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

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. 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 • Enhance understanding of anaerobic degradation • Develop site monitoring techniques • Generate data from polluted sites

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

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

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

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

8. Benzoate as a model system One enzymatic modification BCR

9. Methods N2 Microcosm set up Nitrogen atmosphere O2 Limit oxygen exposure Seal vials Destructive sampling

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:C2H3O2NH4 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. Results Benzoate breakdown Benzoate degraded under anaerobic conditions 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

13. Undiluted 1:10 1:50 1:100 1:250 1:500 1:1000 1:2000 T0 + + + + + - - - T2 + + + + + + + + Benzoate also broken down under aerobic conditions

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. Anaerobic benzoate study

18. Growth conditions Thauera aromatica Thauera ceh Azoarcus evansii Benzoate Aerobic + + Benzoate anaerobic + - + Toluene aerobic + N/A + heptamethylnonane - - - +

19. Origin of pollution B A Contaminated site study Samples courtesy of Shell Inside and outside zone of contamination Look for marker genes 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 Eubacterial Diversity through the sample Complex community Thiomicrospira spp Sulfitobacter spp

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

25. Potential uses of the marker gene system Polluted land Anaerobic lagoon processes 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 Dr Brian Kelleher DCU Mike Spence and Shell QUESTOR