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

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

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

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

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

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

8. Benzoate as a model system One enzymatic modification BCR

9. Nitrogen atmosphere Methods Anaerobic glove bag with nitrogen Inoculation of media within N 2 environment Vials crimped and stored in anaerobic jars N2N2 O2O2

10. Primer development for qPCR BZAQ4FBZAQ4R BZAQ4F/R used as a template to design internal primers

11. HPLC analysis Benzoate degraded over course of experiment Conditions:- 40:60 MeOH:C 2 H 3 O 2 NH 4 Flow rate 0.5ml/min 2.288 5.810 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Minutes 1.002.003.004.005.006.007.00 Results

12. Undiluted1:101:501:1001:2501:5001:10001:2000 T0+++++--- T2++++++++ Semi quantitative approach PCR using primer pair BZAQ4F/R

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

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

15. Pure culture work Azoarcus and Thauera spp Growth conditionsThauera aromaticaThauera cehAzoarcus evansii Benzoate Aerobic++ Benzoate anaerobic+-+ Toluene aerobic+N/A+ heptamethylnonane--- + 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 Sea core samples provided by Dr Brian Kelleher, DCU Benzoyl coa reductase detected in sea core samples Sequence analysis identified marine organisms

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