Understanding the fate of methylated PAHs in the terrestrial environment and groundwater. Project – QUB 10/06/09 Andrew Frazer Project supervisors-:

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Presentation transcript:

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

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

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

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.

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

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

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

Non degrading organism Labelled compound CsCl Degrading organism Non degrading organism

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

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

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. 1- Benzoic acid. 2- Benzoic acid. 3- deuterium labelled benzoic acid. 4- deuterium labelled benzoic acid. 5- P.putida G7 (+ve control). 6- ddH2O (-ve control). M-Generuler 1Kb DNA ladder.

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

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

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

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