This study investigated the natural attenuation of toluene under iron-reducing conditions in soil. Two hypotheses were tested: 1) indigenous soil microorganisms could anaerobically respire and couple the reduction of Fe(III) to the oxidation of toluene, and 2) the process of carbon cycling in soil may change with the addition of toluene. Experiments were conducted using live soil and river water spiked with toluene to replicate subsurface environments. Changes in toluene concentrations, iron levels, and carbon isotopes were analyzed to understand biodegradation and geochemical processes.

1. School of something
FACULTY OF OTHER
School of Civil Engineering
FACULTY OF ENGINEERING
Geochemical Controls during the
Biodegradation of Petroleum
Hydrocarbons in Soil
Rosemary Nmavulem Orlu
Dr Douglas Stewart
Professor Simon Bottrell
cn09ro@leeds.ac.uk

2. Research background
Overall aim of this study:
To investigate the natural attenuation of
toluene under predominantly iron-
reducing conditions using experimental
analogues of subsurface soil
environments.
Hypothesis 1:
Indigenous soil microorganisms in a
previously-contaminated soil may be able
to respire anaerobically and couple the
reduction of Fe (III) to the oxidation of a
hydrocarbon contaminant (toluene).
Hypothesis 2:
The process of carbon cycling in soil may
change as a result of addition of a
hydrocarbon contaminant (toluene).
 Organic compounds that have been sequestered in soil may not necessarily be available for biodegradation
although freshly added compounds may be biodegradable.
 Organic compounds therefore tend to be a source of long-term contamination in soil and groundwater as they
may no longer be degraded regardless of the length of time over which they have been in contact with the soil
matrix.
 The recent discovery of microorganisms capable of conserving energy for growth from iron reduction has led to a
growing interest in the effect of iron (iii) reduction in the bioremediation of aquifer systems contaminated with
hydrocarbons or heavy metals.

3. Stages of experimentation
1. Degradation experiments 2. Sequential chemical extractions 3. Stable carbon isotope analysis
Purpose: To produce
replicates of subsurface
soil environments under
predominantly iron-
reducing conditions
Purpose: To assess operationally-
defined iron pools affected by iron-
mediated toluene degradation
Purpose: To investigate
carbonate transformations
(precipitation and addition)
during iron-mediated toluene
degradation

4. Degradation experiments
 Mesocosm content: live soil and river water successively spiked with toluene
 Analysis: Changes in pH, toluene concentrations and total dissolved (aqueous) iron concentrations
 Nitrogen-filled ‘ballast volumes’ attached to the vessels maintained anaerobic conditions within the
sealed vessels during sampling.
 Toluene analysis: Headspace sampling – ‘salting out’ method.
 Total aqueous iron: Ferrozine method

5. Sequential chemical extractions
Stages of the sequential extraction procedure :
 A 4-step sequence for iron-bound phases
 A 2-step sequence for sulphate-bound iron
 A single step extraction for the total extractable iron content

6. Stable carbon isotope analysis
Stable carbon isotope analysis follows a two-step procedure of acidification followed by cryogenic
distillation of evolved gas in a sealed vacuum line.
A protocol was developed to differentiate between a) the fast and slow reacting soil carbonates based on
the period allowed for the acidification step; and b) carbonate addition and dissolution processes in the
incubated soil material
Carbonate dissolution and re-addition is common during the anaerobic degradation of toluene.
‘a’ – incubated material following carbonate dissolution
‘b’ – incubated material following carbonate addition