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Hanafy Et Al, 2016
Hanafy Et Al, 2016
Amr Abd-EL Mooti EL Hanafy, Yasir Anwar, Saleh Ahmed Mohamed, Saleh
Mohamed Saleh Al-Garni, Jamal Sabir Mohamed Sabir, Osama Abdullah
AbuZinadah, Hussein Al Mehdar, Abdul Wahid Alfaidi & Mohamed Morsi
Mohamed Ahmed
To cite this article: Amr Abd-EL Mooti EL Hanafy, Yasir Anwar, Saleh Ahmed Mohamed,
Saleh Mohamed Saleh Al-Garni, Jamal Sabir Mohamed Sabir, Osama Abdullah AbuZinadah,
Hussein Al Mehdar, Abdul Wahid Alfaidi & Mohamed Morsi Mohamed Ahmed (2016) Isolation
and identification of bacterial consortia responsible for degrading oil spills from the coastal
area of Yanbu, Saudi Arabia, Biotechnology & Biotechnological Equipment, 30:1, 69-74, DOI:
10.1080/13102818.2015.1086282
2015 The Author(s). Published by Taylor & Published online: 30 Oct 2015.
Francis.
Download by: [Cinvestav Del Ipn] Date: 24 July 2017, At: 12:14
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT, 2016
VOL. 30, NO. 1, 69 74
http://dx.doi.org/10.1080/13102818.2015.1086282
spectrometry (GC MS). GC MS analysis shows that Aci- 1% (w/v) of crude oil obtained from Aramco Renery
netobacter baumannii and Pseudomonas spp. can and cultivated at 30 C and 180 r/min for two weeks.
degrade more than 50% of hydrocarbons present in the After incubation, 1 mL of the suspension was taken from
environment.[12,13] each ask and the OD was checked. Samples with OD
In Saudi Arabia, the petroleum sector accounts for over 1.0 at 660 nm were selected for further study.[14,15]
roughly 45% of the budget revenues, 55% of the gross
domestic product (GDP) and 90% of the export earnings
Microscopy
according the 2014 statistics. On the other hand, Saudi
Arabia has an extended coastline on the Arabian Gulf Scanning electron microscope (Hitachi S 5200) was used
(also known as Persian Gulf) and the Red Sea, where most to characterize microbes morphologically. Samples were
oil and petrochemical products are exported, viz. Ras prepared and observed under the microscope for the
Tanura and Yanbu, with continuously increasing risk of presence or absence of agella.[16]
water and soil pollution in these areas. This adds to the
serious environmental damage that Saudi Arabia experi-
Analysis of 16S rRNA
enced in the 1991 Gulf War. As seaoor animals and
plants lie at the basis of the food chain, any damage done GeneJET Genomic DNA Purication Kit (Thermo Scientic)
to the shoreline will ultimately affect the whole shallow- was used for extraction. Genomic DNA was extracted
water ecosystem. Considering all this, it became very according to the manufacturers instructions. Part of each
important to adopt an effective and economic approach extracted DNA sample was used for 16S rDNA amplica-
to overcome such highly probable contamination. tion. The 16S rDNA was amplied using 16F27 and
The aim of this work was to isolate, screen and iden- 16R1525 primers.[17] The polymerase chain reaction (PCR)
tify bacterial strains from the coastal area of Yanbu programme was set at 30 cycles and the amplication
(Saudi Arabia) with potent ability to utilize and degrade was carried out as follows: 92 C for 2 min, 42 C for 30 s,
hazardous compounds resulting from either oil leaks 72 C for 4 min and 4 C incubation at the end of the last
and accidental spills or petrochemical industry waste cycle.[18,19] The amplied fragments were sequenced by
contamination. This area was chosen because it plays an Macrogen (Seoul, South Korea) and a phylogenetic tree
important role in the crude-oil export and petrochemi- was constructed using MEGA version 4.[20,21]
cals industry in Saudi Arabia.
Measurement of degradation ratio of long-chain
Materials and methods hydrocarbons
Gas chromatography mass spectrometry Figure 1. Bacterial isolate S5 from Yanbu soil on nutrient agar
plate (a); Gram staining of isolate S5 with high degradation rate
Metabolic end-products of crude oil degradation by S5 (b); SEM images of isolate S5 at 500 magnication (c) and
(Pseudomonas sp.) culture were determined by GC MS 5000 magnication (d).
(Shimadzu GC-Q2010, Japan) ame ionization detector
(FID) as described in [24]. After two weeks of culture, oil bacterial strains were isolated from the Yanbu region.
was extracted from the liquid medium. Culture was Four of them were selected for further studies, among
done in BHM at 30 C and 180 r/min for two weeks and which isolate S5 showed high degradation activity. An
the remaining oil after degradation was extracted using electron micrograph (SEM) of Pseudomonas sp. S5 is
a separating funnel in the presence of dichloromethane shown in Figure 1. The procedures for its isolation and
(analytical grade). Magnesium sulphate was used in characterization are discussed in greater detail in the fol-
order to remove the remaining moisture. The pro- lowing subsections.
gramme set for GC MS was 60 C for 2 min, then the
temperature was increased by 6 C until it reached
16S rRNA gene sequence analysis
300 C for 15 min. The operating temperature for the
injector was 300 C and for the detector, 320 C. Nitro- Twenty-three bacterial strains were isolated from enrich-
gen was used as a carrier gas. No internal standards ment cultures that were maintained at 30 C for one
were employed. The total time for one GC run was less week. Four isolates that showed higher growth rates on
than 30 min. crude oil were selected for further study. They were des-
ignated as S3, S4, S5 and 4b. After sequencing of the 16S
rRNA gene, data were submitted to the genetic
Results and discussion
sequence database at the National Center for Biotech-
Microorganisms that display remarkable catabolic activi- nology Information (NCBI). The GenBank ID (http://www.
ties are omnipresent and it is unarguably clear now that ncbi.nlm.nih.gov) of these strains are KP262042,
microorganisms can inhabit polluted sites and expedite KP262043, KP262044 and KP262045, respectively. They
the degradation of crude oil that is polluting the soil. were molecularly identied as Pseudomonas aeruginosa
Crude-oil contaminated soils not only affect human (KP262042 and KP262043), Pseudomonas sp. (KP262044)
health but also take a toll on marine life, animals, birds and Nitratireductor sp. (KP262045). The phylogenetic tree
and crops, as well as on naturally occurring desert plants of these four 16S rRNA gene sequences (KP262042,
and the environmental quality as a whole. KP262043, KP262044 and KP262045) was constructed
This study was carried out to screen for microorgan- against 16 different P. aeruginosa, Pseudomonas sp. and
isms in the soil that could potentially utilize crude oil as Nitratireductor sp. from GenBank [20,21] as shown in
a sole carbon and energy source, and can thus be used Figure 2. The optimal tree with a branch length sum of
as potential entities for bioremediation of petroleum- 47.28 is shown. The data showed that the 16S rRNA
contaminated soil breaking down hydrocarbons into car- gene sequences of the three Pseudomonas species lie in
bon dioxide, water and humus.[25,26] In our study, 23 the same cluster with many other P. aeruginosa and
72 A. A.-EL M. EL HANAFY ET AL.
Figure 2. Comparative phylogenetic analysis of the P. aeruginosa (KP262042 and KP262043), Pseudomonas sp. (KP262044) and Nitratire-
ductor sp. (KP262045) isolates with different Pseudomonas and Nitratireductor strains from GenBank.
Pseudomonas sp. strains from GenBank, reecting high aeruginosa), S4 (P. aeruginosa), S5 (Pseudomonas sp.) and
genetic similarity with these strains. On the other hand, 4b (Nitratireductor sp.) showed highest levels of crude-oil
the Nitratireductor sp. isolate (KP262045) was located biodegradation (Table 1), degrading 66%, 65%, 95% and
some distance away from the three other strains, in 70% of the oil, respectively. Strain S5 (Pseudomonas sp.)
another cluster. Similar studies have been conducted by exhibited the highest levels of crude-oil removal (95%)
different scientists to identify oil-degrading bacteria. among all isolates. In a similar study, Leahy et al. [32]
[27 29] determined the hydrocarbon-degrading abilities of bac-
teria spectrophotometrically. Hydrocarbon-degrading
percentages were presented by initial and nal measure-
Hydrocarbon-degrading bacteria screening
ments at 400 nm wavelength. The petroleum-degrading
There are three indicators that conrmed the ability of kinetics of isolated bacteria were spectrophotometrically
these bacteria to biodegrade crude oil: (i) the change in measured at 540 nm.
colour of the culture media from blue to colourless in
the DCPIP test, (ii) the disappearance of crude oil from Crude oil GC MS analysis
the culture media and (iii) the appearance of bacterial As a next step in our study, the growth medium of the
colonies at the bottom of the culture medium. In the most potent oil-degrading isolate (S5) was analyzed by
DCPIP test, the mechanism of biodegradation of crude GC MS (Figure 3). The GC MS proles of the metabolic
oil is evaluated by the introduction of an electron accep- intermediates produced during degradation of crude oil
tor such as DCPIP to the culture medium. In this colouri- by this isolate are shown in Figure 3(b). The major peak
metric technique, the ability of bacterial strains to compounds at retention times of 9.205, 9.925, 10.595,
degrade crude oil is assayed by observing the change in 11.230, 11.825, 12.388, 12.754 and 12.925 min were iden-
DCPIP colour from blue (oxidized) to colourless tied based on a standard compound library as tridecane,
(reduced).[11] Similar works have reported that bacterial hexadecane, heptadecane, tetracosane, heptacosane,
cultures can completely reduce DCPIP at 75, 87, 125 and octacosane, nonacosane and hexatriacontane as shown
138 h for mineral oil, used oil, semi-synthetic oil and syn- in Figure 3(a). These compounds were present in the
thetic oil, respectively.[30] Mariano et al. [31] performed crude oil without biodegradation (control) but in the pres-
the DCPIP assay in order to conrm that diesel oil could ence of isolate S5 all of the compounds were degraded
be degraded by bacterial consortia. Our isolates caused except for hexatriacontane, which was about 25%
a colour change from blue to colourless, indicating that degraded as compared to the control.
they have the ability to degrade crude oil.
Table 1. Growth characteristics and crude oil removal by the
studied bacterial isolates after one week of incubation.
Growth rate and crude-oil removal by the studied
OD Temperature pH Oil degradation
strains Isolates (600 nm) range ( C) range (%)
All bacterial strains were grown in 1% crude oil for one S3 0.56 25 37 6 8 66
week with shaking. After one week, the levels of micro- S4 0.52 25 37 6 8 65
S5 0.93 25 37 6 8 95
bial growth and crude oil biodegradation were analyzed 4b 0.69 25 40 6 10 70
using spectrometry-based methods. Strains S3 (P.
BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 73
Figure 3. GC MS analysis of biodegradation of crude oil (at 30 C, two weeks of incubation) without microorganism (a) and using iso-
late S5 (b). Note: Red stars indicate polyaromatic hydrocarbons (PAHs).
Nearly all the hydrocarbons present in crude oil that a high price. Crude oil spills not only affect the natural envi-
were detected using GC MS analysis were effectively ronment but also pose severe danger to human health.
degraded by the isolate from Yanbu region. Thus, the This highlights the great potential of crude-oil-degrading
results obtained spectrophotometrically were corrobo- bacteria in bioremediation. This study conrmed the pres-
rated by GC MS analysis. The isolate was able to utilize ence of crude-oil-degrading bacteria in Yanbu soil showing
hydrocarbons such as tridecane, hexadecane, heptade- over 90% efcacy of degradation. Of all the isolates, Pseu-
cane, tetracosane, heptacosane, octacosane and nonaco- domonas sp. S5, which showed the highest oil-degrading
sane.[33] The only hydrocarbon that was slightly degraded ability, can possibly be used for bioremediation purposes.
was hexatriacontane, which remained detectable in the This would expedite the process of remediation of the
crude oil after degradation by the bacterial isolates.[34] environment in Saudi Arabia from oil pollution.
Similar GC MS analysis has been performed after degra-
dation of oil by A. baumannii.[12] In another work, isolated Acknowlegments
bacterial organisms were able to reduce at least 50% of This work was supported by the NSTIP strategic technologies pro-
the relative abundance of various hydrocarbons present in gramme in the Kingdom of Saudi Arabia [Project code 12-ENV
crude oil compared to control as evaluated by GC MS 2715-3]. The authors also acknowledge assistance from the Science
analysis.[13] Future research should aim at identication & Technology Unit, Deanship of Scientic Research and Deanship
of Graduate Studies, King Abdulaziz University, Jeddah, KSA.
and study of other groups of microorganisms that can act
on long-chain hydrocarbons, such as soil fungi and actino- Disclosure statement
mycetes, in the Yanbu area.
No potential conict of interest was reported by the authors.
Conclusions
Saudi Arabia is one of the biggest oil exporters in the world Funding
and most crude oil is exported through Yanbu. This signi- NSTIP strategic technologies programme in the Kingdom of
es the importance of the city as an economic hub but at Saudi Arabia: Project code 12-ENV 2715-3.
74 A. A.-EL M. EL HANAFY ET AL.