1. Exploiting the potential for biological reduction in waste and water treatment systems 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 Enhance understanding of anaerobic degradation Develop site monitoring techniques Generate data from polluted sites

4. Anaerobic zone Oxygen concentration Upper layers aerobic Origin of pollution Contaminants may be mobile Contaminants may be very stable Background

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

6. Aerobic degradation Relatively rapid Well studied The full picture? × Anaerobic degradation has potential Relatively new concept

7. BCR Benzoyl coa is a central intermediate Compounds are activated Benzene ring is opened Anaerobic pathway

8. Benzoate as a model system One enzymatic modification BCR

9. Methods Nitrogen atmosphere O2 N2N2 Limit oxygen exposure Seal vials Destructive sampling Microcosm set up

10. Primer design for qPCR Conserved regions exist in the benzoyl coa reductase subunits T. aromatica used as template

11. Chemical analysis HPLC Conditions:- 40:60 MeOH:C 2 H 3 O 2 NH 4 Flow rate 0.5ml/min GC/MS Column temp: 120 °C for 0.5 min followed by ramp 3 °C/min to 140 °C followed by ramp 25 °C/min to 250 °C holding until completion. The spectra were scanned from 60 AMU to 180 AMU.

12. 2.288 5.810 AU 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 1.002.003.004.005.006.007.00 Results Benzoate degraded under anaerobic conditions Benzoate breakdown

13. Benzoate also broken down under aerobic conditions Undiluted1:101:501:1001:2501:5001:10001:2000 T0+++++--- T2++++++++

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

15. Quantitative analysis 16S rDNA Copies increase over time ~6 fold increase

16. BCR gene Copies increase over time ~2.5 fold increase

17. 16s rDNABCRBDO T initial 1.9E+071.00E+054.79E+04 T end 1.09e+082.43E+055.89E+04 Anaerobic benzoate study

18. Growth conditionsThauera aromaticaThauera cehAzoarcus evansii Benzoate Aerobic ++ Benzoate anaerobic +-+ Toluene aerobic +N/A+ heptamethylnonane --- +

19. Samples courtesy of Shell Inside and outside zone of contamination Origin of pollution A B Look for marker genes Contaminated site study Look for relationship

20. BTEX Napthalene

21. 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?

22. PCR study 16s genes detected for both eubacteria and archaea BCR detected within the sea core

23. 16S rDNA DGGE Diversity through the sample Complex community Eubacterial Thiomicrospira spp Sulfitobacter spp

24. Archaeal less complex community Most diversity found at deepest point of core Sea core samples provided by Dr Brian Kelleher, DCU Methanobacteriaceae spp

25. Potential uses of the marker gene system Anaerobic lagoon processes Polluted land Anaerobic sludge process in WWTW

26. Future work Complete BTEX studies Look for BCR gene in samples Apply qPCR to the DNA extracted Possible community structure Phylogenetic analysis of sea core samples

27. Acknowledgements Mike Spence and Shell Dr Brian Kelleher DCU QUESTOR