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14 C-acetate TEAP determination

14 C-acetate TEAP determination. Procedure is based on the biogas profiles produced by a sample when amended with 14 C-acetate in the presence and absence of molybdate – a specific inhibitor of sulfate reduction. Fe(III) reduction. 0.25. 10 mM molybdate. No molybdate. 0.2. 0.15.

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14 C-acetate TEAP determination

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  1. 14C-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

  2. Fe(III) reduction 0.25 10 mM molybdate No molybdate 0.2 0.15 14CO2 (microcuries) 0.1 0.05 0 0 2 4 6 8 10 Time (hour)

  3. Sulfate reduction 1 No molybdate 10 mM molybdate 0.8 0.6 14CO2 (microcuries) 0.4 0.2 0 0 2 4 6 8 10 Time (hour)

  4. 0.3 CH , no molybdate 4 CH , 10 mM molybdate 0.25 4 0.2 14CO2 or 14CH4 (microcuries) 0.15 CO , no molybdate 0.1 2 CO , 10 mM molybdate 2 0.05 0 0 1 2 3 4 5 6 Time (hour) Methanogenesis

  5. 14C-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

  6. Additional factors which control the population • Relative population sizes • Other nutritional sources/requirements • Presence of inhibitors • Genetic regulation • Bioavailability of e- acceptor

  7. Bioremediation Intrinsic remediation Engineered remediation

  8. Isolation/containment • Contaminant mobility • Biodegradation/transformation under intrinsic conditions • Presence of required microbial community • Presence of required nutrients Intrinsic remediation Important Parameters Disadvantages Advantages • Slow • Public perception • Long term monitoring required • Ease • Low cost

  9. Engineered remediation Ex-situ Important Parameters • Accessibility • Biodegradability • Rate of Biodegradation • Is a microbial consortium required • Will microbial end products cause plugging • Disadvantages • Not applicable to large plumes • Not applicable to low level concentration • Cost • Complexity • Advantages • Controlled environment • Ease of monitoring

  10. Engineered remediation In-situ Important Parameters • Extent of contaminant plume • Are environmental parameters conducive • Accessibility of contaminant • Are the appropriate populations present • Disadvantages • Uncontrolled environment • Cost • Complexity • Difficulty of monitoring • Advantages • Applicable to large plumes • Applicable to low level concentration

  11. Microbial or Nutrient injection kPa

  12. Aerobic v’s Anaerobic Aerobic CH3COO- + 2 O2 2 HCO3- + H+DGo = -844 kJ/mol Anaerobic 5 CH3COO- + 8 NO3- + 3 H+ 10 HCO3- + 4 H2O + 4 N2DGo = -792 kJ/mol Thermodynamically O2 respiration is much more favorable

  13. O2 H OH OH Dioxygenase H Dioxygenase Mediated Destabilization of Benzene Ring CO2

  14. Air Injection SO4 Fe(III) NO3 CH4 O2 O2

  15. Monitoring Geochemical Analyses Problems: 1. Homogenous Sample collection 2. Not directly indicative of microbial degradation Microbial Analyses Molecular v’s physiology Problems: 1. Homogenous Sample collection 2. Presence not indicative of activity 3. Known biomarkers are required

  16. Monitoring tools • Stable isotope fractionation • 16S rDNA Probes • Immunoprobes • Functional gene probes

  17. 35ClO4- (75.4%) 37ClO4- (24.6%) Microbial perchlorate reduction Rayleigh fractionation curve Acetate +20 35Cl- - CO2 ClO 4 +10 e- R - = f (a-1) Cl 0 Ro instant d37Cl (%o) -15.75 +/- 0.36 ‰ fractionation - Cl Ro= 37Cl/35Cl ofinitial ClO4- -10 accumulated R = 37Cl/35Cl of residual ClO4- -20 f =fraction ClO4- reacted a = the isotope fractionation factor associated with reduction. 0 0.25 0.5 0.75 1.0 Fraction of perchlorate used Stable isotope fractionation

  18. Advantages: Technically simple Robust Disadvantages: Laborious = costly Long lead time Insensitive Biased by media and electron donor 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 10-9 10-7 10-1 Serial Dilution Culture based methods X X X X X X X X X X X X X X

  19. Treponema pallidum Magnetospirillum magnetotacticum 10 changes Strain SN1 Dechlorospirillum strain WD a proteobacteria Strain DB Azospirillum brasilense Strain TTI Comamonas testosteroni Ideonella dechloratans Strain FL2 Strain FL8 Strain FL9 Dechloromonas agitata Strain CL CKB-type Strain NM Dechloromonas Strain MLC33 Strain JM Strain HZ Strain CL24plus Strain CL24 b proteobacteria Strain CCO Dechloromonas aromatica RCB-type Strain SIUL Strain MissR Rhodocyclus tenuis Strain AH Azospira suillum Strain Iso1 PS-type Strain Iso2 Azospira Strain SDGM Strain PDX Strain KJ Propionivibrio limicola Strain LT-1 Gill symbiont of Thyasira flexuosa Dechloromarinus strain NSS Escherichia coli Pseudomonas stutzeri g proteobacteria Pseudomonas strain PK Strain CFPBD Pseudomonas chloritidismutans Strain PDA Strain PDB Wolinella succinogenes strain HAP-1 e proteobacteria Helicobacter pylori

  20. San Nick Island Sample MPN CKB type RCB type PS type Drainage channel from EOD H-3’ No growth H-6’No growth H-8’ No growth Explosive Ordinance Disposal M-3’* 7.49 + 3.35 x 104negative negative negative M-6’ 7.49 + 3.35 x 104negative negative negative M-9’ No growth 1000 Springs P-3’ No growth P-5.5’ 2.40 + 1.74 x 105negative negative negative P-8’*2.40 + 1.74 x 105negative negative negative pH 8.0 8.7 8.4 8.6 8.4 8.7 8.9 8.8 9.0

  21. MPN's for Longhorn, Texas CKB type Negative Negative Negative Negative Negative RCB type Negative Negative Negative Negative Negative PS type Negative Negative Negative Negative Negative Site 16 Sediment 16 Ground Water* 25 Surface Soil 25 Sediment 25 Ground Water pH 6.6 6.4 4.2 5.6 6.5 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

  22. The biochemistry offers some targets Acetate ClO4- e- Perchlorate reductase ClO2- Chlorite dismutase Cl- e- O2 CO2 H2O Cytoplasm Periplasm 2H++2e- ClO4-/ClO3- Quinone Pool 2H+ QH2 ClO4-/ClO3- Q PcrB PcrA Perchlorate reductase complex PcrC NirT ClO2- Chlorite dismutase complex Cld 4e- Cl- 2H+ O2 6H+ Cytochrome oxidase Cyt o 2H2O ADP + Pi 4H+ 4H+ ATPase ATP Achenbach et al (2006). In Gu, B. and Coates, J.D. (Eds) Perchlorate, Environmental Occurrence, Interactions, and Treatment. Springer Publishers, MA

  23. *These organisms are incapable of reductive (per)chlorate respiration. Cell lysate Whole cells Organism Dechloromonas strain CKB Dechloromonas strain RCB Azospira strain PS I. dechloratans Dechlorospirillum strain WD Dechloromarinus strain NSS Pseudomonas strain PK E. coli* P. stuzeri* Antibody to the Chlorite Dismutase Enzyme Immuno-probe ClO4- ClO2- Cl- O2

  24. 1. Fluorescent tagging. CD antibody can be conjugated to a fluorescent label and visualized using immunofluorescence micrographs. Cell lysate Organism Whole cells Strain CKB perchlorate grown Strain CKB aerobic Functional detection. Antibody only detects cells actively reducing perchlorate. 2. Many uses of the CD Specific Antibody

  25. 3. Immuno-Based Assay for the Rapid Screening of Environmental Samples Positive for perchlorate reducer 0.2 m Filter Environmental groundwater sample or aqueous extract Secondary Goat-anti-rabbit peroxidase IgG Primary rabbit-anti-CD IgG Expressed chlorite dismutase (CD) Analysis oftwo 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. Perchlorate-reducing organism

  26. Environmental groundwater sample containing perchlorate-reducing bacteria Secondary Goat-anti-rabbit peroxidase IgG 1.0 Perchlorate-reducing organism 0.8 0.6 Absorbance (450 nm) 0.4 R2=0.9912 0.2 0 0.4 0.6 0.8 1.0 1.2 0 0.2 D. agitata cell number (x106.ml-1) 4. Rapid ELISA assay for Perchlorate-reducing Bacteria Positive for perchlorate-reducing bacteria Primary rabbit-anti-CD IgG Expressed chlorite dismutase (CD)

  27. Immunoprobe summary Advantages: No culture biases Rapid Simple Cheap Sensitive Functional probe Disadvantages: Not applicable to soil or sediments Potential false positives

  28. Genetic probes targeting the chlorite dismutase gene Lake Fryxell samples Los Alamos well samples SIU Campus samples LakeVida samples Pseudomonas strain PK LakeHoare samples Negative control Soil Pond Well 3 Well 5 Well 7 Well 4 Well 2 WC 12m WC 12m Mat Sediment WC 7m Lake WC chlorite dismutase gene First amplification gives 484 bp cld gene fragment plus spurious products Second amplification gives clean 408 bp cld gene fragment

  29. 10-1 X X X 10-2 X X X 10-3 X X X 10-4 X X X X 10-5 X 10-6 10-7 10-8 10-9 10-7 10-1 Serial Dilution Quantification of Perchlorate-reducing bacteria with MPN-PCR Most Probable Number Counts DNA amplification by polymerase chain reaction (PCR) and analysis by gel electrophoresis

  30. Genetic probe summary Advantages: Rapid Sensitive Will work with soil samples Can be adapted to a functional probe Combined with PR probe can remove false positives Disadvantages: More expensive than immunoprobe Technically involved requires specialized equipment

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