Case study 2 degradation of crude oil by halophilic Archaea

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					Bioremediation
           Bioremediation Defined
• Any process that uses microorganisms, fungi, green plants or their
  enzymes to break down harmful chemicals and pollutants in order
  to return the environment to its original natural condition.




• “Bioremediation is not only about genetics and enzymology but
  also about physiology and ultimately ecology.”—de Lorenzo V:
  Systems biology approaches to bioremediation. Curr Opin
  Biotechnol 2008, 19:579-589.
                     Alleviating Pollution
• Ex situ or in situ intervention
    – Natural attenuation
        • Example: phytoremediation (hyperaccumulators) store
          heavy metals in vacuoles
             – Sebertia acuminata 20% dry weight is nickel.
             – Plants on side of freeways are taking up lead from gas exhaust


    – Bio-stimulation
        • Add nutrients (nitrate/sulfate) that cause blooms of
          naturally occurring microbial bioremediators.
             – Example: bacteria that metabolize polycyclic aromatic
               hydrocarbons or polychlorinated biphenyls

    – Bio-augmentation
        • Genetically Modified Bioremediators
             – Alter organisms to manufacture proteins for desired
               metabolism
                  » Yellow poplar tree given enzyme mercuric reductase
                      thrives in mercury soil, cadmium, TCE
                  » Bacteria gene breaks down TNT is linked to jellyfish gene
                      that glows. Bacteria spread on soil glows green near
                      explosives
                  » Chakrabarty first patented oil eater bacterium. Combined
                      4 plasmids in one bacterial cell gave it the ability to
                      degrade four components of crude oil.
Why do we even need it?
   •   We can’t seem to stop polluting
        – Inorganics
             •   Uranium, technicium, sulfur, slfuric acid
        – Explosives
             •   RDX, TNT
        – Polyaromatic hydrocarbons
             •   creosote
        – Chlorinated hydrocarbons
             •   Trichlorethylene, PCBs, pentachlorophenol
        – Petroleum hydrocarbons
             •   Gas, gas additives (MTBE), deisel

   •   From mid-1980’s up to 90’s numerous
       attempts were made to design GMO for
       environmental release for pollutants and
       heavy metals (USGS).
        – Failures to program: bacteria doesn’t behave in
          a predictable fashion from the lab.
Case Study 1
                         Case Study 1 cont…
•   Most failures at bioremediation are due to failure of introduced organisms to
    thrive in the natural environment or a failure to access the contaminant. This could
    be due to:
     –   Lack of nutrients
     –   Predation or parasitism
     –   Competition (GEM’s tend to compete poorly with indigenous populations).
     –   Immobility of introduced bacteria
     –   Contaminant concentrations below threshold for organism survival
     –   Organisms may feed on alternative substrates (E. coli and Pseudomonas diverge genetically
         from initial inoculum in field trials).

•   A few examples of failed bioremediation attempts:
     – Inoculation of soil with aliphatic hydrocarbon degrading bacteria did not enhance degradation
       of fuel oil
           •   Venosa AD, Wrenn BA (1996) Selective enumeration of aromatic and aliphatic hydrocarbon degrading
               bacteria by a most-probable number procedure. Can. J. Microbiol.42: 252-258
     – A Pseudomonas sp. shown in lab cultures to degrade 1,4-dichlorophenol failed to degrade the
       compound when added to surface soils
           •   Sayler GS, Ripp S (2000) Field applications of genetically engineered microorganisms for
               bioremediation processes. Curr Opin Biotechnol 11:286-289
                Case study 2:
degradation of crude oil by halophilic Archaea
    Defining bioremediation by natural attenuation

– what is the environment? what is the pollutant?

– are bacterial, Archaeal, and/or plant species present that degrade the
  pollutant of interest?

– what conditions (nutrient, temperature, pH, salt etc) are necessary for
  that activity?
       Defining bioremediation by natural attenuation:
hydrocarbon degradation in the Arabian Gulf hypersaline coast

  – what is the environment? what is the pollutant?
      • the hypersaline coast of the Arabian coast
      • crude oil (hydrocarbons)




                                              West, Ian. 2008. Qatar - sabkhas, evaporites and some other
                                              desert features: an introduction.
                                              http://www.soton.ac.uk/~imw/Qatar-Sabkhas.htm
        Defining bioremediation by natural attenuation:
 hydrocarbon degradation in the Arabian Gulf hypersaline coast

     – what is the environment? what is the pollutant?
          • the hypersaline coast of the Arabian coast
          • crude oil (hydrocarbons)
     – are bacterial, Archaeal, and/or plant species present that degrade the
       pollutant of interest?




                                                                          2 Haloferax strains
                                                                          1 Halobacterium strain
                                                                          1 Halococcus strain


environmental samples               grow on minimal mineral plates
                                      with crude oil vapor as sole
                                        carbon/energy source


                                                         (Al-Mailem et al., 2010 Extremophiles)
       Defining bioremediation by natural attenuation:
hydrocarbon degradation in the Arabian Gulf hypersaline coast

  – are bacterial, Archaeal, and/or plant species present that degrade the
    pollutant of interest?
                                         C18 hydrocarbon



                                                         autoclaved control


                                                          Haloferax isolate

                                                          Halobacterium isolate

                                                          Halococcus isolate


                                 gas-liquid chromatography to measure
                                        hydrocarbon degradation
                                          (Al-Mailem et al., 2010 Extremophiles)
       Defining bioremediation by natural attenuation:
hydrocarbon degradation in the Arabian Gulf hypersaline coast

  – what conditions (nutrient, temperature, pH, salt etc) are necessary for
    that activity?
      • increased salt increased hydrocarbon degradation




                                                              Haloferax isolate
                                                              Haloferax isolate
                                                              Haloferax isolate
                                                              Halococcus isolate
                                                              Halobacterium isolate




                                                (Al-Mailem et al., 2010 Extremophiles)
       How could this hydrocarbon degradation activity by
                  Haloarchaea be improved?
•   General strategies for improving microbial bioremediation: stimulation or augmentation
•   Some existing bio-engineering tools
     – University of Minnesota Biocatalysis/Biodegradation
       Database (UMBBD): enzymes, pathways, reactions,
       compounds from hundreds of bacterial species of interest
       (Gao et al., 2010)
     – MetaRouter: tracks possible breakdowns from a starting point
       using all possible reactions (Pazos et al., 2005)
     – in silico modeling of altered strains: ex. Optstrain (Pharkya et
       al. 2004), DESHARKY (Rodrigo et al. 2008)
         How could this hydrocarbon degradation activity by
                    Haloarchaea be improved?

•   How could systems level knowledge help design stimulation or augmentation strategies?


     –    need to know network topology of the pathway being added or altered as well as the
          influence of the environment on that pathway
     –    understand coupling of pathways to better integrate the engineered or altered pathway into
          the rest of the host system
     –    understand demands created by the new flux on resources needed for growth/survival:
          energy, carbon, redox balance, transcriptional and translation capacity
     –    is it possible to compensate by altering regulation by TFs etc, or by adding or deleting other
          pathways?
     –    understand effect of environment on pathway flux
     –    understand role of noise in pathway regulation
     –    if cooperation between multiple microbial species is used, then systems analysis can
          evaluate impact of biodegradative flux on the multispecies consortia

				
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