Accepted Article

DR. MAMADOU MALICK DIALLO (Orcid ID : 0000-0001-8264-2960)

DR. CANER VURAL (Orcid ID : 0000-0003-1400-6377)

PROF. GUVEN OZDEMIR (Orcid ID : 0000-0002-7577-4233)

Article type : JAM - Original Article

Enhanced biodegradation of crude oil in soil by a developed bacterial consortium and

indigenous plant growth promoting bacteria (PGPB)

Authors

Mamadou Malick DIALLO, ORCID ID: 0000-0001-8264-2960

Caner VURAL, ORCID ID: 0000-0003-1400-6377

Hursel CAY, ORCID ID: 0000-0002-2842-1611

Guven OZDEMIR*, ORCID ID: 0000-0002-7577-4233

Department of Biology, Basic and Industrial Microbiology Section, Ege University, 35040,
Izmir, Turkey

*Corresponding author. Phone: +90-232-3111519; E-mail: ozdemirguven@gmail.com

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/JAM.14848
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 Title
Accepted Article
Enhanced biodegradation of crude oil in soil by a developed bacterial consortium and
indigenous plant growth promoting bacteria (PGPB)

Aims
This study aimed to develop an efficient, cost-effective, and eco-friendly bacterial consortium
to degrade petroleum sludge.
Methods and Results
Four bacterial strains belonging to genera Acinetobacter and Pseudomonas were selected to
constitute three different consortia based on their initial concentration. The highest
degradation rate (78%) of 1% (v/v) crude oil after 4 weeks of incubation was recorded when
the concentration of biosurfactant producing isolate was high. alkB, almA, cyp153, pah-
rhdGN, nah, phnAC, and cat23 genes were detected using the PCR method and their
induction levels were optimal at pH 7.0. A crude oil sludge was artificially constituted, and its
bacterial composition was investigated using 16S rRNA gene amplicon sequencing. The
results showed that the soil bacterial community was dominated by plant-growth-promoting
bacteria (PGPB) after crude oil treatment.
Conclusions
Our findings indicate the decontamination of the crude oil contaminated soil was more
effective in the presence of both the constituted consortium and PGPB compared to the
presence of PGPB alone.
Significance and Impact of the Study
This study showed that the PGPB (Taibaiella) present in petroleum uncontaminated soil can
promote the soil decontamination. The addition of both efficient hydrocarbon-degrading and
biosurfactant producing bacteria is also necessary to improve the decontamination.
Keywords: Crude oil sludge, Plant-Growth-Promoting Bacteria, Hydrocarbon-degrading
microorganisms, Biosurfactant production, Biodegradation.

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 Introduction
Accepted Article
Ship accidents, offshore oil rig disasters, pipeline leakages are among the main causes of the
oil spill and spread which result in environmental pollutions. Besides, the petroleum industry
produces huge amounts of crude oil sludge, which is composed of hazardous wastes such as
alkanes, aromatic hydrocarbons, asphaltenes, and nitrogen-sulfur-oxygen (NSO) containing
compounds. Aliphatic and aromatic hydrocarbons can constitute up to 75% of the oil sludge
compounds (Hu et al. 2013). Petroleum sludge is listed as hazardous waste because it is toxic,
cancerogenic, and highly persistent in the environment (Hentati et al. 2013). It was reported
that every 500 tons of refined crude oil generate one ton of petroleum sludge. Therefore,
more than 60 million tons of oil sludge may be generated worldwide every year (da Silva et
al. 2012).
Taking into consideration the produced amount of petroleum sludge, it is necessary to
establish efficient, cost-effective, and eco-friendly techniques to counteract its harmful
effects. In this context, bioremediation is recognized as a recommended process to remove
petroleum pollutants, when compared to physical and chemical methods (Braddock et al.
1997; Margesin et al. 2013; McGenity 2014; Bacosa et al. 2015). Studies have reported the
ability of various bacterial strains belonging to genus Pseudomonas (Zhang et al. 2011),
Acinetobacter (Lin et al. 2014), Rhodococcus (Van Hamme and Ward 2001), Dietzia (Chen
et al. 2017), Bacillus (Ijah and Ukpe 1992), and Alcanivorax (Kasai et al. 2002) to degrade
crude oil. Bacterial aerobic degradation of alkanes, an important component of crude oil and
harmful wastes from the petroleum industry, is initiated by a class of enzymes called Alkane
hydroxylases (AHs) (Van Beilen et al. 2003). Two common AHs enzymes, the integral
membrane alkane monooxygenase (AlkB) and the cytochrome P450, are involved in the
degradation of short and medium chain-length alkanes (Nie et al. 2014). On the other hand, it
is known that the flavin-binding LC alkane monooxygenase (AlmA) and the thermophilic
soluble LC alkane monooxygenase enzymes (ladA) are involved in the oxidation of long-
chain-length alkanes (Throne-Holst et al. 2007; Wentzel et al. 2007). Although, some
bacterial strains contain only one alkane hydroxylase system, it is common to find multiple
enzyme systems in the genomic and plasmid DNA of a microorganism (Rojo 2009).
PAHs are the second most important hydrocarbon family among the constituents of crude oil.
Additionally, they are highly toxic and carcinogenic compounds. Therefore, the
biodegradation of PAHs by microorganisms has been widely investigated (Abdel-Shafy and
Mansour 2016; Liu et al. 2017). Bacteria degrade PAHs aerobically using ring hydroxylating
dioxygenase enzymes, which are encoded by the polyaromatic hydrocarbon ring

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 hydroxylating dioxygenases genes (pah-rhd) (Song et al. 2015). Some pah-rhd genes that
Accepted Article
have been previously identified using polymerase chain reaction (PCR) include nahAc,
phnAc, nagAc, ndoB, bphAc, narA, and nidA (Chikere and Fenibo 2018).
One of the major drawbacks of bioremediation is attributed to the hydrophobic character of
hydrocarbons. The degradation of petroleum hydrocarbons by microorganisms is
considerably influenced by the availability of hydrocarbons (Calvo et al. 2009). Thus, several
studies showed that biosurfactants (BSs) could be used to overcome this hurdle
(Hassanshahian 2014; Mnif et al. 2018) . They are widely used in biodegradation studies due
to their natural character and ability to increase the degradation of hydrocarbons.
The present study focused on constituting a bacterial consortium that can degrade crude oil
sludge at a high rate. Four bacterial strains, which are efficient hydrocarbon-degraders as well
as biosurfactants producers, were isolated from oil-contaminated soils. A consortium was
constituted with the four strains and their ability to degrade crude oil sludge was evaluated.
The result showed that the isolated strains with some PGPB were able to degrade efficiently
crude oil sludge.

Materials and Methods

Isolation of bacterial strains

Bacterial strains were isolated from two different oil-contaminated soils. The first sample was
taken from activated sludge of wastewater treatment plant of a petrochemical industry
(38.47'N, 26.55'E) located Aliağa, Izmir, Turkey. The second sample was collected from oil-
contaminated soils in a car repair workplace area (38.27'N, 27.15'E), located in Bornova,
Izmir, Turkey (Fig. S1).
For bacterial isolation, 5.0 g of each sample was inoculated into 100 ml Bushnell-Hass
medium (BH) (g l-1: KH2PO4, 1; K2HPO4, 1; MgSO4, 0.2; CaCl2, 0.02; NH4NO3, 1; FeCl3,
0.05; yeast extract, 0.05). The cultures were incubated at 30oC by shaking at 160 rpm for 7
days. Then, 5 ml from samples were centrifuged at 3000 g for 5 minutes and the pellets were
suspended in 1 ml of sterile 1x phosphate buffered saline (PBS, pH 7.4). The suspended
pellets were separately used to inoculate flasks containing 100 ml BH medium supplemented
with 2 ml crude oil. The inoculated flasks were incubated at the same conditions mentioned
above. After that, 5 ml aliquots were taken from each sample and centrifuged at 3000 g for 5
min. The obtained pellets were suspended in 1 ml of PBS and transferred to BH agar
supplemented with 1% crude oil. The agar plates were incubated at 30°C for 5 days.

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 After incubation, colonies were further cultured on Nutrient Agar (NA) plates (Merck,
Accepted Article
Germany) and incubated at 30°C for 2 days. Then, single colonies were transferred to new
NA plates. The plates were incubated in the same condition as mentioned above. Single
colonies were then screened for their crude oil degradation abilities.

Selection of hydrocarbon degrading isolates

The isolates that demonstrated ability to grow in presence of crude oil, degrade hydrocarbons
and resist the toxicity of crude oil compounds were selected. Glass tubes (10 ml) containing 5
ml Nutrient Broth (NB) media were inoculated with each isolate and incubated in a shaking
incubator (150 rpm) at 30ºC for 18h. At the end of incubation, 1 ml from each sample was
transferred to 1.5 ml microcentrifuge. The tubes were then centrifuged at 3000 g for
5 minutes and the supernatants were discarded. The pellets were washed twice with sterile
PBS buffer, after which, the appropriate concentration of each isolate (1.5 x 107 CFU ml-1 as
final concentration) was prepared. The inoculum was transferred to 50 ml BH medium
containing 1% (v/v) crude oil and then incubated in a rotary shaker (160 rpm) at 30°C for 4
weeks. Microbial growth was assessed on the 1st, 7th, 14th, 21st, and 28th days by the pour
plate method.

The resistance of the isolates to the toxicity of different concentrations of crude oil was
evaluated. The isolates were individually incubated in 100 ml of BH medium supplemented
with 2%, 5%, 10%, 15% and 20% (v/v) crude oil as sole carbon source. The inoculum was
incubated in the same conditions as mentioned above. At the end of incubation, the bacterial
cells were enumerated by the pour plate method.

Determining the residual hydrocarbons

The degradation rates were determined using the gravimetric analysis method. Briefly,
residual hydrocarbons were extracted by adding an equal volume of hexane/cyclohexane
(2:1) to the medium. The mixture was then shaken for 5 minutes, after which the organic
phase was collected. The extraction process was repeated twice. The organic phase was
evaporated by using a rotary evaporator. The percentage of degraded hydrocarbon was
calculated using the following formula:

(WRO in control culture ― WRO in the sample) x 100

ODR (%) =
WRO in control culture

*ODR= Oil degradation rate, WRO= weight of remaining oil

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 Screening for biosurfactant producing strains
Accepted Article
The production of BSs was screened by growing the isolates in 50 ml of BH medium
supplemented with 1.5 g l-1 NaNO3 as a nitrogen source and 2% glycerol as a carbon source.
Cultivation was carried out at 30°C in a rotary shaker at 200 rpm for 7 days. After incubation,
the presence of BS was evaluated using the emulsification index (EI24%) method. Briefly,
the medium was centrifuged at 5000 g for 10 min, after which 1.5 ml of supernatant was
mixed with an equal amount of kerosene in a 10 ml glass test tubes for 2 min. The emulsion
formed was kept at room temperature for 24 h. The percentage of the emulsion (EI24%) was
calculated according to the following formula:

𝐻𝑖𝑔ℎ𝑡 𝑜𝑓 𝑒𝑚𝑢𝑙𝑠𝑖𝑓𝑖𝑐𝑎𝑡𝑖𝑜𝑛 𝑙𝑎𝑦𝑒𝑟

EI24% = 𝑥100
𝐻𝑖𝑔ℎ𝑡 𝑜𝑓 𝑡𝑜𝑡𝑎𝑙 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

Biosurfactant extraction was performed as previously reported by Silva et al. (2010).

Microbial cells were removed from the culture medium by centrifugation at 5000 g for 30
min. Hydrogen chloride (HCl, 6.0 mol l-1) was used to adjust the pH of the supernatant to 2,
after which an equal volume of chloroform/methanol (2:1) was added. The mixture was
vigorously shaken for 5 minutes after which the organic phase was removed. The extraction
process was repeated twice. The product was concentrated from the pooled organic phase
using a rotary evaporator. The obtained viscous yellowish product was dissolved in methanol
and concentrated again by evaporating the solvent at 45°C. After extraction, the oil
displacement test was carried out to determine BS surface activity. Briefly, 40 μl of the
concentrated extract was pipetted into the center of petri dishes containing a mixture of 50 ml
of distilled water and 50 μl of crude oil. BS activity was defined by the diameter of the
clearing zone formed after the addition of the surfactant.

The family of produced BS was identified by CTAB/Methylene blue agar test. The
CTAB/Methylene blue agar medium contains (g): CTAB, 0.2; methylene blue, 0.005;
glucose, 20; yeast extract, 1; peptone, 2; CaCl2 0.1; and agar 15, which were dissolved in one
liter of BH medium. The plates were inoculated and incubated at 30°C for 3 days. The
formation of dark blue halos around the colonies indicates the production of glycolipids
(Siegmund and Wagner 1991).

Biodegradation Studies
Alkane degradation assay

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 The isolates (1.5 x 107 CFU ml-1 as final concentration) were cultivated separately in 50 ml
Accepted Article
BH medium supplemented with either a mixture of tridecane (C13), tetradecane (C14),
hexadecane (C16) and pristane or hexatriacontane (C36). The starting concentration of each
alkane was equivalent to 500 mg l-1, which made the final concentration of alkane mixture
equivalent to 2000 mg l-1 in the flask. The samples and control flasks were incubated in a
rotary shaker (160 rpm) at 30°C for 7 days. After incubation, the culture medium was
extracted with an equal volume of dichloromethane and 1 ml of the organic phase was used
for Gas Chromatography (GC) analysis.

The concentration of each residual hydrocarbon (C13, C14, C16, and pristane) was calculated
using standard curves. On the other hand, the degradation rate of C36 was estimated by the
gravimetric method. The growth of the isolates in the presence of alkanes was evaluated in
the same conditions as mentioned above. Bacterial cell growth was evaluated on the 1st, 3rd,
5th, and 7th days of incubation by the pour plate method. The counts were converted to log10
CFU ml-1. The results demonstrate the mean values and standard deviations of three trials.

PAH degradation assay

Acenaphthene (ACE), anthracene (ANT), fluorene (FLR) and pyrene (PYR) were used for
PAHs degradation study. The capability of the isolates to degrade 50 mg l-1 of PAHs was
evaluated in flasks containing 50 ml BH medium. Both inoculated samples and control flasks
were incubated in a rotary shaker (160 rpm) at 30°C for 14 days. At the end of incubation, the
residual PAHs were extracted with an equal volume of n-hexane by vortexing for 5 min.
After that, 1 ml of the organic phase was used to quantify the concentration of residual
hydrocarbon by high-performance liquid chromatography (HPLC). Standard curves were
constructed to measure accurately the concentrations of residual PAHs.

Development of consortium for crude oil degradation

The concentration of each strain in the consortium was chosen according to its capability to
degrade hydrocarbons (alkanes, PAHs) or to produce BS. Thus, three different consortia
named Cons1, Cons2 and Cons3 were established according to the initial concentration of the
isolates as shown in Table 1. The biodegradation of 1% (v/v) crude oil in 50 ml BH medium
by each consortium was monitored after 4 weeks. The degradation rates of the crude oil were
determined by the gravimetric method as mentioned above. The consortium which recorded
the highest degradation rate was selected for all subsequent experiments.

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 The effect of pH on the biodegradation activity of the selected consortium was also
Accepted Article
evaluated. The consortium was inoculated into flasks containing BH media with pH 4.5, 5.5,
7.0, 8.5 and 9.5. The flasks were incubated under the same conditions as mentioned above.

Crude oil sludge degradation assay

An artificial crude oil sludge was prepared by adding 10% (v/w) crude oil to unpolluted soil
sample collected from the Botanical Garden and Herbarium Research and Application Center
(38.45'N, 27.23'E), Ege University, Izmir, Turkey. The clean soil sample was collected at a
depth of 0–3 cm under the ground. The degradation of the artificial crude oil sludge by the
consortium was evaluated in flasks under shaken conditions. Five percent of the consortium
was added to flasks containing 100 ml BH medium and supplemented with 5% of artificial
crude oil sludge. Inoculated flasks were incubated in shaken conditions (160 rpm) at 30°C for
4 weeks.
Two sets of flasks, containing the same composition of culture media, were employed as
control. The first set of control was inoculated with crude oil sludge without the consortium,
which allowed assessment of the activity of the indigenous microbial population during the
degradation of TPH. The second set of control flasks contained sterilized soil combined with
crude oil.

Chromatographic Analysis
Gas chromatography (GC) analysis

The residual hydrocarbons analysis was performed in a Gas Chromatography (GC) system
(Agilent 7820A, Santa Clara, California, USA) equipped with a fame ionization detector
(GC-FID). HP-5 column (specification: 30m x 0.32 mm, film of 0.25µm, Agilent, Santa
Clara, California, USA) was used. The operating conditions were as follows: injector
temperature 230°C; detector temperature, 300°C; carrier gas, helium (1 ml min-1); Airflow,
300 ml min-1; Hydrogen flow, 30 ml min-1. The oven temperature program was as follows: 1
min at 70°C, followed by a temperature increase of 10°C min-1 up to 300°C (35 min). The
splitless mode was carried out, and 1µl of residual oil was injected (Diallo et al. 2019).

High-performance liquid chromatography (HPLC) analysis

The quantifications of residual PAH (ACE, PYR, FLR, and ANT) were performed by a
HPLC device (Agilent 1100, Santa Clara, California, USA) equipped with a diode-array
detector (DAD). The separation was carried out with an Eclipse PAH Agilent analytical

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 column (150 × 4.6 mm, 3.5 μm). The separation conditions were conducted using a gradient
Accepted Article
of acetonitrile and water from 50:50 to 100:0 v/v for 16 minutes (Vural et al. 2019). The
injection of 1µl of the sample solution was carried out at flow rate of 1 ml min-1 at 25°C.

Molecular Biological Methods

Characterization of the microbial isolates

The genomic DNA (gDNA) was extracted from the isolates using a ZR Fungal/Bacterial
DNA MiniPrep kit (Zymo Research, USA). Briefly, approximately 3 x 108 CFU ml-1 bacterial
cells were centrifuged at 3000 g for 5 min, the obtained pellets were resuspended in 200 µl of
PCR grade water. Further steps were proceeded according to the manufacturer’s instructions.
DNA was eluted with 50 µl elution buffer. The concentration and purity of DNA were
determined using NanoDrop™ 2000c Spectrophotometer (Thermo Scientifc, USA).11F and
1492R primers were used to amplify the 16S rRNA gene (Table S1).The PCR amplification
was performed using TC-PLUS thermal cycler (TECHNE, USA) at the following conditions:
initial denaturation for 3 min at 94°C; 35 cycles for 30 sec. at 94°C, 30 sec. at 48°C, and 90
sec. at 72°C; the final extension was carried out at 72°C for 5 min. The PCR products were
visualized by electrophoresis on 1% agarose gel using a multispectral imaging system
(BioSpectrum, UK). The PCR fragments (1480 bp) were purified using NucleoSpin Gel and
PCR Clean-up kit (Macherey-Nagel GmBH, Germany). The purified fragments were
sequenced using an automated sequencer ABI Prism Genetic Analyzer (Applied Biosystems,
USA). The sequences were edited with DNA Baser Sequence Assembler software and
analyzed using the BLAST platform in the National Center for Biotechnology Information
(NCBI). Multiple sequence alignment was performed with MEGA 7 software. The
evolutionary history was traced using the Maximum Likelihood methods.

Identification of alkane hydroxylase and aromatic dioxygenase genes

The presence of three alkane hydroxylase (AH) genes (alkB, cyp153 and almA) and four
aromatic dioxygenase (ADO) genes (PAH-ring hydroxylating dioxygenase (pah-rhdGN),
naphthalene dioxygenase (nah), ring-hydroxylating dioxygenase for phenanthrene (phnAC)
and catechol 2-3 dioxygenase (cat23)) were investigated in the gDNA of the isolates using
specific primers (Table S1). The PCR amplification of AH genes was carried out as follows:
initial denaturation for 3 min at 95°C; 35 cycles of 30 sec. at 95°C, 30 sec. at 55°C
for alkB (52°C for cyp153 and 48°C for almA), and 1 min at 72°C; and a final extension at
72°C for 5 min. On the other hand, the amplification of ADO genes was performed as

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 reported previously (Mesarch et al. 2000; Baldwin et al. 2003; Sho et al. 2004; Cébron et al.
Accepted Article
2008). The PCR products were visualized on 1% agarose gel. Sequence analysis was
performed as mentioned above.

Analysis of Soil Bacterial Community by 16S rRNA Gene Amplicon Sequencing

Total gDNA from 0.25 g soil sample was extracted using ZR Soil Microbe DNA Kit™
(Zymo Research, USA) according to the user’s manual. The presence of gDNA was assessed
on 1% agarose gel. The quantity and purity of eluted DNA were evaluated at 260A/280A nm
wavelength using a NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, USA).
To generate the library, the V3-V4 regions of the 16S rRNA gene were amplified with
specific primers followed by purification. The sequencing library for amplicon analysis was
prepared using the Nextera XT DNA Library Prep Kit (Illumina®) and sequenced on an
Illumina Miseq (300 bp paired-end). The qualities of raw 16S rRNA gene sequence reads
were checked using FastQC, while potential chimeric sequences were excluded with
DADA2. The representative sequences were then analyzed with the QIIME2 software
package (Hall and Beiko 2018). The metagenetic sequences have been deposited in the
Sequence Read Archive (SRA) under the accession number SAMN14814826 and
SAMN14814827.

Gene Expression Assays

To analyze the impact of pH on crude oil degradation, the expression level of AH and ADO
gene was evaluated at pH 4.5, 5.5, 7.0, 8.5 and 9.5. In addition, the expression level of AH
genes was also analyzed in the presence of alkanes (C13, C16, C36 and pristane). Total
RNAs were extracted on the 5th day of incubation for alkanes and the 14th day for crude oil
using the JET RNA Purification Kit (Thermo Fisher Scientific, USA). The quantity and
quality of the eluted RNA were confirmed by a NanoDrop™ 2000c Spectrophotometer
(Thermo Scientific, USA). RNAs were converted to cDNA using High Capacity cDNA
Reverse Transcription Kit (Applied Biosystem, USA). The reverse transcription was
performed in a thermal cycler PCR (TechnePlus, UK) as follows: step 1, 10 min at 25°C; step
2, 120 min at 37°C and a final step at 85°C for 5 min. After reverse transcription, the cDNAs
were stored at -20°C. Real-time PCR (rt-PCR) was performed in a LightCycler® 96 System
qPCR (Roche Diagnostics GmBH, Mannheim, Germany) using the LightCycler FastStart
Master SYBR Green I Kit (Roche Diagnostics GmBH, Mannheim, Germany). The assays
were carried out according to the reaction conditions as recommended by the manufacturer.

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 The amplification of ADO genes was performed as previously reported (Mesarch et al. 2000;
Accepted Article
Baldwin et al. 2003; Sho et al. 2004; Cébron et al. 2008), while AH genes were amplified in
the same rt-PCR conditions: 95oC for 15 min; 45 cycles of 10s at 95oC, 10s at 53oC and 9s at
72oC. Melting temperature was performed from 65°C to 95°C at 0.2°C/s melt rates; Cooling
step: 10s at 40°C. The specificity of primers was confirmed by melting curve analysis. The
normalized relative transcript levels were determined using the 2-ΔΔCT method (Livak and
Schmittgen 2001). The ribosomal gene 16S rRNA was employed as a reference gene for
normalizing gene expression data.

Statistical Analysis
One-way ANOVA was performed to compare the growth means of isolates in the presence of
crude oil using GraphPad Prism software. P value < 0.05 was considered significant. The
experimental data are expressed in terms of arithmetic averages with standard deviation (±).

Results
Characterization and growth rates of bacterial strains
In this study, four crude oil-degrading bacteria named 2SA, C1-8, C1-9 and NPK were
isolated from two different oil-contaminated fields. To identify the isolates, phylogenetic
analysis was performed. 2SA, C1-8, C1-9 and NPK were identified as Acinetobacter
haemolyticus (100%) (MH011442), Acinetobacter sp. (100%) (MK332502), Acinetobacter
guillouiae (91%) (MK332503) and Pseudomonas sp. (100%) (MK447603) respectively (Fig.
1). The growth of isolates was monitored in the presence of 1% (v/v) crude oil in 50 ml BH
medium as the sole carbon source. It was observed that the microbial growth increased from
the first day of incubation to reach maximum on the 14th day. No significant difference in the
growth of isolates was noted in the presence of crude oil (p>0.05) (Fig. 2). As shown in the
table 2, the isolates exhibited good degradation activities in the presence of crude oil and pure
hydrocarbons. In this study, all isolates showed high tolerance to crude oil, nevertheless, the
growth of all strains decreased by 3 logs in the presence of 15% and 20% crude oil (Fig. 3).
This result is consistent with those reported by (Taketani et al. 2010) who argues that the
bacterial species isolated from soils chronically contaminated with crude oil are more
resistant to high hydrocarbons concentrations.
Biosurfactant Production
BS production was screened by using the emulsification index (EI24%) test. The results
showed that the strain NPK was the sole isolate that produced BS with an EI24% equivalent

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 to 66.67%. Additionally, the extracted crude biosurfactant displayed good activity with a
Accepted Article
clearance zone of 23.75 ± 1.3 cm2 using the oil spreading method. The activity of the BS
produced by NPK was similar to those reported by (Lan et al. 2015; Das and Kumar 2018;
Lee et al. 2018). CTAB/Methylene blue agar results revealed that the biosurfactant produced
by the isolate NPK might be a glycolipid molecule. Glycolipids consist of a carbohydrate
moiety connected to fatty acids (Hubert et al. 2012).

Hydrocarbon biodegradation assays

In this study, strains 2SA, C1-8 and C1-9 demonstrated the ability to efficiently degrade
alkanes after incubation for 7 days (Table 2). GC analysis showed that the isolate 2SA
completely degraded linear alkanes (C13, C14, and C16) and pristane. Although the isolates
C1-8 and C1-9 slightly degraded pristane, linear alkanes (C13, C14 and C16) were
completely degraded (Fig. S2). However, it was found that the isolate C1-8 was not able to
use C36 alkane as a carbon source. C13 alkane was weakly degraded by the strain NPK.
Isolates 2SA and NPK were able to use PAHs as sole carbon source. The HPLC analysis
showed that the low molecular weight PAHs (LMW-PAHs) (ACE and FLR) used in this
study were significantly degraded by both NPK and 2SA, while their high molecular weight
(HMW-PAHs) counterparts (ANT and PYR) were only partially degraded by the isolate NPK
(Table 2).

Molecular biological assays

PCR amplification allowed the detection of three AH genes in the gDNA of 2SA (alkB, almA
and cyp153) and two AH genes in the gDNA of C1-8 and C1-9 (alkB and almA) (Fig. S3-S5).
The existence of different AHs in the gDNA of isolates 2SA, C1-8 and C1-9 allow these
isolates to have a high ability to degrade alkanes and access to versatile nutrition. The gene
expression results showed that alkB gene in all isolates was highly induced in the presence of
C13 and C16 alkanes. However, alkB gene was weakly expressed in the presence of pristane.
On the other hand, the cyp153 gene of isolate 2SA was the most induced gene in the presence
of pristane (Fig. 5). The almA gene was induced during the degradation of C36 alkane by
strains 2SA and C1-9. On the other hand, the almA genes of 2SA and C1-9 were not induced
in the presence of medium-chain length alkanes. The presence of ADO genes (pah-rhdGN,
nah, phnAC and cat23 genes) in the gDNA of the isolates was evaluated by PCR. The
amplification results showed that the gDNA of NPK and 2SA harbors all the evaluated ADO
genes, whereas only the cat23 gene, which encodes the catechol 2, 3-dioxygenase enzyme,
was amplified in the gDNA of strains C1-8 and C1-9.

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 Development of Consortium for Crude Oil Degradation
Accepted Article
Three different consortia named Cons1, Cons2, and Cons3 were established based on the
initial concentration of the isolates. According to the results, the highest degradation yield of
total hydrocarbons was recorded by Cons2 (78 ± 3.4%) followed by Cons1 (64 ± 2.1%) and
Cons3 (56 ± 2.8%) after 4 weeks of incubation. Therefore, Cons2 was chosen to proceed with
subsequent experiments. The highest degradation of crude oil by Cons2 could be explained
by the initial concentration of 2SA and NPK. While isolate 2SA efficiently degraded
hydrocarbons, it was found that isolate NPK was able to produce BSs. The production of BSs by
NPK allowed access to the hydrophobic hydrocarbons. Similar to our results, many studies in the
literature have reported the advantages of bio-surfactants during the biodegradation of crude oil
(Bezza et al. 2015, Suganthi et al. 2018). The effect of pH on the degradation activity of the
consortium was evaluated. The highest biodegradation yield of crude oil was recorded at pH
7.0. However, the degradation yield of crude oil by the consortium was higher at pH 5.5 (46
± 4.24%) when compared to pH 8.5 (29 ± 4.29%). The hydroxylase enzymes produced by
bacterial cells are activated only over a narrow pH range.

Effect of pH on the expression of hydrocarbon degrading genes

The gene expression study showed that AH genes were highly influenced by pH variations.
The best results were recorded at pH 7.0 for all AH genes with 86, 35, and 63-fold increase in
the expression of alkB, almA and cyp153, respectively (Fig. 5). According to the retention
times of the alkanes (C13, C14, C16, and pristane), as presented in the GC analysis, we can
conclude that the crude oil employed in this study consists mainly of medium-chain length
alkanes. The genes were better induced at acidic media when compared to basic media. The
expression levels of pah-rhdGN, nah, phnAC and cat23 genes were lower when compared to
those of AH genes with 11, 8, 6 and 7-fold respectively at pH 7.0 (Fig. 5 (e)). The induction
of genes could be related to the type and quantity of hydrocarbons. Similarly to AH genes, all
ADO genes were slightly more induced in acidic medium. No gene induction was recorded at
pH 4.5 and 9.5.

Crude oil sludge degradation assay

The efficiency of the constituted consortium on the biodegradation of crude oil sludge was
investigated in lab-scale. During the biodegradation experiments, biotic and abiotic soils were
used as control. The degradation rate of crude oil sludge in the biotic control was equivalent
to 61 ± 3.7% after 4 weeks. This decrease might be caused by indigenous microorganisms.

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 Moreover, GC analysis showed that a wide range of hydrocarbons was used throughout the
Accepted Article
incubation period (Fig. 6). However, the concentration of TPH was reduced up to 93% upon
the addition of the Cons2 consortium and nutrients. This result showed that the bacterial
consortium can efficiently improve the degradation of TPH present in the soil.

Microbial dynamics in the degradation of hydrocarbons

The results of 16S rRNA gene amplicon sequencing indicated that the soil sample was mainly
inhabited by Proteobacteria (28.96%), Chloroflexi (20.70%), and Firmicutes (20.70%) phyla
before treatment. However, after treatment, the bacterial community was dominated by
Proteobacteria (53.83%) and Bacteroidetes (37.61%) phyla, while Firmicutes, Chloroflexi,
Actinobacteria, Tenericutes, Acidobacteria, and Euryarchaeota drastically decreased. γ-
Proteobacteria (28.58%) was the most detected subgroup in the Proteobacteria phylum
followed by α-Proteobacteria (20.78%). On the other hand, the subgroup β-Proteobacteria
decreased from 9.80% to 4.47%) (Fig. 7). Ɛ/δ-Proteobacteria did not perform any role, they
were disappeared from 2.2% to 0% during the degradation process. At the genus level, the
principal dominant bacterial groups were Taibaiella (33.62%), Pseudomonas (20.92%),
Brevundimonas (14.86%) and Acinetobacter (6.30%) (Fig. 8). These microorganisms are
considered as plant growth-promoting bacteria (PGPB) (Almario et al. 2013; Santoyo et al.
2016).

Discussion

Crude oil is composed of thousands of different molecules, therefore, bacterial species can
only degrade a small number of hydrocarbons (Dhote et al. 2018). The complete
biodegradation of crude oil may be possible by establishing a consortium. Microorganisms
constituting the consortium must be able to produce complementary enzymatic patterns
allowing efficient biodegradation of different types of hydrocarbons (Dhote et al. 2017).
However, the biodegradation efficiency of crude oil is not only influenced by the microbial
factor, but also by environmental factors and the amount of crude oil (Bayat et al. 2015). In
this study, a crude oil-degrading consortium constituted with four bacterial strains (2SA, C1-
8, C1-9, and NPK) was established. The isolates were selected based on their ability to
degrade crude oil and hydrocarbons, to resist the toxicity of crude oil compounds, to produce
biosurfactants, and to resist pH variation. The increase in biomass and the decrease in
hydrocarbon concentration indicates that the strains 2SA, C1-8, C1-9, and NPK effectively
degraded crude oil. The efficiency of hydrocarbon-degrading strains may be negatively

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 affected in the presence of a high concentration of crude oil. However, all the selected strains
Accepted Article
showed high tolerance to the toxic effect of crude oil. Some studies reported that the pre-
exposure of the bacterial community to hydrocarbon leads to the selection of populations that
can respond rapidly to subsequent addition of oil (de Quadros et al. 2016; Koolivand et al.
2018).

Alkanes and PAHs are the most important hydrocarbon families among the constituents of
crude oil. Strains 2SA, C1-8 and C1-9 demonstrated ability to efficiently degrade alkanes.
The high degradation ability of alkanes by the strains 2SA, C1-8, and C1-9 might be due by
the presence of different alkane hydroxylase genes (alkB, almA and cyp153) in their gDNA.
The presence of different AH genes in the gDNA of some hydrocarbon-degrading
microorganisms has been described in previous studies (Wang and Shao 2012; Moreno and
Rojo 2017). alkB and cyp153 genes are the most studied genes, they are expressed during the
degradation of short and medium-chain-length alkanes (Van Beilen and Funhoff 2007; Rojo
2009). The Flavin-binding monooxygenase almA enzymes are involved in the oxidation of
long-chain-length alkanes (Wentzel et al. 2007). Although branched alkanes are more
difficult to degrade by microorganisms when compared to their linear counterparts (Moreno
and Rojo 2017), the strain 2SA degraded pristane efficiently. The cyp153 gene was only
detected in gDNA of the isolate 2SA and it was the most induced in the presence of pristane.
On the other hand, the absence of the cyp153 gene in the gDNA of C1-8 and C1-9 could
explain the low degradation ability of pristane by these strains. The same result was reported
by Sevilla et al. (2017) where three cytochrome P450 genes of Alcanivorax borkumensis SK2
were highly induced in the presence of pristane. Even though the gDNA of C1-8 harbors
almA gene, the isolate was not able to use C36 alkane as a sole carbon source. The almA gene
can be involved in the degradation of other kinds of alkanes that are different from long-chain
length alkanes (Liu et al. 2014). The degradation ability of PAHs by the isolates was
evaluated in the presence of LMW-PAHs and HMW-PAHs. Although the strain NPK was the
sole isolate able to degrade partially HMW-PAHs, LMW-PAHs were efficiently degraded by
the isolates 2SA and NPK. Most PAH degrading bacteria use LMW-PAHs as a carbon and
energy source in preference to HMW-PAHs (Molina et al. 1999, Hassanshahian and
Boroujeni, 2016). Co-biodegradation of anthracene, naphthalene, phenanthrene and pyrene
by Acinetobacter johnsonii were evaluated by Jiang et al. (2018). Results showed that
naphthalene was preferentially used prior to other PAHs. The isolates C1-8 and C1-9 were
not able to grow in the presence of PAHs, however, the detection of the catechol 2, 3-

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 dioxygenase gene (cat23) in their gDNA suggests that these isolates might be able to degrade
Accepted Article
other aromatic hydrocarbon families. Catechol 2, 3-dioxygenase enzymes are involved in the
degradation of both monocyclic and polycyclic aromatic hydrocarbons (He et al. 2016). The
genes encoding the aromatic ring-hydroxylating-dioxygenase (RHDα) enzymes, that are
involved in the initial step of the PAH catabolism, have been extensively reviewed and
described in the past several decades (Liang et al. 2019; Verma et al. 2019).

The pH changes affect enzymatic activities by changing the three-dimensional shape of the
active sites (Srinivasarao et al. 2011). In this study, the expression of the degradative genes
was influenced by pH variations. The optimum pH for the degradation of alkanes and PAHS
by the isolates was 7.0. However, better induction of the degradative genes was noted at
acidic media when compared to basic media. Kuppusamy et al. (2016) reported that
Stenotrophomonas (MTS-2) was able to efficiently degrade HMW PAH at pH 5, 6 and 7.
Kim et al. (2005) showed that the acidification of a mycobacterial culture increased the
degradation rates of pyrene and phenanthrene four-fold. However, Murakami et al. (1998)
isolated a catechol 2,3-dioxygenase from Pseudomonas sp. AW-2 that showed maximum
activity at pH 8.0, although the enzyme was stable at pH 6.0–7.5.

After determining the profile of each strain, a consortium was constituted and its efficiency in
degrading crude oil sludge was evaluated in the presence of the indigenous microbial
population. The result showed an improvement of about 34% after the addition of the
consortium in the crude oil sludge. The treatment of the oil contaminated sludge with nutrient
and the exogenous microbial consortium (Cons2) in combination resulted in a higher
degradation rate of TPH when compared to the oil contaminated sludge supplemented with
nutrients alone. Aburto-Medina et al. (2012) reported that the addition of activated sludge
and nutrients to hydrocarbon-contaminated soil enhanced the degradation of hydrocarbon up
to 51.6 ± 8.5% when compared to treatments supplemented only with nutrients (41.3 ±
6.4%). Suja et al. (2014) compared the efficiency of bioaugmentation and biostimulation in
the biodegradation of TPH in crude oil-contaminated soil in laboratory settings. The highest
TPH removal equivalent to (79%) was recorded when microorganisms and nutrients were
added in combination.

The results of 16S rRNA gene amplicon sequencing showed drastic changes in the soil
population after treatment with crude oil. The γ-Proteobacteria class was the most abundant

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 subgroup and at genus level, the principal dominant bacterial groups were Taibaiella,
Accepted Article
Pseudomonas, Brevundimonas, and Acinetobacter. Bao et al. (2017) found that the members
of γ-Proteobacteria and α-Proteobacteria as key players are predominant for the degradation
of petroleum hydrocarbon in soil. Chikere et al. (2019) demonstrated that the predominant
phylum in crude oil-contaminated soil was Proteobacteria followed by Acidobacteria. The
genera Taibaiella and Brevundimonas are considered as PGPB (Zhang et al. 2013; Singh et
al. 2016). The significant increase of PGPB and the decrease of TPH in the biotic control
indicated their ability to degrade hydrocarbons present in the crude oil. However, the
significant increase in abundance of Pseudomonas and Acinetobacter genera with the PGPB
in treated soil suggests that there might be a metabolic relationship between Taibaiella,
Brevundimonas, and Cons2 isolates. The implication of PGPB in the bioremediation of toxic
compounds was previously documented which emphasizes their ability to protect plants from
harmful pollutants (Zhuang et al. 2007; Wu et al. 2019). However, the difference between
our results and the previous studies is the occurrence of Taibaiella and Brevundimonas as
new dominant genera in crude oil degradation. The results showed that Taibaiella and
Brevundimonas genera probably played an important role in the degradation of hydrocarbons.
However, our future work will focus on further experiments to isolate these PGPB, evaluate
their involvement in the degradation of TPH and contribution to plant growth. This study also
showed that the high biodegradation rates of crude oil are dependent on the initial
concentration of hydrocarbon-degrading bacteria as well as the biosurfactant production
ability of the microorganisms in a consortium.

Acknowledgments

The authors acknowledge members of EGEMIKAL Microbiological Analysis Laboratory for

their encouraging supports in organizing experimental works in Gas Chromatography. We
also thank Fatima Shatila for her help.

Conflict of Interest

The authors declare that they have no conflict of interest.

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List of tables

Table 1 Initial concentrations of the isolates on each consortium

Name of isolates Cons1 Cons2 Cons3

(%) (%) (%)
2SA 25 40 35
C1-8 25 15 25
C1-9 25 15 25
NPK 25 30 15

Table 2 Degradation rate of hydrocarbons by each isolate

Isolates’ C13 C14 C16 C36 Pristane ACE ANT FLR PYR Crude oil
name (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)
2SA 100 100 100 70 ± 3 100 76 ± 2.1 ND 81 ± 1.8 ND 42±2.8
C1-8 100 100 100 ND 49 ± 1.6 ND ND ND ND 27 ± 4.2
C1-9 100 100 100 53 ± 2 35 ± 2.1 ND ND ND ND 31 ± 5.7
NPK 40 ± 2.5 ND ND ND ND 100 44 ± 1.4 92 ± 4.01 45 ± 2 27 ± 2.1
ND= Non Degraded hydrocarbons

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List of figures
Accepted Article

Fig. 1 Phylogenetic tree based on the alignment of nucleic acid sequences of the 16s rRNA
gene from strains 2SA, C1-8, C1-9, and NPK.The reference nucleic acid sequences were
retrieved from the NCBI database. Support values are calculated from 1000 bootstrap
replicates and only support above 70% is shown.

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Fig. 2 Growth of the isolates in the presence of crude oil.

Fig. 3 Growth of isolates in the presence of different concnetrations of crude oil. The initial
concentration of each isolate was 6.88 log10 CFU ml-1 ( 1.5 x 107 CFU ml-1).

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Fig. 4 Growth of the isolates in the presence of tridecane (a), hexadecane (b), pristane (c),
and hexatriacontane (d).

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Fig. 5 Expression of alkane hydroxylase genes of 2SA (a), C1-8 (b), and C1-9 (c) in the
presence of alkanes. The effect of pH on the induction of alkane hydroxylase and aromatic
dioxygenase genes during crude oil degradation by the consortium is shown in (d) and (e)
respectively.

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Fig. 6 GC chromatograms of biodegradation of crude oil sludge. (a) = GC chromatograms of

the abiotic control, (b) = GC chromatograms of the biotic control (presence of endogenous
microorganisms only), (c) = GC chromatograms of the sample (presence of Cons2
consortium and endogenous microorganisms).

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Fig. 7 Bacterial taxonomic compositions at phylum level (a) and detailed taxonomic class
compositions (b) of the untreated and treated soil samples (Soil= untreated soil sample,
before incubation; Soil+ Cons2= treated soil samples with Cons2, after incubation) (b). (*The
‘Others’ parameter is the sum of the values below 2%).

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Fig. 8 Detailed genus distributions in untreated soil and treated soil samples before (a and c) and after degradation (b and d). (*The ‘Others’
parameter is the sum of the values below 2%). After incubation in the presence of crude oil, a high variation in the soil bacterial community was
observed with the occurrence of the Plant Growth Promoting Bacteria (PGPB) such as Taibaiella and Brevundimonas genus.

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