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Available online at: www.mbai.org.in doi:10.6024/jmbai.2024.66.1.2455-14
J. Mar. Biol. Ass. India, 66 (1), January-June 2024 89
Abstract
Biosurfactant-producing microorganisms show potential for
bioremediation of oil from soil and water surfaces. These
hydrocarbonoclastic microorganisms release biosurfactants, which play
a significant role in the bioremediation of oil. The present study focuses
on various isolation and screening strategies for biosurfactant-
producing organisms from oil-polluted sites. A total of eight isolates
showing morphological variations were isolated and screened for
biosurfactant production using various tests like Hemolysis assay, Drop
collapse assay, Oil displacement assay, Microtiter plate assay,
Emulsification index assay, and Phenol: Sulphuric acid assay. The potent
biosurfactant producer was identified as
Bacillus halosaccharovorans
with 16s rRNA sequencing. FTIR analysis was performed on the extracted
biosurfactant. To our knowledge, this paper is the first to demonstrate
that
Bacillus halosaccharovorans
is a biosurfactant producer.
Keywords: Biosurfactant, emulsification index, Bacillus
halosaccharovorans, FTIR, marine ecosystem
Introduction
Oil spills exhibit a great threat to marine flora and fauna due to their
inherent chemical composition as well as their ability to obstruct
sunlight penetrating the oceans. It can coat the wings of birds
and render them unable to fly. Even the small concentrations of
Poly aromatic hydrocarbons (PAHs) can have sub-lethal to lethal
damage to marine biology. It can also lead to oxygen deficiency
within the aected marine water (Amir-Heidari
et al.,
2019). In
humans, it can lead to imbalances in the endocrine system, and
respiratory malfunction and may lead to kidney, skin, lung, liver,
or bladder cancer (Deosthali
et al.
, 2021). Barron
et al.,
2020 have
compiled the consequences of the most significant worldwide oil
spills in the past three decades, namely the Exxon Valdez oil spill,
the Hebei Spirit oil spill, and the Deepwater Horizon oil spill. The
Biosurfactant producing bacteria,
Bacillus
halosaccharovorans,
from a marine
ecosystem
Chandan Deosthali1, P. Shete1, N. Patil1 and A. Jain1*
1Smt. Chandibai Himathmal Mansukhani College, Ulhasnagar-421 003, Maharashtra, India.
*Correspondence email: ajcdri@gmail.com
Received: 30 Oct 2023 Revised: 01 Mar 2024
Accepted: 05 Mar 2024 Published: 09 May 2024 Original Article
number of aected biological species speaks of the grave danger
an oil spill poses to the marine environment (Barron
et al.,
2020).
Strategies like the use of booms, adsorbents, skimmers, chemical
dispersants, and in-situ burning are being employed to tackle
the oil spills. However, each of these strategies comes with its
disadvantages and thus the last resort becomes bioremediation
(Dave and Ghaly, 2011; Al-Majed
et al.,
2012; Akpor
et al.,
2014).
Bioremediation by the means of microorganisms enables
the eco-friendly pathway for the elimination of pollutants.
Hydrocarbonoclastic bacteria (a heterogeneous group of
prokaryotes, which can degrade and utilize hydrocarbon
compounds as a source of carbon and energy) produce
amphiphilic molecules called biosurfactants. These biosurfactants
facilitate the bioavailability of oil to bacteria by reducing the
interfacial tension between oil and water surfaces (Sajadi
et al.,
2022).
Biosurfactants have a wide array of applications, other than
oil spill bioremediation. Its antioxidant, antimicrobial, and
wound-healing abilities make it a suitable candidate for the
pharmaceutical industry. The ability to increase the bioavailability
of trace metals and growth-promoting properties has paved its
path in the agricultural industry. The property to reduce surface
tension has enabled biosurfactants to compete with commercially
available chemical surfactants, thus having potential applications
in the detergent and cleaning industry (Bouassida
et al.,
2017).
Material and methods
Sample collection and Storage
Samples were collected at multiple sites, resulting in a total
of five samples contaminated with oil in both water and soil.
Journal of the Marine Biological Association of India Vol. 66, No.1, Jan-Jun 2024
Chandan Deosthali
et al.
90
These samples were collected in sterile containers and were
transported to the lab in an ice bath. The samples were stored
at 4 ℃ till further processing.
Media: Media used for enrichment and isolation of
biosurfactant-producing bacteria is Bushnell and Hass broth/
agar (HiMedia). It is a preferable medium for the growth of
marine microorganisms due to its salt composition and it gives
freedom for the selection of carbon source. It was modified by
adding 3% NaCl for selective isolation of halotolerant bacteria.
Blood required for blood agar plates was purchased from
Central Hospital Blood Bank, Ulhasnagar. All the chemicals
required were purchased from Loba Chemie Pvt. Ltd. and
were of analytical grade.
Enrichment and Isolation: The enrichment of samples
was carried out in three stages. In the first stage, 1g of soil,
1 ml of liquid sample, and 1 ml of suspension (for sample 4)
were inoculated in 100 ml sterile MBHB broths containing
0.5% of phenol as carbon source and were incubated at
room temperature on an orbital shaker for seven days. In
the second stage, 1 ml of inoculum from the 1st stage flasks
were inoculated in sterile MBHB containing 1% phenol as a
carbon source. In the last stage, the 1 ml of inoculum from the
second stage was inoculated in sterile MBHB with 1% spent
ship oil (SSO) as a carbon source and was incubated at room
temperature for seven days on an orbital shaker. For isolation,
sterile MBHA plates with 1% SSO were used. A loopful of broth
was streaked on the plates and they were incubated at room
temperature for 72 hours (Ezebuiro
et al.,
2019).
Retrieval of Cell-Free Supernatant (CFS): The isolates
obtained in pure form were inoculated in sterile MBHB with
1% SSO and were allowed to grow at room temperature for
72 hours in an orbital shaker. The cell mass was separated
by centrifuging at growth media at 6000 rpm at 4℃ for 15
mins. The resulting CFS was used to screen the biosurfactant
production (Abbasi
et al.,
2012).
Screening of Biosurfactant producing microorganisms:
Hemolysis assay was done through a loopful of pure culture from
MBHB was streaked onto blood agar plates. After incubating
at room temperature for 24 hours, plates were examined for
hemolysis patterns. (Ogunshe and Falode, 2021). For the phenol
sulphuric assay, 1 ml of 5% phenol was mixed with 1 ml of CFS
and vortexed thoroughly. This was followed by the drop-wise
addition of conc. H2SO4 along the side of the tube. The formation
of an orange colour indicated a positive response (Suresh
et al.,
2021). For the microtiter plate assay, 100 µL of CFS was added
to wells of a 96-well microtiter plate. A graph paper was kept
under the plate and was observed for distortion of grid lines.
Distilled water in a hydrophobic well exhibits a flat surface.
If biosurfactant is present then the image of the grid will be
distorted due to the concave surface (Saruni
et al.,
2019). For the
drop collapse assay, an oil film was applied to a glass slide to
create a hydrophobic surface. A drop of CFS was placed on the
slide and it was checked whether it remained in bead shape or
collapsed. Loss of shape or flattening of drop indicates a positive
response (Sohail and Jamil, 2019). For the oil displacement assay,
a Petri plate was prepared for this assay by filling it with 20 ml
of distilled water. It was overlaid with 15 µL of oil. 10 µL of CFS
was placed on the oil surface. The formation of a clear zone
resulting from the spread of oil by reduced surface tension
indicated a positive test for biosurfactant (Kurniati
et al.,
2019).
For the Emulsification Index Assay, 2.5 ml of CFS was added
to a tube having an equal volume of SSO and was vortexed
vigorously for 3 minutes. It was left undisturbed for 24 hours at
room temperature. The emulsification index was determined as
a percentage by dividing the height of the emulsified layer by
the height of the liquid column and multiplying it by a hundred
(Zargar
et al.,
2022).
Identification of potent biosurfactant producer: For the
biochemical test, gram nature of potent isolate was identified
and accordingly, standard biochemical tests were carried
out. We utilized Bergey’s manual of systematic bacteriology
to compile and identif y the genus of the organism based
on the obtained results (Singh
et al.,
2016). For 16s rRNA
sequencing, the potent isolate selected was identified by 16s
rRNA sequencing and a phylogenetic tree was generated
(Deosthali and Jain, 2022).
Extraction of crude biosurfactant: The potent isolate was
inoculated in sterile MBHB with 1% SSO and was incubated
on an orbital shaker at ambient temperature for a week. Post
incubation, the cell-free supernatant (CFS) was acquired
through centrifugation of the complete culture medium at
6000 rpm and 4℃ for 15 minutes. Then the CFS was acidified
using 6N HCL till a pH of 2.0 was achieved. It was kept at 4℃
for 24 hours to facilitate maximum precipitation. The pellet was
obtained by centrifuging the content at 4℃, 12500 rpm for 20
mins. The pellet was resuspended in distilled water and the pH
was raised to 7.0 with the help of 1N NaOH. We performed an
extraction on this suspension using methanol. The methanol
portion was transferred to a beaker and left to evaporate at
Table 1. Geographic coordinates of the sampling sites
No. Sampling site Geographical coordinates Sample type
1 HP Petrol Pump 19° 10’ 15.672’’ N 7 3° 4’ 54.696’’ E Soil
2 Indian Oil Petrol Pump 19° 8’ 21. 732’’ N 73° 3’ 0.684’’ E Soil
3 Reti Bunder, Dombivli 19° 13’ 45.0876’’ N 73° 4’ 4.9944’’ E Water
4Below New Kon Bridge,
Kalyan 19° 14’ 45.0384’’ N 73° 7’ 3.7164’’ E Water + Soil
5 Mumbai High 19° 25’ 0.012’’ N 71° 19’ 59.988’’ E Water
© Marine Biological Association of India
Biosurfactant producing marine bacteria
91
37℃. A second round of methanol extraction was carried out,
resulting in the isolation of crude biosurfactants (Das
et al.,
2008). For obtaining the FTIR of crude biosurfactant, 2 mg
of crude biosurfactant was mixed with 20 mg KBr pellet. The
FTIR was performed from a range of 400 to 4000/cm using
IR Prestige-21 (Shimadzu) (Devaraj
et al.,
2019).
Results
From all five dierent samples, eight morphologically dierent
isolates were obtained on MBHA with 1% SSO. These isolates
were subjected to screening methods as described above.
For all the tests a positive control as well as negative control
was maintained with Triton X-100 (1 mg/mL) and Distilled
water respectively. CD08A, CD011A, and CD11C showed a beta
hemolysis pattern whereas other isolates showed an alpha
hemolysis pattern. All the isolates showed a positive reaction
for Phenol: Sulphuric acid test by forming an orange-coloured
product. The Microtiter plate assay relies on biosurfactants’
capacity to decrease surface tension, causing deformation
of the grid positioned beneath it. All the isolates showed a
distortion eect when compared with distilled water. The
CFS containing biosurfactant reduces the surface tension
between oil and water interface enabling the drop to spread
more than that of negative control. In drop collapse assay, the
CFS of CD11C showed the largest diameter of 5 mm which
was 1 mm larger than positive control. The rest of the isolates
showed a diameter within a range of 2-3 mm with negative
control showing a drop diameter of 1 mm. The formation of the
clear zone on the oil surface in the oil displacement assay is
scored as shown in Table 2. CD11C showed the highest score
for oil displacement followed by CD08A and CD11A. Triton
X-100 showed an emulsification index of 38.46%. CD12A and
CD09A showed an emulsification index of less than 40%
whereas the rest of the isolates showed an emulsification
index of more than 40% with CD11C having an E24 of 44.11%.
We identified CD11C as a promising candidate and conducted a
series of biochemical tests on it. The results of the biochemical
tests are mentioned in Table 3. The results concluded that the
organism belonged to the
Bacillus
genus. The results of 16s
rRNA sequencing were compared with the present sequence
database using NCBI BLAST and a phylogenetic tree was also
generated (Fig 1). The isolate CD11C showed 98.17% similarity with
B. halosaccharovorans
. The 16s rRNA sequence was deposited
in NCBI GenBank under the accession number OL989233.
The partially purified biosurfactant (Fig. 2) was subjected to
FTIR analysis (Fig. 3). A broad peak can be observed in the
FTIR graph of CD11C biosurfactant within the range 3000–3500
cm-1, which can be the result of alcohol (-OH) group. Two
sharp peaks within the range 2850 – 3000/cm, indicate the
presence of sp3 C-H stretching. Two sharp peaks at 1350/cm
and 1362/cm indicate the presence of the nitro group in the
compound. A sharp peak at 1126/cm indicates the presence
of the alkoxy (C-O) group. A group of peaks between 980 and
1300 resemble the chemical structures of rhamnose rings
(Bertuso
et al.
, 2022).
Discussion
Studies reflect that around two million tons of oil are introduced
into the aquatic environment per year due to activities carried
out in the sea (Zahed
et al.,
2022). The current strategies
employed for the removal of oil use booms, skimmers, and
chemical dispersants which are ok to be used in deep water.
Table 2. Results for DCT, ODT, and E24 test
Samples Drop Collapse Test Oil Displacement
Te st
Emulsification Index
Assay
CD08A 3 mm + + + + 43.7 5 %
CD08B 2 mm + 43.7 5 %
CD09A 2 mm + 39.39 %
CD10A 3 mm + 4 3.75 %
CD11A 3 mm + + + 42.30 %
CD11B 2 mm + 41.93 %
CD11C 5 mm + + + + + 44.11 %
CD12A 2.5 mm + 35.71 %
NEG CNTRL 1 mm - 0.0 %
POS CNTRL 4 mm + + + + + 38.46 %
Table 3. Results from the Biochemical Test
Tes t Result
Gram Stain Gram Positive
Motility +
Indole -
MR -
VP +
Citrate -
Nitrate Reduction -
Catalase +
Oxidase +
Urease -
LDC +/-
Glucose +
Fructose +
Sucrose +
Lactose +
Xylose +
Mannitol +
Journal of the Marine Biological Association of India Vol. 66, No.1, Jan-Jun 2024
Chandan Deosthali
et al.
92
The use of chemical dispersants is not recommended near
the shore water as it may damage the local flora and fauna
living in shallow waters. Chemical fertilizers were used near
the shoreline during two of the major oil spills
i.e
. in the
Gulf of Mexico-Deepwater Horizon oil spill and in the Gulf of
Alaska-Exxon Valdez oil spill. However, these chemical fertilizers
just disperse the oil and make it available to the local oil-
degrading bacterial community and do not enhance the rate
of its degradation (Atlas and Hazen, 2011). 3.1.6 of Policy and
Guidelines For the use of Oil Spill Dispersants (OSD) In Indian
Waters, published in NOS-DCP (2009) prepared by the Indian
Coast Guard dictates that no oil dispersant shall be used in
shallow water, protected bays, and inlets. However, no direct
application of oil-degrading bacteria has been reported yet.
The potential threats of oil spills on marine biodiversity urge
the scientific community to come up with faster and more eco-
friendly bioremediation solutions. The objective of this research
was to recover bacteria capable of producing biosurfactants and
degrading oil from sites contaminated with petroleum. We aimed
to assess their potential application in the field of bioremediation
technology. Oil-contaminated sites provide a selective strategy
to isolate hydrocarbonoclastic microorganisms which have
the required genetic material to process oil as their carbon
source (Adeleye
et al.,
2022). Harikrishnan
et al.
(2021) isolated
six morphologically dierent isolates on oil agar plates from
oil-contaminated soil samples in Karaikal ONGC, Puducherry,
India (Harikrishnan
et al.,
2021). Jagtap
et al.
(2021) were able
to isolate obligate hydrocarbonoclastic organisms from oil-
contaminated water sites in the Arabian Sea (Jagtap
et al.
,
2021). Devi and Jha successfully isolated 36 oil-degrading
bacteria from refinery sludge samples of an oil refinery (Devi
and Jha, 2020). Thus, it can be implemented that even though
biosurfactant-producing organisms are ubiquitous, the oil-
contaminated sites provide a reliable source to isolate potent
hydrocarbonoclastic, biosurfactant-producing bacteria. The
biosurfactant-producing
Bacillus
species have been reported
before from various sources including soil, rhizosphere, and
marine ecosystems. Bartal
et al.
(2023), isolated dierent
Bacillus
species capable of producing surfactin-like biosurfactants
from the rhizosphere. This includes,
B. cereus,
B. atrophaeus
,
B. subtilis
,
B. pumilis
and
B. velezensis
.
Bacillus licheniformis
Ali5 was reported to remove around 70-79% of motor oil from
sand with the help of biosurfactant produced by it. It resulted
in an additional 32% oil recovery when the biosurfactant was
applied to a sand-packed column (Ali
et al.
, 2019).
Bacillus
cereus
was isolated from marine seawater and showed 96%
degradation of motor oil within 27 days and the biosurfactant
generated exhibited stability across a broad pH spectrum,
temperature, and salinity (Durval
et al.
, 2019). The antifouling
property of biosurfactant extracted from
B. niabensis
was
studied against phytoplanktonic cells and biofilm-forming
bacteria like
Bacillus subtilis
,
Micrococcus
sp.,
Sagittula stellata
and
P. stutzeri
(Alemán-Vega
et al.
, 2020; Sánchez-Lozano
et al.
,
2023). However, the study on the biosurfactant-producing
capabilities of organisms closely related to CD11C which
are
B. simplex
and
B. niabensis
are not well studied for their
bioremediation potential and to the best of our knowledge this
is the first paper shedding light on biosurfactant production
by
B. halosaccharovorans
.
Conclusion
The successful isolation of hydrocarbonoclastic bacteria
confirms the hypothesis that oil-contaminated soil and water
are potential sites for isolating biosurfactant-producing and
oil-degrading bacteria. The bacteria isolated were screened
Fig. 1. Phylogenetic tree for CD11C
Fig. 2. Partially purified biosurfactant
Fig. 3. FTIR analysis of CD11C Biosurfactant
© Marine Biological Association of India
Biosurfactant producing marine bacteria
93
for their biosurfactant-producing ability and the most
promising isolate was identified as
B. halosaccharovorans
using 16s rRNA sequencing. The revelation of rhamnose
rings in the FTIR graph of a partially purified biosurfactant
isolated from CD11C indicates that the biosurfactant produced
might be a rhamnolipid. To the best of our knowledge, no
previous studies have reported the biosurfactant production
by
B. halosaccharovorans
. However, the application of
biosurfactants as degreasing agents on dierent surfaces
and the use of oil-degrading bacteria on the field via spray
application needs to be investigated further. Also, a study
needs to be conducted to investigate and enhance the
bioremediation eectiveness.
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