degradacao petroleo golfo

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Research Article
Received: 10 January 2021 Revised: 19 February 2021 Accepted article published: 2 March 2021 Published online in Wiley Online Library: 13 March 2021
(wileyonlinelibrary.com) DOI 10.1002/jctb.6707
Biodegradation of crude oil using symbiont
crude-oil degrading bacteria isolated from
corals collected at the Persian Gulf
Nasrin Ansari,a Farokh Rokhbakhsh-Zamin,a* Mehdi Hassanshahianb* and
Majid Askari Hesnib
Abstract
BACKGROUND: About 25,000 oil tankers are transported annually through the Persian Gulf in Iran. Therefore, oil pollution in
this region is very high. Research on the use of marine bacteria to degrade crude oil has yielded good results in recent years.
The present study investigates bacteria that are symbiotic with marine corals and their ability to degrade crude oil. Corals were
collected from five regions in the Persian Gulf and enrichedwith decomposing bacteria in ONR7a culture. After biochemical and
molecular identification of bacterial isolates, the ability of the strains to degrade crude oil by spectrophotometric, gravimetric,
and Gas Chromatography- Mass Spectroscopy (GC-MS) methods was investigated.
RESULTS: Finally, 26 bacterial strains were identified from 5 coral samples. The efficient strains that had the highest crude-oil
degradation belonged to genera Cobetia, Shewanella, Alcanivorax, and Cellulosimicrobium. Strains IAUK3568, IAUK3568,
IAUK3502, IAUK3531, and IAUK3552 exhibited a maximum degradation of crude oil of 93.5%, 88.13%, 87.24%, 85.17%, and
77.3%, respectively.
CONCLUSION: The results showed that all bacterial isolates degrade medium-chain alkanes better than long- and short-chain
alkanes. The data obtained in this study confirm that the corals of the Persian Gulf have the necessary bacterial diversity to
decompose crude oil and, consequently, reduce pollution in this region.
© 2021 Society of Chemical Industry (SCI).
Keywords: biodegradation; marine environment; oil pollution; Persian Gulf; corals
INTRODUCTION
Oil and petroleum products are produced and consumed as one
of themain sources of energy around the world. Although consid-
erable efforts have beenmade in the past to reduce oil spills, acci-
dental releases of oil continue to occurs and pollute the
environment.1 Oil spills in aquatic ecosystems cause enormous
economic losses to communities and coastal areas, and the
effects of their damage remain in the environment for a long
time.2
The waters of the Persian Gulf flow into the Indian Ocean
through the Strait of Hormuz and are, therefore, a semi-enclosed
ecosystem. This gulf is a habitat with high oil pollution because
annually, about 25 000 oil tankers move in this gulf, meaning that
oil spills and pollution are frequent in this area.3 The Persian Gulf
maintains diverse ecosystems, like coral reefs, mangrove forests,
and other types of coasts that are affected by oil pollutants. Over
the course of three events (in 1980, 1983, and 1991), about 11mil-
lion barrels of oil entered the waters of the Persian Gulf. These
events have led to pollution in the air, in coastal and mangrove
forests, and underwater, in addition to the oil pollution that is
spreading through countries with these reserves. This will cause
damage to the ecosystem that will last for decades.4
Marine environment habitats have been particularly impacted
by different threats, especially coral reefs, which are one of the
most sensitive ecosystems on earth. Providing a breeding ground
for fish and other marine organisms is one of the reasons why
coral reefs are critically important in marine habitats.2 Due to
the considerable importance of corals in marine ecosystems, their
extinction leads to catastrophic events. In this regard, various
solutions, including bioremediation, are used to discharge marine
pollutants. Bioremediation involves using the ability of live things
to destroy and remove pollutants. As this operation is relatively
inexpensive and stable, it can be implemented in diverse habitats
because it has a relatively minor effect on most systems.5
In addition to bioremediation, there are other ways to remove
oil from the marine environment. For example, we can refer to
physical and chemical methods. Physical and chemical methods
* Correspondence to: F Rokhbakhsh-Zamin, Department of Microbiology,
Kerman Branch, Islamic Azad University, Kerman, 7635131167, Iran, E-mail:
rokhbakhsh@iauk.ac.ir; or M Hassanshahian, Department of Biology, Faculty
of Sciences, Shahid Bahonar University of Kerman, Kerman, 7616913439, Iran.
E-mail: mshahi@uk.ac.ir
a Department of Microbiology, Kerman Branch, Islamic Azad University, Ker-
man, Iran.
b Department of Biology, Faculty of Sciences, Shahid Bahonar University of
Kerman, Kerman, Iran
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https://orcid.org/0000-0001-9899-7168
mailto:rokhbakhsh@iauk.ac.ir
mailto:mshahi@uk.ac.ir
include the use of skimmers, and one of the most common chem-
ical methods for degradation of crude-oil is the use of surfactants
at the surface of seawater. However, these methods create sec-
ondary pollution and are, therefore, not safe for the marine envi-
ronment. Bioremediation methods, which are some of the best
methods for removing crude-oil from the marine environment,
are divided into three groups: biostimulation, bioaugmentation,
and cometabolism.6
The coral reefs of the Persian Gulf are themost significant repos-
itories of biodiversity in the region and are among the most pro-
ductive offshore ecosystems. Corals act as a home and shelter
for fish, crustaceans (such as lobsters and shrimps), mollusks(such
as octopus, tropical spiral snail, and bivalve snail), turtles, and
other sea creatures. The main habitat for corals is frequently shal-
low salty waters; hence, the Persian Gulf is a proper habitat for
such creatures.6 There are about 16 islands, including Kharg and
Lark islands, in the Persian Gulf waters of Iran, which have coral
reefs with 63 species of hard corals. Until 2015, most of the fami-
lies identified were Acroporidae, Poritidae, and Faviidae, but since
2016 (because of climate change and pollution), the Poritidae spe-
cies remains the dominant species in the Gulf.7
The specific aim of the present work was to: (i) isolate crude-oil
degrading bacteria from coral reefs in specific areas with polluted
waters in the Persian Gulf, and (ii) carefully investigate the bio-
technological abilities of the isolated strains to produce biosurfac-
tants and to naturally degrade crude oil.
MATERIALS AND METHODS
The materials used, and experimental methods employed are
listed below.
Sampling
The coral samples were collected from five oil-contaminated sites
at the Persian Gulf. These five stations were Shidver (A sample),
Lavan (B sample), Kish (I sample), Lark (L sample), and Qeshm
islands (N sample). The coral reef samples were collected from a
depth range of about 8–10 m by the SCUBA method. Specimens
were embedded in aluminum foil immediately and every sample
was about 3–4 cm. All coral samples were transported on ice to
the laboratory. To isolate bacteria from collected corals, a piece
of coral (about 2 cm2) was transferred by handling with a sterile
knife. At that time, an emulsion mixture was prepared using the
seawater stored in the internal surfaces of the coral tissue. Macer-
ated coral was used to pursue the study.
Quantification of the number of cultivable bacteria
The Most Probable Number (MPN) and colony-forming units (CFU)
methods were used to determine the frequency of co-coexisting
bacteria. Initially, 100 μl of the sample diluted 10-fold coral was pre-
pared. To identify heterotrophic bacteria, a culture was performed
on Marine Agar medium for 3 days and at a temperature of 30 °C;
for degrading bacteria, a culture was efficiently performed on
ONR7a agar for 7 days and at a temperature of 30 °C. The results
were reported as CFU·g−1. The MPN method for heterotrophic
and hydrocarbon-degrading bacteria was performedin sterile
24-well microplates using sample aliquots with corresponding dilu-
tions and cultivation media. In the first step to correctly identify
degrading bacteria, 1700 μL ONR7a medium was provided in each
well of themicroplates, and then a tenfold dilution series of macer-
ated coral reef samples (until 10-1-10-3) was prepared in ONR7a
medium.8 The wells were inoculated with 100 μL of sample
diluents. Following this, 100 μL of sterile Iranian light crude oil
was applied at each well. Plates were incubated at 30 °C for
21 days. After incubation, the visual evaluation of microbial growth
was performed. On this basis, the number of positive test tubes and
the counts of microorganisms were statistically evaluated. In
another stage (to identify heterotrophic bacteria), each well was
provided with 1700 μL Marine Broth (MB) and 100 μL dilution 10-
3-10-4 of the macerated coral reef sample, and then plates were
incubated at 30 °C for 14 days. All samples from each station were
used for MPN count. MPN counts were properly analyzed with the
computer program MPN calculator version 4.2.9
Isolation and selection of crude-oil degrading bacteria
Crude-oil degrading bacteria were isolated in ONR7a medium
supplemented with 1% (v/v) of crude oil (Iranian light crude oil)
as sole carbon source and energy. ONR7a contained (per liter of
distilled water) 40 g of NaCl, 11.18 g of MgCl2.6H2O, 3.98 g of
Na2SO4, 1.46 g of CaC12.2H2O, 1.3 g of TAPS0 {3- [N tris (hydroxy-
methyl) methylamino]-2 hydroxy propane sulfonic acid}, 0.72 g of
KCl, 0.27 g of NH4Cl, 89 mg of Na2HP04.7H20, 83 mg of NaBr,
31 mg of NaHCO3, 27 mg of H3BO3, 24 mg of SrCl2.6H20, 2.6 mg
of NaF, and 2 mg of FeCl2.4H20. For solid media, Bacterial Agar
(15 g L-1) was added to the solution.
Five ml of coral tissue was mixed into 100 ml of the ONR7a
medium with crude oil (as sole carbon source) and shake for 7
days at 30 °C and 180 rpm. After incubation time one passage
were done into newONR7amediumwith crude oil and inoculums
from the flask were streaked out on agar medium. Different colo-
nies that appeared in ONR7a agar medium were isolated and in
order to eliminate autotrophic bacteria these colonies were cul-
ture into fresh ONR7a media with and without crude oil. Finally,
the isolates with significant growth in ONR7a with crude oil were
selected for further study.10
Screening of crude-oil degrading bacteria using the redox
application of 2,6-Dichlorophenolindophenol (DCPIP)
Screening of potential crude-oil degrading bacteria was carried out
by modified Hanson et al. (1993) method using DCPIP as a redox
indicator.11 In the process of oxidation by bacteria, electrons are
transferred from hydrocarbons to DCPIP (electron receptors). The
change from blue to colorless indicates the ability of bacteria to
use hydrocarbons, which is studied at 600 nm. The inoculum was
prepared by transferring cultures from marine agar slants into
ONRmedium for 24 h at 35 ± 2 °C and180 rpm. Cultureswere then
inoculated into tubes along with DCPIP indicator (0.5% w/v) and
crude oil (1%, v/v) for spectrophotometric analysis. All the tubes
were incubated at room temperature for 144 h. Appropriate con-
trols and substrate/crude oil assays were included. The absorbance
of all the tests wasmeasured by spectrophotometer. Data were col-
lected at a regular time interval of 24 to 144 h.11
Identification of the isolates
Bacterial isolates were examined from both biochemical and
molecular aspects. Based on the Bergey's manual, biochemical
tests (such as Gram staining, catalase, etc.) were performed to
identify bacteria.12,13
Analysis of 16S rRNA was performed to characterize the isolated
strains taxonomically. The total DNA of bacterial strains was extracted
with the Cetyl Trimethyl Ammonium Bromide (CTAB) method. PCR
amplification of 16S rRNA genes was performed using the general
bacteria primer 27F (5-AGAGTTTGATCCTGGCTCAG-3) and universal
reverse primer 1492R (5-TACGYTACCTTGTTACGACTT-3).8 The
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amplification reactionwas carried out in a total volume of 25 μl con-
sisting of 2 mmol L–1 MgCl2 (1μl), 10X PCR reaction buffer
(200 mmol L–1 Tris; 500 mmol L–1 KCl) (2.5 μl), 2 mmol L–1 each
dNTP (2 μl), 0.15 mmol L–1 each primer (1 μl), 1U (0.5 μl) Taq DNA
polymerase (Qiagen, Hilden, Germany), and 2 μl of template DNA
(50 pmol). The total volume was brought up to 15 μl using sterile
milliQ water. The PCR program, consisting of 35 cycles, was per-
formed in a thermal cycler Gene Amp 5700 (PE Applied Biosystem,
Foster City, CA, USA). The temperature profile for PCR was kept at
94 °C for 5 min, 94 °C for 1 min, 54 °C for 1 min, and 72 °C for
1.5 min for 35 cycles, then 72 °C for 10 min and, finally, storage at
4 °C.8
The 16S rRNA amplified was sequenced with Big Dye Termina-
tor V3.1 cycle sequencing kit on an automated capillary sequencer
(model 3100 Avant Genetic Analyzer, Applied Biosystems). Simi-
larity rank from the Ribosomal Database Project RDP) and FASTA
Nucleotide Database Query were used to determine partial 16S
rRNA sequences to estimate the degree of similarity to the other
16S rRNA gene sequences. Analysis and phylogenetic affiliates
of sequences were performed according to the protocol
described by Yakimov et al. (2007).14 The phylogenetic tree was
depicted by MEGA5 software with the neighbor-joining method.
Bacterial growth rate
The bacterial growth schedule was set at 30 °C and 180 rpm for
15 days. The growth of a strain by measuring the turbidity caused
by OD 600 nm (Shimadzu UV-160, Japan) was determined.
Figure 1. The macroscopic image of the 5 coral collected at the Persian Gulf. These five stations were Shidver (A sample), Lavan (B sample), Kish
(I sample), Lark (L sample), and Qeshm island (N sample).
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Estimation of crude oil degradation by isolated strains
The crude oil degradation of each isolated strain was evaluated
using three methods: (i) spectrophotometry, (ii) gravimetric
method, and (iii) gas chromatography-mass spectroscopy (GC-MS).
The remaining crude oil in the medium was calculated by spec-
trophotometry method, as follows: the residual crude oil in the
medium was dissolved in dichloromethane (DCM) and the optical
density was read at 420 nm.15
Furthermore, crude oil degradation was estimated by the
gravimetric method, as follows. After 15 days of incubation of
the strains in the medium with one percent crude oil, the
sample was extracted using dichloromethane in the same man-
ner. The organic phase, which contains crude oil dissolved in
dichloromethane, was exposed to air to evaporate its dichloro-
methane. The degraded crude oil by strains was calculated by
reducing the weight of the un-degraded control flask (blank)
from the degraded flask (experiment) and is expressed as gram
per liter.16
The amount of crude oil not degraded by bacteria from each
sample was checked by GC-MS. Initially, the same volume of
DCM, and then of Na2SO4, was added to degrade the crude oil
and remove the water, respectively. The GC-MS investigation
Figure 2. Enumeration of heterotrophic and hydrocarbon-degrading bacteria by CFU and MPN methods in 5 collected coral. An average of three repe-
titions of each experiment is used in this figure.
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was performed with a Varian 3800model (Coleman-Derr & Tringe)
and SE-54 capillary column (25 m × 0.32 mm × 0.1 μm) equipped
with FID. Helium was considered a carrier gas in the amount of
30 mL min–1and the program of the oven was 1 min at 100 °C
and then, the temperature was increased to 300 °C (2 min) at a
rate of 30 °C min-1. Also, one micro-litter was injected into GC-
MS column.17
The measure of emulsification activity and biosurfactant
production by efficient isolates
In the study of the activity of E24 emulsion, the same volume of
emulsion and hexadecane was placed in the cell-free culture and
Vortex for 2 min. After 24 h at rest, the index emulsion for the emul-
sion layer divided by the total height of the liquid column height
scale in mm was determined as a percentage. Two methods were
Table 1. Screening tests for selection of crude oil degrading bacteria between isolated strain from coral samples
Strain
IAUK
DCPIP
colorless
Quality of growth in ONR
medium with crude oil
OD of growth (600 nm) in ONR
medium with crude oil
Oil-
Spreading
(cm)
Emulsification
activity (E24 %)
Hemolysis in
blood agar (cm)
3501 Delaya+ 2c + 0.338 0.3 0.00 0.0
3502 Fastb + 3 + 1.028 1.4 61.53 2.1
3503 Delay + 1 + 0.294 0.4 7.69 0.0
3504 Fast + 2 + 0.455 0.5 16.60 1.5
3505 Delay+ 1 + 0.190 0.4 6.15 0.4
3506 - + 1 0.471 0.2 61.50 0.0
3507 Fast+ 3 + 0.701 0.4 1.60 2.0
3529 Delay+ 1 + 0.353 0.5 15.00 0.3
3530 - 1 + 0.416 0.5 4.61 0.0
3531 Fast + 3 + 1.061 1.5 50.00 1.8
3542 - 1 + 0.163 0.5 25.70 0.0
3543 Fast + 2 + 0.419 0.5 20.00 1.7
3544 Delay + 2 + 0.419 0.5 0.00 0.0
3545 - 1 + 0.142 0.5 3.30 0.0
3546 Delay + 2 + 0.345 0.6 25.00 0.5
3547 - 1 + 0.149 0.5 0.00 0.0
3548 Fast + 3 + 1.376 1.5 64.90 1.8
3549 - 1 + 0.170 0.5 0.00 0.0
3550 Fast + 2 + 0.520 0.5 1.60 0.6
3551 - 1 + 0.129 0.5 21.42 0.0
3552 Fast + 3 + 1.435 1.7 1.60 1.5
3553 - 1 + 0.088 0.5 53.84 0.0
3565 - - 0.144 0.3 0.00 0.0
3566 Delay + 2 + 0.324 0.4 3.30 0.3
3567 Delay + + 1 0.222 0.5 28.30 0.0
3568 Fast + 3 + 1.376 1.6 33.80 2.1
a Delay means the colorless of DCPIP dye take place at 4 days.
b Fast means the colorless of the DCPIP dye took place at 4 days.
c In the quality test, these scores were given (1+ Weak growth; 2+ moderate growth; 3+ high growth).
Table 2. Biochemical tests for identification of selected isolates
Strain IAUK Morphology Gram Staining TSIa CATALASE OXIDASE Ob Fb NITRATE CITRATE Sc Ic Mc
3502 Coccobacillus Gram-negative alk /alk + - + + - - - - +
3504 Bacilli Gram-negative alk /alk - + + - - - + - +
3531 Coccobacillus Gram-negative alk /alk + _ + + + - - - +
3547 Cocci Gram-negative alk /alk + + - - - - - - -
3548 Bacilli Gram-positive alk /alk + - - - - - - - -
3550 Coccobacillus Gram-negative acid/acid - - + - - + - - +
3552 Bacilli Gram-positive acid/acid + + + - + - + - +
3568 Cocci Gram-positive alk /alk + + + + - + - - +
a Triple sugar iron.
b Oxidation-fermentation.
c Sulfide-indole-motility.
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operated for the screening and selection of the most capable
biosurfactant-producing bacteria between symbiont isolates. The
oil spread technique was carried out according to Youssef et al.
(2004).17 Further, the culture of bacteria in the blood agar medium
was another screening test for biosurfactant production.
Effect of the concentration of crude oil on degradation by
isolated strains
The effect of distinct concentrations of crude oil (1%, 2.5%, 4%,
and 5.5%) on the growth and degradation of crude oil by the
selected bacterial strains was examined using ONR7a medium at
30 °C and 180 rpm for 15 days. Bacterial growth was measured
at 600 nm and crude oil degradation at 420 nm.18
RESULTS
Coral identification
The image of the collected coral samples is presented in Figure 1. This
figure confirmed that all collected corals were stony coral, except for
sample (L), which was soft coral. The results of coral identification
Figure 3. Phylogenetic tree of 16S rDNA sequences of the 5 isolates strains obtained from collected corals at the Persian Gulf. The tree was constructed
using sequences of comparable region of the 16S rDNA gene sequences available in public databases. Neighbor-joining analysis using 1,000 bootstrap
replicates was used to infer tree topology. The bar represents 0.02% sequence divergence.
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show that (A), (B), (I), and (N) coral samples belong to Porites harrisoni
genus and (L) coral samples are related to Sinularia sp. genus.
Measurement of the amount of both types of bacteria in
coral samples according to CFU and MPN
The results for crude-oil degrading and heterotrophic bacteria are
shown in Figure 2. As shown in this figure, the numbers of hetero-
trophic bacteria in l, b, and a coral sample were the highest
(respectively, 4.1 × 107, 3.4 × 107, and 3.1 × 07 CFU·mL−1) com-
pared to other samples. Also, the coral samples l, a, and b had
themaximum abundance of crude-oil degrading bacteria (respec-
tively, 5.2 × 10 5, 4.4 × 10 4, and 3.9 × 10 5 CFU·mL−1). The coral
samples l and a (respectively, 7.5 × 10 5 and 6.4 × 10 5) had the
highest number of crude-oil degrading bacteria (MPN) compared
to other samples. The maximum quantity (MPN) of heterotrophic
bacteria was related to l and a coral samples (respectively,
6.1 × 107 and 5.2 × 107). Also, Figure 2 illustrates that the MPN
values in all samples were higher than the CFU values.
Screening of crude-oil degrading bacteria from coral
samples
Twenty-six crude-oil degrading bacteria were isolated from 5 coral
samples after enrichment cultures were incubated at 30 °C for
2 weeks. Some screening tests were carried out for selecting the
best crude-oil degrading bacteria between 26 isolates.
The results of this screening are shown in Table 1. As shown in
this table, all isolated strains have positive growth in the presence
of crude oil as the sole energy and carbon source. The first screen-
ing test used in this research for the selection of robust degrading
bacteria was DCPIP colorless analysis. As shown in Table 2, three
patterns were seen in these strains. Some strains (such as
IAUK3502, IAUK3504, and IAUK3507) after 24 h turn from blue to
a white color (Fast), but in other strains (such as IAUK3501,
IAUK3503, and IAUK3505), the color change takes place after five
days (Delay). However, some strains (such as IAUK3506, IAUK3530,
and IAUK3542) could not change from the blue color at the end of
the experiment (negative). The data presented in Table 1 con-
firmed that there was a direct relationship between qualitative
and quantitative growth with DCPIP analysis. Strains (such as
IAUK3502, IAUK3531, IAUK3548, IAUK3552, and IAUK3568) that
had the fast patterns in DCPIP analysis also had maximum OD in
ONRmediumwith crude oil as sole carbon source and gave signif-
icant values in quality analysis. The ability of 26 isolates to pro-
duce a biosurfactant was analyzed by oil spreading, hemolysis,
and emulsification activity methods. The results are presented in
Table 2. As shown in this table, hemolysis was weak among most
isolates, although strains IAUK3502 and IAUK3531 had the best
hemolysis (2.1 cm). Also, most isolates had emulsification activity,
and themaximum values of emulsification activity were related to
strains IAUK3502, IAUK3531, and IAUK3548 (E24: 61%, 50%, and
64%, respectively). There was a correlation between high growth
in ONR medium and emulsification activity of isolates.
Identification of bacteria
Eight bacterial strains with the highest rate of growth in crude oil
were selected and first identified by biochemical tests the results
were reported in Table 2. This table confirmed that all strains were
Gram-negative; the majority of strains were catalase-positive and
had motility. Five strains were identified in databases using
sequencing and comparison with 16SrRNA genes. The results of
molecular identification confirmed thatthese five isolates belong
to Cobetia marina strain IAUK3502, Alcanivorax dieselolei strain
IAUK3568, Cellulosimicrobium cellulans strain IAUK3548, Shewanella
Haliotis strain IAUK3552, and Cobetia amphilecti strain IAUK3531.
The 5 bacterial sequences were submitted in the NCBI and the ID
of each mentioned below. MT185142 (strain IAUK3502),
MT185143 (strain IAUK3568), MT185145 (strain IAUK3548),
MT185147 (strain IAUK3552), and MT185154 (strain IAUK3531).
The phylogenic trees of these five isolated strains are illustrated in
Figure 3. This figure shows that strains IAUK3548, IAUK3568, and
IAUK3531 have a high similarity with other sequences that present
Table 4. The rate of alkane degradation in crude oil that calculated
by GC-MS chromatographs for each strains
Strain IAUK Alkane 3502 3531 3548 3552 3568
C9
a 100 100 100 100 100
C10 100 87 100 100 100
C11 61 93 100 95 100
C12 54 53 100 47 100
C13 64 100 100 42 100
C14 63 62 100 56 100
C15 54 81 96 95 100
C16 48 55 93 58 94
C17 46 57 87 22 91
C18 51 31 100 94 89
C19 56 60 49 51 87
C20 57 55 56 45 74
C21 61 36 76 40 64
C22 76 44 69 41 53
C23 58 32 71 41 51
C24 41 25 52 68 46
C25 58 28 49 56 54
C26 66 23 42 53 47
C27 54 15 43 44 42
C28 53 12 39 24 56
C29 48 10 35 16 55
C30 33 7 39 18 38
a The number of carbon.
Table 3. The growth rate of efficient strains and percentage of crude
oil degradation that were measured by three analytical methods
Isolate
IAUK
Growth rate
(OD600 nm)
Percentage of
oil removal
removal
(Spectrometry)
(GC
Method)
(Gravimetric
Method)
3502 1.65 70.40 87.24 60.60
3504 0.77 60.25 66.08 50.20
3507 1.80 30.00 37.13 25.80
3531 1.49 76.70 85.17 77.12
3543 0.44 40.70 54.30 39.60
3546 1.43 39.40 43.50 34.60
3547 1.47 67.57 74.30 57.30
3548 1.53 80.41 88.13 76.50
3550 0.45 70.40 84.13 62.50
3552 0.49 60.70 77.30 58.20
3568 1.90 85.50 93.50 79.50
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in the Gene bank database, although the similarity of different
strains is less in comparison to these three strains.
Crude oil biodegradation by selected strains
Eleven strains were selected to study the degradation of 1% crude
oil at 15 days with constant shaking. Gravimetric, GC-MS, and
spectrometry methods were used to evaluate the growth of
microorganisms and the biodegradation of crude oil, and the
results are included in Table 4. The strains IAUK3568, IAUK3548,
IAUK3502, IAUK3531, and IAUK3552 exhibit maximum degrada-
tion of crude oil (respectively, 93.5%, 88.13%, 87.24%, 85.17%,
and 77.3%). The minimum crude oil degradation between twelve
strains was related to strain IAUK3507 (25.8%). Three methods
were used for calculating crude oil degradation. Between these
three methods, the most precise results were obtained by the
GC-MS method (Table 3) and the GC-MS chromatogram for
IAUK3568 (an example of a strain) compared to blank confirms
that the peaks of crude oil compounds have been significantly
reduced by these strains (Fig. 4).
Ability of degradation strains in different concentrations
of crude oil
The effect of different concentrations of crude oil on degradation
by three efficient strains (IAUK3502, IAUK3568, and IAUK3552)
was determined. These strains were selected because they exhib-
ited maximum degradation of crude oil in the primary screening
analysis. The results are presented in Figure 5. As shown in this fig-
ure, the maximum degradation of crude oil holds place at 2.5%
concentration. Additionally, this figure demonstrates that crude
Figure 4. GC-MS results in tracing the residual crude oil in bacteria. (A) Blank without bacterium and (B) IAUK3568 strain.
Figure 5. Effect of crude oil concentration on its removal by isolates. The star above the bar means statistical significance was found by Duncan's
test (P < 0.05).
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oil degradation decreased when the concentration of crude oil
was increased. The strain that could best degrade powerful levels
of crude oil was strain IAUK3502. These data were statistically con-
firmed because the Duncan's test was performed on the results
and the data was significant (P < 0.05).
Ability to degrade n-alkanes isolates
The ability of n-alkanes (C9-C30) degradtion by selected isolates
from the preceding stages was examined by GC analysis and the
assessed value was compared with the control group that was
not affected by bacteria; the values were summarized in Table 4.
The outcomes have shown that strains IAUK3568, IAUK3548, and
IAUK3552 degraded the most n-alkanes present in crude oil. In
contrast, strains IAUK3531 and IAUK3502 had the minimum deg-
radation of n-alkanes. Because short-chain alkanes dissolve cell
membranes, they are toxic to bacteria. On the other hand, alkanes
with long chain lengths are stable and have little solubility for
these strains. As a result, medium-chain alkanes are the most suit-
able type for crude oil-destroying strains.
DISCUSSION
There is little research on the unique relationship betweenmarine
animals and beneficial microorganisms in the polluted marine
environment. Deep-sea creatures feed directly from freshly set-
tled sediments. Therefore, they are in direct contact with pollut-
ants entering the seawater and, consequently, are linked to the
bacteria accumulated in the contaminated sediment due to their
filter-feeding activity.19
Corals are diverse and complex creatures in marine ecosystems
that serve as shelters for many other marine inhabitants. Destruc-
tive human activities, like oil and sea pollution, have led to the
destruction of corals around the world. An astonishing event on
the Shidver Island in the Persian Gulf showed corals can thrive
and flourish in the presence of crude oil continuously seeping
from natural cracks in the island seabed.20,21
In the current research, the quantity and diversity of crude-oil
degrading bacteria that are in symbiosis with some corals at dis-
tinct zones of the Persian Gulf was investigated. Our research
have shown there is significant diversity in symbiotic degrading
bacteria in the collected corals. In this examination, two different
coral genera were identified.22 Among the five islands where the
corals were collected, the utmost quantity and diversity of sym-
biont crude-oil degrading bacteria belong to corals harvested
from Larak and Shidver islands. The diversity in these two islands
can be attributed to the presence of many oil reservoirs, oil
extraction activities, and the transfer of petroleum products in
these zones, all of which have contributed to the remarkable
oil spill in the Persian Gulf and the selection of symbiont degrad-
ing bacteria.23,24 One more interesting and outstanding result
from the present study is that the mucosal and tissue parts of
Lark Island corals demonstrated more diversity in degrading
crude oil than other corals.25,26
Eleven degrading bacteria were isolated from collected corals
from Lark Island. In the other islands, this value was lower, as fol-
lows: Shidver (2), Lavan (5), Kish (3), and Qeshm (4). This is a result
of the nature of the oil that comes into contact with corals.
Because there are so many oil reservoirs on Lark Island, corals
receive non-volatile oil (which remains the water-soluble compo-
nent) directly. Another interpretation involves the fact that this is
a soft coral, while the others are stony corals. Therefore, the
degrading bacteria can inhabit the tissue of soft coral more
adequately than the tissue of stony corals. The lowest symbiont
degrading bacteria were perceived in corals collected from
Qeshm Island. The minute quantity and variety of degrading bac-
teria in this area can be as ascribed to the low oil production and
transition in this island and it is referred to asan unpolluted
region.27 In this manner, by comparing these two regions (Larak
and Qeshm islands) in the Persian Gulf, it can be concluded that
the level of crude oil contamination caused a direct effect on
the selection of symbiont degrading bacteria in the tissue of
corals. There was limited experimentation on the characterization
and screening of crude-oil degrading bacteria frommarine corals.
Some examples about isolation of crude-oil degrading bacteria
from marine corals was described as follows.
Al-Dahash andMahmoud (2013) examined oil-degrading bacte-
ria in certain coral reef systems in the north of the Persian Gulf
(Kuwait).28 They demonstrated conventional and molecular tech-
niques to distinguish the oil-degrading communities in the tissue
and mucus of coral reefs. The consequences of their research ver-
ified the predominance of bacteria affiliated with Gammaproteo-
bacteria, Actinobacteria, and Firmicutes in the mucus and tissues
of Acropora clathrata and Porites compressa. These bacteria could
degrade a broad range of aliphatic (C9–C28)and aromatic hydro-
carbons (Phenanthrene, Biphenyl, Naphthalene), as well as crude
oil.28
Kellogg (2019) studied the microbiomes of stony and soft deep-
sea corals at Baltimore Canyon. Their outcomes confirmed these
bacterial associates in the tissue of corals played important symbi-
otic roles. These conserved bacterial associates include taxa with
the potential for nitrogen and sulfur cycling, detoxification, and
hydrocarbon degradation.29
Crude-oil degrading bacteria can also be isolated from different
marine animals; for example, Cappello et al. (2012) performed a
study on the prevalence of bacteria in the gills of bivalves in con-
taminated and non-contaminated environments.8 They con-
cluded that the quantity of crude-oil degrading bacteria in
polluted areas was high compared to that in unpolluted regions.
Nevertheless, the frequency of heterotrophic bacteria remains
constant in both environments. In this exploration, crude-oil
degrading bacteria were isolated from coral samples at various
islands of the Persian Gulf. This analysis is the first experience
report on the screening and characterization of crude-oil
degrading bacteria in the tissue of coral in the Persian Gulf. Our
results supported the idea that effective crude-oil degrading bac-
teria exist in the tissue and mucus of marine corals.8
In the initial screening, 26 strains were isolated from five corals,
out of which 11 strains were selected as the most effective. Ulti-
mately, five robust strains that had a high growth rate and crude
oil degradation, as identified by the molecular method. The
results of molecular identification confirmed four diverse genera
(Cobetia, Cellulosimicrobium, Alcanivorax, and Shewanella). These
genera that introduced in the current research also, previously
reported by other researchers as crude oil degrader.
The study conducted by Fakhrzadegan et al. (2019) on bacteria
that decompose from mangrove forests led to the report of Halo-
monas, Alcanivorax, Idiomarina, and Shewanella genera. These are
same genera that were mentioned in this review.27
Sivaraman et al. (2011) reported the isolation of a Hydrocarbo-
noclastic bacterium called Cellulosimicrobium from oil-
contaminated water in India. The same strain was also identified
in the Persian Gulf waters in this study.30
Campos et al. (2015) isolated Alcanivorax dieselolei from the
mucus of the zoanthid Palythoa caribaeorum at Porto de Galinhas,
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Brazil. They confirmed the presence of a hydrocarbon-degrading
bacterium in a reef ecosystem such as Porto de Galinhas. In the
present study, the bacterium was identified from coral isolated
from Qeshm Island.31
The data obtained in this study showed that high concentra-
tions of crude oil can reduce the ability of bacteria to degrade.
Furthermore, high concentrations of hydrocarbons limit the bio-
availability of oxygen, nitrogen, and phosphorus, which reduces
the growth of bacteria and, thus, reduces degradation. Other
experiments in this study confirmed that strains with high emul-
sion activity have the greatest oil degradation effect. Hassansha-
hian et al. (2012) stated that strains with high emulsion activity
(E24) show a high level of hydrophobicity.
32
Narciso-Ortiz et al. (2020) also used a consortium of Acinetobac-
ter bouvetti, Defluvibacter lusatiensis, Xanthomonas, and Shewa-
nella bacteria to remove pollution in the Gulf of Mexico; while
declaring the desired results in their research, they indicated that
in all their experiments, emulsifying activity increased and the
diameter of hydrocarbon droplets decreased over time.33,34
CONCLUSIONS
Persian Gulf corals are resistant to heat, salinity, and various envi-
ronmental pollutions due to their environmental conditions and
strategic location. It is clear that the Persian Gulf is an enormously
rich environment for the screening and isolation of numerous
types of microorganisms with diverse applications. The results of
this study revealed that marine corals collected from five regions
of the Persian Gulf have a high density and diversity of crude-oil
degrading bacteria. Some strains could even degrade elevated
concentrations of crude oil. Therefore, these bacteria can be con-
sumed for the bioremediation of polluted stations at the
Persian Gulf.
ACKNOWLEDGEMENTS
This research is financially supported by Islamic Azad University,
Kerman branch.
CONFLICT OF INTERESTS
There is not any conflict of interest among the authors.
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	Biodegradation of crude oil using symbiont crude-oil degrading bacteria isolated from corals collected at the Persian Gulf
	INTRODUCTION
	MATERIALS AND METHODS
	Sampling
	Quantification of the number of cultivable bacteria
	Isolation and selection of crude-oil degrading bacteria
	Screening of crude-oil degrading bacteria using the redox application of 2,6-Dichlorophenolindophenol (DCPIP)
	Identification of the isolates
	Bacterial growth rate
	Estimation of crude oil degradation by isolated strains
	The measure of emulsification activity and biosurfactant production by efficient isolates
	Effect of the concentration of crude oil on degradation by isolated strains
	RESULTS
	Coral identification
	Measurement of the amount of both types of bacteria in coral samples according to CFU and MPN
	Screening of crude-oil degrading bacteria from coral samples
	Identification of bacteria
	Crude oil biodegradation by selected strains
	Ability of degradation strains in different concentrations of crude oil
	Ability to degrade n-alkanes isolates
	DISCUSSION
	CONCLUSIONS
	ACKNOWLEDGEMENTS
	CONFLICT OF INTERESTS
	REFERENCES