SPE-198799-MS

Characterization of Potential Paraffin Wax Removing Bacteria for

Sustainable Biotechnological Application

A. U. Okoye, Africa Centre of Excellence in Oilfield Chemicals Research, University of Port Harcourt; C. B. Chikere
and G. C. Okpokwasili, Department of Microbiology, Faculty of Science, University of Port Harcourt, Rivers State,
Nigeria

Copyright 2019, Society of Petroleum Engineers

This paper was prepared for presentation at the Nigeria Annual International Conference and Exhibition held in Lagos, Nigeria, 5–7 August 2019.

This paper was selected for presentation by an SPE program committee following review of information contained in an abstract submitted by the author(s). Contents
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Abstract
The presence of paraffin wax precipitation and deposition in tubing surfaces during production is one of the
all-encompassing nuisances in oil and gas industry operations worldwide and it causes major production
problems. The study aimed at acquiring novel insights into the bacterial community diversity capable of
utilizing paraffin wax. Samples were collected from crude oil-polluted site in Gio Community, Tai Local
Government Area, Niger Delta, Nigeria at depth of 0-0.5m (surface polluted soil [SPS]), 1m (sub surface
polluted soil [SPSS]). GPS coordinate points for the North, South, East and West were N40 41′ 39″; N40
41′ 38″; N40 41′ 38″ and N40 41′ 37″ for latitudes respectively. Longitudes for the coordinates were E70
13′ 49″; E70 13′54″; E70 13′ 53″ and E70 13′ 54″ for North, respectively and unpolluted soil (UPS) taken
80m away from polluted site as control. All samples were transported to the laboratory within 6h at 4°C
for analyses. Biodegradation screening was carried out using 2, 6-dichlorophenol indophenol (DCPIP)
(redox potential +0.217 V) indicator to evaluate the biodegradation of hexadecane, paraffin oil and crude
oil by axenic cultures of the bacteria isolated. GC-FID analysis for total petroleum hydrocarbons (TPH)
were 22,146.65ppm for (surface polluted soil [SPS]), 14,087.80ppm (sub surface [SPSS]) and control soil
(UPS) 479.67ppm respectively. Polycyclic aromatic hydrocarbons (PAHs) were 12,209.3ppm for SPS,
3,248.75ppm for SPSS and 22.72ppm for UPS. Total cultivable hydrocarbon utilizing bacterial count
(TCHUB) for SPS, SPSS and UPS were 8.4 × 105cfu/g, 8.0 × 105cfu/g and 3.96 × 104cfu/g respectively.
Pseudomonas spp., Bacillus spp., Acinetobacter sp. and Serratia sp. demonstrated higher biodegradability
of paraffin wax than other 62 TCHUB isolated. These bacteria may probably represent an alternative green
method for scale removal in the oil and gas sector.
Keywords: Bacteria, Biotechnology, Paraffin wax, Biodegradation
2 SPE-198799-MS

INTRODUCTION
The rapid industrialization in the Niger Delta region and increase in anthropogenic activities (exploration,
extraction, refining, transport, and use of petroleum and derivative products) have exposed the soil and
water to hydrocarbons which has adversely affected the ecosystem and food chain.
Crude oil has been an important part of the Nigerian economy for decades, since large reserves were
discovered in the 1950s (Donald et al., 2016). It has been the fundamental source of energy, as well as
serving as primary raw material for the petrochemicals and allied industries (Ite et al., 2013). The increasing
demand and utilization of petroleum products have encouraged its exploration and exploitation in Nigeria
and globally. The increase in world's oil production, refining and distribution has brought with it many
environmental challenges (Onwurah et al., 2007; Ahmed and Fakhhruddin, 2018; Adipah, 2019). Some of
the adverse effects include damage to terrestrial and aquatic life, change in water quality, devastation of the
aesthetic value of beaches, loss of soil fertility, agricultural productivity, considerable environmental and
health hazards (Escalante-Espinosa et al., 2005; Bamidele and Igiri, 2011).
Alkanes are quantitatively the major constituents and most important fraction in crude oil. Their
degradation is widespread among hydrocarbon utilizing bacteria (Rojo, 2010). They are potentially the most
abundant sources of carbon and energy for many hydrocarbonoclastic microorganisms. Alkanes are subject
to both aerobic and anaerobic oxidation (Wang and Shao, 2013). The degraders of alkanes are bacteria that
have a very amenable metabolism, so they can use alkanes and many other compounds as carbon (Smits et
al., 2002; Margesin et al., 2003; Harayama et al., 2004; Zampolli et al., 2014). Hydrocarbons are present
in marine and soil environments, through natural and anthropogenic sources and also occur as by-products
of cell metabolism and death. Alkanes are oxidized by alkane hydroxylases at the terminal or sub-terminal
carbon, converting the alkane into an alcohol. The diversity of alkane hydroxylases includes those that
oxidize low molecular weight alkanes (approximately C1-C4) and are the soluble methane monooxygenase
(SMMO), particulate methane monooxygenase (PMMO), and propane/butane monooxygenase (P/BMO)
enzymes. Acting on mid-weight alkanes (roughly C5-C16) are a group of alkane hydroxylases belonging to
the cytochrome P450 family of enzymes and the membrane-bound non-hemealkB enzymes. Long-chain
alkane monooxygenases (LadA and almA), utilize terminal or sub-terminal oxidation pathway for the
conversion of long-chain alkanes (up to at least C36) to corresponding primary alcohols (Bowman et al.,
2014)
Parafin wax is any saturated, straight chain hydrocarbon with the formula CnH2n+2. Pure paraffin wax
is a petroleum, coal or oil shale by-product that is white or colorless, odorless and tasteless. It consists
of a mixture of hydrocarbon molecules containing between twenty and forty carbon atoms (Viswanathan
and Nallamuthu 2007; 2017). The melting point, without additives, is between 37° to 68° C. It solidifies
at room temperature and begins to melt above approximately 37 °C (99 °F); its boiling point is >370 °C
(698 °F). Universally, it is used for lubrication, electrical insulation, and candles making; dyed paraffin
wax can be made into crayons. It is different from kerosene and other petroleum products sometimes called
paraffin. Isoparaffin, cycloparaffin, and naphtheno-aromatic hydrocarbons are also found in paraffin (Briker
et al., 2003). Solid paraffin, which melts between 5° and 70°C, is primarily obtained from oily distillates
of paraffin-base petroleum.
Many crude oils contain dissolved waxes which can precipitate and deposit under the appropriate
environmental conditions. Waxes are the most important constituent of crude oil (Ganeeva et al., 2016).
Paraffin produced from crude oil consists primarily of long chain, saturated hydrocarbons (linear alkanes/
n-paraffins) with carbon chain lengths of C18 to C75. (Dowman et al., 2014). Paraffin deposition on the
pore walls or the tubing surface of oil production wells causes major production problems. They can build
up in production equipment and pipelines, potentially obstructing flow (reducing volume produced) and
generating other problems (Ehadad et al., 2015).
SPE-198799-MS 3

The presence of paraffin wax precipitation and deposition is one of the all-encompassing problems in oil
industry operations worldwide. It has been acknowledged as one of the critical concerns during production
and transportation of crude oil (Bagdat and Masoud, 2015).
Paraffin wax generate problems in production, transport, operations and can be an issue to tank-bottom
sludge, with wax deposits generating problems from the well head to the refinery (Tukenov, 2014). Presently,
this problem is solved by thermal fluid treatments, the use of chemicals and solvents, or pigging (scraping)
(Aiyjani et al., 2011). Most of these remedial measures are expensive and have limitations. Mechanical
scraping causes loss due to major stop in production. Chemicals and solvents, aside from being harmful
or ecotoxic (Araújoet al., 2010), incompletely liquefy or break up paraffin, which ends up being deposited
somewhere else in the tubings (Zhang et al., 2016). In addition, the crystals stick to the walls of the oil
wells, the casing wall, pump, and other production equipment, which slow the flow of oil and obstruct the
transport in the pipelines (Zhang et al., 2016). According to reports, more than half of the extractable oil
remains underground after the first and second attempts at oil recovery (Pitchel, 2016).
The use of microorganisms represents an ecofriendly and cost-effective way to biodegrade crude oil
derived substances. This ability is highly exploitable during bioremediation processes to ultimately free the
environment from subsequent pollutants derived from these hydrocarbons. However, microbial treatment
method for paraffin deposition has been used as a sustainable substitute to conventional treatment methods
(chemical, mechanical and thermal methods. The diverse metabolic potential of microorganisms and their
interactions with hazardous organic and inorganic compounds have long been known (Gutierrez, 2018).
Microbial methods are environmentally friendly and can be incorporated to detoxify, degrade and prevent
contaminants. The bacteria, required for the attenuation of paraffin deposition problems, should be able to
survive the high temperatures of oil wells, degrade the paraffin under low oxygen and nutrient conditions
while sparing the low carbon chain paraffin (Sood et al., 2008). Microbial biodegradation can retrieve a
wide range of hydrocarbons including heavy oil that remains largely as trapped oil in reservoirs (Head et
al., 2003, Phetcharat et al., 2019).
The aim of the study was to gain novel insights into the bacterial community diversity from a chronically
polluted site capable of utilizing paraffin wax and ascertain their potentials for sustainable biotechnological
applications in the oil and gas sector.

Methodology
Sample collection
Samples were collected from chronically crude oil-polluted site at Gio Community in Tai Local Government
Area (LGA), Rivers State, Nigeria as shown in Fig.1. Soil was collected with soil auger into polyethylene
bags sanitized with 70% ethanol from the crude oil-polluted site at the 4 coordinates. Specific coordinates
of the sampling site for the North, South, East and West were N40 41′ 39″; N40 41′ 38″; N40 41′ 38″ and N40
41′ 37″ for latitudes respectively. Longitudes for the coordinates were E70 13′ 49″; E70 13′54″; E70 13′ 53″
and E70 13′ 54″ for North, South, East and West respectively. The altitudes were 23.87mt (N); 18.66mt (S);
26.47mt (E); 33.64mt (W). The samples were collected at depth of 0-0.5m (surface) and 1m (sub surface)
respectively and unpolluted soil (UPS) to serve as control. All samples were transported to the laboratory
within 6h at 4°C for analyses.
4 SPE-198799-MS

Figure 1—Crude oil-polluted wetland in Gio community, Tai Local Government Area, Rivers State.

Baseline Characterization
○ Physicochemical parameters such as moisture content, nitrate, phosphate, total organic carbon (TOC),
turbidity, temperature, pH and conductivity was determined using methods from APHA (1998).
Gas chromatographic analysis of total petroleum hydrocarbons (TPH) and polycyclic aromatic
hydrocarbon (PAHs) was done to determine the extent of hydrocarbon contamination.
○ Paraffin wax preliminary enrichment
For each sample, 1 g of soil was inoculated into 250 ml Erlenmeyer flask (Fig. 2) containing 100
ml of sterilized Bushnell Haas broth supplemented with 1% (w/v) sterilized paraffin wax (mainly
composed of C18–C48 straight chain alkanes as the sole carbon source) using the method of Zhang et
al. (2016). The setup was incubated in a rotary shaker at 37°C and 150 rpm for 7 days. Ten-fold serial
dilution of paraffin wax enrichment was done using sterile distilled water and plated out in triplicates
using spread plate method for total hydrocarbon utilizing (TCHUB) in Bushnell-Haas agar according
to Wang and Shao (2013); Zhang et al., (2016).
SPE-198799-MS 5

Figure 2—Preliminary paraffin wax enrichment with soil samples.

Enumeration of total culturable heterotrophic bacteria

► One gram (wet weight) of soil sample was homogenized in 0.85% of phosphate buffered saline.
Decimal dilutions (tenfold) of the suspensions were plated out in triplicate on plate count agar (Merck,
Germany) and incubated at 30°C for 24h.

Purification and characterization of hydrocarbon utilizing bacteria

► Discreet colonies of different HUB were randomly picked using a sterile inoculating wire loop and
subcultured by streaking on Luria Bertani (LB) agar plates then incubated at 30°C for 24h. Phenotypic
tests were carried out as described by Bergey and Holt (1994); de la Maza et al. (2013).

Biodegradation (Turbidimetry) screening with redox indicator 2,6-dichlorophenol indophenol

(DCPIP)
Biodegradation screening (turbidimetry test) was used to assess the degradation ability of individual
isolatesat 600nm (Bidoia et al, 2010; Singh and Sedhuraman, 2015; Chikere et al., 2016; Lee et al., 2018).
The assay was carried out using 2, 6-dichlorophenol indophenol (DCPIP) (redox potential +0.217 V)
indicator to evaluate the biodegradation of paraffin oil by axenic cultures of the bacteria isolated. The DCPIP
was prepared by dissolving 1 g of the DCPIP in 1 litre of sterile distilled water (Mariano et al., 2008,
Balogun et al., 2015). The degradation potential of the hydrocarbon utilizing bacteria, were further screened
under aerobic conditions by inoculating a calibrated loopfull of 18h old culture of each isolate into 9 ml of
Bushnell Haas Broth containing 0.5 ml of paraffin oil as the sole carbon source and 0.5ml of 1% DCPIP.
Absorbance of the inoculated medium at 600 nm was determined on days 0, 3, 6, 9 and 12. Biodegradation
was scored by the turbidity of Bushnell Haas Broth after 0-12 days incubation at 30 °C with a concomitant
reduction of the blue dye DCPIP (λmax = 600 nm) to colorless. The growth of the bacterium was measured
by taking the OD readings at 600nm with SP-OPTIMA Spectrophotometer (Optima, Japan).

Polymerase chain reaction and detection of long-chain hydrocarbon degrading gene

Genomic DNA from the recovered axenic bacterial cultures was extracted using Zymo Research Fungal/
Bacteria DNA kit™. DNA samples were quantified using NanoDrop ND-2000 spectrophotometer. Long-
6 SPE-198799-MS

chain hydrocarbon degrading gene (almA) was amplified using the functional primers (ladA FR 5′-
GGCGTSTACGMCRWCTACGGYRGG-3 and ladA RV 5′-GAYCTACCAGGYCGGGTCGTCG-3). The
PCR reaction was carried out using the Solis Biodyne 5X FIREPol Blend Master mix. PCR was performed
in 20 μl of a reaction mixture, and the reaction concentration was brought down from 5x concentration to
1X concentration containing 1X Blend Master mix buffer Buffer (Solis Biodyne), 1.5 mM MgCl2, 200μM
of each deoxynucleoside triphosphates (dNTP)(Solis Biodyne), 20pMol of each primer (Jena Bioscience,
Germany), 2 unit of Hot FIREPol DNA polymerase (Solis Biodyne), Proofreading Enzyme, 5μl of the
extracted DNA, and sterile distilled water was used to make up the reaction mixture. Thermal cycling was
conducted in an Pielter thermal cycler (MJ Research Series) for an initial denaturation of 95°C for 5 minutes
followed by 30 amplification cycles of 30 seconds at 95°C; 1 minute at 50°C and 1 minute 30 Seconds
at 72°C. This was followed by a final extension step of 10 minutes at 72°C. The amplification product
was separated on a 1.5% agarose gel and electrophoresis was carried out at 80V for 1 hour 30 minutes.
After electrophoresis, DNA bands were visualized by ethidium bromide staining. 100bp DNA ladder (Solis
Biodyne) was used as DNA molecular weight marker.

Results/Discussion
Baseline characterization of soil sample
Total petroleum hydrocarbons (TPH) were 22,146.65ppm for (surface polluted soil [SPS]), 14,087.80ppm
(sub surface [SPSS]) and unpolluted soil (UPS) 479.67ppm. Polycyclic aromatic hydrocarbons (PAHs)
were 12,209.3ppm (surface soil [SPS]), 3,248.75ppm for sub surface [SPSS] and for [UPS]) 22.72ppm. The
pollution level at the surface and subsurface far exceeded Department of Petroleum Resources’ (DPR's)
intervention limit of 5000mg/kg and 40mg/kg for TPH and PAHs respectively. This shows the site is
heavily polluted and corroborates the massive impact the pollutant had on vegetation. The results of
the physicochemical parameters (potassium, conductivity, temperature, pH, nitrate, phosphate, and total
organic carbon contents), baseline bacterial counts (total culturable heterotrophic and hydrocarbon utilizing
bacteria) and gas chromatographic analysis of TPH andPAHs are presented in Table 1.

Table: 1—Baseline characteristics of polluted soil samples

Parameters Surface Polluted Soil (SPS) Sub Surface Polluted Soil Pristine Control (US)

Total petroleum hydrocarbon (TPH) 22,146.65ppm 14,087.80ppm 479.67 ppm

Polycyclic aromatic hydrocarbon
12,209.30ppm 3,248.75 ppm 279.72 ppm
(PAH)
Hydrocarbon utilizing bacterial
8.4 × 105cfu/g 8.0 × 105cfu/g 3.96 × 104cfu/g
count (cfu/g)
pH 5.03 4.82 6.12
Nitrate (NO3 )-
8.33mg/kg 6.67 mg/kg 3.84 mg/kg
Phosphate (PO4 ) -
8.11mg/kg 10.834 mg/kg 20.52mg/kg
Temperature 27.6 °C 29.3 °C 30 °C
Electrical Conductivity 141.1 μs/cm 140.73 μs/cm 185.46 μs/cm
Total organic carbon (TOC) 5.64 % 5.068 % 1.97 %
Moisture content 7.93 % 6.76 % 4.88%
Nickel (Ni) 0.38 mg/kg 0.36 mg/kg 0.68 mg/kg
Lead (Pb) 0.54mg/kg 0.41mg/kg 0.0065 mg/kg
Cadmium (Cd) 1.2mg/kg 1.26 mg/kg 0.013 mg/kg
SPE-198799-MS 7

Residual TPH constituents found in the polluted site were hydrocarbon fractions of chain
length C8-C35 (Fig.3.) while PAHs consisted of benzo(b)fluoranthene, acenaphthylene, acenaphthene,
benzo(k) fluoranthene, fluorene, fluoranthene, benzo (a) anthracene, indeno (1,2,3-cd) pyrene,
dibenz (a, h) anthracene, pyrene, phenanthrene, chrysene, naphthalene, benzo (a) pyrene, benzo
(g, h, i) perylene and anthracene as shown in Fig.4. The carcinogenicity of certain PAHs
like benzo[a]anthracene, benzo[a]pyrene, benzo[b]fluoranthene, indeno[1,2,3-c,d]pyrene, anthracene,
benzo[g,h,i]perylene, benzo[e]pyrene, chrysene, fluoranthene, fluorene, phenanthrene and pyrene are a
major concern in oil polluted sites as they are known for their mutagenic, teratogenic properties and are
toxic and/or carcinogenic to humans (Wloka et al., 2017).

Figure 3—TPH abundance in the oil-polluted soil.

Figure 4—Abundance and distribution of PAHs in the oil polluted soil

8 SPE-198799-MS

Analysis of nitrate and phosphate content which gives information about the soil nutrient status showed
the polluted soil was deficient in essential nutrients. This makes the sampling site a potential source for
the recovery of hydrocarbonoclastic long chain alkane degrading bacteria that can be used for the removal
of problematic wax under oligotrophic conditions. Previous analysis of the nutrient content in crude oil
polluted soils has shown that, the presence of the pollutant leads to a significant reduction in macronutrients
like nitrogen and phosphorus (Agbogidi, Eruotor, Akparobi Nnaji, 2007; Wibbe and Blanke, 1999).
The pH of the crude oil polluted soil was acidic and is consistent with previous findings on the influence
of hydrocarbon contamination on soil pH (Ezenne, Nwoke, Ezikpe, Obalum, & Ugwuishiwu, 2014; UNEP,
2011). The ability of hydrocarbon degrading bacterial species to proliferate under this condition is an
important feature that qualifies them for application as potential paraffin wax removal agents. Realistically,
pH conditions during crude oil production and transportation where wax formation becomes a problem are
usually not optimal for bacterial multiplication.

Total Culturable Heterotrophic and Hydrocarbon Utilizing Bacteria

Total culturable hydrocarbon utilizing bacterial count (TCHUB) for SPS, SPSS and UPS were 8.4 × 105cfu/
g, 8.0 × 105cfu/g and 3.96 × 104cfu/g respectively. Total culturable hydrocarbon utilizing bacterial count
(TCHUB) isolated from surface polluted soil (SPS) were Bacillus spp., Pseudomonas spp., Micrococcus
spp., Streptococcus spp., Staphylococcus spp. Rhodococcus spp., Provideencia spp. (Fig 5a) while sub
surface polluted soil (SPSS) had Pseudomonas spp., Bacillus spp., Acinetobacter spp., Serratia spp.,
Aromonas spp., Pseudomonas spp., Proteus spp., Achromobacter spp., Bacillus spp., Acinetobacter spp.,
Serratia spp. and Corynebacterium spp. (Fig 5b).

Figure 5a—Percentage occurrence of total culturable hydrocarbon

utilizing bacterial isolates at the sub s urface polluted soil (SPS)
SPE-198799-MS 9

Figure 5b—Percentage occurrence of total culturable hydrocarbon

utilizing bacterial isolates at the sub s urface polluted soil (SPSS)

Some of the bacterial genera isolated such as Bacillus, Pseudomonas, Klebsiella, Corynebacterium,
Proteus and Micrococcus have been reported by other researchers as hydrocarbon utilizing bacteria isolated
during bioremediation of crude oil contaminated soils (Van Hamme, et al., 2003; Bento et al., 2005; Agarry
et al., 2010; Onuoha, 2013; Chikere et al., 2015). Eziuzor and Okpokwasilli (2009) also isolated Bacillus,
Pseudomonas and Corynebacterium in a study of bioremediation of crude oil polluted mangrove soil
in Port-Harcourt. Previous studies by Balogun et al. (2015), identified Bacillus, Proteus, Psuedomonas,
Micrococcus and Enterobacter spp. as petroleum degrading isolates. The most predominant bacteria species
that utilized paraffin wax in the present study belonged to the genera Pseudomonas and Bacillus. These
bacterial genus have severally been reported to be efficient degraders of long-chain hydrocarbons thereby
making them potentially ideal for wax removal to improve production in the oil industry (Sood and Lal,
2008; Matsumiya, Chung, and Kubo, 2008; Deng et al., 2014).

Long-chain hydrocarbon degrading bacteria screening

The recovered bacterial isolates capable of utilizing hydrocarbons were further screened for their ability
to degrade heavy crude oil and long-chain hydrocarbons using DCPIP (Fig 6a – 6b). The use of DCPIP
to screen for hydrocarbon degradating bacterial species is considered useful and has been demonstrated
to be efficient (Chikere & Obieze, 2018; Varjani & Upasani, 2016). The method is based on the principle
that during microbial oxidation of hydrocarbons, electrons are usually transferred to acceptors such as
oxygen, sulphate and nitrates. When DCPIP is incorporated into a culture media, it serves as an electron
acceptor, hence it can be used to determine hydrocarbons biodegradation by monitoring change in the colour
of DCPIP from blue (oxidized) to colourless (reduced) since decolourization is directly proportional to
hydrocarbons utilization. Pseudomonas spp., Bacillus spp., Acinetobacter sp. and Serratia sp. demonstrated
higher biodegradability as exhibited by complete decolourization of DCPIP after 288 hours (Figs. 6a – 6b
and 7a – 7b).
10 SPE-198799-MS

Figure 6a—Degradation Screening (DCPIP decolorization process through time day 0)

Figure 6b—Degradation Screening (DCPIP decolorization process through time day 12 [288h])
SPE-198799-MS 11

Figure 7a—Long-chain hydrocarbon degradation screening using DCPIP colourimetric method.

Figure 7b—Long-chain hydrocarbon degradation screening using DCPIP colourimetric method

These isolates demonstrated efficient potential for the degradation of paraffin oil and heavy crude during
the period of screening. Their degradation ability and degree of long-chain hydrocarbon utilization differed
from one bacterium to another due to observed differences in their growth curve. The optical density
(O.D) values showed a decrease following the gradual decolourization of DCPIP. The recovered long-chain
hydrocarbon degrading bacterial species have been previously reported as degraders of different fractions
of hydrocarbons.
Acinetobacter sp. has been reported by Kubota et al., (2008) as specializing in only the degradation
of long-chain n-alkanes. (Patel and Bhaskaran, 2018) demonstrated the potential of Pseudomonas in
the removal of paraffin wax from oil wells to boost production. Their study revealed Pseudomonas
nitroreducens depleted 70% of paraffinic crude oil in 10 days. In another study (Abdel-megeed, Al-
harbi, and Al-deyab, 2010) recovered bacterial species including Pseudomonas, Bacillus and Rhodococcus
capable of degrading hexadecane.
12 SPE-198799-MS

Detection of long-chain hydrocarbon degrading gene (almA)

To further screen the recovered long-chain hydrocarbon utilizing bacteria, molecular mechanisms were
employed to detect the presence of long-chain hydrocarbon degrading almA gene. Out of the 22 recovered
putative hydrocarbon degrading bacterial species screened, only one isolate tentatively identified as
Acinetobacter sp. showed the presence of this known gene (Fig. 8).

Figure 8—Long-chain hydrocarbon degrading almA gene screening and detection

The almA gene was first detected in Acinetobacter strains in a study by (Throne-holst, Wentzel, Ellingsen,
Kotlar, and Zotchev, 2007) and represented the first cloned gene shown to be capable of degrading
hydrocarbon chains of length C32 and above. After this first study, the gene have been detected in other
bacterial genus including Parvibaculum, Alcanivorax, Marinobacter, Salinisphaera and Bacillus (Wang and
Shao, 2012). The detection of this gene in our study confirms the recovered isolate as a good resource for
removal of long-chain hydrocarbons including paraffin wax for improved oil production. The inability to
detect the almA gene in all the bacterial isolates that initially degraded paraffin wax indicates the likely
presence of other long-chain alkane degrading genes like LadA that was not screened for in this study

Conclusion
This study shows that the crude oil-polluted site harboured appreciable number and spread of bacterial
species capable of degrading long-chain hydrocarbon fractions. The ability of the recovered bacterial
isolates to degrade heavy crude oil and paraffinic oil as sole source of carbon and energy coupled with
their ability to proliferate under oligotrophic and low pH conditions make them a potential resource for the
removal of paraffin wax in oil wells, thereby, aiding improved oil production.

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