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Exploiting the potential for biological reduction in waste and water treatment processes

Exploiting the potential for biological reduction in waste and water treatment processes Paul Flanagan. Supervisors: Dr C Allen, Dr L Kulakov, Professor M Larkin. Industrial mentor: Dr Geoff Wilcox BP. Objectives. Benzoate dioxygenase.

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Exploiting the potential for biological reduction in waste and water treatment processes

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  1. Exploiting the potential for biological reduction in waste and water treatment processes Paul Flanagan Supervisors: Dr C Allen, Dr L Kulakov, Professor M Larkin Industrial mentor: Dr Geoff Wilcox BP

  2. Objectives Benzoate dioxygenase Can key marker genes be used to predict degradation of pollutants? Benzoyl coa reductase Benzylsuccinate synthase Start date:- October 2007 End date:- September 2010

  3. Potential benefits • Enhanced understanding of anaerobic degradation • Quick and efficient site monitoring • Rapid generation of data involving polluted sites

  4. Aromatic hydrocarbon sources Green plant degradation FUEL Underground storage tanks Microbial formation

  5. Background Origin of pollution Oxygen concentration Anaerobic zone mobile contaminants move away from source readily

  6. Anaerobic degradation Relatively new concept Stability of benzene ring Oxygen + oxygenases

  7. Anaerobic pathway BCR 2 ATP 2 ADP Benzoyl coa formed as a central intermediate Benzoyl coa reductase is a key enzyme in anaerobic degradation pathways

  8. Benzoate as a model system One enzymatic modification BCR

  9. Methods N2 Nitrogen atmosphere O2 Anaerobic glove bag with nitrogen Inoculation of media within N2 environment Vials crimped and stored in anaerobic jars

  10. Primer development for qPCR BZAQ4F BZAQ4R BZAQ4F/R used as a template to design internal primers

  11. Results HPLC analysis Conditions:- 40:60 MeOH:C2H3O2NH4 Flow rate 0.5ml/min Benzoate degraded over course of experiment 0.35 5.810 0.30 0.25 0.20 AU 0.15 0.10 0.05 2.288 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 Minutes

  12. Undiluted 1:10 1:50 1:100 1:250 1:500 1:1000 1:2000 T0 + + + + + - - - T2 + + + + + + + + Semi quantitative approach PCR using primer pair BZAQ4F/R

  13. Preliminary qPCR results 16s and BCR studies carried out 16s DNA BCR DNA Early results indicate: ~4 fold increase in 16s gene ~2 fold increase in BCR

  14. Cloned BCR fragment 484bp inserted into pMOSBlue vector Similarity to T.aromatica BCR

  15. Growth conditions Thaueraaromatica Thaueraceh Azoarcus evansii Benzoate Aerobic + + Benzoate anaerobic + - + Toluene aerobic + N/A + heptamethylnonane - - - Pure culture work Azoarcus and Thauera spp + • Enabling development of • Conditions for delivery • Measurement

  16. Test 2 diverse sites Site within BTEX plume:- Examine the relationship between key genes Is degradation anaerobic? An environmentally different site:- Can key genes be detected?

  17. BCR detected in sea cores Sequence analysis identified marine organisms Benzoyl coa reductase detected in sea core samples Sea core samples provided by Dr Brian Kelleher, DCU

  18. Future work Establish enrichments with BTEX compounds as C sources Qualitative and quantitative analysis of BTEX enrichments DGGE analysis+sequencing Monitor sites of contamination

  19. Supplementary information Benzoyl coa cyclohexa-1,5-diene-1-carbonyl coa Benzene toluene xylene ethylbenzene 1800 580 200 125 solubility mg/ml @ 25°c Resonance energy >100 Kj/mol

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