Petroleum is a major source of energy globally. Wide scale production, transport,
use and disposal of petroleum globally have made it a lead contaminant in both
prevalence and quantity in the environment (Rahman et
al., 2002). The release of these compounds poses a threat to water and
soil ecosystems. Consequently, many techniques are being developed to clean
up petroleum polluted environment. Biodegradation of hydrocarbon-contaminated
soils which exploits the ability of microorganisms to degrade and/or detoxify
organic contamination, has been established as an efficient, economic, versatile
and environmentally sound treatment (Mehrasbi et al.,
2003). The biological treatments are more efficient and cheaper than chemical
and physical ones. In relation to biological treatment, the bioremediation technology
is being employed for the degradation of crude oil in soil matrix through microorganisms
able to transform petroleum hydrocarbons in less toxic compounds. However, the
low solubility and adsorption are two major properties of high molecular weight
hydrocarbons that limit their availability to microorganisms (Millioli
et al., 2009). Compost contributes organic matter to the soil that
may serve as a source of nutrient among other functions for the various microbes
that inhabit the soil. Compost is a very rich source of nitrogen that maintains
and enhances the fertility and productivity of agricultural soils. Studies carried
out both in microcosms and field experiments showed that organic amendments
not only act by improving soil structure and serving as a source of nutrients,
they can also strongly enhance the activities of microflora (Crecchio
et al., 2001). In this study, microorganisms with the capacity to
utilize crude oil as carbon and energy source were isolated from soil composts.
The biodegradation potential of the compost-inhabiting microorganisms was also
estimated in order to assess the applicability of composts in bioremediation
of hydrocarbon-polluted sites.
MATERIALS AND METHODS
Sample collection: Soil samples were collected randomly and homogenized from five different spots at Igando dumpsite (31N 0527762 and UTM 0726400; 31N 0527835 and UTM 0726326; 31N 0527778 and UTM 0726326; 31N 0527723 and UTM 0726340; 31N 0527894 and UTM 0726394) and Olusosun dumpsite at Ojota (31N 054566 and UTM 0728806; 31N 0541634 and UTM 0728963; 31N 0541562 and UTM 0728963; 31N 0541758 and UTM 0728893; 31N 0541732 and UTM 0728700), Lagos, Nigeria, respectively. The soil samples were collected at a depth of 0-10 cm below the soil surface into sterile McCartney bottles and transported to the laboratory. Physicochemical and microbiological analysis commenced immediately upon arrival in the laboratory.
Source of crude oil: The Escravos crude oil (dark brown in colour) used in this study was obtained from Chemistry Department, Faculty of Science, University of Lagos.
Physicochemical analysis of soil samples: The pH of the soil samples
was determined with a pH meter (Jenway 3051) in 1:1 soil solution in distilled
water. The moisture level, organic content, total nitrogen content, potassium
content and available phosphorous were determined at the Department of Chemistry,
University of Lagos, Nigeria as described previously (Obayori
et al., 2008; Onifade et al., 2007;
Jones et al., 1983).
Microbiological analysis: The total heterotrophic bacterial and fungal
counts were enumerated by plating aliquots (0.1 mL) of appropriate diluted soil
samples on nutrient agar and potato dextrose agar containing streptomycin (1
mg 100-1 mL), respectively. Starch casein agar was employed in determining
the population density of actinomycetes according to the method of Kuster
and Williams (1964). The nitrogen fixing bacterial counts were estimated
using the Ashbys mannitol agar. All inoculated plates were incubated aerobically
at room temperature (30°C) counted after 48, 96, 120 and 168 h for bacteria,
fungi, actinomycetes and nitrogen fixers, respectively. Similarly, the population
of hydrocarbon-utilizers was estimated on Mineral Salts (MS) medium formulated
by Kastner et al. (1995). The medium contained
(in g L-1) Na2HPO4: 2.13 g, KH2PO4:
1.30 g, NH4Cl: 0.50 g and MgSO4.7H2O: 0.20
g. Sterile trace elements solution (1.0 mL L-1) of Bauchop
and Elsden (1960) was aseptically added to the medium after sterilization.
The pH of the medium was adjusted to 7.2 and 5.6 for bacterial and fungal estimations,,
respectively. The MS medium was also fortified with 1 mg 100 mL-1
of streptomycin for fungi. Sterile crude petroleum served as the sole carbon
and energy source and made available to the cultures through vapour-phase transfer
(Amund et al., 1987). Plates were counted after
incubation at room temperature for 5-7 days.
Crude oil utilization by the isolates: The ability of the isolates to
utilize crude oil was confirmed by inoculating each isolate in separate cotton
plugged 250 mL Erlenmeyer flasks containing sterile Liquid Minimal Salts (MS)
medium. The liquid MS medium and crude oil were autoclaved separately at 121°C
for 15 min. Sterile crude oil which served as source of carbon and energy was
added at 1% (v/v) to make up a final volume of 100 mL sterile liquid MS medium.
Each isolate was subsequently inoculated in separate flask of the medium. Control
flask containing the liquid MS medium and 1% (w/v) of crude oil but without
organism was also prepared. The flasks were monitored and agitated daily for
a period of 14 days. Isolates with the highest turbidity were selected for further
study. An aliquot of each isolate was transferred to fresh medium containing
1% (w/v) of crude oil to re-confirm their ability to utilize crude oil.
Characterization and identification of the isolates: The isolates were identified on the basis of their cultural, cellular morphology and biochemical characteristics using API kit.
Biodegradation study: The biodegradation study was performed in liquid
MS medium as described by Omotayo et al. (2011).
The growth medium used was prepared as described above. The isolates were inoculated
into each flask and incubated in the dark at about 30°C with constant shaking
for 30 days. Total Viable Counts (TVC) and the emulsification index were monitored
at 6 days intervals. The residual hydrocarbon content before and after biodegradation
was analyzed by gas chromatography.
Emulsification index: In determining the emulsification index, 2 mL
of the medium was centrifuged at 3000 rpm for 15 min to separate the cells.
The supernatant was collected in a test tube while the cells were discarded.
The emulsification stability of the isolates was determined by adding 2 mL of
crude oil to the test tube containing the spent media. The tubes were properly
vortexed at high speed for 2 min and allowed to stand for 24 h. The emulsification
index was calculated as the height of the emulsion divided by the total height
of supernatant with added oil multiplied by 100 (Abbasi
and Amiri, 2008):
Soil microcosm study: Biodegradation of the crude oil polluted soil
sample by axenic and mixed cultures of the isolates was assessed. Two hundred
grams of the soil was placed in 4 aluminum trays (10 cm diameter and height)
and sterilized by autoclaving at 121°C for 1 h. The sterile soil samples
were artificially contaminated with 10 mL crude oil to simulate 5% (v/w) crude
oil pollution (Vidali, 2001). Two of the trays were
each inoculated with 5 mL suspension of the pure isolates containing 109
CFU mL-1 of the isolates. A third tray was inoculated with bacterial
consortium containing 2.5 mL of each inoculum preparation. A fourth tray was
taken as control to determine natural attenuation; it had no inoculum. Sterile
distilled water was added to the soil to achieve a moisture content of 30% of
the water holding capacity of the soils (Vidali, 2001).
The microcosms were kept in the glass-house to minimize loss of moisture via
evaporation and monitored for 30 days. Residual hydrocarbon content of the soil
microcosms were determined by gas chromatography at the end of the biodegradation
Gas chromatographic (GC) analysis: Gas chromatography was used to determine
the residual hydrocarbon present in the media after 30 days of incubation. A
standard profile was first obtained by injecting 1 mL of the hydrocarbon standard
into the GC and a chromatogram was generated to serve as a calibration window
with which the test sample was analyzed. After generating the standard profile,
10 mL of the sample was extracted with 10 mL Hexane and was concentrated to
1 mL (test sample) from which 1 μL was injected into the GC and an equivalent
chromatogram was generated. The peak areas of the standard and the test sample
chromatogram were compared with respect to the concentration of standard of
the sample (Odebunmi et al., 2002). This is given
Substrate specificity test of the isolates: The ability of the isolates to utilize pure hydrocarbon substrates was tested by adding 1% v/v of liquid and 1% w/v of solid hydrocarbons to minimal salts medium to which the isolates had been inoculated in Erlenmeyer flasks. The flasks were incubated with constant shaking at 30°C for 14 days. A control flask was set up for each hydrocarbon. Degradation was monitored by measuring increase in optical density at a wavelength (λ) 520 nm.
Physicochemical parameters of soil samples: The soil from Ojota (pH 6.16) was found to be more acidic compared to the soil from Igando dumpsite (pH 7.72) (Table 1), while the moisture content of the soil from Igando (12.8%) was higher when compared to Ojota soil sample (7.63%). The Total Organic Carbon (TOC), percentage nitrogen and phosphate content and salinity of Igando sample (10.20, 0.22, 0.11 mg kg-1 and 0.11%, respectively) were higher than those of Ojota sample (1.17, 0.06, 0.10 mg kg-1 and 0.06%, respectively). However, the nitrate content of Ojota (0.31) was slightly higher than Igando (0.22).
Enumeration of microbial populations: The microbial populations of Igando soil sample were higher compared to Ojota soil sample. The population per gram of hydrocarbon utilizers in Igando sample were also observed to be higher compared to that of Ojota sample. Likewise, the proportion of hydrocarbon utilizers in the Igando soil sample (0.37%) was found to be higher when compared to Ojota sample (0.34%). The population of nitrogen fixers and actinomycetes were also shown to be higher for Igando soil sample than Ojota soil sample (Table 2).
|| Physicochemical parameters of the soil samples
|PO43-: Phosphate content, NO3:
Nitrate content, TOC: Total organic carbon, K: Potassium content
|| Microbial population of soil samples
|Values are in (CFU g-1)
Characterization and identification of crude oil-utilizing isolates: The isolates were identified using colonial morphology, biochemical tests and the use of API test kit. Tentative identification shows the isolates to be Micrococcus sp., Corynebacterium sp., Bacillus sp., Enterobacter sp., Pseudomonas sp., Alcaligenes sp., Flavobacterium sp., Moraxella sp., Aeromonas sp., Acinetobacter sp., Aspergillus sp. and Penicillium sp. However, Corynebacterium ulcerans, Bacillus badius, Corynebacterium amycolatum and Micrococcus varians were selected for further studies based on their capability to grow well in the crude oil used as carbon and energy source.
Biodegradation study: During the time-course analysis, the Total Viable Counts and Emulsification Index (EI24) were monitored. The results are shown in Fig. 1. The population density of Micrococcus varians shows that there was a considerable period of lag phase of about 15 days before the isolate entered the exponential phase of growth. The population density of Micrococcus varians increased steadily from 7.00x106 CFU to 7.40x1012 CFU mL-1 of medium in 21 days before reaching the stationary phase. The emulsification index rose steadily from 30 to 48.84. Micrococcus varians had a growth rate constant (μ) of 0.027 and grew with a mean generation time (g) of 25.5 h (Table 3).
The population density of Bacillus badius increased steadily in 6 orders of magnitude from 1.99x105 CFU to 4.10x1011 CFU mL-1 of the medium in 24 days before reaching the stationary phase. The emulsification index rose steadily from 30 to 48.23. The growth rate constant (μ) of Bacillus badius in the liquid medium was 0.025 and it grew with a mean generation time (g) of 27.5 h (Table 3).
Corynebacterium ulcerans had a brief lag phase of about 3 days before entering the exponential phase of growth. The population density of the isolate increased from 1.96x107 CFU to 1.20x1012 CFU mL-1 of the medium in 24 days before reaching the stationary phase of growth. The emulsification index rose from 30 to 48.80. Corynebacterium ulcerans had a growth rate constant (μ) of 0.019 and grew with a mean generation time (g) of 36.2 h (Table 3).
The population density of Corynebacterium amycolatum increased from 6.00x105 CFU to 3.60x1011 CFU mL-1 of the medium before reaching the stationary phase. The emulsification index rose from 30 to 48.84. Corynebacterium amycolatum had a growth rate constant (μ) of 0.023 and grew with a mean generation time (g) of 30.0 h (Table 3).
The growth kinetics of the isolates is presented in Table 3.
Micrococcus varians had the lowest doubling time (25.5 h) in the liquid
medium followed by Bacillus badius, Corynebacterium amycolatum
and Corynebacterium ulcerans with mean generation times of 27.5, 30.0
and 36.2 h, respectively.
||Degradation of crude oil by (a) Micrococcus varians (b)
Bacillus badius (c) Corynebacterium ulcerans and (d) Corynebacterium
|| Growth kinetics data of isolates in crude oil liquid culture
The highest number of generations was given by Micrococcus varians
with 16.94 generations followed closely by Bacillus badius with 15.71
generations. Corynebacterium ulcerans and Corynebacterium amycolatum
gave 9.95 and 9.60 generations, respectively.
Gas chromatographic profiles: The gas chromatograms of the biodegradation experiment after 30 days showed reduction in the concentration of residual hydrocarbon present in the media. The concentration of hydrocarbon present at the start of experiment was 3395.82 mg L-1, the value of residual hydrocarbon in liquid cultures of Corynebacterium ulcerans, Bacillus badius, Corynebacterium amycolatum and Micrococcus varians were 400.15, 366.14, 311.59 and 234.24 mg L-1, respectively at day 30 of the experiment. Micrococcus varians gave the highest degradation value of 93.10% followed closely by Corynebacterium amycolatum with a rate of 90.82%. Bacillus badius and Corynebacterium ulcerans gave degradation rates of 89.22 and 88.22%, respectively (Table 4).
|| Residual hydrocarbon content in crude oil liquid culture
medium at the end of 30 day incubation period
|| Residual crude oil in Ojota soil microcosm at the end of
30 day biodegradation
|| Substrate specificity of the isolates
|+: Poor growth (<0.2 μm), ++: Moderate growth (0.2-0.3
μm), +++: Excellent growth (>0.3 μm)
Gas chromatographic analysis was carried out on the microcosm to determine the amount of residual hydrocarbon in the soil after 30 days of degradation. The amount of hydrocarbon present in the control soil at day 0 was 3627.75 mg kg-1, the residual hydrocarbon at day 30 in the control soil was 3167.36 mg kg-1 thus, 12.69% of the total hydrocarbon in the trays was lost due to natural degradation. The residual hydrocarbons in trays of Micrococcus varians, Corynebacterium amycolatum and consortium (Micrococcus varians and Corynebacterium amycolatum) were 490.39, 415.82 and 374.69 mg kg-1, respectively. The consortium had the highest effective biodegradation rate (76.98%) followed by Corynebacterium amycolatum (75.85%) and Micrococcus varians (73.78%), respectively (Table 5).
Substrate specificity tests: The ability of the isolates to degrade different hydrocarbon substrates was observed for a period of 14 days at room temperature. The optical density of the liquid culture medium of the isolates was measured to determine the growth of the isolates in the broth medium. Micrococcus varians, Bacillus badius and Corynebacterium ulcerans had excellent growth on anthracene and engine oil but poor growth on pyrene, toluene, naphthalene, dodecane and xylene. Corynebacterium amycolatum however, had poor growth on all the hydrocarbons tested (Table 6).
In this study, microorganisms capable of degrading crude oil were isolated
from active composted soil samples. The bacterial, actinomycetes and fungal
populations obtained were higher in Igando sample compared to Ojota sample.
The microbial population density of Igando soil sample was higher than that
of Ojota probably because the soil from Igando contained more nutrients compared
to the sample from Ojota. As shown in Table 1 the nitrogen
content, phosphate and total organic carbon of Igando sample were relatively
higher than that of Ojota study site. Anthropogenic impacts, such as changes
in nutrient composition, have the potential to directly or indirectly affect
the bacterial and fungal composition of the soil (Rousk
et al., 2009). Likewise, Igando sample had more moisture which favours
microbial growth than Ojota sample.
The proportion of hydrocarbon utilizers in the Ojota and Igando samples were
0.34 and 0.37%, respectively. It has been reported that population levels of
hydrocarbon utilisers and their population within the microbial community appear
to be a sensitive index of environmental exposure to hydrocarbons (Rahman
et al., 2002). In unpolluted ecosystem, hydrocarbon utilizers generally
constitute about 0.1% of the microbial community and in oil polluted ecosystems
they can constitute up to 100% of the viable microorganisms (Rahman
et al., 2002). The microbial populations quantitatively reflect the
degree or extent of exposure of that ecosystem to hydrocarbon contamination
(Atlas, 1981). The low proportion of hydrocarbon utilizers
compared to the total heterotrophic population indicates that the soil ecosystem
from which the samples were obtained probably had not been exposed to heavy
and consistent crude oil pollution. The compost soil samples used in this study
harboured hydrocarbon utilizers which include Micrococcus sp., Corynebacterium
sp., Bacillus sp., Enterobacter sp., Pseudomonas sp.,
Alcaligenes sp., Flavobacterium sp., Moraxella sp.,
Aeromonas sp., Acinetobacter sp., Aspergillus sp., Penicillium
sp. and other species. The flora reflects the diverse heterotrophic bacteria
present in compost soil and the diversity could be as a result of the varied
sources of the refuse, dumped at the sites. The genera Micrococcus, Bacillus,
Streptomyces, Actinomyces, Azotobacter, Aspergillus, Penicillium and Trichoderma
was mentioned by Ryckeboer et al. (2003)
in their study as part of the microbial flora of compost. Several hydrocarbon
degrading organisms have been isolated from diverse environments; soil and aquatic
sources which are the two major environments affected by hydrocarbon pollution
(Mittal and Singh, 2009) and their isolation is not
restricted to hydrocarbon-bearing environments. The hydrocarbon utilizing isolates
obtained in this study all had varied degree of degradation however, Micrococcus
varians, Bacillus badius, Corynebacterium ulcerans and Corynebacterium
amycolatum appeared to be the fastest growing species in crude oil. The
study of Barathi and Vasudevan (2001) identified the
above genera among hydrocarbon degrading microorganisms. Several other workers
also reported on the above genera as hydrocarbon degrading microorganisms (Atlas,
1981; Leahy and Colwell, 1990; Banat
et al., 2000).
The isolates showed different rates of growth in hydrocarbon liquid media.
The population density of the isolates started from a range of between 1.99x105
and 1.96x107 CFU mL-1 to between 3.60x1011
and 7.4x1012 CFU mL-1. Micrococcus varians had
the highest growth rate constant (0.025) with a mean generation time of 25.5
h compared to the other isolates. This result agrees with that of Rahman
et al. (2002) in which Micrococcus sp. had a very good growth
on crude oil. This was followed closely by Bacillus badius with a mean
generation time of 27.5 h.
The Gas Chromatographic (GC) analysis of the residual crude oil showed that
Micrococcus varians demonstrated the highest degree of crude oil degradation
(93.10%) by having the lowest residual crude oil (234.24 from 3395.82 mg L-1)
at the end of the 30 day incubation period. Bacillus badius, Corynebacterium
ulcerans and Corynebacterium amycolatum exhibited biodegradative
capabilities of 89.22, 88.22 and 90.82%, respectively. The high rate of hydrocarbon
degradation by Micrococcus varians could emanate from the massive growth
and enzyme production responses during the growth of the isolate. This is supported
by the reports of Bogan and Lamar (1996) which showed
that extracellular enzymes of organisms are produced in response to their growth
From the GC analysis of residual hydrocarbon present in the microcosms, the
biodegradation rates of the Micrococcus varians (73.78%) and Corynebacterium
amycolatum (75.85%) were less compared to the biodegradation rates in the
liquid media (93.10 and 90.82%, respectively). This might be due to the bioavailability
of the crude oil to the organisms. The bioavailability of hydrocarbons has been
shown to greatly affect the extent of biodegradation of the hydrocarbons due
to sorption onto soil particles (Leahy and Colwell, 1990).
The bacterial consortium showed a degradation percentage (76.98%) of degradation
of crude oil after 30 days of incubation.
The isolates had good growth on anthracene and engine oil as hydrocarbon substrates
while they exhibited poor growth on the other hydrocarbons tested. The engine
oil is a mixture of alkanes and it contains heavy chains (C18-C40).
The alkanes are the most abundant compounds and are simpler to oxidize. Aliphatic
hydrocarbons are degraded with greater speed but the key step involves oxidation
of the molecule to increase their solubility (Silva et
al., 2006). Biodegradation rates have been shown to be highest for the
saturates, followed by the light aromatics, with high-molecular-weight aromatics
and polar compounds exhibiting extremely low rates of degradation (Leahy
and Colwell, 1990).
An effective degradation of crude oil would require simultaneous action of several metabolically versatile microorganisms with favourable environmental conditions such as pH, temperature and availability of nutrients. The high nitrogen content is a major factor that contributes to the degradation of hydrocarbon in composts. The organisms isolated in the course of this study showed appreciable degree of degradation of the crude oil used. The natural microbial community of the compost soil includes a variety of microorganisms that can degrade, alone or together, most crude oil components. Thus, bioremediation of oil polluted fields could be achieved using indigenous hydrocarbon utilizers of the compost soil and the process could be enhanced by supplementing the polluted environment with compost.