Ibiene et al., 2011 BIOREMEDIATION OF ... - Njafe.org

Nigerian Journal of Agriculture, Food and Environment. 7(3):1-7 

Published September, 2011 Ibiene et al., 2011 

BIOREMEDIATION OF HYDROCARBON CONTAMINATED SOIL IN 

THE NIGER DELTA USING SPENT MUSHROOM COMPOST AND 

OTHER ORGANIC WASTES 

ABSTRACT 

Ibiene* 1 , A. A., Orji 2 , F. A., Ezidi 3 , C. O. and Ngwobia 1 , C. L. 

1 Department of Microbiology, University of Port Harcourt PMB 5232, Port Harcourt, Nigeria. Email: ibieneaa@yahoo.com 

Tel: +2348066720531 

2 Department of Health, Safety and Environment Shell Petroleum Development Company of Nigeria, Rumuobiakani, Port 

Harcourt, Nigeria 

3 National Biotechnology Development Agency, Odi Bayelsa State, Nigeria 

The effect of organic fertilizers (spent mushroom, cow dung and poultry droppings) on the bioremediation of hydrocarbon 

contaminated soil was investigated on a 28 days study period. The hydrocarbon contaminated soil was supplemented with the 

different organic fertilizers and analyzed throughout the study period. Physic-chemical and microbiological parameters like 

soil pH, moisture content, phosphate, nitrate, % total organic carbon (%TOC), total petroleum hydrocarbon (TPH) total 

heterotrophic counts, total hydrocarbon utilizing counts (bacteria an fungi) were studied from baseline to the 28 th day. The 

concentrations of phosphate, nitrate, percentage TOC of the treatments decreased significantly during the study period 

whereas the controls slight decrease in the parameters. pH values of the treatments were within slight acidity to alkalinity as 

the control had acidic pH range during the study period. The percentages of TPH loss in the cow dung option poultry 

droppings option spend mushroom option and control were 97.83%, 98.21%, 99.91% and 27.52%. The hydrocarbon utilizing 

bacterial isolates from the study include Bacillus, Pseudomonas, Kebsiella, Proteus, Flavobacterium, Clostridium, 

Micrococcus, Acinetobacter. The hydrocarbon utilizing fungal isolates from the study were Penicillium, Aspergillus, 

Sacharomyces, Rhizopus, Fusarium and Mucor. This study showed that cow dung, spend mushroom and poultry droppings are 

effective nutrient sources for bioremediation. 

Keywords: Bioremediation, hydrocarbon contamination, organic fertilizers, Niger Delta. 

INTRODUCTION 

The deleterious effects of pollutants on the environment have led to increased awareness and vigilance against 

contamination of the Niger Delta environment. In Nigeria, in relatively recent time, there has been remarkable 

increase in population, urbanization and industrial activities, (Eze and Okpokwasili 2010). Pollution caused by 

petroleum and its derivatives is the most prevalent problem in the environment. The release of crude oil into the 

environment by oil spills is receiving worldwide attention (Millioli et al., 2009). Bioremediation which has 

defined as biological response to environmental abuse has continued to receive research attention across the globe 

(Hammer, 1993). Bioremediation has been described as the use of living microorganisms or microbial processes 

to detoxify and degrade environmental pollution. In order words, it is a technology for removing pollutants from 

the environment thus restoring the original natural environment (Sasikuma and Papmazath, 2003). The long term 

aim of bioremediation designs is to present cost effective designs which reduces the pollutant to a level referred to 

as low as reasonable and practicably possible (ALARP). In order to achieve this cost effectiveness, researchers all 

over the world have begun to pay research attention to the use of organic waste as the source of limiting nutrients 

for effective bioremediation. Cow dung, spend mushroom and poultry droppings are agro-based wastes and are 

usually not well-managed in Nigeria. The objectives of this research study are to monitor the effectiveness of 

spend mushroom, cow dung, and poultry droppings as limiting nutrients sources for bioremediation, and also to 

investigate the microbial population dynamics in the soil undergoing bioremediation. 

MATERIALS AND METHODS 

Study area 

The soil used in this study was collected from Rumuolumeni, Nkpor Village, Obio/Akpor Local Government 

Area of Rivers State, Nigeria. The major occupation of the people, land farming and subsistence agriculture. 

There is no history of crude oil pollution in this environment/area. 

NJAFE VOL. 7 No. 3, 2011 1



Collection of samples 

Samples were collected using soil auger; this was done 15cm deep, at four different points. The four soil samples 

were lumped together in a plastic part and transported to the environmental microbiology laboratory of the 

University of Port Harcourt for bioremediation studies. Spend mushroom used was obtained from Bioresource 

Development Center Odi, Bayelsa State Nigeria. The cow dung was obtained from cattle abattoir, Alakahia, Port 

Harcourt (Nigeria) whereas the poultry droppings were collected from poultry farms in Alakahia, Port Harcourt, 

Nigeria. Five hundred (500) grams of soils in the four containers were supplemented with 50mls of Bonny light 

crude oil containers to simulate conditions for a major spill. Each of these containers were mixed with either 50 

grams of cow dungs, spent mushroom or poultry droppings. One of the four (4) containers was not amended with 

organic fertilizers to serve as control (table 2). 

Table 1: Experimental design 

Experimental set 

Set A 

Set B 

Set C 

Set D (control) 

Test experiment 

500grams of polluted soil + 50 grams of cow dung (CD) 

500grams of polluted soil + 50 grams of poultry droppings (PD) 

500grams of polluted soil + 50 grams of spent mushroom 

500grams of polluted soil + no amendment 

Microbiological analyses of the samples 

Enumeration of total culturable heterotrophic bacterial and fungal counts 

Ten-fold serial dilution was done, one gram of the soil samples was weighed out from each samples and dispensed 

into the first three test tubes containing normal saline. Each tube was shaken for thorough mixing of the mixture. 

1ml was pipetted into another test tube containing 9mls normal saline to give a 10 -1 dilution. The sample was 

diluted serially up to 10 -5 . The same procedure was followed for other samples. The test tubes were covered with 

cotton wool. Exactly 0.1ml aliquots of the dilutions was spread inoculated into Petri dishes containing nutrients 

agar (Oxoid) and potatoes dextrose agar (PDA, Antech) for total culturable bacteria and total cultural fungi 

enumeration respectively (Chikere et al., 2009; Orji, 2011). Enumeration of colonies was done through the 

formula: 

Enumeration of total culturable hydrocarbon utilizing bacterial and fungal counts 

The culturable hydrocarbon utilizing bacteria of the soil samples were enumerated in duplicates on modified 

mineral salt agar (MSA) of mills et al., 1978 using the spread plate method. The vapour phase transfer was used. 

Serial dilution of each sample was carried by suspending 1gram of the sample into 9mls of normal saline and 

0.1ml of the appropriate dilution was inoculated onto a duplicate agar plate. A sterile filter paper (Whatman No 1) 

saturated with crude oil was placed inside the cover of the petri dish, closed, inverted and incubated at 28 0 C for 5- 

8 days. The filter paper saturated with crude oil served as a sole source of carbon (Abu and Ogiji, 1995). 

Enumeration of culturable hydrocarbon utilizing fungi 

The same procedure used for the enumeration of hydrocarbon utilizing bacteria was adopted except that 1ml of 

lactic acid was added for the fungal media to inhibit the growth of hydrocarbon utilizing bacterial (Obire et. al., 

2008). 

Isolate purification 

The colonies formed on MSA for bacteria were further purified by sub-utilizing on nutrient agar. Pure colonies 

were identified based on the size, shape, colour elevation, gram reactions and biochemical characteristics. 

Isolates on MSA for fungi were purified by sub-culturing on potato dextrose agar. Pure fungal isolates were 

further studied using lactophenol stain. A small portion of the fungal growth was picked with a wire loop and 

placed on clean and grease free slide. A drop of lactophenol was added and the preparation was covered with 

cover slip. The slide was observed under x 10 and 40 objectives lenses. (Obire et.al, 2008) 

Physico-chemical analyses 

The parameters studied were the soil pH, nitrate, phosphate, total petroleum hydrocarbon (TPH), total organic 

carbon (% TOC) and moisture content (Jamie and Richard, 1996). The determination of total organic carbon (%) 

was done by titration method using potassium per manganate as oxidant whereas the moisture content was 

determined using the method previously described by Stewart et al., 1974. 

Gas chromatography (GC) analysis 




The extraction of petroleum hydrocarbon was done with dichloromethane (DCM) using cold extraction method 

with ASTM D-3694 heavy machine for 1 hour. Procedurally, 20 grams of dried soil samples was weighed into 

100mls conical flask. Twenty (20) grams of activated anhydrous sodium sulphate and 20 mls of DCM were gently 

added into the barrier containing the test soil sample. 

This was allowed to stand for 1 hour and then filtered into 50mls conical flask using filtration plugged/packed 

with cotton wool. Procedure was repeated on the residual soil until a colourless solution is obtained (Saari et al., 

2007). 

RESULTS AND DISCUSSIONS 

The baseline study showed that total culturable heterotrophic bacteria count was 6.4 x 10 4 Cfu/gram. In addition, 

the total culturable heterotrophic fungal count, total hydrocarbon utilizing bacteria, total culturable hydrocarbon 

utilizing fungal counts were 2.6 x 10 3 Cfu/g, 3.9 x 10 3 Cfu/g and 1.2 x 10 3 Cfu/g respectively. (Table 2). This 

table shows that the hydrocarbon utilizing bacteria in the mangrove soil is relatively adequate for bioremediation 

(Ebuehi et al., 2005).However, the amount of limiting nutrients such as nitrogen and phosphorus present in this 

polluted soil are very low. The total culturable heterotrophic bacterial count in the cow dung, poultry droppings, 

and spend mushroom increased progressively from 4.2 x 10 4 Cfu/g to 6.8 x 10 Cfu/g, 4.9 x 10 4 Cfu/g to 1.12 x 10 6 

Cfu/g and 4.6 x 10 6 to 1.3 x 10 5 Cfu/g respectively (Figure 1). The total culturable hydrocarbon utilizing bacterial 

count for the cow dung, poultry dropping spent mushroom amended soil and control increased from 2.1 x 10 3 

Cfu/g to 2.9 x 10 6 Cfu/g, 1.8 x 10 3 Cfu/g in 3.2 x 10 6 Cfu/g, 2.9 x 10 3 Cfu/g to 5.4 x 10 6 Cfu/g and 3.4 x 10 3 to 2.9 

x 10 5 Cfu/g (figure 2). The total culturable heterotrophic fungal count for the cow dung, poultry droppings spent 

mushroom amended polluted and control increased 2.6 x 10 3 Cfu/g to 6.3 x 10 4 Cfu/g, 2.0 x 103 Cfu/g to 6.6 x 10 4 

Cfu/g, 2.2 x 10 3 to 7.9 x 10 4 Cfu/g and 2.7 x 10 3 Cfu/g to 2.8 x 10 4 Cfu/g respectively (figure 3). The total 

culturable hydrocarbon utilizing fungi count ranged between 1.3 x 10 3 to 5.4 x 10 4 Cfu/g, 1.6 x 10 3 to 6.0 x 10 4 

Cfu/g, 1.4 x 10 3 Cfu/g to 5.3 x 10 5 Cfu/g and 1.2 x 10 3 Cfu/g to 2.9 x 10 Cfu/g for cow dung, poultry droppings, 

spent mushroom and control respectively (Figure 4). Obire et al., 2008 in a bioremediation study using cow dung 

and poultry droppings reported an average heterotrophic fungal count of 32.25 x 10 2 cfu/gram and 46.84 x 

10 5 cfu/gram for cow dung and poultry droppings respectively. 

However, in a laboratory scale bioremediation study, 500grams of the polluted soil was amended with either 

50grams of cow dung or 50grams of water hyacinth recipe and the control experiment. The total culturable 

heterotrophic bacterial and fungal count and hydrocarbon utilizers for the two treatments increased progressively 

from the 28 th day to the 70 th day of the study. The baseline percentage organic carbon (%TOC) was 3.97% 

amendment with organic nutrients increased the total organic carbon to 41.45%, 40.11%, 44.32%, 3.90% for the 

cow dung, poultry droppings, spent mushroom and control respectively (Table 2). There were progressive 

decrease in the %TOC from 41.45% to 26.00%, 40.11%-24.00%, 44.22%-21% and 3.97%-2.85% for cow dung, 

poultry droppings, spent mushroom and control options respectively from day 0 to 28 th day of the study (Figure 

7). The baseline concentration of phosphate was 6.65mg/kg. Thereafter amendment, this increased to 29.00mg/kg, 

34.20mg/kg, 47.79 mg/kg in the cow dung, poultry droppings and spent mushroom respectively. During the 28 th 

days bioremediation study, the phosphate level decreased from 29.00mg/kg-9.25mg/kg, 34.20mg/kg-11.40mg/kg, 

47.79mg/kg-6.70mg/kg, and 6.60-5.00mg/kg (Figure 5) for the cow dung, poultry droppings, spent mushroom and 

control respectively. 

The baseline concentration of nitrate in the polluted soil was 5.8mg/kg, thereafter commandment; the nitrate 

concentration increased to 12.20mg/kg, 14.11mg/kg, 18.50mg/kg, for the cow dung, poultry droppings, and spent 

mushroom options respectively. The concentration of nitrate decreased from 12.20mg/kg to 6.50mg/kg, 14.11- 

7.11, 18.50mg/kg to 5.29, 5.75 to 3.00 for the cow dung, poultry droppings, spent mushroom and control options 

(Figure 6). 

The baseline amount of total petroleum hydrocarbon (TPH) was 639.11± 0.11 PPm. The monitoring of 

bioremediation was done at zero day, 8 th day and 28 th day (Figure 8). On the 8 th day of the study, the TPH in cow 

dung reduced from 633.46 to 374.00PPm. This is about 41.48% reduction or loss in TPH form the environment. 

Still in the cow dung treated polluted soil, the TPH reduced to 13.88PPM at the 28 th day. This represents 97.82% 

loss of TPH. In the poultry dropping option, the TPH reduced to 393.14 PPm on the 8 th day. This represents about 

38.64% loss in TPH. On the 28 th day, the poultry dropping option had only 11.34PPm of TPH left. This represents 

98.20% loss in TPH. In the spent mushroom option, the TPH reduced from 633.46 to 278.72 PPm (on the 8 th day). 

This represents 38.64% percentage loss in TPH. On the 28 th day, the TPH reduced to 0.57 indicating 99.9% loss 

of TPH (Figure 8). The control experimental set-up, without amendment had reduction of TPH from 640.38 (day 




0) to 629.00 (28 th day). This represents 1.77% loss of TPH. On the 28 th day, the TPH in the control of 27.52% 

(Figure 8). 

In a crude oil contaminated agricultural soil at Federal University of Technology, Owerri -Nigeria, the amendment 

of 100grams of contaminated soil with 30 grams of organic nutrient (poultry droppings) led to the loss of 40% 

total petroleum hydrocarbon (Ibekwe et al., 2006). In bioreactor model, Indian scientists established 

bioremediation of diesel hydrocarbon polluted soil using ammonium nitrate (Sharma and Rehman, 2009). In a 

tropical crude oil polluted soil undergoing bioremediation, Chikere et al., 2009b observed that the use of NPK 

fertilizer, urea fertilizer and poultry droppings effectively stimulated bacterial organisms into utilization of crude 

oil. NPK 20:10:10 fertilizer option reduced TPH from 3666.0mg/kg to 89.68mg/kg for 57 days where as urea 

fertilizer option reduced TPH from 3666mg/kg to 162mg/kg for 57 days. In the poultry droppings option, the TPH 

was reduced from 3666.0mg/kg of soil to 135.01mg/kg of soil (Chikere et al., 2009) 

CONCLUSION 

Waste utilization is currently receiving great research attention globally, and the findings of this research work 

identified the usefulness of three wastes (spent mushroom, poultry droppings, and cow dung) in bioremediation of 

crude oil-impacted agricultural soils in the Niger Delta. It is important to state categorically that more funds and 

further research attentions are needed on the pilot scale and in-situ utilization of these wastes. This will enhance 

better understanding of the interactions of environmental factors on the chemicals of concerns (COC’s) and soil 

amendment agents ( i.e. these organic wastes; spent mushroom, poultry droppings, and cow dung). 

Table 2: Baseline properties of the polluted soil 

Parameters 

Values ± SD 

Total culturable heterotrophic bacterial count (Cfu/g) 6.4 x 10 4 ± 0.56 

Total culturable heterotrophic fungal count (Cfu/g) 2.6 x 10 3 ± 0.31 

Total culturable hydrocarbon utilizing bacterial count (Cfu/g) 3.9 x 10 3 

Total culturable hydrocarbon utilizing fungal count (Cfu/g) 1.2 x 10 3 

Nitrate (Mg/kg) 5.8 ± 0.02 

Phosphate (Mg/kg) 6.65 ± 0.04 

Total organic carbon (%TOC) 3.97 ± 0.46 

Total petroleum hydrocarbon (PPm) 639.11 ± 0.11 

Moisture (%) 27 ± 0.08 

pH 5.68 ± 0.05 

Cfu/g; colony forming unit/gram, Mg/kg: milligram per kilogram, PPm, part per million, % percentage 

Table 3: Bacterial and fungal isolates utilizing or degrading Petroleum hydrocarbons 

Hydrocarbon utilizing bacteria 

Bacillus species 

Klebsiella species 

Acinetobacter species 

Farobacterium species 

Micrococcus species 

Clostridium species 

Pseudomonas species 

Hydrocarbon utilizing fungi 

Cladosporium species 

Aspergillus species 

Fusarium species 

Penicillium species 

Mucor species 

Saccharomyces species 




Figure 1: Changes in Total heterotrophic bacterial count during the study period 

Figure 2 : changes in the Total culturable hydrocarbon utilising bacterial count during the study period 

Figure 3 : Total culturable heterotrophic fungi during the study period 

Figure 4: Changes in the count of total culturable hydrocarbon utilizing fungi during the study 




Figure 5: Changes in the concentration of Phophate during the study period. 

Figure 6: Changes in the concentration of Nitrate during the study period 

Figure 7: Changes in the percentage total organic carbon during the study period. 

Figure 8: Changes in the percentage Total Petroleum Hydrocarbon during the study period. 

Figure 9: Changes in the percentage Moisture content during the study period. 




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