1414C-acetate TEAP determinationC-acetate TEAP determination

Procedure is based on the biogas profiles produced by a sample when amended with 14C-acetate in the presence and absence of molybdate – a specific inhibitor of sulfate reduction

0

0.05

0.1

0.15

0.2

0.25

0 2 4 6 8 10

10 mM molybdate

No molybdate

Time (hour)

14C

O2 (

mic

rocu

ries

)

Fe(III) reductionFe(III) reduction

0

0.2

0.4

0.6

0.8

1

0 2 4 6 8 10

No molybdate

10 mM molybdate

Time (hour)

14C

O2 (

mic

rocu

ries

)

Sulfate reductionSulfate reduction

0

0.05

0.1

0.15

0.2

0.25

0.3

0 1 2 3 4 5 6

CH4, no molybdate

CH4, 10 mM molybdate

CO2, no molybdate

CO2, 10 mM molybdate

Time (hour)

14C

O2

or 14

CH

4 (m

icro

curi

es)

MethanogenesisMethanogenesis

1414C-acetate TEAP determinationC-acetate TEAP determination

Problems

•Solid sample collection is required

•Requires supporting geochemical analyses for accuracy

•Requires utilization of radioactive materials

•Requires analyses to be performed on fresh samples

•Requires large amounts of potentially precious samples

Additional factors which control the Additional factors which control the populationpopulation

• Relative population sizes

• Other nutritional sources/requirements

• Presence of inhibitors

• Genetic regulation

• Bioavailability of e- acceptor

BioremediationBioremediation

Engineered remediation

Intrinsic remediation

Intrinsic remediationIntrinsic remediation

•Contaminant mobility

•Biodegradation/transformation under intrinsic conditions

•Isolation/containment

•Presence of required microbial community

•Presence of required nutrients

Important Parameters

Advantages

1. Ease2. Low cost

Disadvantages

1. Slow2. Public perception3. Long term monitoring required

Engineered remediationEngineered remediationEx-situEx-situ

Advantages

1. Controlled environment2. Ease of monitoring

Disadvantages

1. Not applicable to large plumes2. Not applicable to low level concentration3. Cost4. Complexity

Important Parameters

•Accessibility

•Biodegradability

•Rate of Biodegradation

•Is a microbial consortium required

•Will microbial end products cause plugging

In-situIn-situEngineered remediationEngineered remediation

Important Parameters

•Extent of contaminant plume

•Are environmental parameters conducive

•Accessibility of contaminant

•Are the appropriate populations present

Advantages

1. Applicable to large plumes2. Applicable to low level concentration

Disadvantages

1. Uncontrolled environment2. Cost3. Complexity4. Difficulty of monitoring

kPa

Microbial or Nutrient injection

Aerobic v’s AnaerobicAerobic v’s Anaerobic

AerobicCH3COO- + 2 O2 2 HCO3

- + H+ Go = -844 kJ/mol

Anaerobic

5 CH3COO- + 8 NO3- + 3 H+ 10 HCO3

- + 4 H2O + 4 N2 Go = -792 kJ/mol

Thermodynamically O2 respiration is much more favorable

H

OH

OH

HDioxygenase

O2

Dioxygenase Mediated Dioxygenase Mediated Destabilization of Benzene Destabilization of Benzene

RingRing

CO2

O2CH4

NO3

Fe(III)SO4

O2

Air Injection

MonitoringMonitoring

Geochemical Analyses

Problems:1. Homogenous Sample collection2. Not directly indicative of microbial degradation

Microbial Analyses

Molecular v’s physiologyProblems:

1. Homogenous Sample collection2. Presence not indicative of activity3. Known biomarkers are required

Monitoring toolsMonitoring tools

•Stable isotope fractionation

•16S rDNA Probes

•Immunoprobes

•Functional gene probes

Stable isotope fractionation

Microbial perchlorate reduction Rayleigh fractionation curve

-20

-10

0

+10

+20

1.00.750.50.250

Fraction of perchlorate used

-15.75 +/- 0.36 ‰fractionation

ClO4-

instant Cl-

accumulated Cl-

37C

l (%

o)

Acetate

CO2

35ClO4- (75.4%)

37ClO4- (24.6%)

35Cl-

e-

= f (-1) RRo

Ro = 37Cl/35Cl of initial ClO4-

R = 37Cl/35Cl of residual ClO4-

f =fraction ClO4- reacted

= the isotope fractionation factor associated with reduction.

Culture based methods

10-1 10-7Serial Dilution10-9

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

X X XX X XX X XX XX XX

Advantages:Technically simpleRobust

Disadvantages:Laborious = costlyLong lead timeInsensitiveBiased by media and electron donor

AzospiraPS-type

Dechloromonas

CKB-type

RCB-type

Treponema pallidumMagnetospirillum magnetotacticumStrain SN1Dechlorospirillum strain WD

Strain DBAzospirillum brasilense Strain TTI

Comamonas testosteroniIdeonella dechloratansStrain FL2Strain FL8Strain FL9Dechloromonas agitataStrain CLStrain NMStrain MLC33

Strain JMStrain HZ

Strain CL24plusStrain CL24

Strain CCODechloromonas aromatica

Strain SIULStrain MissR

Rhodocyclus tenuisStrain AHAzospira suillum

Strain Iso1Strain Iso2

Strain SDGMStrain PDXStrain KJ

Propionivibrio limicolaStrain LT-1

Gill symbiont of Thyasira flexuosa Dechloromarinus strain NSS

Escherichia coliPseudomonas stutzeriPseudomonas strain PKStrain CFPBDPseudomonas chloritidismutansStrain PDAStrain PDB

Wolinella succinogenes strain HAP-1Helicobacter pylori

10 changes

proteobacteria

proteobacteria

proteobacteria

proteobacteria

pH

8.08.78.4

8.68.48.7

8.98.89.0

Sample MPN CKB type RCB type PS type

Drainage channel from EODH-3’ No growth H-6’ No growthH-8’ No growthExplosive Ordinance DisposalM-3’* 7.49 + 3.35 x 104 negative negative negativeM-6’ 7.49 + 3.35 x 104 negative negative negativeM-9’ No growth1000 SpringsP-3’ No growthP-5.5’ 2.40 + 1.74 x 105 negative negative negativeP-8’* 2.40 + 1.74 x 105 negative negative negative

San Nick Island

pH

6.66.44.25.66.5

MPN's for Longhorn, Texas

MPN

2.40 + 1.74 x 106

2.15 + 0.81 x 106

9.33 + 4.17 x 105

2.40 + 1.74 x 105

1.12 + 0.64 x 103

CKB type

NegativeNegativeNegativeNegativeNegative

RCB type

NegativeNegativeNegativeNegativeNegative

PS type

NegativeNegativeNegativeNegativeNegative

Site

16 Sediment16 Ground Water*25 Surface Soil25 Sediment25 Ground Water

ClO4-

ClO2-

O2

Cl-

H2O

e-

Acetate

CO2

e-

Chlorite dismutase

Perchlorate reductase

PcrA

PcrB

PcrC

ClO4-/ClO3

-

Cld

ClO4-/ClO3

-

ClO2-

Cl-

O2

2H2O

6H+

2H+

Quinone Pool

QH2

NirT

Cyt o

2H+

2H++2e-

4H+

ADP + Pi

ATP

4H+

Cytoplasm Periplasm

Perchlorate reductase complex

Chl

orit

e di

smut

ase

com

plex

Cytochrome oxidase

ATPase

4e-

Q

Achenbach et al (2006). In Gu, B. and Coates, J.D. (Eds) Perchlorate, Environmental Occurrence, Interactions, and Treatment . Springer Publishers, MA

The biochemistry offers some targets

Antibody to the Chlorite Dismutase Enzyme

Dechloromonas strain RCB

Dechloromarinus strain NSS

Dechlorospirillum strain WD

Pseudomonas strain PK

Azospira strain PS

Dechloromonas strain CKB

OrganismWholecells

Celllysate

E. coli*

P. stuzeri*

I. dechloratans

*These organisms are incapable of reductive (per)chlorate respiration.

ClO2-

O2

Cl-

ClO4-

Immuno-probe

Many uses of the CD Specific Antibody

Fluorescent tagging.CD antibody can be conjugated to a fluorescent label and visualized using immunofluorescence micrographs.

1.

Organism Whole cellsCelllysate

Strain CKBperchlorate grown

Strain CKBaerobic

Functional detection. Antibody only detects cells actively reducing perchlorate.

2.

Immuno-Based Assay for the Rapid Screening of Environmental Samples

Secondary Goat-anti-rabbit peroxidase IgG

Primary rabbit-anti-CD IgG

Expressed chlorite dismutase (CD)

Perchlorate-reducing organism

Environmental groundwater sample or aqueous extract

Positive for perchlorate reducer

Analysis of two environmental samples for the presence of the CD enzyme using the ELISA test. The left sample is positive for CD, while the right sample is negative.

0.2 m Filter

3.

Expressed chlorite dismutase (CD)

Perchlorate-reducing organism

Positive for perchlorate-reducing bacteria

Environmental groundwater sample containing perchlorate-reducing

bacteria

Primary rabbit-anti-CD IgG

Secondary Goat-anti-rabbit peroxidase IgG

Rapid ELISA assay for Perchlorate-reducing Bacteria

0

0.2

0.4

0.6

0.8

1.0

Abs

orba

nce

(450

nm

)

0 0.2

D. agitata cell number (x106.ml-1)

0.4 0.6 0.8 1.0 1.2

R2=0.9912

4.

Advantages:No culture biasesRapidSimpleCheapSensitiveFunctional probe

Disadvantages:Not applicable to soil or sedimentsPotential false positives

Immunoprobe summary

Second amplification gives clean 408 bp cld

gene fragment

Pse

udom

onas

str

ain

PK

Los Alamos well samples SIU

Campus samples

Lake Fryxell samples

Neg

ativ

e co

ntro

l

Wel

l 2

Soi

l

Pon

d

Lak

e W

C

Sed

imen

t

WC

7m

WC

12m

LakeHoare samples

WC

12m

Mat

Lak

eVid

a sa

mp

les

Wel

l 3

Wel

l 4

Wel

l 5

Wel

l 7

chlorite dismutase gene

First amplification gives 484 bp cld gene fragment plus

spurious products

Genetic probes targeting the chlorite dismutase gene

Quantification of Perchlorate-reducing bacteria with MPN-PCR

1) Extract DNA from an environmental

sample

2) Sequentially dilute DNA in triplicate

3) PCR amplify the chlorite dismutase

gene from the dilutions

4) Score each dilution for the presence or

absence of the product

5) Quantify the perchlorate-reducing population using the

MPN equation

10-1 10-7Serial Dilution

Most Probable Number Counts

DNA amplification by polymerase chain reaction (PCR)

and analysis by gel electrophoresis

10-9

10-1

10-2

10-3

10-4

10-5

10-6

10-7

10-8

X X XX X XX X XX XX XX

Advantages:RapidSensitiveWill work with soil samplesCan be adapted to a functional probeCombined with PR probe can remove false positives

Disadvantages:More expensive than immunoprobeTechnically involved requires specialized equipment

Genetic probe summary