Biodegradability of Select Polycyclic Aromatic Hydrocarbon Mixtures

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Biodegradability of Select Polycyclic Aromatic
Hydrocarbon Mixtures

• By Jennifer N. Stark
• REU Project
• Professor Autenrieth
• Summer 2005

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What are PAHs?

• Largest and most structurally diverse class of
  hydrocarbons
• --Formed naturally and anthropogenically
• Formation methods include incomplete combustion,
  burning of biomass, slow burning in sediments
• Are hydrophobic and dont solubilize easily in
  water
• Are lipophilic
• HMW more persistent and difficult to degrade

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Environmental Significance of PAHs Studied

• Carcinogenic, toxic, and mutagenic properties
• Partition in fatty tissues when digested

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Characteristics of PAHs Studied
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What is Biodegradation?

• Uses natural biological activity to make
  contaminants less harmful compounds or are
  mineralized
• Microorganisms attack using enzymes
• Example naphthalene dioxygenase

http//www.ch.qub.ac.uk/staff/boyd/fig1.gif
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Sphingomonas paucimobilis EPA505

• Originates in soil, air, and freshwater and
  marine environments
• Able to use fluoranthene as sole source of carbon
  and energy
• Versatile and able to degrade many PAHs
• Produce enzymes needed as catalysts for reactions

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Biodegradation andMonod Kinetics

• Is how fast microbes degrade a substrate
• Monod kinetics-applicable when contaminant is
  adequate food source for organisms
• Rate of biotransformation increases until store
  of contaminant is used up
• First Order Kinetics- substrate concentration
  neglected since its much smaller than the
  saturation constant
• Used for most bioremediation applications
• The rate of degradation is proportional to the
  concentration of the substrate

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Monod Equation for Sole Substrates

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Assumptions for Multisubstrate Kinetics in
Experiment

• Extant kinetics
• Does not account for growth of biomass
• Low initial substrate to biomass ratio
• All PAHs compete for the same enzyme

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Experimental Set-up

• Separated experiments into sole substrate
  experiments and binary mixture experiments
• Performed an initial experiment to determine
  concentrations of PAHs to prepare and sampling
  times needed

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Concentrations of PAHs Determined
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Concentrations of PAHs Present
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Bacteria Inoculation

• Bacteria grown with 800 ml of a nutrient broth
• Nutrient broth incubated for thirty-six hours
  while on horizontal shaker for 160 rpm
• When harvested, bacteria centrifuged and washed
  three times

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Experimental Procedure

• Conducted using 250 mL amber bottles
• reactors were placed on a horizontal shaker
• Required concentration of bacterial solution was
  determined to be .25

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Experimental Procedure

• 7 ml samples taken from the reactor and added to
  the sampling vial
• Sampling vials placed on a rotary shaker for
  twelve hours
• 1 mL sample extracted from each vials and added
  to a GC vial
• Vials analyzed using the GCMS.

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Working with the GC/MS
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Kinetic Parameters Obtained
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Discussion-Sole Substrate Experiments

• First order rate constants comparable
• Comparable affinity constants for naphthalene and
  1-methylfluorene phenanthrene and fluorene.
• Naphthalene constants could be accurately
  estimated but not for others

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Degradation of 1-Methylfluorene
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Degradation of Fluorene
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Degradation of Naphthalene
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Degradation of Phenanthrene
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Discussion-Binary Substrate Experiments

• Model does not fit 1-methylfluorene degradation
• Naphthalene and phenanthrene degradation suggests
  that sequential metabolism occurs

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Results for Binary Mixtures

• Naphthalene degradation predicted by model, not
  phenanthrene
• Kinetic parameters not accurate since more points
  needed for trend line

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Conclusions

• Replicated Anuradha Desais and Petros Petros
  Dimitriou-Christidis experiments
• Observed four compounds had comparable
  degradation rates
• They will likely compete with one another

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Acknowledgments

• My parents and brother
• The NSF-REU Program
• Dr. Autenrieth
• Dr. McDonald
• Anuradha Desai, for all her help and answering my
  (frequent) questions