Biotechnology Letters, Vol 20, No 12, December 1998, pp.

1153–1156

Production of sophorolipids in high

concentration from deproteinized whey
and rapeseed oil in a two stage fed
batch process using Candida bombicola
ATCC 22214 and Cryptococcus curvatus
ATCC 20509
Hans-Joachim Daniel, Matthias Reuss and Christoph Syldatk*
Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany

High concentrations of 422 g sophorolipids l21 were produced using a two-stage cultivation process: first deprotein-
ized whey concentrate (DWC) containing 110 g lactose l21 was used for cultivation of the yeast Cryptococcus curvatus
ATCC 20509, resulting in 34 g dry weight l21, 20 g single-cell oil l21 and reducing the chemical oxygen demand (COD)
from 159 g l21 to 35 g oxygen l21. Afterwards cells were disrupted by passing the cell suspension directly through a
high pressure laboratory homogeniser. After autoclavation, the resulting crude cell extract containing the single-cell oil
served as substrate for growth of Candida bombicola ATCC 22214 and for sophorolipid production in a second stage.
When the single-cell oil was consumed, repeated feeding of 400 g rapeseed oil l21 was started increasing the yield of
sophorolipids to 422 g l21. A simple technique for product isolation, sedimentation, could be used to harvest the crude
sophorolipids.
Keywords: Candida bombicola, Cryptococcus curvatus, whey, lactose, sophorolipids, biosurfactant

Introduction During cultivation, sophorolipids are produced as a mixture

Surfactants and emulsifiers are indispensable components
of daily life. They are widely used in the pharmaceutical,
cosmetic, petroleum and food industries. Almost half of
the surfactants produced (5.2 million t in 1991) are made
for washing and cleaning-agent sector. Most compounds
are synthesized chemically, however also surface-active
molecules of biological origin obtained by bioprocesses
have been described. While their properties are comparable
to those of surfactants prepared chemically, they comprise a
production from renewable resource substrates, such as
whey and the advantage of biodegradibility (Fiechter,
1992). Among biosurfactants, sophorolipids excreted either
by Candida bombicola ATCC 22214 (formerly Torulopsis
bombicola; Cooper and Paddock, 1984) or by Candida apicola
(Stüwer et al., 1987) are subject of particular interest: they
have been already used as compounds in body cleaning
agents (Inoue et al., 1979), act as tumor suppressors (Isoda
et al., 1997) and are reported to enhance the biodegrada-
tion of phenanthrene by a Sphingomonas sp. (Schippers et al.,
1997).
of compounds, basing commonly on acetylated sophorose
(2-O-b-D-glucopyranosyl-b-D-glucopyranose) derivatives,
each linked to a hydroxy fatty acid (Asmer et al., 1988).
The properties and applications of sophorolipids mainly
depend on the abundance of certain structural classes in the
mixtures (Inoue, 1988) like e.g. lactonic or acidic forms.
Many different processes have been developed for produc-
ing sophorolipids. Davila et al. (1997) summarized that
high concentrations of up to 350 g sophorolipids l21 (dry
weight) could be achieved using glucidic substrates, con-
sisting of glucose, lactose, fructose or mannose and/or
lipidic compunds as n-alkanes, vegetable oils or animal
fats.
In our group cultivation and sophorolipid production by
Candida bombicola on cheap media were investigated for
economical reasons. Whey, as a waste product of cheese
industry, leads to high costs in waste water treatment due
to the high COD value, caused by the content of up to
150 g lactose l21. We recently reported that cultivation

© 1998 Chapman & Hall Biotechnology Letters ⋅ Vol 20 ⋅ No 12 ⋅ 1998 1153

H.-J. Daniel et al.
of Candida bombicola on deproteinized whey concentrate
(DWC-20) with lactose as carbon source and repeated feed
of rapeseed oil as lipidic substrate, resulted in high concen-
trations of 280 g sophorolipid l21, but lactose was not
consumed during this cultivation, resulting only in mini-
mal reduction of the chemical oxygen demand (COD) in
waste water treatment (Daniel et al., 1998a). Therefore we
investigated a two-stage process (Daniel et. al, 1998b):
first, Cryptococcus curvatus ATCC 20509 (formerly known as
Candida curvata and Apiotrichum curvatum), originally iso-
lated by Moon et al. (1978), was cultivated on 1:1 (v/v)
diluted deproteinized whey concentrate (DWC-20), there-
by totally consuming the lactose and accumulating up to
60% triglycerides (single cell oil) of its cellular dry weight
as intracellular lipid under nitrogen limitation (Ratledge,
1988; Davies, 1988). After cell disruption, cell debris as
well as single cell oil served as media for growth and
sophorolipid production by Candida bombicola ATCC
22214, but yielded in only 12 g sophorolipids l21. The
reason for the low product concentration was the unfavour-
able C/N-ratio.
In this paper we describe, that this problem could be
overcome by feeding of cheap rapeseed oil during the
production phase, thus leading to high concentrations of
sophorolipids of 422 g l21.
Materials and methods
Organisms and chemicals
Deproteinized whey concentrate (DWC 20 contained 20%
dry weight after evaporation of water) was obtained from
Milei GmbH, Leutkirch-Adrazhofen, Germany and rape-
seed oil from Brändle, Empfingen, Germany. All other
chemicals were purchased from Fluka, Buchs, Switzer-
land.
Candida bombicola (ATCC 22214) and Cryptococcus curvatus
(ATCC 20509) were from the American Type Culture
Collection (ATCC).
Analytical methods
Intracellularly accumulated lipid concentration was deter-
mined according to the method as described in Daniel
et al. (1998b).
Sophorolipid concentrations were evaluated according to
the method described in Daniel et al., 1998b, too, after
extraction of water phase with ethyl acetate/isopropanol
(4:1 (v/v)). Course of sophorolipid production was followed
by TLC using silica gel 60-plates (20 3 20 cm) in
chloroform/methanol/water (65/15/2 (v/v/v)) and visualized
after dipping in acetic acid/conc. sulfuric acid/anisaldehyde
(100/2/1 (v/v/v)) reagent by heating to 150°C for 5 min.
1154 Biotechnology Letters ⋅ Vol 20 ⋅ No 12 ⋅ 1998
Growth was determined in two manners: (i) cells of
Candida bombicola were counted in a Thoma chamber under
microscope (3003), (ii) for dry weight determination of
both yeasts, 5 ml samples were centrifuged in preweighed
tubes. After two washing steps, the cell pellets were dried
in an oven at 105°C for at least 24 h and weighted. For
removing the oil and glycolipids from Candida bombicola in
the second stage, the cells were mixed with 1 ml of
ethanol/butanol/chloroform (10:10:1 (v/v/v)) before the
first centrifugation step.
Determination of lactose based on lactose/glucose test kit
from Boehringer Mannheim, Penzberg, Germany and
chemical oxygen demand (COD) was determined using a
test kit from Merck, Darmstadt, Germany.
General procedure for cultivation and product
isolation
Deproteinized whey concentrate (DWC-20) was sterilized
by a two-step filtration process (Daniel et al., 1998a),
pumping the whey permeate directly into the presterilized
bioreactor. Cultivation of Cryptococcus curvatus ATCC 20509
was performed at 30°C at pH 5.8 in a 3.0 l stirred
bioreactor equipped with a pH-, aeration-, weight-,
temperature- and agitation control. The medium consisted
of 1.5 l sterile filtrated of DWC-20 without any additional
nutrients (DWC-20 contained approximately 100 g l21
lactose, 1 g l21 glucose, 2 g l21 galactose, 100 mg l21
NH41 and further trace elements). After consumption of
lactose, cells were disrupted in 7 stages at 800 bar using a
high pressure laboratory homogeniser Model MINI-LAB,
type 8.30H from APV Homogenizer, Lübeck, Germany.
The medium for Candida bombicola consisted of the result-
ing crude cell extract of Cryptococcus curvatus, containing
cell debris and single-cell oil and was autoclaved in the
bioreactor. The following cultivation was performed at
25°C. During growth phase, pH-value was kept constantly
at pH 4.7, during the production phase at pH 3.3. After
consumption of the single-cell oil, repeated feeding of 100
g rapeseed oil l21 was started (see Figure 1). The adjust-
ment of the pH value in both cultivations was done with
10% NaOH solution or 10% phosphoric acid (v/v)
respectively.
For product sedimentation,850 ml of mixed culture broth
were filled into an 1 l graduate with a diameter of 6 cm
after the end of the cultivation. The level of the sophoro-
lipid phase in this column (bottom phase) was recorded.
Using this method, the sedimentation rate of sophorolipid
could be detected.
Results and discussion
Growth of Cryptococcus curvatus, with a maximum specific
growth rate of mmax 5 0,15 h21 and production of single-

Figure 1a/b Time course of the two-stage cultivation:
(a) Cryptococcus curvatus, consuming lactose from
whey and producing single-cell oil. After cell disruption,
the crude cell extract containing the microbial triglycerides
directly served as a substrate for the second step (b), for
growth of and sophorolipid production by Candida
bombicola. Both cultivations were performed in the same
3-l-bioreactor. Symbols: r dry weight, j lactose, u
sophorolipid in water phase, s single-cell oil content
(each g/l); ↓ indicates additional feeding of 100 g rape-
seed oil l21l. The cultivation was stopped, when the
rapeseed oil was totally consumed and no further
sophorolipid production could be observed.
cell oil by this microorganism are shown in Fig. 1a: after
145 hours 34 g dry weight l21 of biomass containing 20 g
single cell oil l21 were produced from 100 g lactose l21.
During the cultivation the COD value of DWC was
reduced from 159 to 35 g oxygen l21. After cell disintegra-
tion in a high pressure homogeniser, the single cell oil
was released and autoclaved together with the crude cell
debris.
This single cell oil containing crude cell extract was
directly used as medium for growth and initial sophoro-
lipid production by Candida bombicola. At the beginning of
cultivation, the pH-value was constantly kept at pH 4.7.
However, when sophorolipid production started, the pH
was lowered to pH 3.3 according to Fiehler et al. (1997). A
maximum specific growth rate of mmax 5 0.23 h21 was
observed. After two days, a biomass concentration of 16 g
cell dry weight l21 was achieved.
Sophorolipid production started approximately in the mid-
dle of the exponential growth phase and increased sig-
nificantly after repeated feeding of rapeseed oil. Rapeseed
was used for feeding, because its content of oleic acid was
comparable to that of the the single cell obtained from
Cryptococcus curvatus (Daniel et al., 1998b). During the
stationary phase of growth, a continuous excretion of
sophorolipids was observed. The product spectrum, which
Sophorolipid production
was analyzed by TLC according to Asmer et al. (1988) was
dominated by the acetylated lactonized form at the begin-
ning of the cultivation and the deacetylated lactonized
sophorolipid at the end (data not shown).
The single cell oil, resulting from the first stage, was
consumed by Candida bombicola after 29 hours. When
100 g rapeseed oil l21 were added to the cultivation broth,
a short decrease of sophorolipid content could be observed.
This was due to the fact that the sophorolipids were
dissolved into the lipidic phase. An exhaustion of the
lipidic substrate during the cultivation caused an immedi-
ate and steep decrease in O2 consumption and sophorolipid
production. This effect underlined the tight dependence of
the metabolic activity and oil content for the cells.
The specific production rate for sophorolipid formation by
Candida bombicola during the second stage decreased step-
wise from 151 mg sophorolipid gdw21 h21 after the first
feeding of rapeseed oil to 118, 113 and finally to 80 mg
sophorolipid gdw21 h21 after the fourth addition of oil.
Despite this, high concentrations of 422 g dry extracellular
sophorolipids l21 could be obtained, resulting in high
viscosity of the culture broth during the cultivation, so
that subsequent stirring became impossible. Therefore the
cultivation of Candida bombicola was stopped after 410
hours. In total, 400 g rapeseed oil l21 and 20 g single cell
oil l21 were used to produce a content of 422 g sophoro-
lipid l21, indicating a high production efficiency.
Sedimentation in a glass column was investigated as simple
method for product recovery from the culture broth (see
Figure 2). At the beginning, separation started immedi-
ately with a sedimentation rate of 17 ml liquid sophoro-
lipid min21. After 30 minutes, sophorolipids had
Figure 2 Time course of sedimentation of sophoro-
lipids in a 1-l graduate glass column. Symbols: j level of
sophorolipid phase in the graduate (ml); during sedi-
mentation, the upper level of sophorolipid phase was
recorded.
Biotechnology Letters ⋅ Vol 20 ⋅ No 12 ⋅ 1998 1155
H.-J. Daniel et al.
sedimentated as bottom phase and the level of sophorolipid
phase remained constant in the glass column. Extraction
experiments with organic solvents proved that only 5 g
sophorolipid l21 remained dissolved in the top water phase
containing the Candida bombicola cells. The COD value of
the top water phase was 46 g oxygen l21. This indicated
that sedimentation can be used as fast and cheap method
for product recovery in downstream processing of sophoro-
lipids: no organic solvent is necessary to separate the crude
product from culture broth.
With this two-stage process, high amounts of sophoro-
lipids can be easily obtained with the great advantage of
(i) total lactose consumption and (ii) distinct reduction of
the COD value of the initially used DWC-20 whey in
contrast to our previous investigations (Daniel et al. 1998a
and b).
Acknowledgements
Hans-Joachim Daniel was supported by grants of the
Arbeitsgemeinschaft industrieller Forschungsvereinigungen
(AiF) „Otto von Guericke“ 10771 N/I arised by the
Federal Ministry of Economy. Whey was as a gift from
Milei GmbH, Leutkirch-Adrazhofen, Germany, which is
gratefully acknowledged.
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Received: 26 October 1998
Accepted: 10 November 1998