Zhang et al.

Microb Cell Fact (2016) 15:168

DOI 10.1186/s12934-016-0574-8 Microbial Cell Factories

RESEARCH Open Access

Production of lipopeptide biosurfactants

by Bacillus atrophaeus 5‑2a and their potential
use in microbial enhanced oil recovery
Junhui Zhang1, Quanhong Xue1*, Hui Gao1, Hangxian Lai1 and Ping Wang2

Abstract 
Background:  Lipopeptides are known as promising microbial surfactants and have been successfully used in
enhancing oil recovery in extreme environmental conditions. A biosurfactant-producing strain, Bacillus atrophaeus
5-2a, was recently isolated from an oil-contaminated soil in the Ansai oilfield, Northwest China. In this study, we evalu-
ated the crude oil removal efficiency of lipopeptide biosurfactants produced by B. atrophaeus 5-2a and their feasibility
for use in microbial enhanced oil recovery.
Results:  The production of biosurfactants by B. atrophaeus 5-2a was tested in culture media containing eight carbon
sources and nitrogen sources. The production of a crude biosurfactant was 0.77 g L−1 and its surface tension was
26.52 ± 0.057 mN m−1 in a basal medium containing brown sugar (carbon source) and urea (nitrogen source). The
biosurfactants produced by the strain 5-2a demonstrated excellent oil spreading activity and created a stable emul-
sion with paraffin oil. The stability of the biosurfactants was assessed under a wide range of environmental conditions,
including temperature (up to 120 °C), pH (2–13), and salinity (0–50 %, w/v). The biosurfactants were found to retain
surface-active properties under the extreme conditions. Additionally, the biosurfactants were successful in a test to
simulate microbial enhanced oil recovery, removing 90.0 and 93.9 % of crude oil adsorbed on sand and filter paper,
respectively. Fourier transform infrared spectroscopy showed that the biosurfactants were a mixture of lipopeptides,
which are powerful biosurfactants commonly produced by Bacillus species.
Conclusions:  The study highlights the usefulness of optimization of carbon and nitrogen sources and their effects
on the biosurfactants production and further emphasizes on the potential of lipopeptide biosurfactants produced
by B. atrophaeus 5-2a for crude oil removal. The favorable properties of the lipopeptide biosurfactants make them
good candidates for application in the bioremediation of oil-contaminated sites and microbial enhanced oil recovery
process.
Keywords:  Microbial enhanced oil recovery, Biosurfactant, Bacillus atrophaeus, Surface tension, Crude oil removal

Background unsaturated, saturated, or hydrocarbon fatty acids [2].

Biosurfactants are a heterogeneous group of surface- Therefore, biosurfactants reduce surface tension and
active molecules produced by microorganisms, such as interfacial tension in both aqueous solutions and hydro-
bacteria, fungi, and yeasts [1]. The molecular structures carbon mixtures and form micelles and microemulsions
of biosurfactants include a hydrophilic moiety, compris- between the two phases [2, 3]. Such surface proper-
ing an amino acid or peptide, anions or cations, mono-, ties make biosurfactants good candidates for enhancing
di-, or polysaccharides; and a hydrophobic moiety of oil recovery [4, 5]. Bailey et  al. [6] reported that a bio-
surfactant flooding process, using a low concentration
(35–41  ppm) of biosurfactants produced by Bacillus
*Correspondence: xuequanhong6070@163.com
1
College of Natural Resources and Environment, Northwest A & F
mojavensis strain JF-2, resulted in high oil recovery, of up
University, 3 Taicheng Road, 712100 Yangling, China to 35–45 %. In recent years, an increase in concern about
Full list of author information is available at the end of the article

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Zhang et al. Microb Cell Fact (2016) 15:168 Page 2 of 11

environmental protection has caused the development with porous media in coreflooding experiments as a ter-
of cost-effective bioprocesses for biosurfactants produc- tiary-recovery stage. B. subtilis W19 showed high poten-
tion [7]. The use of biosurfactants that have a comparable tial of oil extraction during ex situ MEOR applications in
enhanced oil recovery performance is preferable [4]. which a total of 23  % of residual oil was extracted pro-
Based on the types of biosurfactant-producing micro- duced after biosurfactant and concentrated-biosurfactant
bial species and the nature of their chemical structures, injection [19]. The main drawbacks of lipopeptide biosur-
biosurfactants can be roughly divided into four groups: factants for MEOR are low yields and high production
lipopeptides and lipoproteins, glycolipids, phospholip- costs [20].
ids, and polymeric surfactants [8]. Among these four The aims of this work were to: (1) improve lipopeptide
groups, the best-known compounds are lipopeptides, biosurfactant production yields, through selection of an
produced by Bacillus species, and glycolipids, produced appropriate bacteria strain and optimization of the car-
by Pseudomonas species [9]. In general, mixtures of bon and nitrogen sources in the culture media; (2) char-
cyclic lipopeptides are built from variants of heptapep- acterize the biosurfactants produced by the bacteria
tides and hydroxy fatty acid chains [8], while glycolip- selected; (3) assess the surface activities and potential of
ids are mixtures of rhamnolipid homologs, composed the biosurfactants produced; and (4) determine the feasi-
of one or two rhamnose molecules linked to one or two bility for their use in MEOR.
hydroxy fatty acid chains [10]. The two types of bio-
surfactants improve oil recovery by reducing the inter- Results and discussion
facial tension and altering the wettability of reservoir Effect of carbon source on biosurfactant production
rock [11]. Glycolipids have been extensively studied in Bacillus atrophaeus 5-2a was able to grow and produce
microbial enhanced oil recovery (MEOR) experiments biosurfactants utilizing all of the carbon sources tested,
and lipopeptides, such as surfactin and iturins, have also except paraffin (Table  1). When liquid paraffin was the
been found effective in similar studies [12]. Surfactin is sole carbon source, there was some growth, but it was
known as a powerful microbial surfactant with high sur- lower than that observed with the water-soluble carbon
face activities and has been successfully used in enhanc- sources (Table  1). Several studies have shown, with dif-
ing oil recovery [12–14]. ferent Bacillus strains, that if hydrocarbons (including
Biosurfactants MEOR represents a promising method n-hexadecane and paraffin) are the only carbon source,
to recover a substantial proportion of the residual oil bacterial growth and biosurfactant production is either
from marginal oil fields [15, 16]. Biosurfactants can be completely inhibited [21, 22], or severely limited [16].
implemented in two ways: they can be produced either ex The highest dry cell weights (0.86 and 0.80  g  L−1,
situ to be injected into the reservoir or in situ by indige- respectively) were obtained using maltose and glycerol as
nous or injected microorganisms [15]. The first approach the carbon source. The lowest surface tension (ST) of the
involves the production of biosurfactants above ground culture supernatant (25.82 mN m−1) was obtained when
by fermentation and therefore requires expensive equip- mannitol was the sole carbon source. However, the other
ment, including bioreactor and purification systems [16]. carbohydrate sources tested also decreased ST in the
The second method is more favorable from an economic range of 26.11–26.39 mN m−1, except paraffin. Glucose,
point of view, but the indigenous microorganisms need molasses, and palm oil have been found to be the best
to be identified and their capacity to grow and produce carbon sources for the growth of Bacillus isolates [9, 14].
sufficient amounts of biosurfactants in oil reservoirs Additionally, Bacillus strains were reported to grow uti-
assessed. Unfortunately, this process cannot be com- lizing glycerol and sucrose as the sole carbon sources and
pletely manipulated and this places limitations on the the STs of the culture broths were 27.1 and 27.9 mN m−1,
reservoirs where microorganisms can be used for in situ respectively [16, 23].
treatment [17]. The highest emulsifying activity of the culture was
There have been several successful studies into the obtained using brown sugar as the carbon source
application of biosurfactants during in  situ or ex situ (61.81  %), followed by glucose (58.34  %), glycerol
field tests [12]; Recently, a field study demonstrated that (57.43  %), starch (56.85  %), sucrose (56.76  %), maltose
approximately nine times the minimum concentration (54.80  %) and mannitol (54.11  %). Raw glycerol from
of biosurfactants required to mobilize oil was produced the biodiesel industry has previously been identified
in  situ by a consortium of Bacillus strains, resulting in as a potential low-cost carbon source for biosurfactant
the recovery of substantial amount of oil entrapped in production, with an emulsification efficiency of 67.6  %
the limestone reservoir of the Bebee field, Pontotoc City, against crude oil [24]. Furthermore, Al-Wahaibi et al. [14]
Oklahoma, USA [18]. Additionally, a study tested the found that the biosurfactants produced by Bacillus sub-
interaction of biosurfactant produced by B. subtilis W19 tilis B30 had a high emulsifying activity against various
Zhang et al. Microb Cell Fact (2016) 15:168 Page 3 of 11

Table 1  Dry cell weight (g  L−1), crude biosurfactant yield (g  L−1), oil spreading (cm), emulsification index (%), and  sur-
face tension (mN m−1) obtained for Bacillus atrophaeus 5-2a grown in mineral salt solution with different carbon sources
at 30 °C for 5 days
Carbon source Dry cell weight Crude biosurfactant Oil spreading Emulsification Surface tension
(g L−1) yield (g L−1) (cm) index (%) (mN m−1)

Brown sugar 0.56 ± 0.0071c 0.95 ± 0.071b 18.4 ± 0.10b 61.81 ± 0.98a 26.12 ± 0.085c

Sucrose 0.37 ± 0.028e 0.74 ± 0.085c 18.1 ± 0.16c 56.76 ± 0.25c 26.32 ± 0.035b
Glucose 0.33 ± 0.021e 0.53 ± 0.071d 17.2 ± 0.12e 58.34 ± 0.33b 26.38 ± 0.035b
Maltose 0.86 ± 0.035a 0.82 ± 0.085bc 18.2 ± 0.10bc 54.80 ± 0.18d 26.11 ± 0.028c
Starch 0.51 ± 0.014cd 0.71 ± 0.071c 17.7 ± 0.12d 56.85 ± 0.13c 26.39 ± 0.099b
Mannitol 0.48 ± 0.0071d 1.11 ± 0.042a 19.6 ± 0.071a 54.11 ± 0.085d 25.82 ± 0.028d
Glycerol 0.80 ± 0.014b 0.72 ± 0.028c 17.8 ± 0.12d 57.43 ± 0.14bc 26.32 ± 0.057b
Paraffin 0.14 ± 0.028f 0.06 ± 0.028e 8.2 ± 0.16f 0.00 ± 0.00e 40.49 ± 0.057a
Values are presented as the mean ± standard deviation (n = 3). Different superscript letters within a column indicate significant differences (P < 0.05) by Duncan’s
multiple range test

hydrocarbons when glucose and molasses were used as The lowest ST, which corresponded to the highest
the carbon sources. crude biosurfactant yield and the biggest the diameter of
The amount of biosurfactants produced varied from oil spreading, was obtained when urea was used as the
0.53 to 1.11  g  L−1 and the diameter of oil spreading sole nitrogen source (26.43 mN m−1). The other nitrogen
ranged from 17.2 to 19.6  cm, depending on the carbon sources tested also offered good results in terms of ST
source used (Table  1). The highest crude biosurfactant (26.65–29.51 mN m−1), crude biosurfactant yield (0.42–
yield and diameter of oil spreading were obtained when 0.73 g L−1) and diameter of oil spreading (14.2–19.2 cm)
mannitol was used as the carbon source. In the second for the culture supernatant. These results agree with
place, the crude biosurfactant yield and diameter of oil Makkar and Cameotra [25] who reported that the maxi-
spreading reached 0.95  g  L−1 and 18.4  cm, respectively, mum amount of biosurfactant, and ST values between
with brown sugar as the carbon source. These results 29 and 29.5 mN  m−1, were produced by a thermophilic
are in agreement with the ST results obtained for B. B. subtilis when urea or nitrate ions were supplied as the
atrophaeus 5-2a, but in contrast with the highest emulsi- nitrogen sources.
fying activity (achieved with brown sugar). This indicates The highest emulsifying activity was observed when
that various types of biosurfactants with different prop- (NH4)2SO4 and NaNO3 were used (61.16 and 61.23  %,
erties were synthesized by this strain, depending on the respectively), followed by KNO3 (61.14  %), urea
carbon source used. (60.54  %), beef extract (59.50  %), peptone (59.47  %) and
NH4Cl (59.34  %). This is in agreement with the results
Effect of nitrogen source on biosurfactant production reported by Dastgheib et al. [22], in which sodium nitrate
Bacillus atrophaeus 5-2a was able to utilize all of the was the best substrate for emulsifier production, followed
nitrogen sources tested (Table  2); growth was accom- by urea, yeast extract and peptone.
panied with biosurfactant production. The highest dry Among all of the carbon and nitrogen sources tested,
cell weight (0.78  g  L−1) was obtained using urea as the brown sugar and urea were found to be the most suitable
nitrogen source in the culture. For biosurfactant produc- carbon and nitrogen sources regarding the amounts of
tion, the nitrogen source can be inorganic (e.g., NaNO3, crude biosurfactant, diameter of oil spreading, emulsify-
NH4Cl, (NH4)2SO4, NH4NO3 or urea) or organic (e.g., ing activity and ST. They are also inexpensive and easily
beef extract, tryptone, or yeast extract). In previous stud- available, making their potential application in MEOR
ies, some B. subtilis strains could not use (NH4)2SO4 or economically feasible. Therefore, brown sugar and urea
KNO3 for microbial growth or biosurfactant production; were selected as the carbon and nitrogen sources for the
however, they could use NaNO3, NH4NO3 or KNO3 [21, remaining experiments.
25]. In this study, the fact that B. atrophaeus 5-2a could
grow and produce biosurfactants using all of the nitro- Comparison of the optimal media for biosurfactant
gen sources tested indicates that it is more competi- production
tive than previous Bacillus strains tested for industrial The potential use of Bacillus strains for biosurfactant
applications. production has been widely described in the literature
Zhang et al. Microb Cell Fact (2016) 15:168 Page 4 of 11

Table 2  Dry cell weight (g L−1), crude biosurfactant yield (g L−1), oil spreading (cm), emulsification index (%), and surface
tension (mNm−1) obtained for  Bacillus atrophaeus 5-2a grown in  mineral salt solution with  different nitrogen sources
at 30 °C for 5 days
Nitrogen source Dry cell weight Crude biosurfactant Oil spreading Emulsification Surface tension
(g L−1) yield (g L−1) (cm) index (%) (mN m−1)

Beef extract 0.64 ± 0.021e 0.47 ± 0.014c 16.5 ± 0.10f 59.50 ± 0.34c 27.64 ± 0.028b

Peptone 0.87 ± 0.057d 0.66 ± 0.028ab 18.8 ± 0.10b 59.47 ± 0.36c 26.65 ± 0.057d
Corn steep liquor 0.63 ± 0.028e 0.42 ± 0.085c 14.2 ± 0.16 g 10.41 ± 0.57d 29.51 ± 0.035a
Urea 0.99 ± 0.028c 0.78 ± 0.028a 19.2 ± 0.10a 60.54 ± 0.38ab 26.43 ± 0.021e
NH4Cl 1.41 ± 0.014a 0.55 ± 0.071bc 16.9 ± 0.10e 59.34 ± 0.18c 27.64 ± 0.014b
(NH4)2SO4 1.22 ± 0.014b 0.66 ± 0.085ab 17.2 ± 0.12d 61.16 ± 0.25a 27.42 ± 0.092c
NaNO3 0.85 ± 0.0071d 0.73 ± 0.042a 17.6 ± 0.12c 61.23 ± 0.59a 27.38 ± 0.099c
KNO3 0.47 ± 0.014f 0.53 ± 0.099bc 16.7 ± 0.16e 60.14 ± 0.19bc 27.60 ± 0.057b
Values are presented as the mean ± standard deviation (n = 3). Different superscript letters within a column indicate significant differences (P < 0.05) by Duncan’s
multiple range test

[14, 16, 20]. To the authors’ knowledge, however, no stud- the amount of biosurfactant (~0.77  g  L−1) was similar
ies have examined the production of biosurfactants by to the values reported by other authors using different
B. atrophaeus. In the study, B. atrophaeus 5-2a demon- substrates.
strated a higher ability to produce biosurfactants in the The biosurfactants produced using the BB and BU
BB medium than the BU medium. Its production of bio- media were able to create low STs of the supernatant,
surfactants in the BU medium was assessed to ascertain at 25.47 and 26.52 mN  m−1, respectively (Table  3). The
its potential to ferment cheaper raw materials (i.e., urea results show that urea is an efficient nitrogen source.
and brown sugar). There is evidence that the nitrogen source plays an
essential part in the biosurfactant production process
Biosurfactant yield and surface tension [26]. Elazzazy et  al. [27] showed that urea and NaNO3
The crude biosurfactant dried yield (after acid precipi- were the most efficient nitrogen sources for Virgibacillus
tation) was 1.01 g L−1 in the BB medium and 0.77 g L−1 salarius KSA-T; their culture produced a biosurfactant
in the BU medium, corresponding to a yield per gram of with minimal ST (29.5 mN  m−1) and maximum emul-
cell dry weight of 0.75 g g−1 and 0.81 g g−1, respectively sifying activity (82 %). Additionally, Ghribi and Ellouze-
(Table  3). Although the BB medium produced a higher Chaabouni [28] found that biosurfactant production in
yield than the BU medium, the nitrogen sources (beef their culture was highest using urea. Although there was
extract and peptone) in the medium meant it was more no significant difference between sodium nitrate, ammo-
expensive than the BU medium, which only contained nium nitrate, yeast extract, peptone or urea on biosur-
urea as a nitrogen source. In other studies, crude biosur- factant production, urea was chosen as the cheaper
factant yields of 0.30–2.3 g L−1 have been achieved using nitrogen source, in comparison to sodium nitrate [21,
a mineral medium supplemented with date molasses and 25].
NH4NO3 as carbon and nitrogen sources [14, 17]. Sousa
et al. [23] found 0.44 g L−1 of biosurfactant was produced Emulsifying activity
by B. subtilis LAMI005 using a mineral medium contain- The emulsifying activity of the biosurfactants produced
ing raw glycerol and (NH4)2SO4. In the present study, using the BB and BU media was appreciable, against

Table 3  Dry cell weight (g L−1), crude biosurfactant yield (g L−1), oil spreading (cm), emulsification index (%), and surface
tension (mN m−1) obtained from Bacillus atrophaeus 5-2a in BB and BU media
Medium Dry cell Crude biosurfactant Oil spreading Emulsification Surface tension Yield
weight (gL−1) yield (gL−1) (cm) index (%) (mN m−1) (g g−1)

BB 1.34 ± 0.014a 1.01 ± 0.016a 19.9 ± 0.071a 54.73 ± 0.085b 25.47 ± 0.042b 0.75

BU 0.95 ± 0.028b 0.77 ± 0.014b 19.1 ± 0.10b 59.49 ± 0.33a 26.52 ± 0.057a 0.81
Values are presented as the mean ± standard deviation (n = 3). Different superscript letters within a column indicate significant differences (P < 0.05) by Duncan’s
multiple range test. BB for the fermentation medium used brown sugar, beef extract and peptone as the carbon and nitrogen sources; BU for the optimal medium
used brown sugar and inorganic nitrogen urea as the carbon and nitrogen sources. The same as below, unless otherwise specified
Zhang et al. Microb Cell Fact (2016) 15:168 Page 5 of 11

paraffin oil (Table 3). A significantly higher emulsification authors have described similar results, in terms of surface
index (E24, 59.49 %) was obtained using the BU medium activity [30, 31], and performance [14, 29], following heat
compared to the BB medium (E24, 54.73 %) (P < 0.05). The treatment.
emulsification properties of a biosurfactant are of practi- There were minimum deviations in the diameter of
cal importance; good emulsification properties increase oil spreading and ST over the pH range of 6–13, and the
the potential environmental and industrial applica- emulsification activities of the biosurfactants were stable
tions of biosurfactants [5]. Formation of an oil-in-water above pH 7.0. Higher stability was observed under alka-
emulsion often leads to an improvement in the effective line compared to acidic conditions and the minimum ST
mobility ratio [12]. The cell-free broth produced by the was obtained at pH 6.0 (Fig. 2b). Under an acidic pH (pH
BU medium could probably enhance oil recovery, based 2.0 and 5.0) the biosurfactants showed much less activ-
on the results observed with paraffin oil. ity; the diameter of oil spreading and emulsification index
decreased, and the ST increased, due to precipitation of
Chemical characteristics of the biosurfactants the biosurfactants. These results indicate that increased
TLC showed four compounds with Rf values of 0.47, pH has a positive effect on surface activity and stability
0.57, 0.75 and 0.8, respectively, when ninhydrin reagent of the biosurfactants. Some reports have confirmed the
was sprayed, indicating the presence of amino acids. No stability of biosurfactants produced by Bacillus strains at
compounds were observed when sprayed with phenol– different pH values, but mostly under alkaline conditions
sulfuric acid, confirming the absence of sugar moiety. [5, 14].
The above results confirm the lipopeptide nature of the The surface activity of the biosurfactants produced
biosurfactants. Similar results for other lipopeptide bio- using both the BB and BU media varied with salinity of
surfactants, produced by B. subtilis, have been reported 0–50 % (w/v); when the salinity was lower than 9 %, the
[5, 24]. diameter of oil spreading, emulsification index and ST of
The FT-IR spectra of the biosurfactants produced the cell-free supernatants were constant. The diameter
by B. atrophaeus 5-2a show a characteristic band at of oil spreading and emulsification index decreased, and
3308.28  cm−1, indicating the presence of an –NH bond the ST increased, with higher salt concentrations; how-
(Fig.  1). The bands at 1652.40  cm−1 and 1540.97  cm−1 ever, the activity remained high at a salinity of 15 % (w/v).
indicate the presence of the –CO–N bond, while Even at the highest salt concentration (50  %, w/v), the
the bands at 2959.92–2928.66  cm−1 and 1456.85– biosurfactants produced in the BB and BU media still had
1387.09 cm−1 reflect the stretch (–CH) of CH2 and CH3 reasonable oil spreading activity and the STs were 36.84
groups, respectively, in the aliphatic chains. The absorp- mN m−1 and 38.65 mN m−1, respectively (Fig. 2c). Over-
tion peak, located at 1736.07 cm−1 indicates the presence all, relatively high stability, with respect to salinity, was
of ester carbonyl groups (–CO bond). The stretching observed in comparison with other studies that used B.
modes of the –NH, –CO–N and –CO bonds, and the subtilis, Nocardiopsis sp. B4 and Serratia marscecens [14,
–CH3 and –CH2 fractions, fall within the same range of 31, 32].
wave numbers as previously found; this indicates the sim- The biosurfactants produced by B. atrophaeus 5-2a
ilarity in structure of the biosurfactants produced by B. were stable over a range of environmental factors and
atrophaeus 5-2a with lipopeptides previously described maintained their surface activities. Oil reservoirs are
in the literature [5, 24]. harsh environments, with the potential of high salin-
ity and a wide range of pH values; the observed stabil-
Biosurfactant stability ity of the biosurfactants assessed in this study, over the
Biosurfactants are “green chemicals” used to enhance oil pH range of 6–13 and salinity concentrations of 0–15 %,
recovery. To use biosurfactants for ex situ MEOR, they indicates that they would be suitable for oil recovery in
need to be stable across a range of temperatures, pH and most reservoirs. These results show that the biosur-
salinities, to ensure wide applicability [14, 29]. The sur- factants from B. atrophaeus 5-2a are good candidates for
face activities of the biosurfactants produced using both application in MEOR.
the BB and BU media were quite stable over a wide range
of temperatures, from 20 to 120 °C (Fig. 2a). Heating the Removal of crude oil from filter papers and sand
cell-free supernatant up to 100  °C (or autoclaving it at Application of biosurfactants for MEOR is one of the
121  °C) had no significant effect on the surface activity most promising methods for recovering a substantial
of the biosurfactants. There were no significant differ- proportion of residual oil and has been receiving more
ences in the diameter of oil spreading, ST or emulsifica- and more attention recently [12]. Both of the superna-
tion activities before and after heating (P < 0.05). Several tants from the BB and BU media were able to remove
Zhang et al. Microb Cell Fact (2016) 15:168 Page 6 of 11

Fig. 1  FT-IR absorption spectra of biosurfactants produced by Bacillus atrophaeus 5-2a from ‘BB’ (a) and ‘BU’ media (b). BB for the fermentation
medium used brown sugar, beef extract and peptone as the carbon and nitrogen sources; BU for the optimal medium used brown sugar and
inorganic nitrogen urea as the carbon and nitrogen sources. The same as below, unless otherwise specified

the majority of crude oil adsorbed on filter paper and cell-free broth containing a biosurfactant produced by
sand (Fig.  3). The removal efficiencies from the filter B. subtilis PT2. Pereira et  al. [16], who removed crude
paper and sand by the supernatant of the BB medium oil from contaminated sands, found that three strains
were 94.3 and 94.0 %, respectively; that is, 7.4- and 10.1- of B. subtilis were effective in oil recovery from sand
fold that of the control. For the supernatant from the pores, with rates between 19 and 22  %. The fermenta-
BU medium, they were 93.1 and 90.0  %, respectively tion broths from the present study that contained bio-
(7.3- and 9.7-fold that of the control) (Table  4). Porn- surfactants from B. atrophaeus 5-2a were clearly highly
sunthorntawee et  al. [9] reported that 61.6  % of crude efficient in the crude oil removal tests, which is promis-
residual oil adsorbed in sand was removed using a ing for MEOR.
Zhang et al. Microb Cell Fact (2016) 15:168 Page 7 of 11

Fig. 2  Stability studies for biosurfactants produced by Bacillus atrophaeus 5-2a in ‘BB’ and ‘BU’ media, under different conditions of temperature (a),
pH (b), and salinity (c). Values represented the mean ± standard deviation (n = 3). D for oil spreading (cm); E24 for emulsification index (%); and ST
for surface tension (mN m−1)

Conclusions reduce the surface tension of the culture supernatant to

Bacillus atrophaeus 5-2a produced a potent biosur- 26.52 mN  m−1, and exhibited appreciable emulsifica-
factant with high surface activity and emulsification tion activity against paraffin oil (E24, 59.49  %). The bio-
property, when using a cheap mineral salt medium con- surfactants produced by the strain 5-2a from both the
taining brown sugar and urea as the carbon and nitro- BB and BU media remained stable under harsh condi-
gen sources, respectively. The biosurfactant was able to tions, including wide ranges of pH, temperature, and
Zhang et al. Microb Cell Fact (2016) 15:168 Page 8 of 11

Fig. 3  Photos showing the removal efficiency of crude oil adsorbed on filter paper and sand by fermentation both from BB and BU media

Table 4  Crude oil removal efficiencies of fermentation both containing biosurfactants from BB and BU media
Medium Crude oil removal

Filter paper Sand

Removal efficiency (REp %) REp/RECtrl Removal efficiency (REs %) REs/RECtrl

Ctrl 12.8 ± 0.19c – 9.3 ± 0.042c –

BB 94.3 ± 0.049a 7.4 94.0 ± 0.092a 10.1
BU 93.1 ± 0.12b 7.3 90.0 ± 0.057b 9.7
Values are presented as the mean ± standard deviation (n = 3). Different superscript letters within a column indicate significant differences (P < 0.05) by Duncan’s
multiple range test

salinity. They removed  ≥90  % of crude oil from artifi- Northwest China [33]. The oil spreading method was used
cially contaminated filter paper and sand. TLC and Fou- to select the potential biosurfactant-producing strains, as
rier transform infrared spectroscopy showed that the described by Youssef et al. [34], with minor modifications.
biosurfactants produced were a mixture of lipopeptides. Based on its oil spreading activity, Bacillus atrophaeus 5-2a
This study demonstrated the potential and feasibility of was selected for further study; it was identified as Bacillus
the lipopeptides produced by B. atrophaeus 5-2a for atrophaeus KP314029 by 16S rRNA gene sequencing [33]
application in MEOR. Investigations by laboratory-scale and was used for the present work. The purified culture
sand-pack columns are warranted to further assess the was maintained on beef extract peptone agar medium and
applicability of the lipopeptides in field applications. deposited in the China Center for Type Culture Collection
(CCTCC; strain number CCTCC M 2014673).
Methods The basal mineral salt solution (MSS; pH 7.0) used
Bacteria, media and oil contained (g  L−1): MgSO4·7H2O, 0.3; KH2PO4, 5.0;
Several bacteria were isolated from oil-contaminated surface K2HPO4·3H2O, 5.0; and NaCl, 5.0. The fermentation
soils near kowtow machines and oil tanks, adjacent to wells medium (BB; pH 7.0) used contained (g L−1): beef extract,
Hua-119 and Hao-129 in Ansai oilfield, Shaanxi province, 3.0; peptone, 10.0; NaCl, 5.0; and brown sugar, 10.0.
Zhang et al. Microb Cell Fact (2016) 15:168 Page 9 of 11

Crude oil was obtained from a depleted oil well (Hua- Analytical methods
20-4) in Ansai oilfield. The oil sample was taken at Bacterial cells were harvested by centrifuging (10,000×g)
1208  m depth in a low-permeability reservoir called for 10  min at 4  °C (Eppendorf, 5804R, Germany) and
Chang 6 (37°04′38  N, 109°02′58 E). The temperature in the dry cell weight (g  L−1) was determined after drying
the reservoir was approximately 40 °C and the well depth at 110  °C for 24  h. The cell-free supernatant was taken
reached 1283–1286  m. The oil sample was stored in a for the crude biosurfactant yield, oil spreading, emul-
plastic bucket at 4 °C until use. sion index and ST analyses. Data are expressed as the
mean ± standard deviation (n = 3). Comparison of group
Effects of carbon and nitrogen sources on biosurfactant means was conducted using Duncan’s multiple range test
production (considered significant at P  <  0.05). The analyses were
Biosurfactant production by the culture of Bacillus performed using SAS 9.2 (SAS Institute Inc, Cary, NC,
atrophaeus 5-2a was evaluated using a MSS with different USA). The experiments were performed in triplicate.
carbon and nitrogen sources. Eight carbon source treat-
ments (brown sugar, sucrose, glucose, maltose, starch, Oil spreading analysis
mannitol, glycerol and paraffin) were analyzed at final Oil spreading analysis tested the displacement activity
concentrations of 10.0  g  L−1 in the MSS media, which of the fermentation broth, measured using the method
contained NaNO3 (2.0 g L−1) and (NH4)2SO4 (1.0 g L−1) of Youssef et  al. [34], with minor modifications. A large
as the nitrogen sources. Eight nitrogen source treatments plastic tub (25 cm diameter) was filled up with 3000 mL
were assessed: beef extract, peptone, corn steep liquor, of clean water and two drops of paraffin oil were added to
urea, NaNO3, NH4Cl, (NH4)2SO4 and KNO3; each was the surface of the water. Then, one drop of fermentation
added to create a final concentration of 3.0  g  L−1 in the broth was added to the surface of the liquid paraffin. The
MSS media and brown sugar (10.0 g L−1) was used as the diameter of the clear zone created on the paraffin oil sur-
carbon source. The initial pH of the media during each face was measured. The larger the diameter of the clear
treatment was adjusted to 7.0. zone, the higher the surface activity of the test solution.
To obtain a seed inoculum, the pure culture of B.
atrophaeus 5-2a was transferred to 100 mL of BB medium Emulsification index
and incubated at 30  °C with shaking (120  rpm) for 3 d, Emulsifying activity was determined by adding 5  mL of
creating a cell density of 1010 colony-forming units paraffin oil to 5 mL of the cell-free supernatant in a glass
m L−1. For each treatment, 5 % seed inoculum was trans- tube, then mixing it with a vortex for 2 min and incubat-
ferred to 600 mL tissue culture vessels containing 100 mL ing it at ambient temperature for 24 h. The emulsification
of the treatment medium. The cultures were incubated at index (E24;  %) was calculated as the height of the emul-
30 °C, with shaking (120 rpm), for 5 days. After fermenta- sion layer (mm) divided by the total height of the liquid
tion, the samples were collected and the dry cell weight, column (mm) and multiplied by 100 [35]:
crude biosurfactant yield, oil spreading, emulsification
HE
index and surface tension (ST) were analyzed. E24 % = × 100
HT
Effects of the optimal media on biosurfactant production where HE and HT are the height of the emulsion layer
The ability of the Bacillus atrophaeus 5-2a culture to and the total height of liquid column, respectively.
produce biosurfactants was further evaluated using two
media. The first was the BB medium, in which the cul- Surface tension
ture presented the best results regarding biosurfactant ST of the culture supernatants was measured with a digi-
production. The second medium (hereafter known as tal surface tensiometer (JYW-200A, Chengde, Shandong,
BU) used brown sugar and inorganic nitrogen urea as China), using the ring method previously described [36].
the carbon and nitrogen sources, and was pH 7.0 (g L−1): For calibration, the ST of distilled water was first meas-
MgSO4·7H2O, 0.3; KH2PO4, 5.0; K2HPO4·3H2O, 10.0; ured. All ST readings were taken in triplicate and an
NaCl, 5.0; urea, 3.0; and brown sugar, 10.0. The brown average value was used to express the ST of each sample.
sugar and urea as the carbon and nitrogen sources were
used to assess the biosurfactant production with an eco- Dried weight measurement of biosurfactants
nomically viable medium, to test its potential application The biosurfactants were extracted using the acid pre-
in MEOR. The cultures were incubated at 30  °C, with cipitation method described by Nitschke and Pastore
shaking (120 rpm), for 5 days. Then, the dry cell weight, [37]. Briefly, the cell-free supernatant was adjusted to
crude biosurfactant yield, oil spreading, emulsion index pH 2.0 using 6 M HCl and left overnight at 4 °C for com-
(E24) and ST were analyzed. plete precipitation of the biosurfactants. The precipitate
Zhang et al. Microb Cell Fact (2016) 15:168 Page 10 of 11

was collected by centrifugation (10,000×g) for 10 min at Removal of crude oil from filter paper and sand
4 °C and washed twice with acidified water (pH 2.0). The The potential use of the biosurfactants for MEOR was
crude biosurfactants were oven-dried at 110 °C for 24 h assessed using artificially contaminated filter paper and
and weighed. sand. Assessment of the oil removed from artificially
contaminated filter paper was carried out using the
Characterization of the biosurfactants method of Zhang et  al. [38]. For removing the oil from
Thin layer chromatography artificially contaminated sand, sand (0.25–0.50 mm frac-
The biosurfactants were preliminarily characterized by tions) was taken from the Weihe River and 90  g of the
thin layer chromatography (TLC). The biosurfactant sand, contaminated with 10 % crude oil, was transferred
extract (5  mg) was hydrolyzed with 6  M HCl in sealed to a 600  mL tissue culture vessel containing 150  mL of
tubes, maintained at 110  °C for 24  h. The hydrolysate cell-free supernatant. After 4  days of static incubation
was separated on home-made silica gel plates using at 40  °C in the dark, the mixtures were filtered through
CH3CH2CH2CH2OH:CH3COOH:H2O (4:1:1, v/v/v) as sterile cotton wool using washing solution, to separate
the developing solvent system. The compounds separated the sand and crude oil. The crude oil covered sterile cot-
by TLC were visualized by spraying with ninhydrin 0.5 % ton wool was extracted with 60  mL hexane, dried by
(w/v, in water) to identify those with free amino groups. vacuum-rotary evaporation at 40 °C, cooled in a vacuum
Phenol–sulfuric acid (prepared by mixing 95 mL ethanol, desiccator to ambient temperature and then weighed (m).
5 mL of sulfuric acid and 3 g of phenol) was used to iden- Control columns were prepared in the same way, with the
tify the sugar moieties. The plates were heated at 110 °C addition of 150 mL distilled water. The crude oil removal
for 5  min until the appearance of the respective colors efficiency (REs %) was calculated as follows:
[5].
m
REs % = × 100
10
Fourier transform infrared spectroscopy
The structural groups of the biosurfactants were iden- where m is the mass (g) of crude oil removed from the
tified using fourier transform infrared (FT-IR) spec- artificially contaminated sand after the fermentation broth
troscopy analysis. The FT-IR spectrum of the dried treatment, and 10 is the original mass of the crude oil.
biosurfactants was recorded on a TENSOR 27 FT-IR
Authors’ contributions
spectrometer, equipped with a DLATGS detector JHZ carried out the experiments, analyzed the data and drafted the manu-
(Bruker, Germany); for this, 1 mg of dried biosurfactants script. QHX conceived and supervised the study and reviewed the final manu-
was mixed with 100  mg of KBr and pressed down with script. HG assisted in bacterial isolation and identification experiments. HXL
and PW took oil-contaminated soils and crude oil samples, and participated in
7500 kg for 30 s to obtain translucent pellets. The FT-IR the design of the study and coordination. All authors read and approved the
spectra, with a resolution of 4  cm−1, were acquired final manuscript.
between 400 and 4000 wave numbers (cm−1).
Author details
1
 College of Natural Resources and Environment, Northwest A & F Univer-
Biosurfactant stability sity, 3 Taicheng Road, 712100 Yangling, China. 2 College of Earth Sciences
The stability (activity) of the biosurfactants was studied and Resources, Chang’an University, 710055 Xi’an, China.

under a wide range of temperatures, pH and salt con- Acknowledgements

centrations [29]. The stability studies were performed The study was supported by the Boqin Biological Engineering Co., Ltd.
using the cell-free supernatant (obtained by centrifuga- (Sanyuan, Shaanxi Province, China). FT-IR data were collected at the Labora-
tory of the College of Natural Resources and Environment, Northwest A & F
tion at 10,000×g for 10  min at 4  °C). In the first set of University (Yangling, Shaanxi Province, China). We thank Dr. Hong-hong Zhang
tests, the supernatant was maintained at different con- for technical assistance.
stant temperatures, in the range of 20–100 °C for 3 h, and
Competing interests
then allowed to cool to ambient temperature. In addition, The authors declare that they have no competing interests.
the supernatant was subjected to autoclave conditions
(121 °C, 15 psi for 30 min) as another temperature treat- Availability of data and materials
The datasets supporting the conclusions of this article are included within the
ment. In the second set of tests, the pH of the superna- article.
tant was adjusted to various pH values, ranging from pH
2 to 13, using HCl (1 N) and NaOH (1 N). In the final set Funding
The study was supported by the Boqin Biological Engineering Co., Ltd.
of tests, NaCl was added to the supernatant at different (Sanyuan, Shaanxi Province, China).
concentrations 0–50 % (w/v). In each series of tests, the
diameter of the clear zone, the emulsification index and Received: 25 April 2016 Accepted: 28 September 2016

ST were measured.
Zhang et al. Microb Cell Fact (2016) 15:168 Page 11 of 11

References 20. Gurjar J, Sengupta B. Production of surfactin from rice mill polishing
1. Chakraborty S, Ghosh M, Chakraborti S, Jana S, Sen KK, Kokare C, Zhang L. residue by submerged fermentation using Bacillus subtilis MTCC 2423.
Biosurfactant produced from Actinomycetes nocardiopsis A17: characteri- Bioresour Technol. 2015;189:243–9.
zation and its biological evaluation. Int J Biol Macromol. 2015;79:405–12. 21. Abdel-Mawgoud AM, Aboulwafa MM, Hassouna NAH. Optimization
2. Georgiou G, Lin SC, Sharma MM. Surface active compounds from micro- of surfactin production by Bacillus subtilis isolate BS5. Appl Biochem
organism. Bio/Technology. 1992;10:60–5. Biotechnol. 2008;150:305–25.
3. Neu TR. Significance of bacterial surface-active compounds in interaction 22. Dastgheib SMM, Amoozegar MA, Elahi E, Asad S, Banat IM. Bioemulsi-
of bacteria with interfaces. Microbiol Rev. 1996;60:151–66. fier production by a halothermophilic Bacillus strain with potential
4. Souayeh M, Al-Wahaibi Y, Al-Bahry S, Elshafie A, Al-Bemani A, Joshi S, applications in microbially enhanced oil recovery. Biotechnol Lett.
Al-Hashmi A, Al-Mandhari M. Optimization of a low-concentration Bacil- 2008;30:263–70.
lus subtilis strain biosurfactant toward microbial enhanced oil recovery. 23. Sousa M, Melo VMM, Rodrigues S. Sant’ana HB, Gonçalves LRB. Screen-
Energy Fuel. 2014;28:5606–11. ing of biosurfactant-producing Bacillus strains using glycerol from the
5. Bezza FA, Chirwa EMN. Production and applications of lipopeptide biodiesel synthesis as main carbon source. Bioprocess Biosyst Eng.
biosurfactant for bioremediation and oil recovery by Bacillus subtilis CN2. 2012;35:897–906.
Biochem Eng J. 2015;101:168–78. 24. Faria AF, Teodoro-Martinez DS, Barbosa GNO, Vaz BG, Silva IS, Garcia JS,
6. Bailey SA, Kenney TM, Schneider D. Microbial enhanced oil recovery: Tótola MR, Eberlin MN, Grossman M, Alves OL, Durrant LR. Production and
diverse successful applications of biotechnology in the oil field. SPE structural characterization of surfactin (C14/Leu7) produced by Bacillus
72129, Proceedings of SPE Asia Pacific improved oil recovery conference. subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel indus-
Society of petroleum engineers, Richardson. 2001. try. Process Biochem. 2011;46:1951–7.
7. Gudiña EJ, Fernandes EC, Rodrigues AI, Teixeira JA, Rodrigues LR. Biosur- 25. Makkar RS, Cameotra SS. Biosurfactant production by a thermophilic
factant production by Bacillus subtilis using corns steep liquor as culture Bacillus subtilis strain. J Ind Microbiol Biotechnol. 1997;18:37–42.
medium. Front Microbiol. 2015;59:1–7. 26. Wu JY, Yeh KL, Lu WB, Lin CL, Chang JS. Rhamnolipid production with
8. Gudiña EJ, Rangarajan V, Sen R, Rodrigues LR. Potential therapeutic appli- indigenous Pseudomonas aeruginosa EM1 isolated from oil-contaminated
cations of biosurfactants. Trends Pharmacol Sci. 2013;34:667–75. site. Bioresour Technol. 2008;99:1157–64.
9. Pornsunthorntawee O, Arttaweeporn N, Paisanjit S, Somboonthanate P, 27. Elazzazy AM, Abdelmoneim TS, Almaghrabi OA. Isolation and charac-
Abe M, Rujiravanit R, Chavadej S. Isolation and comparison of biosur- terization of biosurfactant production under extreme environmental
factants produced by Bacillus subtilis PT2 and Pseudomonas aeruginosa conditions by alkali-halo-thermophilic bacteria from Saudi Arabia. Saudi J
SP4 for microbial surfactant-enhanced oil recovery. Biochem Eng J. Biol Sci. 2015;22:466–75.
2008;42:172–9. 28. Ghribi D, Ellouze-Chaabouni S. Enhancement of Bacillus subtilis lipo-
10. Aparna A, Srinikethana G, Smithab H. Production and characterization of peptide biosurfactants production through optimization of medium
biosurfactant produced by a novel Pseudomonas sp. 2B. Colloids Surf B. composition and adequate control of aeration. Biotechnol Res Int.
2012;95:23–9. 2011;2090–3138:653–4.
11. Al-Sulaimani H, Al-Wahaibi Y, Al-Bahry S, Elshafie A, Al-Bemani A, Joshi 29. Gudiña EJ, Pereira JFB, Rodrigues LR, Coutinho JAP, Teixeira JA. Isolation
S, Ayatollahi S. Residual-oil recovery through injection of biosurfactant, and study of microorganisms from oil samples for application in micro-
chemical surfactant, and mixtures of both under reservoir temperatures: bial enhanced oil recovery. Int Biodeterior Biodegrad. 2012;68:56–64.
induced-wettability and interfacial tension effects. SPE Reservoir Eval Eng. 30. Ghojavand H, Vahabzadeh F, Roayaei E, Shahraki AK. Production and
2012;15:210–7. properties of a biosurfactant obtained from a member of the Bacillus
12. Sen R. Biotechnology in petroleum recovery: the microbial EOR. Prog subtilis group (PTCC 1696). J Colloid Interf Sci. 2008;324:172–6.
Energy Combust. 2008;34:714–24. 31. Khopade A, Biao R, Liu X, Mahadik K, Zhang L, Kokare C. Production and
13. Schaller KD, Fox SL, Bruhn DF, Noah KS, Bala GA. Characterization of stability studies of the biosurfactant isolated from marine Nocardiopsis sp.
surfactin from Bacillus subtilis for application as an agent for enhanced oil B4. Desalination. 2012;285:198–204.
recovery. Appl Biochem Biotechnol. 2004;113–116:827–36. 32. Ibrahim ML, Ijah UJJ, Manga SB, Bilbis LS, Umar S. Production and partial
14. Al-Wahaibi Y, Joshib S, Al-Bahry S, Elshafie A, Al-Bemania A, Shibulal B. characterization of biosurfactant produced by crude oil degrading bacte-
Biosurfactant production by Bacillus subtilis B30 and its application in ria. Int Biodeterior Biodegrad. 2013;81:28–34.
enhancing oil recovery. Colloids Surf B. 2014;114:324–33. 33. Zhang JH, Xue QH, Gao H, Lai HX, Wang P. Bacterial degradation of crude
15. Banat IM, Franzetti A, Gandolfi I, Bestetti G, Martinotti MG, Fracchia L, oil using solid formulations of Bacillus strains isolated from oil-contami-
Smyth TJ, Marchant R. Microbial biosurfactants production, applications nated soil towards microbial enhanced oil recovery application. RSC Adv.
and future potential. Appl Microbiol Biotechnol. 2010;87:427–44. 2016;6:5566–74.
16. Pereira JFB, Gudiña EJ, Costa R, Vitorino R, Teixeira JA, Coutinho JAP, 34. Youssef NH, Duncan KE, Nagle DP, Savage KN, Knapp RM, Mclnerney MJ.
Rodrigues LR. Optimization and characterization of biosurfactant produc- Comparison of methods to detect biosurfactant production by diverse
tion by Bacillus subtilis isolates towards microbial enhanced oil recovery microorganisms. J Microbiol Method. 2004;56:339–47.
applications. Fuel. 2013;111:259–68. 35. Jadhav VV, Yadav A, Shouche YS, Aphale S, Moghe A, Pillai S, Arora A,
17. Al-Bahry SN, Elshafie AE, Al-Wahaibi YM, Al-Bemani AS, Joshi SJ, Al-Maaini Bhadekar RK. Studies on biosurfactant from Oceanobacillus sp. BRI 10
RA, Al-Alawi WJ, Sugai Y, Al-Mandhari M. Microbial consortia in Oman oil isolated from Antarctic sea water. Desalination. 2013;318:64–71.
fields: a possible use in enhanced oil recovery. J Microbiol Biotechnol. 36. Xia WJ, Dong HP, Yu L, Yu DF. Comparative study of biosurfactant pro-
2013;81:141–6. duced by microorganisms isolated from formation water of petroleum
18. Youssef N, Simpson DR, Duncan KE, McInerney MJ, Folmsbee M, reservoir. Colloid Surface A. 2011;392:124–30.
Fincher T, Knapp R. In-situ biosurfactant production by Bacillus strains 37. Nitschke M, Pastore GM. Production and properties of a surfactant
injected into a limestone petroleum reservoir. Appl Environ Microbiol. obtained from Bacillus subtilis grown on cassava wastewater. Bioresour
2007;73:1239–47. Technol. 2006;97:336–41.
19. Al-Sulaimani H, Al-Wahaibi Y, Al-Bahry SN, Elshafie A, Al-Bemani A, Joshi 38. Zhang JH, Xue QH, Gao H, Ma X, Wang P. Biodegradation of crude oil
S, Zargari S. Optimization and partial characterization of biosurfactants by fungal enzyme preparations from Aspergillus spp for potential use in
produced by Bacillus species and their potential for ex situ enhanced oil enhanced oil recovery. J Chem Technol Biotechnol. 2016;91:865–75.
recovery. SPE J. 2011;16:672–83.