Principles & Applications of BTEX Bioremediation

Pedro J.J. Alvarez, Ph.D., P.E., DEE

University of Iowa

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?

“Water, water everywhere, nor any drop to drink”The Rime of the Ancient Mariner, Samuel Taylor Coleridge

Contaminants of Concern: BTEX

Importance: • Relatively high solubility = High migration potential • Toxicity: Benzene can cause leukemia at 5 µg/l• Volatile, hydrophobic, biodegradable

CH3

CH3CH3

CH3

CH3

CH2CH3

CH3

CH3

Benzene

m-Xylene p-Xyleneo-Xylene

Toluene Ethylbenzene

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.

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.

010203040506070

8090

100

B T E p-X m-X o-X N

% S

train

s th

at d

egra

ded

com

poun

dFrequency Analysis of Biodegradation

Capabilities of 55 Hydrocarbon Degraders

Gülensoy and Alvarez (1999). Biodegradation. 10:331-340

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.

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] > 50 g/L)

Benzene Degradation by Pseudomonas CFS-215: Toluene enhanced enzyme induction

1510500

10

20

30

40

50

Time (days)

Ben

zene

Con

cent

ratio

n (m

g/L)

T = 0.1 mg/L

T = 50 mg/L

T = 0

ControlControl

Alvarez and Vogel (1991) Appl. Environ. Microbiol., 57: 2981-2985

Cometabolic Degradation of o-Xyleneby Denitrifying Toluene Degraders

504030201000

2

4

6

8

10

ActiveControls

TOLUENE

days

mg/

l

504030201000.0

0.5

1.0

1.5

2.0

ActiveControls

o-XYLENE

days

mg/

l

Alvarez and Vogel (1995) Wat. Sci. Technol., 31: 15-28

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.

Benzene degradation to CO2 and CH4 under methanogenic conditions

0

-0.25

-0.50

0.25

0.50

0.75O2H2O

NO3-N2

HS-

CH4

benzene

H2

SO42-

CO2

CO2

H+

EH°´volts

Half ReactionReduction Potential Hierarchy

OxidizedReduced

C6H6 + 4.5 H2O 2.25 CO2 + 3.75 CH4Go’ = -(30 e-/mol) (96.63 kJ/volt) (-0.24 -(-0.29) volts)Go’ = -133 kJ/mol of benzene, or -4.5 kJ/e- equiv transferred(barely feasible)

Benzene degradation to CO2 under aerobic conditionsC6H6 + 7.5 O2 6 CO2 + 3 H2OGo’ = -(30 e-/mol) (96.63 kJ/volt) (+0.82 -(-0.29) volts)Go’ = -3,200 kJ/mol of benzene, or -107 kJ/e- equiv transferred (24 x more feasible)

Electron Tower

The electron tower concept

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.

Anaerobic BTEX DegradationRates are much slower because anaerobic electron acceptors (e.g., NO3

-, Fe+3, 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.

CS-CoAO

Source: Wiedemeier et al., 1999

In aquifers, electron acceptors are used in sequence. Those of higher oxidation potential are used preferentially:

O2 > NO3- > Mn+4 > Fe+3 > SO4

-2 > CO2

Requirements for Biodegradation (contd)5. Favorable environment (continued):

d) Adequate temperature (rates double for ∆T = +10°C).

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 > migration rate

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)

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

When is engineered bioremediation feasible?

6 5 4 3 2 1 - log Kh (cm/s)

2

1

0

-1

-2

-3

log

k; (p

er d

ay)

Feasible

withEnhancement

Feasible

Not feasible

Feasibility depends on:

1) Kh distribution of nutrients and e- acceptors (Kh > 10-5 m/s)

2) Adsorption bioavailability (depends on Kow and foc, problem for PAHs)

3) Potential degradation rate (half life < 10 days)

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) ES&T, 27(10) 1974-1979

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)

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

Air Curtain

Treated Water

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)

Benzoate addition as auxiliary substrate (1 mg/L) stimulated benzene attenuation through 1-D “biobarrier”

0

50

100

150

200

Effl

uent

Ben

zene

(µg/

L)

0 1 2 3 4 5 6 7 8 9 10Time (days)

with benzoate

Sterile control

Not amended

Alvarez P.J.J., L. Cronkhite, and C.S. Hunt (1988). Environ. Sci. Technol. 1998; 32(5) 634-639

COO-

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

Bioremediation experienced many up- and downturns

1950’s: Microbial infallibility hypothesis (Gayle, 1952)

1970’s: Regulatory pressure stimulates development. Adding bacteria to contaminated sites becomes common practice. Failure to meet expectations (e.g., DDT accumulation) prompts a major downturn.

1980’s: 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.

1990’s: 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.

VolatilizationVolatilization

Aerobic Unsaturated ZoneAerobic Unsaturated Zone

Oxygen ExchangeOxygen Exchange

Aerobic Aerobic uncontaminateduncontaminatedgroundwatergroundwater

DissolutionDissolution

Aerobic ProcessesAerobic Processes

Anaerobic coreAnaerobic core

Mixing, DilutionMixing, Dilution

AdvectionAdvection

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

PE

Fluxo da água subterrânea

Atenuação Natural

Plume

Source

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.

Plume Dimensions Reflect Natural Attenuation

MEDIAN PLUME DIMENSIONS

132 ft

1000 ft

500 ft

700 ft

200 400 600 800 10000

BTEX Plumes(604 Sites)

TCE Plumes (88 Sites)

Other chlorinated solvent plumes(29 Sites)

Salt Water Plumes (chloride)(25 Sites)

Feet

Con

cent

ratio

n

“safe”

Con

cent

ratio

n

“safe”

What is Risk-Based Corrective Action?

Clean source only to a level that will result in an acceptable risk at the potential receptor’s location (e.g., property boundary)

Need a mathematical model to calculate the required Co

Co =?

receptor

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.

)(2)(

)(2)(

)(2)2/(

)(22)/(

)(2)/41(

8),( 2/12/12/12/12/1

2/1

xZerf

xZerf

xYyerf

xYyerf

vtvvtxerfcCtxC

zzyyx

xo

)/(exp4112

exp2/1

vxtkv

xs

x

x

Analytical Solution of the Advection-Dispersion-Sorption Equation with First-Order Decay, for Constant Rectangular

Source (Domenico, 1987)

Variable Baseline Value Lp (%) (day-1) 0.0005 -24Co (ppb) 25,000 +7

Z (m) 3 +7Y (m) 10 +7x (m) 10 -1

foc 0.01 -17

n 0.3 +17b (g/cm3) 1.86 -17

Vw (m/day) 0.044 +33

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

Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez (1999). Proc. 6th International Petroleum Environmental Conference, Houston, TX.

Frequency Distribution for (n=79)

0.100.050.00

100

50

0

(day-1)

Den

sity

Mean = 0.0112 day -1

Median = 0.005 day-1

(t1/2 = 139 days)

How variable are biodegradation rates in the field, and What are “reasonable” parameters for RBCA models?

Lovanh, N., Y.-K. Zhang, and P.J.J. Alvarez (1999). Proc. 6th International Petroleum Environmental Conference, Houston, TX.

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?

EPISTEMOLOGY OF BIOREMEDIATIONepisteme = knowledgeTheory 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)

Epistemology’s Uncertainty PrincipleReductionism simplifies the system, enhances hypothesis testing, and interpretation It also augments lab artifacts and hinders the relevance of the information we obtain

Holism Reductionism

High

LowExpt. c

ontrol,

Lab artifa

ctsComplexity, Relevance

High

Low

Scale: Field Microcosms Cells Extracts Genes Disciplines: Ecology

BiogeochemistryPhysiology

Biochemistry Genetics

Molecular Biology

ImplicationsQuantitative 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)

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

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

ConclusionsIndigenous 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.

Lets Take a Break!

TYPES OF MICROBES USED

A. Indigenous MicroorganismsUsed in most applications (99%)Pseudomonas have wide catabolic capacityMay need to enhance proliferation/enzyme induction

B. Acclimated StrainsPreselected naturally occurring bacteriaGenerally 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

H o w d o e s o n e p r o v e b i o d e g r a d a t i o n i s o c c u r r i n g i n s i t u ?1 . D o c u m e n t l o s s o f c o n t a m i n a n t s a t f i e l d s c a le .

S h o w t h a t d e c r e a s e i n c o n c e n t r a t i o n s i s n o t s o l e l y t h e r e s u l t o f p l u m e m i g r a t i o na n d d i l u t i o n . e . g . , s h o w t h a t c o n t a m i n a n t f l u x d e c r e a s e s a l o n g f l o w p a t h , u s i n gw e l l t r a n s e c t s .

2 . G e o c h e m i c a l i n d i c a t o r s t o d e m o n s t r a t e i n d i r e c t l y t h e t y p e o f d e g r a d a t i o np r o c e s s e s a c t i v e a t t h e s i t e . L o o k f o r O 2 , N O 3

- , a n d S O 42 - l e v e l s b e l o w b a c k g r o u n d i n t h e c o r e o f t h e p l u m e ,

a n d F e ( I I ) a n d C H 4 l e v e l s a b o v e b a c k g r o u n d . A l s o , h i g h e r C O 2 a n d a l k a l i n i t y .

H 2 l e v e l s c a n r e f l e c t d o m i n a n t r e d o x p r o c e s s e s :0 . 1 n M d e n i t r i f i c a t i o n0 . 2 – 0 . 8 n M F e ( I I I ) r e d u c t i o n1 . 0 – 4 . 0 n M s u l f a t e r e d u c t i o n> 5 . 0 n M m e t h a n o g e n e s i s

M u l t i l e v e lW e l lC l u s t e r

F l u x= v C

Stable Isotope Analysis: Carbon atoms in BTEX are mainly 12C with some heavy isotopes (13C) Isotope fractionation results because light (12C) isotope bonds are preferentially biodegraded compared to heavy isotope (13C) bonds No isotope fractionation results from abiotic processes (dilution, sorption, etc.). Thus, biodegradation results in isotopical depletion of 13C for dissolved inorganic carbon and daughter products (13C = -20 to –30 per mil), and get and enrichment of 13C for residual contamination of parent compound. 3. Laboratory or in situ microcosms showing BTEX degradation

(look for mineralization or accumulation of metabolites)

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.

k

KS Contaminant Concentration, C

Spe

cific

deg

rada

tion

rate

dC

/dt/X

2k

CKX C k

dtdC

S-

Monod’s Equation

Why First-Order Degradation Rates?Monod’s Equation, When C << KS

SS K C X k- CKC X k - dt

dC

SK X k

dt

dCC

(not constant)

con

cen

tra

tion

cell

distancecell

cell

CKCqq

2/1

maxdeg

cellbulkt CCkq

0.0

0.2

0.4

0.6

0.8

1.0

05

1015

2025

30

05

1015

Also, Mass Transfer Limitations Are Conducive to

First-Order Kinetics (even if C > Ks)

0

20

40

60

80TO

LUE

NO

(mg/

L)

0 30 60 90 120 150

Tiempo (Días)

102 células/mL

107 células/mL

Simulaciones empleadas:k = 0.28 g-T/g-células/díaKS = 8.6 mg-T/LY = 0.6 g- células/g-T

Alta concentración microbiana = Taza más rápida

¿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.