1. Understanding the fate of methylated PAHs in the terrestrial environment and groundwater. Project – QUB 10/06/09
Andrew Frazer
Project supervisors-: Dr C Allen, Dr L Kulakov, Prof M Larkin, Dr J Quinn
Industrial mentors: Dr. Mike Spence & Dr. Gordon Lethbridge,
Shell Global Solutions

2. Project Summary
Investigate degradation of methylated vs non methylated PAHs
Develop Stable Isotope Probing (SIP) to utilise deuterium labeled substrates
Broad application to waste treatment process monitoring
Start date – September 2006
End date – September 2009

3. Polycyclic Aromatic Hydrocarbons
(PAHs)
naphthalene
anthracene
Benz[a]anthracene
Benzo[a]pyrene
Toxic (Mutagenic, Carcinogenic)
Major economic and environmental importance.

4. PAH Biodegradation.
Microorganisms
Large, toxic, insoluble compounds
Small, non toxic, soluble compounds
Metabolism and degradation of smaller, simple, fused ring PAHs has been investigated in detail
Microbial degradation of larger 4 and 5 ringed PAHs and methylated PAHs is less well understood.

5. Why investigate methylated PAHs?
NDO
naphthalene
cis-1,2-dihydroxynaphthalene
2-methylnaphthalene ?

6. What is the problem?
How are these compounds degraded in the environment?
Culture based techniques
In vitro
< 1% of organisms applicable
Non culture based techniques
In situ
Entire communities of degrading organisms
can be investigated

7. Stable Isotope Probing (SIP)
What is it ?
How does it work?

8. Non degrading organism
Labelled compound
CsCl
Degrading organism
Non degrading organism

9. How do we separate molecules?
dH2O from siphon
=CONTROL!
DENSITY GRADIENT
Less dense,
non-enriched DNA
More dense, isotopically enriched DNA
Collection of gradient fractions

10. UV absorbance of gradient fractions. d2 – labelled DNA
A260nm B
Fraction number

11. Soil enrichment cultures.
naphthalene
benzoic acid
C13 naphthalene unavailable
Small amounts of DNA obtained from soil
“Blind” gradient collection
1% agarose gel showing PCR products for benzoic acid soil enrichment cultures using benzoate dioxygenase primers (BAF1 and BAR2). KEY Benzoic acid. 2- Benzoic acid. 3- deuterium labelled benzoic acid. 4- deuterium labelled benzoic acid P.putida G7 (+ve control) ddH2O (-ve control). M-Generuler 1Kb DNA ladder.

12. Deuterium vs 13C SIP d2 benzoic acid 13C benzoic acid
d2 benzoic acid pre CsCl centrifugation
13C benzoic acid pre CsCl centrifugation
d2 benzoic acid heavy fraction
13C benzoic acid heavy fraction
d2 benzoic acid light fraction
13C benzoic acid light fraction

13. Methylated vs non methylated PAHs
Wide range of SIP substrates now available!
naphthalene
1 methyl naphthalene
2 methyl naphthalene

14. Summary to date
Successfully labelled PAH degrading bacteria and demonstrated SIP using pure cultures
Demonstrated 13C vs deuterium SIP using benzoic acid soil enrichment cultures
Developed methods for lab comparison of methylated/non-methylated PAH degraders
Obtained soil samples and on site chemistry from PAH polluted site

15. Future work d2 vs. C13 SIP publication
SIP experiment to investigate degradation of methylated and non methylated PAHs in contaminated land samples
“bio prospecting” experiments using deuterium labeled substrates