydrology of the site?
What is the vertical and horizontal distribution of
the contaminants?
What are the concentrations of the
contaminants?
What is the lithology of the subsurface?
Steps in Performing an ISB
Historical background
Obtain fresh core and groundwater samples
Run a screening study to determine if adapted
organisms are there, their numbers, and whether they
can degrade the contaminant
Duration: 1- 4 weeks
Cost: Minimal
Optimize the conditions to determine the best electron
acceptor, optimal nutrient mixture, and evaluate the
rate and extent of biodegradation
Duration: 14-20 weeks
Cost:$10,000 - $100,000
Steps in Performing an ISB, cont’d.
Perform a soil-nutrient interaction study
Duration: 1-5 days depending on
conductivity
Cost: Minimal
Engineering Designs
Construction
Implementation: monitoring soils and/or
groundwater to show biodegradation is
occurring
Closure
Indicators of an Acclimated Microbial
Community
Depressed oxygen levels in the area of
the spill
Increased numbers of colonies capable of
growing on the contaminant
Increased levels of ATP
Demonstration in microcosm studies that
have degradation of the contaminants
If appropriate, increased levels of salts in
the groundwater
How to Enhance the Indigenous
Microbial Community
Add nutrients: nitrogen, phosphorus, trace
metals, co-substrates
Add electron acceptor: oxygen, nitrate,
humates, etc.
Information Required for In Situ
Bioventing
Adapted microorganisms are present
Radius of influence of the wellspermeability of the soils to gases
Likely flow paths
Presence of natural biogenic gases:
carbon dioxide, methane, and oxygen
Evidence of in situ respiration