ISOLATION AND IDENTIFICATION OF PAH DEGRADING BACTERIA FROM CONTAMINATED SOILS

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ISOLATION AND IDENTIFICATION OF PAH DEGRADING
BACTERIA FROM CONTAMINATED SOILS
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Introduction
PhD started 03 Jul 2006
International student from Iran
Initially self
funded
UniSA Presidents Fellowship 1st January
2008 CRC CARE Scholarship 1st January 2008
Project title Bioavailability of Polycyclic
Aromatic Hydrocarbons (PAHs) and its Impact on
Bioremediation
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Introduction

• PAHs are ubiquitous environmental pollutants
• Bioremediation may be used for PAH removal
• Effective for 4-ring PAHs
• Generally not effective for 5 ring PAHs
• Limiting factor for PAH bioremediation is
  bioavailability
• ??? Risk of residual PAHs following
  bioremediation

Aim Efficacy of PAH bioremediation
Factors that influence PAH bioavailability for
bioremediation Whether residual PAHs
(non-degradable) are bioavailable for human
health or not?
CRC CARE Milestones Program 1 Target 1.2
Methods for measuring speciation, bioavailability
and toxicity of contaminants Program 2 Target
2.2 Bioremediation technology for organics
present in contamination mixtures Milestone
2.2.2 Laboratory based development of
bioremediation technology for organic
contaminants. Milestone 2.2.3 Isolation of
novel organic contaminant detoxifying
microorganisms. Milestone 2.2.4
Characterisation of contaminant catabolic
pathways.
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PhD Overview
DEGRADATION
ISOLATION
PAH-SOIL
ENRICHMENT
B
X 3
C
T
BIOAVAILABILITY
BIOACCESSIBILITY
GC-FID ANALYSIS
MICROCOSM
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PAH Enrichment Cultures

• liquid culture enrichment (BSM)

Phenanthrene (250 mg L-1) Pyrene (250 mg L-1)
Benzoapyrene (BaP) (50 mg L-1)
CONTROL
BENZOaPYRENE
PHENANTHRENE
PYRENE
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Isolation of PAH Degraders

• PAH degrading bacteria on BSM-Phenanthrene/Pyrene
  agar plates

• Growth observation (clearing zone )

• 
  shape
• 
  size
• 
  color
• visual characterizations opacity
• 
  elevation
• 
  pigment

• Identification using 16SrRNA

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Identification of PAH degraders
Sequencing of the 16SrRNA gene from crude DNA
extraction (NCBI (http//www.ncbi.nlm.nih.gov)
and Greengenes (http//greengenes.lbl.gov)
databases
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PAH Degrading Capability
Mixed bacterial cultures Pure cultures

• Degradation

• Determination of PAH degradative capability
• Phenanthrene
• Anthracene
• Pyrene
• Benzaanthracene
• Dibenza,hanthracene
• Benzoapyrene
• Degradation monitored over a time course period
• Phenanthrene (2 days)
• Benzoapyrene (98 days)
• Determination of microbial numbers (MPN)
• at each sample point time

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Phenanthrene Degradation Mixed culture
Phenanthrene enriched microbial community from
soil B
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Phenanthrene Degradation Pure Culture
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Conclusions

• Enrichments
• PAH degrading microbial communities were
• enriched on phenanthrene, pyrene and
• benzoapyrene
• Isolation of PAH degrading pure cultures
• Eleven pure cultures isolated both on
• Phenanthrene and Pyrene
• PAH degradation
• Phenanthrene was completed degraded by
• pure cultures of Sphingomonas sp. and
• Burkholderia sp.
• Pyrene was completely degraded by pure
• cultures of Rhodanobacter sp. and Bosea sp.

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FUTURE RESEARCH
DEGRADATION
ISOLATION
PAH-SOIL
ENRICHMENT
B
X 3
C
T
BIOAVAILABILITY (HUMAN HEALTH)
BIOACCESSIBILITY
GC-FID ANALYSIS
MICROCOSM EXTRACTION
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Acknowledgements
Funding UniSA Presidents Fellowship 1st
January 2008 CRC CARE Scholarship 1st January
2008
Supervisors Dr Albert Juhasz (Principal) Professor
Ravi Naidu (Associate Supervisor) Dr Megharaj
Mallavarapu (Associate Supervisor)