Water Air Soil Pollut (2016) 227: 325

DOI 10.1007/s11270-016-3031-8

Production and Application of Gordonia westfalica GY40

Biosurfactant for Remediation of Fuel Oil Spill
Supattra Laorrattanasak & Witchaya Rongsayamanont & Nichakorn Khondee &
Nanthorn Paorach & Suwat Soonglerdsongpha & Onruthai Pinyakong &
Ekawan Luepromchai

Received: 31 May 2016 / Accepted: 2 August 2016 / Published online: 12 August 2016
# Springer International Publishing Switzerland 2016

Abstract This study aims to produce and apply a
biosurfactant from Gordonia westfalica GY40 for en-
hancing fuel oil solubilization and degradation in sea-
water. The immobilization of G. westfalica GY40 cells
on chitosan flakes increased biosurfactant yield, and we
achieved a biosurfactant concentration as high as
1.85 g L−1 when using 2 % soybean oil as the carbon
source. The critical micelle dilution (CMD) value of
cell-free broth was 25 % and the lowest surface tension
was 35 mN m−1. The cell-free broth was able to solubi-
lize and disperse fuel oil, at efficiencies corresponding
to biosurfactant concentrations and CMD values. The
surface activity of cell-free broth was stable under wide
S. Laorrattanasak : W. Rongsayamanont : N. Khondee
International Postgraduate Programs in Environmental
Management, Graduate School, Chulalongkorn University,
Bangkok, Thailand
S. Laorrattanasak : W. Rongsayamanont : N. Khondee :
O. Pinyakong : E. Luepromchai
Research Program on Remediation Technologies for Petroleum
Contamination, Center of Excellence on Hazardous Substance
Management (HSM), Chulalongkorn University, Bangkok,
Thailand
N. Paorach : O. Pinyakong : E. Luepromchai (*)
Bioremediation Research Unit, Department of Microbiology,
Faculty of Science, Chulalongkorn University, Bangkok, Thailand
10330
e-mail: ekawan.l@chula.ac.th
S. Soonglerdsongpha
Environmental Technology Research Department, PTT Research
and Technology Institute, PTT Public Company Limited,
Ayutthaya, Thailand
ranges of salinity, temperature, and pH. For the oil
degradation test, cell-free broth at 0.5× CMD was added
along with polyurethane foam-immobilized Gordonia
sp. JC11, an efficient oil-degrading bacterial inoculum,
to fuel oil spiked seawater. The system removed 81 % of
1 g L−1 fuel oil in nutrient seawater medium within
6 days. When tested with three seawater samples col-
lected along the Thai coastal area, the addition of both
biosurfactant and immobilized Gordonia sp. JC11 was
able to remove 60–70 % of 1 g L−1 fuel oil, while the
natural attenuation (control) removed only 26–35 % of
fuel oil. The application of cell-free broth reduced the
extraction and purification steps. In addition, the simple
production of G. westfalica GY40 biosurfactant and
Gordonia sp. JC11 inoculum suggested that they are
suitable for cleaning-up oil spills in seawater.
Keywords Biosurfactant . Gordonia . Oil dispersant .
Oil spill . Biodegradation
1 Introduction
Biosurfactants from the class Actinobacteria have be-
come popular due to their diverse chemical structures,
which include trehalose-comprising surfactants, non-
trehalose containing glycolipids, lipopeptides, and poly-
meric biosurfactants (Kügler et al. 2015). In this class,
bacteria of the genus Gordonia can produce extracellu-
lar and/or cell-bound biosurfactants, some of which
have been identified as trehalose lipids, lipopeptides,
and polymeric glycolipids (Dogan et al. 2006; Franzetti
325 Page 2 of 13
et al. 2008, 2009a, b; Saeki et al. 2009; Jackisch-
Matsuura et al. 2014). Biosurfactants from the genus
Gordonia have been applied for cleaning-up petroleum
contaminations and are normally produced using petro-
leum hydrocarbons such as diesel oil, heptadecane, and
hexadecane as carbon substrates (Dogan et al. 2006;
Franzetti et al. 2008, 2009a, b; Saeki et al. 2009;
Jackisch-Matsuura et al. 2014). However, large-scale
production of these biosurfactants is hindered due to
the use of hazardous substrates.
Various vegetable oils and glycerol have been used
for biosurfactant production by many bacteria and
yeasts (Silva et al. 2014). However, only Pizzul et al.
(2006) investigated the potential use of rapeseed oil for
biosurfactant production by various Gordonia strains.
They reported that Gordonia sp. APB grown on 0.1 %
rapeseed oil reduced the surface tension of the culture
medium by 20.4 %, while Gordonia rubripertincta
DSM 46038 lowered the surface tension to less than
10 %. Thus, the extent of biosurfactant production from
vegetable oil varies within the Gordonia genus. In this
study, Gordonia westfalica GY40, a local isolate, was
investigated for its ability to produce extracellular
biosurfactant from palm oil, soybean oil, and glycerol.
These substrates are low-cost materials and are widely
available. To increase production, G. westfalica GY40
cells were immobilized on chitosan flakes following the
procedure of Khondee et al. (2015). Chitosan is a natu-
ral, non-toxic, and biodegradable polymer, which allows
high density of bacterial cells to attach, thereby increas-
ing biosurfactant yield and facilitating continuous
biosurfactant production (Khondee et al. 2015). We then
characterized the cell-free broth containing biosurfactant
of G. westfalica GY40 for petroleum solubilization and
dispersion to determine its potential application for oil
spill remediation.
Oil spills in seawater commonly occur around the
world and affect the marine ecosystem. The role of
biosurfactants is to enhance the dispersal of petroleum
in seawater and increase its bioavailability for microor-
ganisms via solubilization. This process leads to the
subsequent removal of petroleum through microbial
degradation (Silva et al. 2014). The application of
Gordonia biosurfactant for enhanced oil spill removal
has been reported by Saeki et al. (2009). The
biosurfactant was prepared by spray drying the sterilized
culture broth of Gordonia sp. JE-1058, and was applied
as a powder at 0.5 g L−1 to seawater contaminated with
5 g L−1 crude oil. The results showed that the indigenous
Water Air Soil Pollut (2016) 227: 325
marine bacteria degraded more oil in the presence of
biosurfactant powder than with the addition of nutrients
(Saeki et al. 2009). Nonetheless, the success of oil
bioremediation depends on the type and amount of oil,
environmental factors, and microbial inoculants (Mercer
and Trevors 2011). This study focused on fuel oil, which
is the main fuel for cargo ships and generally contains
high percentage of aromatics. The biodegradation of
aromatics is considerably slow, for example even
though 100 % of light-medium linear alkanes were
degraded within the first month after the Prestige fuel
oil spill, only about 35 % of aromatics were depleted
over the same time period (Gallego et al. 2006). To
accelerate fuel oil biodegradation, G. westfalica GY40
biosurfactant, produced in this study, was applied along
with an oil-degrading inoculum, Gordonia sp. JC11
immobilized on polyurethane foam (PUF), which has
been used to treat oily bilge water from small fishing
vessels (Chanthamalee et al. 2013). Although, the bac-
terium has limited ability to produce biosurfactants, it
can degrade both saturates and aromatics in various oil
samples (Chanthamalee and Luepromchai 2012).
The aim of this study is to identify a non-hazardous
substrate for producing G. westfalica GY40
biosurfactant, and to use the biosurfactant for increasing
fuel oil solubilization, dispersion, and biodegradation.
The process of applying the biosurfactant as cell-free
broth, along with PUF-immobilized Gordonia sp. JC11,
to contaminated seawater was simple and economical,
and is a convenient remediation approach for potentially
cleaning-up fuel oil in seawater.
2 Materials and Methods
2.1 Bacteria, Media, and Chemicals
G. westfalica GY40 was isolated from agricultural soil
and originally used glycerol as the carbon source. It was
deposited at the MSCU culture collection, Thailand
Bioresource Research Center (TBRC), as MSCU
0607. The bacterium, as free cells, produced extracellu-
lar biosurfactant at 0.41 g L−1 after cultivation in basal
medium containing 2 % bottom glycerol and 0.75 %
palm oil, for 7 days. It was maintained on 25 % (w/v)
Luria-Bertani (LB) agar and sub-cultured monthly.
Gordonia sp. JC11 (TISTR 1944 or MSCU 0424) was
maintained in nutrient seawater (NSW) broth containing
100 mg L−1 fuel oil following Chantamalee and
Water Air Soil Pollut (2016) 227: 325
Luepromchai (2012). NSW consisted of 1.0 g NH4NO3,
0.02 g ferric citrate, 0.02 g K2HPO4, 0.5 g yeast extract,
800 mL synthetic seawater, and 200 mL distilled water
per liter.
Food grade palm oil and soybean oil were used.
Bottom glycerol (or waste glycerol) is the residual waste
after recovering methanol and glycerol from the by-
product of biodiesel manufacturing, and was obtained
from Thai Oleochemicals Company Limited. Fuel oil
with a viscosity of 630 cP was obtained from PTT
Public Company Limited. The compositions of fuel oil
from thin layer chromatography and flame ionization
detection (TLC-FID) analysis were 15 % saturates, 62 %
aromatics, 15 % asphaltene, and 8 % resin. Slickgone
NS Type 2/3 and sodium dodecyl sulfate (SDS) were
used as the control dispersant and surfactant, respective-
ly. All other chemicals were of analytical grade.
2.2 Bacterial Immobilization
G. westfalica GY40 was immobilized on squid pen
chitosan flakes (1–2 mm diameter) following the proce-
dure of Khondee et al. (2015), in a 250-mL flask.
Briefly, a 24-h bacterial inoculum (1.0 OD600) was
transferred into 50 mL of 25 % (w/v) LB medium
containing 8 % (w/v) sterilized chitosan at a concentra-
tion of 2 % (v/v) and cultured at 30 °C and 200 rpm in a
shaker for 3 days. The immobilized bacterium was used
immediately for biosurfactant production. Gordonia sp.
JC11 was immobilized on 0.5 cm × 1 cm × 2 cm PUF
strips as described in Chantamalee and Luepromchai
(2012). The PUF-immobilized bacterium could be
stored at 4 °C for at least 2 months before being used
as the inoculum for fuel oil degradation in seawater.
2.3 Biosurfactant Production
After immobilization, the medium (25 % LB) was re-
placed with basal medium and a carbon source was
added. Basal medium (BM) for biosurfactant production
contained 7.0 g L−1 NH4SO2, 1.0 g L−1 K2HPO4,
0.5 g L−1 KH2PO4, 0.1 g L−1 KCl,
0.5 g L−1 MgSO4·7H2O, 0.01 g L−1 CaCl2, 0.01 g L−1
FeSO4·7H2O, 0.1 g L−1 yeast extract, and 0.05 mL trace
elements (consisted of 0.26 g L−1 H3BO3, 0.5 g L−1
CuSO4·5H2O, 0.5 g L−1 MnSO4·H2O, 0.06 g L−1
MoNa2O4·2H2O, and 0.7 g L−1 ZnSO4·7H2O). The
medium was adjusted to pH 7.5. There were four types
of carbon sources: 2 % glycerol, a mixture of 2 %
Page 3 of 13 325
bottom glycerol and 0.75 % palm oil, 2 % palm oil,
and 2 % soybean oil. Previously, Khondee et al. (2015)
reported that bottom glycerol acted as the main carbon
source, while palm oil was an inducer for biosurfactant
production by Bacillus sp. GY19. Since, our study used
different bacterial strains, we selected soybean oil as the
alternative carbon source. Glycerol and palm oil on their
own were used as controls. Biosurfactant production
was carried out by incubating the flask at 30 °C and
200 rpm in a shaker for 7 days. After incubation, the
culture broth was separated from the immobilized cells
by pouring it out of the flask. The cell-free broth was
obtained by centrifuging the culture broth at 8000 rpm
for 10 min. The extent of biosurfactant production was
determined from crude biosurfactant yield and
biosurfactant activity, i.e., oil displacement and surface
tension reduction of cell-free broth. To reuse the
chitosan-immobilized G. westfalica GY40, fresh basal
medium and carbon source were added to the flask after
separating culture broth, and further incubated under the
same conditions. The process was repeated until
biosurfactant production decreased. All experiments
were carried out in triplicates.
2.4 Biosurfactant Characterization
Biosurfactant in cell-free broth was extracted and con-
centrated to measure biosurfactant yield and determine
its composition, following the procedure of Khondee
et al. (2015). Briefly, the pH of the cell-free broth was
adjusted to 2.0 and extracted by chloroform-methanol
(2:1). The organic solvent was later separated and evap-
orated in a rotary evaporator. The viscous yellowish
product was dissolved in methanol, filtered, and con-
centrated again at 45 °C. The amount of crude
biosurfactant was measured by weighing the final ex-
tracted product. Preliminary colorimetric analysis was
carried out to determine the chemical compositions of
the crude biosurfactant. Total lipid, protein, and sugar
content were determined by sulfo-phospho-vanillin test,
Bradford assay, and phenol-sulfuric acid test,
respectively.
The surface tension (ST) and critical micelle di-
lution (CMD) of the cell-free broth were measured
with a digital tensiometer (Kruss, K10ST, Germany)
at 25 °C using the plate method. CMD value was
determined from a plot of surface tension vs. serial
dilution of samples. Dispersion activity was deter-
mined by the oil displacement technique according
325 Page 4 of 13
to Bharali et al. (2011). In general, 10 μL of cell-free
broth was dropped onto the surface of the fuel oil
layer, which was formed by adding 8 μL of fuel oil
on 20 mL of seawater in a Petri dish (diameter of
80 mm). The dispersant to oil ratio (DOR) was 1:0.8.
Oil displacement ð%Þ ¼ ðDiameter of clear zone = Diameter of oil before testingÞ  100
Oil solubilization was carried out by mixing
25 mL of cell-free broth and 100 mg fuel oil in
a 100-mL flask. The sample was placed in a
shaker at 200 rpm for 1 day. Next, 10 mL of the
aqueous layer was extracted by chloroform and
analyzed for the amount of solubilized oil by
TLC-FID. Percent oil solubilization was calculated
based on the total amount of oil added. The sta-
bility of biosurfactant in cell-free broth was deter-
mined based on their surface activities under dif-
ferent environmental conditions including varying
NaCl concentrations (0–10 % w/v), temperature
(50–121 °C), and pH (2.0–12.0). Acidic pH was
adjusted by 3 M HCl and alkaline pH was adjust-
ed by 0.1 M NaOH. The surface tension of cell-
free broth was measured after incubating the sam-
ple at each experimental condition for 24 h. The
surface tension of cell-free broth at 121 °C was
measured after autoclaving the sample for 15 min.
The cell-free broth could be stored in a plastic
bottle at 4 °C for at least 1 month.
2.5 Biodegradation of Fuel Oil
Biodegradation of fuel oil was investigated in a batch
microcosm experiment that consisted of 40 mL seawater
containing 1000 mg L−1 fuel oil in a 250-mL flask.
PUF-immobilized Gordonia sp. JC11 was added at
0.1 g per flask as the oil-degrading inoculum. To in-
crease the solubilization and dispersion of fuel oil,
10 mL of biosurfactant in cell-free broth was added to
achieve a final concentration of 0.5× CMD. Control was
uninoculated microcosms. The microcosms of
Gordonia sp. JC11 alone (JC11), Gordonia sp. JC11
with biosurfactant in cell-free broth (JC11 + BSF), and
control were set in triplicates and placed in a shaker at
Water Air Soil Pollut (2016) 227: 325
The DOR was later varied to determine the optimum
amount of cell-free broth as fuel oil dispersant. The
diameter of the clear zone on the oil surface was
measured to calculate oil displacement efficiency
using the following equation:
30 °C and 200 rpm. At each sampling time-point, a
piece of PUF and 1 mL of seawater were collected to
determine bacterial number. The residual oil was then
extracted from the whole flask containing PUF and
seawater with an equal volume of chloroform. The
amount of oil was analyzed by TLC-FID. The numbers
of Gordonia sp. JC11 on PUF and in aqueous solution
were counted by the drop plate technique. The colony
color of Gordonia sp. JC11 is orange; hence, it was easy
to distinguish them from other bacteria in the seawater.
Since nutrients and seawater properties might affect
biodegradation, this experiment compared the extent of
fuel oil biodegradation in nutrient seawater medium and
three seawater samples collected from different ports
along the eastern shoreline of Thailand. The properties
of seawater samples are given in Table 1. All chemical
and physical parameters were analyzed by standard
methods. The number of fuel oil-degrading bacteria
was estimated by the plate count technique using
NSW + fuel oil agar.
2.6 Analytical Methods
The number of bacteria attached to chitosan and PUF
were analyzed after dislodging the cells from the
immobilizing materials. Briefly, the immobilized cells
were filtered through a sterilized stainless steel sieve,
washed twice with 0.85 % (w/v) NaCl and rehydrated in
0.85 % (w/v) NaCl solution for 2 min. The mixture was
sonicated for 2 min and shaken vigorously on a vortex
mixer for 3 min. The process was repeated twice. The
supernatant was separated and the number of bacteria
was measured by the drop plate technique using LB
agar.
The amount of fuel oil in seawater was determined by
TLC-FID as described in Chantamalee et al. (2013). The
Water Air Soil Pollut (2016) 227: 325 Page 5 of 13 325

Table 1 Properties of seawater samples

Seawater sample/properties Nutrient seawater mediuma Bangsan, Chonburi Maptaphut, Rayong Samed Island, Rayong

Date of sampling – Jun. 1, 2013 Jun. 7, 2013 Aug. 2, 2013

Appearance Light yellow and Clear and odorless Light yellow and Clear and odorless
odorless odorless
pH 7.8 7.7 8.2 8.2
Salinity (%) 3.4 3.4 3.4 3.9
Total nitrogen (mg L−1) 100 <0.03 <0.03 <0.03
Total phosphorus (mg L−1) 400 0.2 <0.1 <0.1
Total petroleum hydrocarbons – 4.61 3.14 6.44
(mg L−1)b
COD (mg L–1) 3023 2070 3070 1675
Fuel oil-degrading bacteria – 4.48 4.74 4.53
(log CFU mL−1)
a
Nutrient seawater medium was used as the control
b
According to the Pollution Control Department, Thailand, the standard value of petroleum hydrocarbons in seawater is <0.005 mg L-1 .

amount of fuel oil was calculated from the total peak areas resin) and compared to the internal standard, stearyl alco-
of all fractions (i.e., saturates, aromatics, asphaltene, and hol. The percent oil removal was calculated as follows:

h . i
Fuel oil removal ð%Þ ¼ 100  ðFuel oil ðT 0 Þ − Fuel oil ðT x ÞÞ Fuel oil ðT 0 Þ

T0 and Tx refer to amount of fuel oil on day 0 and the
day of analysis, respectively.
3 Results and Discussion
3.1 Effect of Various Substrates on Biosurfactant
Production
Carbon substrates play an important role in
biosurfactant production, and different bacterial strains
prefer different carbon substrates. Chitosan-
immobilized G. westfalica GY40 produced more
biosurfactant from all tested substrates (range, 0.61–
1.85 g L−1) compared to free cells (0.41 g L−1). Among
substrates, 2 % soybean oil gave both the highest
biosurfactant yield (Fig. 1a) as well as the highest sur-
face activities (Fig. 1b). The cell-free broth from the
medium containing soybean oil had 84 % oil displace-
ment, and reduced surface tension from 59.6 to
35.8 mN m−1 (Fig. 1b). However, G. westfalica GY40
in medium containing 2 % glycerol had higher growth
than other substrates. The bacterial numbers on chitosan
flakes from glycerol medium increased around 2 mag-
nitudes to 1.7 × 109 CFU g−1 chitosan on day 7 (Fig. 1c),
while cell-free broth had the lowest surface activity
(Fig. 1b). The results suggested that the production of
biosurfactant by G. westfalica GY40 was suppressed by
hydrophilic compounds. The requirement for a hydro-
phobic substrate was similar to other bacteria of the
Gordonia genus, where petroleum hydrocarbons and
rapeseed oil are normally used for biosurfactant produc-
tion (Dogan et al. 2006; Franzetti et al. 2008, 2009a, b;
Saeki et al. 2009; Jackisch-Matsuura et al. 2014; Pizzul
et al. 2006). In media containing vegetable oils, i.e., 2 %
bottom glycerol + 0.75 % palm oil, 2 % palm oil, and
2 % soybean oil, the increase in bacterial numbers was
only 1 magnitude and the number of bacteria from each
vegetable oil was not much different with the average of
5.0 × 108 CFU g−1 chitosan after incubation. The carbon
325 Page 6 of 13 Water Air Soil Pollut (2016) 227: 325

Fig. 1 Effect of various a 2.0

substrates on biosurfactant

Biosurfactant yield (g L-1)

production by chitosan-
immobilized G. westfalica GY40 1.6
as determined from biosurfactant 1.2
yield (a), surface tension
reduction and percent oil 0.8
displacement of culture broth
after 7-day incubation (b), and 0.4
bacterial number on chitosan
flakes indicates bacterial growth 0.0
2% Glycerol 2% Bottom 2% Palm oil 2% Soybean oil
during biosurfactant production
glycerol +
(c). The reduction in surface ten- 0.75% Palm oil
sion was determined from the
differences between surface
tension of the basal medium at
b Oil displacement Surface tension reduction

Surface tension reduction (mN m-1)

100 30
59.6 mN m−1 and culture broth
after 7 days incubation

Oil displacement (%)

80
25
60
20
40
15
20

0 10
2% Glycerol 2% Bottom 2% Palm oil 2% Soybean oil
glycerol +
0.75% Palm oil
c
1.0E+10 Day 0 Day 7

1.0E+08
(CFU g-1 chitosan)
Bacterial number

1.0E+06

1.0E+04

1.0E+02

1.0E+00
2% Glycerol 2% Bottom 2% Palm oil 2% Soybean oil
glycerol+
0.75% Palm oil

from vegetable oils was probably used to produce more biosurfactant yield could be due to the low solubility
biosurfactant than new bacterial cells. Consequently, we of soybean oil; thus, the bacterium has to produce more
selected 2 % soybean oil for biosurfactant production by biosurfactant to solubilize this substrate. Similarly,
G. westfalica GY40. Ferraz et al. (2002) reported that linoleic acid increased
Vegetable oils act as fatty acid precursors of the the rate of biosurfactant production by Serratia
lipophilic moiety in various biosurfactants (Kim et al. marcescens, while Prieto et al. (2008) reported that
2002; Khondee et al. 2015). Oleic and linoleic acids soybean oil promoted rhamnolipid production by Pseu-
(C18) are major fatty acids in soybean oil, while domonas aeruginosa. Besides soybean oil, oleic acid is
palmitic acid (C16) is the major fatty acid in palm oil also found in aquaculture wastewater, which has been
(Svensson 2010). The longer hydrophobic chain of the used as a substrate for biosurfactant production by
biosurfactant from soybean oil could be responsible for P. aeruginosa DG2a (Pepi et al. 2013). It is thus inter-
its higher surface activity. At the same time, high esting to determine the application of aquaculture
Water Air Soil Pollut (2016) 227: 325
wastewater for biosurfactant production to further de-
crease the cost of G. westfalica GY40 biosurfactant.
3.2 Reusability of Chitosan-Immobilized Cells
for Biosurfactant production
Chitosan-immobilized G. westfalica GY40 was reused
for four cycles by changing the medium, while the same
immobilized cells were maintained inside the flask. In
the first cycle, the cell number on the chitosan flakes and
surface tension reduction were similar to the previous
experiment with 2 % soybean oil as the carbon source
(Figs. 1b, c and 2). The second production showed that
the cell number increased from 5.1 × 10 8 to
2.4 × 109 CFU g−1 chitosan, while the surface tension
reduction value slightly decreased from 23.8 to
22.4 mN m −1. However, by the third and fourth
production cycles, both cell numbers and surface
tension reduction values were decreased. These results
indicated that the bacterial cells started detaching from
the chitosan surface, led to lower biosurfactant
production and higher surface tension. The results
suggest that these immobilized cells can be reused for
two cycles. However, Khondee et al. (2015) reported
that chitosan-immobilized Bacillus sp. GY19 is reusable
for lipopeptide production in a stirred tank fermenter, at
least five times. The high reusability is due to the fact
that the mixing in the stirred tank fermenter provides
good mass transfer without causing any clogging prob-
lems due to biofilm formation on the chitosan surface
Cell number (CFU/g)
Surface tension reduction (mN/m)
1.0E+10
Bacterial number (CFU g-1 chitosan)
1.0E+08
1.0E+06
1.0E+04
1.0E+02
1.0E+00
1 2 3 4
Production cycles
Fig. 2 Reusability of chitosan-immobilized G. westfalica GY40
cells for biosurfactant production with 2 % soybean oil as the
substrate. The reduction in surface tension was determined from
the differences between surface tension of basal medium at
59.6 mN m−1 and culture broth after 7 days incubation. At the
end of each production cycle, the culture broth was replaced with
new medium
Page 7 of 13 325
(Khondee et al. 2015). Thus, G. westfalica GY40
immobilized cells should be used in a stirred tank fer-
menter to increase their reusability and for long-term
biosurfactant production. Moreover, the application of
another fermentation method, such as fed-batch (Morya
et al. 2013; Yao et al. 2015; Maddikeri et al. 2015)
coupled with the repeated-batch strategy used in this
study, might enhance the concentration of biosurfactant
in each production cycle.
3.3 Chemical Compositions and Properties
of Biosurfactant
The crude biosurfactant produced from G. westfalica
GY40 with soybean oil as the carbon source contained
37 % lipid, 8 % protein, and 32 % sugar indicating that it
was a potential glycolipid biosurfactant. Franzetti et al.
(2008) also hypothesized that the biosurfactant pro-
duced from Gordonia sp. BS29 was in the glycolipid
class using preliminary chemical characterization. Gly-
colipids showed many interesting functional properties
including emulsification, pore formation, anti-
adhesiveness and anti-biofilm formation, detergency,
wetting, and flotation (Inès and Dhouha 2015). To indi-
rectly obtain the critical micelle concentration of the
biosurfactant, this study determined the CMD value of
the cell-free broth from 2 % soybean oil medium.
Figure 3a shows that the CMD of G. westfalica GY40
was 25 % at the lowest surface tension of 35 mN m−1
(Fig. 3a). From the biosurfactant yield in Fig. 1a, this
CMD corresponded to a CMC of 460 mg L−1. The
effectiveness of G. westfalica GY40 biosurfactant was
comparable to other Actinobacteria. For example, the
surface tension of the Gordonia amicalis cell-free broth
Surface tension reduction (mN m-1)
was 37 mN m−1 (Jackisch-Matsuura et al. 2014) and
30
25 various Rhodococcus species produce biosurfactants
20 with CMC values of 37–250 mg L−1 and a surface
15 tension range between 19 and 30 mN m−1 (Mnif and
Ghribi 2015).
10
In general, the solubility of fuel oil in water ranges
5
from 6 to 1400 mg L−1 (Tesoro Tesoro Corporation
0 2013). The fuel oil used in this study is considered
hydrophobic with a high amount of aromatic hydrocar-
bons (62 %) and high viscosity (630 cP). The higher
viscosity of fuel oil results in lower dissolved concen-
tration of hydrocarbons in water (Chao et al. 2012). In
this study, only 17 % of fuel oil (680 mg L−1) dissolved
in water (Fig. 3b). The cell-free broth was able to in-
crease fuel oil solubilization and the efficiency
325 Page 8 of 13
corresponded to its biosurfactant concentrations. The
undiluted cell-free broth (4× CMD) was able to solubi-
lize 66 % of 4 g L−1 fuel oil (2640 mg L−1). The percent
solubilization was much higher than the samples at
CMD (25 % cell-free broth) and 0.4× CMD (10 %
cell-free broth), which solubilized 35 and 22 % of the
oil, respectively (Fig. 3b). When compared to a com-
mercial surfactant, the cell-free broth solubilized fuel oil
better than SDS at the same concentration (1× CMC or
1× CMD) (Fig. 3b). The fuel oil solubilization activity
of SDS was almost similar to water at <20 %
(800 mg L−1). The low activity of SDS was probably
due to its higher hydrophilicity than the G. westfalica
GY40 biosurfactant. According to Khondee et al.
(2015), the GY40 biosurfactant tail should have 18
carbons from soybean oil substrate. In contrast, the
hydrophobic part of SDS consists of 12 carbons, which
make it more hydrophilic than GY40 biosurfactant. To
a
60
55
Surface tension (mN m-1)
50
45
40
35
30
25
20
0 25
BSF dilution (%)
b 80
100
Fuel oil solubilization (%)
80
60
40
20
0 0
Water SDS BSF
(10%)
Fig. 3 Surface tension (a) and solubilization activity (b) of the
diluted culture broth (BSF) containing G. westfalica GY40
biosurfactant. The initial concentration of fuel oil was 4 g L−1.
BSF (100 %) is the undiluted culture broth. Sodium dodecyl
sulfate (SDS) at CMC (0.2 %) was used as a reference surfactant
Water Air Soil Pollut (2016) 227: 325
further increase the solubilization of fuel oil, the
biosurfactant could be extracted from cell-free broth
for the preparation of concentrated solution (>4× CMD).
The cell-free broth and Slickgone NS (a commercial
dispersant) had comparable fuel oil displacement effi-
ciencies (75–90 %) when applied at 1:0.8–1:10 DOR
(Fig. 4). Nonetheless, the cell-free broth had decreased
efficiency at 1:25 DOR, while the efficiency of
Slickgone NS did not change (Fig. 4). Thus, the cell-
free broth should be applied as a dispersant at 1:10
DOR. To lower the application dose, the cell-free broth
could be spray dried to obtain concentrated
biosurfactant powder like Saeki et al. (2009), who pro-
duced JE1058BS, a solvent-free oil spill dispersant from
Gordonia sp. JE-1058. Nonetheless, the direct use of
cell-free broth from G. westfalica GY40 would be more
convenient and cost effective for on-site production of
the dispersant.
3.4 Stability of Biosurfactant
The stability of the biosurfactant in cell-free broth was
tested to evaluate the possibility for an ex situ applica-
tion. Results showed that the biosurfactant was stable
within a wide range of NaCl concentrations (2–10 %)
and high temperatures (50–121 °C) (Fig. 5a, b). More-
over, the presence of NaCl led to a decrease in surface
tension from 35 to 32 mN m−1. The GY40 biosurfactant
BSF Slickgone NS
50 75 100 100
Fuel oil displacement (%)
60
40
20
BSF BSF 1:0.8 1:1 1:2 1:5 1:10 1:25
(25%) (100%) Dispersant to oil ratio (DOR)
Fig. 4 Oil displacement efficiency against fuel oil of cell-free
broth containing G. westfalica GY40 biosurfactant (100 % BSF)
and Slickgone NS Type 2/3 at different dispersant to oil ratios
(DOR). Note: There was no fuel oil displacement when using DI
water
Water Air Soil Pollut (2016) 227: 325 Page 9 of 13 325

a between hydrogen ions from HCl and the anionic polar

45
ends of the biosurfactant molecules (Déziel et al. 1999).
Surface tension (mN m-1)

40 Thus, the loss of biosurfactant molecules caused a de-

crease in its capability to reduce surface tension. The
35
results confirmed that the biosurfactant could be applied
30 to seawater as well as to other alkaline environments.
25
3.5 Fuel Oil Biodegradation in Nutrient Seawater
20 Medium
0 2 4 6 8 10
NaCl (%) The application of biosurfactant from G. westfalica
b
45 GY40 (0.5× CMD cell-free broth) to remove fuel oil at
1 g L−1 was initially carried out with nutrient seawater
Surface tension (mN m-1)

40
medium that provided the optimal conditions for the oil-
35 degrading bacterium, Gordonia sp. JC11. The removal
of fuel oil by biodegradation occurred more rapidly in
30 the presence of biosurfactant, with 63 % oil removed on
25 day 2 and 80 % on day 6 (Fig. 1a). During the same
period, the immobilized bacteria alone could remove 39
20 and 70 % on days 2 and 6, respectively. The uninocu-
50 60 70 80 90 100 110 120
lated control set showed around 10–20 % fuel oil re-
Temperature ( C)
c moval at the end of experiment. Consequently, the major
45 mechanism for fuel oil removal was expected to be
Surface tension (mN m-1)

biodegradation by Gordonia sp. JC11. However, there

40
35
30
25
20
2 4 6 8
pH
Fig. 5 Effect of a NaCl; b temperature; and c pH on biosurfactant
produced from G. westfalica GY40. The surface tension of undi-
luted culture broth was measured after exposure to different
conditions
is postulated to be a glycolipid; thus, its hydrophilic
domain would contain a hydroxyl group that confers
anionic properties. Consequently, positive sodium ions
from NaCl can bond with the negatively charged hydro-
philic part of the biosurfactant molecule resulting in the
enhancement of biosurfactant solubilization and micelle
formation (Israelachvili 1994). At alkaline pH, the
biosurfactant had high stability with surface tension
values around 34–35 mN m−1. In contrast, the surface
tension increased up to around 40 mN m−1 in acidic
conditions (Fig. 5c). Between pH 2.0 to 6.0, the
biosurfactant tends to precipitate due to bonding
was also a reduction in fuel oil due to adsorption on the
PUF, and the solubilization of fuel oil in biosurfactant
micelles, as seen from the higher fuel oil removal (31 %)
in the treatment containing both PUF-immobilized bac-
teria and cell-free broth (JC11 + BSF) than other treat-
ments on day 0 (Fig. 1a).
10 12 G l yc o l i p i d b i o s u r f a c t a n t s f r o m t h e c l a s s
Actinobacteria are reported to have antimicrobial prop-
erties (Franzetti et al. 2010). However, the number of
bacteria on PUF with and without biosurfactant was
almost similar at 8 log CFU g−1 PUF, which confirmed
that the biosurfactant was not toxic to the oil-degrading
bacterium (Fig. 6b). This may be due to the fact that
both bacteria used in this study were from the genus
Gordonia. Many researchers select a single bacterium
with both biosurfactant production and hydrocarbon
degradation activities for petroleum remediation.
However, biosurfactant is usually produced as a
secondary metabolite; thus, a longer incubation time is
required for hydrocarbon reduction. Similar to this
study, Hua et al. (2004) found that the addition of
extracted biosurfactant produced by Candida antarctica
T-34 increased the biodegradation of 8 % crude oil by
the same yeast in batch cultures from 53 to 75 % within
6 days. Another advantage of producing the
325 Page 10 of 13 Water Air Soil Pollut (2016) 227: 325

biosurfactant separately is the possibility to use low-cost a

substrates. For example, Noparat et al. (2014) produced 100 JC11 JC11 + BSF Control
biosurfactant from Sphingobacterium spiritvorum AS43
80

Fuel oil removal (%)

using molasses and applied the extracted biosurfactant
for the degradation of 5 % used lubricant oil. They 60
found that S. spiritvorum AS43 with additional
biosurfactant degraded 25 % more oil than the treatment 40
with only bacterial cells.
Besides solubilization, the biosurfactant emulsified 20
the fuel oil, as seen in Fig. 6c as a milky medium.
0
Hydrocarbon biodegradation can happen via three
0 2 4 6 8
mechanisms viz. the utilization of (a) soluble organic Time (days)
molecules, (b) pseudosolubilized hydrocarbons by sur-
factants, and (c) stabilized emulsions (Nievas et al. b
2008). Thus, the emulsification process we observed in 9.0 JC11 JC11 + BSF
Fig. 6c also enhanced fuel oil biodegradation. Different

Gordonia sp. JC11 number

petroleum hydrocarbons give different degradation re- 8.5

(log CFU g-1 PUF)

sults due to varying compositions of hydrocarbons.
Chanthamalee and Luepromchai (2012) reported that 8.0
PUF-immobilized Gordonia sp. JC11 can remove
53 % of 1 g L−1 waste lubricant in nutrient seawater 7.5
medium after 5 days. In this study, the ability of
Gordonia sp. JC11 without biosurfactant to degrade fuel 7.0
oil was around 70 % (Fig. 6a). It is therefore possible to 0 2 4 6 8
increase the degradation of the waste lubricant as well as Time (days)
other petroleum hydrocarbons by applying the
biosurfactant along with the bacteria. c

3.6 Fuel Oil Biodegradation in Various Seawater

Samples

Indigenous microorganisms in seawater may play an JC11 + Control

important role in petroleum degradation and many en-
vironmental parameters can affect the activity of
biosurfactant. Thus, we further tested the biosurfactant
and immobilized bacteria with three seawater samples
collected along the eastern shoreline of Thailand. The
characteristics of each seawater sample are shown in
Table 1. All collected seawater samples had much lower
nitrogen and phosphorus contents than the nutrient sea-
water medium, but their pH and salinity were in the
same range at 8 and 3.4 %, respectively (Table 1). The
seawater samples were slightly contaminated with pe-
troleum hydrocarbons (3–6 mg L−1) and contained a
low number of petroleum-degrading bacteria (4.5–4.7
log CFU mL−1) (Table 1). Thus, the seawater was spiked
with 1 g L−1 fuel oil. The bioaugmentation experiment
was conducted by adding 0.5× CMD cell-free broth and
PUF-immobilized bacteria to the seawater and
JC11
BSF
Fig. 6 Removal of 1 g L−1 fuel oil from nutrient seawater medium
after adding PUF-immobilized Gordonia sp. JC11 and cell-free
broth containing biosurfactant (JC11 + BSF) (a). Control was un-
inoculated medium, while JC11 was a microcosm without
biosurfactant. The changes in bacterial numbers and example of
microcosms are shown in b and c, respectively
incubated for 6 days. Percent petroleum removals were
60, 76, and 60 % in bioaugmented seawater samples
from Bangsan, Maptaphut, and Samed Island, respec-
tively (Fig. 7a). On the other hand, the uninoculated
control set removed between 27 and 35 % fuel oil
(Fig. 7a), which was almost two times higher than the
control nutrient seawater medium (Fig. 6a). The highest
Water Air Soil Pollut (2016) 227: 325 Page 11 of 13 325

a by indigenous bacteria at an average of 30 to

100 Control JC11 + BSF
65 % (Fig. 7a). Regardless of seawater samples,
the added bacteria were able to survive and grow
Fuel oil removal (%)

80
well. The numbers of Gordonia sp. JC11 on PUF
60 from all samples were almost the same as in the
nutrient seawater medium at 8–9 log CFU g−1
40 PUF after 6 days (Fig. 7b). Some bacteria leached
from PUF into the aqueous phase at 7–8 log
20
CFU mL−1. The high bacterial number suggested
0 that they played a major role in fuel oil removal.
Bangsan Maptaphut Samed Compared to the nutrient seawater medium in the
Seawater sample
b previous experiment, all seawater samples had
on PUF In aqueous
lower oil removal efficiency. This corresponded
(log CFU g-1 PUF or log CFU mL-1)

9 with the low level of nutrients in natural seawater

Gordonia sp. JC11 number

8 (Table 1). To increase biodegradation of fuel oil in

7 the environment, the biosurfactant can be applied
6 with a small amount of fertilizer to promote the
5 activity of oil-degrading bacteria. For example,
4 Gallego et al. (2006) reported that the application
3 of oleophilic fertilizer S200 enhanced the depletion
2
of hydrocarbons ranging from 10 % up to 30 %
depending on the composition of fuel oil and at
1
Bangsan Maptaphut Samed the same time increased the growth of hydrocarbon
degrading microbial populations.
Seawater sample
Fig. 7 Effect of seawater samples on the removal efficiency of
1 g L−1 fuel oil after 6 days incubation. Percent removal of fuel oil
was monitored after adding PUF-immobilized Gordonia sp. JC11 4 Conclusions
and cell-free broth containing biosurfactant (JC11 + BSF) (a).
Control was uninoculated seawater. The number of Gordonia sp. In conclusion, a glycolipid biosurfactant could be
JC11 on PUF and in aqueous phase was determined at the end of
the experiment (b) conveniently produced from chitosan-immobilized
G. westfalica GY40 with soybean oil as the carbon
substrate. The cell-free broth contained sufficient
concentrations of the biosurfactant that could be
oil removal efficiencies for both control and bioaugmen- used to solubilize and disperse fuel oil. The pres-
tation treatments were found for Maptaphut seawater ence of biosurfactant also enhanced the activity of
samples, which corresponded to the highest number of oil-degrading bacteria, Gordonia sp. JC11, for fuel
petroleum-degrading bacteria in the seawater at the be- oil degradation in various seawater samples. Con-
ginning of the study (Table 1). The results indicated that sequently, the biosurfactant from G. westfalica
the indigenous bacteria degraded some parts of the fuel GY40 could be used as a dispersant during a fuel
oil and the application of biosurfactant could further oil spill or applied together with Gordonia sp.
stimulate their oil degradation activity. Similarly, Saeki JC11 for efficient fuel oil biodegradation in coastal
et al. (2009) reported that the biosurfactant-based reme- environment. Future research should include the
diation agent prepared from the culture broth of scale-up production of this biosurfactant and the
Gordonia sp. JE-1058 increased the number of marine determination of biosurfactant activity for other
bacteria and had low toxicity toward marine larval petroleum hydrocarbons.
species.
The addition of oil-degrading bacteria along
Acknowledgments The research was partially funded by the
with the biosurfactant resulted in a greater than PTT Research and Technology Institute and the Center of Excel-
twofold increase in the extent of fuel oil removal lence on Hazardous Substance Management (HSM).
325 Page 12 of 13
References
Bharali, P., Das, S., Konwar, B. K., & Thakur, A. J. (2011). Crude
biosurfactant from thermophilic Alcaligenes faecalis: feasi-
bility in petro-spill bioremediation. International
Biodeterioration & Biodegradation, 65, 682–690.
Chanthamalee, J., & Luepromchai, E. (2012). Isolation and appli-
cation of Gordonia sp. JC11 for removal of boat lubricants.
Journal of General and Applied Microbiology, 58, 19–31.
Chanthamalee, J., Wongchitphimon, T., & Luepromchai, E.
(2013). Treatment of oily bilge water from small fishing
vessels by PUF-immobilized Gordonia sp. JC11. Water, Air,
and Soil Pollution, 224, 1601.
Chao, M., Shen, X., Lun, F., Shen, A., & Yuan, Q. (2012). Toxicity
of fuel oil water accommodated fractions on two marine
microalgae, Skeletonema costatum and Chlorela spp.
Bulletin of Environmental Contamination and Toxicology,
88, 712–716.
Déziel, E., Lépine, F., Dennie, D., Boismenu, D., Mamer, O. A., &
Villemur, R. (1999). Liquid chromatography/mass spectrom-
etry analysis of mixtures of rhamnolipids produced by
Pseudomonas aeruginosa strain 57RP grown on mannitol
or naphthalene. Biochimica et Biophysica Acta (BBA) -
Molecular and Cell Biology of Lipids, 1440, 244–253.
Dogan, I., Pagilla, K. R., Webster, D. A., & Stark, B. C. (2006).
Expression of Vitreoscilla hemoglobin in Gordonia amarae
enhances biosurfactant production. Journal of Industrial
Microbiology and Biotechnololgy, 33, 693–700.
Ferraz, C., Araújo, Á. A., & Pastore, G. M. (2002). The influence
of vegetable oils on biosurfactant production by Serratia
marcescens. Applied Biochemistry and Biotechnology, 98,
841–847.
Franzetti, A., Bestetti, G., Caredda, P., La Colla, P., & Tamburini,
E. (2008). Surface-active compounds and their role in the
access to hydrocarbons in Gordonia strains. FEMS
Microbiology Ecology, 63, 238–248.
Franzetti, A., Caredda, P., La Colla, P., Pintus, M., Tamburini, E.,
Papacchini, M., & Bestetti, G. (2009a). Cultural factors af-
fecting biosurfactant production by Gordonia sp. BS29.
International Biodeterioration & Biodegradation, 63, 943–
947.
Franzetti, A., Caredda, P., Ruggeri, C., La Colla, P., Tamburini, E.,
Papacchini, M., & Bestetti, G. (2009b). Potential applications
of surface active compounds by Gordonia sp. strain BS29 in
soil remediation technologies. Chemosphere, 75, 801–807.
Franzetti, A., Gandolfi, I., Bestetti, G., Smyth, T. J. P., & Banat, I.
M. (2010). Production and applications of trehalose lipid
biosurfactants. European Journal of Lipid Science and
Technology, 112, 617–627.
Gallego, J., González-Rojas, E., Peláez, A., Sánchez, J., García-
Martínez, M., Ortiz, J., Torres, T., & Llamas, J. (2006).
Natural attenuation and bioremediation of Prestige fuel oil
along the Atlantic coast of Galicia (Spain). Organic
Geochemistry, 37, 1869–1884.
Hua, Z., Chen, Y., Du, G., & Chen, J. (2004). Effects of
biosurfactants produced by Candida antarctica on the bio-
degradation of petroleum compounds. World Journal of
Microbiology and Biotechnology, 20, 25–29.
Water Air Soil Pollut (2016) 227: 325
Inès, M., & Dhouha, G. (2015). Glycolipid biosurfactants: poten-
tial related biomedical and biotechnological applications.
Carbohydrate Research, 416, 59–69.
Israelachvili, J. (1994). The science and applications of emul-
si on s —an over vi ew. Co l l o i d s a nd Su rf a c e s A :
Physicochemical and Engineering Aspects, 91, 1–8.
Jackisch-Matsuura, A. B., Santos, L. S., Eberlin, M. N., Faria, A.
F. D., Matsuura, T. M., Grossman, J., & Durrant, L. R.
(2014). Production and characterization of surface-active
compounds from Gordonia amicalis. Brazilian Archives of
Biology and Technology, 57, 138–144.
Khondee, N., Tathong, S., Pinyakong, O., Müller, R.,
Soonglerdsongpha, S., Ruangchainikom, C., Tongcumpou,
C., & Luepromchai, E. (2015). Lipopeptide biosurfactant
production by chitosan-immobilized Bacillus sp. GY19 and
their recovery by foam fractionation. Biochemical
Engineering Journal, 93, 47–54.
Kim, H.-S., Jeon, J.-W., Lee, H.-W., Park, Y.-I., Seo, W.-T., Oh,
H.-M., Katsuragi, T., Tani, Y., & Yoon, B.-D. (2002).
Extracellular production of a glycolipid biosurfactant,
mannosylerythritol lipid, from Candida antarctica.
Biotechnology Letters, 24, 225–229.
Kügler, J. H., Le Roes-Hill, M., Syldatk, C., & Hausmann, R.
(2015). Surfactants tailored by the class Actinobacteria.
Frontiers in Microbiology, 6, 212.
Maddikeri, G. L., Gogate, P. R., & Pandit, A. B. (2015). Improved
synthesis of sophorolipids from waste cooking oil using fed
batch approach in the presence of ultrasound. Chemical
Engineering Journal, 263, 479–487.
Mercer, K., & Trevors, J. T. (2011). Remediation of oil spills in
temperate and tropical coastal marine environments. The
Environmentalist, 31, 338–347.
Mnif, I., & Ghribi, D. (2015). Microbial derived surface active
compounds: properties and screening concept. World Journal
of Microbiology and Biotechnology, 31, 1001–1020.
Morya, V. K., Park, J. H., Kim, T. J., Jeon, S., & Kim, E. K. (2013).
Production and characterization of low molecular weight
sophorolipid under fed-batch culture. Bioresource
Technology, 143, 282–288.
Nievas, M. L., Commendatore, M. G., Esteves, J. L., & Bucalá, V.
(2008). Biodegradation pattern of hydrocarbons from a fuel
oil-type complex residue by an emulsifier-producing micro-
bial consortium. Journal of Hazardous Materials, 154, 96–
104.
Noparat, P., Maneerat, S., & Saimmai, A. (2014). Application of
biosurfactant from Sphingobacterium spiritivorum AS43 in
the biodegradation of used lubricating oil. Applied
Biochemistry and Biotechnology, 172, 3949–3963.
Pepi, M., Focardi, S., Lobianco, A., Angelini, D. L., Borghini, F.,
& Focardi, S. E. (2013). Degradation of fatty acids and
production of biosurfactant as an added value, by a bacterial
strain Pseudomonas aeruginosa DG2a isolated from aqua-
culture wastewaters. Water, Air, & Soil Pollution, 224, 1772.
Pizzul, L., Castillo, M. d., & Stenström, J. (2006). Characterization
of selected actinomycetes degrading polyaromatic hydrocar-
bons in liquid culture and spiked soil. World Journal of
Microbiology and Biotechnology, 22, 745–752.
Prieto, L., Michelon, M., Burkert, J., Kalil, S., & Burkert, C.
(2008). The production of rhamnolipid by a Pseudomonas
aeruginosa strain isolated from a southern coastal zone in
Brazil. Chemosphere, 71, 1781–1785.
Water Air Soil Pollut (2016) 227: 325
Saeki, H., Sasaki, M., Komatsu, K., Miura, A., & Matsuda, H.
(2009). Oil spill remediation by using the remediation agent
JE1058BS that contains a biosurfactant produced by
Gordonia sp. strain JE-1058. Bioresource Technology, 100,
572–577.
Silva, R. D. C. F., Almeida, D. G., Rufino, R. D., Luna, J. M.,
Santos, V. A., & Sarubbo, L. A. (2014). Applications of
biosurfactants in the petroleum industry and the remediation
of oil spills. International Journal of Molecular Sciences, 15,
12523–12542.
Page 13 of 13 325
Svensson, M. (2010). Surfactants based on natural fatty acids. In
Kjellin, M. & Johansson, I. (Ed.), Surfactants from renewable
resources (pp. 3–19). Chichester: Wiley
Tesoro Corporation. (2013). Fuel oil [Material Safety Data Sheet].
https://tsocorpsite.files.wordpress.com/2014/08/utility-fuel-
oil.pdf. Accessed 4 November 2015.
Yao, S., Zhao, S., Lu, Z., Gao, Y., Lv, F., & Bie, X. (2015). Control
of agitation and aeration rates in the production of surfactin in
foam overflowing fed-batch culture with industrial fermenta-
tion. Revista Argentina de Microbiología, 47, 344–349.