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Bioresource Technology
Volume 254, April 2018, Pages 174-179

Improved polycyclic aromatic hydrocarbon degradation in a

crude oil by individual and a consortium of bacteria
Smita Kumari a, b, Raj Kumar Regar a, c , Natesan Manickam a, b

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https://doi.org/10.1016/j.biortech.2018.01.075 Get rights and content

Highlights
• The bacteria isolated and characterized have ability to biodegrade multiple
PAHs.

• Development of five-member bacterial consortium for the HMW-PAH

degradation.

• A Rhamnolipid JBR−425 biosurfactant enhanced the PAH biodegradation

up to 10–15%.

Abstract
In this study, we report the ability of Stenotrophomonas maltophilia, Ochrob actrum
anthropi, Pseudomonas mendocina, Microb acterium esteraromaticum and Pseudomonas

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aeruginosa to degrade multiple polycyclic aromatic hydrocarbons (PAHs) present in crude

oil. The PAHs in the crude oil sample obtained from Digboi oil refinery, India were
estimated to be naphthalene (10.0 mg L −1), fluorene (1.9 mg L −1), phenanthrene
(3.5 mg L−1) and benzo(b)fluoranthene (6.5 mg L −1). Exposure of individual bacteria to
crude oil showed high rate of biodegradation of specific PAHs by M. esteraromaticum,
81.4%-naphthalene; P. aeruginosa, 67.1%-phenanthrene and 61.0%-
benzo(b)fluoranthene; S. maltophilia, 47.9%-fluorene in 45 days. However, consortium of
these bacteria showed enhanced biodegradation of 89.1%-naphthalene, 63.8%-fluorene,
81% of phenanthrene and 72.8% benzo(b)fluoranthene in the crude oil. The degradation
was further improved up to 10% by consortium on addition of 40 μg mL −1 rhamnolipid
JBR-425 biosurfactant. These results suggest that the developed bacterial consortium
has significant potential in PAH remediation.

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Keywords
Bacterial consortium; HMW- polycyclic aromatic hydrocarbons; Crude oil; Biosurfactant

1. Introduction
Polycyclic aromatic hydrocarbons (PAHs) contamination is a major concern for
environment and health (Downward et al., 2014). PAHs are ubiquitous pollutants which
are introduced into the environment both through natural and anthropogenic activities

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(Abdel-Shafy and Mansour, 2016). Due to their hydrophobic nature, they persist in the
environment for a long period and serve as a secondary sink for further contaminations
(Duran and Cravo-Laureau, 2016). Many compounds such as pyrene, benzo(a)pyrene and
chrysene are carcinogenic, mutagenic and teratogenic and thus (Balachandran et al.,
2012) United State Environmental Protection Agency (US-EPA) has considered 16 PAHs
as a priority pollutant. The major constituents of crude oils are naphthenes, asphaltenes,
waxes, asphalts, aromatic hydrocarbons, resins and other volatile compounds such as
benzene, toluene, ethyl benzene and xylene (Sammarco et al., 2013). Besides these,
natural and accidental spillages of crude sludge from oil drill-sites of coastal areas cause
serious hazards to terrestrial habitat and marine ecosystems (Tornero and Hanke, 2016).
To restore the contaminated sites, bioremediation may provide a viable, economical and
complete solution. Previously, the implementation of phytoremediation (Jeelani et al.,
2017), physico-chemical and thermal desorption methods have been extensively reported
for reduction in PAH load in the environment. Bioremediation using microorganism is
found to be an ecofriendly and economically suitable technology to efficiently degrade PAH
from sites of contamination. However, for the complete degradation of PAH, their
availability to the microorganism was found limited due to its hydrophobic nature and high
adsorbing potential to soil (Kalmykova et al., 2014). Several studies have reported using
chemical surfactant (Adrion et al., 2016) and biosurfactants (Bezza and Chirwa, 2016,
Hazra et al., 2012) for enhancing the solubility and thereby the accelerating rate of PAHs
degradation. Many researchers have reported pure culture bacteria, such as
Mycob acterium sp., (Hennessee and Li, 2016), Stenotrophomonas sp., (Kumari et al.,
2017) and Sphingomonas sp., (van Herwijnen et al., 2003) for the degradation of HMW-
PAHs. Genes and enzymes involved in biodegradation of pyrene and phenanthrene
suggests the presence of multicomponent oxygenases, dehydrogenases and followed by
lower pathway degradative enzymes (Kim et al., 2007, Kumari et al., 2017) in M.
vanb aalenii PYR-1 and Stenotrophomonas sp. Although studies have been reported for
PAH degradation by individual strains (Lyu et al., 2014), the rate of degradation efficiency
was found enhanced by a consortium of bacteria due to their efficient synergism and
coordinated metabolic activities (Xu et al., 2013). Bioremediation of PAHs was studied as
a microcosm in a limited field conditions employing the mixture of bacteria in consortium
manner (Tauler et al., 2016). Application of consortium of bacteria having good potential in
degradation of alkanes, but having poor degradation efficiency for other high molecular
weight PAHs present in crude oil was also reported (Chen et al., 2017, Pugazhendi et al.,
2017).

Worldwide, efforts are under way for optimizing factors such as salinity, temperature and
pH which may influence the biodegradation rate of PAHs, particularly in field conditions. To
meet such harsh conditions in the field, extremophilic bacteria, such as Acidiphilum
cryptum, Acidophilic thiobacilii were studied for the degradation at acidic pH 2.0 and

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Mycob acterium sp. strain MHP-1 and PYR-1 were found to degrade at alkaline pH of 9.0
(Habe et al., 2004). In view of the health hazards and ecological threat posed by the
contamination of PAHs, there is an urgent need for developing successful and cost-
effective bioremediation processes.

Here, we report the studies to establish a specialized HMW-PAH degrading consortium

consisting of five bacterial species belonging to four different genera, isolated by
functional substrate clearance. The bacteria were studied for their degradation potential
on individual PAHs utilizing them as sole source of carbon and energy. More significantly,
biodegradation of a crude oil collected from an oil refinery was studied for the degradation
of at least 4 PAHs present in it to develop feasible microbiological processes for
restoration of oil polluted environments.

2. Materials and methods

2.1. Crude oil, chemicals and media

Crude oil used in the study for PAH degradation was procured from Digboi refinery,
Tinsukia, Assam, India. Naphthalene (98%), phenanthrene (98%), benzo(b)fluoranthene,
and fluorene (98%) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Solvents,
acetonitrile, n-hexane, dichloromethane and diethyl ether used are of high performance
liquid chromatography (HPLC) grade and procured from Thermo Fisher Scientific,
Waltham, MA, USA. Luria Bertani (LB) used for culture purpose was purchased from BD
Difco, NJ, USA. Minimal salt medium (MSM) used for culture growth and maintenance had
(g L−1) KH2PO4 3.4 g, Na2HPO4 19.6 g, (NH4)2SO4 1 g, MgSO4 (97.4 mg) and trace element,
such as CuSO4 1 g, ZnSO4 1 g, FeSO4 1 g, CaCl 2 1 g, H 3BO4 1 g purchased from Fisher
Scientific, Waltham, MA, USA.

2.2. Isolation and characterization of bacteria

Soil samples for isolation of potential PAH degrading bacteria were collected from
contaminated soil of Mathura oil refinery, Mathura, India and a tyre wastes dump site
(Qaiser Bagh, Lucknow, India). The soil slurry was used for screening of bacteria by a
previously reported method (Kumari et al., 2017) with a minor modification. One gram of
soil from the sample was mixed with liquid minimal medium and kept on rocker with
gentle mixing for 24 h. After a brief wait for the settlement of soil, the supernatant was
spread on to minimal agar plates prior to spray with pyrene and/or phenanthrene to
examine the substrate clearance zone around the colonies. The positive colonies
exhibiting clearance were taken for detailed characterization and stored at −80 °C in 15%
glycerol for further studies. For the identification of bacteria, genomic DNA was isolated
from pure culture by Nucleo-pore gDNA mini kit (Genetix Biotech Asia Pvt. Ltd. New Delhi,
India), polymerase chain reaction (PCR) was performed to amplify 16S rRNA gene.

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Universal primer 27F (5′AGAGTTTGATCTGGCTCAG-3′) and 1522R (5′

AAGGAGGTGATCCANCCRCA-3′) was used to amplify the DNA fragment coding for 16S
rRNA gene in PCR (Mastercycler epigradient S, Germany) following the conditions
reported previously (Kumari et al., 2017). Further, the PCR product was purified (Sigma
GenElute TM Gel Extraction Kit), cloned in pGEM-T Easy vector vector (Promega, Madison,
USA) and clone was confirmed through blue white screening. From each plate, 10 clones
were randomly selected for plasmid isolation, (Nucleopore, Genetix Biotech Asia Pvt. Ltd.),
restriction digestion by EcoRI confirmed the presence of 16S rRNA gene and further
plasmid was sequenced using Sanger’s dideoxy nucleotide sequencing procedure. The
16S rRNA gene sequences were aligned and BLAST against the 16S rRNA reference
database EzBioCloud (Yoon et al., 2017) and the phylogenetic tree was constructed using
MEGA version 7.0 (Kumar et al. 2016). For biochemical tests, such as indole test,
catalase, and citrate utilization, protocols followed according to manual of common
bacterial identification (Dong and Cai, 2001).

2.3. PAH degradation by individual bacteria and consortium in liquid medium

The ability of five isolated bacteria to degrade major PAHs present in crude oil was
evaluated in liquid medium in liquid culture conditions. Briefly, inoculums were prepared
by growing individual bacteria in LB broth to early log phase, and the culture diluted with
sterile minimal medium broth to adjust to a bacterial count 2 × 10 4 CFU mL −1. First five
sets of flasks in duplicates were inoculated with individual bacteria with 2% v/v crude oil as
a sole carbon source in 250 mL Erlenmeyer flasks containing 50 mL minimal medium.
Two sets of experiments were performed for consortium studies that consist of a mixture
of five strains, i.e. IITR07, IITR46, IITR47, IITR48 and IITR87 with or without 40 µg mL −1
rhamnolipid JBR-425. The critical micelles concentration (CMC) was taken based on the
previous studies (Manickam et al., 2012). Flasks were incubated at 200 rpm in a shaker
(New Brunswick, Edison, NJ, USA) at 37 °C for 45 d. The flasks without addition of
bacteria, but with crude oil in same condition were set as a control. After every 15 d of
interval, one set of flasks was removed and the contents were extracted using n-hexane:
dichloromethane (1:1) to analyse the residual PAHs. The solvent fraction was evaporated
at room temperature, reconstituted in acetonitrile: methanol (1:1) and a suitable aliquot
was analysed on Ultra High Performance Liquid Chromatography (UHPLC), and
degradation percentage was calculated by the formula given below.

2.4. Evaluation pH for optimum degradation

Effect of pH on bacterial growth was examined in an effort to optimize the conditions for
maximizing the degradation. The pH selected were 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, and 7.5 and
50 mL of minimal medium culture was adjusted to the desired/required pH using 1 M HCl

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solution (Pawar, 2015). An initial inoculum of 0.1 OD 600 having 1.7 × 10 8, 1.8 × 10 8, 1.2 ×
108, 1.6 × 10 8 and 1.5 × 10 8 colony forming units (CFU mL −1) with respective to
Ochrob actrum anthropi, Pseudomonas mendocina, Microb acterium esteraromaticum,
Pseudomonas aeruginosa and Stenotrophomonas maltophilia was added to the flasks
containing the 100 mM pyrene which is the most preferred substrate by all the above
bacteria. Growth of the bacterium was measured at 0, 7, 14, 21 and 28 d of intervals by
enumerating the bacterial colonies on LB agar plate for CFU count.

2.5. Analytical methods

Qualitative and quantitative analysis of PAHs was performed using Ultra High
Performance Liquid Chromatography (UHPLC) system (Thermo Scientific, Waltham,
USA). The separation of ions was achieved using Extent C18 column (C18, 15 cm ×
2.1 mm × 1.8 μ particle size). An aliquot of 10 μL of filtered sample was injected into the
system. The column flow rate was maintained at 0.4 mL min –1 in isocratic mode, mobile
phase consisted acetonitrile:water (80:20 ratio v/v) and the total run time of the analysis
was 15 min per sample using a UV detector with the wavelength at 254 nm.

2.6. Statistical analysis

The results were expressed as mean ± standard deviation (SD) of at least three
independent experiments. Statistical analysis was performed using the Graph Pad
Prism5 software. The data were compared by one way analysis of variance (ANOVA) and
Tukey’s post hoc test. Data were summarized as mean SD or ns P > 0.05 or ∗ P < 0.05 or
∗∗ P < 0.01 as compared to controls.

2.7. Nucleotide sequence submission

The 16S rRNA gene sequence of PAH-degrading strains IITR07, IITR46, IITR47, IITR48
and IITR87 were submitted in the NCBI-GenBank under the accession number
KP772231, MF321766, MF321767, MF321768 and KJ933407respectively. All these strains
were also deposited in the Microbial Type Culture Collection (MTCC) and Gene Bank, at
Institute of Microbial Technology, Chandigarh, under the accession number MTCC12722
(IITR07), MTCC12718 (IITR46), MTCC12719 (IITR47), MTCC12720 (IITR48) and
MTCC12721 (IITR87).

3. Results and discussion

3.1. Isolation and characterization of PAH degrading bacteria

PAH degrading bacterial isolates designated as IITR07, IITR46, IITR47, IITR48 and IITR87
were enriched using PAHs as a sole source of carbon and energy. All the five bacteria
showed degradation potential by metabolizing the phenanthrene and pyrene by exhibiting
clearance zone around the substrate on minimal medium agar plates after incubation for

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3 d at 37 °C. Biochemical characterization of bacteria revealed that four of them were

Gram-negative and IITR47 was Gram positive. All bacteria were catalase positive and
indole negative. Strains IITR07, IITR46 and IITR48 were found to utilize citrate. The 16S
rRNA gene sequences analysis led to their identification and characterisation as
Ochrob actrum anthropi IITR07, Pseudomonas mendocina IITR46, Microb acterium
esteraromaticum IITR47, and Pseudomonas aeruginosa IITR48, which is also supported
by the phylogenetic tree classification. A combined phylogenetic tree was constructed
using 16S rRNA sequence of isolated strains along with the reported PAH degraders
showing similarity (Fig. 1). In the combined phylogenetic tree, M. esteraromaticum IITR47
forms a separate branch along with P. mendocina IITR47 and P. aeruginosa IITR47.

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Fig. 1. Phylogenetic neighbour-joining tree based on 16S rRNA gene sequence of strain IIT R07,
IIT R46, IIT R47, IIT R48 and their relationship with reported PAH degrading member of
Ochrobactrum, Pseudomonas, Microbacterium, and Stenotrophomonas respectively using MEGA
version 7. T he bootstrap test was based on 1000 replicates and values are shown next to the
branches.

3.2. Effect of pH on growth

All the five bacteria were examined for growth under a range of 4.0–7.5 pH in minimal

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medium in the presence of 100 mM pyrene as sole carbon source. Maximum growth was
obtained at 7.5 pH for S. maltophilia, O. anthropi, P. mendocina, and P. aeruginosa while M.
esteraromaticum was found to grow better in a pH range of 6.5 (Fig. 2). According to Kim et
al. (2005), the increased degradation rate of pyrene and phenanthrene by M. vanb aalenii
PYR-1 was also found in the pH range of 6.5 to 7.5. All the bacteria showed poor growth
below the 5.5 pH, as observed in case of pyrene degradation (Fig. 2).

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Fig. 2. Growth evaluation of PAH degrading strains IIT R07, IIT R46, IIT R47, IIT R48 and IIT R87
on a wide range of pH (4.0–7.5). T he strain was culture in presence of 100 mM of pyrene as a
sole source of carbon and energy in minimal medium for 28 d. Values are mean of three
observations ± standard deviation (SD).

3.3. Degradation of PAH by individual bacteria and consortium

PAH degradation of crude oil was examined in liquid medium at every 15 d of interval up to

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45 d and the residual PAHs quantified. The concentration of major PAHs present in the
Digboi crude oil was 10, 1.9, 3.5 and 6.5 mg L −1 of naphthalene, fluorene, phenanthrene
and benzo(b)fluoranthene respectively. The degradation efficiency of five bacteria
individually and in a consortium, was studied in presence of biosurfactant. Among the five
isolates, highest degradation was achieved for naphthalene, 80.40% by M.
esteraromaticum and fluorene, 47.9% by S. maltophilia as shown in Fig. 3. Similarly,
phenanthrene, 67.1% and benzo(b)fluoranthene, 61.2% degradation was achieved by P.
aeruginosa in 45 d (Fig. 3). In case of naphthalene (64.9%) and fluorene (35.5%)
degradation by S. maltophilia and M. esteraromaticum was obtained which was
significantly less when compared to the other degraders. Similarly, it was also observed
that P. mendocina poorly degraded phenanthrene and benzo(b)fluoranthene 40.9% and
34.8%, respectively, as shown in Fig. 3. However, for naphthalene degradation a
biosurfactant producing Streptomyces sp. AM2 was found to show as high as 81.03%
(Ferradji et al., 2014) which is similar to the strain M. esteraromaticum studied.

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Fig. 3. Evaluation of PAH biodegradation percentage of individual strain and a consortium with
or without biosurfactant (rhamnolipid) in liquid medium containing 2% crude oil incubated for 45
d. Degradation of PAH in a consortium with rhamnolipid JBR-425 degrade naphthalene 97.3%,
fluorene 76.2%, phenanthrene 96.5% and benzo(b)fluoranthene 84.2%. Among the individual
strains, highest degradation of naphthalene 80.4% (IIT R47), phenanthrene 67.1%
benzo(b)fluoranthene 61.2% (IIT R48) and fluorene 47.9% (IIT R87), was achieved in 45 d. Values

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are mean of three observations ± standard deviation (SD). Shown data were significantly different
from control (* P < 0.05, ** P < 0.01).

A significant increase in the degradation of PAH was achieved when the consortium
comprising of five bacteria was employed in 45 d when compared to the earlier studies
(Patowary et al., 2016). Also shown in previous reports, the application of biosurfactant
could significantly enhance the rate of dissolution and hence increase in the rate of
degradation of high and low molecular weight PAH from crude oil (Zhao et al., 2011,
Sartoros et al., 2015). Degradation of naphthalene, fluorene, phenanthrene and
benzo(b)fluoranthene by the consortium (CNSM + BS) in the presence of rhamnolipid
JBR-425 was 97.3%, 76.2%, 96.5% and 84.2%, respectively, with significantly higher
degradation as compared to consortium of bacteria without biosurfactant (CNSM) was
achieved (Fig. 3) with their initial concentration of 10.3, 1.9, 3.5 and 6.5 mg L −1 (Table 1)
from the crude sludge used in the study. Under the studied conditions in the report, the
consortium along with biosurfactant (CNSM + BS) showed 1.5 to 2.0-fold when compared
to the consortium in the absence of the surfactant. Many biosurfactant producing bacteria
such as Pseudomonas sp., Streptomyces and Rhodococcus sp., have the extraordinary
characteristic to enhance the rate of dissolution and thus increasing the availability of
PAHs to the microorganisms (Ferradji et al., 2014).

Table 1. Estimation of polyaromatic hydrocarbons (PAHs) concentration of crude oil sample

collected from Digboi, Assam, before and after 45 d of degradation within in liquid medium.

Compound Initial Final residual concentration (mg L−1)

concentration
IITR07 IITR46 IITR47 IITR48 IITR87 CNSM CNSM + BS
(mg L−1)

Naphthalene 10.0 1.8 3.5 1.5 1.8 2.6 1.9 0.2

Fluorene 1.9 1.2 1.1 1.2 0.7 1.0 0.7 0.4

Phenanthrene 3.5 1.3 2.1 1.4 1.0 1.8 0.6 0.1

Benzo(b)fluoranthene 6.5 3.0 4.2 3.5 1.2 3.0 1.9 1.0

CNSM = consortium without surfactant, CNSM + BS = consortium with surfactant

In summary, under the studied conditions, it was observed that the consortium of bacteria
has proven a better model, since 97% of naphthalene degradation in 45 d was achieved
and also enhanced degradation of other PAHs. Similarly, M. esteraromaticum, when grown
individually resulted in 80% of naphthalene degradation within 45 days. Many researchers

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have reported the enhanced degradation of mixed PAHs, by a bacterial consortium

because of their enzymatic and metabolic function was stimulated by the communities
involved as consortium rather than single strain (Wu et al., 2013, Wanapaisan et al.,
2018). Therefore, the study supports the use of potential microbial consortium for
biodegradation of PAHs present in crude oil exploiting them for the restoration of
contaminated sites with polycyclic aromatic hydrocarbons.

4. Conclusion
Our results proved that the use of bacterial consortium in the presence of low
concentrations of rhamnolipid JBR-425 enhanced degradation of all four PAHs tested. Our
effort also has led to the isolation of a Gram positive Microb acterium esteraromaticum not
studied in detail for high molecular weight PAHs degradation. In view of the large-scale
contamination of PAHS in various ecosystems worldwide, we have optimized degradation
efficiency of bacteria under wide pH range. Therefore, the study using the bacterial
consortium along with surfactant augmentation may prove to be significant in facilitating
the large-scale remediation of oil sludge contaminated sites.

Acknowledgement
Authors are thankful to Council of Scientific & Industrial Research (CSIR), New Delhi, India
for financial support under the Network Project INDEPTH, No. BSC0111. We are also
thankful to Dr. Deka Boruah for providing crude oil sample from Digboi Oil Refinery,
Assam, India. SK acknowledges UGC, University Grants Commission (UGC), India, for
providing the fellowship and Academy of Science and Innovative Research (AcSIR), India
for PhD enrolment. This manuscript carries CSIR-IITR communication number 3516.

Appendix A. Supplementary data

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Supplementary data 1.

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