Microbial Bioremediation of Hydrocarbons in Soil and Coastal
Environments
Rachel Blumhagen
May 29, 2008
Hydrocarbons • Petroleum is a complex mixture of hydrocarbons.
– Alkanes (CnHn+1), monoaromatics, and polycyclic aromatic hydrocarbons (unsaturated).
octaneexamples of PAH
bicyclo[2,2,1]heptane
Created by Csari with ACD Chemsketch
Oil Spills
http://www.pacificariptide.com/pacifica_riptide/oil_spill/index.html4
www.oceanservice.noaa.gov
High contamination of crude oil •recalcitrant •harms animals and endangers local species •severely changes the ecosystem
http://www.waterencyclopedia.com/Oc-Po/Oil-Spills-Impact-on-the-Ocean.html
Hydrocarbons in the Environment“petroleum hydrocarbon impacted sites across the United States
results from a wide range of past industrial, military, and petroleum production, and distribution practices”.
-Total Petroleum Hydrocarbon Working Group
Ecosystem
-prokaryotic and eukaryotic degradation
Human Activities
-physically cleaning up and removinglarge amounts of contaminant
Ways of Clean Up and Restoration of Ecosystem
biodegradation
Biodegradation• Microbiological process by which complex hazardous compounds
are broken down biologically into smaller molecules (Singh and Ward, 2004).
“Hydrocarbon degrading microorganism”
Hydrocarbons(electron donor and
carbon source)
Oxygen or Inorganic substrate
(electron acceptor)
Biotransformation
less complex metabolites
Mineralization
Inorganic minerals, H2O, CO2, CH4
TCA cycle
Van Hamme et al. 2003
FIG. 1. Schematic of alkanedegradation in gram-negative bacteria, showing the locations and functions of the alk gene products. The products include AlkB (alkane hydroxylase), AlkF and AlkG (rubredoxins), AlkH(aldehyde dehydrogenase), AlkJ(alcohol dehydrogenase), AlkK (acyl-CoA synthetase), AlkL (outer membrane protein that may be involved in uptake), AlkN (a methyl-accepting transducer protein that may be involved in chemotaxis), AlkT (rubredoxinreductase), and AlkS (positive regulator of the alkBFGHIJKL operon and alkSTgenes).
Many transcriptional regulators facilitate gene regulation
MetabolismElectron transport system
Most metabolic genomics done on Pseudomonas putida(OCT plasmid).
Physiological adaptations to using hydrocarbons as substrate
Hydrocarbons are hydrophobic!
To prevent disruption of the membrane:-create biofilms-alter hydrophobic character of cell surface-change lipo-polysaccharide components of membrane
To facilitate uptake of substrate:-biosurfactants: amphiphilic compounds that increase availability
by emulsification of oil substrate-chemotaxis: facilitates movement to higher concentrations of
substrates
Hydrocarbon degrading microbes are present in most terrestrial and coastal environments
(this explains the faults of bioaugmentation)
Biotic Factors:-microbial community with ability
to degrade hydrocarbons
Abiotic Factors: -Oxygen availability-Temperature-Soil type-Nutrients
Rate of Biodegradation of Oil
Chemical Nature of Contaminant:
-short vs. long chain alkanes-size of aromatics
Bioremediation: process of adding nutrients to accelerate rate of biodegradation.
Nutrients• Application of nutrients to exploit microbial degradation of contaminants
(biostimulation) considered a better approach because:– less expensive – not as time consuming – low environmental impact– reduces toxicity
• Limiting macronutrients mainly used: Nitrogen and Phosphorus
• Along with other biodegradation factors to be considered, concentration of nutrients will affect capacity to stimulate hydrocarbon community.– Too little could result in no effect.– Too much could have negative side effects.
Bioremediation of an Experimental Oil Spill
MacNaughton et al. 1999
Four treatments:-No oil-Oil alone-Oil + nutrients-Oil + nutrients + inoculum.
Found rate of biodegradation of both alkanes and aromatics was highest among nutrient treatments.
Significantly greater increase in biomass in nutrient treatments compared to non-oiled and naturally attenuated treatments.
Increase in gram negatives in response to nutrients.
Phylogenetic anaylsis of inoculumtreated plot gave little resemblance to composition of inoculum.
“Who” does this?
Phylogenetic relationships of bands sequenced from previous DGGE analysis.
Conclusions:
• Addition of nutrients changes community composition, seen by an increase in gram negatives.
• Inoculated bacteria did not compete favorable with indigenous population of bacteria, seen by lack of inoculated microbes in inoculumplots.
MacNaughton et al. 1999
Changes in community composition over time
Species Diversity Overall
Hydrocarbon degrading species
Time after contamination
Num
ber o
f mic
roor
gani
sms
Time at which majority of oil has been degraded
Proliferates once contamination occurs while community diversity becomes more homogenous.
Once majority of hydrocarbon substrate is catabolized, population prior to contamination returns.
Microcosm used to simulate oil spill contaminated sediment
(Roling et al. 2002)
Experiment of Nutrient-Enhanced Oil Spill Bioremediation
Amount N in residing seawater Daily Carbon dioxide productionC
umul
ativ
e
KEY white: TPH, black: TRH, hatched: alkanes (11-33), grey: PAH
(Roling et al. 2002)
Phylogenetic analysis between treatments
(Roling et al. 2002)
Microcosms not affected by oiling or nutrient treatments
Oiled, untreated control
Oiled, 4% N treatment-gamma proteobacteria dominates
Conclusions of this study:• Oil degradation rates are limited by
macronutrients: nitrogen and phosphorus.• Nutrient concentration threshold exists at which
biodegradation rate is optimal.• Decrease in community diversity in response to
oil however subsequent rapid recovery to pre-oiled level of diversity.
• Biases of microcosms:– heterogeneous distribution of contaminant.– uneven distribution of nutrients.
Biodegradation over 6 distinct geographical locations across the US
Within 50 days, greater than 80% of applied crude oil had been degraded
across all soils.
Hamamura et al. 2006
Five of the soils showed faster sequential depletion of
shorter chain before long chain n-alkanes
DGGE Analysis and Phylogenetic Analysis
Emergent in 4 out of the 7 soils (g+)
Differences in geographic and physiochemical properties of soil influence patterns of degradation as well as microbial populations which will respond to contamination.
Why?
Community diversity prior to oil spill (heterogeneous, high diversity seen among soil habitats)
Community diversity after contamination (more homogeneous, less diverse, dominated by phyla capable of degrading hydrocarbons)
WARNING: ROMANTIC INTERPRETATION OF COMMUNITY DIVERSITY
MAY BE MISLEADING
TAKE HOME MESSAGE:The ecosystem will over time harvest hydrocarbons into energy and contribute
to the carbon cycle…there is hope!
Success of bioremediation efforts
Biotic Factors:-microbial community with ability
to degrade hydrocarbons
Abiotic Factors: -Oxygen availability-Temperature-Soil type-Nutrients
Chemical Nature of Contaminant:
-short vs. long chain alkanes-size of aromatics
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