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