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Klebsiella oxytoca: an efficient pyrene-degrading bacterial strain isolated

from petroleum-contaminated soil

Article in Archives of Microbiology · May 2022

DOI: 10.1007/s00203-022-02850-9

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Archives of Microbiology (2022) 204:248
https://doi.org/10.1007/s00203-022-02850-9

ORIGINAL PAPER

Klebsiella oxytoca: an efficient pyrene‑degrading bacterial strain

isolated from petroleum‑contaminated soil
Abdulkhaleg M. Alfaify1 · Mushtaq Ahmad Mir2 · Sulaiman A. Alrumman1

Received: 20 November 2021 / Revised: 21 February 2022 / Accepted: 15 March 2022

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022

Abstract
Polycyclic aromatic hydrocarbons (PAHs) are the hazardous xenobiotic agents of oil production. One of the methods to
eliminate hazardous compounds is bioremediation, which is the most efficient and cost-effective method to eliminate the
harmful byproducts of crude petroleum processing. In this study, five pure bacterial isolates were isolated from petroleum-
contaminated soil, four of which showed a robust growth on the PAH pyrene, as a sole carbon source. Various methods viz
mass spectroscopy, biochemical assays, and 16S RNA sequencing employed to identify the isolates ascertained the con-
sistent identification of Klebsiella oxytoca by all three methods. Scanning electron microscopy and Gram staining further
demonstrated the characterization of the K. oxytoca. High-performance liquid chromatography of the culture supernatant of
K. oxytoca grown in pyrene containing media showed that the cells started utilizing pyrene from the 6th day onwards and by
the 12th day of growth, 70% of the pyrene was completely degraded. A genome search for the genes predicted to be involved
in pyrene degradation using Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed their presence in the genome
of K. oxytoca. These results suggest that K. oxytoca would be a suitable candidate for removing soil aromatic hydrocarbons.

Keywords Biodegradation · Pyrene · Polycyclic aromatic hydrocarbons · Contaminated soil · Petroleum

Introduction vegetation, aquatic animals and eventually to humans.

Environmental hazards are associated with many pollutants
including those produced during oil production. The most
significant environmental pollution is the contamination of
the soil by hazardous hydrocarbons including low molecular
weight (LMW) and high-molecular weight (HMW) hydro-
carbons (Molina-Barahona et al. 2005; Robles-González
et al. 2008). Polycyclic Aromatic Hydrocarbons (PAHs)
composed of two or more benzene rings accumulate inces-
santly in soil, sediments, and water due to their long half-life
and human activities (Ghosal et al. 2016; Singare 2016).
From the soil and water, the hazardous hydrocarbons enter
Communicated by Erko Stackebrandt.
* Mushtaq Ahmad Mir
mmir@kku.edu.sa
1
Department of Biology, College of Science, King Khalid
University, Abha, Saudi Arabia
2
Department of Clinical Laboratory Sciences, College
of Applied Medical Sciences, King Khalid University,
P. O. Box 3665, Abha 61421, Saudi Arabia
Because of their toxic, carcinogenic, mutagenic, and tera-
togenic nature, PAHs are of public health and environmen-
tal concern (Haritash and Kaushik 2009). Furthermore, the
fused benzene ring structure of PAHs renders them hydro-
phobic, stable and, hence, recalcitrant pollutants (Kanaly
and Harayama 2000). Many strategies including physical
(adsorption, volatilization, photolysis) and chemical treat-
ments are in use to remove PAHs (Robles-González et al.
2008; Gan et al. 2009; Perelo 2010). Although these tech-
niques are well developed, they are expensive, complex, and
have a regulatory burden.
The most cost-effective and reliable mechanism for bio-
degradation involves microorganisms that remove xenobi-
otic contaminants including crude oil (Dua et al. 2002; Cap-
pello et al. 2007; Singh et al. 2008). There are several recent
reports about the biodegradation of the PAHs especially of
LMW composed of fewer than four benzene rings (Tian
et al. 2008; Hesham et al. 2014). Biodegradation of LMW
PAHs, especially of naphthalene has been studied exten-
sively in Pseudomonas (Vila et al. 2001; Kim et al. 2005).
As hydrocarbon contamination of the soil has increased
worldwide, the local environment is certainly affected by

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this worsening environmental problem. Therefore, there is
an urgent need to find a safe method to remove PAH con-
tamination from the environment. There are reports of few
bacterial genera having the ability to degrade HMW hydro-
carbons. PAHs of HMW like benzo[a]pyrene, fluoranthene,
and pyrene are reported to be degraded by the actinomycetes
group of bacteria, especially strains belonging to the Myco-
bacterium and Rhodococcus genera (Rehmann et al. 1998;
Ghosh et al. 2014; Bourguignon et al. 2014; Subashchan-
drabose et al. 2019). Therefore, identification of the local
key microorganisms from contaminated soil that play an
important role in the degradation processes of HMW PAHs
is one of the most effective and efficient ways to remove
toxic PAHs from the environment. In this study, we iso-
lated and identified Klebsiella oxytoca from the petroleum-
contaminated soil, which could efficiently degrade pyrene.
Materials and methods
Chemicals and soil samples
Pyrene (purity 99%) was purchased from Sigma-Aldrich,
USA. HPLC-grade acetone was purchased from Honeywell,
Inc. USA. Chemicals for mineral basal media (MBM) were
purchased from different suppliers. Pyrene was dissolved in
acetone at 10 mg/ml and the final concentration of pyrene
used in liquid broth media was 0.1 mg/ml.
The oil-contaminated soil samples were collected in ster-
ile 50 ml falcon tubes from three different sites located on
Banimalek road in Abha, Saudi Arabia. Sample A was col-
lected from the site where gasoline is spilled from trucks,
sample B was from a gasoline station, while sample C was
collected from an oil exchange station. All the samples were
stored at 4 °C until further use.
Growth media and culturing conditions
Tryptic soy broth (TSB) was purchased from Sigma-Aldrich,
USA and nutrient broth was purchased from TM Media,
India. Minimal base medium (MBM) [1 g ­(NH4)2SO4,
0.8 g ­K2HPO4, 0.2 g ­KH2PO4, 0.2 g ­MgSO4⋅7H2O, 0.1 g
­CaCl2⋅2H2O, and 5 mg ­FeSO4⋅7H2O in 1 l of distilled water,
pH 7.2] supplemented with pyrene was used for the suspen-
sion of oil-contaminated soil samples. In brief, one gram of
each contaminated soil sample was suspended in 100 ml of
MBM supplemented with 0.1 mg/ml of pyrene in 250 ml
flasks (Wu et al. 2013). The samples were incubated at 37 °C
for 7 days at 200 rpm (Jin et al. 2017). Subsequently, 10 ml
from each culture were sub-cultured into 90 ml of the same
media under similar growth conditions. After two more sub-
culturing under similar conditions, 100 µl from each culture
were plated on MBM-agar plates coated with 1 ml solution
13
Archives of Microbiology (2022) 204:248
of pyrene (0.1 mg/ml) dissolved in acetone. All the plates
were incubated at 37 °C for 2–3 days. After two weeks of
further incubation, the markedly large-sized colonies show-
ing distinct morphology and color were re-streaked on pyr-
ene-coated MBM-agar plates. Isolates, which consistently
produced a bigger size colony with distinct morphology and
color, were selected for pyrene degradation study.
Growth kinetics using BD MGIT 960 BACTEC
For growth kinetics, the Becton Dickinson MGIT 960
BACTEC Automated Mycobacterial Detection system in
BSL3 mycobacterial laboratory at the Southern Region
Military Hospital, Khamies Mushayet, Saudi Arabia, was
used. The cells were inoculated into 7 ml of MBM (control)
or MBM supplemented with 0.01% pyrene at a very low
density ­(106 cfu/ml) in a test tube. The test tube contains a
fluorophore at its bottom, which fluoresces in the presence
of ­CO2 produced by the growing bacterial cells. The tubes
were incubated at 37 °C in the BACTEC system. Fluorescent
readings were recorded regularly at intervals of 24 h. The
fluorescence signal is displayed in the form of growth units,
which were plotted against the time.
Bacterial isolate identification methods
VITEK® MS and V ­ ITEK® 2 COMPACT from bioMé-
rieux were used for the identification of bacterial isolates.
­VITEK® MS uses Matrix-Assisted Laser Desorption Ioni-
zation Time-of-Flight (MALDI-TOF) technology to obtain
the protein fingerprinting from whole bacterial cells, which
it later uses to search the protein fingerprinting database
of bacterial isolates. ­VITEK® 2 COMPACT works on bio-
chemical tests measuring carbon source utilization, enzy-
­ ITEK® MS
matic activities, and antibiotic resistance. Both V
®
and ­VITEK 2 COMPACT in BSL3 mycobacterial labo-
ratory at the Southern Region Military Hospital, Khamies
Mushayet, Saudi Arabia, were used as per the manufacturer’s
instructions.
Gram staining and scanning electron microscopy
Gram staining was performed on bacterial cells grown in
tryptic soy broth (company––) to an O­ D600 of 0.4. Cells were
heat-fixed on a glass slide and flooded with crystal violet for
one min. After rinsing the slide with water, the cells were
treated with iodine for one min. Subsequently, the slide was
rinsed with water, and cells were flooded with counter stain
safranin for one min. The slide was rinsed with water and
observed under 100 × objective of Nikon microscope, fitted
with 10MP USB 2.0 color CMOS C-mount camera.
For scanning electron microscopy (SEM), the bacterial
broth was centrifuged and fixed in 2.5% glutaraldehyde
Archives of Microbiology (2022) 204:248
buffered to pH 7.2 with 0.1 M Na–phosphate buffer for
24 h at room temperature, and then washed thoroughly with
Na–phosphate buffer three times. The sample was then post-
fixed with 1% osmium in Na–phosphate buffer for 1 h and
dehydrated successively with different concentrations of
ethanol of 30%, 50%, 70%, 95%, and 100%. For each etha-
nol volume, the sample was incubated for 10 min except
100% ethanol, which was for 1 h. Subsequently, the sample
was immersed in a critical point dryer and mounted onto an
aluminum metal stub. The sample was coated with a thin
layer of gold by Edward’s sputtering coater and examined
with a JEOL JSM 6360-LV (Tokyo, Japan) microscope in
the Electron Microscope Unit of King Khalid University
(KKU), Pathology department, College of Medicine.
DNA extraction and 16S rRNA sequencing
The genomic DNA of the bacterial isolates was extracted
using the ­DNeasy® Plant Mini Kit (QIAGEN). After check-
ing the purity and intactness of the genomic DNA on an 1%
agarose gel, it was sent to Macrogen, Korea (https://​dna.​
macro​gen.​com/​eng/) for 16S rRNA sequencing. In brief,
PCR product of ~ 1.5 kb amplified from genomic DNA
using universal flanking primers 27F (5′AGAG ​ TTT​ GAT​ CC​
TGG​CTC​AG3′), 1492R (5′TAC​GGY​TAC​CTT​GTT​ACG​
ACTT3′) were purified and sequenced by internal primers
785F (5′GGAT ​ TAG​ ATA​ CCC
​ TGG ​ TA3​ ′), 907R (5′CCGT​ CA​
ATTCMTTT​RAG​TTT3′) in both directions using an ABI
3730 automated sequencer (Macrogen, Seoul, Korea).
Sequence alignment, phylogenetic tree analysis
and gene search
The 16S rRNA sequences of each bacterial isolate obtained
were aligned and compared with the known 16S rRNA
sequences in the GenBank database using the basic local
alignment search tool (BLASTN) available at the National
Center for Biotechnology Information website (NCBI)
(http://​www.​ncbi.​nlm.​nih.​gov/​blast). The nearest neigh-
boring sequences in the database with high homology per-
centage scores were aligned using MEGA-11 (Molecular
Evolutionary Genetic Analysis, version 11). To determine
the taxonomic position of the isolate, a phylogenetic tree
was constructed with MEGA-11 using a maximum likeli-
hood algorithm. KEGG (Kyoto Encyclopedia of Genes
and Genomes) was used to identify and locate the genes
involved in the catabolism of PAHs in the genome of a ref-
erence strain K. oxytoca CAV1374 (GB; CP011636). Other
bacterial genomes taken for the search of genes involved in
degradation and metabolism of PAHs, and other xenobiot-
ics were Klebsiella sp. LTGPAF-6F (GB; CP017450), Kleb-
siella michiganensis M1 (GB; CP008841), and Klebsiella
pneumoniae subsp. pneumoniae KPNIH24 (GB; CP008797).
Page 3 of 9 248
High‑performance liquid chromatography
Pyrene degradation by the K. oxytoca (A12) and Brevibac-
terium frigoritolerans (AA11) was estimated by high-per-
formance liquid chromatography (HPLC) of the superna-
tant of the bacterial culture grown in MBM supplemented
with pyrene as a sole source of carbon. In brief, 1 ml each
of an overnight nutrient-broth-grown bacterial culture was
inoculated into 100 ml of MBM supplemented with 0.1 mg/
ml pyrene in triplicates. For the spontaneous degradation
of pyrene, 100 ml of MBM supplemented with 0.1 mg/ml
pyrene in a separate flask was included as a control. All the
triplicate cultures of each isolate including the control were
incubated at 37 °C for 12 days at 150 rpm. 2 ml samples (in
triplicates) for each bacterial isolate were withdrawn at the
regular time intervals of 3, 6, 9, and 12 days. After spin-
ning down the cells, the concentration of the pyrene in the
culture supernatant of each sample was determined using
HPLC coupled with Shimadzu UV–VIS Detector SPD-10A,
wherein 10 µl sample was subjected to 254 nm ultraviolet
detection at 30 °C. Average values calculated at each time
point were normalized with the control and presented as
percentages.
Results
Enrichment, isolation, and phenotypic
characterization of bacteria growing in the presence
of pyrene
To isolate the bacterial strains that could degrade the
more complex PAH, pyrene, soil samples collected from
various oil-contaminated sites were dissolved in pyrene-
supplemented MBM. The mixtures were incubated over-
night at 37 °C. Sub-culturing several times in the same
media enriched the pyrene-degrading bacterial isolates.
The cultures were then plated on MBM-agar plates coated
with pyrene. A total of 24 prominent colonies of small
(42%), smooth (88%), and creamy (54%) morphology were
observed (Table 1).
Five among the twenty-four bacterial isolates consist-
ently produced bigger size colonies on the pyrene agar plate.
Besides colony size, these five isolates showed consistency
in other physical characteristics of texture and color. The
fives isolates (AA11, AA12, AA18, AA21, and AA23) were
chosen for pyrene broth growth assay.
Growth kinetics of five isolates in pyrene broth
To further ascertain the ability of five isolates to utilize
pyrene as a sole carbon source, the growth assay was set
up in BACTEC automated mycobacterial detection system.
13
248 Page 4 of 9 Archives of Microbiology (2022) 204:248

Table 1  Phenotypic Isolate Isolation site Size Texture Color Isolate Isolation site Size Texture Color
characteristics of bacterial
isolates growing in presence of AA1 A S Smooth Cream AA13 B S Smooth Colorless
pyrene
AA2 A S Smooth Cream AA14 B S Smooth Cream
AA3 A S Smooth Cream AA15 B M Smooth Yellow
AA4 A S Smooth Cream AA16 B S Smooth Cream
AA5 A L Smooth Dark cream AA17 B S Smooth Cream
AA6 A M Smooth Cream AA18* B L Smooth Milky
AA7 A S Smooth Cream AA19 C S Smooth Cream
AA8 A M Smooth Cream AA20 C M Smooth Cream
AA9 A M Smooth Yellow AA21* C L Rough Light yellow
AA10 A L Smooth Milky AA22 C L Rough Colorless
AA11* A L Smooth Orange AA23* C L Smooth Buff
AA12* A L Rough Colorless AA24 C S Smooth Colorless

Size of colonies; S small, M moderate, L large

*Bacterial isolates chosen for pyrene growth assay

Identification of bacterial isolates by various

methods

To identify the isolates, we took several approaches viz bio-

Fig. 1  Growth profile of bacterial isolates individually grown in
MBM containing pyrene as a sole source of carbon. The growth units
(Y axis) observed in BACTEC were plotted against the time in days
(X axis)
Taking the advantage of fluorescence signal produced by
a fluorophore in response to the production of ­CO2, the
growth of each isolate was monitored over time. All five
isolates were individually inoculated in MBM supple-
mented with pyrene. For control, the isolates were inoc-
ulated in MBM alone. The growth of each culture was
recorded at regular 24-h time intervals for 12 days. It is
clear from Fig. 1 that all the isolates took 5 days to accli-
matize to the pyrene growth media, except AA11 that took
10 days. Furthermore, among all the five isolates AA23
showed relatively rapid growth contrary to slow growing
AA11. None of the isolates showed any growth in minimal
media alone (data not shown), suggesting that the pyrene
was the sole source of carbon and energy.
chemical assays, mass spectral fingerprinting of whole bac-
terial protein, and 16S rRNA sequencing. Bacterial strains
differ in their sources for carbon utilization, corresponding
enzymatic activities, and resistance to a particular antibi-
otic. The VITEK2 compact system from bioMérieux uses
reagent cards that contain substrates for several different
enzymes to measure either turbidity or colored products of
substrate metabolism. The reagent cards have 64 wells, each
containing an individual test substrate. Substrates measure
various metabolic activities such as acidification, alkalini-
zation, enzyme hydrolysis, and growth in the presence of
inhibitory substances. Comparison of the test results of a
bacterial strain to a reference database of bacterial isolates
identifies the bacteria rapidly. Furthermore, we took advan-
tage of the ­VITEK® MS from bioMérieux to identify the
bacteria rapidly.
In addition to the biochemical methods and mass spec-
tral fingerprinting, 16S rRNA sequencing of each bacterial
isolate was performed. The 16S rRNA sequences of each
bacterial isolate were blasted against the known 16S rRNA
sequences in the GenBank database using BLASTN. The
first hit showing > 99% identity to query sequence identi-
fied the bacterial strain. The strains identified by VITEK2
compact system (S1), ­VITEK® MS (S2), and the 16S rRNA
sequencing are listed in Table 2. The nucleotide sequences
of 16S rRNA were submitted to the GenBank under the
accession numbers given in Table 2.
The unique bacterial isolate that was identified as, Kleb-
siella oxytoca by all three methods, was AA12 (Table 2),
which was also found taxonomically related to genus Kleb-
siella (Fig. 2D). We believe that AA18 and AA23 might

13
Archives of Microbiology (2022) 204:248 Page 5 of 9 248

Table 2  Bacterial isolate Isolates VITEK® MS VITEK® 2 COMPACT​ 16S rRNA sequencing
identification by various
methods Organism Organism Organism Identity (%) Accession Number

AA11 NA R. radiobacter B. frigoritolerans 99.87 MG571760.1

AA12 K. oxytoca K. oxytoca K. oxytoca* 99.93 MG571761.1
AA18 K. oxytoca K. oxytoca K. michiganensis 99.93 MG571764.1
AA21 B. subtilis S. lentus P. oryzihabitans 99.81 MG571765.1
AA23 K. oxytoca K. oxytoca K. sp. 99.53 MG571766.1

Fig. 2  Phylogenetic trees of five

bacterial strains: phylogenetic
analysis of 16S rRNA gene
of each isolate and its related
microorganisms by maximum
likelihood method. The numeri-
cal value on branch points
indicates the bootstrap value out
of 100

13
248 Page 6 of 9 Archives of Microbiology (2022) 204:248

be replicates of AA12, though their identification by 16S
rRNA sequencing is different. Bacterial isolate AA18 shared
99.93% identity with Klebsiella michiganensis and clustered
with Klebsiella oxytoca in a single clade (Fig. 2C). Similarly,
bacterial isolate AA23 being 99.53% identical to Klebsiella
sp. clustered well with Klebsiella oxytoca (Fig. 2E). The
AA11 and AA21 isolates were identified as different bac-
terial species by all the three methods used in this study,
which corroborate very well with the phylogenetic analysis,
wherein AA11 and AA21 are taxonomically closely related
to genus Bacillus and Pseudomonas, respectively (Fig. 2A,
B). Consistent identification of AA12 isolate as K. oxytoca
by all three methods led us to characterize it further.
Characterization of K. oxytoca
To ascertain the identification of K. oxytoca, Gram staining
of the K. oxytoca grown in TSB was carried out wherein
safranin was used as a counterstain. The Gram staining
demonstrated that the bacterial strain is a Gram-negative
bacillus (Fig. 3). Similarly, the cells grown in TSB and sub-
sequently fixed with glutaraldehyde and osmium tetraoxide
were observed as rod-shaped cells under scanning electron
microscope (SEM) consistent with the identification as K.
oxytoca.
against days of incubation. The pyrene concentrations varied
in K. oxytoca culture in the first week of the experiment,
probably due to the varying degree of solubilization, as a
thin layer of pyrene was observed on the surface of media
and along the sides of the culture flask. It is clear from the
degradation curves (Fig. 4) that K. oxytoca began pyrene
degradation on the 6th day of incubation and continued
thereafter. By the 12th day, the pyrene concentration in K.
oxytoca culture reduced to 30.5% to that of the 3rd day. Con-
trarily, B. frigoritolerans did not show any degradation of
pyrene even up to the 12th day.
Discussion
PAHs are the major environmental pollutants causing
diverse effects of toxicity, mutagenicity and carcinogenicity
on human health. Their clearance from the environment is
of paramount importance. Bioremediation or using micro-
organisms (mainly bacteria) for degrading PAH compounds
has been an environmentally safe and effective method to

Klebsiella oxytoca efficiently degraded pyrene

In order to investigate the pyrene-degrading efficiency of K.

oxytoca, the strain was grown in pyrene-supplemented MBM
at 37 °C in a shaking incubator (rpm; 200) for 12 days. To
compare its degradation efficiency with sluggishly grow-
ing bacterial isolate, AA11 (B. frigoritolerans) was grown
under similar conditions. For spontaneous degradation of
pyrene, MBM supplemented with pyrene was incubated
under similar conditions. The samples were withdrawn on
consecutive days of 3, 6, 9, and 12. The amount of pyrene in
Fig. 4  Pyrene degradation. Graph showing the concentration of pyr-
the culture broth of each strain was determined using HPLC
ene in the culture medium of K. oxytoca (AA12) grown over the time
after normalization with that of pyrene control in the media. in MBM containing pyrene as a sole carbon. Error bars are the stand-
The amount of pyrene in culture supernatant (%) was plotted ard deviations of three biological replicates

Fig. 3  Light microscopic (A)

and scanning electron micro-
scopic (B) images of K. oxytoca
(AA12) cells. Light microscopic
images were captured with
× 100 objective and a scale bar
of 2 μm

13
Archives of Microbiology (2022) 204:248 Page 7 of 9 248

dispose of the PAHs (Peng et al. 2008; Shahsavari et al. humans, which reflects the presence of different sets of
2016). Biodegradation of PAH has been under study for genes in its genome involved in its colonization to differ-
more than just the last decade. Numerous PAHs-degrading ent habitats. Different species of Klebsiella happen to have
strains have been isolated and characterized. At the same ample potential for the degradation of diverse pollutants,
time, the number of PAHs compounds degraded by these including pyrene and benzo[a]pyrene (Ping et al. 2014).
isolates has increased, too. Also, the pathways and mecha- The operons responsible for xenobiotic biodegradation and
nisms for PAHs degradation by several strains have been elu- metabolism are well conserved in K. oxytoca (Fig. 5). Due
cidated. The contaminated soil sample from petrochemical to the presence of several dioxygenase genes, K. pneumo-
refinery fields displayed a high diversity of microbial PAH- niae has an ability to oxidatively catabolize aromatic rings
degrading activities. An effective enrichment technique is an of HMW PAH of pyrene, chrysene, and benzo(a)pyrene
efficient method to discover the promising strains that could (Rajkumari et al. 2018). Several other genes predicted to
utilize PAHs as the sole carbon source. be involved in PAH catabolism, viz, aromatic ring-opening
In this study, an effective enrichment technique was 4,5-DOPA dioxygenase extradiol (ygiD), extradiol catechol
employed to isolate and identify efficient pyrene-degrading dioxygenase, catechol 1, 2-dioxygenase (catA), muconol-
bacterial strains. Only five (AA11, AA12, AA18, AA21, and actone (catB), muconolactone δ-isomerase (catC), catechol
AA23) out of twenty-four isolates maintained the morpholo- 2,3-dioxygenase, toluate 1,2-dioxygenase (benC), 3,4 dihy-
gies of large size, and color upon successive streaking on droxyphenylacetate 2,3-dioxygenase, benzoate dioxygenase
pyrene agar plates. All of them, except AA11, grew intensely large subunit (benB) and small subunit (benA) (Rajkumari
in pyrene broth, after five days of acclimatization to the new et al. 2018) are well conserved in amino acid sequence,
growth condition. Biochemical analysis, mass spectral fin- gene length and their organization in the K. oxytoca genome
gerprinting of whole proteins and 16S rRNA sequencing of (Fig. 5B). Another operon containing genes predicted to be
these isolates identified three of them (AA12, AA18, and involved in other types of xenobiotic biodegradation and
AA23) as replicas of K. oxytoca (see “Results”). The identi- metabolism (Fig. 5A) is also well conserved in K. oxytoca,
fication of the other two isolates (AA11 and AA21) was not
consistent between the three methods employed. With such
ambiguity, the identification by 16S rRNA was considered
authentic. Some of these bacterial species had been reported
earlier to be able to degrade PAHs (Singh et al. 2008; Sub-
ashchandrabose et al. 2019; Tian et al. 2008). Since only one
isolate AA12 was identified by all the three methods as K.
oxytoca, further characterization of AA12 isolate was car-
ried out using Gram staining and scanning electron micros-
copy. It was found that cells were Gram-negative and rod
shaped in their morphology.
Quantitation of the pyrene remaining in culture superna-
tant by HPLC was used to monitor the pyrene degradation by
K. oxytoca over time. The HPLC data analysis showed that
K. oxytoca took five days to acclimatize to new growth con-
ditions and by the 12th day, 69.5% of pyrene from the cul-
ture media was removed successfully. Similar to this study,
other bacterial strains isolated from soil took ten days to start
decomposition of PAHs (Vila et al. 2001).
Hydrocarbon catabolic microorganisms use the pollutants
as energy sources and remove contamination of petrochemi-
cals and petroleum products by a process known as bioreme-
diation. Analysis of hydrocarbon catabolic genes from dif-
ferent bacterial species reveals sequence–structure–function
relationships of diverse catabolic genes. Different gene sets
from different microorganisms are involved in the biodeg- Fig. 5  Operons involved in PAH degradation. Genes above and below
radation of PAHs (Ma et al. 2013; Yu et al. 2014; Xu et al. the line are in sense and antisense strand of chromosomal DNA,
respectively. ORF color indicates the metabolic pathways, blue; car-
2016).
bohydrate metabolism, crepe; xenobiotic biodegradation and metab-
Klebsiella oxytoca is a versatile microorganism tolerating olism, light green; basal cellular processes, pink; regulatory factors,
different microhabitats of soil, plants, and animals including dark green; lipid metabolism

13
 248 Page 8 of 9
suggesting that the enzymes required for catabolism of
pyrene and other PAHs are encoded in the genome of K.
oxytoca. These operons are well conserved in most of the
genomes of K. pneumoniae and K. oxytoca. In conclusion,
a highly efficient PAH-degrading strain, K. oxytoca was
isolated and identified, which would be environmentally
safer alternative tools to protect the environment from PAH
pollution.
Acknowledgements The authors thank Faris Saif Al Masoud and Sul-
tan Ahmad Alkahtani of Microbiology Laboratories, Southern Region
Armed Forces Hospital, Khamis Mushait, Saudi Arabia, for allowing
us to use ­VITEK® MS and ­VITEK® 2 COMPACT. We also thank Prof.
Refaat A. Eid, Department of Pathology, College of Medicine, King
Khalid University, Abha, Saudi Arabia, for performing SEM.
Author contributions AMA and MAM performed the experiment.
MAM and SAA conceived and designed the study. MAM analyzed
the date and wrote the manuscript. All authors read and approved the
manuscript.
Funding The authors extend their appreciation to the King Abdul-Aziz
City for Science and Technology (KACST) for funding this work under
Grant No. 1-17-01-010-0012.
Declarations
Conflict of interest The authors declare no potential conflicts of inter-
est in research, authorship, and/or publication of this article.
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