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Soil Fungal Population Study Related to Oil Pollution along
Different Distances from Kawrgosk Oil Refinery of Erbil-Iraq
Nashmeel Saeed Khudhur* and Nareen Q. Faqi Abdulla**
*Department of Environmental Science, College of Science, University of Salahaddin -
Erbil, Iraq
** Department of Biology, College of Science, University of Salahaddin - Erbil, Iraq
ABSTRACT
Fungal populations’ inhabitant in soils along different distances from Kawrgosk Oil Refinery was
analyzed in relation to the soil physicochemical characteristics and residual oil contents in sixteen sites.
In the soil samples, a total of fourteen species belonging to twelve fungal genera were isolated. The total
number of isolated fungi from these sites was 243×103 cfu.g-1 dry soil (colony forming unit). Maximum
population of fungi 41×103 cfu.g-1 dry soil was observed in site 6 (250-m away from the refinery centre),
while the minimum population of fungi 2×103 cfu.g-1 dry soil was isolated in the refinery centre. The
most frequently isolated fungi were yeasts 96×103 cfu.g-1 dry soil with a percentage of occurrence 41.03
%, followed by Aspergillus ochraceous 43×103 cfu.g-1 dry soil (18.38 %), Rhodotorula sp. 24×103 cfu.g-1 dry
soil (10.26 %), Penicillium spp. 23×103 cfu.g-1 dry soil (9.83 %) and A. niger 12×103 cfu.g-1 dry soil (5.13
%). While the least frequently isolated fungal species was A. terreus 1×103 cfu.g-1 dry soil (0.43 %).
Investigation of the isolation of fungi from these sites showed that population of fungi was significantly
different. Pearson’s correlation among soil physicochemical properties, oil residues and fungal
population in all of the studied sites, showed different results. The correlation between total cfu of fungi
with oil residue was negative by r value of -0.092; the correlation between total cfu of fungi with silt and
clay contents, pH, total P, K, and S were also negative by r values of -0.005, -0.135, -0.290, -0.090, -0.255
and -0.227 respectively at 0.05 level of significance. While, the correlation between total cfu of fungi
with moisture, sand, EC, total organic C and total N were positive by r values of 0.005, 0.143, 0.355,
0.161 and 0.152 respectively.
KEYWORDS: Oil pollution, Soil, Fungal population, Kawrgosk Oil Refinery.
INTRODUCTION
ndustrialisation, urbanization and increased population have increased
environmental pollution rapidly (Yadav and Vyas, 2013). These industries are
more hazardous to the environment upon its existence within the limits of the
cities, or its existence within urban area, and or near the agricultural terrines
such as Kawrgosk Oil Refinery, west of Erbil city. Naturally, soil is the richest
reservoir of microorganisms and a key component of ecosystems because
environmental sustainability depends largely on a sustainable soil ecosystem
(Adedokun and Ataga, 2006). Among these microorganisms fungi are one of the
dominant groups present in soil which represent the main reservoir of fungi (Rane and
Gandhe, 2006). Soil fungi can be grouped into three general functional grouped based
on how they get their energy, decomposers, mutualists and pathogens (Brady and Weil,
2002 and Tugel et al., 2000). Fungal hyphae physically bind soil particles together,
creating stable aggregates that help increase water infiltration and soil water holding
I
1
capacity (Ingham, 2007). Whenever soil is polluted, the ecosystem is altered and
agricultural activities are affected (Igwo-Ezikpe et al., 2009). The demand for crude oil
as a source of energy and primary raw material for industries has increased. This has
led to an increase in production, transportation and refinery which have therefore
resulted in grossing pollution of the environment (Adesina and Adelasoye, 2014). Tons
of hydrocarbons enter the environment through oil spill, tank leakages or wastewater
disposal (Wang et al., 1994). Oil spills occur at all stages of production, transportation,
and handling of petroleum products. This could results from pipeline ruptures,
accidents, and dumping of waste engine oil (Odjegba and Atebe, 2007). These
pollutants are toxic and hazardous to life. Their release into the environment has led
to many environmental problems that are of global concern (Wang et al., 1994).
Nevertheless, some of the soil microorganisms that participated in soil processes such
as transformation of nutrients are active hydrocarbon degraders (Pandey et al., 2005).
Crude oil occurs naturally as a complex mixture of hydrocarbon and non-hydrocarbon
compounds which contains a measurable toxicity towards living organisms (Adesina
and Adelasoye, 2014). Generally, soil conditions of agricultural land, microorganisms
as well as plants are damaged or altered by any contact with crude oil. Beyond 3% oil
concentration in an environment, crude oil becomes increasingly toxic to soil biota and
crop growth (Onuoha et al., 2003). One major environmental concern of soil
contaminated with crude oil or petroleum product is an increase in organic carbon of
the soil with a concomitant decrease in soil nitrate and phosphorous, thus imposing a
condition that impaired oil degradation in the soil (Okolo et al., 2005). Different
species and different life stages of organisms have been demonstrated to have different
susceptibilities to pollution. The scale of pollution depends on the quantity of oil and
the damage done to the environment. In heavily polluted areas, there will be an
immediate detrimental effect on the life of flora and fauna (Obire and Anyanwu, 2009).
Due to higher increase in oil industry activities in Kurdistan Region of Iraq, since
there are a little information of the environmental status of the areas around the
refinery locations, and the concerns of a possible environmental pollution that will
cause health and life threats to living organisms particularly fungi, this study was
carried out.
MATERIALS AND METHODS
A/ Study area
Erbil Refinery is located in Khabat district, at Kawrgosek, 40 km west of Erbil
city, and it occupies a land of 2.5 Km² to the left of upper Zab River. The refinery is
composed at this stage of three production lines for crude Oil refining and the
production and storage and distribution and supply of petroleum products as per
applied standards, which represent the first plant for crude Oil refining in Kurdistan
region. Construction of this refinery started in 2005. The refinery produces the
following oil products: naphtha, kerosene, gasoil (desel), fuel oil, gasoline (automobile’s
benzine) and liquid gas (after operating the second production line). These products
are stored and distributed in storage tanks and then transported through loading
2
stations by tankers, or may be pumped through a pipe to Erbil Depot according to the
request (KAR website, 2015). The studied sites in the present study were different
distances at Kawrgosk Oil Refinery (Figure 1). Sixteen sites according to distance
from the refinery centre were taken for this study which were: site 1: 0-m (Oil Refining
centre), site 2: 50-m, site 3: 100-m, site 4: 150-m, site 5: 200-m, site 6: 250-m, site 7:
300-m, site 8: 350-m, site 9: 400-m, site 10: 450-m, site 11: 500-m, site 12: 1000-m, site
13: 3000-m, site 14: 5000-m and site 15: 10000-meters away from the refinery centre
and the last site (site 16) was the College of Science Greenhouse which was used as
control.
B/ Soil samples collection
During December 2014, soil samples (0-10 cm depth) from these sites were
collected into polyethylene bags. From each site three samples were taken. The soil
samples were then taken to the laboratory for analysis. Soils were air-dried, crushed
and sieved, for various physico-chemical parameters, through 2-mm stainless sieve to
remove debris (Pansu and Gautheyrou, 2006). For fungal study, soil samples with
three replicates were taken from each study site with disinfected spatula and the
samples were placed in sterilized packets then stored in a cool box until they reached
the laboratory. In the laboratory the samples were milled and sieved twice to remove
large stones and debris to obtain soil samples with small particles. The samples then
were processed in an isolated process of fungi using the standard plate method.
C/ Soil physicochemical analysis
Soil moisture content was determined by gravimetric method in which the samples
were dried to constant weight as described by (Jaiswal, 2003). Particle size
distribution and soil texture was determined by hydrometer method according to
(Ryan et al., 2001). The pH and EC of the soils were determined using a calibrated pH-
meter (JENWAY 3505) and an electrical conductivity meter (JENWAY 4510) in 1:1
(soil: water suspension) in line with the method proposed by (Ryan et al., 2001).
Walkly-Black procedure (1934) was followed for determination of soil total organic
carbon (Pansu and Gautheyrou, 2006). The micro-Kjeldahl procedure was used as
mentioned by (Ryan et al., 2001) for estimation of total nitrogen. Ascorbic acid
combined with potassium antimonyl tartrate of Murphy and Riley (1962) which
described by (Pansu and Gautheyrou, 2006) was used for determination of total
phosphorus. Total potassium was determined by flame photometric method according
to (Pansu and Gautheyrou, 2006). Total sulfur was determined by turbidimetric
method as described by (Pansu and Gautheyrou, 2006).
D/ Media used for counting, isolation and identification of fungi
For counting, isolation and identification of soil total fungi, potato dextrose agar
was prepared by adding 39 g of powdered medium into distilled water and bringing the
volume to 1 litre. The medium composition is as follow: [potato, 200 g; dextrose, 20 g;
and agar, 20 g] as described in (Onuorah, 1982). Then mixed thoroughly and gently
heating and bringing to boiling in flasks. The medium was then autoclaved. Then 0.2
mg Chloramphinicol was added (Cheesbrough, 1992).
E/ Isolation techniques
3
Serial dilution was prepared by adding one gram of each soil sample to 9 ml of
sterile distilled water in sterilized test tubes, shaken well, a serial dilution of (10-3)
were made in the same method. One ml of (10-3) dilution was poured in each Petri dish
containing prepared medium (PDA + Chloramphinicol) by sterile pipette, each sample
made by three replication plates and incubated at 25 °C for 10 days (Basu, 1980).
F/ Diagnosis and identification of the fungal genera
The fungal isolates were transferred to sterilized plates for purification and
identification. The grown fungi were mounted on a slide, stained with lactophenol-
cotton blue to detect fungal structures (Basu, 1980), covered with a cover slip,
examined under microscope and identified on the basis of their colony morphology and
spore characteristics (Rajankar et al., 2007).
G/ Keys used for identification of the fungal genera
The texts (Books) used for identification of soil fungi, depending on their
taxonomic keys are as follows: (Larone, 1995; Pitt and Hocking, 1997; Guarro et al.,
1999; Howard, 2002; Watanabe, 2002; Ulhan et al., 2006; McClenny, 2007 and
Pornsuriya et al., 2008).
H/ Statistical analysis
Statistical analysis for the obtained data was performed using descriptive
statistics and ANOVA accompanied with Duncan’s test for comparing of the means at
the level of significant of 0.05 by SPSS version 11.5 and Microsoft excel 2010. Person’s
correlation was done to test the relationship among fungal population, oil residues and
the soil physicochemical characteristics from the different sites at (p<0.05) level was
considered statically significant.
RESULTS
The results of soil physical properties are presented in (table 1). The highest
moisture content (5.73±0.017) was observed at site 13 which characterized by clay loam
texture class, and the lowest content of moisture (0.50±0.035) was at site 11 which has
a sandy loam soil class. The results showed significant differences (p<0.05) between
the sites 2, 3, 4, 9, 12, 13, 14 and 15 as compared with the control soil; however the soil
texture class for these sites were respectively: silt loam, loam, sandy loam, sandy loam,
silty clay loam, clay loam, clay, clay and loam for garden soil (control). Soil chemical
properties were represented in (table 2). The pH of the soils were ranged from
7.39±0.049 to 8.47±0.017 in the site 15 (10-Km away from the refinery centre) and site
2 (50-m away from the refinery centre), indicating neutral to slightly alkaline soils.
There were no significant differences between pH of the soils from different sites when
compared with control. The highest EC of 231±1.155 µS.cm-1 was obtained in site 8
(350-m away from the refinery centre) and the lowest EC of 14±1.155 µS.cm-1 was in
site 11 (500-m away from the refinery centre); however there were significant
differences between the sites 6, 7 and 8 in comparing with control soil. The highest
total organic carbon was 44.822±0.577 and 44.822±0.058 g.Kg-1 in both the sites 10
4
(450-m away from the refinery centre) and 9 (400-m away from the refinery centre),
whereas the lowest organic carbon content was 7.804±0.001 and 7.804±0.001 in both
the sites 3 (100-m away from the refinery centre) and 1 (the refinery centre).
Significant differences between all the studied sites were observed except for the sites
1, 3 and 7. Total nitrogen was ranged between 0.756±0.000 and 2.313±0.000 g.Kg-1 in
both the sites 7 (300-m away from the refinery centre) and 6 (250-m away from the
refinery centre) respectively. Statistical analysis revealed no significant differences
between the studied sites regarding to total nitrogen content of the soil samples when
compared with control. Moreover, total phosphorus content of the studied soils showed
no significant differences between all of the sites and the range was between
0.014±0.001 and 0.023±0.002 g.Kg-1 from the sites 4 (150-m away from the refinery
centre) and 11 (500-m away from the refinery centre) respectively. Total potassium of
the studied soils were between 16.4±0.115 and 29.5±0.058 g.Kg-1 in both the sites 6
(250-m away from the refinery centre) and 16 (control) and there were no significant
differences between the studied sites in comparing with garden soil. The highest total
sulfur content 26.7±0.115 g.Kg-1 was recorded at site 15 (10-Km away from the refinery
centre) and zero values were recorded in sites 4, 5 and 6 as the lowest level. Significant
differences were obtained between the studied sites except for the sites 2, 4, 5, 6 and
14. The residual oil content of the soil samples were determined and presented on the
(figure 2). The highest oil residue was in site 14 (5-Km away from the refinery centre)
which was 0.0022 ppm; whereas, the lowest oil residue 0.0007 ppm was in site 5 (200-
m away from the refinery centre) and there were no significant statistical differences
between the studied sites in comparing with control. The results of fungal study were
tabulated in (table 3) which shows the identity and the total colony forming units (cfu)
of fungi on PDA medium. A total of twelve different fungal genera and fourteen species
were isolated. All fungi were identified on the basis of their cultural and morphological
characteristics. The total number of isolated fungi from the sixteen selected sites was
243×103 cfu.g-1 dry soil. Maximum population of fungi 41×103 cfu.g-1 dry soil was
observed in site 6 (250-m away from the refinery centre), while the minimum
population of fungi 2×103 cfu.g-1 dry soil was isolated in the refinery centre (figure 3).
The most frequently isolated fungi were yeasts 96×103 cfu.g-1 dry soil which have a
percentage of occurrence of 41.03 % (figure 4), followed by Aspergillus ochraceous
43×103 cfu.g-1 dry soil (18.38 %), Rhodotorula sp. 24×103 cfu.g-1 dry soil (10.26 %),
Penicillium spp. 23×103 cfu.g-1 dry soil (9.83 %) and A. niger 12×103 cfu.g-1 dry soil (5.13
%). While the least frequently isolated fungal species was A. terreus 1×103 cfu.g-1 dry
soil (0.43 %) as shown in (figure 4). The microscopic nature of some of the isolated
fungal genera was shown in (figure 6). Moreover, results of statistical multiple
comparisons between the studied sites when compared with control showed significant
differences except for the sites: the refinery centre, 100-m away from the refinery
centre, 500-m away from the refinery centre and 1-Km away from the refinery centre.
After computation of pearson’s correlation between the physical and chemical
properties with the oil residues and fungal population in all of the studied sites (table
4), a significant negative correlation was obtained between sand and clay content of
the studied soils, as well as a significant positive correlation was obtained between
5
total phosphorus and total sulfur contents of the studied soils at 0.05 level of
significance. Moreover, the correlation between total cfu of fungi with oil residue was
negative by r value of -0.092 (figure 5); the correlation between total cfu of fungi with
silt and clay contents, pH, total P, K, and S were also negative by r values of -0.005,
-0.135, -0.290, -0.090, -0.255 and -0.227 respectively at 0.05 level of significance. While,
the correlation between total cfu of fungi with moisture, sand, EC, total organic C and
total N were positive by r values of 0.005, 0.143, 0.355, 0.161 and 0.152 respectively at
0.05 level of significance.
DISCUSSION
The highest moisture content (5.73±0.017) was observed at site 13 which
characterized by clay loam texture class and the observation is close to those of
(Umanu and Dodo, 2013) at different contaminated soils from Ota, Nigeria. Regarding
to moisture changes, (Strickland and Rousk, 2010) states that moisture regimes are
apt to change both in degree and frequency across wide geographical regions in the
face of global climate changes. In general, it has been proposed that fungi will exhibit
less of a response to changes in moisture because of their chitinous cell walls make
fungi more resistant and resilient to changes in moisture content of soil.
Soil pH is one of the main parameter for determining the extent of pollution, it was
ranged between 7.39 to 8.47, indicating neutral to slightly alkaline soils, and this
finding come in agreement with those obtained by (Yadav and Vyas, 2013) in Jodhpur-
Rajasthan and observation of (Benal et al., 2014) from petroleum hydrocarbon
contaminated site in Manglia. In general, fungi have been found to be more acid-
tolerant leading to increased fungal dominance in acidic soils. However, this does not
appear to be a conclusive pattern since alterations in pH can result in increased,
decreased, or unchanged levels of fungal dominance (Strickland and Rousk, 2010).
Works conducted across a land-use gradient in the Southeastern U.S. found the
relative abundance of fungal taxa were more strongly related to soil P and C:N ratios
than any other edaphic characteristic (Lauber et al., 2008). Soil electrical conductivity
is a measure of soluble salt content in the soil and is used as an overall indicator of the
level of macro- and micronutrients in the soil. The highest EC of 231±1.155 µS.cm-1
was close to the observation of (Yadav and Vyas, 2013) who found 250 µS.cm-1 as
highest EC in Mogra polluted site. However, low EC values were observed in sites 1,
11, 12, 13 and 15 and this may be an indicator of contamination which affected soil
structure and modified its physicochemical properties (Hawrot and Nowak, 2006). The
reduction in salt concentration which are suitable terminal electron acceptors may
affecting the indigenous microbial growth and metabolism (Ujowundu et al., 2011).
This could be the reason for the lower fungal growth in contaminated soil samples.
Total organic carbon ranged between 7.804 and 44.822 g.Kg-1 which is in agreement
with finding of (Benal et al., 2014; Kucharski and Jastrzębska, 2005 and Ekhaise and
Nkwelle, 2011) and higher than the observations of (Hawrot and Nowak, 2006) in six
polluted sites in Jodhpur-Rajasthan who found a range of 1.0-3.8 g.Kg-1 and this may
refer to the nature of the studied soil. The obtained total nitrogen (0.756-2.313 g.Kg-1)
and total phosphorus (0.014-0.023 g.Kg-1) in this study are lower than the obtained
6
results by (Pečiulytė and Volodkienė, 2009) in the chemical factory near the city of
Kėdainiai in Central Lithuaniaand, and this may refer to their studied sites because
the factory’s major product by the present is nitrogen phosphorous fertilizer. However
present findings of total nitrogen contents were similar to the observations of (Ekhaise
and Nkwelle, 2011) from the mechanic workshops.
The highest observed oil residue of 0.0022 ppm at site 14 was lower than that
observed by (Umanu and Dodo, 2013) which was 10.10 ppm in contaminated soils of
Nigeria, and this may refer to the type of oil source in these area in comparing with
the present site, whereas, they obtained zero values in non-contaminated soils,
however the lowest oil residue value of 0.0007 ppm at site 5 (200-m away from the
refinery centre) by the present study possibly indicate no contamination with oil in
this site according to the statement of (Umanu and Dodo, 2013). On the other hand
site 14 characterized by only yeast population of 16×103 cfu.g-1 which may have no the
sufficient capacity to degrade the oil content in this area, while site 5 which has the
lowest oil residue, characterized by Aspergillus niger (5×103 cfu.g-1) and Penicillium
spp. (4×103 cfu.g-1) which have a specialized enzyme system for degrading of petroleum
compounds; in this regard (Gesinde et al., 2008) reported that indigenous
microorganisms are more capable of degrading indigenous crude oil due to the fact
that native microorganisms are best adapted to intrinsic environmental conditions.
Fungal communities in soils are an important component, because they participate
in regulating microbial activity in polluted soils (Pečiulytė and Volodkienė, 2009). By
the present study, a total of twelve different fungal genera and fourteen species were
isolated and this is similar with the observations of (Dawoodi et al., 2015) who isolated
thirteen fungal genera from oil-contaminated soils with different oil contamination
from Khuzestan, Iran and the thirteen isolated fungal genera included Aspergillus,
Penicillium, Fusarium, Acremonium, Candida, Rhodotorula, Mucor, Aureobasidium,
Cunninghamella, Rhizopus, Alternaria, Beauveria and Paecilomyces. Investigation of
the isolation of fungi from oil-contaminated environments showed that abundance and
fungal population in different stations were significantly different and this in
consistence with the present findings. The total number of isolated fungi from the
sixteen selected sites was 243×103 (2.43×105) cfu.g-1 dry soil, but (Umanu and Dodo,
2013) isolated slightly higher count 4.3 to 8.3×105 cfu.g-1 and this may either refer to
the moisture content in the studied soils as stated by (Eze and Okpokwasili, 2008), or
it may refer to the organic matter and nitrogen content in the soils (Pečiulytė and
Volodkienė, 2009). Maximum population of fungi 41×103 cfu.g-1 dry soil was observed in
site 6 (250-m away from the refinery centre), while the minimum population of fungi
2×103 cfu.g-1 dry soil was isolated in the refinery centre and the difference in count of
the sites may be due to the difference in sites receiving domestic effluents and
agricultural runoff (Allamin et al., 2014) who reported that variation in counts in sites
will be due to the industrial and domestic discharges to the sites; moreover, the lower
fungal population may refer to the least amount of total organic carbon in the refinery
center (table 2) which affect the population and diversity of fungi. The most frequently
isolated fungi were yeasts 96×103 cfu.g-1 dry soil which have a percentage of occurrence
of 41.03 % (figure 3), followed by Aspergillus ochraceous 43×103 cfu.g-1 dry soil (18.38
7
%), Rhodotorula sp. 24×103 cfu.g-1 dry soil (10.26 %), Penicillium spp. 23×103 cfu.g-1 dry
soil (9.83 %) and A. niger 12×103 cfu.g-1 dry soil (5.13 %), in this circumstance our
results come in agreement with those found by (Nkwelang et al., 2008) who found that
the major genera of fungi in polluted soils of Calabar-Nigeria were Aspergillus,
Penicillium and Mucor; as well as agree with findings of (Olukunle, 2013) in Ondo oil-
polluted soils and water who isolated mostly Penicillium italicum, Aspergillus niger, P.
oxalicum, Streptothorix atra, Articulosporium inflata, Geniculosporium serpens, A.
flavus, Halosprangium pavum, A. fumigates and A. rapens. However, (Benal et al.,
2014) identified Aspergillus, Penicillium, Fusarium, Rhizopus, Mucor, Cladosporium,
Morssonina, Chaetomium, Curvularia, Helminthosporium, Alternaria and
Trichoderma species in petro-polluted soil in Manglia. For the fungal isolates observed
by (Eze et al., 2014), Penicillium spp. had the highest occurrence of 33.3% while Mucor,
Rhizopus, Aspergillus and Cephalosporium spp. had the least occurrence of 16.7% in
Nigeria. However, (Balaji et al., 2014) isolated lower counts of Aspergillus ochraceous,
Penicillium spp. and A. niger in the grounds of several automobile stations at different
places within the city of Chennai, India polluted by PAHs, and the dominant fungal
species by their study were Mucor racemosus (6.3 cfu.g-1), Rhizopus stlonifer (7.2 cfu.g-
1), Aspergillus niger (3.4 cfu.g-1) and Penicillium chrysogenum (5.3 cfu.g-1); since they
grown the fungal isolates on medium containing 1 % (w/v) of the following carbon
sources such as diesel, kerosene, petrol, grease, motor oil and contaminated soil from
garages. Whereas, (Umanu and Dodo, 2013) isolated Penicillium chrysogenum,
Aspergillus sp. and Candida sp. dominantly in contaminated soils from Nigeria.
Furthermore, (Hawrot and Nowak, 2006) mostly isolated Penicillium sp. and
Aspergillus sp. in soils contaminated with fresh and old crude oil spills at different
location at the Badia Region-Jordan; whereas (Allamin et al., 2014) mainly isolated
Aspergillus, Penicillium and Rhizopus species. These organisms have been previously
implicated with petroleum product degradation. Moreover, results of statistical
multiple comparisons between the studied sites when compared with control showed
significant differences, except for the sites the refinery centre, 100-m away from the
refinery centre, 500-m away from the refinery centre and 1-Km away from the refinery
centre and no differences between these sites come in agreement with the observation
of (Allamin et al., 2014) who found no significant differences between five studied sites
each with 1-Km intervals in Kukawa local government area of Borno State, Nigeria
according to Turkey’s HSD at P < 0.05 during December 2012; however significant
differences between all the other sites come in agreement with (Allamin et al., 2014)
who revealed that the counts between five sample sites in Nigeria were significantly
different (P≤0.05).
Moreover, the correlation between total cfu of fungi with oil residue was negative
by r value of -0.092; and this may refer to the ratios of carbon and nitrogen in the
studied sites as stated by (Lauber et al., 2008). Moreover, the correlation between total
cfu of fungi with silt and clay contents, pH, total P, K, and S were also negative by r
values of -0.005, -0.135, -0.290, -0.090, -0.255 and -0.227 respectively at 0.05 level of
significance, however, (Pečiulytė and Volodkienė, 2009) also obtained a negative
correlation between fungal population and pH by r values of -0.71; as well as they
8
obtained negative correlations between number of fungal genera with total P and total
K by r values of -0.71 and -0.70 respectively in the studied soils of chemical factory in
Kėdainiai city. The possible stress factor behind this negative correlation that the
microorganisms can be subjected to could be substrate availability as a result of the
higher proportion of phosphorus, potassium and sulfur in the unavailable form that is
not substrate for microorganisms (Witter et al., 1993). While, the correlation between
total cfu of fungi with moisture, sand, EC, total organic C and total N were positive by
r values of 0.005, 0.143, 0.355, 0.161 and 0.152 respectively at 0.05 level of significance,
and (Pečiulytė and Volodkienė, 2009) also obtained positive correlations between
fungal population with moisture content, organic matter and total nitrogen by r values
of 0.46, 0.74 and 0.77 respectively in the studied soils of chemical factory in Kėdainiai
city. When considering the response of fungi to changes in moisture, (Frey et al., 1999)
found that fungal biomass and fungal dominance were both positively related to soil
moisture in cultivated settings. Moreover, (Adeboye et al., 2011) obtained a high
positive correlation (r = 0.84, P ≤ 0.0001) between soil organic carbon and total
nitrogen and microbial properties at the 5-10 cm soil depth in southern Nigeria.
CONCLUSION
The study concluded that, the detected oil residues were lower than that observed
in the previous studies conducted in oil polluted soils in different parts of the world
and this may possibly indicate no contamination for nowadays in the studied area
because the refinery is still new-established and newly works and not faced the level of
soil contamination. Soil fungi are important component because they participate in the
regulation of soil microbial activity in polluted areas. Although oil pollution in the
studied area around the oil refinery is low, it influences the community of soil fungi to
some degree and the fungal population was negatively correlated with oil residue. The
results of this study will be helpful to decision-makers, planners, scientists, and the
local communities to protect the environment in areas surrounding various oil
refineries established in Kurdistan Region of Iraq for the future.
ACKNOWLEDGMENT
The authors are grateful to departments of Environmental Science and Biology for
providing facilities of this study in both the advanced laboratory for research studies
and Mycology Laboratory, respectively.
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Figure 1: Map showing A. Iraq, B. Kurdistan Region and C. Erbil City with the
studied area.
Table 1: Physical properties of the studied soils.
Sit
e
No
.
Distance (m) Moisture
%
Particle size distribution
Sand
%Silt % Clay % Texture
class
10 (Oil Refining
Centre) 0.84±0.023hi 26.89 32.78 40.34 Clay
2 50 4.34±0.017c34.66 54.88 10.45 Silt Loam
3 100 3.27±0.035d48.31 43.94 7.75 Loam
4 150 3.39±0.040d63.77 36.23 0.00 Sandy Loam
5 200 1.63±0.017fg 54.26 43.20 2.54 Sandy Loam
6 250 2.30±0.017e69.29 30.71 0.00 Sandy Loam
7 300 1.68±0.017f46.60 45.77 7.63 Loam
8 350 0.96±0.032hi 52.04 40.39 7.57 Sandy Loam
9 400 5.50±0.058ab 68.25 31.75 0.00 Sandy Loam
10 450 1.16±0.023gh 57.00 40.47 2.53 Sandy Loam
11 500 0.50±0.035i59.80 35.18 5.03 Sandy Loam
12 1000 (1-Km) 5.04±0.012b18.38 42.12 39.49 Silty Clay
Loam
13 3000 (3-Km) 5.73±0.017a17.79 39.78 42.43 Clay Loam
14 5000 (5-Km) 4.25±0.662d24.28 23.50 52.22 Clay
15 10000 (10-Km) 3.75±0.012d22.08 36.36 41.56 Clay
16 Control (garden
soil) 2.09±0.017ef 48.93 33.19 17.87 Loam
Table 2: Chemical properties (mean ± S.E.) of the studied soils.
Si pH EC Total Total – N Total – P Total – K Total – S
13
te
No
.µS.cm-1 organic C
g.Kg-1 g.Kg-1 g.Kg-1 g.Kg-1 g.Kg-1
18.24±0.023bc
d21±4.619g7.804±0.001i1.545±0.006cd 0.017±0.006 17±0.000k15.4±0.058d
28.47±0.017a55±2.887f21.811±0.577d1.528±0.000cd 0.018±0.001 19.7±0.058i1.853±0.015k
38.37±0.040a
b53±1.732f7.804±0.001i2.298±0.000a0.019±0.001 22±0.000ef 6.896±0.002i
48.11±0.012de 57±1.732ef 11.806±0.058h1.455±0.000d0.014±0.001 19.9±0.058i0.0±0.000l
57.97±0.012ef 64±1.155de 23.812±0.173bc 1.468±0.000d0.016±0.000 20.7±0.173h0.0±0.000l
67.84±0.023fg
h76±0.577c24.812±0.012b2.313±0.000a0.016±0.002 16.4±0.115l0.0±0.000l
78.16±0.058cd 167±4.041b9.805±0.001i0.756±0.000e0.018±0.002 21.5±0.231g7.424±0.115h
87.79±0.058gh 231±1.155a16.808±0.173f1.675±0.000c0.019±0.003 22.5±0.115d15±0.000e
97.89±.046fg 59±2.887ef 44.822±0.058a1.549±0.000cd 0.018±0.002 23.6±0.115c14.6±0.058f
10 7.78±0.017gh 70±1.155cd 44.822±0.577a1.529±0.000cd 0.02±0.001 25.4±0.173b19.8±0.058c
11 7.7±0.115h14±1.155i22.811±1.155cd 2.293±0.058a0.023±0.002 19.1±0.058j15.2±0.115de
12 7.78±0.115gh 25±1.155g14.807±0.577g1.459±0.173d0.018±0.006 21.7±0.058fg 20.4±0.173b
13 7.76±0.058gh 27±1.732g18.809±0.002e1.529±0.058cd 0.02±0.006 16.4±0.058l7.701±0.115g
14 7.53±0.017i75±2.887c17.809±0.001ef 2.295±0.001a0.016±0.002 16.7±0.115kl 2.068±0.017k
15 7.39±0.049i 27±2.887g12.806±1.155h1.457±0.000d0.021±0.001 22.3±0.173de 26.7±0.115a
16 8.31±0.023ab
c75±1.155c9.648±1.732i1.845±0.012b0.018±0.001 29.5±0.058a4.396±0.115j
Figure 2: Residual oil content (ppm) in the studied sites. Different letters mean
significant differences among the studied sites.
14
Table 3: Fungal flora isolated from sixteen sites located at different distance from
Kawrgosk Oil Refinery. Total cfu.g-1 dry soil expressed as (mean ± S.E.) and multiplied
by 103.
Site No.
Fungi 1234567891
01
11
21
31
41
51
6
Total no. of
species isolated
from all sites
Alternaria raphani 0 0 0 0 0 0 0 0 3 0 0 0 2 0 0 0 5
Aspergillus niger 0 0 0 0 5 0 2 1 0 0 0 0 0 0 0 4 12
A. ochraceous 0 0 2 0 0 31 1 2 0 7 0 0 0 0 0 0 43
A. terreus 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1
Circinella muscae 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 3
Cladosporium oxysporum 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 4
Cunninghamella elegans 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 6
Fusarium sp. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0 6
Mycelia sterilia 0 0 0 0 0 0 0 3 0 0 0 0 0 0 0 0 3
Penicillium spp. 0 6 3 0 4 1 2 1 6 0 0 0 0 0 0 0 23
Rhizoctonia solani 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 5
Rhodotorula sp. 0 0 0 0 0 0 4 0 0 0 0 0 10 0 10 0 24
Taeniolella exitis 0 0 0 0 0 2 0 0 0 1 0 0 0 0 0 0 3
Yeasts 2 16 0 8 8 7 0 19 0 5 6 4 0 16 5 0 96
Total cfu.g-1 dry soil of
isolated fungal species
from each site
2±0.577k
22±1.154d
6±0.000i
8±0.000h
17±1.154ef
41±0.000a
13±0.577g
26±0.000b
9±0.577h
13±1.154g
6±0.000i
4±0.577j
18±0.577e
16±0.000f
24±0.000c
9±0.000h
234±1.426
Figure 3: Total cfu of fungi per a gram of dry soil along different distances from
Kawrgosk Oil Refinery.
Figure 4: Percentage (%) of occurrence of fungal isolates from the studied sites.
15
Table 4: Person’s correlation among physical and chemical parameters, total cfu of
fungi and residual oil content in the studied soils.
Moisture
Sand
Silt
Clay
pH
EC
Total organic
C
Total – N
Total – P
Total – K
Total – S
Total cfu of
fungi
Residual oil
Moisture 1
Sand -0.405 1
Silt 0.016 -0.095 1
Clay 0.382 -0.921** -0.300 1
pH -0.127 0.192 0.489 -0.367 1
EC -0.345 0.283 0.150 -0.330 0.064 1
Total organic C 0.100 0.447 -0.077 -0.399 -0.290 -0.055 1
Total – N -0.067 0.183 -0.482 0.013 -0.147 -0.242 0.028 1
Total – P -0.135 -0.162 0.177 0.086 -0.324 -0.138 0.155 0.096 1
Total – K -0.148 0.261 0.156 -0.312 0.198 0.229 0.152 -0.216 0.203 1
Total – S -0.048 -0.295 -0.021 0.291 -0.479 -0.127 0.165 -0.211 0.662** 0.275 1
Total cfu of fungi 0.005 0.143 -0.005 -0.135 -0.290 0.355 0.161 0.152 -0.090 -0.255 -0.227 1
Residual oil 0.029 -0.131 -0.408 0.285 -0.286 0.080 0.211 0.235 0.210 0.326 0.048 -0.092 1
** Correlation is significant at the 0.01 level (2-tailed).
Figure 5: Correlation between total cfu.g-1 of isolated fungal species and oil residue
from different studied sites.
16
Figure 6: The isolated fungal genera on PDA medium in the studied soils. A/
Circinella muscae; B/ Aspergillus sp.; C/ Penicillium sp.; D/ Rhizopus sp. and E/
Cunninghamella elegans.
17