




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 4 (2), pp. 001-005, February, 2016. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

 

Full Length Research Paper 

 

Biodegradation of Lagoma crude oil using pig dung 

 
Yakubu, M. Bello 

 
School of Science, College of Education, PMB 39, Minna, Nigeria. E-mail: bellyms@yahoo.com. Tel: +234-

66-222205, +234-805- 046-7560. 
 

Accepted 12 November, 2015 
 
Pig dung bacteria were isolated and screened for crude oil degrading capabilities. The pig dung was also 
investigated for enhancement of crude oil biodegradation. Addition of chicken manure to oil polluted soil (at 
10% (v/w) pollution level) stimulated the biodegradation of lagoma crude oil used in the present study. In the soil 
amended with pig dung, 68.2% of the crude oil was degraded, whereas only 50.7% of same oil was degraded in 

the unamended soil. The pH of the amended soil rose from 6.2 to 7.2. Pig dung was found to contain 1.7 x 10
6
 

cfu g
-1

 crude oil degrading bacteria, and 1.8 x 10
8
 cfu g 

-1
 aerobic heterotrophs. The crude oil utilizing bacteria 

were identified as species of Pseudomonas, Proteus, Klebsiella, Bacillus and Micrococcus. Pig dung could 
therefore be an option for crude oil pollution mitigation project. 
 
Key words: Pig dung, biodegradation, crude oil, pollution, nutrients, amended soil. 

 
INTRODUCTION 

 
Oil released into the environment is a well-recognized 
problem in today's world. Oil spills affect many species of 
plants and animals in the environment, as well as 
humans (Plohl et al., 2002) . The involvement of microor-
ganisms in the degradation of petroleum and its products 
in the environment has been established as an efficient, 
economic, versatile, and environmentally sound treatment 
(Margesin and Schinner, 2001). The microorganisms 
implicated in oil degradation are widely distributed in 
nature and have been isolated from soil and water ecosy-
stems with their oil degrading potentials investigated.  

The search for effective and efficient methods of oil 
removal from contaminated sites has intensified in recent 
years, because microbial degradation that is responsible 
for clearing untreated oil spills is slow (Grangemard, et 
al., 2001). One promising method that has been 
researched into is the application of chemical fertilizers to 
augment for the mineral elements, particularly nitrogen 
and phosphorous’ limitations in the soil during biodegra-
dation (Margesin and Schinner, 1999). These fertilizers, 
especially in developing countries are not sufficient for 
agriculture, let alone for clearing oil spills.  

They also tend to result in soil hardening and fertility 
decline. It therefore, necessitates the search for cheaper 

and environmentally friendly options of enhancing crude 
oil biodegradation. The present study is therefore to 

investigate the potential of pig dung bacteria for crude oil 

 
 
 

 
degradation, as well as utilizing the pig dung for the 

probable stimulation of crude oil biodegradation in the 

soil. 
 
MATERIALS AND METHOD Collection 

and processing of samples 

 
The crude oil used was lagoma light crude oil, which was collected 
in sterile sampling bottles from Kaduna Refining and Petrochemical 
Company, Kaduna, Nigeria. The soil sample used was collected 
from the main campus of Wuhan University, Wuhan, PR China, 
transported to the laboratory and processed for use in the bioreme-
diation studies. The sample was air dried and sieved through 2 mm 
mesh size.  

The pig dung used was collected fresh from a pigsty at Eastlake, 
Wuhan, China. The dung was collected in thick polythene sheets 
and transported to the laboratory, and then sun dried for several 
days. After drying, the samples were ground and stored in the 
laboratory for further use. The biological and physicochemical 
properties of the pig dung used in this work were determined (Ijah 
and Antai, 2003) 

 
Isolation and identification of bacteria 
 
Isolation of bacteria was done following the modified methods of 
Solano-Solano et al. (2000). Ten grams of air-dried pig dung were 
thoroughly suspended in 100 ml sterile tap water, and then the 
supernatant was serially diluted with sterile tap water. Zero point 

one milliliter each of a serially diluted (10
-7

, 10
-5

, 10
-3

 and 10
-1

) pig 



 
 
 

 
Table 1. Biological and physicochemical properties of pig 

dung. 
 

Descriptor Value
m

 

pH 7.3 ± 0.2 

Moisture (%) 8.6 ± 0.9 

Nitrogen (%) 2.1 ± 0.6 

Phosphorous (%) 1.6 ± 0.3 

Calcium (%) 0.2 ± 0.1 

Magnesium (%) 0.5 ± 0.1 

Sodium (%) 1.9 ± 0.9 

Potassium (%) 1.8 ± 0.3 

Aerobic heterotrophic bacteria 1.8 x 10
8
 CFU g

-1
 

Crude oil utilizing bacteria 1.7 x 10
6
 CFUg

-1
 

 
m

Mean of three determinations. 
 

 
dung samples were spread inoculated onto nutrient agar (NA) and 
mineral salts medium supplemented with the crude oil (oil agar, OA) 
for the isolation of bacteria and enumeration of crude oil degrading 
bacteria respectively. The mineral salts medium (MSM) of Bushnell 
and Haas (1941) was employed, which has the following compo-  
sition: (1.0 g KH PO , 1.0 g K HPO , 1.0 g NH NO , 0.2 g MgSO .7H 
O, 0.05 g FeCl , 0.02 g CaCl .2H O in 1000 ml of distilled water at 
pH 7.0). The inoculated NA plates were incubated at room 

temperature (28 ± 2
o
 C) for 48 h, while the OA plates were 

incubated for 3 to 5 days at the same temperature. The bacterial 
colonies that appeared on the plates were counted, and distinct 
colonies were picked and purified by repeated sub-culturing.  

A number of microscopic and biochemical tests were carried out 
for identification purposes. The tests included gram reaction, shape, 
spore, motility, catalase, indole production, oxidase, methyl red-
voges proskauer (MR-VP), gelatin liquefaction, nitrate reduction, 
urease, citrate, starch hydrolysis, O/F of glucose, pigmentation and 
utilization of the following carbohydrates – glucose, sucrose, fruc-
tose, maltose, xylose, arabinose, mannitol and inositol. The isolates 
were identified by comparing their characteristics with those of 
known taxa, as recommended by Cowan and Lisbon (Ijah and 
Antai, 2003). 

 

Utilization of crude oil by the bacterial isolates 
 
The ability of the bacterial isolates to utilize crude oil as the only 
source of carbon and energy was determined by the method of 
Okpokwasili and Okorie (1988). 0.1 ml of 24 h old nutrient broth 
culture was inoculated into each test tube containing 10 ml of sterile 
MSM of Bushnell and Haas and 1% (v/v) crude oil. Control test 
tubes were set up containing 10 ml of MSM with 1% (v/v) crude oil, 
but had no added bacteria. The tubes were incubated at room 
temperature for sixteen days on an orbital shaker (HYA, Scientific 
Instrument Manufacturer, Wuhan) at 120 rpm. At the end of the 
incubation period, the growth of the isolates was determined by 
visual observation of the oil medium for turbidity, as compared to 
the control tubes (Olesnicky et al., 2002; Okpokwasili and Okorie, 
1988).  

The extent of degradation of the incorporated crude oil by the 
bacterial isolates was determined by the gravimetric analysis 
method of Odu (Ijah and Antai, 2003). The amount of crude oil left 
after the incubation time was determined by extracting the residual 
oil with 50 ml of toluene from the 10 ml culture. The mixture was 
separated using separatory funnel and then filtered off with What 

 
 
 
 

 
man filter paper. The optical density (absorbance) was read on a 
spectrophotometer (UV-120-02, Shimadzu, Japan) at 410 nm 
wavelength. Using a previously prepared standard curve, the weight 
of the crude oil was determined. The amount of crude oil degraded 
was calculated by subtracting the weight of residual crude oil from 
weight of the added (initial) crude oil, divided by the weight of the 
initial crude oil and then multiplied by 100. 

 

Measurement of crude oil biodegradation in soil amended with 

pig dung 
 
The rate of bacterial utilization of crude oil in the soil was assessed 
by using the the gravimetric method. 100 g of soil contained in 
screw-capped bottles in triplicates were each treated with 10% (v/w) 
crude oil. 10 g of pig dung were added to each bottle, and the soil 
moisture corrected by the addition of 10 ml of sterile double distilled 
water (Roman et al., 1996). A control experiment, without added 
chicken manure was set up. All the bottles were incubated for 16 
days at room temperature. At four days’ interval, the amount of 
weight loss of the crude oil was determined. 20 g of oil-polluted soil 
were weighed into sampling bottle and 100 ml of CCl4 added to 
each bottle. The oil-solvent mixes were separated using a separa-
tory funnel. The extracts were dried by adding 0.1g anhydrous 
Na2SO4 (Facundo, 2000; Mulligan et al., 2001), and then filtered 
through Whatman No. 1 filter paper. The CCl4 was allowed to 
evaporate off at room temperature in a fume hood. Weight loss was 
determined as described above.  

Gas chromatographic analysis was employed to confirm the 
results of the gravimetric method. The extractable crude oil was 
recovered and analyzed on gas chromatograph (hp, Paw) equipped 
with a CHROMOSORB, PA-AW 80 – 100 capillary column and 
flame ionization detector (FID). The operational parameters were: 

injection temperature of 400
o
C, helium carrier flow of 28 ml per 

minute, injection 1 µl. The oven temperature was set initially at 

50
o
C min

-1
. The major hydrocarbon compounds of the crude oil 

were identified on the basis of their retention time and by comparing 
them to those of analytical standards.  

To determine the pH, 10 g of the sample was mixed with 25 ml of 
sterile water in a beaker, stirred and allowed to stand for 30 min. 
The pH was then taken with the pH meter (HC21006, China). The 
mixture was stirred again, allowed to stand and pH retaken. A 
triplicate determination was performed. 

 

RESULTS 
 
The biological and physicochemical properties of the pig 
dung are presented in Table 1. Twenty-seven bacterial 
isolates were obtained from the pig dung as shown in 
Table 2. The identification of the isolares revealed them 
to belong to the species of Staphylococcus, 
Pseudomonas, Proteus, Acinetobacter, Campylobacter, 
Streptococcus, Klebsiella, Enterobacter, Bacillus, 
Micrococcus and Escherichia. Bacillus species were the 
most frequent isolated bacteria, which constituted about 
15% of the total isolates. The results in Table 2 reveal the 
extent of growth, as well as the amount of crude oil 
degraded by the bacterial isolates from pig dung. Twelve 
(44.4%) isolates, out of the total isolates were able to 
utilize crude oil as sole source of carbon and energy. A 
Bacillus sp degraded the crude oil at a relatively high rate 
of 63.5% after the incubation time of 16 days. The other 
crude oil utilizers belong to the genera of Pseudomonas, 
Micrococcus, Proteus, and Klebsiella. It is however, 



 
 
 

 
Table 2. Extent of growth and degradation of crude oil by 

bacterial isolates from pig dung. 
 

 Growth in Crude oil 
 crude oil degraded 

Bacterial isolates MSM (%)
m

 

PGB07 Bacillus sp +++ 63.5 ± 1.4 

PGB12 Pseudomonas ++ 43.4 ± 2.2 

aeruginosa ++ 40.6 ± 1.3 

PGB19 Micrococcus sp ++ 39.7 ± 0.5 

PGB27 Bacillus sp ++ 37.6 ± 0.6 

PGB03 Bacillus sp + 22.8 ± 1.5 

PGB05 Pseudomonas sp + 15.5 ± 0.2 

PGB09 Micrococcus sp + 13.2 ± 0.8 

PGB11 Proteus sp + 13.2 ± 0.6 

PGB15 Klebsiella sp + 13.0 ± 0.4 

PGB24 Micrococcus sp + 11.5 ± 1.2 

PGB23 Proteus sp + 11.5 ± 1.1 

PGB17 Klebsiella sp - 0 

PGB01 Escherichia coli - 0 

PGB02 Bacillus sp - 0 

PGB04 Campylobacter sp - 0 

PGB06 Acinetobacter sp - 0 

PGB08 Enterobacter sp - 0 

PGB10 Proteus sp - 0 

PGB13 Campylobacter sp - 0 

PGB14 Enterobacter sp - 0 

PGB16 Streptococcus sp - 0 

PGB18 Staphylococcus sp - 0 

PGB20 Staphylococcus sp - 0 

PGB21 Campylobacter sp - 0 

PGB22 Pseudomonas sp - 0 

PGB25 Streptococcus sp - 0 

PGB26 Staphylococcus sp   
 

+++ Heavy growth; ++ moderate growth; + little growth; - no 

growth. 
m

Mean of three determinations. 

 

 
Table 3. The amount of crude oil degraded in soil amended 

with pig dung. 
 

Incubation Weight loss (%)
m

 of crude oil in soil 

time (days) Pig dung  No pig dung 

4 13.5 ± 0.5  10.4 ± 0.2 

8 33.7 ± 1.8  24.3 ± 0.6 

12 56.9 ± 2.2  38.6 ± 1.4 

16 68.2 ± 1.9  50.7 ± 1.6 
 

m
Mean of three determinations. 

 

 

shown that fifteen of the isolates were not able to 
degrade the crude oil.  

The results of the weight loss of oil from the soil treated 

with crude oil and amended with pig dung are presented 

 
 
 
 

 

in Table 3. At the end of the incubation period, 68.2% of 
crude oil was degraded in soil amended with pig dung as 
against 50.7% of same oil in unamended soil after 
incubation period.  

These were confirmed by the results obtained from gas 
chromatograph in Figure 1 (crude oil amended with pig 
dung) and Figure 2 (control; crude oil not amended with 
pig dung), where it is shown that the bacterial population 
contained in pig dung utilized almost all the components 
of the crude oil. The fractions of isoprenoids (pristane and 
phytane) were however, not attacked by the bacterial 
community in the pig dung after 16 days of incubation. 
The pattern nonetheless shows that the microorganisms 
first attacked the lower and higher hydrocarbon chains 
and those of middle length were attacked later in the 
course of incubation. 

The pH values obtained in soils treated with crude oil 
and amended with pig dung reveal that the pH of control 
soil ranged between 5.6 and 5.8 (Table 4) . While that of 
the unamended soil ranged between 5.7 and 6.0 after 
sixteen days of incubation. The pH of the amended soil 
increased from 6.2 at time zero of sampling to 7.2 over 
same period, which is from acidic to slightly alkaline pH 
(Table 4).  

There were significant (P < 0.05) differences among the 

treatments and the period of incubation with respect to 
weight loss of the crude oil. A linear correlation was 
observed between the gas chromatographic rates of 
biodegradation and weight losses of crude oil in soils 
amended with chicken manure. 

 

DISCUSSION 
 
Many types of microorganisms have been reported to be 
present in pig dung, which include bacteria and fungi 
(Laukova, 2000; Mansour et al., 1999). This is in addition 
to the mineral nutrients, which include nitrogen and 
phosphorous in the sample for microbial growth and 
metabolism. In this present study, eleven genera of 
bacteria were identified from the pig dung sample used, 
which were mainly enterics. Though, Bacillus species 
was the most predominant isolated bacterial species, it 
prevalence could be attributed to the fact that it forms 
spores, which help microorganisms to withstand harsh 
conditions, such as sun drying employed in this work. 
Isolation of Bacillus species from animal dung could also 
be attributed to its ubiquitous distribution in nature. 
Mansour et al. (1999) reported the isolation of Bacillus, 
Acinetobacter, Staphylococcus and Enterobacter among 
other bacteria from pig dung.  

There are volumes of literature on bacterial degradation 
of crude oil in the ecosystem. However, what is inte-
resting is the source of the oil degrading bacteria in the 
natural ecosystem. It is interesting to find that pig dung, in 
addition to being rich in mineral elements nitrogen and 
phosphorous (Diez et al., 2001)] necessary for crude oil 
biodegradation, also contain bacteria with varying de- 



    

 Table 4. The pH reactions of soil treated with crude oil and amended with pig dung. 
      

    pH values
m

  

  Incubation Non polluted, not  Crude oil polluted soil 
  time (days) amended soil Crude oil polluted soil amended with pig dung 

  0 5.6 ± 0.1 5.7 ± 0.1 6.2 ± 0.1 

  4 5.7 ± 0.0 5.8 ± 0.0 6.9 ± 0.0 

  8 5.8 ± 0.3 5.9 ± 0.1 6.9 ± 0.2 

  12 5.9 ± 0.1 6.0 ± 0.1 7.1 ± 0.4 

  16 5.8 ± 0.2 5.9 ± 0.2 7.2 ± 0.1 
 

m
Mean of three determinations.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. Gas chromatograph of lagoma light crude oil 

amended with pig dung after, 4, 8, 12 and 16 days of 

incubation at 28
o
C. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Gas chromatograph of lagoma light crude oil 

(control) not amended with pig dung after, 4, 8, 12 and 

16 days of incubation at 28
o
C. 

 

 

grees of crude oil degrading capabilities. This capability is 
shown here to be further widespread among the bacteria 
present in the pig dung. The oil-utilizing bacteria isolated 
from the pig dung in the present work have previously 
been implicated in crude oil biodegradation, 

 

 

though from different sources (Ijah and Antai, 2003; Ijah, 
1998). 

Stimulated biodegradation of crude oil is at present 

being encouraged because it ensures rapid remediation 

of oil-polluted ecosystems (Ijah and Antai, 2003). Most 



 
 
 

 

often, chemical fertilizers are used to enhance microbial 
degradation of crude oil in the natural environments. 
Mineral elements have been reported to be essential for 
crude oil biodegradation (Mitchell, 1999; Atlas, 1995). 
Apart from the oil utilizing bacteria in animal dung, if 
manure is spread back on the land, not only will it add 
much needed nutrients, but also organic matter, which is 
not found in chemicals. Therefore, the addition of pig 
dung to the soil enhanced the rates of crude oil 
biodegradation.  

Also, the bacterial consortium in the pig dung attacked 
and degraded almost all the components of lagoma light 
crude oil, except the fractions of isoprenoids (pristane 
and phytane) that were resistant to bacterial attack. Not a 
single bacterium can degrade all the component fractions 
of any crude oil, and significant oil biodegradation is 
normally achieved by microbial synergy. Pig dung could, 
therefore, harbour both primary and secondary crude oil 
biodegraders. As such, the use of pig dung may be a 
promising potential in integrated oil spill abatement.  

Furthermore, the pig dung raised the pH of the soil to a 
range between 6.2 and 7.2, which is ideal for most crude 
oil utilizing bacteria (Vidali, 2001) . This indicates that the 
pig dung had a buffering effect on the soil, and since 
strong acidity is a limitation in biodegradation, it would 
have contributed to the enhanced crude oil degradation in 
the soil. The use of pig dung to stimulate crude oil bio-
degradation in the soil could be one of the severally 
sought environmentally friendly ways of eliminating 
petroleum hydrocarbon in the natural ecosystem. 
 

 
REFERENCES 
 
Atlas MR (1995). Bioremediation of petroleum pollutants. Inter. 

Biodeterior. Biodegrad. 35: 317-327. 
Bushnell LD, Haas HF (1941). The utilization of certain hydrocarbons by 

microorganisms. J. Bacteriol. 41: 653-673. 
Diez JA, de la Torre AI, Cartagena MC (2001). Waste management 

evaluation of the application of pig slurry to an experimental crop 
using agronomic and ecotoxicological approaches. J. Environ. Qual. 
30: 2165-2172.  

Grangemard I, Wallach J, Marget-Dana R, Peypoux F (2001).  
Licenysin:  a  more  efficient  cation  chelator  than  surfactin.  Appl. 
Biochem. Biotechnol. 90:199-210. 

Ijah UJJ (1998). Studies on relative capabilities of bacterial and yeasts 
isolates from tropical soil in degrading crude oil. Waste Manage. 18: 
293-299. 

Ijah UJJ, Antai SP (2003). The potential use of Chicken-drop 

microorganisms for oil spill remediation. The Environmentalist. 23: 

89-95 

 
  

 
 

 
Laukova A (2000). In vitro treatment of different isolates from cattle 

dung and pig slurry by nisin. Acta Vet Brno. 69: 147-151. 
Mansour MA, Bottefroy D Linder A (1999). Inhibition of Bacillus 

lincheniformis spore growth in milk by nisin, monolaurin, and pH 
combinations. J. Appl. Microbiol, 86: 311-324. 

Margesin R, Schinner F (2001). Bioremediation (natural attenuation and 
biostimulation) of Diesel-Oil-Contaminated soil in an Alpine Glacier 
Skiing area. Appl. Environ. Microbiol. 67(7): 3127-3133. 

Margesin R, Schinner F (1999). A feasibility study for the in situ 
remediation of a former tank farm. World J. Microbiol. Biotechnol. 15: 
615-622. 

Mitchell JG (1999). In the wake of the spill: ten years after Exxon  
Valdez. Natl. Geogr. 195(3): 96-117.  

Okpokwasili GC, Okorie BB (1988). Biodeterioration potentials of 
microorganisms isolated from engine lubricating oil. Tribol. Inter. 21: 
215-217. 

Olesnicky EC, Miller L, Fukunaga J, Laskin AI (2002). Biodegradation of 
brominated biphenyls by members of the genus Sphingomonas (C). 
In: American Society for Microbiology. 102nd General meeting, Salt 
lake city, Utah. pp. 19-23.  

Plohl K, Leskovšek H, Bricelj M (2002). Biological degradation of motor 
oil in water. Acta Chim. Slov. 49: 279-289.  

Roman R, Caballero R, Bustos A, Diez JA, Cartagena MC, Vallejo A. 
Caballero A (1996). Water and solutes movement under conventional 
corn in central Spain: I. Water balance. Soil. Sci. Soc. Am. J. 60: 
1530-1536.  

Solano-Serena F, Marchal R, Casarégola S, Vasnier C, Lebeault JM, 
Vandecasteele JP (2000). A Mycobacterium strain with extended 
capacities for degradation of gasoline hydrocarbons. Appl. Environ. 
Microbiol. 66(6): 2392-2399.  

Vidali M (2001). Bioremediation. An overview. Pure Appl. Chem. 73(7):  
1163-1172. 


