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
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
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
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