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ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue IX September 2025
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Assessment Of Degradative Potentials Of Bacteria Isolated From
Palm Oil-Polluted Site Using Spectrophotometric Method
1
Nwakoby Nnamdi Enoch,
2
Ejimofor Chiamaka Frances,
3
Ikechukwu Harmony Iheukwumere,
4
Ezeogo
James Item and
5
Abba Oluchukwu
1,3
Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University Uli, Anambra State,
Nigeria.
2
Department of Biological Science, Chukwuemeka Odumegwu Ojukwu University Uli, Anambra State,
Nigeria.
4
Department of Science Laboratory Technology, Federal Polytechnic Ngodo Isuochi, Abia State,
Nigeria.
5
Department of Microbiology, Federal University Gusau, Zamfara State, Nigeria.
DOI: https://doi.org/10.51244/IJRSI.2025.120800306
Received: 02 Sep 2025; Accepted: 08 Sep 2025; Published: 08 October 2025
ABSTRACT
Bioremediation is one of the current approaches in environmental microbiology or environmental
biotechnology that has been exercised for the reduction and removal of hydrocarbon pollutants.
Microorganisms, typically bacteria that have particular metabolic capacities, are essential for the
biodegradation of hydrocarbon pollutants. This study was undertaken to assess ex situ degradative potentials
of Pseudomonas species isolated from palm oil effluent-polluted site using spectrophotometric method. Soil
sediments were collected from different points at palm oil effluent disposal site located at different local palm
oil producers at Uli community, Ihiala Local Government, Anambra State. The samples were analyzed for the
presence of palm oil effluent degrading bacteria using a modified mineral basal medium. The bacterial isolates
were characterized based on their cultural characteristics, microscopy, and biochemical characteristics. The
hydrocarbon adaptation utilization potentials of the bacterial isolates were evaluated using spectrophotometric
method. The biodegradative potentials of the bacterial isolates were evaluated using hydrocarbon
supplemented modified mineral basal medium and spectrophotometer. The Gram negative bacteria isolated
were Pseudomonas species. The optical diameter of the adapted bacterial isolates showed that the isolates
adapted to the hydrocarbon medium while the biodegradative potentials of the isolates showed that the
hydrocarbon was biodegraded as revealed in the weight loss, which increased as the day of degradation
increased. The study has shown that Pseudomonas species are good hydrocarbon utilizing bacteria, which can
be optimized in bioremediation of palm oil effluent-polluted site.
Keywords: Palm oil, effluent, Bacteria, degradation
INTRODUCTION
Palm oil mill effluent (POME) which is also known as Palm oil effluent (POE), Palm oil slurry (POS) or Palm
oil sludge (POS) is a perennial crop with the most common species “Elaeis guineensis” grown extensively in
West Africa's humid tropical and subtropical region, where it originated from (Bambang et al., 2012),
however the world's largest producers of crude palm oil are actually Indonesia, Malaysia, Thailand, Columbia
and Nigeria (Ohimain and Izah, 2014; Izah et al., 2016). Among all territorial name been given, Palm oil mill
effluent seems to summarize it's content which is defined as the voluminous liquid waste that originates from
the sterilization and clarification forms in milling oil palm. It is a wastewater produced from palm oil milling
exercises which require successful treatment before release into nature because of its exceptionally polluting
properties (Ismail et al., 2010).
Thus, POME is being treated via palm oil mills before evacuating it into the streams and rivers. Palm oil mill
effluent is termed to be a highly polluting material and researchers have done so much in their studies to find
ways of removing its threat to the environment (Awotoye et al., 2011; Izah and Ohimain, 2016). The
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composition of the effluents are from different sources and are obtained from palm oil, water, sand and solid
(suspended and dissolved). Water which composed of 93-95%, solidly composed of 3- 4% and oil composed
of 0.5-2% are various composition as a percentage of total sludge (Madaki et al., 2013). Biodegradation also
known as “biological breakdown” is the process by which organic substances are decomposed by
microorganisms (mainly aerobic bacteria) into simpler substances such as carbon dioxide, water and
ammonia. The use of biotechnological forms including microorganisms, with the target of taking care of
environmental contamination issues, is quickly developing where POME and its side effects are concerned.
The process by which microorganisms such as bacteria, fungi and other biological activity act on material by
naturally disintegration is called Biodegradation. The biological treatment relies immensely upon a
consortium of microorganism's activities, which operate the organic substances present in the POME as
enhancements and in the end debase these organic 0issues into a simple by-product, for example, methane,
carbon dioxide and hydrogen provided, and water (Mohammadreza and Soheila, 2014).
According to Liew et al. (2015), the past few decades, various methods have been recorded for the treatment
of POME. Anaerobic or facultative ponding method, tank digestion and mechanical aeration, tank digestion
and facultative ponds, physico-chemical and biological care, and decanter and facultative ponds are the most
refined treatment plans that have been widely applied in POME degradation. However, these approaches have
some disadvantages, such as the prolonged period of retention, greenhouse gases output, large land area
requirement and inconsistent nutrient removal (Ganapathy et al., 2019; Affandi et al., 2014).
Biological treatment such as ponding system is a conventional treatment that involves aerobic and anaerobic
processes which involves microorganisms such as bacteria, molds, algae, yeasts, and fungi to degrade lipids in
the POME (Ganapathy et al., 2019; Rupani, et al., 2019). One of the unique ways through which
microorganisms obtained energy is by catalyzing energy that causes chemical reactions involving breaking
bonds and moving electrons away from the pollutants, during this process, the organic contaminant is
oxidized in this form of reaction, while the chemical that acquires the electron is reduced.
Several researchers had succeeded in isolating and characterization of bacteria and fungi from palm oil
effluent-contaminated soil (Kwute and Ijah, 2014; Adegbola et al., 2020; Popoola et al., 2022). Some of the
microorganisms identified were Bacillus, Pseudomonas, Micrococcus, Aspergillus, Penicillium and so on.
These microorganisms survived in such environment due to their high degradative potentials. The energy
obtained from this transition is then invested in growth and metabolism of the biodegrading microbe
(Ramadan et al., 2012; Loretta et al., 2016). The aim of this study is to assess an ex situ degradative potentials
of Pseudomonas species isolated from palm oil-polluted site using spectrophotometric method.
MATERIALS AND METHODS
Study Area
The study was conducted at Umuaku, Uli, Ihiala Local Government Area, Anambra State. Uli is a village
located between latitudes 5.47°N and 5.783°N and longitude 6.52°E and 6.87°E on the South eastern part of
Nigeria. Uli extends westward to the confluence of the rivers of Atammiri and Eyinja, and across Usham lake
down to the lower Niger region. Uli has rainforest vegetation with two seasonal climatic conditions: rainy
season and dry season, which is characterized by the harmattan between December and February. Uli is
characterized by double maxima of rainfall with a light drop in either July or August known as dry spell or
August break. The annual total rainfall is about 1,600 mm with a relative humidity of 80 % at dawn.
Sample Collection
The soil surface was carefully scrapped out using sterile spoon. Soil auger was used to a plough depth of 15
cm in the sampling sites (palm oil effluent-contaminated sites), and soil sample was drawn (up to 10 samples)
from each sampling unit into a sterile tray. The samples were thoroughly mixed and foreign materials such as
roots, stones, pebbles and gravels were carefully removed. The soil sample was then reduced to half by
quartering the sample. Quartering was carried out by dividing the soil sample into four equal parts and the two
opposite quarters were discarded and the remaining two quarters were mixed. The process was repeated for
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the rest of soil samples used for this study. The samples were carefully labeled and then kept in a disinfected
cooler, to maintain its temperature and stability of the number of the isolates. The samples were transported to
the laboratory for analysis within 2 h.
Sterilization of materials
As stated in prescott et al. (2005), conical flasks (pyrex), prepared media and other plastic materials were
sterilized by autoclaving at 121℃ for 15 minutes at a pressure of 15 psi. Glass wares such as pipettes, glass
spreader, petri dishes, measuring cylinder, and other glass materials were sterilized in the laboratory hot air
oven at a temperature of 160 ℃ for 1hr before use.
Isolation of Palm Oil Degrading Bacteria from the Samples
Serial dilution
A ten-fold serial dilution of the samples was carried out by adding 1g or 1ml respectively of sediment and
water samples aseptically in test tubes containing 9ml of 0.85% of physiological saline solution labeled 10
-1
to
10
-10
dilution factors with the aid of a sterile pipette in a repeated manner. With another sterile pipette, 0.1
aliquots of the appropriate dilutions (dilutions that produce colony counts between 30 – 300 colonies) were
spread plated on the surfaces of the solidified media in triplicates with the aid of a glass spreader. Precisely,
10
-3
dilutions were spread plated. The spreader was sterilized after each successive spreading by dipping it in
70% ethanol and then passing it through flame of a Bunsen burner (Bahig et al., 2008; Chikere et al., 2009;
Kafilzadeh et al., 2012).
Enrichment, culturing and isolation of palm oil degraders
The palm oil degraders were isolated from sediments of the three sampling sites using modified mineral basal
agar (4g K
2
HPO
4
, 1.0g (NH
4
)
2
SO
4
, 0.1g MgSO
4
, 1.8g K
2
HPO
4
, 0.1g FeSO
4
, 0.1g NaCl, 0.2ml CaCl
2
, 15g
Agar agar and distilled water 1000ml at pH 7±0.2) enriched with xylene, anthracene and pyrene as hole
carbon and energy source. The medium was sterilized by autoclaving at 121
o
C at a pressure of 15psi for 15
minutes. Thereafter, 0.2 ml acetone solution containing 0.1% w/v of the selected hydrocarbons (xylene,
anthracene and pyrene) were aseptically pipetted and uniformly spread on the agar surface of the pre-dried
petri dish plates. The acetone was allowed to evaporate under sterile condition before inoculating with 0.1ml
of diluted sediment and water samples. The inoculated plates were sealed using adhesive tape and foil to
prevent contamination and photolysis and later placed in black polythene bags, and then incubated in the dark
at 28 ± 0.2 ℃ for 24 – 48h (John and Okpokwasili, 2012).
Purification and maintenance of cultures
Colonies that developed on hydrocarbon-coated plates were replicated onto fresh hydrocarbon-coated agar
plates and incubated for 14 days. Isolates that grew on these plates were selected as xylene, anthracene and
pyrene degraders and sub-cultured on Bjou bottles where they are preserved at 4℃ in refrigerator (John et al.,
2012).
Cultural and Morphological Characteristics
After sub-culturing and incubation, culturing morphological properties such as shape, elevation, margin, optic,
texture, colour, size and surface characteristics of the selected bacterial strains were observed and noted
(Prescott et al., 2005).
Gram staining
This technique divides bacteria into Gram positive and Gram negative groups. A smear of the isolates was
made on a clean dry grease free slide, using a sterile wire loop. The smear was air dried and heat fixed by
passing over flame quickly three times. It was then covered with 0.5% crystal violet solution for 1minute and
rinsed with distilled water. The slide was flooded with 1% Gram’s iodine (which served as a mordant that
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fixes the dye inside the cell). The iodine was washed off after one minute and 95% ethanol was used to
decolorize the smear for 15 seconds. The smear was counter stained with 0.1% safranin dye solution for one
minute. It was then washed off and the slide air-dried, and observed under the microscope using oil immersion
objective lens after placing a drop of oil immersion. Gram positive and negative reactions were indicated by
purple and red colors respectively (Cheesbrough, 2010).
Spore staining
According to the method of HPA (2007), smears of the isolates were prepared and fixed on a slide. The
underside was vapor heated and flooded with 5% malachite green solution. Heating would continue until
visible water condensate forms under the slide with evaporation at the top. It was washed using distilled
water. Smears were counter stained with 0.5% safranin solution for 10 seconds. Slides were washed, dried and
observed under oil immersion objective lens after pacing a drop of immersion oil. A green space within the
cells would indicate the presence of spores.
BIOCHEMICAL CHARACTERISTICS
Catalase test
As stated in cheesbrough (2010), the test identifies organisms that produce the enzyme catalase. A drop of
30% freshly prepared hydrogen peroxide (3ml H
2
O
2
in 7ml H
2
0) was placed on a clean slide and loopful of
isolate was transferred into it and emulsified. The appearance of gas bubbles indicates positive reaction. The
reagent was shaken before the test to expel any dissolved oxygen and avoid false positive result.
Indole test
As stated in cheesbrough (2010), the tryptone-broth was prepared and 5ml was dispensed into each test tubes
and sterilized. The isolates were inoculated into the test tube and incubated at 28℃ for 48h. After incubation,
5 drops of Kovac’s reagent (4 – p – dimethyl – aninobenzaldehyde) were added to the tubes, shaken gently
and allowed to settle. A red coloration in alcohol dye indicates a positive result for the reaction.
Motility test
A directional and purposefully movement of the organisms demonstrate motility. Nutrient broth was
supplemented with 0.2% agar, dispensed into test tubes and sterilized by autoclaving at 121℃ and 15psi for
15minutes. The inoculated test tubes were incubated for 24h. Diffused growth, which spreads throughout the
medium, indicates motility. Non-motile organisms grew along the line of inoculation (Cheesbrough, 2010).
Citrate test
As stated in Prescott et al. (2005), the test was used to determine organisms that could utilize citrate as a sole-
carbon source for metabolism. Slant of Simmon’s citrate agar were prepared according to manufacturer’s
instructions. The slants were inoculated by streaking over the surface with a loopful of an 18h old culture and
incubated at 37℃ for 48h. Positive results were indicated by the growth on agar and a change in color from
green to blue and absence of color change indicates negative results.
Starch hydrolysis test
To determine the abilities of the isolates to hydrolyze starch, 50 μl of liquid cultures of each isolates were
dropped on starch – based solid medium containing per litre, 3g meat extract, 10g starch and 15g agar
(Cheesebrough, 2010).
Gelatin test
As stated by Prescott et al.(2005), gelatin agar medium was composed of 40g/l of gelatin, 30g/l of tryptic soy
broth and 100ml of distilled water. A small inocula of the isolates was stabbed to about three-quarter of the
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way to the bottom of a tube of deep agar with the inoculating needle. The separate stab tubes for each of
isolates were incubated at 37℃ for 24 – 48h. The incubated stab and the un-inoculated control tubes were
placed into the refrigerator for approximately 30 minutes. The inoculated stab tubes were compared with the
control by tapping the tubes gently.
Hydrogen sulfide production test
As stated in Prescott et al.(2005), The test determines that ability of organisms to reduce sulfur compounds
Triple sugar ion agar slants were prepared and each isolates were inoculated into test tubes by streaking the
inocula across the top of the slants and stabbing the slant tubes to the
bottom. Tubes were incubated at 28℃ for 24h. Positive result is indicated by the formation of black color
coupled with displacement of the agar slant and red to yellow color observation.
Sugar fermentation
As stated in Prescott et al.(2005), the test determines the ability of the isolates to ferment glucose, sucrose,
lactose, mannitol, maltose, xylose, arabinose and saccharose and also ability to produce gas. The fermentation
medium contained 1% peptone water and 5 drops of 0.2% bromothymol blue indicator solution. Then 9ml of
the medium was dispensed into clean dry test tubes in which Durham tubes been dropped (inverted and
without air space) and sterilized by autoclaving at 121℃ and 15psi for 15 minutes. 1ml of the sterile 5% test
sugar solution was added to medium and inoculated with a loopful of the test organisms and incubated at 30℃
for 24h. A change in color of the medium (from blue to yellow) was recorded as positive reaction, while
presence of gas in Durham tubes indicates gas production.
Oxidase test
As stated in Prescott et al.(2005), the test identifies any organism that produces the enzyme oxidase. A loopful
of isolates was transferred into pieces of Whatman No.1 filter paper, impregnated with a solution of freshly
prepared oxidase test reagent (N,N,N’,N’ tetra-methyl-phenylene diamine) and smeared. Oxidation of the
phenylene diamine in the reagent to dark purple or blue color within 10 seconds indicates a positive result.
Casein hydrolysis test
The casein hydrolysis was observed by zones of clearing after 24h of incubation. For this purpose, 50 μl liquid
cultures of each isolates were dropped on casein-based solid medium containing (per litre) 10 g casein and
15g agar. After 24h of incubation, the inhibition zones were determined (Cheesbrough, 2010).
Hydrocarbon Adaptation Utilization Test
In order to screen and select the best and strongest degrading bacterial isolate, different organisms were tested
by growing 5ml of each desired isolates in large test tubes containing 25 ml of the modified mineral basal
medium with 1ml of xylene, anthracene and pyrene hydrocarbons which were dissolved in acetone and added
to each tube autoclaving. Thereafter, the test tubes were incubated at room temperature (28±2℃) for five days.
Bacteria that started growing fast with high turbidity in the vicinity of the medium containing aromatic
compounds measured at 600nm using a UV – VIS spectrophotometer (Astell, UV – Vis Grating, 752W) were
selected as the candidate of xylene, anthracene and pyrene degrading bacteria. Cultures without increase in
turbidity over initial optical density (OD) and uninoculated control were scored as no growth (-) while
cultures with increased turbidity significantly greater than the control were scored as growth (+) (John et al.,
2012).
Degradation Assay
By adopting the methods of Bennet et al. (2012) and John and Okpokwasili (2012), as modified in this study,
the degradation rates of bacterial isolates were determined using hydrocarbon supplemented modified mineral
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basal medium (4g K
2
HPO
4
, 1.0g (NH
4
)
2
SO
4
, 0.1g MgSO
4
, 1.8g K
2
HPO
4
, 0.1g FeSO
4
, 0.1g NaCl, 0.2ml
CaCl
2
, 15g Agar agar and distilled water 1000ml at pH 7±0.2). Precisely, 1ml of 48 h old cultures of each
organisms were introduced into 28 sterile 200ml capacity conical flasks (7 sets of 4 flasks) containing 100ml
of sterile modified mineral basal medium supplemented with 1 ml of xylene or 1ml of anthracene or 1g of
pyrene, respectively as source of carbon at 24℃ for 24 days. During incubation, representative samples from
the three days sets of flasks were withdrawn at intervals of 0, 4, 8, 12, 16, 20 and 24 days and the residual
hydrocarbons were determined spectrophotometrically using ethyl acetate as the extraction solvent. For each
sample, 5 ml
ethyl acetate was added and vigorously shaken manually. The organic and aqueous layers from media were
separated by centrifugation at 5000rpm for 20 minutes. The aqueous layers were discarded while the organic
layers were analyzed with UV – VIS spectrophotometer at 240 nm wavelength (Astell UV – Vis Grating, 752
W). The percentages of biodegradation of the hydrocarbons were determined as follows:
% degradation =
𝑎−𝑏
𝑎
×
100
1
Where a = the absorbance of the medium before incubation; b is the maximum absorbance of the medium
after each 4
th
day of the incubation period.
Statistical Analysis
The difference in the absorbance of the bacterial isolates was determined using students’‘t’ test and values of
P that exceeded 0.05 (P > 0.05) were considered not significant.
RESULTS
Characterization of Bacterial Isolates in the Impacted Soil
The result of characterization of the bacterial isolates in the impacted soil is presented in Table 1. The result
revealed that the bacterial isolate Y appeared cream white on Nutrient agar while isolate Y appeared fussy
white on the same agar. The edge, elevation, surface, optical nature, and size of the isolates appeared entire,
convex, smooth, opaque, and small, respectively. Both isolates were Gram negative, motile, rods, and non-
spore formers. The biochemical characteristics of the isolates revealed that both isolates were positive to
catalase, citrate, oxidase, gelatin, casein, starch, and glucose, while maltose and sugar alcohols were not
utilized.
Table 1: Cultural and morphological characteristics of the bacterial isolates
Parameter
Isolate X
Isolate Y
Appearance on Nutrient Agar
Pale yellow
Pale green
Edge
Entire
Entire
Elevation
Convex
Convex
Surface
Smooth
Smooth
Optical Nature
Opaque
Opaque
Size
Small
Small
Gram Reaction
Negative
Negative
Cell Morphology
Rods
Rods
Motility
Motile
Motile
Endospore
Absent
Absent
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Bacterium
Pseudomonas species
Pseudomonas species
Table 2: Biochemical characterization of the bacterial isolates
Parameter
Isolate X
Isolate Y
Catalase
+
+
Citrate
+
+
Indole
_
_
Oxidase
+
_
H
2
S
_
_
Gelatin
+
+
Casein
+
+
Starch
+
+
Glucose
+
+
Maltose
_
_
Dulcitol
_
_
Inositol
_
_
Xylitol
_
_
Bacterium
Pseudomonas sp.
Pseudomonas sp.
Optical Diameter of the Adapted Isolates and Weight Loss of hydrocarbon During Degradation
The result of optical diameter of the adapted isolates is presented in Table 3. The result revealed that at day 0,
the bacterial isolates recorded the least optical diameter of 0.0021 and 0.0039, respectively. Meanwhile, as the
day progresses, the optical diameter increased but the increment was not significant. Meanwhile, isolate Y
showed higher optical diameter as the day increased. Similarly, the weight loss of hydrocarbon during
degradation is presented in Table 4. The result revealed that at day 0, there was zero weight loss. Also, the
weight loss during degradation increases as the day increases with the highest weight loss recorded at day 4 of
degradation.
Table 3: Optical diameter of the adapted isolates
Day
Pseudomonas X
Pseudomonas Y
O
0.0021
0.0039
1
0.0282
0.0377
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2
0.0562
0.084
3
0.1310
0.1960
4
0.2520
0.3900
Table 4: Weight loss of the hydrocarbon during degradation
Day
Weight (%)
Pseudomonas X
Pseudomonas Y
0
0.00
0.00
1
0.58
0.69
2
0.95
1.91
3
2.53
2.98
4
3.97
4.95
DISCUSSION
Environmental contamination has threatened the ecosystem in all ramifications. Various contaminants
basically hydrocarbons alter the normal structure of soil and vital soil microorganisms that aid in nutrient
recycling and soil fertility. The characteristic features of the bacteria isolated from the palm oil-polluted soil
sediment in this study corroborate with the report of several researchers (Jeremiah et al., 2014; Jeremiah et al.,
2018; Imo and Ihejirika, 2021; Popoola et al., 2022) who evaluated potentials of certain bacterial species in
degrading complex compounds in palm oil. In this study, Pseudomonas species was isolated while Adegbola
et al. (2020) and Popoola et al. (2022) isolated Bacillus species, Micrococcus species, Pseudomonas,
Aspergillus species as palm oil effluent degraders. The ability of the bacterial isolates to utilize various sugars
and sugar alcohol such as starch, glucose, maltose, dulcitol, inositol, and xylitol could be attributed to their
high degradative potentials. This observation agrees with the findings of several researchers (Popoola and
Onilude, 2017; Jeremiah et al., 2018; Popoola et al., 2022) who documented that Bacillus species utilize
breakdown carbohydrate as source of carbon and energy for metabolism. The increase in the optical diameter
of the bacterial isolates as the degradation day increases could be attributed to their ability to utilize the
products of degradation as source of energy and carbon for optimum proliferation. This observation
corresponds to the report of Kwute and Ijah (2014) that investigated potentials of microorganisms in
degrading palm oil effluent and recorded a high number of the isolates at the highest day of degradation. The
loss in weight of the hydrocarbon as the degradation day increases could be attributed to biodegradation of the
bacterial isolates where the major components are utilized as source of energy and carbon. This observation is
in line with the reports of several researchers (Mohammadreza and Soheila, 2014; Jeremiah et al., 2014;
Kwute and Ijah, 2014) who evaluated biodegradative potentials of different microorganisms.
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CONCLUSION
This study has investigated the ex-situ assessment of biodegradtive potential of bacteria isolated from diesel
contaminated sites and revealed that Pseudomonas species had ability to degrade complex compounds in palm
oil effluent as energy and carbon sources. The study also showed that biodegradation increases with time, and
there is always reduction in the weight of hydrocarbon as biodegradation increases. Therefore, Pseudomonas
species can be optimized for environmental sanitation of palm oil effluent-polluted soil for optimum
productivity.
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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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Page 3390
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