The effect of temperature on the

bioavailability and microbial degradation of

Polycyclic Aromatic Hydrocarbons in Soil

Eleanor Danaher

11315676

8th

April 2015

This research project was submitted to University College Dublin in

partial fulfilment of the requirements for a B.Sc. (Hons.) Degree in

Environmental Biology.

School of Biology and Environmental Science

Supervisor:

Dr. Evelyn Doyle

Summary

Polycyclic aromatic hydrocarbons (PAHs) are compounds consisting of benzene rings and are

ubiquitous in the environment. These compounds receive a lot of attention due to their high

toxicity, carcinogenicity and recalcitrance. Due to these characteristics it is important that they

be removed from the environment. Conventional techniques (e.g. incineration) used in the

removal of these compounds are expensive and not environmentally sound. Bioremediation is

regarded as a much ‘greener’ and cost-effective technique for the removal of these compounds

from the environment. However, the efficacy of this method still remains somewhat

unpredictable. In order for bioremediation strategies to be successful, complete information on

the factors that control PAH degradation must be fully understood. These factors range from the

in situ microbial communities and soil type to the pH and temperature of the soil.

One issue that affects the success of bioremediation is low bioavailability of the

compounds in the soil. If these PAHs are not available in the soil for the microorganisms, then

degradation will not occur. This project aims to examine the effect of temperature on the

bioavailability and biodegradation of two PAHs fluoranthene, a 4-ring PAH and benzo[a]pyrene,

a 5-ring PAH. These contaminants were added to soil in a microcosm based experiment and

incubated at different temperatures. The bioavailability and degradation was assessed by gas

chromatography (GC) after 1, 21 and 42 days of incubation. The results obtained in this project

indicated that temperature had an effect on the degradation of these PAHs. However, the

bioavailability of the compound appeared to be largely influenced by the compound itself. The

effect that temperature has on the removal of these compounds must be factored in when

remediation projects are being designed in order to achieve greater levels of success in the

eradication of these toxic compounds from soil.

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Table of Contents 1. Introduction ..................................................................................................................................... 1

1.1 Polycyclic Aromatic Hydrocarbons .............................................................................................. 1

1.2 Bioremediation ........................................................................................................................ 4

1.3 Bioavailability ......................................................................................................................... 5

1.4 Degradation of PAHs .............................................................................................................. 7

1.5 Project Aims.................................................................................................................................. 8

2. Materials and methods .................................................................................................................. 10

2.1 Soil sampling and location .......................................................................................................... 10

2.2 Physicochemical analysis ............................................................................................................ 10

2.3 Microcosm setup and experimental design ................................................................................. 11

2.4 PAH extraction from soil ............................................................................................................ 12

2.5 Gas chromatography analysis of PAHs ...................................................................................... 13

2.6 Dehydrogenase activity ............................................................................................................... 13

2.7 Enumeration of Bacteria (Total heterotrophic counts) ................................................................ 14

2.8 Growth and isolation of PAH degraders ..................................................................................... 14

2.9 DNA Extraction from Soil .......................................................................................................... 15

2.10 Nucleic Acid quantification ...................................................................................................... 16

2.11 Agarose gel electrophoresis ...................................................................................................... 16

2.12 Quantitative real-time PCR (qPCR) .......................................................................................... 17

2.13 Determining bioavailability of fluoranthene and benzo[a]pyrene ............................................ 18

3. Results .......................................................................................................................................... 19

3.1 Determination of the effect of temperature on microbial degradation of PAHs in soil .............. 19

3.2 Determination of microbial activity in the soil ........................................................................... 22

3.3 Enumeration of bacteria in control, fluoranthene and benzo[a]pyrene amended soil ................. 23

3.4 Determination of abundance of Gram positive gene RHDα ....................................................... 26

3.5 Effect of temperature on bioavailability of fluoranthene and benzo[a]pyrene .......................... 29

4. Discussion .................................................................................................................................... 33

5. Bibliography ................................................................................................................................. 42

6. Acknowledgements ....................................................................................................................... 49

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1. Introduction

1.1 Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are a group of compounds consisting of two or

more benzene rings which are fused together linearly, angularly or in a cluster arrangement.

While the simplest form is naphthalene, which consists of two rings, they can comprise up to

6 rings. PAHs are considered to be of high molecular weight (HMW) if they consist of four

or more fused rings (Table 1.1). Generally, PAHs are organic pollutants that are widely

distributed in the environment; they are toxic and very persistent (Cerniglia, 1992).

PAHs are naturally found and arise from sources such as forest fires. However, their

levels have increased over the past 100 to 150 years due to increasing levels of industrial

activity (Wild and Jones, 1995) and, according to Dubey and Narayanan (2010),

anthropogenic sources are now the primary sources of PAHs in atmospheric pollution. For

example, power generation, refuse burning and coke production alone provide 50% of the

annual benzo[a]pyrene emissions, which are widely used as a standard of PAH emissions

(Wick et al, 2011). PAHs arise synthetically due to the incomplete combustion of coal, oil,

gas and garbage (Lu et al, 2008). As a consequence of the location of industries in urban

areas, PAH concentrations can be 10 to 100 times higher in urban industrial soils than in

remote soils (Wild and Jones, 1995). The concentrations and type of PAHs found in soils are

also dependent on the type of industry. Juhasz and Naidu (2000) reported total PAH

concentrations of 5863mg kg-1 at a creosote production site, 18,704mg kg-1 at a wood

preserving site, 821mg kg-1 at a petrochemical site and 451mg kg-1 at a gas manufacturing

plant site. Benzo[a]pyrene is used as a marker in setting air quality and emission standards.

Benzo[a]pyrene is used because it is the most common PAH in ambient air (EPA, 2014).

Creedon et al (2010) reported the principal source of PAHs in air to be combustion of solid

fuels in residential areas with 80% in 1990 and 77% in 2006. Road transport emissions were

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also significant, contributing to 15% in 1990, however by 2006 this had been reduced to 7%.

While there are directives for the limit of PAH concentration in ambient air (2004/107/EC)

and food (2006/1881/EC) the EU currently has no concentration limits set for these PAHs in

soil. Ireland currently follows guidelines set by the Dutch Ministry of Housing, Spatial

planning and the Environment (VROM, 2001) which applies a target concentration of 1mg

total PAHs kg-1 soil with an intervention value of 40mg kg-1 (Storey, 2012)

PAHs are toxic and are considered priority pollutants by the European Union and the

US EPA. There have been studies conducted associating certain cancers with exposure to

PAHs (Samantha et al, 2002). The carcinogenic properties of these compounds are more

closely associated with high molecular weight PAHs (ATSDR, 1995). Studies by Bamforth

and Singleton (2005) have also shown that there are particular PAH metabolites which

interact with DNA and can cause mutations, producing malignancies and heritable genetic

damage in humans. PAHs are not thought to be genotoxic without the activation by

mammalian enzymes. This reaction proceeds via the cytochrome P450 monooxygenase,

oxidizing the aromatic ring to form epoxide and diol-epoxide reactive intermediates. These

intermediates may then undergo one of four different mechanisms of hydrolysis or oxidation

before combining with or attacking DNA to form covalent adducts with the DNA. These

adducts can cause mutations and result in tumours. PAHs have also been shown to have

toxic effects on plants, invertebrates and microorganisms (Winther-Nielson et al, 1997).

A major concern with PAHs is their recalcitrance in the environment. One of the main

causal factors for their persistence in the environment is their low aqueous solubility due to

their hydrophobic properties. PAHs adsorb onto soils and particulates, which influences their

bioavailability and biodegradation (Juhasz and Naidu, 2000). Their fate is mainly dependant

on the molecular weight of each PAH. In a gaseous or water dissolved phase LMW PAHs

can undergo rapid photochemical degradation (Miller and Olejnik, 2001) and, if they become

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attached to particulate matter, they can undergo biological processes leading to their partial or

complete removal from the environment. However, due to their hydrophobic properties,

HMW PAHs become bound to particulate matter and over time become less available to

microorganisms, becoming very difficult to degrade and posing harmful to the environment

(Cerniglia, 1992).

While most PAHs enter the environment via the atmosphere, sediment and soil are the

primary environmental repositories for these compounds (Dabestani and Ivanov, 1999).

Due to the dangers of these compounds, rapid remediation is required (Urgun et al, 2006). As

these compounds can occur naturally in the environment, there are microorganisms which

have the ability to breakdown these carcinogenic contaminants. Due to these microorganisms,

contaminated sites can be remediated through microbial and environmental manipulations,

known as bioremediation (Wick et al, 2011).

Table 1.1 High molecular weight PAHs and their physicochemical properties. (Doyle et al,

2008)

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1.2 Bioremediation

Bioremediation is a method that manipulates biological processes in order to reduce the

concentration of contaminants in the environment (Bamforth and Singleton, 2005). It is

defined as ‘the process whereby organic wastes are biologically degraded under controlled

conditions to an innocuous state’ (Mueller et al, 1997). Bioremediation can occur in situ and

ex situ. The ability to treat soils contaminated by PAHs in situ is of great interest as it is cost

and time effective. A range of bioremediation strategies exist including natural attenuation,

bioaugmentation, phytoremediation and biostimulation. Bioaugmentation is the addition of

known PAH degrading microbial communities to the soil whereas biostimulation and natural

attenuation rely exclusively on the native communities of the soil.

Natural attenuation is solely dependent on the microbes present in the environment

and can be a slow process. Lu et al (2011) reported that most PAH degrading microbes are

naturally present in most environments, meaning that if PAHs are bioavailable, they will be

broken down over time. However, a HMW PAH such a benzo[a]pyrene has been reported to

have a half- life of up to 10 years (Daugherty, 1997) so natural attenuation does not provide

a rapid enough solution.

Phytoremediation employs plant-influenced microbial, chemical, and physical

processes to remediate contaminated soils and is an effective in situ treatment. Denys et al

(2006) conducted a field study over 2 years and reported a 60% degradation in 3- ring PAHs;

however, 5- and 6- rings were more recalcitrant and phytoremediation did not prove effective

for these high molecular weight compounds.

Biostimulation has been shown to be effective in increasing the rate and extent of degradation

of these toxins. Biostimulation includes the addition of fertilizers, aeration techniques and

composting. Nelson et al reported a degradation of 16,144kg of PAH contaminated mass

from a petroleum hydrocarbon site over a period of 30 months with the addition of fertilizer

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into the soil. Civilini (1994) reported a degradation of 81.63% for benzo[a]anthracene and

98.63% for fluorene over a 15 day period with incubation at 45°C with the use of

composting.

Regardless of the remediation strategy used, success is highly influenced by a number

of environmental factors including pH, nutrient requirements of the microbes, the availability

of water and oxygen, PAH bioavailability, salinity, temperature and the adaptation of the

microorganisms population (Balba et al, 1991).

1.3 Bioavailability

According to Semple et al, (2004) bioavailability is defined as ‘that which is freely available

to cross an organism’s cellular membrane from the medium the organism inhabits at a given

time’. Bioavailability greatly controls microbial metabolism and degradation of PAHs in soil.

In order to successfully design a bioremediation process reliable estimates of PAH

bioavailability are extremely important, while also determining the severity of their adverse

effects (Wick et al, 2011). Figure 1.1 displays the unavailable and bioavailable compound in

soil.

Bioavailability of PAHs can be assessed using a number of approaches with the

persulfate oxidation method proposed by Cuypers et al (2000) the most commonly used. The

basis for this test is that when persulfate is added to soil and heated it decomposes and forms

sulfate radicals (SO4-). The sulfate radicals react with the available organic matter and any

sorbed PAHs (Kislenko et al, 1996). The amount of PAH that remains in the soil following

the treatment can then extracted and detected by GC. The amount of PAH extracted is thus a

measure of PAH that is not available for microbial degradation.

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Bioavailability of PAHs is affected by the physical properties of the PAH itself.

PAHs with a lower molecular weight (LMW) are generally thought to be more bioavailable

than higher molecular weights (HMW) and will degrade quicker. HMW PAH compounds

are less bioavailable due to their low solubility and hydrophobicity, whereas LMW PAHs

have higher solubility and volatility (Stokes et al, 2006; and Harmsen 2007). The

bioavailability will also change with time and weathering stage. The PAH will become less

bioavailable as it gets sorbed to the soil by minerals and organic matter (Uyttebroek et al,

2007). Studies have shown that some microorganisms have evolved in order to overcome

decreased bioavailability including the production of biosurfactants. These biosurfactants

increase the PAHs solubility, making it more accessible to microbial degradation (Van Dyke

et al, 1991). Fungi can overcome the issue of bioavailability through the production of

extracellular enzymes and mycelial growth. They have cell bound enzymes with broad

specificity which can detoxify the PAH (Potin et al, 2004).

Figure 1.1 Conceptual diagram of bioavailable and non-bioavailable compound (Adapted

from Okere and Semple, 2012)

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1.4 Degradation of PAHs

There are a number of different organisms able to degrade PAHs including bacteria, fungi

and algae. Bacteria accounts for the largest group of degraders (Cerniglia et al, 1992; Kastner

et al, 1994). There are a number of bacteria genera and species that can degrade 2- or 3-ring

PAHs, but few genera have shown ability to degrade HMW PAHs (Table 1.2). However,

some Pseudomonas species have been found to degrade 4-ring and 5-ring PAHs (Juhasz and

Naidu, 2000). The pathways, enzymes and genes associated with PAH degradation has been

elucidated for pure cultures of bacteria and fungi. However, bacteria have received more

attention (Table 1.2). For bacteria there are two steps involved in the biodegradation of PAHs

in soil. Firstly, the physical uptake of the PAH by a microbial cell and secondly the biological

metabolism of the PAH by the cell. The first step is dependent on the physical availability of

the compound to the cell and highlights the significance of bioavailability for biodegradation.

The second step can proceed only if the microbial cell possesses the ability to degrade the

PAH (Semple et al, 2004). If the microbial cell possesses an adequate metabolic pathway,

then the degradation of the PAH can proceed as long as it is in a form which the organism

can utilise, usually an aqueous phase. Where the PAH is not accessible to the microorganism,

limited biodegradation of the PAH will occur (Semple et al, 2003). Viamajala et al (2007)

found that biodegradation of PAHs is improved when the temperature of a soil environment

is higher. This may be because higher temperature influences solubility of PAHs so they

become more bioavailable. In a study conducted by Trably and Patureau (2006) PAH

contaminated sludge was examined in aerobic bioreactors at different temperatures (35°C,

45°C and 55°C). There was more than an 80% loss observed for the lighter PAHs (fluorene,

phenanthrene and anthracene) at all temperatures but rates were inversely correlated with

increasing molecular weight. The authors attributed this to decreasing bioavailability of the

heavier PAHs which were only degraded by 50%. This study showed that increasing the

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temperature from 25°C to 55°C increased biodegradation of the heavier PAHs to 80% (60%

increase). However, at 55°C high abiotic losses were observed for all PAHs, which was due

to volatilization. Optimal conditions were found to be at 45°C. This study concluded that by

increasing temperatures, bioavailability of PAHs was increased and led to higher

biodegradation.

Fungi are a large component of soil biomass and may also have a considerable effect on PAH

transformation. In comparison to bacteria that use PAHs as a source of carbon and energy,

fungi tend to detoxify PAHs by means of enzymatic oxidation (Johnsen et al, 2005). PAH

metabolites created by fungi are generally more water soluble and chemically reactive,

rendering them more accessible to native soil bacteria. (Potin et al, 2004).

Bacteria genera PAH compounds degraded

Acidovorax phenanthrene, anthracene

Alcaligenes phenanthrene, fluorene, fluoranthene

Arthrobacter benzene, naphthalene, phenanthrene

Mycobacterium phenanthrene, pyrene, benzo[a]pyrene

Pseudomonas Phenanthrene, fluoranthene, fluorine, benzo[a]pyrene

Rhodococcus pyrene, benzo[a]pyrene

Sphingomonas phenanthrene, fluoranthene, anthracene

Table 1.2 Some genera of PAH-degrading microorganisms (adapted from Frick et al, 1999)

1.5 Project Aims

Composting is one of the most successful approaches used to remediate soil contaminated

with HMW PAHS (<4 rings). Kobayashi et al (2009) showed a 73.1% removal of

benzo[a]pyrene after a treatment time of 14 days with the addition of manure compost. The

composting process involves the degradation of organic material by a succession of

microorganisms and is accompanied by changes in temperature. It is not clear if the success

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of composting in the removal of PAHs is due to stimulation of microbial activity or an

increase in bioavailability of PAHs at higher temperatures or the interaction of both.

The overall aim of this project was to examine the effect of temperature on the

bioavailability and degradation of two PAHs; the 4-ring PAH flouranthene and the 5-ring

PAH benzo[a]pyrene in soil. The rate and extent of degradation was examined at 22°C, 45°C

and under changing temperature conditions similar to those observed during a typical

composting process. Bioavailability was also examined using the persulfate oxidation

method. The level of biological activity in the soil at the different temperatures was

determined using dehydrogenase and the number of total culturable and PAH utilizing

bacteria was also assessed. In addition, the effect of temperature on the abundance of a key

functional gene involved in bacterial PAH degradation, a ring hydroxylating dioxygenase

(RHD) was determined using quantitative PCR.

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2. Materials and methods

2.1 Soil sampling and location

Soil was collected from Rosemount on the UCD Belfield Campus. The soils were dug using a

trowel; the top few centimeters were scraped off and the soil collected. These soils were

sieved using a 2.00mm lab test sieve to remove any large particles or stones.

2.2 Physicochemical analysis

2.2.1 Organic matter content

Soil organic matter content was determined by placing the dried soils into a furnace and

burning off all the organic matter. The soil samples were placed in crucibles which were

weighed before placing them into a furnace at 400°C for 24hours and then reweighed. The

difference in weights was calculated as a percentage and this was the organic matter content.

2.2.2 Soil moisture content

Soil moisture content was found by weighing 3 x 5 g samples and then placing them in an

oven overnight to dry at 80°C. The samples were then reweighed and the difference between

the two weights was the moisture content of the soils, which was then calculated as a

percentage. A calculation was then done to determine the amount of water needed to be

added to the soil to ensure the samples were kept at a moisture content of 28%.This was

achieved by watering the soils daily.

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Figure 2.1 Collection site of soil on the UCD campus

2.3 Microcosm setup and experimental design

Fluroanthene (200mg kg-1 dry matter, made up in acetone) was added to the soil in a tray,

which was well mixed into the soil by hand ensuring all clumps of soil were removed and the

soil received an even mix with the fluroanthene. 18 microcosm pots were prepared and

labelled; there were 3 replicates for each soil sample. 9 microcosms of soil contaminated with

fluoranthene were incubated at 22°C and 9 were incubated at 45°C. Samples were taken on

days 1, 21 and 42. These were placed in a freezer at -20°C.

This was repeated with another tray of soil and benzo[a]pyrene (32mg kg-1). 15

microcosm pots were prepared and labelled for benzo[a]pyrene. It was sampled on days 1 and

42, while 3 replicates were sampled on day 42 that had been held under changing temperature

conditions. Control soil was also set up which contained acetone (22mg kg-1). 18 microcosms

were labelled and set up in the same manner as fluoranthene. A sampling pattern of days and

treatments is provided in Table 2.1.

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Treatments Day 1 Day 21 Day 42

Control (no PAH) 45°C X X X

Benzo[a]pyrene 45°C X X

Fluoranthene 45°C X X X

Control(no PAH) 22°C X X X

Benzo[a]pyrene 22°C X X

Fluoranthene 22°C X X X

Benzo[a]pyrene changing temperature X

Table 2.1 Sampling pattern undertaken

2.4 PAH extraction from soil

2.4.1 Shaking method used for fluoranthene

1.2g of soil was weighed into 50mL centrifuge tubes and 120µl of internal standard pyrene

(2000mg 1-1) added. Samples were placed in a fume hood to allow evaporation of acetone for

10 minutes. Then 10mL of acetone was added to each sample. Tubes were capped and placed

on an orbital shaker for 30 minutes at 250 rpm. Subsequently, 10mL of hexane was added to

each sample and were shaken at 250rpm for an additional 30minutes. After that time,

deionised water was added up to 40mL and shaken for an additional 5 minutes at 200rpm to

facilitate separation of the hexane phase. Tubes were left to stand for 5 minutes for soil

contents to settle out, after which time 1mL of the upper hexane phase was collected and

filtered through a 0.2µm polytetrafluoroethylene (PTFE) filter into a labelled gas

chromatography (GC) vial.

2.4.2 Soxhlet extraction used for benzo[a]pyrene

6g of soil sample along with 6g of anhydrous sodium sulphate (Sigma-Aldrich) was weighed

out and 150µl of internal standard pyrene (2000mg l-1) was added. The soil was mixed and

placed in a fume hood for approximately 5 minutes to allow for the evaporation of acetone.

Soil was then transferred to a cellulose extraction thimble (Whatman Ltd.) and placed in a

Soxhlet extraction system (Foss Soxtec Avanti 2055). The PAH was extracted using an

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acetone:hexane mixture (1:1 v/v) under the following conditions: 60 minutes boiling at

135°C; 120 minutes solvent rinsing at 135°C. The soil extract was then evaporated to less

than 10ml, transferred to a volumetric flask and made up to a final volume of 10ml with the

same acetone:hexane mixture. 1ml was removed and filtered through 0.2µm PFTE filter into

a labelled GC vial.

2.5 Gas chromatography analysis of PAHs

Gas chromatography was used to calculate the concentration of PAHs in extracted samples.

The gas chromatographer used in the experiment was an Agilent Technologies 6890N gas

chromatograph coupled with a flame ionization detector (FID). Samples were injected onto a

HP-5 fused polysiloxane capillary column (Agilent J&W) and a temperature gradient of 50°C

to 300°C increasing at 8°C/min was applied. The carrier gas used was He (90 kPa) and make

up of gas was N (100-150 kPa). The injection volume was 1µl with a split flow of 1:10. The

injector temperature was maintained at 250°C and a detector temperature of 300°C was used.

The instrument was calibrated using serial dilutions of known concentrations of PAHs. PAH

concentration was calculated based on peak area and comparison with a standard solution of

the appropriate PAH. For calculation of average PAH concentration, triplicate samples were

used for a given treatment and given sampling day. Moisture content of the soil samples was

taken into account in the calculation of final PAH concentration.

2.6 Dehydrogenase activity

Total microbial activity in soil was assessed utilizing a modification of the Thalmann (1968)

method which is based on the colorimetric estimation of triphenyltetrazolium chloride (TTC)

reduction to triphenylformazan (TPF) in soil after 24 hour incubation at 30°C. A 1g sample

of soil was transferred to a 50mL screw top centrifuge tube and 1ml of TTC buffer was added

(0.8% w/v in Tris-HCl Buffer). TTC buffer was prepared using 0.4g TTC and 50ml tris-HCl.

Samples were then placed on a shaker incubator (150rpm) and incubated in the dark at 30°C

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for 24h. After incubation 8ml acetone (Sigma-Aldrich) was added; tubes were shaken

vigorously by hand and further incubated for 2 hours in the dark at ambient temperature.

Subsequently, tubes were centrifuged at 4000rpm for 3 minutes and 1ml of the top layer of

this mixture was then transferred into a cuvette. Absorbance was read at OD546nm.

Dehydrogenase activity was calculated against a standard curve of TPF (Sigma-Aldrich) and

expressed as µg TPF in 1g of dry weight of soil.

2.7 Enumeration of Bacteria (Total heterotrophic counts)

Plate counts of the soils were undertaken by mixing 1g of soil sample with 9ml of sterile

ringers solution in a test tube inside a laminar flow cabinet. This was the 10-1 dilution. 1ml of

this was then pipetted to another test tube containing 9ml of ringers solution, which was the

10-2 dilution. This step was repeated to give the 10-3, 10-4 and 10-5 dilutions. 20g of nutrient

agar was added to 700ml of deionised water in a 1 litre duran bottle. This was then placed on

the stirrer and heated to mix thoroughly prior to autoclaving at 121°C and 15 lb/in2 for 2

hours. Subsequently, 3.5ml of cycloheximide was filtered into the bottle. Plates were poured

in the laminar flow cabinet. 0.1ml of the dilutions from 10-3-10-5 of each soil sample were

spread (in replicates of 3) onto the surface of the nutrient agar plates. Plates were then placed

in an incubator at the temperature of the original microcosm. Total growth was observed after

seven days of incubation and bacterial colony forming units were recorded.

2.8 Growth and isolation of PAH degraders

2.6 g of Bushnell Haas agar was added to 800ml of deionised water in a 1 litre duran bottle. 2

bottles were prepared and autoclaved at 121°C and 15lb/ in2 for 2 hours. 4ml of

cycloheximide was filtered into each bottle in the laminar flow cabinet. 16ml fluoranthene

was added to one bottle, and 10ml of benzo[a]pyrene was added to the second bottle. Plates

were then poured. 5g soil was then added to 95ml ringers and serial dilutions made of both

benzo[a]pyrene and fluoranthene amended soil. Dilutions were made up to 10-5. 0.1ml of the

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dilutions from 10-3-10-5 of each soil sample were spread (in replicates of 3) onto either the

fluoranthene or benzo[a]pyrenesupplemented plates. Plates were incubated for 1 week at

22°C or 45°C depending on their prior incubation period.

2.9 DNA Extraction from Soil

DNA was extracted from the soil using a modification of the Griffiths method (2000). All

materials were sterilized by autoclave. 0.5g soil was added to a 2ml polyethylene micro-

centrifuge tube containing 0.5g of sterile 0.1mm glass beads and 0.5g of sterile 0.5mm

zirconia beads. 0.5ml of CTAB (hexadecyltri-methyl ammonium bromide, Sigma-Aldrich)

extraction buffer was also added to each tube. The tubes were firmly capped and centrifuged

at 3,000rcf for 10 seconds at 4°C followed by 10 minute incubation in a water bath at 70°C.

After incubation, 0.5ml of phenol:chlorophorm:isoamyl alcohol (Sigma-Aldrich 25:24:1

v/v/v) was added, tubes were placed in a Hybaid Ribolyser and shaken at 5.5m/s for 30

seconds. After bead, beating tubes were centrifuged at 16,000rcf for 5 minutes at 4°C and the

upper aqueous layer transferred to a clean sterile Eppendorf tube. Residual phenol was

removed by addition of an equal volume of chlorophorm:isoamyl (Sigma-Aldrich, 24:1 v/v)

and centrifugation at 16000rcf for 1 minute. This treatment was repeated twice. Subsequently

1μl glycogen (Roche), 60μl of 3M sodium acetate (Sigma-Aldrich) and 1ml of 95% ice cold

ethanol were added to each tube to precipitate DNA. Overnight incubation at -20°C increased

the yield of precipitated DNA. Following incubation, precipitated DNA was centrifuged at

15,000rcf for 15 minutes at 4°C. The resulting pellet was rinsed in 70% ethanol and

centrifuged at 15,000rcf for 15 minutes, this step was then repeated. DNA pellets were dried

in a vacuum and re-suspended in sterile deionized water. DNA extracts were purified using

High Pure PCR Product Purification Kit according to the manufacturer’s instructions.

Purified DNA samples were quantified using a Nanodrop ND-1000 spectrophotometer

(Thermo Scientific) as described in Section 2.10. DNA extracts were diluted to a final

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concentration of 20ng µl-1. For quantitative PCR (qPCR) DNA templates were diluted to a

final concentration of 15ng µl-1. All prepared DNA templates were checked for DNA

concentration and stored at -20°C.

2.10 Nucleic Acid quantification

Purified nucleic acids (2 µl) were quantified using a NanodropTM ND-1000 (Thermo

Scientific) at a wavelength (λ) 260nm. The 260/280 nm ratio of a sample was used to assess

the quality of the DNA present, with values of ~1.8 accepted as “pure” and values within the

range of 1.0-2.2 considered acceptable for further use. It should be noted that some degree of

caution must be exercised when using readings from a spectrometer as readings usually do

not entirely discriminate nucleic acids from other contaminants, in particular humic acids.

Therefore, known concentrations of nucleic acid (based on spectrometer readings) were run

on 1% agarose gel with reference to a size ladder of known concentration to confirm

spectrometer readings.

2.11 Agarose gel electrophoresis

The presence of nucleic acids was confirmed by agarose gel electrophoresis. Agarose (Roche

Diagnostics) gels were prepared in 1xTAE buffer (40mM Tris, 20mM acetate 2mM EDTA,

pH 8.0). Concentrations of 1.2% (w/v) were used. Ethidium bromide was added at a

concentration of 0.25µg ml-1. Wells were loaded with DNA samples and a molecular weight

DNA ladder was included in each run. DNA samples were prepared by mixing with an equal

volume of loading buffer (20mM EDTA, 50% (v/v) glycerol, 0.05% (w/v) bromophenol

blue). Gel electrophoresis was carried out at 85V in 1xTAE running buffer for 25 minutes

using Bio-Rad Powerpack 300. Gels were visualized using UV illuminator (UVP,

Cambridge) and images captured and stored with ImageStore 5000 Gel Documentation

software.

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2.12 Quantitative real-time PCR (qPCR)

2.12.1 Standards for real-time PCR calibration

Alpha (α) subunits of a typical PAH ring hydroxylating dioxygenase from Gram positive

Rhodococcus ruber were amplified using PAH-RHDα GP primers previously by Fengxiao

Zhu (personal communication).

2.12.2 Quatitative PCR assay

Quantitative PCR (qPCR) was carried out in a 12.5µl reaction volume containing 6.25µl of

LightCycler®FastStart DNA MasterPLUS SYBR Green I mix (Roche), 0.25µM of each primer,

0.25µl ROX-low and 1µl of DNA template at a concentration of 15ng µl-1 . The reaction

mixture was brought up to a final volume of 12.5µl with sterile nucleotide free water

provided within kit. Reactions were performed in Lightcycler® capillary tubes. Amplification

was carried out in a Lightcycler® Carousel-Based System using the following temperature

profiles: 5 min of denaturation at 95°C followed with 50 cycles of 4 steps. These steps were:

30s of denaturation at 95°C, 30s of annealing at temperatures of 54°C and 30s of elongation

at 72°C. The intensity of SYBR Green-DNA complexes was measured during 10s step at

80°C to facilitate primer dimer dissociation. The final step consisted of 7 min at 72°C.

Melting curve analysis was performed at the end of the run by measuring the SYBR Green

signal intensity during 0.5°C temperature increment every 10s, from 51°C to 95°C. The

presence of PCR products of appropriate size were confirmed on an ethidium bromide stained

1% (w/v) agarose gel. Standard curves were included in each experiment. Cycle threshold

values were determined using the second derivative function. All analyses were performed

using Lightcycler Relative Quantification Software version 1.0 (Roche, UK).

18

2.13 Determining bioavailability of fluoranthene and benzo[a]pyrene

Bioavailability was determined by use of the potassium persulfate oxidation method proposed

in Cuypers et al (2002). 1.25g of soil was weighed into a 50mL centrifuge tube. Potassium

persulfate (K2S2O8) and deionised water was added to give 12 g persulfate per gram of

organic matter and an aqueous persulfate concentration of 0.0357 g/ml. The tubes were

placed in a water bath at 70°C and 120rpm for 3 hours and also shaken by hand every hour

after which time a reddish/brown colour could be observed (Figure 2.2). The samples were

then centrifuged for 3min at 3000rcf. The supernatant was discarded and the remaining

sludge was incubated at 45°C for 2 hours. The weight of the soil was recorded and PAHs

were extracted as detailed in Section 2.4.

Figure 2.2 Before (left) and after (right) pictures of persulfate oxidation of expanded organic

matter in the soil. A reddish/brown colour can be seen after the oxidation has taken place.

19

3. Results

3.1 Determination of the effect of temperature on microbial degradation of PAHs in soil

Soil was amended with fluoranthene (200 mg kg-1) and incubated at 220C and 450C in a

microcosm based experiment as outlined in Section 2.3. Individual microcosm pots were

destructively sampled on days 1, 21 and day 42 and the level of fluoranthene determined.

Fluoranthene was extracted from the soil in triplicate and quantified by GC-FID (Section 2.4-

2.5). A typical GC chromatogram is shown in Figure 3.1.

By day 21, 21% and 33% fluoranthene had been degraded at 220C and 450C,

respectively and no significant difference (P<0.01) in degradation was observed at the

different temperatures (Figure 3.2). By day 42 however, almost all the fluoranthene had been

removed, with only 3% remaining in the soil incubated at 220C while 40% still remained at

450C and these levels were significantly different (P<0.001). Previous studies have shown

that no degradation was observed in an abiotic control (Fengxiao Zhu, personal

communication).

Figure 3.1 Typical GC output for PAHs and their retention times analysed using an Agilent

Technologies 6890N GC with a flame ionization detector (Adapted from Storey, 2012).

20

Figure 3.2 Level of fluoranthene remaining in soil after 1, 21 and 42 days at different

incubation temperatures. Columns represent the mean of 3 replicates with error bars

representing standard error of the mean. Columns with different letters are significantly from

each other at P <0.05.

Having seen the effect of temperature on the 4-ring PAH fluoranthene, soil was then

amended with benzo[a]pyrene (32 mg kg-1) and incubated at 220C, 450C and a changing

temperature regime designed to simulate conditions occurring during composting. The

degradation of benzo[a]pyrene was then examined. Individual microcosm pots were

destructively sampled on days 1 and 42. Day 21 was not included in this experiment as

previous results had shown that degradation of benzo[a]pyrene was slower than fluoranthene

and degradation would not be expected at this time point. As benzo[a]pyrene is very

hydrophobic, with a half-life of up to 10 years, and known to be difficult to extract from a

complex environment such as soil, the first experiment undertaken was to determine the

extraction efficiency of the Soxhlet and shaking extraction methods employed in this study.

Although the shaking method successfully extracted 94% of fluoranthene added to soil, this

method was only capable of removing 43% of benzo[a]pyrene (Table 3.1). However, the

21

more vigorous Soxhlet method removed 75% benzo[a]pyrene, and this method was used for

all subsequent extractions of this compound.

Benzo[a]pyrene (32 mg kg-1) was added to soil in acetone and left for 1 day at the

different temperatures prior to extraction. 75% of benzo[a]pyrene was recovered from soil

incubated under the 3 different temperature regimes and no significant difference was

observed between the replicates (Fig 3.3). After day 42, benzo[a]pyrene levels had decreased

to 20.5mg kg-1 in soils incubated at 220C but these levels were not significantly different to

day 1. However, the levels remaining in soil incubated at 450C and under changing

temperature conditions were significantly different from those present initially (P=0.0025).

On day 42 benzo[a]pyrene had decreased from 32mg kg-1 to 13.8mg kg-1 in soil incubated at

450C and to 14.7mg kg-1 in the soil incubated under changing temperatures conditions. Levels

of benzo[a]pyrene in soil on day 42 were not significantly different from each other

regardless of incubation conditions.

Extraction efficiency (%)

Method

Fluoranthene Benzo[a]pyrene

200mg kg-1 32mg kg-1

Soxhlet continuous

extraction

- 75%

Shaking extraction 94% 43%

Table 3.1 Comparison of extraction efficiencies of two methods used during the experiment.

Each value represents a mean of three measurements.

22

Figure 3.3 Level of benzo[a]pyrene remaining in soil after days 1 and 42 at different

incubation temperatures. Columns represent the mean of 3 replicates with error bars

representing standard error of the mean. Columns with different letters are significantly from

each other at P <0.05.

3.2 Determination of microbial activity in the soil

Biological activity in the PAH amended soils was compared at the end of the experiment by

measuring dehydrogenase activity (Figure 3.4). Dehydrogenase activity was highest in soils

incubated at 220C and no significant difference was observed in the level of activity observed

in unamended, or fluoranthene and benzo[a]pyrene amended soils. At 450C the

benzo[a]pyrene amended soil had significantly higher (P<0.0001) levels of dehydrogenase

activity compared to the unamended control and fluoranthene amended soils.

23

Figure 3.4 Dehydrogenase activity of control, fluoranthene and benzo[a]pyreneamended

soils at 220C and 450C. Columns represent the mean of 3 replicates with error bars

representing standard error of the mean. Columns with different letters are significantly from

each other at P <0.05.

3.3 Enumeration of bacteria in control, fluoranthene and benzo[a]pyrene amended soil

Total heterotrophic and PAH utilizing bacteria in the soils incubated under the different

temperature regimes after 42 days were then determined using serial dilutions and a spread

plate technique (Section 2.7). The plates were then incubated at the same temperature as the

original microcosm.

The total heterotrophic counts are displayed in Figure 3.5. When soil was incubated

at 220C there was no significant difference observed in the number of heterotrophic bacteria

in unamended soil or soil containing fluoranthene or benzo[a]pyrene. A similar result was

observed when soils were incubated at 450C. The only significant difference observed in the

number of heterotrophic bacteria was between fluoranthene amended soil at 220C and

unamended soil at 450C (P <0.05). Some of the growth observed during the incubation period

is displayed in Figure 3.6.

24

Figure 3.5 Enumeration of culturable microorganisms using spread plate technique. Columns

represent the mean of 3 replicates with error bars representing standard error of the mean.

Columns with different letters are significantly from each other at P <0.05.

Figure 3.6 Fluoranthene on the left and benzo[a]pyrene on right. Growth observed after two

days incubation at 45°C.

PAH utilizing bacteria were enumerated using a Bushnell Haas minimal medium

(Section 2.8). Appropriate dilutions of soil from fluoranthene and benzo[a]pyrene amended

microcosms were inoculated onto plates of minimal medium supplemented with the same

PAH used to amend the soil. For example, fluoranthene amended soil was plated on minimal

medium + fluoranthene whereas soil from the benzo[a]pyrene microcosms were plated on

minimal medium + benzo[a]pyrene. Fluoranthene and benzo[a]pyrene utilizing bacteria were

also enumerated in the unamended control soil by plating this out on minimal medium +

25

fluoranthene and minimal medium + benzo[a]pyrene plates. Plates were then incubated at the

same temperature as the original microcosm.

The results, shown in Figure 3.7a displayed no significant differences within the

treatment levels. However, there was a significant difference between the controls and the

treatments (P<0.005). The number of PAH utilising bacteria in both fluoranthene amended

soils at the two incubation temperatures were significantly lower than those detected in the

unamended control soils. While the number of PAH utilising bacteria shown in Figure 3.7b

displayed no significant differences between the unamended control and benzo[a]pyrene and

there was also no significant difference observed within the levels (P>0.3141). Some of the

growth observed during the incubation period is displayed in Figure 3.8.

(a) (b)

Figure 3.7 Number of PAH utilizing bacteria enumerated at different incubation

temperatures in (a) unamended control and fluoranthene on minimal medium plates

supplemented with fluroanthene and (b)unamended control and benzo[a]pyrene on minimal

medium plates supplemented with benzo[a]pyrene. Columns represent the mean of 3

replicates with error bars representing standard error of the mean. Columns with different

letters are significantly from each other at P <0.05.

26

(a) (b)

Figure 3.8 Growth observed after 7 days full incubation. Figure a) displays growth of

fluoranthene amended soil on fluoranthene plate at 45°C. Figure b) displays growth of

benzo[a]pyrene amended soil on benzo[a]pyrene plate at 45°C.

It was noted that there appeared to be growth of white rot fungi present on the

benzo[a]pyrene soil that had been incubated under the changing temperature regime (Figure

3.9). However, no further studies were conducted to determine the type of fungal growth

present.

Figure 3.9 White rot observed on the benzo[a]pyrene soil incubated under changing

temperature conditions.

3.4 Determination of abundance of Gram positive gene RHDα

The first step of aerobic PAH degradation is typically catalysed by ring hydroxylating

dioxygenases/monooxygenases. Quantitative PCR was carried out to determine the

abundance of the α-subunit of a ring hydroxylating dioxygenase (PAH RHDα) gene

27

associated with Gram positive bacteria. DNA was extracted in triplicate from the 51

microcosms using a modification of the Griffiths method and the concentration of DNA

quantified using a nanodrop (Section 2.9). DNA was then purified and the concentration of

DNA determined again. Concentrations of DNA varied from 300ngμl-1 pre clean-up to

15ngμl-1 post clean-up (Table 3.2). These samples were then amplified using PCR and run on

an agarose gel to confirm the presence of the target plasmid (Section 2.11). A sample of a gel

displaying the target plasmid is shown in Figure 3.10.

Copy numbers of the gene varied from 7,027 per ng DNA in fluoranthene amended

soil on day 1 at 22°C to 18,755 per ng in fluoranthene amended soil on day 42 at 22°C

(Figure 3.11a). However, the only significant differences observed were among the

unamended controls (P=0.0033). Figure 3.11b displays the gene abundance in

benzo[a]pyrene amended and control soils, and there were no significant differences observed

in these samples apart from the day 42 control (P=0.0048).

Samples at 45oC Pre clean up ng ul-1 Post clean up ng ul-1

BaP R1 D42 300 15

BaP R2 D42 278 14.9

BaP R3 D42 178 15

Flu R1 D42 250 15.8

Flu R2 D42 287 15

Flu R3 D42 216 15.4

Table 3.2 Typical DNA concentration pre and post clean-up. Final concentrations were

aimed to be at 15ng ul-1

28

Figure 3.10 Agarose gel of samples from the various microcosms for determination of gene

presence using PCR. Lanes 1-10 samples and positive and negative controls.

(a)

(b)

Figure 3.11 RHDα gene copy numbers in Gram positive bacteria in a) fluoranthene

amended soil at temperatures 22°C and 45°C on days 1, 21 and 42 and b) benzo[a]pyrene

amended soil at temperatures 22°C and 45°C on days 1 and 42. Columns represent the mean

of 3 replicates with error bars representing standard error of the mean. Columns with

different letters are significantly from each other at P <0.05.

29

3.5 Effect of temperature on bioavailability of fluoranthene and benzo[a]pyrene

As fluoranthene and benzo[a]pyrene are known to adsorb to components in soil, the

bioavailability of these PAHs at the different temperatures was assessed over time using

persulfate oxidation method as described in (Section 2.13). Potassium persulfate (K2S2O8)

and deionised water were added to soil and tubes were placed in a water bath at 70°C and

120rpm for 3 hours, shaken by hand every hour after which time a reddish/brown colour

could be observed (Fig. 2.2).

On day 1 when no degradation had occurred, 10% and 19% fluoranthene was

sequestered at 22°C and 45°C, respectively, indicating that 89.7% and 80.6% was available

for microbial degradation (Fig. 3.12a). By day 21, degradation 79% of the compound

remained in the soil at 22°C, and 16% of this was sequestered while 62% was bioavailable.

At 45°C, 66% of compound remained in the soil, 24% of which was sequestered and 41%

bioavailable (Fig 3.7b). By day 42, only 3% of fluoranthene remained in the soil at 22°C of

which 1.5% was sequestered and 1.5% bioavailable. 40% remained in the soil incubated at

45°C of which 20% was sequestered and 20% bioavailable (Fig 3.7c).

(a) (b)

0

20

40

60

80

100

120

140

22°C 45°C

Flu

Per

cen

tage

(%)

bioavailable

sequestration

0

20

40

60

80

100

120

22°C 45°C

Flu

Per

cen

tage

(%)

bioavailable

sequestration

30

(c)

Figure 3.12 The percentage of sequestered and bioavailable fluoranthene at 22°C and 45°C

on days (a) 1, (b) 21 and (c) 42. Columns represent the mean of 3 replicates with error bars

representing standard error of the mean.

Benzo[a]pyrene is considered more hydrophobic and recalcitrant than fluoranthene due to its

higher molecular weight. Similar to fluoranthene, the bioavailability of benzo[a]pyrene was

assessed through persulfate oxidation and the results showed no significant differences at the

different incubation temperatures. On day 1 there was 6% sequestration occurring at 45°C in

comparison to 9.5% at 22°C and the changing temperature regime (Fig 3.8a). However, on

day 42 there was 6.4% sequestered at 45°C which was slightly higher than the levels of

sequestration at 22°C (4.9%) and the changing temperature regime (4.5%) (Fig 3.8b).

(a)

0

20

40

60

80

100

120

22°C 45°C

Flu

Per

cen

tage

(%)

bioavailable

sequestration

0.0

20.0

40.0

60.0

80.0

100.0

120.0

22°C 45°C Changingtemp

BaP

Per

cen

tgae

(%)

bioavailable

sequestration

31

(b)

Figure 3.13 The percentage of sequestered and bioavailable benzo[a]pyrene at 22°C, 45°C

and changing temperature on day (a) 1 and (b) 42. Columns represent the mean of 3

replicates with error bars representing standard error of the mean.

The amount of the two PAHs that were bioavailable, sequestered and degraded is summarised

in Figures 3.14a and 3.14b. For fluoranthene, on day 42 at 22°C there was a large amount of

degradation and a small portion of the compound is sequestered in comparison to 45°C where

there is a larger portion of the compound sequestered and not as much evidence of

degradation (Fig 3.14a).

On day 42 for benzo[a]pyrene, there was little degradation observed at 22°C.

Although levels of sequestration in all 3 incubation temperatures appear low, the soils

incubated at 45°C and changing temperature displayed a much higher level of degradation

compared to the soil incubated at 22°C (Fig 3.14b).

0.0

20.0

40.0

60.0

80.0

100.0

120.0

22°C 45°C Changingtemp

BaP

Per

cen

tgae

(%)

bioavailable

sequestration

32

(a)

(b)

Figure 3.14 Summary graph displaying the percentage level of sequestration,

bioavailability and degradation as a percentage of the total amount of PAH added on day 1 of

(a) fluoranthene day 42 and (b) benzo[a]pyrene day 42.Columns represent the mean of 3

replicates with error bars representing standard error of the mean.

0

20

40

60

80

100

120

22°C 45°C

Flu

Per

cetn

age(

%)

degradation

bioavailable

sequestration

0.0

20.0

40.0

60.0

80.0

100.0

120.0

22°C 45°C Changingtemp

Bap

Per

cen

tage

(%)

degradation

bioavailable

sequestration

33

4. Discussion

Environmental pollution is a worldwide problem. Although every country emits a diverse

range of chemicals in different quantities, the main driving factors behind these emissions

remain the same. These factors include industrialization, urbanization and population growth

(Philip et al, 2005). Contamination with organic compounds such as PAHs occurs worldwide

due to both anthropogenic and natural sources. While natural sources include the incomplete

combustion of organic matter from volcanoes, waste incineration and forest fires (Doyle et al,

2008), they contribute lesser amounts than their counterpart. Anthropogenic sources of PAH

pollution are the largest contributor and include oil and petrochemical production as the main

industrial sources (Dubey and Narayanan, 2010). These contaminations can occur due to

spillages which have taken place either accidentally or intentionally.

Although bioremediation is considered to have considerable potential for the

remediation of contaminated sites, its effectiveness is influenced by many different factors

making it difficult to find one bioremediation strategy effective for all PAH contaminated

sites. As well as factors such as soil pH, moisture and organic matter content and temperature

influencing the success in the remediation of these chemicals the compound itself also plays a

large role. Characteristics such as melting and boiling point, thermodynamic stability due to

negative resonance energy as well as hydrophobicity and low vapour pressure all impact the

outcome of bioremediation (Juhasz and Naidu, 2000). As the molecular weight of PAHs

increases so does their hydrophobicity, recalcitrance and toxicity (Urgun et al, 2006). Thus,

lower molecular weight PAHs are easier to remove and are less likely to accumulate in

comparison to higher molecular weight PAHs. As bioavailability is a prerequisite for the

remediation of these soils, these PAH characteristics can greatly affect this process

(Cerniglia, 1992). If a compound is sequestered and unavailable to microbes, limited

34

degradation will occur. This study examined the effect of temperature on the bioavailability

and biodegradation of fluoranthene and benzo[a]pyrene in soil using a microcosm based

approach. Microcosms have enabled scientists to investigate soil ecology in less complex

ecosystems than in natural ones and have become a major research tool in recent decades

(Kampichler et al, 2001). There are some limitations and disadvantages attached to

microcosm experiments such as their distance from reality and scale problems (Lukkari et al,

2006). However, a comparative study recently conducted by Tingey et al (2008) provided

evidence that results from microcosms displayed similar patterns to field experiments.

Fluoranthene is a 4-ringed PAH that occurs as a natural component of fossil fuels and

is also formed during their combustion (Mitra et al, 2013). It appears as light yellow needles

with a melting point of 111°C and a solubility 0.26mg l-1. While benzo[a]pyrene is a 5-ringed

PAH which appears as yellow crystals, it has a melting point of 179°C and a solubility of

0.0038mg l-1 (Doyle et al, 2008). It derives mainly from wood burning, coal tar and

automobile exhaust fumes. These two PAHs were chosen because both are considered

priority pollutants (Samantha et al, 2002) and previous studies have shown that both

compounds, particularly benzo[a]pyrene, represent a significant challenge to soil microbes.

Degradation occurs within a reasonable time frame allowing them to be studied within the

time constraints of the current study. Sawulski et al (2014) recorded 90% removal of

fluoranthene after 20 days. While the same study noted less than 20% removal of the initial

concentration of benzo[a]pyrene after 20 days, 42 days would be sufficient to see a certain

level of degradation.

All the soil used in this experiment was taken from the same area of UCD and

therefore the microbial composition, pH, organic matter, nutrient content and porosity were

identical. It was understood that this soil had no previous exposure to PAH contamination.

However, during the course of this study results indicated that this soil may have been

35

previously exposed to PAHs. Evidence of this appeared in the results of the unamended

controls in which there appeared to be high RHDα gene copy numbers which were not

significantly different from soils amended with fluoranthene and benzo[a]pyrene.

In this study, incubation temperatures of 220C and 450C were used. These

temperatures were on either side of reported optimal degradation temperatures and this was

done in order to observe the differences between the two temperatures. Some studies have

shown optimal temperatures of PAH degradation to occur at 300C (Bishnoi et al, 2008) while

Trably and Patureau (2006) suggest an optimal temperature of 450C. These authors have also

reported that volatilization of these compounds occurs at 550C. During this experiment there

was also a triplicate of benzo[a]pyrene incubated under a changing temperature regime. This

changing temperature was designed to simulate the changing temperature which occurs

during the lifecycle of compost. The changing temperature regime occurred over the course

of 42 days. It began at 220C and gradually increased to 480C. The temperature remained at

480C for fifteen days and was then incrementally returned to 220C at which it remained at for

the last seven days. Although compost can reach temperatures of up to 650C (Jenkins, 2012),

these temperatures were not used in this study in order to prevent volatilization.

The addition of compost to contaminated soil has been used for remediation of soil

polluted with a variety of chemicals (Yuan et al, 2009). It is not clear if the success of the

composting process is due to stimulation of microbial activity or an increase in bioavailability

of PAHs at higher temperatures or the interaction of both. Courtney and Mullen (2008) report

high nutrient content and the ability to increase aeration as factors which contribute to

success in the remediation of contaminated soils. However, as the aim of this study was to

examine the effect of the changing temperature, soil was not amended with compost, only

changing temperature was simulated. While the results obtained in this study indicated that

soil incubated at 45°C and changing temperature had a significantly higher level of

36

degradation in comparison to soil incubated at 22°C (P=0.0025), there appeared to be no

differences in the bioavailability of benzo[a]pyrene at these different temperatures.

Wu et al (2014) investigated the effect of multiple factors affecting the

bioavailability of PAHs in compost amended contaminated soil. These factors included soil

type, compost type, contact time and the ratio of compost addition. However, results showed

that only soil type and contact appeared to have significant effects. HMW PAHs also became

more sequestered in comparison to LMW. Although, when LMW PAHs were also present

with HMW PAHs, competitive sorption occurred during the initial stages of incubation,

enhanced by compost addition. This resulted in less sequestration of HMW PAHs.

Interestingly, previous studies have indicated that naphthalene increased the degradation of

PAHs with a greater ring number (Couling et al, 2010). A further study involving the

addition of compost and a LMW PAH such as naphthalene to examine the effect of

degradation on the benzo[a]pyrene amended soil would be interesting to investigate. These

studies indicate how diverse the effects of composting can be. It is crucial that further study is

conducted to examine the effect of compost on the removal of these PAHs before using this

remediation strategy in situ. Risk assessment must also be conducted as adding and mixing

non-contaminated compost with contaminated soil would result in a far greater quantity of

contaminated material (Semple et al, 2001).

There was a significantly higher level of degradation observed in the fluoranthene amended

soil in comparison to the benzo[a]pyrene amended soil. This result was not unexpected as

fluoranthene has a lower molecular weight than benzo[a]pyrene and is therefore degraded

more easily. A similar result was observed in a previous study by Sawulski et al (2014). After

20 days incubation, this study recorded a 90% removal of fluoranthene, while there was only

a 20% removal of the initial concentration of benzo[a]pyrene. Limiting factors for the

degradation of benzo[a]pyrene are physical, chemical and environmental. While it may be

37

degraded it usually occurs via co-oxidation or co-metabolism through less recalcitrant

compounds (Couling et al, 2010). Subsequently, benzo[a]pyrene may not be transported into

the cell or may not be an inducer for transport of degradative enzymes (Pinyakong et al,

2003). However, where these factors are not limiting, the bioavailability will decrease the

rate and extent of degradation (Juhasz Naidu 2000). Benzo[a]pyrene adsorbs or covalently

attaches to organic matter which restricts degradation (Pignatello and Xing, 1996).

Interestingly, Erickson et al., (1993) reported a decrease in sorption of HMW with increasing

contact time in soils. Had this study been conducted for longer there may have been a

decrease observed in the sequestration and an increase in bioavailability over time. However,

it appears that without an additional carbon source or presence of a LMW PAH the extent of

degradation may not have increased.

The measurement of dehydrogenase can be used to assess the effect of soil

amendments on the biological activity in the soil (Margesin et al, 2003). Significantly higher

levels of dehydrogenase activity was observed after 42 days at 22° in all soils (P<0.0001).

For fluoranthene the higher levels of activity observed at 22°C may reflect the fact that

degradation of the PAH was occurring rapidly and may explain why degradation at 45°C was

significantly lower. For benzo[a]pyrene dehydrogenase activity was highest at 22°C;

however, at 45°C activity in benzo[a]pyrene amended soil was also significantly higher than

the control and fluoranthene amended soil. The changing temperature regime involved eight

days at 22°C and four days at 45°C, which may explain the higher level of degradation in the

changing temperature regime. The level of activity also suggests that overall these PAHs did

not have a toxic effect. However, this may not be true for all organisms present in the soil,

which may have affected the degradation rates (Weissenfiels et al, 1992).

While the dehydrogenase tests provided information on the biological activity in the

soil, culture work was also carried out to examine the level of culturable bacteria present.

38

Although there are limitations associated with culture-based techniques, including the fact

that a large percentage of microorganisms are not easily cultivated (Kirk et al, 2004), and the

microorganisms that are successfully cultured may not represent the microorganisms which

are of importance in an environmental context (Nichols, 2007), it is still useful to see whether

there are differences between treatments to give us an insight into possible toxicity. There

was no significant difference in the total bacteria enumerated at 22°C in comparison to 45°C.

Interestingly, when PAH utilizing bacteria were cultured, the unamended controls were

significantly higher in comparison to the fluoranthene amended soils at both temperatures (P

<0.05). This result may indicate that fluoranthene has a toxic effect on certain microbial

populations in the soil.

It must be noted that the concentration of benzo[a]pyrene on the minimal medium

plates were lower than fluoranthene due to its toxicity. However, in comparison to

fluoranthene, benzo[a]pyrene displayed no significant differences between 22°C and 45°C or

between the unamended controls when PAH utilizing bacteria were cultured. Previous

studies conducted by Storey (2012) successfully cultured bacteria on minimal medium and

benzo[a]pyrene. However, when these colonies were isolated, it was found that they were

not capable of degrading benzo[a]pyrene. This implies that small amounts of nutrients were

likely transferred from the enrichment culture and that these bacteria were capable of

withstanding the relatively low concentration of benzo[a]pyrene present. To date, only one

known bacterial species isolated Mycobacterium vanbaalenii PYR-1 is capable of degrading

benzo[a]pyrene (Heitkamp et al, 1988; Kanaly and Harayama, 2010).

While the agar plates were supplemented with cycloheximide to prevent fungal

growth there was a white rot observed on the benzo[a]pyrene amended soil under changing

temperature (Fig 3.10). Fungi have been previously suggested to play an important role in the

initial attack of high molecular weight PAHs, producing metabolites which then become

39

available as substrates for bacteria (Sack et al, 1997). While no further studies were

conducted to identify this fungal growth, many studies have been conducted on the

degradation of benzo[a]pyrene by the presence of fungus. One group of white-rot fungus that

has been previously investigated for the degradation of PAHs is Phanerochaete

chyrsosporium, which produce lignin peroxidises or ligninases. While it is reported that this

fungus can grow within a wide temperature range, optimal temperatures have been reported

at 39°C (Cookson, 1995). Additionally, the optimal moisture content is reported to be 40-

45% (EPA, 1999). While this study maintained the soil moisture content at 28%, it would

have been interesting to see the results had another triplicate been kept under the same

conditions with a higher moisture content. Previous studies have shown fungal communities

respond more rapidly to presence of benzo[a]pyrene than bacterial communities (Sawulski et

al, 2014) and this could be due to the non-specific nature of fungal PAH degradation

(Winquist et al, 2014). From this it may suggest that more fungal than bacterial degradation

occurred which would also explain the slow rate of degradation as fungal degradation is

thought to be slower than bacterial degradation (Field and De Jong 1992; Barr and Aust

1994).

The first step of aerobic PAH degradation is typically catalysed by ring hydroxylating

dioxygenases/monooxygenases (Jouanneau et al, 2011). Quantitative PCR was carried out in

order to determine the abundance of the α-subunit of a ring hydroxylating dioxygenase (PAH

RHDα) gene copy numbers from Gram positive bacterial populations. Although studies have

reported high levels of the RHD gene from Gram negative bacteria in soils, many studies

have reported that it is the gene associated with Gram positive bacteria that appears to

respond significantly during PAH degradation, in particular Actinobacteria (Sawulski et al,

2014).

40

While gene copy numbers in fluoranthene displayed an increasing trend in the soil

incubated at 22°C from day 1 to day 42, no significant differences were observed (P <0.05).

Although studies conducted by Sawulski et al (2014) were over a 20 day period, a similar

increasing trend was observed. The RHD gene abundance in the benzo[a]pyrene amended

soil remained constant throughout all treatments and no significant differences were

observed. This result is similar to findings in previous studies and it appears that

benzo[a]pyrene does not have a significant effect on the presence of this gene (Sawulski et al,

2014). However, only one known PAH degrading gene was examined in this project, Cao et

al (2015) predicts that there are one hundred and thirty-eight genes involved in the

degradation of PAHs, including 14 dioxygenase genes. It must be noted that molecular based

approaches do have intrinsic biases including the vital PCR step which may alter the true

microbial community structure (Pinto et al, 2012). However, as all samples in this study were

prepared in a similar manner, equivalent biases would have been introduced to all samples.

As a result of this, suitable comparisons can still be made (Kennedy et al, 2005).

As previously stated, bioavailability is a prerequisite for the degradation of PAHs.

While bioavailability is affected by a number of factors, this project focused on the effect of

temperature. Previous studies have shown that an increase in temperature has an effect on the

bioavailability of PAHs and can enhance bioremediation of contaminated soil (Trably and

Patreau 2006). Raising the temperature of the contaminated soil can decrease adsorption,

increasing solubility and therefore the availability of the compound for microorganisms to

degrade (White et al, 1999). While the results obtained in this project indicated that

temperature had an effect on the bioavailability of fluoranthene it appeared not to have an

effect on the bioavailability of benzo[a]pyrene. Furthermore, the degradation of fluorathene

was significantly higher at lower temperatures, while a contrasting result was exhibited with

41

benzo[a]pyrene, in which significantly higher levels of degradation occurred at 45°C and

changing temperatures.

Overall, this study has provided results on the effect of temperature on the

bioavailability and degradation of two PAHs fluoranthene (4-ringed) and benzo[a]pyrene (5-

ringed). These findings have shown the contrasting manner in which these two PAHs can be

degraded and how they both act differently under the same conditions, which may be due to

the fact that benzo[a]pyrene has a higher molecular weight than fluoranthene.

These results are indicative of the issues related to bioremediation. While temperature

appeared to have a different effect on the two compounds, this is only one of many factors

that will affect the process. It is therefore essential to analyse all physicochemical properties

of soil as well as the compound’s characteristics, in order to achieve successful remediation

of PAH contaminated soils.

42

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6. Acknowledgements

I would like to thank Dr. Evelyn Doyle for all the help and guidance throughout this project. I

would also like to say a big thank you to Fengxiao Zhu for all the technical support and

guidance during the course of the lab work. To John Flynn for all his help with my GC issues

and to all of lab 2.45 for their patience and help throughout the year. I would also like to give

special thanks to Ber and Ger Danaher for all their support throughout all my years in

education, in particular this year, when I was not easy to live with. And finally to Mac, who

managed to always cheer me up when all I wanted to do was give up.