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European Journal of Applied Sciences – Vol.10, No.3
Publication Date:June 25, 2022
DOI:10.14738/aivp.103.11378. Ezenwelu, C. O., Ezeonyejiaku, C. D., Udemezue, O. I., Nwobodo, V. O., &Oparaji, E. H. (2022). Assessment of Emulsification
Potentials and Bio- Diversities of Bacterial Isolates from Bonny Loading Jetty Crude Oil Polluted Soil, Bonny Island, Rivers
State.European Journal of Applied Sciences, 10(3). 396-407.
Services for Science and Education – United Kingdom
Assessment of Emulsification Potentials and Bio- Diversities of
Bacterial Isolates from Bonny Loading Jetty Crude Oil Polluted
Soil, Bonny Island, Rivers State
Ezenwelu, Chijioke O.
Applied Biochemistry Department, Nnamdi Azikiwe University
Awka, Anambra State. (08037669536)
Ezeonyejiaku, C.D.
Department of Zoology, Nnamdi Azikiwe University
Awka, Anambra State, Nigeria
Udemezue, Onyeka, I.
Department of Microbiology and Brewery
Nnamdi Azikiwe University, Awka, Anambra State
Nwobodo Valentine, O.
Applied Biochemistry Department, Nnamdi Azikiwe University
Awka, Anambra State. (08037669536)
Oparaji, Emeka H.
Department of Chemical Sciences
Spiritan University, Nneochi, Abia State
ABSTRACT
Surfactant producing microorganisms are indispensable in utility; In the present
study, contaminated homogenized soil samples from Bonny Island River showed
the following physicochemical properties: pH (5.67), Chloride ion (Cl-
) (321±0.2
mg/g),Potassium ion (K+) (5.26±0.01mg/g), Magnesium ion(Mg2+) (2.19±0.05
mg/g), Iron II ion(Fe2+) (38.73±0.13ppm), Calcium ion (Ca2+) (4.12±0.1mg/g),
Copper II ion (Cu2+) (3.2±0.01ppm), Lead II ion (Pb2+) (0.21±0.02 ppm), Phosphate
ion (PO3) (6.24±0.1 mg/g), Total petroleum hydrocarbons(TPH 364±0.05 Mg/g),
Total organic carbon (TOC 56.5±0.05 mg/g) and Total organic matter contents
(TOM) (63.45±0.2) .Prior to the emulsification screening carried out; microbial
isolation and total heterotrophic counting (CFU/g) carried out showed a total of
twelve microbial distinct isolates from the polluted soil with a microbial
population of 2.5 x 108 and 2.1 x 108 CFU/g of the soil. Dilution folds of 10-1 and 10-
2 from the Bushnell-Hass media showed microbial population of 0.33 x 104 and
0.78 x 104CFU/g respectively. Klebsiella sp. showed emulsification indices
of43.3%, 51.2%, 50 and 48.9%with diesel and coconut oil, kerosene and crude oil
respectively. Pseudomonas sp. showed emulsification indices of 40, 43, 54 and
45.6% with diesel, coconut oil, kerosene and crude oil respectively. Bacillus sp.
showed emulsification indices of 46, 45, 32 and 37% with diesel, coconut oil,
kerosene and crude oil respectively. The present study have shown the great
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397
Ezenwelu, C. O., Ezeonyejiaku, C. D., Udemezue, O. I., Nwobodo, V. O., &Oparaji, E. H. (2022). Assessment of Emulsification Potentials and Bio- Diversities of Bacterial Isolates from Bonny Loading Jetty Crude Oil Polluted Soil, Bonny Island, Rivers State. European Journal of Applied Sciences,
10(3). 396-407.
URL: http://dx.doi.org/10.14738/aivp.103.11378
potential of utilization of the bacterial consortium in oil recovering from various
damp oil-wells and in cost efficient bioremediation process.
Keywords: Surfactants, bioremediation, emulsification, microbes
INTRODUCTION
The use of biological materials in phytoremediation and bioremediation offer promising
technique for sustainable waste management (Gerhardt et al., 2009). These biological
processes are important and effective over physicochemical processes because their end
products are non-toxic and eco-friendly (Pradecptet al., 2011).Emulsifying agents such as bio
surfactants are surface active bio molecules produced extracellularly or as part of a cell
membrane by a variety of microorganisms ranging from yeast (non-filamentous fungi)
bacteria and filamentous fungi with wide range of applications. Recently, interests in
microbial emulsifiers (surfactants) have increased because of their advantages when
compared to the synthetic or chemical surfactants. Advantages which include their ability to
display a high level of biocompatibility, biodegradation, biodigestibilty, ease of preparation
and ability to serve in a wide range of physicochemical parameter such as: temperature, pH
and salt concentration (Chen et al., 2007).
Due to their unique functional properties, biosurfactants are used in several industries
including organic chemicals, petroleum/petrochemicals, mining, metallurgy, agrochemicals,
foods/beverages and the pharmaceuticals (Vijayakumar and Saravan, 2015). The interfacial
surface tension reducing ability of surfactant makes them able to play important role in oil
recovery and remediation of spilled crude oil. During these exercise, biosurfactants aid the
bioavailability of hydrophobic substrates through solubilization/ desorption. They also
regulate the removal and attachment of microorganisms from surface interface.
In Nigeria, within the coastal region, there are large surface areas contaminated with
petroleum hydrocarbons mainly due to accidental spills or leaks from underground deposits
(Onugbolu and Adieze, 2018). This has caused significance negative impacts and hazards for
agro ecosystems and human health (Baldanet al., 2015).
Biodegradation of petroleum hydro carbons is complex and generally requires different
microbial species or consortia with specific enzymatic capabilities that accelerate the rate of
petroleum degradation. Nevertheless, the efficacy of bioremediation through microbial action
is generally limited by the low availability and solubility of petroleum hydrocarbons due to
their hydrophobicity and adsorption into solid particles and weathering of the hydrocarbon
minerals (Chai-weiet al., 2013). Also microbial presence initially and their eutrophication
during the process of oil recovery and environmental cleaning play much important role to
the remediation process.
Bacteria among those in the Acinetobacter genera may produce substances with emulsifying
characteristic that can degrade petroleum hydrocarbons base on the bioavailability of these
bacteria in the polluted site (Gallego et al., 2011). Emulsifying agents such as biosurfactants
have advantages over chemical surfactants such as improved emulsification, biodegradability
and environmental compatibility along with low toxicity and ability to continue their activity
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European Journal of Applied Sciences(EJAS) Vol.10, Issue 3, June-2022
Services for Science and Education – United Kingdom
under extreme pH, temperature and salinity condition (Soounzaet al., 2013). There are few
studies on the use of emulsifiers producing bacterial for the recovery of chronologically
contaminated soil in Nigeria as most petroleum facilities in Nigeria are above 50 years in use
(Antiquated) making them a risk for hydrocarbon spills due to rusted pipe line and other
anthropogenic activities (Onugbolu and Adieze, 2018). The use of emulsifying bacteria with
potential physiologic activities on weathered petroleum hydrocarbon may significantly
contribute to mid- long term hydrocarbon degradation. This present study is aimed at
assessing the emulsification potentials of bacterial isolates and their biodiversities in a
petroleum loading site at Bonny Island using microbial load measurement and corresponding
breakpoints by standard procedures.
MATERIALS AND METHODS
Sample Collections
Soils samples were collected from a crude oil polluted site from bonny island, Rivers state
Nigeria. The collected soil were pooled together in a clean asceptic container and transferred
to the laboratory.
v Soil Analysis
Soil sample from the polluted site prior to microbial isolations were subjected to various
physicochemical soil profiling test as described in journal of ATSDR, (2010).
The following tests were carried out:
Soil pH Profiling Test
Conductance Test
Determination of Macro and Micro Contents of the Soil
Total organic carbon contents
Total petroleum hydrocarbon contents of the soil
Heavy Metals Identification such as Fe, Hg, As, Cd, Cu and Pb using the atomic absorption
spectra (AAS).
Isolation of the Organisms from Soil
Soil samples containing debries were pooled together from the polluted site into clean plastic
cans and were also to be taken to the laboratory for isolation processes as described by
Onugbolu and Adieze, 2016.
Heterotrophic Counting
Total heterotrophic biomass from both the nutrient media and the mineral salt agar was
counted from the grown media plate as described as follow:
TCU/g= ������ �������� × �������� ������ × ������ �� ��������� ����
Screening of Isolates for Biosurfactant Production
Prior to the screening for biosurfactants, the isolates identified was inoculated into 10ml of
broth medium each and the incubated at 37oC for 72 h. The culture media was centrifuged at
3000 revolutions per minute (r.p.m.) for 30 minutes.