biopile bioremediation of petroleum hydrocarbon contaminated soils
TRANSCRIPT
Biopile Bioremediation of Petroleum Hydrocarbon Contaminated
Soils from a Sub-Arctic Site
Jessica Snelgrove
Department of Civil Engineering and Applied Mechanics
McGill University, Montreal
October, 2010
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the
requirements for the degree of Master of Engineering
© Jessica Snelgrove, 2010
i
ABSTRACT
Petroleum contamination of several hundred sites in the northern arctic and sub-arctic
regions of Canada has occurred as a result of petroleum oil exploration and use of petroleum
fuels for heating, transportation and electricity generation. Petroleum contamination can persist
in the ground for long periods of time and be a source of long-term environmental
contamination. Bioremediation is a non-disruptive and often cost-effective technology for
remediation of petroleum-contaminated sites that involves the microbial degradation of
hydrocarbon compounds. Biopiles allow for rapid ex-situ treatment of petroleum-hydrocarbon
contaminated soils. Two pilot scale biopiles (300 kg soil each) were construct using soils
contaminated with approximately 1 500 mg/kg total petroleum hydrocarbons (TPH) from
Norman Wells, North West Territories. Both systems were supplied with oxygen to stimulate
aerobic conditions, and monitored in an enclosed room maintained at a temperature of 15oC, the
ambient summer temperature in Norman Wells. One biopile was amended with ammonium
nitrate at a ratio of 100:5:1 (C:N:P) to determine the effects of nutrients on TPH biodegradation.
The research showed that biodegradation occurred within both biopile systems. Analysis of the
hydrocarbon fractions, TPH chromatograms, and oxygen consumption and carbon dioxide
production supported biodegradation versus volatilization. However, an absolute confirmation
of whether these loses were due to biodegradation (or to what extent) are not possible to be
reported here. Analysis of the inorganic nitrogen and aggregation of the soils helped provide
insight into the process of biodegradation in both biopile systems. Overall 42% of the total
petroleum hydrocarbons were removed from the nutrient amended biopile and 38 % in the
ii
control biopile. For the F2 (>C10-16) fraction, both systems had less than 200 mg/kg soil and for
the F3 (>C16-34) fraction around 700 mg/kg soil.
iii
RESUME
La contamination de pétrole de plusieurs cent sites dans les régions du nord, arctiques et
subarctiques de Canada est arrivée à la suite de l'exploration de pétrole de pétrole et à la suite de
l'usage de carburants de pétrole pour le chauffage, la génération de transport et électricité. La
contamination de pétrole peut persister dans le sol pour les périodes longues de temps et est une
source de contamination écologique à long terme. Bioremediation est une technologie non-
perturbateur et souvent rentable pour le redressement de sites pétrole-contaminé qui impliquent
la dégradation microbienne de composés d'hydrocarbure. Les biopiles tiennent au compte du
traitement d'ex-situ rapide de pétrole-hydrocarbure a contaminé des sols. Deux biopiles à
l'échelle pilote (300 sol de kg chacun) étaient les sols d'utilisation de construction contaminé
avec approximativement 1 500 mg/kg hydrocarbures de pétrole totaux (TPH) desNorman Wells,
le Nord Territoires d'Ouest. Les deux systèmes ont été fournis avec l'oxygène pour stimuler des
conditions aérobiques, et contrôlé dans une pièce enclose maintenue à une température de 15oC,
la température d'été ambiante dans Norman Wells. Une biopile a été modifiée avec le nitrate
d'ammonium à une proportion de 100:5:1 (C:N:P) déterminer les effets de nutriments sur TPH
biodegradation. La recherche a montré à ce biodegradation est arrivé dans les deux systèmes de
biopile. L'analyse des fractions d'hydrocarbure, de chromatogrammes de TPH, et de
consommation d'oxygène et de production de dioxyde de carbone a soutenu biodegradation
contre volatilization. Toutefois, une confirmation absolue de savoir si ces pertes étaient dues à la
biodégradation (ou dans quelle mesure) ne sont pas possible d'être rapportée ici.L'analyse de
l'azote et l'agrégation inorganique des sols aidés fournit la perspicacité dans le processus de
biodegradation dans les deux systèmes de biopile. Général 42% des hydrocarbures totaux de
iv
pétrole a été enlevé du nutriment la biopile modifiée et 38 % dans la biopile de contrôle. Pour le
F2 (>C10-16) fraction, les deux systèmes ont eu moins que 200 sol de mg/kg et pour le F3
(>C16-34) fraction autour de 700 sol de mg/kg.
v
ACKNOWLEDGEMENTS
The completion of this thesis and research is something I could not have done without the support
system from McGill, my friends, and family. I’d like to thank Professor Ghoshal for taking me on as a
Masters student and providing me with the opportunity to work on this research. He was a sounding
board for ideas and an endless resource for information pertaining to the research and was extremely
supportive as I dealt with learning the ins and outs of the lab. Together we were able to interpret the data
and develop this thesis.
I couldn’t have done this research without the help of the members in my research group.
Wonjae Chang helped greatly by teaching me lab techniques and protocol methods, and was an overall
source of encouragement and reassurance. As well, Ali Akbrai, Simon Dagher, and Salman Hafeez
helped in developing lab techniques and acquiring data. As a group we shared ideas and results and
helped each other whenever possible in the lab. Their strength was also appreciated when moving 300 kg
barrels of soil!
The technicians at McGill were a great resource. Diana Brumelis provided help in familiarizing
myself with the Environmental Lab, and John Bartczak was a great help in the design and construction of
the biopiles and the storage of the soil. Bill Cook’s help with the maintenance of the cold room was
always appreciated, especially for his fast response to the problems that would occur in the middle of the
night! Jorge Sayat was always there to make sure that my computer needs were met and that. A huge
thanks to Ranjan Roy and Andrew Golztajn in the Chemical Engineering Department for all their help
with my lab techniques and development of lab protocols.
I have all my friends and family to thank for listening to me discuss all aspects of my research
over the past two years. They provided support and advice, and were always there to offer me well
deserved breaks! Without the support of my Mom, I wouldn’t have been able to do this.
vi
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................................... i
RESUME ..................................................................................................................................................... iii
ACKNOWLEDGEMENTS .......................................................................................................................... v
TABLE OF CONTENTS ............................................................................................................................. vi
LIST OF FIGURES ................................................................................................................................... viii
LIST OF TABLES ........................................................................................................................................ x
1.0 INTRODUCTION ............................................................................................................................ 1
1.1 Petroleum Contamination in the Arctic .............................................................................................. 1
1.2 Clean up Standards ............................................................................................................................. 3
1.2 Application of Bioremediation............................................................................................................ 4
1.3 Objectives .......................................................................................................................................... 5
1.4 Approach ............................................................................................................................................ 6
2.0 LITERATURE REVIEW ................................................................................................................. 8
2.1 Biopiles ............................................................................................................................................... 8
2.2 Factors and Conditions Affecting Bioremediation ........................................................................... 10
2.2.1 Bacteria ...................................................................................................................................... 10
2.2.3 Temperature ............................................................................................................................... 11
2.2.4 Nutrients ..................................................................................................................................... 12
2.2.5 Electron Acceptors ..................................................................................................................... 13
2.2.6 Water content ............................................................................................................................. 14
2.2.7 pH ............................................................................................................................................... 15
2.2.8 Soil Type ..................................................................................................................................... 15
3.0 MATERIALS AND METHODS .......................................................................................................... 17
3.1 Laboratory Setup ............................................................................................................................... 17
3.1.2 Biopile Tanks ............................................................................................................................. 18
3.2 Experimental Design ......................................................................................................................... 20
3.2.1 Biopile Size ................................................................................................................................ 20
3.2.1 Microcosm Experiment .............................................................................................................. 21
vii
3.2.2 Temperature ............................................................................................................................... 22
3.2.3 Nutrient Addition ....................................................................................................................... 23
3.2.4 Moisture Content........................................................................................................................ 24
3.2.5 Air Flow ..................................................................................................................................... 24
3.3 Sampling ........................................................................................................................................... 25
3.4 Analytical Methods ........................................................................................................................... 25
3.4.1 Total Petroleum Hydrocarbon (TPH) Extraction ...................................................................... 25
3.4.2 Inorganic nitrogen extraction .................................................................................................... 27
3.4.3 Plate Counting of Bacteria ........................................................................................................ 28
3.5 Statistical Analysis ........................................................................................................................... 30
4.0 RESULTS AND DISCUSSION ........................................................................................................... 30
4.1 Analysis of Changes in Soil Parameters ........................................................................................... 30
4.1.1 Assessment of Changes in Moisture Content ............................................................................ 30
4.1.2 Carbon Dioxide Production and Oxygen Consumption ............................................................ 33
4.1.3 Hydrocarbon Degradation ........................................................................................................ 36
4.2 Continuing the Experiment Beyond Day 65 ..................................................................................... 46
4.2.1 Oxygen and Carbon Dioxide Levels .......................................................................................... 46
4.2.2 Continued TPH Degradation ..................................................................................................... 48
4.3 Aggregation and Soil Properties ....................................................................................................... 55
4.2.1 Plate Counting ........................................................................................................................... 56
4.2.3 Nutrient Cycling ......................................................................................................................... 57
5.0 CONCLUSIONS ................................................................................................................................... 64
6.0 REFERENCES ..................................................................................................................................... 65
APPENDIX A: PHYSIOCHEMICAL CHARACTERISTICS OF SOIL ................................................. 68
APPENDIX B: SUMMARY OF XRD ANALYSIS .................................................................................. 69
viii
LIST OF FIGURES
Figure 1: Arctic Oil Resources (AMAP Secretariat, 2003) ......................................................................... 2
Figure 2: Cold Temperature Room ............................................................................................................... 7
Figure 3: Typical Biopile Setup (United States Environmental Protection Agency, 2004)......................... 9
Figure 4: Photograph of the pilot-scale biopile showing the air supply tubes and gas sampling tubes. .... 19
Figure 5: Sampling ports and air design .................................................................................................... 19
Figure 6: Temperature profile of Norman Wells, NWT (Environmental Canada, 2009) .......................... 23
Figure 7: Surrogate recovery, Blank vs Control Day 129 .......................................................................... 27
Figure 8: Gravimetric moisture content, Control vs Nutrient amended Biopile ....................................... 31
Figure 9: Air dried moisture content, Control vs Nutrient amended Biopile ............................................. 32
Figure 10: CO2 production in the control and nutrient amended biopiles ................................................. 34
Figure 11: O2 production in the control and nutrient amended biopiles ..................................................... 35
Figure 12: Correlation between % O2 and % CO2 in the control (left) and nutrient amended (right) biopile
.................................................................................................................................................................... 36
Figure 13: TPH analysis by layer, nutrient amended biopile ..................................................................... 37
Figure 14: TPH in control and nutrient amended biopiles ........................................................................ 38
Figure 15: F2 hydrocarbon fraction in control and nutrient amended biopiles .......................................... 39
Figure 16: F3 hydrocarbon fraction in the control and nutrient amended biopiles .................................... 41
Figure 17: Chromatogram: day 0 vs. day 15 - Control ............................................................................. 43
Figure 18: Chromatogram: day 0 vs. day 15 – Nutrient amended ............................................................ 43
Figure 19: Chromatogram: day 15 vs. day 65 - Control ........................................................................... 44
Figure 20: Chromatogram: day 15 vs. day 65 - Nutrient amended ........................................................... 44
Figure 21: UCM Area, Control biopile ...................................................................................................... 45
Figure 22: UCM Area, Nutrient amended biopile ..................................................................................... 46
ix
Figure 23: Percentage carbon dioxide, beyond day 65 .............................................................................. 47
Figure 24: Percentage oxygen, beyond day 65 .......................................................................................... 47
Figure 25: F2 hydrocarbon fraction, beyond day 65 .................................................................................. 48
Figure 26: F3 hydrocarbon fraction, beyond day 65 .................................................................................. 49
Figure 27: TPH, beyond day 65 ................................................................................................................. 49
Figure 28: TPH, entire 150 day system ...................................................................................................... 50
Figure 29: Chromatogram: day 65 vs day 80 - Control ............................................................................. 51
Figure 30: Chromatogram: day 65 vs day 80 - Nutrient amended ............................................................ 51
Figure 31: Chromatogram: day 80 vs day 109 - Control .......................................................................... 52
Figure 32: Chromatogram: day 80 vs day 109 - Nutrient amended ........................................................... 52
Figure 33: Chromatogram: day 109 vs day 150 - Control ........................................................................ 53
Figure 34: Chromatogram: day 109 vs day 150 - Nutrient amended ........................................................ 53
Figure 35: UCM, entire 150 day system - nutrient amended ..................................................................... 54
Figure 36: UCM, entire 150 day system - control ..................................................................................... 55
Figure 37: Inorganic nitrogen - control, coarse aggregates ........................................................................ 59
Figure 38: Inorganic nitrogen - control, medium aggregates ..................................................................... 59
Figure 39: Inorganic nitrogen - nutrient amended, medium aggregates .................................................... 60
Figure 40: Inorganic nitrogen - nutrient amended, coarse aggregates ....................................................... 60
Figure 41: Inorganic and organic nitrogen in control biopile .................................................................... 63
Figure 42: Inorganic and organic nitrogen in nutrient amended biopile .................................................... 63
x
LIST OF TABLES
Table 1 : Summary of Tier 1 levels (mg/kg) for surface and subsurface soils (CCME, 2008) .................... 4
Table 2: Genera of Hydrocarbon-Degrading Bacteria (Wong et al., 1997) ............................................... 11
Table 3: Major Metabolic Sequences (Baker and Herson, 1994) .............................................................. 14
Table 4: TPH (mg/kg) in Microcosms ....................................................................................................... 21
Table 5: Comparison of an F2 compound to a F3 compound (OSHA, 2007) ........................................... 40
Table 6: Total indigenous heterotrophic and hydrocarbon degrading bacteria .......................................... 56
1
1.0 INTRODUCTION
1.1 Petroleum Contamination in the Arctic
It is estimated that out of the 1 300 000 tonnes of petroleum that is released world wide
into the sea, 260 000 tonnes of that occur off of the shores of North America. Forty eight percent
of this is fuel, and twenty nine percent is crude oil. And although the most concentrated spills
come from tankers, these accidents only amount to 5% of the oil that is entering the waters.
Most of it is runoff of oil and fuel from land. In Canada, twelve spills of over 4 000 liters are
reported each day, with five of them being on land (Mudge, 2009). In Canada alone, there are an
estimated 2 400 hydrocarbon contaminated sites (Filler et al., 2008).
In the Arctic, many petroleum products are used, stored and transported as petroleum is
the primary energy source. Spills may occur in a variety of forms due to infrastructure failure,
human error, or natural hazards. Whenever fuel is moved or stored, oil spills may occur (Mohn
et al., 2001). Crude oil spills from ruptured pipelines in the Arctic are one of the largest sources
of terrestrial petroleum pollution, followed by shoreline spills from tankers or resupply vessels
(Filler et al., 2008). A major source of this contamination is associated with the 42 early warning
radar stations built across the Arctic during the cold war. Other sources are abandoned mines
that disposed of drums of oil, such as at Nanisivik where 2 000 drums of used oil was disposed
of within a landfill (Filler et al., 2008). There is also chronic spillage occurring within
settlements. Between 1971 and 2006, diesel fuel spills from tank farms in Rankin Inlet amounted
to 289 000 litres. A large percentage of these spills occurred in the 1970s and 1980s when
environmental regulations were not as strict, and the sites may not have been cleaned up to
today’s standards (Filler et al., 2008).
2
Another source of contamination are the oil seeps that are associated with the Arctic oil
reserves. Figure 1 illustrates where Arctic oil resources and production are in North America,
and illustrates potential areas of contamination associated with these natural resources.
Figure 1: Arctic Oil Resources (AMAP Secretariat, 2003)
Past and future spills are associated with the vast petroleum reserves in the Arctic and there is an
ongoing need to optimize treatment technologies for contaminated soils in polar areas (Braddock
et al., 1997). This paper will address oil contaminated soil from Norman Wells that was
provided to McGill University by Imperial Oil Limited.
3
1.2 Clean up Standards
Oil spills are a concern in the Arctic environment for various reasons. Due to their
reduced nature and volatility, they pose a fire and explosion hazard. As well, the majority of
petroleum hydrocarbon constituents are toxic to some degree (CCME, 2008) and it is possible
for such contamination to enter biological food chains and threaten both indigenous organisms
and the well-being of human populations (Whyte et al., 1999). The lighter hydrocarbons are
more mobile in the ground, water, and air and can disperse further and create problems at
distances far from the point of release (CCME, 2008). An oil spill on land has the potential to
affect both terrestrial and aquatic environments as it disperses. The larger and branched chain
hydrocarbons are very persistent in the environment. Reports have shown the persistence of
refined hydrocarbons in Alaska twenty eight years after the spill (Braddock, 1997). As well,
there are the aesthetic issues to consider as hydrocarbons may affect the taste, odour or
appearance of water, air or soil (CCME, 2008). Guidelines in different countries are
established to help with clean up standards (Filler et al., 2008).
Within Canada, the guidelines used for petroleum contaminated soil is set by the CCME
(Canadian Council of Ministers for the Environment) and the Canada-Wide Standards for
Petroleum Hydrocarbons in Soil. It provides a tiered framework with conservative risked-based
standards corresponding to defined land uses, exposure scenarios and site characteristics (Filler
et al., 2008). For Tier 1, levels are applied to contaminated sites, providing a generic/national
level that protects human health and the environment. Tier 2 cleanup levels are adjustments to
Tier 1 levels based on site specific information. Tier 3 levels are developed from site-specific
ecological or human health risk assessments when the assumptions inherent in Tier 1 values are
not appropriate for the site (CCME, 2008). Tier 3 is usually applied to large sites undergoing
4
long-term remediation due to the large costs involved (Filler et al., 2008). The diversity of
petroleum-hydrocarbons is addressed by breaking them into the four broad physio-chemical
fractions, as defined by the equivalent carbon numbers. Table 1 lists the maximum
concentrations of petroleum hydrocarbons for Tier 1 levels for coarse and fine grained sands.
Table 1 : Summary of Tier 1 levels (mg/kg) for surface and subsurface soils (CCME, 2008)
Soil Type
Land Use
F1 ECN1
C6-10
F2 ECN
>C10-16
F3 ECN
>C16-34
F4 ECN
>C34-50+
Fine-grained
surface
Agricultural
Residential/Parkland
Commercial/Industrial
210
210 (170)3
320 (170)3
150
150
260 (230)3
1 300
1 300
2 500
5 600
5 600
6 600
Coarse-grained2
surface
Agricultural
Residential/Parkland
Commercial/Industrial
30
30
320 (240)3
150
150
260 (230)3
300
300
1 700
2 800
2 800
3 300
1 ECN = Equivalent carbon number
2Coarse refers to coarse-textured soil with a median grain size > 75 µm
3Where applicable, for protection of potable groundwater
1.2 Application of Bioremediation
Due to the remoteness and unique characteristics of Arctic regions, conventional physio-
chemical technologies can be costly and difficult to implement. Bioremediation has been
proposed as a cleanup technology because it may be the most logistical and economical
favourable solution (Whyte et al., 1999).
5
Bioremediation occurs when micro-organisms are used to degrade or transform organic
contaminants to non-toxic compounds. These organisms can be biostimulated through electron
acceptors, moisture and nutrient addition. Once optimum conditions are met, remediation of the
contaminants that are bioavailable to micro-organisms can occur in a relatively short time
without future containment of the soil (Kratzke et al., 1998).
Bioremediation has been implemented widely, even to large oil spills like the Exxon
Valdez spill in Alaska. Bioremediation was used extensively to accelerate the natural
degradation of residual oil, helping to minimize the ecological impact (Bragg et al., 1994).
Along the Atlantic coast of Spain, heavy fuel oil was spilled from the oil tanker Prestige in 2002.
Due to unique characteristics along the shoreline, mechanical removal of the fuel was not always
practical, and bioremediation was recommended as an alternative (Gallego et al., 2006). Both of
these sites contaminated the soil on shore and relied on biostimulation of the micro organism
population by applying additional nutrient sources to the soil in order to accelerate
bioremediation. Other sites have needed to inoculate the soil with additional hydrocarbon
degrading micro-organisms if the initial population was too small, a process referred to as
bioaugmentation. Studies on bioaugmentation have been done by Thomassin- Lacrois et al.
(2002) and Mohn et al. (2001).
1.3 Objectives
The overall objectives of this research were to assess the rate and extent of bioremediation
of petroleum contaminated soils Norman Wells at ambient summer temperatures.
The specific objectives of this research included the following:
6
• Evaluate volatilization vs. bioremediation of aerated soils at a constant temperature
of 15oC, with comparisons being made between a control biopile with no nutrient
addition, and a biopile amended with nutrients.
• Evaluate the extent of bioremediation that is possible.
• Indentify possible rate limiting factors that may affect the rate and extent of
bioremediation.
1.4 Approach
Studies have demonstrated that biopiles allow for rapid ex-situ treatment of petroleum
hydrocarbon contaminated soils (Delille et al., 2008; Mohn, et al., 2001). Access to excavated
soils in northern sites may be limited to the summer months, so it is efficient to have a
remediation system that requires low maintenance. Biopiles can be an effective remediation
method for arctic sites (Filler et al., 2008).
The laboratory facilities at McGill contain a cold room (Figure 2) where cool temperatures
can be simulated, and controlled studies of actively aerated biopiles under cold conditions can be
carried out. Pilot-scale biopiles were constructed, and contaminated soil was supplied by
Imperial Oil Limited for experiments.
7
Figure 2: Cold Temperature Room
To promote biodegradation conditions, electron acceptors were applied to both biopiles in
the form of oxygen. One of the biopiles was amended with ammonium nitrate in the C:N:P ratio
of 100:5:1. The other biopile did not have any nutrients added to assess the effects of nutrient
amendment on biodegradation. The experiment was conducted at 15oC to provide favourable
temperature conditions for the micro-organisms. The experiment was conducted over 150 days
to observe the rate and length of biodegradation. Total petroleum hydrocarbons, bacteria
population counts, and inorganic nitrogen parameters were monitored to assess what areas may
limit biodegradation.
8
2.0 LITERATURE REVIEW
2.1 Biopiles
Biopiles have been used to facilitate bioremediation by creating piles of petroleum
contaminated soils above ground and stimulating aerobic microbial activity through aeration.
Normally this can be achieved with the indigenous micro organism population. Within these
piles aerobic microbial activity degrades the petroleum based constituents adsorbed to soil
particles (Kratzke et al., 1998).
Biopiles are considered relatively easy to construct and need only a few basic
requirements. In order to protect the subsurface environment, biopiles are typically built on an
impermeable base to reduce the potential of migration of leachate. Leachate collection pipes
may also be added to the biopile. In order to aerate the biopile, a perforated piping network is
installed above the base and is connected to a blower. The system of piping and pumps forces
air into the pile under positive pressure, or draws air through the system under negative pressure.
The ability to maintain even aerobic conditions in the soil will dictate the size and shape of the
biopile. Although biopiles do not usually exceed a height of 2.4 meters, a tall biopile of over 3
meters will require another level of aeration. Typically biopiles are covered with an impermeable
membrane to prevent the release of contaminates and contaminated soil to the environment. The
covers protect the soil from wind and precipitation helping to maintain more consistent moisture
content, and helps retain heat (Kratzke et al., 1998). Exhaust pipes are included in the design for
air release if the system is being aerated. These pipes would be monitored to ensure that the
concentration of contaminants in air did not exceed the applicable guideline. Figure 3 illustrates
the biopile design.
9
Figure 3: Typical Biopile Setup (United States Environmental Protection Agency, 2004)
There are several guidelines that are typically followed when building biopiles. These
include toxic metal concentrations below 2 500 mg/kg soil, chlorinated or recalcitrant organic
compounds are present in negligible amounts, and the total volume of soil to be treated is greater
than 191 m3(Kratzke et al., 1998).
Beyond these guidelines, there are several other parameters that may be adjusted to help
optimize the bioremediation of the soil. These include moisture content, pH, aeration,
temperature, nutrients, and the bacteria community. These are discussed in section 2.2.
10
2.2 Factors and Conditions Affecting Bioremediation
The effectiveness of bioremediation is dependent on the site conditions and soil properties
found at the contamination site. Temperature, pH, water content, nutrients, electron acceptors,
bacteria, and contaminant characteristics all play a significant role in bioremediation. These
factors are discussed below along with the challenges that are associated with Arctic sites.
2.2.1 Bacteria
Bacteria can obtain carbon from two different sources: either organic compounds or
carbon dioxide, and can obtain energy from either chemical compounds or substrate and
sunlight. Chemical compounds can either be organic or inorganic sources of carbon. Microbes
obtain energy from chemicals through oxidation-reduction processes and use this energy to
synthesize new cells and maintain old cells already formed. End products of metabolism are
water, carbon dioxide and new cell mass. In most bioremediation systems, the source of both
carbon and energy is the contaminant itself (Wong et al., 1997).
Bacteria can be classified based on what carbon source they use and the source of their
energy. Autotroph bacteria use carbon dioxide are and heterotrophs derive carbon from organic
compounds. Bacteria that derive energy from photosynthesis are phototrophs, and those that
derive it from chemical substances are chemotrophs. The bacteria that play a key role in
bioremediation are those that obtain their carbon and energy from organic compounds, referred
to as chemoheterotrophs (Wong et al., 1997). Table 2 lists the genera of hydrocarbon consuming
bacteria isolated from soil. These bacteria have the potential to use petroleum hydrocarbons as
their carbon source.
11
Table 2: Genera of Hydrocarbon-Degrading Bacteria (Wong et al., 1997)
Achromobacter
Acinetobacter
Alcaligenes
Arthrobacter
Bacillus
Brevibacterium
Chromobacterium
Corynebacterium
Cytophaga
Ervinia
Flavobacterium
Micrococcus
Mycobacterium
Nocardia
Proteus
Pseudomonas
Rhodoccus
Sarcina
Serratia
Sphingomonas
Spirillum
Steptomyces
Vibrio
Xanthomonas
Laboratory studies have confirmed that hydrocarbon degrading bacteria from the generas
Acinetobacter, Sphingomonas, Pseudomans, or Rhodoccus are present in contaminated arctic
soil, and that after hydrocarbon spillage their numbers usually increase (Aislabie et al., 2006).
Even with the presence of hydrocarbon degrading bacteria, hydrocarbons persit in soils at
cold climate sites indicating that in situ rates of hydrocarbon degradation are slow. Their activity
is likely limited by the low temperature, low moisture and nutrient limitations common of the
Arctic.
2.2.3 Temperature
Soil temperature obviously plays a direct role in biodegradation. Microbial activity is
related to temperature, and generally metabolic reactions increase with increasing temperature.
Typically biological processes increase with temperature up to a maximum temperature which
enzyme denaturisation leads to cell inhibition and death. Typically, biodegradation rates with
temperature, and can be calculated with the van’t Hoff Arrhenius equation shown below (Baker
and Herson, 1994). In the equation, Ea represents the activation energy of a chemical reaction.
12
As well, soil temperature may affect the physiochemical state of the contaminants and the
soil matrix by affecting reaction rates, or the phase and volume of the water and contaminants. It
can influence soil volume, oxidation-reduction potentials, and the water structure within the
matrix (Baker and Herson, 1994).
Typically low temperatures near the surface of the soil are associated with little or no
biodegradation of many organic substrates. This can lead to persistent organic contamination in
the arctic (Alexander, 1994). Despite the limitations that low soil temperature in the Arctic may
provide, micro activity and biodegradation has been show in soil temperatures ranging as low as
-12oC (Leahy and Colwell, 1990). Although most studies involve the degradation of organic
pollutants at incubation temperatures ranging from 20 – 35oC, the optimal range for
biodegradation will vary by species. Local environmental conditions select for populations with
a low optimal temperature for biodegradation (Margesin and Schinner, 1999).
2.2.4 Nutrients
In order to synthesise certain molecules, cells require macronutrients. Nitrogen is used in
cells for the synthesis of proteins and nucleic acids. Phosphorus is required in microbial cells for
the synthesis of adenosine triphosphate (ATP), nucleic acids, and cell membranes. Soils that
have intrinsically low nitrogen and phosphorus will require nutrient addition to allow for a
sufficient increase in biomass and significant hydrocarbon degradation (Baker and Herson,
1994).
13
From compositional analysis of microbial biomass, carbon, nitrogen, and phosphorous
are present in the ratio of 106:16:1(Ferguson et al., 2003). This “Redfield Ratio” is often sited as
the optimal C:N:P ratio for nutrient amendment. However this ratio does not take into account
that the majority of carbon that is mineralized is converted to carbon dioxide and then lost from
the system. As well, nitrogen can be lost in the system through nitrification-denitrification
processes. In field and laboratory studies, C:N ratios ranging from 14:1 to 560:1 have been
proposed as suitable for biodegradation (Ferguson et al., 2003).
Typically soils of polar regions are generally low in nutrients. After an oil spill, the
addition of hydrocarbons can lead to further depletion of the available nitrogen and phosphorus
when they are assimilated during biodegradation. It is typical of bioremediation projects to add
nutrients to the soil to biostimulate the nutrients. By treating the soil with nitrogen and
phosphorus, the cell growth rate can increase and the microbial lag phase can decrease
(Walworth et al., 2007). Nutrients range from organic sources such as cod bone meal (Walworth
et al., 2003) to inorganic sources such as ammonium chloride (Walecka-Hutchison and
Walworth, 2006; J. Walworth et al., 2007), and diammonium phosphate (Thomassin-Lacroix et
al., 2002).
2.2.5 Electron Acceptors
Biodegradation and the break down of organic compounds for carbon and energy occurs
through catabolism, a complex series of couple oxidation-reduction reactions. As the reaction
proceeds electrons are removed from and added to intermediates along the path, releasing energy
that is conserved in the form of ATP. ATP is a higher-energy phosphate bond that is used by the
cell for biosynthetic reactions. Catabolism can be divided into two groups: fermentation and
14
respiration, and are differentiated primarly based on the terminal electron acceptor (Baker and
Herson, 1994). This is summarized in Table 3 below.
Table 3: Major Metabolic Sequences (Baker and Herson, 1994)
Type of Metabolism Electron Donor Terminal Electron Acceptor
Fermentation1 Organic compound Organic compound
Respiration
Aerobic respiration
Anerobic respiration
Organic or inorganic
Inorganic compound
Oxygen
Nitrate, sulfate
1Has not been shown to be significant in the bioremediation of contaminated environments
Aerobic resipration occurs when oxygen is the terminal electron acceptor. Some aerobic
oganisms can carry out both aerobic and anerobic respiration but will preferetially use oxygen as
a terminal acceptor until it is depleted. Aerobic respiration provides greater energy compared to
anaerobic respiration. This occurs because of the differences in reduction potentials between the
electron donor and the terminal electron acceptor. In order, the most oxidizing electron
acceptors are: oxygen, nitrate, sulfate and then carbon dioxide. Aerobic respiration is thus more
efficiet and preferred for bioremediaiton (Baker and Herson, 1994).
2.2.6 Water content
Water plays an important role in biodegradation. An adequate supply of water is required
to meet the physiological requirements of micro organisms and to provide a medium for the
transport of nutrients and metabolic by-products to and from the micro organisms. However
excess moisture may cause a problem in areas where it pools, creating anoxic areas and reduced
biodegradation (Baker and Herson, 1994).
15
Within the soil, water is present in three forms: gravitational, capillary and hygroscopic.
The gravitational water is the water that primarily occupies the macro pores within the soil
matrix. It can move freely through the soil by gravitational forces. As it moves through the soil,
it may displace air and create anoxic conditions, or cause the leaching of materials into lower soil
layers. Capillary water is contained within the micro pores of the soil matrix. This determines
the field capacity of the soil, which is defined as the amount of water remaining within the soil
after gravitational water has drained away (Wong et al., 1997). The amount of capillary water is
dependent on the soil texture, as soils with a greater percentage of clay have more water than
sandy soils which are well drained. Hygroscopic water is the water that is attached to the surface
of the soil matrix through hydrogen bonding or dipole interactions. This water is not considered
to be biologically available (Baker and Herson, 1994).
2.2.7 pH
Generally microorganisms are limited to environments with pH values ranging from 6.0
to 8.0. As well, pH in the soil can affect the availability of macronutrients. An increase in pH
has lead to the decrease in the availability of calcium, magnesium, sodium, potassium, ammonia,
nitrogen, and phosphorous. A decrease in soil pH also results in decreasing the availability of
nitrate and chloride (Baker and Herson, 1994) . When adding nutrients to soil, it is important to
verfiy the effect the pH of the soil will have on their availability.
2.2.8 Soil Type
Soil permeability is a key component in the success of bioremediation, providing space
16
for air and water to move and transport nutrients and contaminants throughout the soil. The
voids in porous soil can be classified as macro voids, large pores with a very mobile phase, and
micro voids, small pores with an immobile phase. Dispersion and convection of air are the
dominate transportation methods within macro voids. These methods occur faster than diffusion,
which is the main transportation method in the immobile phase. When analyzing expected
bioremediation times, it is important to consider that organic contaminant is diffusing out of the
aggregate while oxygen and nutrients are diffusing in. Both of these affect bioremediation
periods (Dhawan et al., 1993).
Soils that are more permeable can transport and distribute nutrients and electron
acceptors more effectively in the soil, providing more surface area and space for distribution.
Non-cohesive soils such as gravel and sand are thus more favourable (Baker and Herson, 1994).
Fine soil particles found in silt and clay, form aggregates within which there is very slow or no
convective flow (Dhawan et al., 1993).
One study showed that due to the transportation kinetics, the bioremediation process was
restricted mostly to the macro pores of the system in the initial year of a treatment. It had not
had time to penetrate into the soil aggregates sufficiently (Gogoi et al., 2003). It is important to
consider how nutrients are applied and aeration rates based on the permeability of the soil.
17
3.0 MATERIALS AND METHODS
The soil used in the experiments came from Norman Wells, North West Territories. This
area has been used for petroleum resources since the early twentieth century with commercial oil
activities starting in the 1920s at Norman Wells. In 1933, a refinery was built to provide fuel for
local use, and during World War II, a 925 kilometre pipeline was built to transport oil to the
Alaska Highway to provide fuel for the military. This pipeline was abandoned in the 1940s, but
further expansion of the field took place in the early 1980s with a pipeline being built south to
Alberta (Imperial Oil, 2006; Town of Norman Wells).
3.1 Laboratory Setup
3.1.1 Soil Handling and Storage
In November 2007, three plastic-lined wooden sea cans containing approximately 3 200
kg of crude-oil contaminated soils from Norman Wells, North West Territories were received.
Upon receipt, the soils were transferred into plastic-lined 45 gallon plastic drums with minimum
head space, sealed, and placed in storage at -4oC.
In preparation for this biopile experiment, five plastic drums were removed and thawed.
They were emptied onto plastic lining and thoroughly mixed with shovels to homogenize the
soil. They were then transferred back into the plastic drums with three of the drums being
returned to storage and two of the drums taken up to the cold room to be used immediately in the
biopile experiment.
18
3.1.2 Biopile Tanks
Two stainless steel pilot-scale biopile reactors were fabricated and constructed as sealable
rectangular tanks, as shown in Figure 4. The dimensions of the tank, shown in Figure 5, are 1.0
m in length, 0.6 m wide and 0.35 m deep, holding a volume of 0.21 m3. Soil is filled up to 5 cm
from the top of the tank to ensure that there is minimal headspace in the reactor. The tanks are
connected to a supply of dry air, and the flow is regulated with valves and monitored with flow
meters. The air is fed into the bottom of the tank using perforated stainless steel air supply tubes
that run along the length of the tank. These tubes inside the tank are 0.6 m long and 16 mm in
diameter with perorations 3 mm in diameter, spaced 50 mm apart. The ratio of the area of the
perforation to pipe cross sectional area is 0.14. The perforations face down to prevent soil from
entering the air supply tubes under gravitational pressure. Exhaust air from the tank is passed
through an activated carbon column to ensure that volatized hydrocarbons are removed. There
are six soil gas sampling ports along the length of the tank that are fitted with perforated tubes
for collecting soil gas. They are spaced to ensure that the sampling in one port is independent of
the soil gas composition in the other ports.
19
Figure 4: Photograph of the pilot-scale biopile showing the air supply tubes and gas sampling tubes.
Figure 5: Sampling ports and air design
20
3.2 Experimental Design
3.2.1 Biopile Size
Previous studies have been done on biopiles of various sizes. They have ranged from
small microcosms 400 g (Braddock et al., 1997) to pilot scale biopiles 4 kg (Delille et al., 2008),
to field studies of biopiles that contain 4 800 m3 of soil (Filler et al., 2001). Microcosms are
convenient to set up in multiple units, but their small volume can be limiting. The heterogeneous
nature of soil is difficult to capture in a small volume. As well, the mass transfer processes
relating to oxygen, hydrocarbon distribution, and moisture transportation vary with scale. It is
unrealistic to conduct the field scale biopile in the constraints of the laboratory, so for this study
300 kg was chosen, a mass used in other studies as well (Braddock et al., 1997; Walworth et al.,
2007). At this mass, the soil can still be broken down into layers to see if there is any variance
with depth, depending on the proximity of the soil to the air supply. As well the entire soil
sample will maintain the same temperature and not create pockets of warmer air that may
influence the bacteria in that area. With larger biopiles, the inner core has a higher temperature
than the outer core, which is closer to the atmospheric temperature.
One of the important factors governing the dimensions of a biopile is maintaining aerobic
conditions throughout the entire biopile. The design of the biopiles allows for this. In many
cases the air supply may be warmed to create thermally enhanced biopiles, but this was not
chosen for the purpose of this study. The air tanks were kept in the cold room so that they were
the same temperature as the soil. A low air flow rate was used, allowing the air to equilibrate
with its surroundings.
21
3.2.1 Microcosm Experiment
In order to determine nutrient doses for the pilot scale biopiles, microcosm experiments
were done with three nutrient systems prior to the initiation of the experiment. Three jars were
left as untreated controls (no nutrient addition), and the rest were amended with ammonium
nitrate, three jars at a 100:9:1 (molar ratio based on TPH), and three jars at 100:5:1. Each jar
contained 500 g of soil, and was opened every two to three days and stirred to provide aeration.
The TPH results are shown in Table 4.
Table 4: TPH (mg/kg) in Microcosms
Control 100:5:1 100:9:1
Day 0 2018 (±160)
Day 32 1728 (±127) 1511 (±170) 1593 (±200)
Using the two way ANOVA test, data between the control, 100:5:1 and 100:9:1 nutrient
ammendments were analyzed for signifcant differences between day 0 and day 32. There was a
signifcant difference between TPH, F2 and F3 fractions between day 0 and day 32 for all
systems. There was no signifcant difference between TPH concentrations on day 32 between
systems.
22
Based on the microcosms, there was no reason to suspect that a higher nutrient dose
would be more effective on a larger scale. The design of the pilot scale biopiles was based on
the 100:5:1 ratio, similar to other experiments done by Walworth and Braddock.
3.2.2 Temperature
Figure 6 shows the surface temperature profile of Norman Wells, at Latitude 65oN,
Longitude 126oW, from March till November. The plotted temperatures are mean daily average
temperatures from 1970 to 2007. From the beginning of June to the beginning of August, the
temperatures are around 15oC or above. The cold room was programmed to maintain a
temperature of 15oC throughout the duration of the experiment to represent the temperature at the
site in the summer months. The experiment extended beyond 60 days to observe further changes
in the system.
23
Figure 6: Temperature profile of Norman Wells, NWT (Environmental Canada, 2009)
3.2.3 Nutrient Addition
Nutrients were added to the soil at a C:N:P ratio of 100 :5 :1. It was assumed that
petroleum hydrocarbons were the only available source or carbon in the soil, and preliminary
testing done on the soil indicated levels of contamination were around 1500 mg TPH/kg soil.
Soil characterization was conducted when the soils were initially received. Based on the soil
characterization, it was assumed that there was sufficient phosphorus in the soil and it was
readily available to micro organisms. The soil characterization can be found in Appendix A.
24
Nitrogen was added in the form of ammonium nitrate, with 68 grams of ammonium
nitrate was dissolved in 1 litre of water and then filter sterilized at room temperature.
The nutrients were added to the soil by layer, filling the tank with one third of the total
soil, and then spraying one third of the nutrients on top, mixing it throughout the layer using a
trowel. The next layer was placed on top and the nutrients added.
3.2.4 Moisture Content
By adding 1 litre of water to the biopile tank, the total moisture content was only changed
by 1%. This was done since the initial moisture content already provided 66% of the water
holding capacity (WHC).
Both the control and nutrient amended biopile were monitored throughout the experiment
to ensure that the moisture content did not change significantly, and that the supply of air was not
drying out the soil by measuring the moisture content with each sampling.
3.2.5 Air Flow
In order to maintain aerobic conditions, dry air was supplied to both the control and
nutrient amended biopile at a rate of 2 mL/day. Previous work determined this was sufficient to
maintain oxygen levels at 80% of ambient (atmospheric) levels.
25
3.3 Sampling
Soil samples were taken from the biopile using sterilized soil probes. Biopiles were
divided into three sampling layers of approximately 10 cm deep: top, middle, and bottom. From
each layer, the sample was comprised of composite soil samples from five randomly chosen
areas in that biopile layer. The soil samples were then placed in sterilized plastic bags, sealed,
and stored in a freezer at -20 oC until use.
Carbon dioxide and oxygen levels were measured with a portable ATX 620 Multi-gas
monitor equipped with an infrared end electrochemical sensor (Industrial Scientific Co.). The
monitor provides suction to the sampling port, drawing in air from the biopile. At each of the six
sampling ports, nine readings were taken over a period of three minutes during each sampling
event.
3.4 Analytical Methods
3.4.1 Total Petroleum Hydrocarbon (TPH) Extraction
TPH extraction was done according to the Canadian Council of Ministers of the
Environment (CCME) and their reference method for the Canada-wide standard for petroleum
hydrocarbons in soil. F2, F3 and F4 extractable hydrocarbons, in the range C10 to C50, were
determined by extracting 10 grams of soil sample and 10 grams of sodium sulphate with 140 mL
of a 50:50 hexane: acetone solvent in a Soxhlet apparatus (Gerhardt, SCP Science, Soxtherm 200
Automatic). Each sample was spiked with 2 µL of o-terapheynol as a surrogate in order to
assess the recovery of the solvent. With each run, a blank sample (no soil) spiked with the
surrogate was also analyzed in order to identify any contamination that may have occurred in the
processing of the samples. The hexane: acetone solvent was used because it allows extraction of
26
wet soils. The recovered solvent is dried using sodium sulphate and ran through columns with
silica gel to remove polar material. The recovered solvent is then concentrated through nitrogen
blow down, until there is a final extraction volume of approximately 2 mL. Samples are then
filtered and diluted with toluene. (CCME, 2001).
TPH was analyzed for each sampling day by layer. The samples were analyzed in the
GC (Agilent, 6890 N Network GC), fitted with a flame ionization detector. Hydrogen and air
provide the flame for the oven, maintaining the temperature at 250 oC. Helium is used as the
inert carrier gas that transports the evaporated solvents through the columns. As the carrier gas
passes the solvent through the column, the solvent adsorbs onto the column walls. Each
molecule will have a different travel time through the column, and the retention time of the
solvent in the column is recorded. In order to quantify the amount of total petroleum
hydrocarbons in the sample, standards were included in every tenth run to create a calibration
curve. In the sampling queue, each run began with toluene being injected to ensure that no
contamination had carried over from the previous run. Methanol was injected and run in the GC
after every five samples to ensure the needle and column were clean. Each sample was run with
two injections – each from a separate vial. Any discrepancies in the two values would indicate a
mechanical error in the GC, or an error in the vial preparation – such as excessive head space
allowing volatilization. At the end of the GC runs, the chromatograms were quantified. By
integrating the area under the curve, the total amount of hydrocarbons were calculated.
Any data that had less than 80% recovery of the surrogate was discarded, an example of
the chromatogram for a run comparing the surrogate recovery in the blank sample to a soil
sample is shown below in Figure 7, allowing for visual comparison. The amount of surrogate
recovered was also quantified during integration of the chromatograms.
27
Figure 7: Surrogate recovery, Blank vs Control Day 129
3.4.2 Inorganic nitrogen extraction
The extractant reagent was 0.01 M calcium sulphate prepared with deionized water. To
extract nitrate, nitrite and ammonia (inorganic nitrogens) from the soil, 30 mL of extractant was
added to 3 grams of soil in an Erlenmeyer flask and placed on a shaker for 5 minutes. This
method was developed from the method presented by Carter et al (1993). The suspension was
then filtered with 0.45 µm filters and stored in the freezer until analysis. For each sampling day,
composite soil samples were prepared from the top, middle and bottom layers and then separated
into coarse and medium aggregates using the #10 sieve. Analysis of each aggregate size for
inorganic nitrogen was done in triplicate. A blank sample of deionized water was also processed
along with the samples.
Surrogate spike
sample blank
28
Analysis of nitrate and nitrite was done with on the ion chromatogram (Metrohm, 819 IC
Detector). Ion chromatography allows the separation of ions and polar molecules based on the
charge properties of the molecule and the rate at which they pass through a column (Fritz and
Gjerde, 2009). Based on the retention time of standards, the chromatograms were used to
determine the concentration of nitrate and nitrite. In each sample queue, the standards were run
to account for any drift in the calibration curve. For every five samples run, a sample of
deionized water was analyzed to ensure there was contamination.
In order to analyze the ammonia concentration in the samples, Nessler’s Reagent was
used to create a colourimetric reaction. The Nessler’s Reagent was prepared with potassium
iodide, mercuric chloride, and potassium hydroxide, and combined with Reagent #1 (from
LaMotte) which contained sodium potassium titrate. In the presence of ammonia, the reagents
react and produce a yellow tint in the solute (LaMotte, 2009). The absorbance of this colour
(490 nm) was then measured on the spectrometer (Evolution 300, UV vis Spectrophotometer)
and compared to the calibration curve to find the concentration of ammonia in the solute. Each
time samples were analyzed, standards were included in the analysis. A sample of deionized
water amended with the reagents was used to create the base line.
3.4.3 Plate Counting of Bacteria
In preparation, sterilized glass and plastic petri dishes were prepared. Glass dishes were
prepared using the Bushnell agar, and plastic petri dishes with the R2A agar. All material used
during the procedure was autoclaved for sterilization.
The R2A agar was used as the medium to grow heterotrophic bacteria. Using sterilized
sieves, composite soil samples from each biopile system were separated into coarse and fine
29
grained samples. Each system was analyzed for population counts in coarse and medium
aggregates, by preparing triplicate samples.
Ten grams of soil was added to an Erlenmeyer flask, with 95 mL of distilled water
(dilutent). Sterilized glass beads were added to help with mixing, and the flask was capped. The
bottles were placed on the mechanical shaker for 10 minutes.
This first dilution represents a 10-1
dilution. For the dilution series, a 1 ml sample
was added to 9 mL dilution blank (distilled water). This sequence was continued up to a dilution
of 10-7
.
For addition onto the agar plates, four dilutions were selected: 10-4
, 10-5
, 10-6
, 10-7
and 0.1
mL of aliquot was transferred onto a separate plate, beginning with the highest dilution. The
suspension (0.1 mL aliquot) was spread on the agar surface using a sterile glass spreader for each
plate. Between transfers, the spreader was kept submerged in a beaker of ETOH (95% ethanol)
and excess ETOH was burned off prior to use. After the transfer, the plates were inverted and
placed in the incubator at 15oC. After 2 weeks, the number of CFU formed on the plates was
counted and recorded. The plates remained in the incubator and monitored to ensure that no
further growth occurred.
For the Bushnell agar, the procedure was the same. However, the Bushnell agar does not
contain a source of carbon for the bacteria, which is why it can be used to enumerate
hydrocarbon degrading bacteria. Before inverting the plates, 10 µL of arctic diesel was added to
the lid.
30
3.5 Statistical Analysis
Statistical analysis of the data was done using SigmaPlot 11.0 software. Comparisons
between biopile systems and time frames to determine significant differences were done using
one-way analysis of variance (ANOVA) and t-tests (α = 0.05).
4.0 RESULTS AND DISCUSSION
4.1 Analysis of Changes in Soil Parameters
4.1.1 Assessment of Changes in Moisture Content
For each sampling day, the gravimetric moisture content was measured in each layer,
with three samples analyzed per layer. Moisture content is an important parameter to measure to
ensure that the air being injected into the biopiles does not dry out the soil to the point where it is
detrimental to bacterial growth. This would only be a problem in the vicinity near the air
injection site or if very high air injection rates were used. Ten grams of soil were weighed per
sample and dried in an oven at 100 oC for twenty four hours, cooled in a desicator, and then
reweighed. There was no significant difference in the moisture content between layers so the
data is presented as one data point per day in Figure 8.
In Figure 8, the gravimetric moisture content in both the nutrient amended and control
biopile is plotted throughout time. The nutrient amended biopile had a slightly higher moisture
content due to the one litre of water that was used to mix and distribute the nutrients throughout
the soil. In the control system, the moisture content varied between 14.7% to 15.1%. In the
nutrient amended system, the moisture content varied between 15.2% and 16% which was not a
31
significant change. Both of these systems mainted a moisture content that was 65% of the water
holding capacity of the soil. A moisture content that is between 40 – 85% of the water holding
capacity is considered to be ideal (Mohn et al., 2001). Any lower, and moisture would need to
be added to maintain proper bacterial growth. For values greater than 85%, additional drainage
considerations beyond the standard design would need to be included (Mohn et al., 2001). The
air dried moisture content was also measured on several days to verify the moisture content. The
soils were dried at 15oC for forty eight hours in the cold room, and the results are summarized in
Figure 9.
Figure 8: Gravimetric moisture content, Control vs Nutrient amended Biopile
32
Figure 9: Air dried moisture content, Control vs Nutrient amended Biopile
The air dried samples have a slightly higher moisture content then the samples dried in
the oven, showing the oven is a more extensive method of drying out samples.
There was minimal change in the moisture content of the biopiles with time. Additional
moisture sources compensate for any drying due to air injection. In the field, moisture may come
from precipitation, but in both the field and laboratory the bioremediation process helps maintain
moisture levels. While microogranisms metabolize hydrocarbons they produce water (Hinchee
& Brockman, 1995). This is illustrated in the stoichiometric equation for the microbial
degradation of n-hexane. For every mole of n-hexane degraded, seven moles of water are
produced.
33
4.1.2 Carbon Dioxide Production and Oxygen Consumption
The amount of carbon dioxide and oxygen produced in the soil was measured thirteen
times during the 65 day period, with six readings taken at each sampling port. There was
minimal variation in the readings between sampling ports, and the six data points were used to
represent one point per sampling day in the graphs. Figures 10 and 11 below show the
percentage of carbon dioxide and oxygen in the biopile systems with time. In the ambient air of
the cold room, the concentration of oxygen was 21 % and 0.05 % for carbon dioxide. In the
biopile systems, oxygen levels were lower and carbon dioxide levels higher, indicating that
cellular respiration was occurring. The highest levels of carbon dioxide occurred during the first
forty days of the treatment, at which time they then began to level off. However, after day 40,
carbon dioxide levels were still approximately 0.5 %. Oxygen levels were also significantly
depleted in the first forty days, and remained below 21 % after day 40.
34
Figure 10: CO2 production in the control and nutrient amended biopiles
35
Figure 11: O2 production in the control and nutrient amended biopiles
In Figure 12, the percentage of oxygen and carbon dioxide were correlated. In the control
biopile the correlation coefficient was 0.9475 and in the nutrient amended biopile it was 0.9675.
Thus there is a strong correlation between the production of carbon dioxide and consumption of
oxygen, indicating that aerobic biological activity is occurring. Again, if looking at the
mineralization stoichiometry for n-hexane, for every 9.5 moles of oxygen, 6 moles of carbon
dioxide are produced. This is equivalent to 304 g of oxygen consumed for 264 g of carbon
36
produced at a ratio of 1.15. In the nutrient amended biopile, the average ratio was 1.89 ± 2.85
and in the control biopile the ratio was 1.26 ± 1.63.
4.1.3 Hydrocarbon Degradation
The total petroleum hydrocarbons (TPH) were measured in each layer of the soil,
analyzing three samples per layer. There was no significant difference between layers, so the
data was combined to provide nine data points per sampling day and represent the biopile system
as a whole. Figure 13 below demonstrates this similarity, showing the TPH for each layer for the
first four sampling days in the nutrient amended biopile.
Figure 12: Correlation between % O2 and % CO2 in the control (left) and nutrient amended (right) biopile
37
Figure 13: TPH analysis by layer, nutrient amended biopile
Figure 14 below quantifies the amount of TPH in the biopile systems and how it changed with
time. Although there is no significant difference between the two systems (nutrient amended and
control), there is a statistical difference between day 0 and day 65 in both systems. There is a
continuous decrease in the amount of TPH in both systems with time. The most significant
decrease in the biopiles occurs within the first fifteen days. Other sampling days showed a
gradual decrease in TPH.
38
Figure 14: TPH in control and nutrient amended biopiles
To further analyze the TPH content of the biopiles, the GC/FID analysis can be broken
down into the F2 and F3 fractions of hydrocarbons in the soil. The F2 fraction represents the
semi-volatile fraction, comprised of aromatics and aliphatic sub fractions in the>C10 to C16
range, whereas in the F3 fraction contains aromatics and aliphatics in the >C16 to C34 range
(CCME, 2008). Prior to biopile experimentation, soils samples were sent to Maxxam Analytics
to analyze for aromatic compounds. None were detected.
Figure 15 below shows the amount of F2 in the biopile systems with time. In comparison
to the TPH and F3 graphs, there is a very rapid decline in the first forty days. Since this is the
39
semi-volatile fraction, there is the chance that in the first forty days there was volatilization
occurring. However, initial analysis of the activated carbon did not detect any hydrocarbons.
Figure 15: F2 hydrocarbon fraction in control and nutrient amended biopiles
Figure 16 shows the amount of F3 hydrocarbon fraction in the soil with time. The
decrease in F3 hydrocarbons is at a slower rate than F2 and is gradual with time. Statistical
analysis (ANOVA and t-test) showed that there was a significant difference with time, but not
between the biopile systems. Again, as with the TPH, the large decrease between day 0 and day
15 is visible in this graph. The decrease in the F3 fraction of hydrocarbons in the soil indicates
that bioremediation occurred. The F3 fraction is heavier and less volatile than the F2 fraction, so
in order for them to be removed from the system, it is necessary for them to be consumed by
bacteria. In Table 5 below, decane and heptadecane are compared to illustrate the differences in
40
compounds from two different hydrocarbon fractions. Heptadecane is heavier than decane, and
the vapour pressure of decane is larger. The larger the vapour pressure, the more volatile the
compound is. Within the F2 fraction, the initial decrease may be volatilization, but as the
experiment continues heavier and less volatile compounds will be left in this fraction. As the F2
fraction is depleted of the lighter hydrocarbons, only the heavier hydrocarbons are available for
biodegradation.
Table 5: Comparison of an F2 compound to a F3 compound (OSHA, 2007)
F2 - Decane F3 - Heptadecane
Molecular Formula C10H22 C17H36
Molecular Weight (g/mol) 142 240
Vapour Pressure (kPa at 25°C) 0.17 < 0.1
Assuming that the average weight of the petroleum spilt is Assuming that the average
weight of the petroleum spilt is 300 g/mol and that decane and heptadecane are both present
as 1% by weight, and using the air flow rate of 2 mL/day, the amount of each compound that
is volatilized can be calculated based on Raoult’s law (Ghoshal and Luthy, 1998) At the air
flow rate if air-NAPL (non-aqueous phase liquid) equilibrium is attained, 1.72x10-7
mol of
decane are removed per day, compared to 1.02 x10-7
mol of heptadecane. A maximum of 1.7
times more moles of the lighter F2 fraction are removed during volatilization.
41
Figure 16: F3 hydrocarbon fraction in the control and nutrient amended biopiles
To further analyze the difference between day 0 and day 15, and day 15 and day 65, the
chromatograms were examined. In the following Figures (17 through 20), there is a comparison
of day 0 vs day 15, day 15 vs day 65 for the nutrient amended and control biopiles.
A direct comparison of the GC chromatogram of Day 0 and Day 15 is shown in Figures
17 and 18. The comparisons of the two GC profiles showed there are significant reductions in
different hydrocarbon fractions. The decrease in resolved peak areas including n-alkanes,
appeared in the ranges of both F2 (>C10-C16) and F3 (>C16-C34). As well, there is a decrease
in the humped portion of the chromatogram. This area is the unresolved complex mixture
42
(UCM). It represents compounds that cannot be separated by the GC and contains branched
isoprenoids, cyclic alkanes, steranes, hopanes and other difficult to degrade components (Mills et
al., 2003). The UCM compounds are of higher molecular weights and are unlikely to volatilize,
therefore the decrease in this area indicates there was biodegradation. The decrease of the UCM
area and the increased retention time of the centroid are all characteristic of biodegradation
(Mills et al., 2003). Figures 18 and 19 show the direct comparison between day 15 and day 65.
Although no significant decrease in the UCM in either biopiles, there are decreases in the
resolved peak areas in the nutrient amended biopile. As the petroleum is biodegraded, the UCM
becomes the dominant feature which is indicative of biodegradation (Mills et al., 2003).
43
Figure 17: Chromatogram: day 0 vs. day 15 - Control
Figure 18: Chromatogram: day 0 vs. day 15 – Nutrient amended
44
Figure 19: Chromatogram: day 15 vs. day 65 - Control
Figure 20: Chromatogram: day 15 vs. day 65 - Nutrient amended
45
The value of the UCM can be quantified by integrating the area of the resolved peaks and
subtracting it from the total resolved area. This is shown in Figure 21 and 22 below. In both the
control and nutrient amended systems there was a significant decrease in the UCM area with
time. As discussed previously, the UCM is composed of molecules with higher molecular
weights that are less likely to volatilize. This is indicative that these compounds were
biodegraded. However, there was no significant difference between the control and nutrient
amended biopile.
Figure 21: UCM Area, Control biopile
46
Figure 22: UCM Area, Nutrient amended biopile
4.2 Continuing the Experiment Beyond Day 65
Within the first 65 days of the experiment, there were significant differences in the total
petroleum hydrocarbons with time and significant carbon dioxide production and oxygen
consumption with time. The study was continued on until day 150 at 15oC in order to observe
further changes in the system.
4.2.1 Oxygen and Carbon Dioxide Levels
After day 65 there is no significant change in either oxygen or carbon dioxide levels with
time. However, they are still above the background levels of the room indicating that
bioremediation may still be occurring.
47
Figure 23: Percentage carbon dioxide, beyond day 65
Figure 24: Percentage oxygen, beyond day 65
48
4.2.2 Continued TPH Degradation
In Figures 25 through 27 below, the graphs for F2, F3 and TPH are shown for days 65
through to 150. Overall, the hydrocarbon levels seem to be decreasing at a much slower rate
than in the initial 65 days for both the nutrient amended and the control biopiles, with the change
between day 65 and day 150 being very small. Compounds that are more resistive to
bioremediation take longer to be removed from the system, but can still occur (Peters et al.,
2005). However, if the entire system throughout the 150 days as a whole, as for TPH shown in
Figure 28, the graph fits the exponential decay graph with a first order reaction. Biodegradation
would still be occurring, but at much slower rates as the time increases. The curve that fits the
control biopile are y = (r2 = 0.8591) and for the nutrient amended biopile it is
y = (r2 = 0.8687).
Figure 25: F2 hydrocarbon fraction, beyond day 65
49
Figure 26: F3 hydrocarbon fraction, beyond day 65
Figure 27: TPH, beyond day 65
50
Figure 28: TPH, entire 150 day system
Again, the chromatograms for the control and nutrient amended systems can be analyzed.
There is not a large difference in the UCM between day 65 and day 80 (Figures 29 and 30), but
there are decreases in the resolved peaks. Between day 80 and day 109, there is a significant
difference in the UCM in both biopiles. As well, in the above graphs, there was a faster rate of
decrease between day 80 and day 109 (Figures 30 and 31) for all hydrocarbon fractions.
Between day 109 and day 150 (Figures 32 and 33), there wasn’t a decrease in the UCM, but the
resolved peaks did decrease a small portion. So although the rates of bioremediation may have
decreased significantly after day 65, there is still evidence in the chromatograms that
bioremediation is occurring.
51
Figure 29: Chromatogram: day 65 vs day 80 - Control
Figure 30: Chromatogram: day 65 vs day 80 - Nutrient amended
52
Figure 31: Chromatogram: day 80 vs day 109 - Control
Figure 32: Chromatogram: day 80 vs day 109 - Nutrient amended
53
Figure 33: Chromatogram: day 109 vs day 150 - Control
Figure 34: Chromatogram: day 109 vs day 150 - Nutrient amended
54
In the UCM quantitative analysis, there is not a clear difference between day 43 and day
150 in the control biopile, but the nutrient amended biopile does show a significant decrease in
the UCM. Again, although bioremediation may not be occurring at as fast a rate as it was
initially, there is still evidence that it is occurring. These can be referred to in Figures 35 and 36
below.
Figure 35: UCM, entire 150 day system - nutrient amended
55
Figure 36: UCM, entire 150 day system - control
4.3 Aggregation and Soil Properties
Soil analysis done by Maxxam Analytique (sieve analysis) and AGAT (X-ray diffraction)
provided insight into the different soil properties. Based on the results from Maxxam, the soils
contained 3.5% gravel (>2.0 mm), 72% sand (0.05 – 2.0 mm) and 24.5 % silt/clays (<0.05 mm),
as described by the U.S. Department of Agriculture’s classification system. Under the Unified
Soil Classification system, soils containing over 12% fines (No. 200 sieve) are classified as
“sand with fines”. As well, the soil is considered well graded.
To better understand the soil mineralogy, the soil samples were sent to AGAT
Laboratories for a bulk and clay X-ray diffraction (XRD) analysis. The combined total (bulk and
56
clay) XRD results indicate that the sample consists of significant amounts of quartz, but also
notable amounts of the clays illite, chlorite, and kaolinite. The clay fraction (i.e. fraction less
than 3 microns in size) is approximately 18% of the total volume of the sample. Appendix B
shows the percentages of each mineral in the fractions. As discussed earlier, clay in soil can
influence bioremediation, affecting the distribution of oxygen, nutrients, and contamination.
Analysis of the soil by Wonjae Chang, a member of the laboratory team, found that 18%
of the soil was coarse and 72% medium aggregates. This was done by mechanical separation of
the soil. Based on these results and the literature available, bacteria population and nutrients
were analyzed based on the medium and coarse aggregates in the biopiles.
4.2.1 Plate Counting
Analysis of the initial bacteria in the soil prior to nutrient addition was done based on
composite samples from the biopiles, counting both the heterotrophic and hydrocarbon degrading
bacteria. The samples were gently separated with a sterilized #60 sieve to obtain the coarse and
medium aggregates and incubated at 15oC. The results are summarized below.
Table 6: Total indigenous heterotrophic and hydrocarbon degrading bacteria
Heterotrophic Bacteria Hydrocarbon Degrading Bacteria
Coarse (> 75 µm) 2.18x106 ± 2.66x10
5 2.76x10
5 ± 1.34x10
5
Medium (<75 µm) 5.14x106 ± 1.85x10
6 4.19x10
6 ± 3.04x10
6
57
Although the mean averages of the bacteria counts indicate that the medium aggregates
contain more bacteria, it is difficult to confidently assess this due to the high standard deviations
typical of plate counting. However, it is noted that prior to nutrient addition and stimulus by
oxygen, the population of bacteria were high enough to support bioremediation. It has been
noted that a minimum heterotrophic plate count of 103 CFU/gram of soil is considered to be
effective. Typical population densities in soils range from 104 to 10
7 CFU/gram of soil (Leahy
and Colwell, 1990).
4.2.3 Nutrient Cycling
As mentioned previously, nitrogen and phosphorus are usually present in rate limiting
amounts in the soil, with the bulk of nitrogen and phosphorus contained in the organic fraction of
the soil which is not directly available to the bacteria (Walworth et al., 2003). It would need to be
mineralized first and then converted to inorganic nutrients; since mineralization rates are slower
than hydrocarbon degradation rates, hydrocarbon degradation rates can be limited by this
(Walworth et al., 2003). Several studies have been done comparing organic nutrients to
inorganic nutrients, but there has been no significant difference in petroleum losses between the
nutrient sources (Walworth et al., 2003).
Studies have shown that the addition of aqueous soluble fertilizer salts leads to salts
quickly partitioning into soil water and increasing the concentration of salt. This decreases
osmotic potential, where an osmotic potential decrease of 0.5 MPa can reduce degradation by
fifty percent. A large number of studies suggest that the addition of nitrogen fertilizers should be
58
added as an estimate of the concentration of nitrogen in soil solution, at a recommend value less
than or equal to 2 500 mg N/kg water (Walworth et al., 1997). At the ratio of 100:5:1 and a
moisture content of 15%, the nitrogen in the soil is less than 2 500 mg N/kg water.
A study by Chang and Weaver (1997) indicated that ammonia was consumed by micro
organisms first. Bacteria can easily able to use ammonia for direct incorporation into amino
acids, while nitrates need to first be reduced to ammonia for use. Certain bacteria are able to use
nitrate or nitrogen bacteria, but for others energy must first be used to convert nitrates to
ammonia (King et al., 1998).
The biopile systems were analyzed for inorganic nitrogen content, including nitrate
(NO3-), nitrite (NO2
-), and ammonia/ammonium (NH4
+/NH3
+). For each sampling day, the
samples were analyzed in triplicate for the coarse and medium aggregates and the data was
cumulated based on the values for the soil aggregation. Various samples were sent to Maxxam
Analytique to confirm results. These graphs are shown below.
59
Figure 37: Inorganic nitrogen - control, coarse aggregates
Figure 38: Inorganic nitrogen - control, medium aggregates
60
Figure 39: Inorganic nitrogen - nutrient amended, medium aggregates
Figure 40: Inorganic nitrogen - nutrient amended, coarse aggregates
61
In the control biopile, there are limited amounts of inorganic nitrogen in the soil and it is
difficult to notice a difference in concentrations of inorganic nitrogen within the aggregates due
to the large error bars associated with nitrogen measurement (Walecka-Hutchison and Walworth,
2007). It does appear that the amount of nitrate is increasing within the system and reflecting a
slight increase in the inorganic nitrogen. The amount of nitrite in this system (and in the nutrient
amended system) is very low. Nitrite usually does not accumulate in soil unless the conditions
are very alkaline or there are exceptionally high ammonium levels. Nitrite oxidizers are very
susceptible to environmental stresses, and higher levels would indicate environmental stresses
(Duncan et al., 1998). The background ammonia levels are also very low.
For the nutrient amended biopile, the addition of ammonium nitrate greatly increased the
amount of inorganic nitrogen in both the coarse and medium aggregates. In both coarse and
medium aggregates, they are below the recommended 2500 mg N/kg water, so that should not
negatively affect the environments in either aggregate size. From day 0 to day 65, the amount of
ammonia decreased and the amount of nitrate increased in both aggregate sizes. Under the
aerobic conditions of the biopile, nitrification took place. Nitrification occurs in two phases, as
ammonium oxidation and nitrite oxidation by different groups of autotrophic chemolithotrophic
bacteria. Nitrite never builds up in the system as the second part of the phase occurs rapidly
(Duncan et al., 1998). The lag time between ammonia depletion and nitrate increase may have
occurred as ammonia was incorporated into the microbial biomass as the hydrocarbon was being
degraded. Since the rate of nitrate accumulation increased rapidly over a short period of time,
this indicates the initial population of nitrifying micro organisms was quite high (Chang and
Weaver, 1997). Initially, the amount of nitrate, and ammonia in the two aggregate sizes were
similar, but as nitrification occurred nitrate levels built up significantly more in the coarse
62
aggregates and resulted in a higher level of inorganic nitrogen in the coarse aggregates. As was
noted earlier, the medium aggregates had a slightly higher bacteria population, but they are not
receiving more nutrients. The smaller population of micro organisms in the coarse aggregates
may not be able to consume nitrates as fast as they are produced. Overall, the biopiles were
under aerobic conditions, but the medium aggregates may be limited in oxygen due to their
structure. As was discussed earlier, the medium aggregates have micro voids and the nutrient
mixture and oxygen may take longer to diffuse into these areas. This may become the limiting
factor and explain the build up of nitrates.
In the nutrient amended biopile, and to a lesser extent in the control biopile, the amount
of inorganic nitrogen in the systems increased. There was no additional source of nitrogen that
was added to the biopile, so it was assumed that the total amount of inorganic nitrogen would
have decreased as it was taken up by bacteria. In the figures below, the amount of inorganic
nitrogen is plotted along with the amount of organic nitrogen in the biopiles (organic nitrogen
was sent to Maxxam Analytique), using composite samples of medium and coarse aggregates. In
the control system, there is a clear decrease of organic nitrogen along with an increase in
inorganic nitrogen. As well, this is shown in the nutrient amended biopile system, with there
being a decrease between day 0 and day 43 in the organic nitrogen. If organic nitrogen is being
transformed into ammonia through mineralization, then there is always a constant supply of
inorganic nitrogen that is available to the micro organisms and the system is not nitrogen limited.
This may explain why biodegradation was able to occur in the control system at a similar rate to
the nutrient amended system. The final values of nitrate and ammonia do not account for any
nitrogen that may have been incorporated into microbial biomass while the oil was being
degraded (Chang and Weaver, 1997).
63
Figure 41: Inorganic and organic nitrogen in control biopile
Figure 42: Inorganic and organic nitrogen in nutrient amended biopile
64
5.0 CONCLUSIONS
Throughout the 150 day experiment, the nutrient amended and control biopiles were
monitored for oxygen consumption and carbon dioxide production, and the soils sampled for
analysis of the total petroleum hydrocarbons and inorganic nitrogen.
Based on the increased consumption of oxygen and production of carbon dioxide it was
evident that a biological process was occurring. Further analysis of the hydrocarbons in the soil
supported bioremediation by analysis of the different hydrocarbon fractions and losses in the
unresolved complex mixture.
Overall 42% of the total petroleum hydrocarbons were removed from the nutrient
amended biopile and 38 % in the control biopile. For the F2 fraction, both systems had less than
200 mg/kg soil and for the F3 fraction around 700 mg/kg soil. This is in accordance with the
CCME standards (based on the standards for fine grained soil, as 72% of the soil composition is
sand). Although removal rates decreased as time went on, bioremediation continued as after the
initial 65 days. However, an absolute confirmation of whether these loses were due to
biodegradation (or to what extent) are not possible to be reported here.
Nutrient amendment did not stimulate the micro organisms to produce a faster removal
rate in the nutrient amended biopile. Both the control and the nutrient amended biopile
demonstrated similar removal patterns. Nutrient analysis showed that the inorganic nitrogen in
the system was not being consumed as fast as it was produced. Potentially organic nitrogen was
being converted in both biopile systems, providing a continuous source of inorganic nitrogen.
65
6.0 REFERENCES
Aislabie, J., Saul, D. J., & Foght, J. M. (2006). Bioremediaation of hydrocarbon-contaminated
polar soils. Extremophiles, 10, 171-179.
Alexander, M. (1994). Biodegradation and Bioremediation. San Diego, CA: Academic Press.
AMAP Secretariat. (2003). Arctic Monitoring and Assessment Programme Retrieved May 26,
2009, from http://www.amap.no/
Baker, K. H., & Herson, D. S. (1994). Bioremediation. New York, NY: McGraw-Hill.
Braddock, J., Ruth, M., Catterall, P., Walworth, J., & K., M. (1997). Enhancement and inhibition
of microbial activity in hydrocarbon contaminated arctic soils: implications for nutrient
amended bioremediation. Environmental Science and Technology, 31(7), 2078-2084.
Bragg, J. R., Prince, R. C., Harner, E. J., & Atlas, R. M. (1994). Effectiveness of bioremediation
for the Exxon Valdez oil spill. Nature Publishing Group, 368, 413-418.
CCME. (2001). Reference Method for the Canada-Wide Standard for Petroleum Hydrocarbons
in Soil - Tier 1.
CCME. (2008). Canada-Wide Standard for Petroleum Hydrocarbons in Soil: Scientific
Rationale.
Chang, Z. Z., & Weaver, R. W. (1997). Nitrification and utilization of ammonium and nitrate
during oil bioremediation at different soil water potentials. Soil and Sediment
Contamination, 6(2), 149-160.
Delille, D., Duval, A., & Pelletier, E. (2008). Highly efficient pilot biopiles for on-site
fertilization treatment of diesel oil-contaminated sub-Antarctic soil. Cold Regions Science
and Technology, 54, 7-18.
Dhawan, S., Erickson, L. E., & Fan, L. T. (1993). Model Development and Simulation of
Bioremediation in Soil Beds with Aggregates. Ground Water, 31(2), 271-283.
Duncan, K., Hettenbach, E. J. S., Potter, W., & Sublette, K. (1998). Nitrogen Cycling and Nitric
Oxide Emissions in Oil-Impacted Prairie Soils. Bioremediation Journal, 1(3), 195-208.
Ferguson, S. H., Franzmann, P. D., Revill, A. T., Snape, I., & Rayner, J. L. (2003). The effects of
nitrogen and water on mineralisation of hydrocarbons in diesel-contaminated terrestrial
Anarctic soils. Cold Regions Science and Technology, 37(2), 197-212.
66
Filler, D. M., Lindstrom, J. E., Braddock, J. F., Johnson, R. A., & Nickalaski, R. (2001). Integral
Regions Science and Technology. Cold Regions Science and Technology, 32, 143-156.
Filler, D. M., Snape, I., & Barnes, D. L. (2008). Bioremediation of Petroleum Hydrocarbons in
Cold Regions Cambridge: Cambridge University Press.
Fritz, J. S., & Gjerde, D. T. (2009). Ion Chromatography (4th ed.). New York: Wiley-VCH.
Gallego, J. R., Gonazalez-Rojas, E., Pelaez, A. I., Sanchez, J., Garcia-Martinez, M. J., Ortiz, J.
E., et al. (2006). Natural attenuation and bioremediation of Prestige fuel oil along the
Atlantic coast of Glaicia (Spain). Organic Geochemistry, 37(1869-1884).
Ghoshal, S., & Luthy, R. G. (1998). Biodegradation Kinetics of Naphthalene in Nonaqueous
Phase Liquid-Water Mixed Batch Systems: Comparison of Model Predictions and
Experimental Results. Biotechnology and Bioengineering, 57, 356-366.
Gogoi, B. K., Dutta, N. N., Goswami, P., & Mohan, T. R. K. (2003). A case study of
bioremediation of petroleum-hydrocarbon contaminated soil at a crude oil spill site.
Advances in Environmental Research, 7, 767-782.
Hinchee, R. E., & Brockman, F. J. (1995). Microbial Processes for Bioremediation. Colombus,
Ohio: Battelle Press.
Imperial Oil. (2006). The story of Imperial Oil. Retrieved April 14, 2009, from
http://www.imperialoil.ca/Canada-English/ThisIs/Story/TI_S_StoryOfIOL.asp
King, R. B., Long, G. M., & Sheldon, J. K. (1998). Practical Environmental Bioremediation:
the Field Guide. Boca Raton, Florida: CRC Press.
Kratzke, R., Major, W., Fahnestock, F. v., & Wickramanayake, G. (1998). Biopile Design,
Operation, and Maintenance Handbook for Treateing Hydrocarbon-Contaminated Soils.
Columbus, Ohio: Battelle Press.
LaMotte. (2009). Water Testing Kits. Retrieved February 24, 2009, 2009
Leahy, J. G., & Colwell, R. R. (1990). Microbial Degradation of Hydrocarbons in the
Environment. Microbiological Reviews, 54(3), 305-315.
Margesin, R., & Schinner, F. (1999). Biological decontamination of oil spills in cold
environments. Journal of Chemical Technology and Biotechnology, 74, 381-389.
Mills, M. A., Bonner, J. S., McDonald, T. J., & Autenrieth, C. A. P. R. L. (2003). Intrinsic
bioremediation of a petroleum-impacted wetland. Marine Pollution Bulletin, 46, 887-899.
Mohn, W. W., Radximinski, C. X., M.-C.Fortin, & K.J.Reimer. (2001). On site bioremediation
of hydrocarbon-contaminated Arctic tundra soils in inoculated biopiles. Applied
67
Microbiology Biotechnology, 57, 242-247.
Mudge, S. M. (2009). Methods in Environmental Forensics. Boca Raton, FL: CRC Press.
OSHA. (2007). Chemical Sampling Information. Retrieved December 20, 2009, from
http://www.osha.gov/dts/chemicalsampling/toc/toc_chemsamp.html
Peters, K. E., Walters, C. C., & Moldowan, J. M. (2005). The Biomarker Guide: Biomarkers and
isotopes in petroleum systems and Earth (2 ed. Vol. 2). New York, New York:
Cambridge University Press.
Thomassin-Lacroix, E. J. M., Reimer, M. E. K. J., & Mohn, W. W. (2002). Biostimulation and
bioaugmentation for on-site treatment of weathered diesel fuel in Arctic soil. Applied
Micorbiological Biotechnology, 59, 551-556.
Town of Norman Wells. Visit Norman Wells. Retrieved April 14, 2009, from
http://www.normanwells.com/visit/proud_history.html
United States Environmental Protection Agency. (2004). How to Evaluate Alternative Cleanup
Technologies for Underground Storage Tank Sites. Retrieved December 7, 2009, from
http://www.epa.gov/swerust1/pubs/tums.htm
Walecka-Hutchison, C. M., & Walworth, J. (2006). Assessment of C:N Ratios and Water
Potential for Nitrogen Optimixation in Diesel Bioremediation. Bioremediation Journal,
10, 25-35.
Walecka-Hutchison, C. M., & Walworth, J. L. (2007). Evaluating the effects of gross nitrogen
mineralization, immobilization and nitrification on nitrogen fertilizer availability in soil
experimentally contamined with diesel. Biodegradation, 18, 133-144.
Walworth, J., Pond, A., Rayner, I. S. J., Feguson, S., & Harvey, P. (2007). Nitrogen requirements
for maximinzing petroleum bioremediation in a sub-Antarctic soil. Cold Regions Science
and Technology, 48, 84-91.
Walworth, J. L., Woolard, C. R., & Harris, K. C. (2003). Nutrient amendments for contaminated
peri-glacial soils: use of cod bone meal as a controlled release nutrient source. Cold
Regions Science and Technology, 37, 81-88.
Walworth, J. L., Woolard, C. R., & Reynolds, J. F. B. C. M. (1997). Enahncement and
inhibiition of soil petroleum biodegradation through the use of fertilizer nitrogen: an
approach to determining optimum levels. Journal of Soil Contamination, 6, 465-480.
Whyte, L. G., Bourbonniere, L., Bellerose, C., & Greer, C. W. (1999). Bioremediation
Assessment of Hyrdocarbon-Contaminated Soils from the High Arctic.
Wong, J. H. C., Lim, C. H., & Nolen, G. L. (1997). Design of Remediation Systems. Boca Raton,
FL: Lewis Publishers.
68
APPENDIX A: PHYSIOCHEMICAL CHARACTERISTICS OF SOIL
Analysis done by Maxxam Analytique
Physiochemical Parameter Values
Soil particle composition Gravel: 3.5% Sand: 72%
Silt & Clays: 24.5%
Well graded soils
Max. Water Holding Capacity (WHC) 32%
Moisture content (gravimetric) 21 % (~65% of Max. WHC)
Soil pH 7.4
Nutrients Concentration (mg/kg soil)
Nitrate (N): N-NO3- ND
Nitrite (N): N-NO2- ND
Nitrogen ammonia: N-NH3 ND
TKN (Total Kjeldahl Nitrogen) 1400
Total phosphorous 490
69
APPENDIX B: SUMMARY OF XRD ANALYSIS
Analysis done by AGAT Technologies
Soil
Fraction
Weight
%
Quartz Plagio.
Feldspar
Calcite Dolomite Kaolinite Chlorite Illite Total
Clay
Bulk
fraction
Clay
fraction
Bulk &
Clay
81.96
18.04
100
56
4
47
5
0
4
7
1
6
15
1
13
6
41
12
2
10
3
9
43
15
17
94
30