FUTA Journal of Research in Sciences, 2013 (1): 82-86

MICROBIOLOGICAL AND PHYSICOCHEMICAL PROPERTIES OF

DIESEL SIMULATED SOIL
1*
E. Stephen, A. S. Usman, M.O. Okolo, E.A. Akogu2 and O.P. Abioye3
Department of Microbiology, Kogi State University, Anyingba, Kogi State, Nigeria.
Department of Science Laboratory Technology, Kogi State Polytechnic, Lokoja, Nigeria
Department of Microbiology, Federal University of Technology, Minna, Niger State, Nigeria.
*Corresponding author: psychsea07@gmail. Com, +234-8036347036
ABSTRACT
Microbiological and physicochemical properties of diesel simulated soil (11 % v/w) was studied for a period of
8 weeks. The results revealed lower counts in bacteria and fungi in diesel polluted soil (DPS) compared to oil
free soil (OFS). There were significant differences (P<0.05) in both bacteria and fungi counts obtained from
diesel free soil and diesel polluted soil. Ten organisms were isolated in the course of this study. These were
Aspergillus flavus, A.niger, Bacillus cereus, Candida tropicalis, Micrococcus luteus, Pseudomonas aeruginosa,
Proteus mirabilis, Staphyloccoccus aureus, Geotrichum candidum and Rhizopus nigricans. Diesel pollution
increased the organic carbon, organic matter and nitrogen contents of the soil. There were no significant
differences in pH, moisture, organic carbon, organic matter and nitrogen at 5% probability level. The result
obtained indicated that diesel pollution had altered the microbial and physicochemical properties of the soil.
Keywords: oil free soil, diesel polluted soil, physicochemical properties

INTRODUCTION found in the ecosystem. Diesel contains low

There has been a rapid increase in population molecular weight compounds and high
worldwide resulting in increased demand for proportion of saturated hydrocarbons that are
hydrocarbon products such as diesel, petrol, usually more toxic than long chained
kerosene, pesticides and industrial chemicals hydrocarbons. Diesel pollution is on the
(Chakrabarty et al., 1998). Although many of increase in Nigeria, as well as other
the chemicals are utilized or destroyed, a high developing countries (Stephen et al., 2011).
percentage is released into the air, water and The increase use of diesel generators has
soil representing a potential environmental increased the consumption which invariably
hazard (Alexander, 1995; Stephen et al., results in diesel contamination of soil and
2011). Processing and distribution of water bodies when such generators are
petroleum hydrocarbons as well as the use of serviced, and the oil is discarded carelessly in
petroleum products lead to contamination of the environment. This often results in
soil (Ayotamuno et al., 2006). Changes in soil distortion of soil physical, biological and
properties due to contamination with chemical properties. This study, therefore, was
petroleum- derived substances can lead to undertaken to assess the effect of diesel oil on
water and oxygen deficits as well as shortage soil microorganisms and the physicochemical
of available forms of nitrogen and phosphorus properties of the oil contaminated soil.
(Wyszkowska and Kucharski, 2000). MATERIALS AND METHODS
Hydrocarbon spill is a serious threat to the Sample collection
ecology (Schafer et al., 2009). According to The experimental design consisted of six
Dorn et al. (1998), hydrocarbon contains perforated earthenware pots containing 9 kg of
substances that are toxic to the flora and fauna soil each. Three pots served as the control
82
E. Stephen et al., FUTA J. Res. Sci., Vol 9, No. 1, Apr (2013) pp 82-86

(Diesel free soil, DFS) while the other three flavus had the highest frequency of occurrence
pots were simulated with one litre of diesel (14.58%) followed by A. niger and
each (Diesel polluted soil, DPS) to achieve an Pseudomonas aeruginosa (12.50%). The least
11.11% pollution level. The soil samples were frequency of occurrence was observed in
collected bi-weekly for a period of 8 weeks Geotrichum candidum (6.25%).
(May –June, 2012) and homogenized before Table 2 show the mean physicochemical
analysis. The pots were perforated to increase properties of the soil samples analysed. The
aeration and to avoid water logging. pH of all the samples tends towards alkalinity.
Laboratory/ statistical methods The values obtained for pH, organic carbon,
The pH of the soil was determined at ambient organic matter content, nitrogen and moisture
temperature using glass electrode pH and were higher in diesel polluted soil than in
conductivity meter (Hannia, Italy) in 1:1 water diesel free soil while phosphorus concentration
to soil ratio. Nitrogen was determined by the was higher in diesel free soil than in diesel
micro Kjedahl method (Ibitoye, 2006) while polluted soil. There were no significant
phosphorus was determined as described by differences (P>0.05) in the pH, organic
Murphy and Riley (1962). The ignition carbon, organic matter content, nitrogen and
method of Akinsanmi (1975) was used to moisture between the diesel free soil (DFS)
determine the organic matter content, while and diesel polluted soil (DPS). However, a
the dry weight method was used to determine significant difference was observed in the
the moisture content. Microbiological analysis phosphorus concentration between diesel free
was carried out following the procedure soil (DFS) and diesel polluted soil (DPS).
described by Harrigan and McCane (1990). 50
Frequency and occurrence of organisms was
45
determined using the approach of Dung and
Total viable bacteria counts (x10 6 cfu/g)

40
Stephen (2010). Descriptive statistics and
35
paired T-test was performed using SPSS
version 16. Experimental precision achieved 30
DFS
was reported at p ≤ 0.05 levels. 25
DPS
RESULTS 20

Figure 1 shows the total viable count of 15

bacteria obtained from the soil samples 10
analysed. The count in diesel free soil (DFS) 5
ranged from 1.1x 107 to 4.2 x107 cfu/g and 8.0
0
x106-2.6x107 cfu/g for diesel polluted soil 0 14 28 42 56
(DPS). DFS had higher counts than DPS. Tim e (Days)

There were significant differences (P>0.05) in

Figure 1: Total viable bacteria counts obtained from
the bacterial counts obtained from DFS and soil samples analysed
DPS. Figure 2 shows the fungal count
20
obtained from the soil samples- DFS and DPS.
The counts ranged from 3.0 x106 to 13.0x106 18

cfu/g for DFS, 1.0 x 106 – 7.0 x106 cfu/g for 16

DPS. The fungi counts were higher in DFS 14

than DPS. There were significant differences

Fungal counts

12
(P>0.05) in the fungal counts obtained from 10
DFS
DPS
DFS and DPS. Table 1 show the frequency 8
and occurrence of microorganisms isolated in
6
this study. The bacteria were Bacillus cereus,
4
Micrococcus luteus, Pseudomonas aeruginosa,
Proteus mirabilis and Staphylococcus aureus 2

while the fungi include Aspergillus flavus, A. 0

0 14 28 42 56
niger, Candida tropicalis, Geotrichum Tim e (Days)
candidum and Rhizopus nigricans. Aspergillus

83
E. Stephen et al., FUTA J. Res. Sci., Vol 9, No. 1, Apr (2013) pp 82-86

Figure 2: Fungi counts obtained from soil samples analysed

Table 1: Frequency and occurrence of microorganisms in soil samples analysed

Isolates DFS DPS Total Percentage
Aspergillus flavus 4 3 7 14.58
A. niger 3 3 6 12.50
Bacillus cereus 3 2 5 10.41
Candida tropicalis 3 2 5 10.41
Geotrichum candidum 2 1 3 6.25
Microccoccus luteus 2 2 4 8.33
Proteus mirabilis 2 2 4 8.33
Pseudomonas aeruginosa 4 2 6 12.50
Rhizopus nigricans 2 2 4 8.33
Staphylococcus aureus 2 2 4 8.33
Total 48 ≈100
_________________________________________________________________________________
DFS: Diesel free soil, DPS: Diesel polluted soil

Table 2: Physicochemical characteristics of soil sample analysed (mean ± standard error of

mean).
DFS DPS
pH 7.14±0.18 7.34±0.08

Organic Carbon (%) 1.14±0.08 1.43±0.30

Organic matter (%) 2.01±0.14 2.47±0.52

Nitogen (%) 0.25±0.02 0.32±0.06

Moisture (%) 17.60±3.40 21.57±1.87

Phosphorus (ppm) 7.83±0.39 a, 7.44±0.36 b

a,b
: means denoted by different superscripts along the same row are significantly (p<0.05) different.

DISCUSSION microbial counts in polluted soil and attributed

Higher bacteria and fungal counts were it to harmful metabolites and toxic compounds
observed in Diesel free soil (DFS) compared in the environment as a result of petroleum
to Diesel Polluted Soil (DPS). This could be degradation. Ten organisms were isolated in
attributed to non- exposure of DFS to Diesel the course of the study. These were Bacillus
which did not distort the biological and cereus, Micrococcus luteus, Pseudomonas
physical properties of the soil. This is in aeruginosa, Proteus mirabilis,
agreement with Leahy and Colwell (1990) and Staphyloccoccus aureus, Aspergillus flavus,
Stephen et al. (2011). These researchers A.niger, Candida tropicalis, Geotrichum
reported higher bacteria counts in unpolluted candidum and Rhizopus nigricans. These
soil than polluted soil. The low microbial organisms have been isolated elsewhere from
counts in DPS may be due to inability of some hydrocarbon polluted soil (Ijah and Abioye,
organisms to withstand the high concentration 2003; Okereke et al., 2007; Stephen et al.,
of the diesel (11.11% pollution level) in the 2011;Stephen and Egene, 2012). Ijah and
environment. This finding is similar to that of Abioye (2003) reported the predominance of
Bossert and Bartha (1994) who observed low Bacillus spp in kerosene spilled site in Minna,
84
E. Stephen et al., FUTA J. Res. Sci., Vol 9, No. 1, Apr (2013) pp 82-86

Niger State, Nigeria while Okereke et al. phosphorus soluble and brought some into
(2007) reported consistent isolation of solution (Ayotamuno et al., 2006).
Pseudomonas, Bacillus spp and CONCLUSION
Staphylococcus spp in oil spill sites in Egema, The results of this study show that diesel
Imo State, Nigeria. According to Chikere et al. polluted soils had high organic carbon, organic
(2009), hydrocarbon polluted soils are matter, nitrogen and moisture than diesel free
dominated by Gram negative bacteria such as soil. This is an indication that pollution of the
Pseudomonas spp and Proteus spp. environment with diesel accidentally or due to
Aspergillus flavus and A. niger have been human activities can enter the food chain and
reported as fungi that degrade hydrocarbons. seriously affects animal and human health;
Kuiper et al. (2004), Okereke et al. (2007), distort the microbial population and
Nkweenleng et al. (2008) and Chikere et al. consequently the soil physicochemical
(2009) reported the crude oil degradative properties. Hence, there is a need to enlighten
abilities of A. flavus and A. niger isolated from the public against the danger posed by
oil spilled sites. The pH of the soil samples indiscriminate disposal of diesel oil in the
tend towards neutrality. This range of pH has environment.
been reported by Bossert and Bartha (1994) REFERENCES
and Stephen and Egene (2012) to favour the Alexander, M. (1995). How toxic are
degradation of diesel oil. Similar pH values chemicals in the soil? Environmental
were observed by Stephen et al. (2011) and Science and Technology 29: 2713-2717.
Stephen and Egene (2012) from diesel and Akinsanmi, O. (1975). Certificate
waste lubricating oil polluted soils. Agricultural Science. Longman, Nigeria. Pp
The higher organic carbon and organic matter 104-112.
contents observed in DPS compared to DFS Ayotamuno, M. J., Kogbara, R. B. and
may be as a result of the addition of the diesel Egwuenum, P. N. (2006). Comparism of
to the soil. Similar result was reported by corn and elephant grass in the
Stephen et al. (2013) in spent lubricating oil phytoremediation of a petroleum
polluted soil subjected to phytoremediation. hydrocarbon contaminated agricultural soil
Organic carbon serves as source of energy for in Port-Harcourt, Nigeria. Journal of
the metabolism of the diesel from the soil. Agriculture and Environment 4 (2&4): 218-
Nitrogen content of DPS was slightly higher 222.
than DFS. This agrees with the findings of Bossert, I. and Bartha, R. (1994). The fate of
Stephen and Ijah (2011) and Stephen and petroleum in soil ecosystem. In : Petroleum
Egene (2012). These investigators reported Microbiology, Macmillan, New York,
increase nitrogen contents of soil polluted by U.S.A, pp 435-473.
hydrocarbons. The reason could be due to Chakrabarty, T., Subrahmanyam, P. V. R.
higher organic matter content in the diesel and Sundaresan, B. B. (1998).
polluted soil. Biodegradation of recalcitrant industrial
The moisture content in DFS was lower than waste. Journal of Applied Sciences 2: 172-
DPS. This observation may be due to low 184.
permeability and infiltration of water in the Chikere, C. B., Okpokwasili, G. C. and
diesel polluted soil compared to the diesel free Chikere, B. O. (2009). Bacterial diversity
soil (Edema et al., 2011). in a tropical oil polluted soil undergoing
The phosphorus concentration was higher in bioremediation. African Journal of
DFS than DPS. This observation is contrary to Biotechnology 8(11): 2535-2540.
that of Stephen and Egene (2012) who Dorn , P. B., Vipond, J. P., Salanitro, T. E.
reported higher phosphorus content in spent and Wisniewski, H. L. (1998). Assessment
lubricating oil polluted soil compared to oil of the acute toxicity of crude oil in soils
free soil and attributed it to the presence of the using earthworms, microtoxic and plants.
oil. The higher phosphorus content in DFS Chemosphere
relative to DPS may be due to the existence of 37: 845-860.
reduced condition in the soil that made

85
E. Stephen et al., FUTA J. Res. Sci., Vol 9, No. 1, Apr (2013) pp 82-86

Dung, J. A. and Stephen, E. (2010). hydrocarbon utilizing micro-organism in

Occurrence and frequency of Fungi isolated tropical soil polluted with oily sludge.
from the soil of Academic areas of African Journal of Biotechnology 7 (8):
University of Ilorin, Ilorin, Nigeria. 1075-1080.
International Journal of Applied Biological Okereke, J. N., Obiekezie, S. O. and Obasi,
Research 2(1): 7-13. K. O. (2007). Microbial flora of oil spilled
Edema, C. U., Idu, T. E. and Edema, M. O. sites in Egbema, Imo State, Nigeria. African
(2011). Remediation of soil contaminated Journal of Biotechnology 6(4): 991-993.
with polycyclic aromatic hydrocarbons from Schafer, A.N., Snape, I. and Siciliano, S.D.
crude oil. African Journal of Biotechnology (2009). Influence of liquid water and soil
10 (7):1146-1149. temperature on petroleum hydrocarbon
Harrigan, W. F. and McCane, M. E.(1990). toxicity in Antarctic soil. Environmental
Laboratory methods in Food and Dairy Toxicology and Chemistry 28: 1409-1415.
Microbiology, 8th ed.Academic press, Stephen, E. and Ijah, U. J. J. (2011).
London. Comparison of Glycine max and Sida acuta
Ibitoye, A. A. (2006). Laboratory Manual on in the phytoremediation of waste lubricating
Basic Soil Analysis (2nd ed). Foladave oil polluted soil. Nature and Science 9(8):
Nigeria 190-193.
Limited, Akure, pp 30-37. Stephen, E., Okolo, M. O. and Akogu, E. A.
Ijah, U. J. J. and Abioye, O. P. (2003). (2011). Microbiological properties of oil
Assessment of physicochemical and impacted soil 36 months after diesel spill.
Microbiological Properties of soil 30 International Journal of Education, Science,
months after kerosene spill. Journal of Mathematics and Environmental Studies
Research in Science and Management 1(1): 3(1): 77-82.
24-30. Stephen, E. and Egene, U. M. (2012).
Kuiper, I., Lagendijk, E. L., Bloemberg, G. Microbiology and physicochemical
V. and Lugtenberg, J. J. B. (2004). properties of soil polluted with lubricating
Rhizoremediation: A beneficial plant- oil in Anyingba, Kogi State, Nigeria.
microbe interaction. The American Nigerian Journal of Technological Research
Phytopathological Society 17 (1): 6-15. 7(2): 49-52.
Leahy, J. C. and Colwel, R. R. (1990). Stephen, E., Yakubu, S. A., Omebije, P. E.,
Microbial degradation of hydrocarbons in Edegbo, E. and Makolo, D. (2013).
the environment. Journal of Physicochemical properties of Spent
Environmental Microbiology 54:305-315. lubricating oil polluted soil subjected to
Murphy, J. and Riley, J. P. (1962). A Phytoremediation. Journal of Environment
modified Single Sowton method for the and earth Sciences 2(1): 1-4.
determination of phosphorus in natural Wyszkowska, J and Kucharski, J. (2000).
water. Analytical Chemistry 27, 31-36. Biochemical properties of soil contaminated
Nkweenleng, G., Kamugu, H. F. L., Nkeng, by petrol. Polish Journal of Environmental
G. E. and Antai, S. P. (2008). Studies on Studies 9(6): 476-485
the diversity, abundance and succession of

86