Application of crude oil degrading micro-organisms immobilized in

cellulosic material for bioremediation of crude oil contaminated soil

Stanley chinedu Mamah a , Henry Adimula b , Nkiruka Ogbodo a , Grace Adams c ,
Moses Chukwu a , Charles Ugwu a , Okahu Alpheus a , Joan Amanjide a
a
Department of Chemical Engineering, Alex Ekwueme Federal University, Ebonyi State, Nigeria
b
Industrial Safety and Environmental Technology Department, Petroleum Training Institute Effrun,
Nigeria
c
National Agency for Science and Engineering Infrastructure (NASENI), Abuja, Nigeria
*Corresponding author: stanley.chinedu@funai.edu.ng, douglascms@yahoo.com
In this research, crude oil degrading microorganisms were produced and immobilized in a local
cellulosic material (coconut fibre) and the immobilized product was applied in the
bioremediation of soil contaminated with crude oil. Coconut fibre (carrier) was collected,
processed and characterized for selected relevant parameters (physico-chemical properties) that
makes it suitable as carrier for the crude oil degrading microorganisms. Crude oil degrading
microorganisms were produced from stock culture through enrichment method and
characterized. A bioreactor was designed and locally fabricated. The crude oil microorganisms
were mass produced via the use of a bioreactor from the consortium of the three best crude oil
degrader. Petroleum Hydrocarbon degrading bacteria were immobilized in coconut fibre which
was applied in bioremediation of crude oil contaminated soil. A parcel of land with an area
measurement of 144 m2 was contaminated with 380 litres of crude oil (Escravos light) and also
treated. Two weeks after contamination, the land was tilled to a depth of 15cm with garden fork
and a consortium of petroleum degrading bacteria immobilized in coconut fibres (ratio of 1:1)
was applied to the soil. Soil samples were collected on a weekly interval for laboratory analysis
of residue hydrocarbon content, physico-chemical properties, microbial count of the treated soil
using Gas Chromatography (GC) and other methods of analysis. The results showed that about
91.29% of hydrocarbon content in the original crude oil was removed after seven weeks
treatment. The percent of total petroleum hydrocarbon (TPH), poly aromatic hydrocarbons
(PAHs), benzene, toluene, Ethyl benzene, xylene, (BTEX), degraded in the contaminated soil
were 83.01%, 95.86%, and 95.02% respectively. Results obtained demonstrated the effectiveness
of immobilized hydrocarbon degrader in treatment of crude oil polluted.
Keywords: Bioremediation, degrading microorganisms, cellulosic material, crude oil
contaminated soil, bioreactor
1.0 Introduction
The Nigerian petroleum industry has contributed substantially to the growth of the
Nigerian economy since oil production began in Nigeria in 1958, two years after the discovery of
oil in Oloibiri, in the Niger Delta (Ukhurebor et al., 2023). The various activities of the
petroleum industry: prospecting, exploration, production and transportation of both the crude oil
and refined products have come with the inevitable impacts on the Nigerian environment
creating enormous challenges for the industry (Ukhurebor et al., 2021). One of the major
environmental challenge of the Nigerian oil industry is the issue of onshore and offshore
pollution by crude oil. Various causes including; accidents, sabotage, and equipment failure,
have been blamed for the large scale pollution caused by oil of the Nigeria environment (Umar
et al., 2021). Whatever the cause(s), however, the results have been the deterioration of the
Nigeria environment which has resulted in deep socioeconomic impacts on the people especially
of the Niger Delta and other oil producing areas of the country. Farmlands have been degraded
and the aquatic systems polluted impacting negatively on their ability to sustain production of
crops and aquatic life which resulted in the loss of livelihood and quality of life of the people
(Amnesty International, 2018).

The increasing clamour for sustainable development and the need to better protect the
environment to preserve its ability to sustain the plant and animal ecosystem dependent on it has
been driving efforts in environmental protection and management (Hu et al., 2023). Various
policy interventions such as the Oil Pollution Act of 1990 ( US EPA, 1990), the Federal
Environmental protection Agency Act Cap 131 LFN 1990 and The Environmental Impact
Assessment (EIA) decree No 86 of 1992, , have been made over the years to stem the increasing
devastating effects of the petroleum industry on the Nigerian environment with very little
achieved thus far.

Several physical, chemical and biological approaches have been developed over the years to
contain, clean up and ameliorate the damaging effects of oil spill/pollution (Asif et al., 2022).
Each of these approaches have achieved some measure of success.. Biological treatments
involve breakdown of contaminates into non-toxic form using microbiological process (Varjani,
Upasani and Pandey, 2020).

The low cost and environmental friendliness of biological methods have often served to
recommend such processes as the preferred methods of intervention. Bioremediation has been
particularly useful as it exploits the ability of natural indigenous or exogenous microorganisms to
degrade petroleum hydrocarbons. Hence, bioremediation can be defined as the application of
living organism to eliminate environmental contaminants from soil, water as well as gases. A
number of microorganisms are able to utilize the carbon in petroleum hydrocarbon as carbon and
energy source. The process of bioremediation is a slow process sometimes spanning several
months before appreciable quantities of the contaminants are degraded. A number of approaches
including biostimulation (involving the addition of nutrients; N, P, K to stimulate microbial
growth) and bioaugmentation (which introduces exogenous microorganisms to supplement the
native population of microorganisms) have been used to enhance the rate of bioremediation.

In a previous work three bacteria isolates, Streptococcus thermophilus, Micrococcus

varians and Bacillus sp. which showed potential for petroleum hydrocarbon degradation were
immobilized in ground cellulosic carrier: coconut fibers and groundnut husks (Agiri, Gabriel,
Mamah, 2015). The bacteria loads of the immobilized materials were counted and indicated that
the isolated bacteria survived in the coconut fibers as well as in groundnut husks. Population of
isolated bacteria immobilized within coconut fibers including that of groundnut husks declined
slightly after 120 days of culturing. The immobilized bacteria systems showed potentials in
petroleum hydrocarbon degradation. At the end of twenty one days of utilization of immobilized
bacteria in lab, degrading of Forcados crude, petroleum hydrocarbon residual concentration
ranged from 14.87 to 34.93% compared to 58.97% of the original concentration remaining in the
control.

In the present work, a cell culture bioreactor was designed and fabricated locally and used to
optimally mass produce petroleum hydrocarbon degrading microorganisms. The crude oil
degrading bacteria was produced from previous cell stock cultures and a consortium of the three
best petroleum hydrocarbon degrading microorganism was made. Immobilization of degraders
onto the coconut fibre was carried out and the immobilized crude oil degrading microorganisms
were utilized in the treatment of crude oil sludge/ drilling cuttings contaminated soils. General
characterization of the crude oil sludge/ drill cutting was carried out and samples collected from
the bioremediation test site were analyzed for some selected parameters such as total petroleum
hydrocarbon (TPH), poly aromatic hydrocarbons (PAHs), total organic content, hydrocarbon
utilizing bacteria (HUB), total heterotrophic bacteria (THB), and other physico-chemical
properties.
2.0 Materials and Method

2.1 Materials

Haier Thermocool refrigerator, Complete grinding and pellet making machine (24 R Tim super Diesel
Engine R175AN), TEC generator (LL8GF-4A, 6.0KW model), Oven,Infra red sterilizer, Cell culture
bioreactor, garden shade, hand arguer rain boots, watering cans. Reagents; Sodium Chloride ,Potassium
Chloride,Potassium dihydrogen orthophosphate, Sodium Nitrate, Deionized Water, Cobalt (II)
Sulphate, Calcium Carbonate, Copper Sulphate Hydrate, Boric acid, Zinc Sulphate heptahydrate,
Magnium Oxide , Manganous Sulphate Monohydrate,Ferrous sulphate heptahydrate,
Hydrochloric acid, Petroleum ether, Ammonium Nitrate, Nutrient Agar, Ethanol absolute, and
Agar No.1, all purchased from BDH, England.

2.2 Methodology
2.2.1 Characterization of the Coconut Fibre
The coconut was characterized for its water content, nitrogen content, phosphorus content,
porosity and pH.

2.2.2 Water Content Determination

The water content is the ratio of the weight of water to that of the solids in a given mass of
material. This ratio is expressed as percentage. In a typical determination a crucible, cleaned and
oven dried, was weighed (W1). The crucible was then filled with the dried meal of the cellulosic
materials, and weighed (W2). The crucible containing the cellulosic material was then kept in an
oven at a temperature between 105oC to 110oC for 24 hours. The final constant weight (W3) of
the container with the dried sample were then determined. The water (moisture) content W (%)
was determined from the relationship:

Water (moisture) content W (%) = [(W2-W3)/ (W3-W1)] x 100.

2.2.3Total Nitrogen determination

2.00g of ground coconut fibres and 9ml of Conc. Tetraoxosulphate (IV) acid were mixed and
heated gently using hot plate till white fumes was noticed. Afterwards, it was cooled, filtered
and the filtrate poured into 100ml in a volumetric flask. 25ml of the digest was taken from the
flask and made up to 50ml with distilled water. 5ml of 12M potassium hydroxide was poured
and the solution filtered.

2.2.4 Evaluation of Phosphorus content

1g of ground coconut fibre was weighed into 250ml flask and 4ml of Tetraoxo hydrogen chlorate
(VII) acid, 2ml of concentrated Trioxonitrate (V) acid and 2ml concentrated tetraoxosulphate
(IV) acid was introduced into the fume chamber. It was then heated using hot plate till dense
white fume was noticed and allowed to cool. 50ml distilled water was added and it was heated
for 30 second. Thereafter, it as filtered after cooling. 0.2112g of ascorbic acid and Phosphate
reagent B was prepared by adding 40ml of Phosphate reagent A to the ascorbic acid.
2.2.5 Preparation of Binding Agent (Clay)

Clay was used as adhesive for the cellulosic material. The clay was collected from Eku, Delta
state. It was sun dried crushed using mortar and pestle to unequal sizes. The unequal particles
were sieved using sieve size of 0.4mm to get uniform particle sizes.
2.2.6 Sterilization of MSM and Trace Element Solutions

MSM and Trace element were prepared into separate containers and sterilized using auto clave
(YX-280A) at 0.165Mpa for fifteen minutes. This is to ensure contaminated free solutions.
2.2.7 Enrichment of Crude Oil Degrader Microbes

Into three conical flasks containing 2 ml of previously stocked crude oil degrading microbes
labeled C, F, and G, 100mi Mineral Salt Medium (MSM) was added. After which, sterile crude
oil (1 ml) was added. The cover was loosely placed to allow for air and incubated at 30oC.
Further enrichment was achieved, after microbe growth was observed in13 days. The MSM (2
ml) broth culture was transferred from each of growth flasks into fresh MSM (100 ml) in sterile
conical flasks and labeled C2, F2, and G2 respectively and were all incubated at 30 oC. The covers
were loosely placed to allow for air.
Growth was observed after incubating the MSM broth culture for 17 days after which the MSM
(5ml) broth culture (5ml) was transferred from each of the growth flasks into fresh MSM
(1000ml) in sterile conical flasks and labeled C3, F3 and G3 respectively. They were covered
with cotton wool and aluminum foil and incubated at 30 oC for 30 days. This was repeated five
times and a total of fifteen litres of oil degrader microbes are in stock.
2.2.8 Immobilization of Microbial Isolates on Sterile Coconut Fibre

The immobilisation method of simple mixing reported by (Mishra et al., 2001) was adopted. The
consortium 15 ml made from the isolates C, F, and G was transferred onto a sterile coconut fibre
(carrier material) (1:1 v/w) and mixed. The seeded carriers were stored in the refrigerator for
future use.
2.2.9 Production of Immobilized Microbial Isolates pellets

This process involved compressing or molding a material into the shape of a pellet. Various
ratios of crude oil degrading microbial isolates, coconut fibre (Carrier), binding agent (pellet
composition) were mixed until the appropriate mixture with the desired shape was obtained
which was in the ratio of 2.4:1:0.6 for the microbial isolates, coconut fibre and binding agent
respectively.
2.1.10 Field Test Procedure:
A parcel of land measuring 144 m 2 was contaminated with 380 litres of crude oil (Escavos light).
One week after contamination, the soil was tilled to a depth of 15cm with garden fork and shade.
After tilling, a consortium of crude oil degrading microorganism immobilized in coconut fibres
in a ratio of 1:1 was applied to the soil. Nutrient (fertilizer NPK 15-15-15) was added to the soil
thrice weekly and tilled twice a week to a depth of 15cm using garden fork after addition of
nutrient. Soil samples were collected on a weekly interval with soil auger for laboratory analysis
of physico-chemical properties of the soil, microbial count and other parameters.
2.2.11 Bacteria Cell Loading
The bioreactor was sterilized with appropriate disinfectant and wash severally with sterile
distilled water. Water was put into the thermal jacket through the inlet hose and connect the air-
pipe of bioreactor into the air-outlet of the air pump. Thereafter, the bioreactor was loaded with
bacteria culture and cover the bioreactor with lids. The heater was set to the required
temperature, switch on the heating system and agitation system by push of the knobs on the
panel. After the power is switched on the air pump started to supply air and oxygen evenly in the
bioreactor.
2. 2.12 Immobilization of Microbes into Coconut Fibre (Carrier)
About 20 litres of the harvested hydrocarbon degrading bacteria was immobilized into about
20kg of coconut fibre in a plastic container, stirred with wooden stirrer and stored.
2.2.13 Field Test of HC Degrader on Crude Oil/Drill Cuttings

Samples of crude oil sludge mixed with drill cuttings were collected with six plastic buckets (20
litres) from an Oil Servicing Company in Delta State. A parcel of land was cleared at the
Petroleum Training Institute Effurun, Delta State. A polythene measuring 3.5m x 2.6 m was laid
on the soil and the crude oil sludge /drill cutting was put on it. Sample of crude oil/drill cutting
were collected with hand arguer for characterization and analysis. After tilling the soil, a
consortium of crude oil degrading microbes seeded in coconut fibres (20 litres in 20kg coconut
fibre mill) in the ratio of 1:1 was applied to the crude oil/ drilling cutting and nutrient (NPK 15-
15-15 fertilizer) solution was added at interval of 4 days with tilling using garden fork and
samples collected for analysis. The untreated and treated crude oil sludge/drill cutting samples
collected were analyzed for, physico-chemical properties, microbial count and hydrocarbon
content using the Gas Chromatography Flame Ionization Detection (GC -FID) and other analysis
methods.
2.2.14 Gas Chromatographic Analysis of Drill Cuttings
The Parameters determined include: Total Phenolic hydrocarbon, (TPHs) poly aromatic
hydrocarbon (PAHs) benzene, toluene, Ethyl benzene, xylene, (BTEX), heavy metals, pH,
Temperature, Heterotrophic bacteria count, Hydrocarbon degrading bacteria, Hydrocarbon
degrading fungi, total organic compounds.
2.2.15 Estimation of Total Heterotrophic Bacteria (THB) by Plate Count Technique

1.0 gm. of the soil sample was rehydrated with 9ml sterile distilled water in a MacCartney bottle
containing some glass chips (i.e. 10-1 w/v in the first instance). Hundred folds serial dilution of
the suspension was carried out five times in a set of MacCartney bottles each containing 9.9ml
sterile distilled water. Same dilution was carried out on each of the water samples. 1 mm of each
diluent was plated out respectively in duplicates employing the use of nutrient agar medium
(sterile) kept in molten form. Pour plate method was adopted.

The culture plates, having allowed the agar medium to set, were incubated aerobically at 28 oC
for 36-48 hours. Thus enumerating for only aerobes and facultative heterotrophic bacteria. The
culture plates were observed for growth and selected for count after the expiration of the
incubational period. The culture plate on which the number of colonies was less than 300,
starting from the least to the erml diluted and its duplicate for each sample was selected. The
averaged count was multiplied by the dilution factor of that dilution and expressed as cell
forming unit (CFU) per gram or milliliter of the original sample. This is a viable count.

2.2.16 Estimation of Total Heterotrophic Fungi (THF) By Plate Count Method.

The same procedure used for the estimation of THB was used also for the estimation of THF.
The differences are, however, as follows:

a) Sabourand Dextrose agar (SDA) was employed as the culture medium.

b) The culture plates were incubated aerobically at 28 oC for 5-7 days (until the plates
showed no further increase in the number of fungal colonies).
2.2.17 Estimation of Hydro-Carbon Degrading Bacteria (HDB) By Plate Count Method.

Culture Medium: Bushnell-Haas broth in which 15g/l agar, 30mg/l fungusol miconazale Nitrate
B.P (2%) and varying concentrations of NaCl for fresh and salt-water environment were
incorporated. 15ml/l sterile crude oil was also added as carbon source.

2.2.18 Estimation of Hydro-Carbon Degrading Fungi (HDF) By Plate Count Method.

Culture Medium: Bushnell-Haas broth in which 15g/l agar, 50mg/l streptomycin and varying
concentrations of NaCl for fresh and salt-water environment were incorporated. 15ml/l sterile
crude oil was also added as carbon source.

3. 0 Results and Discussion

The results of the characterization and identification of the isolates showed that the microbial
isolates that could utilize the hydrocarbon were: Streptococcus thermophilus, Leuconostoc
mesenteriodes, , Aerococcus viridans, Micrococcus roseus ,Flavobacterium thalpophilium,
Micrococcus varians and Bacillus sp. (Agiri et al., 2011). Consortium of bacteria was created
consist of Streptococcus thermophilus, Micrococcus varians and Bacillus sp.
The examined soil contaminated with petroleum collected from crude oil storage tank in the
petroleum Training Institute Effurun showed a high pollution. The soil initially contained
15623.88 mg/kg, 11.87mg/Kg and 1342.24 mg/Kg of TPH, BTEX and PAHS respectively. The
tables showed the determined values of total petroleum hydrocarbon (TPH), total organic carbon
(TOC), Poly aromatic hydrocarbons (PAHS) and Benzene, toluene, Ethyl benzene and Xylene
(BTEXs), and pH of the uncontaminated and contaminated soil after seven weeks of treatment.
Others were Total Heterotrophic Bacteria count, Hydrocarbon Utilizing Bacteria count, Nitrate
and Total Phosphorus. The trend in degradation process as observed from the data before and
after biological treatment showed a gradual reduction in the total amount of total petroleum
hydrocarbon (TPH), total organic carbon (TOC), Poly aromatic hydrocarbons (PAHS) and
benzene, toluene, ethyl benzene and xylene (BTEXs) present in both the uncontaminated and
contaminated soils over a treatment period of seven weeks. About 91.29% of hydrocarbon
content in the original crude oil was removed after seven weeks treatment with the consortium of
bacteria immobilized in the cellulosic agricultural harvest material (coconut fibre). The percent
of TPH, PAHS, BTEX degraded in crude oil in the contaminated soil were 83.01%, 95.86%, and
95.02% respectively after seven weeks of treatment as shown in Fig.3.d, Fig.3e and Fig.3f.
The pH, TOC, Nitrate and total phosphorous levels in soil were raised slightly after remediation
as shown in Fig. 3g, Fig3h and Fig3i. This could be attributed to the amount of fertilizer added as
nutrient, Nitrogen and phosphorous for microbial growth.
The result also show that there a gradual increase in the amount of total heterotrophic bacteria
Fig.3j and an increase in the amount of the hydrocarbon utilizing bacteria was recorded (Fig.3k).
The increase in the amount of the hydrocarbon utilizing bacteria was slight initially and suggests
that bacteria start degrading after about three weeks of treatment. This is in agreement with
various stages of hydrocarbon microbial degradation process of lag, growth, exponential,
decline. Since preliminary experiments (the results are not given) revealed the presence of
hydrocarbon degraders in the soil, the growth of microorganisms was stimulated by the addition
of N and P sources, aeration and mixing. In this way by application of bioremediation
techniques, the concentration of TPH, PAHS, and BTEX were reduced to appropriable levels
during the seven weeks of the experiment. The changes in the number of microorganisms shows
that some of microorganisms were members of the indigenous micro flora and most were
introduced into the system with the immobilized crude oil degrader added to the soil in order to
stimulate biodegradation. The highest number of microorganisms was attained between 3-6
weeks of the process. The total number of bacteria in the soil during bioremediation was in range
103–107 CFU/g. The number of hydrocarbon-degrading bacteria was 10 6–107 CFU/g, and
hydrocarbon-degrading fungi 105–106 CFU/g. On the basis of the results presented in Table 3.3,
it is apparent that a stable microbial community was formed during the bioremediation
experiment.
The relationship between oil removal efficiency and bioremediation time is shown in Fig. 3a.
The oil removal efficiency of uncontaminated soil sample was approximately 9%. The removal
is linked to oil volatilization as well as aerial oxidation. These samples contains native
microorganisms, the oil removal efficiency improved linearly on the first week and it then
leveled off around 37% of oil removal. This implies that indigenous microbe contributed to the
degradation of 25% of petroleum hydrocarbon, in comparison to t removal efficiency of the soil
sample into which immobilized hydrocarbon micro-organism was added.
The soil samples incorporated with the consortium of hydrocarbon degrading microorganism
immobilized in coconut displayed higher oil removal efficiency. The removal efficiency for
TPH, PAHS and BTEX reached up to 60% after three weeks of incorporation. The isolated
microorganisms adapted rapidly to the environment efficiently degrading hydrocarbon
compound. Fig. 3b displays that the biodegradation ability of all the petroleum degrading
bacteria on the soil follow the same pattern. Hence, degradation efficiency of TPH, PAHS and
BTEX gradually increased, which is an indication that the contaminated soil offered a suitable
environment for bacterial growth. 83.0% of TPH, 95.86% of PAHS and 95.0% of BTEX were
degraded during the first six weeks of bioremediation while the degradation efficiency of the
blank sample was only 27%. It is evidenced that the hydrocarbons were degraded majorly by the
petroleum-degrading bacteria.
Fig.3e indicates the number of bacteria as a function of time. The number of bacteria improved
gradually, as bioremediation lasted. The number of bacteria in the inoculated sample was more
than the number of bacteria in the pristine sample. This shows that the indigenous microbes were
inefficient in degrading oil and that the inoculation of petroleum-degrading active bacteria shows
potential in removing hydrocarbons contaminant.
The microbial population increase at the time of remediation process except for control sample
was an evidence of their capacity to use petroleum hydrocarbon as carbon as well as energy
source.. In report by (Apul et al., 2022) the relative significance of bacterial to fungal
populations in hydrocarbons degradation has been controversy. Agitated enrichments, bacteria
tends to have more predominant roles, while other researchers are of the opinion that fungi can
be predominant in undisturbed land surface and water. This issue also reflected in this research
as it is not certain to clearly determine whether bacteria or fungi played the predominant roles
during the remediation process. Many micro-organisms have been known to have the capacity to
use hydrocarbon as source of energy as well as carbon, hence such microorganism are naturally
widely distributed.
The bioremediation reaction rate order was investigated and results represented in Fig.3b.
Once degradation of the crude oil commences, the petroleum hydrocarbon being eliminated with
time and the shape of removal curve is a function of petroleum hydrocarbon type, concentrations,
organism responsible and various environmental factors. In first order reaction, plot natural
logarithm (PAHs, TPH and BTEX) verse time (t) is straight line and slope gives value of rate
constant. The regression coefficients (R2) obtained from the linear plot were 0.910, 0.952 and
0.962 for PAHs, TPH and BTEX respectively. This indicates very strong relationship between
rate of degradation of PAHs, TPH and BTEX with respect to time as seen in Fig. 3 a. From the
plot in Fig.3 d - Fig 3 f , the PAHs, TPH, and BTEX concentrations reduced with time owing to
the growth of hydrocarbon degrading bacteria. The graph in Fig. 3c graphically satisfied the
conditions for first order reaction with a rate constant of 0.499 week -1, 0.256 week-1 and
0.454week-1 and half-life of 1.389 day1, 2.708 days1 and 1.527day1 for PAHs TPH and BTEX
respectively. Bioremediation efficiency of 95.86%, 83.00% and 95.02% were achieved for
PAHs, TPH, and BTEX respectively as seen in Fig. 3a. This is an indication that remediation
process was very efficient as well as effective in clean-up of soil polluted with crude oil.
Microbial growth and activity are readily affected by pH, temperature, and moisture. Although
microorganisms have been also isolated in extreme conditions, most of them grow optimally
over a narrow range, so that it is important to achieve optimal conditions. The temperature
readings of the experiments were in the range of 28–32 oC, which is the ambient temperature in
Nigeria. Low temperature will affect microbial growth and propagation, and under normal
circumstances, rates of degradation decrease accordingly. This is a result of primarily decrease in
the rates of enzymatic activity. The optimum temperature is typically in the range of 31 to 41 oC.
At temperature above this norm, enzymatic activities are inhibited as proteins denature. There
was no significant change in the pH throughout the experiment. It implies that bioremediation
process is pH dependent. The pH value showed a sharp depression at 5.6 as seen in Fig 3g. The
simultaneous production and accumulation of acidic metabolic products particularly during the
first week of the experiments may have accounted for the decrease in the pH which may be
responsible for the decrease in the bacterial population. The pH in the 3rd week through the sixth
week was within the range of 6.8 -7.1 indicating enhanced microbial activity.

Fig 3 (a) Plot of removal efficiency of hydrocarbon (%) against time, (b) Plot of Log of Total
Hydrocarbon Utilizing Bacteria Count (HUBC) and Log of Total Heterotrophic Bacteria Count
(HBC) against Period (weeks)
c d

Fig. 3 ( c) Plot of Total Hydrocarbon against period,(d) Plot of poly aromatic hydrocarbons
against period
e f
Fig. 3 (e) Plot of total benzene, toluene, ethylbenzene and xylene against period, (f) Plot of total
organic carbon against period
g h

Fig. 3 (g) Plot of pH against period, (h) Plot of concentration of heavy against period
j
i

Fig. 3(i) Plot of total heterotrophic bacteria count against period, (j) Plot of hydrocarbon utilizing
bacteria count against period
k

Fig. 3(k) Plot of Nitrate concentration against period, (l) Plot of Phosphorus against period
 n
m

Fig. 3 (m) Plot of total petroleum hydrocarbon degraded per week, (n) Plot of poly aromatic
hydrocarbon degraded per week

Fig. 3 (o) Plot of Benzene, Toluene, Ethylbenzene and Xylene degraded per week
The quality of soil was not adversely affected by remediation with the immobilized products.
The results show that the consortium of these isolates has high proficiency in bioremediation
processes.
Crude oil being a complex mixture of hydrocarbon and non-hydrocarbon molecules when spilled
affects the soil adversely as shown in Fig4a. After one week of contamination, the plants and
probably some others microorganisms could have been destroyed as the soil is laid bare.
Treatment of the contaminated soil with the immobilized microorganism in coconut fibre is
intended to reduce these hydrocarbon molecules which are toxic and major pollutants to the
immediate environment. The potentiality of the bacteria in degrading hydrocarbon is based on
the fact as it serves as energy source to the hydrocarbon utilizing bacteria. Based on the ease of
application of the immobilized microorganism on the crude oil contaminated soil Fig.4b and
steady recovery of the soil and consequent growth of plants Fig4c and Fig4d after seven weeks
and thirteen weeks of treatment respectively. Therefore, bioremediation an effective, low cost

a b
process for rapid remediation of contaminated soil could be adopted as a process for sustainable
ecosystem management.

c d

Fig 4 (a)Parcel of crude oil contaminated land after one week, (b) Site after application of crude oil
degrading microorganism (c) Site after four weeks of treatment (d) Crude oil contaminated site, seven
weeks after treatment
To test the effect of petroleum degrading bacteria on oil removal, active bacteria were introduced
to the crude/drill cutting put on about 9 m 2 polythene lining on the soil. A control sample, made
up of crude oil/drill cuttings (22 litres plastic bucket) put on 1m 2 land but containing no
petroleum hydrocarbon degrading microorganisms.
Fig5a show the relationship between oil residual concentration of petroleum hydrocarbon verse
bioremediation time. The hydrocarbon groups lost may be linked to oil volatilization as well as
aerial oxidation. But most importantly, the concentration of petroleum materials as can be seen
from the plots increased linearly during the days this indicates that the immobilized petroleum
hydrocarbon degrading bacteria contributed to removal of petroleum hydrocarbons, in
comparison to residual concentration of petroleum hydrocarbon in the control and so, after 16
days of treatment, about 77.94%, 55.56% and 48.92% of PAHs, Aliphatic and TPH respectively
have been removed from the drill cuttings compared to 33.21%, 11.26% and 11.69 %
respectively of the control.
The crude oil sludge/drill cutting samples inoculated with immobilized petroleum degrader
displayed higher oil removal efficiency. The TPH and PAHs removal efficiencies were 64.75%
and 40.55% after 8 days of inoculation. The immobilized petroleum degrader rapidly adapted to
the environment efficiently degrading hydrocarbons compounds. Fig5b shows that the
degradation efficiency of TPH and PAHs increased gradually, which indicates that the crude
oil/drill cutting provides an appropriated environment for bacterial growth. Between 48.92% and
77.94% of hydrocarbons were degraded during the first 16 days of bioremediation while the
degradation efficiency of the control was only 11.26% - 33.21%. It is clear that the hydrocarbons
were degraded mainly by the petroleum-degrading active bacteria.

a b

Fig5 Plot of residual concentration of aliphatics, TPH, PAHs against time (days) in , (a) control
sample (b) contaminated sample
4.0 Conclusion

The studies have developed bacterial products with a batch bioreactor which has distinct merits
over conventional method of bioremediations. The product can be quickly applied for
remediation of crude oil polluted site and the deployment showed that it is effective in the
remediation of soils contaminated with crude oil having removed about 91.29% content of crude
petroleum contaminants in seven weeks compared to removal of about 17.36% crude petroleum
content by free residence bacteria cells. The resultant products have great potentials for usage as
preferred product to remediate crude oil polluted sites. And can serve as an effective and fast
remediation tool for cleaning up crude petroleum polluted site at low cost.
Acknowledgement
We wish to express our profound gratitude to the PTDF for providing the fund for this research.
We also thank the management of the Petroleum Training Institute, Effurun, Delta State for their
support and encouragement.
References

Agiri, Gabriel, Mamah, S. (2015) ‘Performance evaluation of oil degrading microbes isolated from crude
oilcontaminated soil in Niger delta area of Nigeria’, Elixir International Journal, 80(30945–30948).

Apul, O. G. et al. (2022) ‘Biodegradation of petroleum hydrocarbons in a weathered, unsaturated soil is inhibited by
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