150 likes | 262 Vues
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.
E N D
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 • 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. Small, non toxic, soluble compounds Large, toxic, insoluble compounds
Why investigate methylated PAHs? NDO cis-1,2-dihydroxynaphthalene naphthalene 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
What is it ? How does it work? Stable Isotope Probing (SIP)
Labelled compound CsCl Degradingorganism Non degrading organism
How do we separate molecules? dH2O from siphon =CONTROL! DENSITYGRADIENT Less dense, non-enriched DNA More dense, isotopically enriched DNA Collection of gradient fractions
UV absorbance of gradient fractions. d2– labelled DNA A C A260nm B Fraction number
C13 naphthalene unavailable Small amounts of DNA obtained from soil “Blind” gradient collection Soil enrichment cultures. 1 2 3 4 5 6 M benzoic acid naphthalene 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 • d2vs. 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