Nigerian Journal of Basic and Applied Science (2009), 17(1).

ISSN 794-5696

Effects of Lead Concentrations on Biostimulation of Oil-Contaminated Soil

*1M.N. Almustapha, 2S.I. Kaura, 1L.G. Hassan, 3M.B.Yerima, 4I. Abdulkadir, 5A.Salihu
1
Department of Pure and Applied Chemistry, Usmanu Danfodiyo University, Sokoto
2
Department of Petroleum Resources, Nigeria
3
Department of Microbiology, Usmanu Danfodiyo University, Sokoto
4
Department of Chemistry, Ahmadu Bello University, Zaria
5
Department of Biochemistry, Ahmadu Bello University, Zaria
[*Corresponding author: [email protected] GSM: +2348035548611]

ABSTRACT: The inhibitory effects of lead on bioremediation were studied. The addition of
fertilizer as a nutrient in oil contaminated soil enhanced the microbial activity. Different
concentrations of lead influenced the rate as well as activities of microorganisms and the overall
biodegradation processes. About three times decrease in number of cells per milliliter was observed
at lead concentration of 1mg/g soil compared to control. Therefore, for successful bioremediation
lead concentrations must be minimized for the stimulation of microorganisms by nutrient addition as
well as for biodegradation.

KEY WORDS: Bioremediation, heavy metals, Lead, microorganisms Oil contaminated soil

INTRODUCTION biodegradetion processes (Leahy and Colwell,

The extensive use of petroleum products has 1990; Atlas and Bartha, 1992). The presence of
resulted in the contamination of all recalcitrant compounds such as heavy metals in
compartments of the environment. Thus, the sites contaminated with crude oil and other
need for urgent and sustainable action to organic wastes may create problems, especially
remediate these contamina-ted sites (Wilson & for authorities that depend on bioremediation as
Jones, 1993). Other activities related to the main strategy for pollution alleviation. In
petroleum exploration, production, general, available technologies for metal
transportation, processing, storage and use also remediation in soil are often destructive and
contribut to the environmental problems (Sarkar costly, and usually total removal of all metals is
et al., 2005). sometimes not feasible as it lasts for several
years (Al-Saleh and Obuekwe, 2005).
Different methods are employed for the
treatment of contaminated lands (Lewis et al., To warrant application, the bioremediation
2004). These include: physical, chemical and process should demonstrate that removal of
biological methods (Singh et al., 2005). contaminants is the primary effect of
Biological treatment process (Bioremediation) biodegrade-tion, and that the degradation rate is
is a promising technology currently in use with greater than the normal rate of decontamination.
the advantages of cost effectiveness the The difficulties of developing bioremediation
potential to remediate the environment without strategies lie in achieving better results in the
causing much damage (Riser-Roberts, 1998; field as in the laboratory (Bento et al., 2005).
Kim et al., 2001). In this process, contaminants
are destroyed or reduced by microorganisms This study used laboratory-based experiments
(Sabate et al., 2004). to ascertain the effect of lead at varying
concentra-tion on bioremediation of crude-oil
A number of factors affect the biodegradation contaminated soil.
of hydrocarbons in the environment. These
include the composition of the hydrocarbons MATERIALS AND METHODS
(contami-nants), physical state of the Exactly 10.0g of the homogenized soil sample
hydrocarbons, temperature, oxygen, nutrients (some of its properties are as shown in Table 1)
availability, nature and distribution of the were measured in 500 cm 3 Erlenmeyer flask
microorganisms. Others like pH, salinity, containing 100 cm3 sterile mineral salt medium
pressure, water activity etc. affect both the and 1% (v/v) crude oil (Adebusoye, et al.,
distribution of microorganisms and 2007). Each flask was shaken at room

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temperature on a rotary shaker at 170 rpm for 2 Model 2380) and the concentration was found
weeks (Adebusoye et al., 2007; Singh and Lin, to be below sublethal levels (<17.5 μg/g).
2008). The composi-tion of the mineral medium
(g/L) was: 1.0g K2HPO4, 1.0g NH4NO3, 0.2 g RESULTS
MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.1 g NaCl, In this report, a preliminary biodegradation
0.01g FeCl3.6H2O. Each experimental set was study was carried out to determine the
repeated thrice according to the following: degradation capabilities of indigenous
microbial cultures using fertilizer amendment at
Group A: Garden soil + crude oil. two different lead concen-trations. Investigation
Group B: Garden soil + Fertilizer (N & P) + of the culture compositions using TLC-FID
Crude oil. (Iatroscan) reveal varying amount of
Group C: Contaminated soil (of high heavy hydrocarbons in form of saturates and
metals concentration) + Crude oil. aromatics. Thus, culture composi-tion, together
Group D: Contaminated soil (of high heavy with agitation and aeration for 2 weeks allowed
metals concentration) + Fertilizer (N compositional degradation of the crude–oil
& P) + Crude oil. contaminated soil by micro organisms (Figure
Group E: Contaminated soil (of low heavy 1). Hydrocarbon components from Iatroscan
metals concentration) + Crude oil. analysis and the microbial population present in
Group F: Contaminated soil (of low heavy each culture set are presented in Table 2. To
metals concentration) + Fertilizer (N further confirm the inhibitory effects of lead on
& P) + Crude oil. biodegradation, lead as lead was used at
varying concentrations to determine the
After the incubation period, the residual oil in population of degrading microorganisms (Fig.
each flask was harvested by liquid-liquid 2).
extraction method, where 80cm3 of chloroform
were mixed with the culture medium and was DISCUSSION
transferred to a 250cm3 separatory funnel. The Both indigenous and extraneous
culture flask was rinsed several times with microorganisms can utilize crude oils as source
chloroform and transferred to the funnel. The of carbon and energy and break them down to
chloroform phase was filtered and collected simpler non-toxic compounds such as CO 2 and
using anhydrous sodium sulfate to absorb H2O. This process takes a long time as the
residual water. The extracted aqueous phase degradation efficiency is considerably low
was re-extracted several times. All chloroform under natural conditions (Chaîneau et al.,
extracts were pooled and evaporated in a 2003). Thus, bioremediation in soil can be
vacuum rotary evaporator at 35 oC. The residual promoted by stimulation of the indigenous
oil and its degraded products were determined microorganisms, by introducing nutrients into
using TLC-FID (Iatroscan); however, the soil (biostimulation) (Bento et al., 2005).
dissolving the extracts in n-hexane prior to this
analysis precipitated all the asphaltenes Nutrients are added for the growth and
(Polars). The number of hydro-carbon maintenance of microorganisms. By providing
degrading microorganisms was determined appropriate nutrient balance, it is possible to
using the most probable number (MPN) achieve high rate of hydrocarbon degradation
technique (Mandri and Lin, 2007). (Rehm, 1993). The use of appropriate
concentrations and nutrient ratios can avoid a
Effect of lead nitrate on hydrocarbon situation where growth is limited by depletion
degradation of one essential nutrient while all other
The effects of different concentrations of lead nutrients may be present in excess (Maier et al.,
(0, 0.1, 0.5, 1.0 mg g-1 dry soil) on microbial 2000).
population in the soil samples were determined
in cultures containing the various lead- Addition of fertilizer (N & P) enhances
amendments in hydrocarbon substrates. The biodegradation of oil in soil. Nitrogen-
presence of heavy metals in the soil samples containing fertilizer has stimulating effect. It
was analyzed prior to this addition by Atomic enhances the rate of microbial degradation
Absorption Spectrophoto-meter (Perkin Elmer (Atlas, 1981; Leahy and Colwell, 1990; Shabir

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et al., 2007). Nutrients favour microbial organisms in samples with high concentration
growth; while nutrient limitation reduced the of lead.
overall microbial activity in the soil thereby
preventing them from functioning at optimal Lead at certain concentrations can be inhibitory
levels (Turco and Sedowasky, 1995). to microbial activity (Laskowski et al., 1994;
Roane et al., 1996) and its presence in high
Although the nutrient concentrations sufficient concentrations in contaminated sites can
for near-maximum growth of hydrocarbon- prolong the rate of bioremediation. This decline
degrading microorganisms are small (only 2.0– in the number of hydrocarbon-degraders was
10.0 mg/L), the effectiveness of biostimulation reflected in the amount of the hydrocarbon
requires continuous contact between the added substrate degraded, which decreased with
nutrients and the oil within the bioremediation increase in the level of lead in each culture (Al-
zone. Maximizing the residence time of Saleh and Obuekwe, 2005). In some cases,
nutrients is the key to achieving rapid and cost- however, the addition of some metals at low
effective clean up (Li et al., 2007). This also is concentrations has been observed to stimulate
in agreement with the work of Gallego et al., microbial activity as well as biodegradation.
(2001); who postulated that addition of For instance, copper and cadmium (both at 0.01
inorganic N and P enhanced the rate of mg total metal/dm3) were reported to enhance
degradation of diesel oil by 90%; hence benzoate biodegradation rate by 185 % and 2-
addition of nutrients is a standard practice for chlorophenol biodegradation rate by 168 %
increasing biodegradation. Other processes such (Zhang et al., 2009). Therefore, the success of
as addition of woodchips, tilling and watering bioremediation is dependent on the level of
can enhance biodegradation (De-qing, et al., metabolic and genetic adaptation of the
2007). microbial populations to their environment.
Thus, an increase in the population density of
Different concentrations of lead affected the the microbes could ensure rapid degradation of
microbial composition as indicated by reduced the pollutants (Bento et al., 2005).
number of hydrocarbon degrading micro-

Table 1: Some basic properties of the soil used in the study

Sample pH Organic carbon Total N Sand (g/kg) Silt (g/kg) Clay
(g/kg) (g/kg) (g/kg)
Soil 6.30 15.10 2.38 610 320 70

Figure 1: Effects of group composition on degradation of Crude oil

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Table 2: Leavels of Residual extracts and MPN count of microorganisms in samples

Group Saturates (%) Aromatics (%) MPN (CFU/ml)
A 27.29 30.01 9.3x104
B 20.33 24.99 5.4x105
C 46.89 31.75 1.2x104
D 41.98 30.03 1.8x104
E 40.16 30.21 3.5x104
F 37.46 30.08 4.1x104
Control 48.20 32.54 Not measured
MPN = Most probable Number CFU = Colony Forming Unit

Figure 2: Inhibitory effects of different concentration of Lead on Microbial count.

Atlas, R.M. (1981). Microbial degradation of

CONCLUSION Petroleum Hydrocarbons: an Environ-
The presence of inhibitory substances such as mental Perspective, Microbio. Rev. 45:
heavy metals has significant effect on 180-209.
biodegradation. Significant reduction in Atlas, R.M and Bartha, R. (1992). Hydrocarbon
microbial population as well as bioremediation Biodegradation and Oil Spill
was observed at varying concentration of lead. Bioremediation, Adv. Microbial Eco. 12:
The effectiveness of bioremediation depends on 287-327.
the success of identifying the rate-limiting Bento, F.M., Camargo, F.A.O., Okeke, B.C.
factors and optimizing them in the feasibility and Frankenberger, W.T. (2005).
and biotreatability studies. Comparative bioremediation of soils
contaminated with diesel oil by natural
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