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Title: Production and applications of lipopeptide biosurfactant

for bioremediation and oil recovery by Bacillus subtilis
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Author: Fisseha Andualem Bezza Evans M. Nkhalambayausi
Chirwa
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http://dx.doi.org/doi:10.1016/j.bej.2015.05.007
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Production and Applications of Lipopeptide Biosurfactant for

Bioremediation and Oil Recovery by Bacillus subtilis CN2.
FissehaAndualem Bezza1, Evans M. Nkhalambayausi Chirwa1*
1

Water Utilization and Environmental Engineering Division, Department of Chemical Engineering,

University of Pretoria, Pretoria 0002, Republic of South Africa. *Tel: +27-12-420 5894,
Email: Evans.Chirwa@up.ac.za
*

Corresponding author. Tel.: +27 12 420 5894.

Graphical abstract
fx1
Highlights

Lipopeptide biosurfactant was produced by Bacillus subtilis CN2 strain.

The biosurfactant increased the degradation of PAHs in the motor oil by more than
100%.
The biosurfactant recovered up to 84.6% of motor oil adsorbed to a sand sample.
The biosurfactant showed stability at extreme temperatures, pH and salinity.

Abstract
Petroleum compounds are recalcitrant to microbial degradation and persist in ecosystems
because of their high hydrophobicity. Lipopeptide biosurfactant was produced by
hydrocarbon degrading and biosurfactant producing strain isolated from creosote
contaminated soil. The isolate CN2 was identified as Bacillus subtilis using 16S rRNA gene
sequencing. In a used motor oil degradation assay carried out in an Erlenmeyer flask using
microbial consortium, the biosurfactant enhanced the degradation of used motor oil
polycyclic aromatic hydrocarbon (PAH) components up to 82 % in 18 days of incubation,
more than twofold compared to the degradation with no biosurfactant supplementation. The
biosurfactant was tested for its oil recovery potential and it recovered 85% of used motor oil
from contaminated sand in 24h. Tandem mass spectrometry (LC-MS/MS), Thin-layer
1

chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) analysis showed
that the biosurfactant produced is lipopeptidal of molecular mass 1472Da. The lipopeptide
showed emulsifying activity against a variety of hydrocarbons and attained emulsion indices
of 81 and 84% with Hexane and cyclohexane respectively. The lipopeptide showed stability
over a temperaturerange of (25125C), pH range of ( 512) and salinityrange of (520 %,
w/v). The results reveal the potential of the biosurfactant for use in bioremediation of
hydrocarbon contaminated environmental media and washing of petroleum sludge
contaminated soil.
Key words: Bioremediation, Biosurfactant, Chromatography, Environmental Preservation,
Waste Treatment, PAHs.
1 Introduction
The release of contaminants, such as petroleum and its byproducts, into the environment is
one of the main causes of global pollution and has become a focus of great concern both in
industrialized and developing countries due to the broad environmental distribution in soil,
groundwater, and air [1]. The presence of different types of automobiles and machinery has
resulted in an increase in the use of lubricating oil. Spillage of used motor oils such as diesel
or jet fuel contaminates our natural environment with hydrocarbons [2].New motor oil
contains a higher percentage of fresh and lighter (often more volatile and water soluble)
hydrocarbons that would be more of a concern for acute toxicity to organisms. Used motor oil
contains more metals and heavy polycyclicaromatic hydrocarbons (PAHs) that would
contribute to chronic hazards includingmutagenicity and carcinogenicity [3]. PAHs represent
the group of compounds in oil that has received the greatest attention due to their
carcinogenic and mutagenic properties [4].Thousand million gallons of waste engine oil is
generated annually from mechanical workshops and discharged carelessly into the
2

environment [5, 6] According to USEPA (1996), only 1 litre of used engine oil is enough to
contaminate one million gallons of freshwater. Used engine oil also renders the environment
unsightly and constitutes a potential threat to humans, animals, and vegetation [7].
Compared to physico-chemical methods, biological methods have gained increased
acceptance in cleaning up the hydrocarbon contaminated sites because these are
environmentally friendly, cost-effective, and efficient [8].However, the general low
bioavailability of hydrocarbons, which are highly recalcitrant molecules that can persist in the
environment due to their hydrophobicity and low water solubility, is a hindrance to microbial
degradation[9,10]. Release of biosurfactants is one of the strategies used by microorganisms
to influence the uptake of PAHs and hydrophobic compounds in general [11].
Microbial surface active agents or biosurfactants are amphiphilic molecules consisting of
hydrophobic and hydrophilic moieties that tend to interact with surfaces of various polarities
and reduce surface and interfacial tensions[12].By reducing surface and interfacial tensions,
biosurfactants increase the solubility and area of contact of insoluble compounds, their
mobility, bioavailability and subsequent biodegradation [13].The hydrophobic portion of the
molecule is long-chain fatty acids, hydroxyl fatty acids or a-alkyl-b-hydroxyl fatty acids. The
hydrophilic moiety can be a carbohydrate, amino acid, cyclic peptide, phosphate, carboxylic
acid or alcohol[14]. Biosurfactants have several advantages over chemical surfactants such as
low toxicity, high biodegradability, better environmental compatibility, high foaming
capability, higher selectivity, specific activity at extreme temperature, pH, salinity, and the
ability to synthesize them from renewable food stocks [15,16]. Many types of
biosurfactantshave been synthesized from bacteria belonging to a wide variety of genera,
such as Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Bacillus, Rhodococcus
andEnterobacter[17].Biosurfactants are classified as glycolipids, lipopeptides, phospholipids,

fatty acids, neutral lipids, and polymeric or particulate compounds [18]. Lipopeptides are
among the most commonly isolated and characterized biosurfactants. Among the many
classes of biosurfactants, lipopeptides represent a class of microbial surfactants with
remarkable surface properties and biological activities, such as surplus crude oil recovery,
efficient removal of petroleum hydrocarbons andheavy metals from contaminated soils
[19].The bacterial genera, namelyBacillus, Pseudomonas and Arthrobacter are reported to
produce such lipopeptide biosurfactants [20]. The lipopeptides produced by numerous
Bacillus spp. are classified into three familiesdepending on their amino acids sequence:
surfactins, iturins and fengycins [19]. There are many reports describing the effect of
exogenously added microbial biosurfactants in enhancing the bioremediation of crude oilpolluted soils by indigenous microbes [21,22, 8]
The aim of this study is to isolate potent hydrocarbon-degrading and biosurfactant-producing
bacterial strain and to assess the abilities of the biosurfactant produced in assisting the
biodegradation of representative PAHs in motor oil and assess its potential application for
motor oil recovery from spiked sand. For the bioremediation purpose, potential hydrocarbon
degrading microbial consortia isolated from creosote contaminated soil using coal tar
creosote as a source of carbon and energy was used. B.subtilisCN2 was isolated as an
efficient degrader and biosurfactant producer. The lipopeptidebiosurfactant produced by the
isolate was characterized and its efficacy in enhancing motor oil biodegradation in liquid
culture was investigated.

2. Materials and Methods

2.1 Materials
Used motor oil was obtained from a local automobile garage. The used oil was collected in
well-washed pots and kept at 4 C before undergoing the experiment. All chemicals used
wereof analytical grade from Merck, USA.
2.2 Microorganisms
Coal tar creosote -contaminated soil was collected from a wood treatment plant in South
Africa. Microbial consortium was obtained via selective enrichment in 250 mL Erlenmeyer
flasks. Five grams of the contaminated soil sample was inoculated into to 100 mL of mineral
salt medium (MSM) accordingtoTrummler et al. [23]. Creosote 5 % (v/v) was used as a sole
carbon and energy source. Enrichment was conducted at 30 C and 120 rpm rotary shaker
and kept for about 7 days. After 7 days of incubation, 10 mL of enriched culture was
transferred into another flask containing 100 mL fresh sterile MSM with Creosote 5 % (v/v)
and incubated. A total of five subsequent enrichments was carried out to selectively isolate
the PAH degrading microbialconsortium.This microbial consortium wasmaintained in MSM
supplemented with 25% glycerol at 20C for further use in biodegradation experiments.
Isolation and purification of predominant bacterial isolates in the consortium was performed
through spread and streak plate techniques onLuria-Bertani (LB) agar. Aliquots from the
culture dilutions (100 L) were spread on the LBagar plates and incubated at 37C for 3 days.
On the basis of the colony morphology 13 bacterial isolates were obtained.The isolates were
subjected to 16S rRNA gene sequencing analysis.Strain B.subtilisCN2 wasselected due to its
effective biosurfactant productionpotential. Themineral salt medium (MSM) was composed
of 6.0 g L-1(NH4) 2SO4, 0.4 g L-1 MgSO4 x 7H2O, 0.4 g L-1 CaCl2 x 2 H2O, 7.59 g L-1
Na2HPO4 x 2 H2O, 4.43 g L-1 KH2PO4, and 2ml L-1 of trace element solution. The trace
5

element solution consisted of; 20.1 g L-1 EDTA(disodium salt), 16 g L-1 FeCl3 x 6 H2O, 0.18
g L-1 CoCl2 x 6 H2O, 0.18 g L-1 ZnSO4 x 7H2O, 0.16 g L-1 CuSO4x 5 H2O, and 0.10 g L-1
MnSO4 x H2O.
2.3 Identification of bacterial strain.
Genomic DNA was extracted from purified colonies according to the protocol described inthe
Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI, USA).
16SrRNA genes were amplified by a reverse transcriptase-polymerase chain reaction (RTPCR) using primers pA and pH1 (Primer pA corresponds to position 8-27; Primer pH to
position1541-1522 of the 16S gene under the following reaction conditions: 1 min at 94C,
30 cyclesof 30s at 94C, 1 min at 50C and 2 min at 72C, and a final extension step of 10
min at72C).
PCR fragments were then cloned into pGEM-T-easy (Promega) PromegaWizardGenomic
DNA Purification Kit (Version 12/2010). Partially sequenced amplified 16S rDNAfragment
was compared with other gene sequences in Gen Bank using(http://www.ncbi.nlm.nih.gov)
and aligned with gene sequence of our isolates. Thealigned sequences were used to construct
a distance matrix, after the generation of 1,000bootstrap sets that was subsequently used to
construct a phylogenetic tree using theneighbor-joining method MEGA version 6 software
[24].
2.4Screening methods for potential biosurfactant-producing bacteria
Biosurfactant-producing isolates were screened using the drop collapse method and the oil
spreading test.
In the drop collapse method, 2 L of mineral oil was added to each well of a 96-well micro
titer plate lid. The lid was equilibrated for 1 h at room temperature, and then 5 l of the
6

culture was added to the surface of oil [25]. The shape of the drop on the surface of oil was
inspected after 1 min.If the drop remainedbeaded; the result was scored as negative. If the
drop collapsed, the result was scored as positive. Cultures were tested in triplicate. Distilled
water and MSM without cells were used as negative controls
For the oil spreading test, 50 mL of distilled water was added to a large petri dish (25 cm
diameter) followed by the addition of 20 l of crude oil to the surface of the water. Ten
microliters of culture were then added to the surface of oil [26]. The diameter of the clear
zone on the oil surface was measured and related to the concentration of
biosurfactant.Cultures were tested in triplicate. Distilled water and MSM without cells were
used as negative controls
2.5Biosurfactant production and recovery byBacillus subtilis strain CN2
Strain CN2 was found out to be efficient biosurfactant producer after drop collapse and oil
spread test. The bacterial isolate CN2 was inoculated in 25mL sterile nutrient broth at
37Cshaking at 150 rpm in a rotary shaker Labcon SPL-MP 15 Orbital Shaker
(LabconLaboratory Services, South Africa) for 24 hours. Biosurfactant production by
B.subtilisCN2 was carried out in1000mL conical flasks containing 500 ml of MSM
(Trummler et al., 2003) supplemented with glycerol 4 % (v/v).Theflask was inoculatedwith 5
ml of the inoculum prepared overnight at 37 C and 150 rpm and pH 7 and incubated at
37C,pH 7and 150 rpm for 96h.
After growth for 96h the pH was adjusted to 7 , cells were removed by centrifugation (12,000
rpm at 4 C for 20 min) to obtain cell-free supernatants and crude biosurfactant was
precipitatedfrom the supernatant by adding 6 N HCl to pH of 2.0. The acid precipitate was
recovered bycentrifugation (12,000 rpm at 4 C for 20 min) and was further extracted with
chloroform andmethanol (2:1).The solvent was evaporated in a vacuum. The residue was
7

dissolved in methanol and filtered through a 0.22 mm filter (Millipore)..The crude extracts
were purified through a Silica gel column (silica gel 60 (63- 200 mesh); Merck KGaA).The
samples were eluted first with chloroform to remove yellow pigment and impurities of
hydrocarbons, then with a mixture of chloroform and methanol 80:20 v/v (100 mL), then
35:65 v/v (100 mL) collecting two fractions after discarding the first chloroform eluted
fraction. The fractions eluted were collected and the solvents were evaporatedin a rotary
evaporator at 40 C.Eluted fractions were characterized by thin layer chromatography (TLC)
2.6Characterization of the biosurfactant
2.6.1 Thin layer chromatography (TLC)
After the crude extracts were purified through Silica gel column (silica gel 60 (63- 200
mesh); Merck KGaA), 5mg of the extract was dissolved in methanol and analysed using thin
layer chromatography (TLC) on silica gel 60 plates (F254; Merck ).Chromatogramswere
developed with the solvent systemchloroform:methanol:water

(65:15:4,v/v).Spots were

revealed by spraying with: a) ninhydrin 0.35 % (w/v, in acetone) for detection of compounds
with free amino groups; b)p-anisaldehyde for detection of the sugar moieties,Prepared by
mixing 100 mL acetic acid, 2 mL of sulfuric acid and 1 mL of p-anisaldehyde.The reagents
were sprayed and the plates were heated at110C for 5 min until the appearance of the
respective colours [27]
2.6.2Fourier transform infrared spectroscopy (FTIR)
The chemical structure and components of the crude biosurfactant sample were determined
using Fourier transform infrared (FTIR) spectroscopy (Perkin Elmer 1600 FTIR) equipped
with an Attenuated Total Reflectance (ATR) Crystal Accessory (Perkin Elmer, Connecticut,
USA). In this test, 1 mgof crude biosurfactant was mixed with 100 mg of KBr and pressed
8

with load for 30 s, to obtain translucent pellets.The IR scan was performed over 4004000
cm-1 wavenumber range with a resolution of 2 cm-1. The reflectance spectra were recorded
and averaged over 32 scans, using the total internal reflectance configuration with a
HarrickTM Mvp-pro cell consisting of a diamond crystal. Spectra were viewed in OMNIC
software.
2.6.3 Mass spectrometric analysis
LC-MS/MS was performed using a Waters Acquity UPLC system coupled to a Waters
Synapt G2 mass spectrometer. Prior to analysis the MS was calibrated in both positive and
negative ion resolution mode over the mass range of 100 - 2000 Da typically using sodium
formate clusters. In this method, 5 L of extract was injected onto a Waters C18 BEH 1.7 m
(2.1 x 100 mm) column. The mobile phase consisted of A: Water (0.1 % formic acid), B:
Acetonitrile (0.1% formic acid) run with a 20-min gradient of (30 %, vol/vol Acetonitrile for
5 min; 30 100 %, vol/vol Acetonitrile for 8 min and 100 %, vol/vol Acetonitrile for 2 min;
100 30 %, vol/vol Acetonitrile for 1 min; 30 %, vol/vol Acetonitrile for 4 min ) at a flow
rate of 0.4 L/min. The eluent flowed directly into the ESI source. The MS instrument was
operated in MSE mode collecting both low energy (4V) precursor and high energy (ramp: 2040V) product spectra. A 2 ng/L solution of leucineenkephalinwas used as the lock mass
with a constant flow rate of 5 L/min. Mass spectra were attained in both positive and
negative ion mode using the following parameters: scan time of 0.5 seconds; cone voltage of
30 V, capillary voltage of 2.8 kV; source temperature 100C; desolvation temperature 300C;
cone gas 100 L/h; desolavtion gas 500 L/h.

2.7. Surface-activity determination

2.7.1 Surface tension
The surface tension of the culture broth supernatant and biosurfactant solution was measured
according to the Ring method as previously described [28]. Using a Du Nouy tensiometer
(KrssTensiometer, K11 model - Germany), equipped with a 1.9 cm platinum ring. Surface
tension values represent the average of three independent measurements performed at room
temperature (25C). The biosurfactant concentration expressed in terms of critical micelle
dilution (CMD) was estimated by measuring the surface tension for varying dilutions of the
sample. When measuring the CMD the cell free broth were diluted 10 times CMD-1 or 100
times CMD-2 with Phosphate buffer solution (10 mMKH2PO4/K2HPO4 and 150 m MNaCl
with pH adjusted to 7.0) and the surface tension of each sample was measured as described
above [16]. All measurements were done in triplicate.
2.7.2 Emulsification Activity
The cell-free broth of the culture supernatant was centrifuged and tested for its emulsifying
ability by a modified method [29]. Five millilitre of the hydrocarbons (hexane , cyclohexane,
used motor oil) was added to 5 mL of the Cell-free broth in a screw cap test tube and
vortexed at high speed for 2 min at room temperature and then incubated at 25C for 24h.
The emulsification capacity measured as an emulsification index (E24) was calculated by
dividing the measured height of the emulsion layer by the total height of the mixture and
multiplying by 100. All the experiments were carried out in triplicates and results were
reported as mean Standard errors.
2.7.3Stability assay

10

The stability studies were carried out with respect to the effect of temperature, pH, and
salinity on emulsification capacity and surface tension of the biosurfactant. The analysis was
done in triplicates using the 72 -hour cell-free culture broth (0.71% w/v) obtained by
centrifuging the culture sample at 12000rpm for 20 minutes. To determinethe thermal
stability of the biosurfactant, cell-freebroth was maintained at a constant temperature in the
range20125 C for 30 min and cooled at room temperature. Todetermine the effect of pH on
the activity, the pH of the biosurfactantwas adjusted at a value in the range 2.011.0.
Theeffect of NaCl on the activityof the biosurfactant was investigated by adding NaCl in
therange 520% (w/v). Ineach case, E24and surface tension was determined as described
above. Hexane was used as a representative hydrocarbon in the E24 stability studies [30].
2.8 Biodegradation of motor oil
2.8.1 . Microorganisms
The PAH-degrading microbial consortiumthat was enriched from creosote-contaminated soil
sample (described insection 2.2.) was used in the biodegradation experiments.16S rRNA gene
sequence analysis revealed that the culturable isolates were predominantly composed of
Bacillus subtilis, Paenibacillus dendritiformis, Pseudomonas aeruginosa , Ochrobactrum
intermedium. This population was subcultured fortnightly on Trummler et al.[23] MSM with
5% v/v used motor oil as asole source of carbon and energy for 4 weeks. To obtain
microbial inoculum, the consortium was precultured in MSM supplemented with 1.0 % (v/v)
of motor oil in flasks on a rotary shaker (with shaking at 180 r.p.m) at 35 C and pH 7 for 5
days. After 5 days, the culture broth was centrifugedand the pellets were resuspended in
MSM at OD

600

of 0.8 to obtain a highly concentrated microbial suspension for the

experiments.
2.8.2 Experimental setup
11

Shake flask biodegradation experiments were carried out in 250 mL Erlenmeyer flasks with
150 mL of sterilized MSM (pH 7) supplemented with 3% (v/v) of used motor oil. Sterilized
MSM was inoculated with 2% (v/v) microbialsuspension and theculture flasks were
incubated in an orbital shaker at 180 rpm and 35 C for3 weeks. Biodegradation experiments
were conducted in three different sets using the above MSM.
(i) Bacterial cells (2%v/v) + used motor oil (3% v/v) + MSM;
(ii) Bacterial cells (2%v/v) + usedmotor oil (3% v/v) + biosurfactant (50 mL cell free
supernatant of 96 hr culture , after diluting further with Phosphate buffer solution to obtain a
concentration of 0.15 % (w/v))+ MSM and
(iii) Uninoculated abiotic control ; used motor oil ( 3%,v/v) + MSM.
The flasks were incubated in an orbital shaker at 180 rpm, and 35 Cfor 3 weeks.All the
experiments were carried out in duplicates and results were reported as mean Standard
errors. Samples were withdrawn from each flask at a 4 day interval and aliquots of
appropriate dilutions were plated (in triplicates) onto nutrient agar for total viable counts
(TVC).Periodically, samples were extracted twice with hexane and analyzed on GC MS.
2.9 Gas chromatographic analysis of residual motor oil.
The contents of the whole flasks were extracted with hexane, dried over anhydrous sodium
sulfate, redissolved in hexane, and analyzed using a Clarus 600 T GC/MS (PerkinElmer,
Connecticut,USA) equipped with a PerkinElmer Elite 5MS capillary column (30 m 0. 25
mm ID 0. 5

fixed phase) with helium as a carrier gas. The oven temperature was kept

initially at 60C for 5 min, followed by an increase to 300C at a rate of 15 C/min.

Degradation of selected peaks was identified by searching for closest matches in theWiley
Mass Spectral Libraries. The MS was operated in the electron impact (EI) ionization mode to
12

determine the appropriate masses for the selected ion monitoring ranged from 100 to 600amu
(atom to mass unit). All samples were performed in duplicates and the results were expressed
as mean value.The Residual hydrocarbon was extracted from flasks using hexane after 1, 9,
and 18 days of growth and analyzed by GC.The biodegradation of petroleum hydrocarbons
and fractions (aliphatic, aromatic, resin and asphaltene) were analysed asdescribed by Mishra
et al. [31].
After

solvent

evaporation,

the

residual

TPH

was

fractionated

into

alkane,

aromatic,asphaltene, and NSO (nitrogen, sulphur, and oxygen-containing compounds)

fractions on a silica gel column. For this purpose, sampleswere dissolved in n-pentane and
separated into soluble and insolublefractions (asphaltene). The soluble fraction was loaded on
a silica gelcolumn and eluted with different solvents. The alkane fraction waseluted with 100
mL of hexane followed by the aromatic fraction, whichwas eluted with 100 mL of toluene.
Finally, the NSO fraction was elutedwith methanol and chloroform 200 mL (1:1).The
aromaticfractions were analysed by gas chromatography (GC-FID). Degradation values from
the uninoculated abiotic control were used to account for abiotic losses. All the experiments
were carried out in duplicate and the mean values were used as results. For the quantification
of the monitored hydrocarbons, standard calibration curves were constructed from dilutions
of the oil sludge, and the calibration curves for each representative peak were used to
calculate percentage degradations and expressed as percentages of respective controls.

13

2.10 Application of the biosurfactants in recovery of spiked used motor oil from sand
The applicability of biosurfactants produced by B. subtilisCN2 in oil recovery was evaluated
using artificially contaminated sand [16]. Samples of 150 g of sand were mixed with 15 g of
used dense motor oil in 250 mL Erlenmeyer flasks by shaking and allowed to age for 2
months.Afterwards, 200 mL of cell free broth was added to one flask and distilled water to
another flask (control). The samples were incubated on a rotary shaker (150 rpm) for 24 h at
30C and then were centrifuged at 6000 rpm for 10 min for separation of the laundering
solutionand the sand. The amount of oil residing in the sand after the action of
biosurfactantwasgravimetrically determined as the amount of material extracted from the
sand by hexane [32]. Control assays were performed using demineralized water at the same
conditions. All the experiments were performed in triplicateand results are expressed as the
average standard deviation"
3 Results and discussion
3.1 Isolation and Screening of Bacterial Isolates for Biosurfactant Production
Among 13 bacterial isolates obtained, 7 were found to be potential biosurfactant producer
when tested by drop-collapse and oil spread test for biosurfactant production. Bacterial
isolate CN2 screened was found to possessmaximum biosurfactant production ability. The
positive result obtained from the above confirmatory assays confirmed the biosurfactant
production by the strain. Based on the 16S rDNA gene sequences and using the GenBank
BLAST tool, isolate CN2 (Fig. 1) was found to be closely related to Bacillus subtilis, with a
percentage of similarity of 100%. The 16S rRNA gene sequence of strain CN2 was aligned
automatically to reference sequences of the genus Bacillusobtained from the GenBank
(http://www.ncbi.nlm.nih.gov/BLAST/), and a phylogenetic tree was constructed ( Fig. 1)
Based

on

the

neighbor-joining

method

using

the

software

MEGA

version
14

6.0[24].Thecomplete 16S rDNA sequence of the strain CN2 was deposited in the GenBank
database under the accession number KP793927.

3.2 Chemical characterization

3.2.1 FTIR Characterization
As presented in Fig. 2, the FT-IR spectrum of the of the purified biosurfactant isolated from
B. subtilis CN2 showed strongly absorbing bands at 3,286 cm1 as a result of NH stretching
indicating the peptide groups. The presence of an aliphatic chain was indicated by the CH
modes from 2936 to 2882 cm1 and 1412.6 1328 cm1. The strong absorption band at
1,648.3 cm1 is due to the amide band (CO stretching in the peptide bond).The CO
stretching and COC, ester groups were present at the 1,209 and 1,108 cm-1 ranges,
respectively. The IR absorption pattern revealed the presence of peptide and carboxyl groups
that indicated their lipopeptide nature [33, 34, 35, 36].Comparing with a standard sample of
commercial surfactin from Sigma-Aldrich by Sousa et al. [33],the presence of (Amide
,peptide and aliphatic) functional groupswasrevealed,thusconfirming the lipopeptidal nature
of the biosurfactant.

3.2.2 Thin-layer chromatography (TLC) analysis of purified biosurfactant

Thin-layer chromatography characterization revealed a pink spot with Rfvalue of 0.62 (Fig.
3) when sprayed with ninhydrin reagent, indicating the presence of amino acids. No spot was
observed when sprayed with p-anisaldehyde confirming the absence of sugar moiety. The
above result confirmed the lipopeptidal nature of the biosurfactant. Similar results were
reported by Sriram et al. [36].

15

3.2.3 Mass Spectrometry

As shown in LCMSMS spectral analysis of the purified fraction (Fig. 4), a series of
protonated ions were observed for the fraction of the purified sample eluting at 0.59 min, at
m/z 1030, 1064, 1098, 1132,1166,1200, 1235,1268,1302, 1336, 1370, 1404,1438 and 1472.
These peaks differ by 34Da, suggesting a series of homologous molecules with different
length of fatty acid chain. The difference of 17Da, 34 Da observed between peaks is due to
NH3 loss.
These spectra are very similar to those reported by Ishikawa et al.[37]. A study on the amino
acid sequence in iturin by Ishikawa et al.[37] indicated that the difference between two peaks
is 17.In a McLafferty rearrangement mechanismwhich isa reaction observed in mass
spectrometry, the cleavage happens between alpha and beta atoms and two hydrogens are
transferred to the alkoxy part including alpha atoms[38].Lipopetides can consist of short
linear chains or cyclic structures of amino acids, linked to a fatty acid via ester or amide
bonds or both.In a cyclic lipopeptide an ester or an amide is formed if a carbonyl group,
laying in the C - terminal amino acid residue/residues,isbonded to an alcohol or an amine
with a long chain and the alcohol or amine would have two or more gamma Hydrogen. As a
result, for ester structure the OH2 would be added to C-terminal part and its mass would be
increased by 18.01; and to the amide structure the NH3 would be added to C-terminal part
and its mass would be increased by 17.01 [38]. Thus we can deduce that the biosurfactant
produced by the strain B.subtilis CN2 is the cyclic lipopeptide iturin of molecular mass
1472Da.

16

3.3 Physical properties of the biosurfactant

3.3.1 Surface activity
The isolate CN2 lowered the surface tension of culture broth from 72 to 32 mN/m after72 h
incubation. The biosurfactant concentration in terms of critical micelle dilution (CMD) in the
cell free broth was 20CMD at the minimum surface tension, indicating that the biosurfactant
concentration in the cell free broth was 20 times its CMC. The emulsification capacity of
theculture broth was nothighest at this point and continued to increase with further growth
and biosurfactant production. This reveals that the biosurfactant concentration became
sufficient for micelle formation after 72 h of fermentation, beyond which constant surface
tension is observed. The surface tension versus biosurfactant concentration plot was used to
determine the CMC as the point of interception between the two lines that best fit the decline
and the constant plateau of surface tension. The Lipopeptide

has a critical micelle

concentration (CMC) of 18510mg L corresponding to minimum surface tension of 31 1

mN/m determined after column purification and serial dilution of the biosurfactant with
Phosphate buffer solution (10 mM KH2PO4/K2HPO4 and 150 mMNaCl with pH adjusted to
7.0).
The CMC obtained in this study is comparable with the CMC reported in the literature for
surfactin by Fox and Bala. [39] who reported 0.15 ,0.16 and 2.9 g L1 depending on media
used and 200 mg L1 reported by Ismail et al. [40]. Thimon et al. [41] reported that iturinic
lipopeptides have close surface tension values , which are significantly higher than that of
surfactin, the CMC values are quite variable from 9 M for surfactin to 80, 160 and 170 M
for iturin C, bacillomycin D and bacillomycin L respectively which are iturinic compounds.
The CMC values of iturine reported by Thimon et alare comparable to the CMC value of 127
M of the iturin produced by CN2 strain in the current study.Hamley[42]has reported
critical micelle concentration of 2540 10M for iturin in water.Cooper et al. [43] reported
a CMC of 0.025 g L1 for crystalline surfactin, some other values (13, 22, and 17 mg L1)
have also been reported [44].

17

An efficient biosurfactant can reduce the surface tension of water and air from 72 mN/m to
less than 30 mN/m[40,45]. Accordingly, B. subtillisCN2 is a promising biosurfactant
producer with

a potential for use in environmental remediation and oil recovery

strategies.Abdel-Mawgoud et al. [44] demonstrated the high efficiency of a crude

biosurfactant produced by B. subtilis BS5 based on the reduction of the surface tension of
water from 70 to 36 mN/m. Vazet al. [46]and Ghojavand et al.[47] reported surface tensions
reductions to 29 mN/m and 27 mN/m respectively.For the B. subtillis CN2, the highest
amount of biosurfactant produced was7150 mg L1 of culture medium obtained when using
glycerol as the carbon source at concentration of 6% (w/v). Gudia et al.[48] reported about
1300 mg L1 of biosurfactant production by Bacillus subtilis #573 strain using corn steep
liquor (CSL) culture medium consisting of 10% (v/v) of CSL. Lin et al. [49] and Rahman et
al. [50] reported highest iturin productions of 121.28 mg/L and 4000mg/L respectively from
B. subtillis strains.
3.3.2 Emulsification activity
In addition to surface activity, good emulsification property of a biosurfactant gives it
additional environmental and industrial applications .The biosurfactant showed appreciable
emulsification indices, with hexane (822%), cyclohexane (841%), and used motor oil
(762.5%). The emulsions formed were stable for over 6 weeks. The results show that the
biosurfactant could emulsify different hydrocarbons, which confirmed their applicability for
hydrocarbon polluted media bioremediation through dispersing and enhancement of the
availability of the recalcitrant hydrocarbons [51].Ghojavand et al.[47] studied the
emulsification index of a biosurfactant synthesized by a member of the B. subtilis group
(PTCC 1696) with several typical hydrocarbons, n-hexane, n-heptane, cyclohexane and nnonane. The maximum emulsification activity of the biosurfactant was reported to be 64.4%
with cyclohexane.For the isolate CN2 , the highest emulsifying activity was obtained with
18

glycerol and glucose while the use of hydrocarbons as the sole carbon source showed the
least biosurfactant production and very low emulsificarion. This is in agreement with the
results reported by other authors for different B. subtilis isolates [52,16]. The CMC of the
biosurfactant produced by the strain CN2 reported here is higher (i.e. less active) than
previous studies. However, the emulsification index and yield of the biosurfactant

is

comparably higher when compared with Bacillus subtillisproduced biosurfactants from

soluble carbon sources such as glycerol and glucose [47,45,46].
3.3.3 Stability
3.3.3.1 Temperature stability
The applicability of the biosurfactant in several fields also depends on their stability at
different pH, salinity, and temperature. The stability of the biosurfactant was tested over a
widerange of temperature. The biosurfactant produced by B.subtilis CN2was shown to be
thermo stable. The emulsification activity and surface tension of the biosurfactant remained
constant throughout the studied range of temperature (25-125C) with average surface
tension value of 311 mN/m and average E24 value of 821% .Heatingof the supernatant to
120

for

half

an

hour

caused

no

significant

effect

on

the

biosurfactantperformances.Khopadea et al .[15] reported that heating of the biosurfactant to

100 C caused no significant effect on the biosurfactant performance.Heating at 125C for
30 min resulted in a slight increase in surface activity similar result was reported by AbdelMawgoud et al.[44]. Abdel-Mawgoud et al. suggested that this increase in activity may be
attributed to the heat-dependent coagulation and precipitation of substances such as proteins
contaminating the surfactin solution; which might have been coextracted with surfactin
during extraction steps

19

3.3.3.2 pH stability
The biosurfactantdemonstrated a stable E24and surface tension over the pH range of 512and
an appreciable decrease of E24 at pH <5 and pH >13 (Fig. 5).At pH 13, the value of
emulsification activity (E24) maintained was 92%. For pH values lower than 5, the samples
become turbid, and showed reduction in emulsification activity and surface tension due to
partial precipitation of the biosurfactant. Similar results of increasing surface activity with
increasing pH were reported byKhopade et al.[18] and Abouseoudet al. [30].Abouseoud et
al.[30] reported that pH increase from 5 to 12 decreased surface tension from 34 to 30 mN/m
, and increased emulsion stability (15% increase of E24).Increase in emulsion stability with
increasing pH (Fig. 5) could be caused by a better stability of fatty acids-surfactant micelles
in the presence of NaOH andthe precipitation of secondary metabolites at higher pH
values[30,18].Extreme pH valuescould possibly transform weak surface-activespecies into
more active emulsifiers by increasingionization [53, 30].

3.3.3.3 Effect of salinity

Theemulsification activityand surface tension ofthe biosurfactant remained constant
throughout the studied range of salinity (5 -20%, w/v)with average surface tension value of
311 mN/m and average E24 value of 821%. Abouseoud et al. [30]. reported a similar effect
on effectiveness upon the addition of 20 % sodium chloride, for a biosurfactant produced by
Pseudomonas fluorescens Migula 1895-DSMZ. Khopadea et al [15] reported that

little

changes were observed in increase in concentration of NaCl up to 9% (w/v) while at higher

concentration of NaCl, the biosurfactant retained 80% of its activity. The biosurfactant
produced byB. subtilis CN2 has stability at alkaline pH and high salinity; such a biosurfactant
may be useful for bioremediation of spills in the marine environment because of its stability
in alkaline condition and in the presence of salt [15].

20

3.4 Biodegradation of used motor oil

There was a significant increase in cell density in the culture broths in the 18 days of
incubation, with an associated decrease in different components of the used engine oil,
demonstrating that the consortium can utilize used motor oil as the sole carbon and energy
source. High turbidity and enhanced bacterial growth were observed in the biosurfactant
supplemented culture than the one without biosurfactant supplementation (Figure 6).The total
viable cell count increased(from 6.4 log (CFU) mL-1 at the day of incubation toto9.99 log
(CFU) mL-1)on day 15 in the flask supplemented with the biosurfactant...In the microcosm
with no biosurfactant supplementation the total viable cell count were considerably lower by
up to two orders of magnitude ( increase from 6.4 to 7.77 log ( CFU) mL-1 compared to
increase from 6.4 to 9.99 log ( CFU) mL-1) in both nutrients and biosurfactant supplemented
flask. Even though both microcosms were supplied with nutrients from the mineral salt
medium the greater bacterial growth in those microcosms supplemented with both nutrients
and biosurfactant (compared with nutrients alone) suggests that by increasing hydrocarbon
bioavailability the microbial growth was enhanced. The biosurfactant increased the surface
area of the hydrophobic water-insoluble substrate and improved their bioavailability, thereby
enhancing the growth of bacteria and the rate of bioremediation [54]. One biological strategy
that can enhance contact between bacteria andwater-insoluble hydrocarbons is emulsification
of thehydrocarbon. Biosurfactants help to disperse the oil and increase thesurface area for
growth, Unless the surface area of the hydrophobic substrate isincreasedexponential
bacterialgrowth is not possible, and biomass will only increase arithmetically [55].
Fresh motor oil usually contains very small amounts of polycyclic aromatic hydrocarbons
(PAHs). On the other hand, used oil may contain minute quantities of additive, metals such as
lead, zinc, barium and magnesium resulting from engine wear and higher percentages of

21

alkylbenzenes, naphthalene, methyl naphthalene and higher polycyclicaromatic hydrocarbons

(PAHs) as a result of pyrosynthesis [56,57]. The degradation of representative PAHs and
methylated derivatives was monitored during the shake flask treatment. The representative
PAHsmonitored

were

Naphthalene;Acenaphthene;

Naphthalene

2-methyl-,

Fluorine;Phenanthrene;Fluoranthene;Naphthalene, 1-phenyl-; Naphthalene, 2-phenyl- and 9Allylanthracene. Biodegradation tests of the motor oil components inthe laboratory scale
microcosm

experiment

showed

maximum

degradation

in

the

culture

with

biosurfactantamendment (Bacterial cells + used motor oil + biosurfactant + MSM) than the
one without biosurfactant (Bacterial cells + used motor oil + MSM). Furthermore, there was
no significant removal observed in the abiotic controls (less than 3.1 1. 2%), confirming that
the removal was due to biological activity.
The biosurfactant amendment more than doubled the degradation of the PAHs, mainly
enhanced degradation of the more hydrophobic PAHs was observed (Figure7A).At day18 of
incubation there was considerable disappearance of the peaks in the biosurfactant amended
sample, whereas in the unamended microcosm persistence of the PAHs was observed. Mainly
the more hydrophobic PAHs showed the least degradation in the microcosm without
biosurfactantsupplementation (Figure7B). Enhanced bacterial growth was observed in the
biosurfactant amended microcosm.
One of the main factors affecting the biodegradation efficiency of complex oily compounds is
the low availability of contaminants for microbial attack. An alternative to expand
bioavailability and contaminant metabolism is increasing substrate solubilisation by using
biosurfactants [58].Biosurfactants can increase the aqueous solubilisation of poorly soluble
compounds, including Polyaromatic hydrocarbons and enhancehydrocarbon bioremediation
by two mechanisms [59]. The first includes the increase of substrate bioavailability for

22

microorganisms, while the other involves interaction with the cell surface which increases the
hydrophobicity of the surface allowing hydrophobic substrates to associate more easily with
bacterial cells. By reducing surface and interfacial tensions, biosurfactants increase the
surface areas of insoluble compounds leading to increased mobility and bioavailability of
hydrocarbons. The enhanced biodegradation levels obtained with biosurfactant demonstrated
that that they represent the most efficient accelerators for hydrocarbon biodegradation
through

enhancing

their

bioavailability

and

facilitating

their

degradation

by

microorganisms[22].Investigation of surface tension and emulsifying activity of the culture

medium demonstrated that the microbial consortium were able to produce biosurfactants in
mineral salt medium containing motor oil as the sole carbon source. The highest
emulsification index (> 90 % with hexane) was observed in the biosurfacatnt supplemented
microcosm and high surface tension reduction, lower than 35 mN/m was observed in both
biosurfcatnt supplemented and non-supplemented microcosms.Most of the members of the
consortium used in this study like, Bacillus subtilis, Paenibacillus dendritiformis,
Pseudomonas aeruginosa, Ochrobactrum intermedium have been confirmed as potent
biosurfactant producers in the screening tests conducted. Thus the additional initial
biosurfactants added to the culture medium could be enough to facilitate the bacterial access
to the crude oil followed with more biosurfactant production which resulted in the high
emulsification index observed. Thavasi et al.[21] observed maximum crude oil degradation
and

biosurfactant

production

with

Pseudomonas

aeruginosa

(89%,

with

0.1%

biosurfactant).Similarstudies ofbiosurfactant enhanced degradation have been reported

bymany authors. Chandankere et al. [8] reported that an effective biosurfactant-producer and
hydrocarbon degrading bacterial strain, Bacillus methylotrophicusUSTBadegraded 92% of
crude oil in 2 weeks time and

produced biosurfactant during the course of hydrocarbon

degradation that had the ability to decrease the surface tension of water from 72 to 28 mN/m.

23

A biosurfactant from L. delbrueckiicultured with peanut oil cake as the carbon source was
tested in biodegradation experimentswith crude oil. While significant oil degradation
occurred when the biosurfactant and fertilizers were added ,the biosurfactant alone was also
capable of promoting biodegradation to a large extent without added fertilizers[60].Crude oil
biodegradation with biosurfactnt from Pseudomonas cepacia CCT6659 cultivated with 2%
soybean waste frying oil and 2% corn steep liquor as substrates showed 83% biodegradation
activity in the first 10 days of the experiments when the biosurfactant and bacterial cells were
used together[61]. In additionto increasing bioavailability biosurfactant or biosurfactant
producing bacteria reduce thetoxicity level of heavy metals in the used motor and this
supports the growth of other hydrocarbon degrading bacterial strains to supplement the
process of oil degradation. The usefulness of biosurfactants for bioremediation of heavy
metal contaminated media is mainly based on their ability to form complexes with metals.
The anionic biosurfactants create complexes with metals in a nonionic form by ionic bonds
[59].
3.5 Used motor oil removal from the contaminated sand.
Petroleum hydrocarbon compounds bind to soil components and are difficult to remove due
to their hydrophobicity. Biosurfactants can emulsify hydrocarbons enhancing their water
solubility, decreasing surface tension and increasing the displacement of oil substances from
soil particles [62,32].The results showed 84.67.1 and 15 3% used motor oil recoveries
from the contaminated sands in the cell free supernatant from B.subtilis CN2 and control
(distilled water) respectively.This result is comparable with the ones reported by Liu et
al.[63]in which biosurfactant from Bacillus subtilis BS-37 recovered 96% of crude oil from
oil-sand at the concentration of 300 mg L1, surfactin (LB) and surfactin (G) solution. Joshi
and Desai [64]reported 30.2234.19% additional oil recovery over the water flood residual
oil saturation using crude biosurfactant obtained from Bacillus strains by sand pack column
24

studies. Al-Wahaibi et al. [65]reported 1726% extra oil recoveryusing crude biosurfactant
obtained fromB. Subtilis B30. The current study showed that,thebiosurfactatnt produced by
B.subtilisCN2 has potential application in soil washing andenhanced microbial oil recovery.
Conclusion
Biosurfactants are promising for enhanced remediation of environmental media contaminated
by hydrophobic and recalcitrant organic pollutants. In the current study it has been shown
that the biosurfactant from B.subtilisCN2 strain

increased the pseudosolubilization and

emulsification of highly hydrophobic used motor oil sludge, thereby increasing the
bioavailability and its subsequent degradation. The biosurfactant from the CN2 strain

also

showed an interesting potential application in soil washing or enhanced microbial oil

recovery. The findings demonstrate that this biosurfactant can have a remarkable potential for
environmental application and enhanced oil recovery at harsh environmental conditions of
extreme salinity, alkalinity and thermal conditions.
5 Acknowledgements
This research was funded by the National Research Foundation (NRF) of South Africa
through the Incentive Funding for Rated Researchers Grant No. IFR2010042900080 awarded
to Prof. Evans M.N. Chirwa of the University of Pretoria. We thank Prof Fanus Venter of the
Department of Microbiology and Plant Pathology, University of Pretoria, for his assistance
with the DNA sequencing and characterization of bacteria from the soil.

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Figure 1.

Phylogenetic tree based on 16S rRNA sequence, constructed by the neighborjoining method, showing the position of strain CN2 among related members of
the genus Bacillus. Reference strain organisms are included and sequence
accession numbers are given in parentheses. Bootstrap values from 1000
replicates.

Figure 2

Fourier transform infrared spectra (FTIR) spectra of the biosurfactant produced

by the Bacillus subtilis CN2

Figure3

Thin-Layer Chromatography (TLC) of the biosurfactant obtained from Bacillus

subtilis CN2 after treatment with ninhydrin 0.35 % (w/v, in acetone) revealed a
pink spot with Rf value of 0.62 indicating the presence of amino
acids.Chromatograms were developed with chloroform: methanol: water
(65:15:4 v/v)

Figure 4

Tandem mass spectrometry (LC-MS/MS) Mass spectra of the fraction of the

biosurfactants produced by Bacillus subtilis CN2 eluting at 0.569 min.

Figures 5 Influence of pHon the surface tension values (mN/m) and emulsifying indexe
(%)of 72 hour cell-free broth of Bacillus subtilis CN2,obtained by centrifuging
the culture at 12000rpm for 20 minutes.The error bars represents standard errors
ofthree independent experiments (n = 3).
34

Figure 6 The growth kinetics of the consortium on MSM supplied with used motor oil (3%
v/v) as the sole carbon source .With 0.15% w/v biosurfactant supplementation (
diamond ) , withno biosurfactant supplementation ( square ).Data are expressed
as the mean standard error of two independent experiments performed in
duplicate.Other process parameters: pH, 7; temperature, 35 C; agitation speed,
180 rpm.
Figure 7 Percentage of residual monitored PAHs in used motor oil (3% v/v) amended flasks,
as the sole source of carbon and energy,

at day 0 (blue); day 9 (red) and day 18

(green): (A) With (0.15% w/v) biosurfactant supplementation;

(B) with no

biosurfactant supplementation. Data are expressed as the mean standard error of

two independent experiments performed in duplicate. Error bars denote standard
errors of duplicate samples. Other process parameters: pH, 7.5; temperature, 35 C;
agitation speed, 180 rpm.

35