ISSN 0003-6838, Applied Biochemistry and Microbiology, 2009, Vol. 45, No. 6, pp. 588–592. © Pleiades Publishing, Inc.

, 2009.
Original Russian Text © I.N. Gogotov, A.I. Miroshnikov, 2009, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2009, Vol. 45, No. 6, pp. 654–658.

The Influence of Growth Medium Composition

and Physicochemical Factors on Biosurfactant Production
by the Bacterium Bacillus licheniformis VKM B-511
I. N. Gogotova and A. I. Miroshnikovb
a Institute
of Basic Biological Problems, Russian Academy of Sciences,
Pushchino, Moscow Oblast, 142290 Russia
e-mail: i.n.gogotov@rambler.ru
b Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Oblast, 142290 Russia

Received May 4, 2008

Abstract—The ability of Bacillus licheniformis strain VKM B-511 to grow and synthesize biosurfactants
under both aerobic and anaerobic conditions has been demonstrated. Yields of biosurfactants, emulsion indices
and surface tension were considerably higher in culture liquor and preparations derived from cultures grown
anaerobically at a C/N ratio of 1 : 24, pH 7.0, and temperature of 30°C. Biosurfactant production by B. lichen-
formis also depended on concentrations of NaCl and Na2S in the medium and on water characteristics, reaching
4.58 g/l for bacteria grown anaerobically on a medium containing anolyte fraction of water.
DOI: 10.1134/S0003683809060027

INTRODUCTION The objective here is to study the effect of culture

Surface-active agents produced by many microor-
ganisms, biosurfactants, have attracted much attention
in recent years, both in theoretical and practical aspects
[1–5]. This has been due to their wide application in oil-
producing and mineral-resource industries, chemical,
pharmaceutical and food sectors and as well as in
removing environmental pollutants, such as carbohy-
drates or heavy metals. Microbe-produced biosurfac-
tants compare favorably to synthetic ones in emulsify-
ing activity. However, unlike the synthetic ones, they
are nontoxic, biodegradable, and are obtained from
renewable sources, which qualities hold much promise
for microbe-produced biosurfactants in the develop-
ment of modern environment-friendly technologies.
There are two large classes of biosurfactants—those
synthesized chemically and those produced by
microbes. Currently, five groups of biosurfactants are
known, pertaining to glycolipids, lipopeptides, fatty
acids, neutral lipids, lipopolysaccharides, and polysac-
charide–lipid complexes [1]. The majority of the stud-
ied biosurfactant producers, including rhodococci, have
the ability to synthesize them under aerobic conditions
and in the presence of carbohydrates in the medium
[3, 4]. Microorganisms that produce biosurfactants
anaerobically are poorly investigated. The ability to
produce biosurfactants anaerobically has yet been con-
firmed only in Desulfovibrio desulfuricans [6],
Clostridium pasteurianum [7], and Bacillus lichenifor-
mis JF-2 [8]. Synthesis regulation, properties and com-
mercial potential of their biosurfactants are poorly
investigated.
medium composition and external factors on biosurfac-
tant synthesis by a formerly unexamined strain
B. licheniformis VKM B-511 both under aerobic and
anaerobic conditions.
METHODS
Object of investigation. We used the strain Basillus
licheniformis VKM B-511 (ATSS 14580b, DSM 13,
INFO 1220), obtained from the collection of VKM
IBFM, Russian Academy of Sciences, that is able to
grow both under aerobic and anaerobic conditions.
Culturing of B. licheniformis. Aerobic culturing of
B. licheniformis VKM B-511 was done in 250- or 500-ml
flasks on a shaker (150 rpm, 37°ë) or in bioreactors
with working volumes of 250 or 500 ml at 37°ë. The
culture was grown and maintained on a medium with
the following constituents (g/l): sucrose – 21.0,
äç2êé4 – 4.0, Na2HPO4 – 6.0, MgSO4 · 7ç2é – 1.0,
CaCl2 · 2ç2é – 0.08, aspartic acid – 1.0, and trilon B –1.3.
For growing and maintenance of bacteria under
anaerobic conditions, a medium with the following com-
position was used (g/l): glucose – 15.0, NaNO3 – 1.5,
NaCl – 50.0, Na2S × 9 H2O – 0.5, and yeast extract – 1.5.
The media were sterilized at 121°ë for 30 min. The cul-
tures were grown at the initial pH value of 7.2 and
maintained in an active state by monthly transfers. The
bacteria were stored at +4°ë on an A 5 medium with
potato agar. To examine anaerobic biosurfactant pro-
duction, the bacteria were grown on a mineral medium
modified by us [8] in 1-liter bottles, sealed with rubber

588
 THE INFLUENCE OF GROWTH MEDIUM COMPOSITION 589

Table 1. The effect of temperature on growth, emulsion index, and yield of biosurfactants in B. licheniformis VKM B-511
A600 Yield of biosurfactants, g/l E24, %
Temperature, °C
aerobically anaerobically aerobically anaerobically aerobically anaerobically
20 0.137 0.222 0.71 0.95 27.8 32.4
30 0.141 0.270 0.80 1.20 41.0 46.1
40 0.134 0.284 0.74 1.06 34.2 33.0
Note: The bacteria were grown for 5 days under aerobic or anaerobic conditions. Tables 1–4 and Figs. 1 and 2 show values of E24 and the
surface tension (σs) defined for the cellular supernatant.

Table 2. The influence of NaCl and Na2S on the growth, chemical composition, emulsion index, and surface tension of bio-
surfactants derived from the supernatant of B. licheniformis VKM B-511
Yield of bio- Na2S · 9H2O, Yield of bio-
NaCl, g/l A600 E24, % σs, mN/m E24
surfactants, g/l g/l surfactants, g/l
0 0.105 0.36 29.6 48.1 0.1 0.80 35.4
30 0.094 0.40 22.5 49.8 1.0 0.77 35.3
60 0.112 0.86 31.2 51.7 2.0 1.29 41.8
90 0.137 1.17 44.5 60.2 3.0 1.0 38.5
Ar n.m. n.m. n.m. n.m. Ar 0.80 31.6
Note: The bacteria were grown anaerobically for 5 days. The color of biosurfactants varied with increasing Na2S concentration from white
(argone blow) to deep grey (the addition of 3 g/l of Na2S). Surface tension was measured by the du Nouy anchor-ring method [10].

corks. Kerosene (0.5% by volume) was used as a source
of carbon and energy. For the absorption of oxygen
from the solution, the medium was vacuumed ahead of
time and Na2S was added. Bacterial concentration was
calculated from optical density at 600 nm. Calibration
curve for the calculation of the biomass was built as a
function of optical density. For sterilization of kero-
sene, we used Millex-GV4 filters by Millipore, USA.
To estimate the effects of pH, temperature, and NaCl on
biosurfactant synthesis, the culture was grown at
parameter values set out in Tables 1–3.
Analytical methods. Biosurfactants were extracted
from a 24-h culture by centrifugation (10000 g,
30 min). The biosurfactants of B. licheniformis were
isolated from culture liquid through acidation with
hydrochloric acid, followed by pelleting at +4°ë, which
resulted in precipitation of the emulsifier [9]. 1N HCl
was added to the supernatant until the pH was lowered
to 2.0, and the mixture was left standing for 12 h at 4°ë.
The resulting amorphous precipitate was pelleted by
centrifugation (10000 g, 20 min), followed by suspen-
sion in a minimum volume of distilled water, where-
upon the pH was adjusted to 7.0 with NaOH. The sus-
pension thus obtained was dried with an Ikar infrared
dryer (Russia) and the preparations were stored at room
temperature. Such processing allows obtaining dry
crude biosurfactant.
Emulsifier activity of biosurfactant-containing prep-
arations was measured by a method that uses kerosene
as the substrate for emulsification [1]. Five milliliters of
the substrate to be emulsified were added to 5 ml of the
studied solution, containing biosurfactant, and the mix-
ture was inverted for 2 min. Percent emulsion index
(Ö24) was measured in 24 h and was defined by the ratio
of the emulsion layer height to total liquid height in the
tube. Supernatant of the culture liquid was obtained by
centrifugation (5000 g, 20 min); the cell suspension

Table 3. The effect of the approach to sterilization of media with catholyte or anolyte fractions on the growth, yield of bio-
surfactants, and emulsion index in samples of B. licheniformis VKM B-511
A600 Biosurfactants, g/l E24, %
Water quality AC Filter
AC Filter AC Filter
SP CS SP CS
Distilled water 0.270 0.221 2.59 2.90 36.0 14.0 14.3 1.2
Catholyte fraction 0.297 0.296 1.42 1.73 11.3 4.1 9.1 1.3
Anolyte fraction 0.257 0.208 2.96 4.58 13.2 3.5 9.3 1.1
Note: The culture was grown anaerobically for 5 days. SP is supernatant, CS is cell suspension, and AC is autoclaving. The filter used was
Millex-GV4 by Millipore.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 45 No. 6 2009

590 GOGOTOV, MIROSHNIKOV
g/l mN/m Ö24
0.80 60 60
0.78 58
0.76 56
0.74
54
III
0.72 II 52
0.70 I 50
0.68 48
0.66 46
0.64 44
1 2 3 1
Fig. 1. The influence of the source of carbon (1–3) in culture
medium on the (I) yield, g/l, (II) emulsion index, E24, and
(III) surface tension, σ of biosurfactant samples from
B. licheniformis VKM B-511. The culture was grown aero-
bically in the presence of 0.1% aspartic acid (30°C, 24 h,
150 rpm). 1 is fructose, 2 is sucrose, and 3 is glucose.
was diluted in distilled water to the volume from which
cells were pelleted.
Effectiveness of the biosurfactants produced by
B. licheniformis VKM B-511 was estimated by measur-
ing surface tension (σ, mN/m) with a VZ-246 glass cap-
illary viscometer (Russia) or an MT-6 hand-operated
tensiometer by Sigma (USA) via the du Nouy anchor-
ring method [10].
Preparation of electrochemically activated water
(ECA). ECA-bidistilled water was prepared using a
previously described installation. A three-chamber dia-
phragm electrolyzer is the central unit of the facility.
All chambers of the electrolyzer are separated from
each other by membranes with a pore size of 4 μm
(Vladipor, Russia). Thirty-square-centimeter plate
electrodes are used; the cathode material is titanium
and the anode material is platinized titanium. Catholyte
and anolyte fractions, subsequently used for the prepa-
ration of culturing media, were obtained in flow regime
at the flow rate of 1.2 ml/min.
RESULTS AND DISCUSSION
In experiments with different strains of B. licheni-
formis they were shown to be capable of producing bio-
surfactants [11–14] on glucose or fructose (strains F2.2
and 86) and sucrose (strain BL86) as sources of energy.
Our data, related to the influence of fructose, sucrose,
and glucose on the yield of biosurfactants, surface ten-
sion, and emulsion index, indicate that these parameters
reach a maximum when B. licheniformis VKM B-511
is grown on a sucrose-containing medium. Notably, the
overall yield of biosurfactants was 2-fold higher in the
strain B-511 than in other tested strains of this bacte-
rium.
It is known that, apart from the source of carbon, the
production of biosurfactants by B. licheniformis is
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
g/l mN/m Ö24
1.0 III 60 60
58
0.8 50 50
56
II 40 40
54 0.6
52 30 30
50 0.4
20 20
48
0.2 10 10
46 I
44 0 0 0
2 3
Fig. 2. The effect of nitrogen source (1–3) in culture
medium on the (I) yield, g/l, (II) emulsion index, E24, and
(III) surface tension, σ of biosurfactant samples from
B. licheniformis VKM B-511. The culture was grown aerobi-
cally in the presence of 2% sucrose (30°C, 24 h, 150 rpm). 1 is
ammonium nitrate, 2 is glutamic acid, and 3 is aspartic acid.
influenced by the nature of the nitrogen source [15].
Regulatory action of amino acids on lipopeptide syn-
thesis has been reported also with reference to the prep-
arations surfactin, iturin [16], bacillomicin, and myco-
subtilin [1]. Our experiments with B-511 demonstrated
(Fig. 2) that growing this strain on media with glutamic
and aspartic acids increases the yield of biosurfactants
by 3- and 5-fold, respectively. Thus, supplementing the
medium with these aminoacids, the structural units or,
possibly, precursors of lichenysin A significantly influ-
ences the production of this biosurfactant and its activ-
ity in different strains of B. licheniformis [17, 18].
Very little is known about the ability of bacteria to
produce biosurfactants anaerobically. With respect to
B. licheniformis, this has been shown only for the strain
JF-2 grown on a medium with glucose, yeast extract,
and nitrate [8]. Our trials with a previously unstudied
strain, B. licheniformis VKM B-511, revealed that it
produces biosurfactants in higher yields anaerobically
than it does aerobically (Figs. 1–3, Tables 1–4). Despite
a slower growth rate, under anaerobic conditions, the
strain synthesized biosurfactants that exhibited
increased values of the emulsion index and surface ten-
sion. Notably, this bacterium produced twice as rapid
growth as B. licheniformis JF-2 [8] on the same
medium and, importantly for its practical use, more
biosurfactants.
The yield of biosurfactants, surface tension, and
emulsion index of the strain B-511, apart from being
influenced by the nature of the sources of carbon and
nitrogen, depended also upon the ratio of these ele-
ments in the medium. The maximum yield of biosurfac-
tants (3.03 g/l) was produced by this strain at a C/N
ratio of 1 : 24 (Fig. 3). The temperature and pH value
also had a strong influence on biosurfactant production
and the emulsion index. The maximum yield of biosur-
factants and a high emulsion index were shown for this
Vol. 45 No. 6 2009
 THE INFLUENCE OF GROWTH MEDIUM COMPOSITION 591

strain, when grown both under aerobic and anaerobic A600 g/l
conditions, at pH 7.0. 0.20 1 2 3.5
Among known Rhodococcus spp. strains there are 3.0
both psychrophilic, that grow and produce biosurfac- 0.15
tants at 5–20°ë, and mesophilic, producing the most 2.5
effective growth at 30°ë and producing biosurfactant 2.0
synthesis at 20°ë [17]. The studied strain, similar to 0.10
B. licheniformis JF-2, exhibited a higher yield of bio- 1.5
surfactants and emulsion index, both under aerobic and
anaerobic conditions, at 30°ë (Table 1). 0.05 1.0
Earlier studies have revealed the ability of 0.5
B. licheniformis JF-2 [8] to grow and produce biosur-
0 0
factants on a medium containing up to 10% NaCl. Our 1:6 1:12 1:24 1:53
investigation of B-511 demonstrated (Table 2) that this N/C
strain was similarly able to grow and synthesize biosur-
factants in the presence of up to 9% NaCl in the Fig. 3. The effect of the N/C ratio (the ratio of nitrogen to
carbon concentration) on the chemical composition of bio-
medium. The preparation lichenisin A (biosurfactant of surfactants (g/l) in the cellular supernatant of B. lichenifor-
B. licheniformis) is known to contain sulfur-bearing mis B-511. 1 is A600 and 2 is the yield of biosurfactants. The
amionoacids [19]. An investigation of the effects of culture was grown aerobically on a medium containing 2%
Na2S and an inert gas Ar on growth and biosurfactant sucrose (30°C, 24 h, 150 rpm).
production in B-511 (Table 2) indicated that the maxi-
mum stimulation of the biosurfactant production and
emulsion index was achieved by the addition of Na2S rate of B-511 was produced by the catholyte fraction, as
into the culture medium (at 2 g/l). compared with distilled water and anolyte water fraction.
It has been shown that, after electrochemical activa- Autoclaving of media based on the catholyte frac-
tion (ECA), water solutions acquire changes to their tion does not influence the growth rates of the slow-
physicochemical properties and biological activity. growing mycobacteria, as shown in [25]. In contrast to
This phenomenon is widely used in medicine, agricul- these data, our investigations of B. licheniformis VKM
ture, and industry [20, 21]. However, the reasons for an B-511 have shown that the growth rate of this bacte-
increased activity of such solutions remain uncertain. rium is much higher on a catholyte fraction-containing
In recent years, much attention has been paid to the medium, sterilized by both autoclaving and filtering
electrochemical activation of distilled and bidistilled through a Millex-GV4 (Millipore, USA) (Table 3).
water, as well as to efforts to account for the activity of However, the autoclaving of media considerably low-
catholyte and anolyte fractions of such water. It is sug- ered the emulsion indices of biosurfactants derived
gested in [22] that another factor, apart from the prod- from supernatants of cultures grown both on catholyte-
ucts of hydrolisis, is involved in the occurrence of acti- and anolyte-containing media. This has not been
vated distilled and bidistilled water, namely, changes in accounted for yet and requires further studies.
water structure. It is also believed [23–24] that ECA of Examination of the yield, surface tension, and Ö24 of
solutions results from the formation of hydrogen perox- the biosurfactant preparations from B. licheniformis
ide in catholyte and anolyte fractions due to free-radical VKM B-511 grown both aerobically and anaerobically,
reactions with the participation of hydrated electrons, as related to the time of culture (Table 4), showed that
and consequently, the activation of water may be the maximum yield of biosurfactants was produced
achieved by the addition of ç2é2. However, it has under anaerobic conditions.
recently been shown [20] that the hydrogen peroxide
occurring during the activation of a medium does not
account for the biological activity of the catholyte frac- Table 4. The growth, yield of biosurfactants, σs, and emul-
tion and has no influence on cell growth in Escherichia sion index of B. licheniformis B-511 grown under aerobic
coli K12. Our experiments aimed at testing the effect of and anaerobic conditions
catholyte and anolyte water fractions on biosurfactant syn-
Culture condi-
thesis by the bacterium B. licheniformis VKM B-511 tions
I II III IV V
showed (Table 3) that, in contrast with the catholyte
fraction and distilled water, the anolyte fraction stimu- Aerobic 1.760 0.69 41.0 47.1 62.9
lates biosurfactant production but not the emulsion Anaerobic 0.455 2.59 46.1 35.3 n.m.
index nor the bacterial growth. This may be related to Note: The bacteria were grown for 6 days. N.m. means that the
the ionogenicity and charge of the molecules of biosur- value was not measured. Surface tension was measured via
the du Nouy anchor-ring method [10]. I is growth, A600, II is
factant; however, further studies are required to prove yield of biosurfactants, g/l, III is E24 of culture liquid, IV is
this hypothesis. As was the case with E. coli K12 [20], σs of the supernatant, mN/m, and V is σs of the acid residue,
the most pronounced stimulating effect on the growth mN/m.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 45 No. 6 2009

592 GOGOTOV, MIROSHNIKOV
Thus, our experiments revealed the optimal culture
conditions for the strain B. licheniformis VKM B-511
that provide an increased yield of biosurfactants under
both aerobic and anaerobic conditions. It was shown that,
under aerobic conditions, the studied strain (B-511), sim-
ilar to B. licheniformis BAS50 [14], produces a higher
yield of biosurfactants when grown on media with
aspartic and glutamic acids, than on media with nitro-
gen as ammonium or nitrate. In contrast to other
B. licheniformis strains, the studied strain does not need
the additional supply of microelements for growth and
produces biosurfactants more efficiently when the cul-
ture is grown anaerobically on a medium containing
anolyte water fraction.
REFERENCES
1. Desai, J.D. and Banat, I.M., Microbiol. Mol. Biol. Rev.,
1997, vol. 61, no. 1, pp. 47–64.
2. Gogotov, I.N. and Khodakov, R.S., Prikl. Biokhim. Mik-
robiol., 2008, vol. 44, no. 2, pp. 207–212.
3. Mukherjee, S., Das P., and Sen, R., Trends Biotechnol.,
2006, vol. 24, no. 5, pp. 509–515.
4. Pirog, T.P., Shevchuk, T.A., Voloshina, I.N., and
Karpenko, E.V., Prikl. Biokhim. Mikrobiol., 2004,
vol. 40, no. 1, pp. 27–36.
5. Kostina, E.G., Revin, V.V., Atykyan, N.A., and Gogo-
tov, I.N., in Nauka i innovatsii v respublike Mordoviya.
Materialy IV nauchno-praktich. konf. (Science and Inno-
vations in the Republic Mordovia, Proc. IV Sci.-Pract.
Conf.), Saransk: Mordov. Gos. Univ. Im. N.P. Ogareva,
2005, pp. 583–588.
6. Aeckersberg, F., Bak, F., and Widdel, F., Arch. Micro-
biol., 1991, vol. 156, no. 1, pp. 5–14.
7. Cooper, D.G., Zajic, J.F., Gerson, D.F., and Man-
ninen, K.I., J. Ferment. Technol., 1980, vol. 58, no. 1,
pp. 83–86.
8. Javahery, M., Jenneman, G.E., McInerney, M.J., and
Knapp, R.M., Appl. Environ. Microbiol., 1985, vol. 50,
no. 5, pp. 698–700.
9. Clark, J.B., Munnecke, D.M., and Jenneman, G.E., Dev.
Ind. Microbiol., 1981, vol. 22, no. 5, pp. 695–701.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
10. Abramzon, A.A., Zaichenko, L.P., and Raingol’d, S.I.,
Poverkhnostno-aktivnye veshchestva. Sintez, analiz,
primenenie (Surfactants: Synthesis, Analysis and Appli-
cation), Leningrad: Khimiya, 1988.
11. Cooper, D.C. and Goldenberg, B.G., Appl. Environ.
Microbiol., 1987, vol. 53, no. 2, pp. 224–229.
12. Horovitz, S., Gilbert, J.N., and Griffin, W.M., J. Industr.
Microbiol., 1990, vol. 6, pp. 243–248.
13. Thaniyavarn, J., Roongsawang, N., and Kameyama, T.,
Biosci. Biotechnol. Biochem., 2003, vol. 67, no. 6,
pp. 1239–1244.
14. Lin, S.-Ch., Sharma, M.M., and Georgiou, G., Biotech-
nol. Prog., 1993, vol. 9, no. 1, pp. 138–145.
15. Yakimov, M.M., Fredriuckson, H.L., and Timmis, K.N.,
Appl. Environ. Microbiol., 1995, vol. 61, no. 6,
pp. 1706–1713.
16. Besson, F. and Michel, G., Biotechnol. Lett., 1992,
vol. 14, no. 6, pp. 1013–1018.
17. Pirog, T.P., Voloshina, I.N., Ignatenko, S.V., and
Vil’danova-Martsishin, R.I., Biotekhnologiya, 2005,
issue 6, pp. 27–36.
18. Inerney, M.J., Javahery, M., and Nagle, D.P., J. Industr.
Microbiol., 1990, vol. 5, no. 1, pp. 95–102.
19. Yakimov, M.M., Fredrickson, H.L., and Timmis, K.N., Bio-
technol. Appl. Biochem., 1996, vol. 23, no. 1, pp. 13–18.
20. Miroshnikov, A.I., Masalimov, Zh.K., and Bruskov, V.I.,
Mol. Biofiz., 2004, vol. 49, no. 1, pp. 32–37.
21. Prilutskii, V.I. and Bakhir, V.M., Elektrokhimicheski
aktivirovannaya voda: anomal’nye svoistva, mekhanizm
biologicheskogo deistviya (Electrochemically Activated
Water: Abnormal Properties and Mechanism of Biologi-
cal Action), Moscow: Vseross. Nauch.-Issled. Inst. Med.
Tekhn., 1997.
22. Petrushanko, I.Yu. and Lobyshev, V.I., Biofizika, 2001,
vol. 46, no. 3, pp. 389–401.
23. Kloss, A.I., Dokl. Akad. Nauk SSSR, 1988, vol. 303,
no. 6, pp. 1403–1407.
24. Bruskov, V.I., Masalimov, Zh.K., and Chernikov, A.V.,
Dokl. Akad. Nauk, 2002, vol. 384, no. 6, pp. 821–824.
25. RF Patent No. 2 106 879, Byull. Izobret., 1998, issue 8,
p. 1.
Vol. 45 No. 6 2009