Transcript Case study 2: degradation of crude oil by halophilic Archaea

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
Haloferax isolate
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