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

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

Potential benefits Enhance understanding of anaerobic degradation Develop site monitoring techniques Generate data from polluted sites

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

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

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

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

Benzoate as a model system One enzymatic modification BCR

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

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

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.

AU Results Benzoate degraded under anaerobic conditions Benzoate breakdown

Benzoate also broken down under aerobic conditions Undiluted1:101:501:1001:2501:5001:10001:2000 T T

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

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

BCR gene Copies increase over time ~2.5 fold increase

16s rDNABCRBDO T initial 1.9E E E+04 T end 1.09e E E+04 Anaerobic benzoate study

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

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

BTEX Napthalene

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?

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

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

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

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

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

Acknowledgements Mike Spence and Shell Dr Brian Kelleher DCU QUESTOR