Overview

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Principles Applications of BTEX Bioremediation
Pedro J.J. Alvarez, Ph.D., P.E.,
DEE University of Iowa

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Prospectus

• What are BTEX and why care about them?
• What is needed to biodegrade them?
• How to exploit biodegradation for site cleanup?
• What are the more serious technical and political
  challenges related to BTEX bioremediation?
• What is epistemology and how can it help us
  address some of these challenges?

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Water, water everywhere, nor any drop to
drinkThe Rime of the Ancient Mariner, Samuel
Taylor Coleridge
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Contaminants of Concern BTEX
Importance Relatively high solubility High
migration potential Toxicity Benzene can
cause leukemia at 5 µg/l Volatile, hydrophobic,
biodegradable
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Requirements for Biodegradation

• 1. Existence of organism(s) with required
  catabolic potential.
• Xenobiotic will be degraded to an appreciable
  extent only if the organism has enzymes that
  catalyze its conversion to a product that is an
  intermediate or a substrate for common metabolic
  pathways.
• The greater the differences in structure between
  the xenobiotic and the constituents of living
  organisms (or the less common the xenobiotic
  building blocks are in living matter), the less
  likelihood of extensive transformation or the
  slower the transformation.

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Requirements for Biodegradation (contd)

• 2. Presence of organism(s) in the environment.
• BTEX degraders are commonly found, but
  differences in relative abundance of dissimilar
  phenotypes may lead to apparent discrepancies
  in the biodegradability of a given BTEX compound
  at different sites.
• Depending on the relative abundance of different
  strains,
• B could degrade earlier than T at one site, but
  the opposite may be observed at other sites.

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Frequency Analysis of Biodegradation Capabilities
of 55 Hydrocarbon Degraders
100
90
80
70
60
50
Strains that degraded compound
40
30
20
10
0
B
T
E
p-X
m-X
o-X
N
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Requirements for Biodegradation (contd)

• 3. Compound must be accessible to organism
• a) Physicochemical aspects (bioavailability).
• Desorption, dissolution, diffusion, and mass
  transport
• b) Biochemical aspects.
• Membrane permeability (important for
  intracellular enzymes), uptake regulation.

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Requirements for Biodegradation (contd)

• 4. If catabolic enzymes involved are not
  constitutive, they must be induced
• Inducer(s) must be present above specific
  treshold (e.g., T gt 50 mg/L)

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Benzene Degradation by Pseudomonas CFS-215
Toluene enhanced enzyme induction
Control
T 0
Benzene Concentration (mg/L)
T 0.1 mg/L
T 50 mg/L
Time (days)
Alvarez and Vogel (1991) Appl. Environ.
Microbiol., 57 2981-2985
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Cometabolic Degradation of o-Xylene by
Denitrifying Toluene Degraders
TOLUENE
mg/l
Active
Controls
days
o-XYLENE
mg/l
Active
Controls
days
Alvarez and Vogel (1995) Wat. Sci. Technol., 31
15-28
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Requirements for Biodegradation (contd)

• 5. Environment conducive to growth of desirable
  phenotypes and functioning of their enzymes
• a) Presence of recognizable substrate(s) that
  can serve as energy and carbon source(s) (e.g.,
  the BTEX) and limiting nutrients (N and
  P, trace metals, etc.).
• b) Moisture (80 of soil field capacity, or 15
  H2O on a weight basis, is optimum for vadose zone
  remediation. Need at least 40 of field
  capacity).
• c) Availability of e- acceptors (e.g., O2 for
  oxidative reactions) or e- donors (e.g., H2 for
  reductive transformations). The e- acceptor
  establishes metabolism mode and specific
  reactions.

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The electron tower concept
Half Reaction
Reduction Potential Hierarchy
E

H
volts
Oxidized
Reduced
-0.50

H
H
2
Benzene degradation to CO2 and CH4 under
methanogenic conditions

C
H
4.5 H
O

2.25 CO
3.75 CH
benzene
CO
6
6
2
2
4
2
D
Go

-(30 e-/
mol
) (96.63 kJ/volt) (-0.24 -(-0.29) volts)
-0.25
D
Go

-133 kJ/
mol
of benzene, or -
4.5 kJ/e

equiv
transferred
-
CH
CO
4
2
(barely feasible)
HS
-
SO
2-
4
Benzene degradation to CO2 under aerobic
conditions
0

C
H
7.5 O


6 CO
3 H
O
6
6
2
2
2
D
Go

-(30 e-/
mol
) (96.63 kJ/volt) (0.82 -(-0.29) volts)
D
107 kJ/e
-
equiv
transferred
Go

-3,200 kJ/
mol
of benzene, or -
Electron Tower
(24 x more feasible)
0.25
0.50
NO
-
N
2
0.75
3
O
H
O
2
2
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Aerobic BTEX Degradation

• BTEX are hydrocarbons (highly reduced) so their
  Oxidation to CO2 is highly feasible
  thermodynamically (fuel)
• Aerobic BTEX biodegradation is fast (O2 diffusion
  is often rate-limiting)
• Aerobic BTEX degraders are ubiquitous (e.g.,
  Pseudomonas)
• Need oxygenase enzymes (i.e., enzymes that
  activate O2 and add it to carbon atoms in the
  BTEX molecule)
• The ring must be dihydroxylated before ring
  fission. Once the ring is opened, the resulting
  fatty acids are readily metabolized further to
  CO2.

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Anaerobic BTEX Degradation

• Rates are much slower because anaerobic electron
  acceptors (e.g., NO3-, Fe3, SO4-2, and CO2)
  are not as strong oxidants as O2.
• Benzene, the most toxic of the BTEX, is
  recalcitrant under anaerobic conditions
  (i.e., it degrades very slowly after TEX, or
  not at all)
• Anaerobic degradation mechanisms are not fully
  understood. Benzoyl-CoA is a common
  intermediate, and it is reduced prior to ring
  fission by hydrolysis. The oxygen in the evolved
  CO2 is from water.
• Anaerobic BTEX degradation processes (e.g.,
  denitrifying, iron-reducing, sulfidogenic, and
  methanogenic) are important natural attenuation
  mechanisms.

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In aquifers, electron acceptors are used in
sequence. Those of higher oxidation potential
are used preferentially O2 gt NO3- gt Mn4 gt Fe3
gt SO4-2 gt CO2
Source Wiedemeier et al., 1999
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Requirements for Biodegradation (contd)

• 5. Favorable environment (continued)
• d) Adequate temperature (rates double for ?T
  10C).
• e) Adequate pH (6-9).
• f) Absence/control of toxic substances (e.g.,
  precipitation of heavy metals, dilution of toxic
  conc.).
• g) Absence of easily degradable, non-target
  substrates that could be preferentially
  metabolized (ethanol?).
• 6. Time.
• Without engineered enhancement, benzene
  half-lives on the order of 100 days are
  common in aquifers.
• Want degradation rate gt migration rate

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What is Bioremediation?

• It is a managed or spontaneous process in which
  biological, especially microbiological, catalysis
  acts on pollutants, thereby remedying or
  eliminating environmental contamination present
  in water, wastewater, sludge, soil, aquifer
  material, or gas streams. (a.k.a.
  biorestoration).
• Ex Situ (Above ground)
• In Situ (In its original place, below ground)
• Engineered Systems (biostimulation vs.
  bioaugmentation)
• Natural Attenuation (intrinsic/passive)

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Why Use Bioremediation?

• Can be faster and cheaper (at least 10x less
  expensive than removal incineration, or pump
  and treat)
• Minimum land and environmental disturbance (e.g.,
  generation of lesser volume of remediation
  wastes)
• Can attack hard-to-withdraw hydrophobic
  pollutants
• Done on site, eliminates transportation cost
  liability
• Environmentally sound (natural pathways)
• Does not dewater the aquifer

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When is engineered bioremediation feasible?

• Feasibility depends on
• 1) Kh ? distribution of nutrients and e-
  acceptors (Kh gt 10-5 m/s)
• 2) Adsorption ? bioavailability (depends on Kow
  and foc, problem for PAHs)
• 3) Potential degradation rate (half life lt
  10 days)

2
Feasible
1
with
Feasible


Enhancement
0
log k (per day)
-1
Not feasible
-2
-3
6 5 4 3 2
1

- log
K

(cm/s)
h
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• Bioventing
• Used to bioremediate BTEX trapped above water
  table
• Vacuum pumps pull air through unsaturated soil
• Need to infiltrate water (with nutrients) to
  prevent desiccation

Source MacDonald and Rittmanm (1993) EST,
27(10) 1974-1979
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• Water Circulation Systems
• Used to bioremediate BTEX in saturated zone
  (Raymond)
• Contaminated water is extracted, treated
  (air-stripping, activated carbon, or
  biodegradation), and recycled.
• Some is amended with nutrients and reinjected
  (pulsing is better).
• Clogging near injection well screens and
  infiltration galleries can be a problem
  (bacterial growth, mineral precipitation) but
  pulsing reduces clogging (may need occasional
  Cl2, H2O2)

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• Air Sparging
• Injection of compressed air directly into
  contaminated zone stimulates aerobic degradation,
  strips BTEX into unsaturated zone to be removed
  by vapor-capture system
• Not effective when low-permeability soil traps or
  diverts airflow

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• Biobarriers
• Containment method that prevents further
  transport (hydraulic or physical controls on
  groundwater movement may be required to ensure
  that BTEX pass through barrier
• Biologically active zone is created in the path
  of the plume by injecting nutrients and electron
  acceptors (could use oxygen-releasing compounds,
  or inject compressed air and form an air curtain)

Treated Water
Air Curtain
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Benzoate addition as auxiliary substrate (1 mg/L)
stimulated benzene attenuation through 1-D
biobarrier
200
Sterile control
150
Effluent Benzene (µg/L)
Not amended
100
-
C
O
O
50
with benzoate
0
0
1
2
3
4
5
6
7
8
9
10
Time (days)
Alvarez P.J.J., L. Cronkhite, and C.S. Hunt
(1988). Environ. Sci. Technol. 1998 32(5)
634-639
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Bioremediation Market

• According to the Organization for Economic
  Cooperation Development), the global market
  potential for environmental biotechnology doubled
  in the past 10 years to 75 billions in the year
  2000
• In USA, we have 400,000 highly contaminated
  sites, and NRC estimates the cleanup cost to be
  on the order of 1,000 billions
• In USA, the current bioremediation market is only
  about 0.5 billions

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Bioremediation experienced many up- and downturns

• 1950s Microbial infallibility hypothesis
  (Gayle, 1952)
• 1970s Regulatory pressure stimulates
  development. Adding bacteria to contaminated
  sites becomes common practice. Failure to meet
  expectations (e.g., DDT accumulation) prompts a
  major downturn.
• 1980s It becomes clear that fundamental
  processes need to be understood before a
  successful technology can be designed. This
  realization, along with the fear of liability and
  Superfund, stimulates the blending of science and
  engineering to tackle environmental problems.
• 1990s Many bioremediation and hybrid
  technologies are developed. However, decision
  makers insist on pump and treat, and Superfund is
  depleted. Poor cleanup record and high costs
  stimulate paradigm shift towards natural
  attenuation and RBCA.

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Aerobic Unsaturated Zone
Volatilization
Oxygen Exchange
Anaerobic core
Dissolution
Advection
Aerobic uncontaminated groundwater
Aerobic Processes
Mixing, Dilution
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
46
Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Atenuação Natural
PE
Fluxo da água subterrânea
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Plume
Source
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What is Monitored Natural Attenuation?

• MNA is the combination of natural biological,
  chemical and physical processes that act without
  human intervention to reduce the mass, toxicity,
  mobility, volume, or concentration of the
  contaminants (e.g., biodegradation, dispersion,
  dilution, sorption, and volatilization).
• Success depends on adequate site
  characterization, a long-term monitoring plan
  consistent with the level of knowledge regarding
  subsurface conditions at the site, control of the
  contaminant source, and a reasonable time frame
  to achieve the objectives.
• MNA should not be a default technology or
  presumptive remedy. The burden of proof (e.g.,
  loss of contaminants at field scale, and
  geochemical foot-prints) should be on proponent,
  and evidence of its effectiveness should
  emphasize biodegradation.

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Plume Dimensions Reflect Natural Attenuation
MEDIAN PLUME DIMENSIONS
BTEX Plumes (604 Sites)
132 ft
1000 ft
TCE Plumes (88 Sites)
Other chlorinated solvent plumes (29 Sites)
500 ft
Salt Water Plumes (chloride) (25 Sites)
700 ft
200
400
0
600
800
1000
Feet
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Concentration
safe
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• What is Risk-Based Corrective Action?
• Clean source only to a level that will result in
  an acceptable risk at the potential receptors
  location (e.g., property boundary)
• Need a mathematical model to calculate the
  required Co

receptor
Co ?
Concentration
safe
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Analytical Solution of the Advection-Dispersion-So
rption Equation with First-Order Decay, for
Constant Rectangular Source (Domenico, 1987)

• Models are useful analytical tools, and can be
  used to demonstrate that natural attenuation is
  occurring
• Limited predictive capability (order-of-magnitude
  accuracy) groundwater flow and microbial
  behavior rarely follow simplifying assumptions.

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Sensitivity Analysis Effect of Doubling a
Variable on Plume Length (Lp)

• Variable Baseline Value ?Lp ()
• ? (day-1) 0.0005 -24
• Co (ppb) 25,000 7
• Z (m) 3 7
• Y (m) 10 7
• ?x (m) 10 -1
• foc 0.01 -17
• n 0.3 17
• ?b (g/cm3) 1.86 -17
• Vw (m/day) 0.044
  33

Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez
(1999). Proc. 6th International Petroleum
Environmental Conference, Houston, TX.
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Frequency Distribution for ? (n79)
How variable are biodegradation rates in the
field, and What are reasonable parameters for
RBCA models?
Mean 0.0112 day -1 Median 0.005 day-1
(t1/2 139 days)
100
Density
50
0
0.10
0.05
0.00
? (day-1)
Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez
(1999). Proc. 6th International Petroleum
Environmental Conference, Houston, TX.
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Current Status of Bioremediation

• We have made significant advances towards
  understanding the biochemical and genetic basis
  for biodegradation. However, bioremediation
  is still an underutilized technology.
• Bioremediation is not universally understood, or
  trusted by those who must approve it. To take
  full advantage of its potential, we need to
  communicate better the capabilities and
  limitations of bioremediation, and answer
• What is being done in the subsurface, Why, How,
  and Who is doing what?
• How fast is it being done, and can we control it
  and make it go faster?
• When can we meet cleanup standards in a
  cost-effective manner?
• Can we reasonably predict that what we want to
  happen, will happen?

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EPISTEMOLOGY OF BIOREMEDIATION

• episteme knowledge
• Theory of the method and basis we use to acquire
  knowledge, including the possibility and
  opportunity to advance fundamental understanding,
  sphere of action, and the philosophy of the
  scientific disciplines that we rely upon.
• Reductionism
• System analysis through separation of its
  components (eliminates
  complexity to enhance interpretation).
• Based on the premise that a system can be known
  by studying its components, and that an idea can
  be understood if we understand its concepts
  separately.
• Used increasingly in bioremediation research to
  investigate mechanisms.
• Holism
• The totality of a system is greater than the sum
  of its parts (synergism antagonism)

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Epistemologys Uncertainty Principle

• Reductionism simplifies the system, enhances
  hypothesis testing, and interpretation
• It also augments lab artifacts and hinders the
  relevance of the information we obtain

High Low
High Low
Complexity, Relevance
Expt. control, Lab artifacts
Holism Reductionism
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Implications

• Quantitative extrapolation from the lab to the
  field is taboo. (interpolate but do not
  extrapolate)
• Rely more on holistic disciplines (e.g., ecology,
  biogeochemistry) and iterate more between the
  field and the lab, between basic and applied
  research.
• Multidisciplinary Research (interstices)
• Aurea mediocridad (San Ignacio de Loyola)

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Pay attention to detail. You never know who is
watching your work, and where your next
promotion or demotion will come from.

• Bioremediation is seldom a straight line to an
  imagined goal (many branching decision points
  requiring flexibility and versatility)
• Remedial technologies are rapidly evolving. Be
  committed to life-long learning, and be aware
  that imagination and creativity could more
  important than knowledge

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Conclusions

• Indigenous microorganisms can often destroy BTEX
  and other common groundwater contaminants,
  making bioremediation (often) technically
  feasible.
• The pendulum recently swung towards natural
  attenuation. This can save money but take much
  longer to achieve cleanup and appear as if
  officials are walking away from contaminated
  sites. Early public involvement is critical to
  minimize such controversy.

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Lets Take a Break!
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TYPES OF MICROBES USED A. Indigenous
Microorganisms Used in most applications
(99) Pseudomonas have wide catabolic
capacity May need to enhance proliferation/enzyme
induction B. Acclimated Strains Preselected
naturally occurring bacteria Generally not
needed for BTEX Often fail to function in situ
common reasons - Conc. of target compound too
low to support growth - Other substances and
organisms inhibit growth - Microbe uses other
food than target contaminant - Target compound
not accessible to microbe C. Genetically
Engineered Microbes (GEMs) Could combine
desirable traits from different microbes -
Ability to withstand stress degrade
recalcitrant compounds - Not needed for BTEX,
many technical political constraints
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Análisis de varianza de las interacciones BTEXN

• Las capacidades de degradación fueron mas amplias
  cuando los BTEXN fueron alimentados como mezcla
  que separadamente (particularmente cuando el T
  estaba presente)
• Las interacciones negativas (e.g., inhibición
  competitiva, toxicidad) fueron estadísticamente
  significativas cuando se alimentó 1 mg/L a cada
  una.
• Por estadística de Kappa se encontró una
  correlación significativa entre las habilidades
  para degradar T y E, p-X y m-X, y p-X y o-X. La
  falla de degradar B fue correlacionada con la
  inhabilidad para degradar o-X.

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Monods Equation
k
Specific degradation rate dC/dt/X
X
C
k


dC
k
-


2
C
K
dt
S

KS
Contaminant Concentration, C
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Why First-Order Degradation Rates? Monods
Equation, When C ltlt KS
X
k
C
dC


k
X


-
-


C





K
K
dt
C
S
S
dC
- lC

dt

(not constant)
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Also, Mass Transfer Limitations Are Conducive to
First-Order Kinetics (even if C gt Ks)
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Alta concentración microbiana Taza más rápida
Simulaciones empleadas k 0.28
g-T/g-células/día KS 8.6 mg-T/L Y 0.6 g-
células/g-T
80
60
107 células/mL
TOLUENO (mg/L)
40
102 células/mL
20
0
0
30
60
90
120
150
Tiempo (Días)
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Por qué es tan difícil limpiar acuíferos?
Detectar la contaminación en aguas subterráneas
es como buscar una aguja en un pajar. Los puertos
de muestreo pueden ser demasiado profundos, no
muy profundos o en un lugar equivocado.