Downstream processing of mannosylerythritol lipids produced by ...

Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 DOI 10.1002/ejlt.200401122 373
Udo Rau a
La Anh Nguyen b
Harald Roeper c
Helmut Koch c
Siegmund Lang a
a Technical University
Braunschweig,
Institute of Biochemistry and
Biotechnology,
Braunschweig, Germany
b C12 P4 Khu tap the Kim Giang,
Hanoi, Vietnam
c Cargill R&D Europe,
Vilvoorde, Belgium
1 Introduction
Surfactants are amphiphile molecules consisting of a
hydrophilic and a hydrophobic domain. The non-polar
hydrophobic part is frequently a hydrocarbon chain. The
polar component appears in many variations [1]. Biosurfactants
are structurally diverse compounds, mainly
produced by oil-, fatty acids- and/or hydrocarbon-utilising
microorganisms, which exhibit surface activity.
Among the biosurfactants, the mannosylerythritol lipids,
as a representative of glycolipids, have gained reasonable
attraction in recent years [2] due to their manifold applications
such as pharmaceuticals [3, 4], as chemical tool
for purification of proteins [5], or as anti-agglomeration
agent of ice-water slurry [6]. Bioremediation of petroleum
hydrocarbons as environmentally friendly decontamination
technology is also an interesting field of application
[7, 8].
Mannosylerythritol lipid (MEL), i.e. 2,3-di-O-alka(e)noyl-ß-D-mannopyranosyl-(1?4)-O-meso-erythritolpartially
acetylated at C4 and/or C6, contains mannose and
the sugar alcohol erythritol as permanent hydrophilic
moiety, and acetyl groups as well as fatty acids as variable
hydrophobic groups (Fig. 1). The different degrees of
acetylation are reflected by four different types of MEL A-
D [9]. MEL can be produced by Ustilago maydis [10],
Schizonella melanogramma [11], Kurtzmanomyces sp.
[12], and different strains of the genus Candida [9, 13, 14].
Most investigations related to cultivation were performed
in shake flasks. Only few reports exist about bioreactor
production of MEL [15, 16]. We used strains of Pseudo-
Correspondence: Udo Rau, Technical University Braunschweig,
Institute of Biochemistry and Biotechnology, Spielmannstr. 7,
D-38106 Braunschweig, Germany. Phone: 149 531 391–5740,
Fax: 149 531 391–5763, e-mail: U.Rau@tu-bs.de
Downstream processing of mannosylerythritol
lipids produced by Pseudozyma aphidis
Pseudozyma aphidis DSM 14930 was used for the bioreactor production of mannosylerythritol
lipids (MEL). A scheme is presented for the isolation and purification of
MEL by using different solvents. Adsorption experiments of MEL at Amberlite XAD
resins were performed. Up to 93% (wt-%) MEL could be transferred from the culture
suspension into a newly formed, highly viscous solid phase by heating the culture
suspension to 100 7C. This phase contained on average 87% (wt-%) MEL and
could be simply isolated by pouring off the supernatant, without producing solvent
waste.
Keywords: Mannosylerythritol lipid, MEL, downstream processing, analysis.
zyma aphidis for the production of MEL in shake flasks as
well as in a bioreactor [17, 18]. To date, in spite of interesting
applications, a real breakthrough of MEL did not
take place, mainly due to high production costs. Beside a
high yield and productivity of the bioreactor process, the
subsequent downstreaming of the product is economically
crucial. Therefore, an easy-to-handle procedure is
required for an economic isolation and purification of
MEL. The current report describes such a process.
Fig. 1. Molecular structure of mannosylerythritol lipid.
The length and saturation of the fatty acid residues (R2,
R3) depend on the substrate and microorganism used.
For example, if Pseudozyma aphidis DSM 14930 is grown
on soybean oil: R2 = R3 = C7-C14 fatty acids, saturated
and unsaturated. R4, R6 = acetyl or H.
2 Material and methods
2.1 Microorganism
Pseudozyma aphidis DSM 14930 was isolated from soil,
identified by and deposited at the Deutsche Stammsammlung
von Mikroorganismen und Zellkulturen
(DSMZ), Braunschweig, Germany. Stock cultures were
grown for 3 d at 30 7C on agar medium containing 24 g
potato dextrose L 21 and 20 g agar L 21 . They were stored
at 4 7C and renewed every 4 wk.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.de
Research Paper

374 U. Rau et al. Eur. J. Lipid Sci. Technol. 107 (2005) 373–380
2.2 Media and cultivation conditions
The seed culture contained 100 mL medium in 500 mL
baffled shake flasks and was inoculated from agar slants.
The medium for precultivation contained (all media data
are related to 1 L deionised water): 30 g glucose, 1 g
NH 4NO 3, 0.3 g KH 2PO 4, 1 g yeast extract, pH = 6.0 (not
controlled). After 2 d on a rotary shaker at 110 rpm and
30 7C, the preculture was transferred into the following
bioreactor medium: 80 mL soybean oil (density
0.84 g mL 21 ), 2 g NaNO 3, 0.2 g KH 2PO 4, 0.2 g
MgSO 4 7H 2O, 1 g yeast extract, pH = 6.2 (not controlled),
temperature 27 7C. A 72-L bioreactor (Sartorius BBI Systems
GmbH, Melsungen, Germany) equipped with three
Rushton turbine impellers was used. The bioreactor was
filled with only 30 L medium due to foam formation. As
initial impeller speed and aeration rate, 300 rpm and
720 L h 21 , respectively, were applied. Soybean oil as antifoam
agent and carbon source was additionally supplied
throughout the cultivation. The bioreactor cultivation is
described in detail elsewhere [18].
2.3 Analyses
In order to quantitatively detect MEL, triglycerides and
fatty acids, 3 mL culture suspension was acidified with
two drops of 5 N HCl to pH 2 and was subsequently
extracted three times with 3 mL methyl tertiary butyl ether
(MTBE). The mixture was vortexed for 1 min and centrifuged
for 5 min at 6000 rpm. The organic phases were
combined and analysed either by TLC or HPLC. The distinct
separation of single spots for qualitative TLC analysis
was carried out by the development of Silica gel 60
F 254 (Merck, Germany) plates with the ternary solvent
system CHCl 3/MeOH/H 20 (65:15:2). HPLC was performed
on a silica gel column (Nucleosil 100-5; CS-Chromatographie
Service GmbH, Langerwehe, Germany) with
an evaporative light scattering detector (ACS Mass
Detector model 750/14; Houston, TX, USA) using a gradient
solvent program consisting of various proportions of
CHCl 3 and CH 3OH (from 99:1 to 0:100, vol/vol) at a flow
rate of 1 mL min 21 [17]. The data of MEL, soybean oil,
fatty acids and cell protein are mean values from at least
two independent determinations. Differences of individual
data varied between 2% and 7%. Due to non-uniform
accumulation of storage material inside the cells, bio dry
mass showed higher deviations from the mean value and,
therefore, up to a fourfold determination was carried out.
2.4 Surface tension
The influence of MEL on the surface tension of water at
25 7C was measured with a tensiomat (Lauda Wobser
GmbH, Lauda-Königshofen, Germany) using the ring
method. The detailed description of this method is published
elsewhere [17].
2.5 Downstream processing of MEL
2.5.1 Adsorption at XAD
Different types of Amberlite XAD (Rohm and Haas, Philadelphia,
PA, USA) were used as adsorbent. XAD-4 and
XAD-16 are cross-linked polymers with different groups at
their surface for the preferred adsorption of organic compounds
with low and low-to-medium molecular weights,
respectively. XAD-7HP can adsorb non-polar compounds
from aqueous systems as well as polar compounds from
non-polar solvents. Adsorption tests were carried out as
follows: 10 mL culture suspension was added to 4 mL
Amberlite beads. After 24 h of stirring, the beads were
separated by filtration (glass fibre filter) and rinsed with
10 mL water. The rinsing solution was extracted by the
addition of 10 mL MTBE under vigorous mixing. The
beads were divided and subsequently extracted with
10 mL of either MTBE or methanol. The organic phases
were analysed and compared by TLC.
2.5.2 Extraction with organic solvents
The thoroughly mixed culture suspension was first
extracted three times with MTBE. Aqueous and organic
phases were separated by centrifugation. The collected
organic phases were evaporated and vacuum dried
(60 7C, 3000 Pa). After solving the extract in methanol, it
was subsequently treated also three times, but with
cyclohexane. The again evaporated and dried extract still
contained minor amounts of soybean oil and fatty acids.
These compounds were removed by using a mixture of
n-hexane, methanol and water (1:6:3, pH 5.5) as well as
subsequent threefold extraction of the aqueous phase by
n-hexane. The lyophilised aqueous phase resulted in a
pure MEL fraction as transparent, brown-coloured, highly
viscous fluid.
2.5.3 Heating
After heating of the culture suspension to 100–121 7C for
1–20 min, two MEL-containing phases, a solid sticky and
an aqueous one, were formed. About 90% (wt/vol) of MEL
was transferred into the solid phase by this procedure,
which could be easily separated by pouring off the cell
debris-containing aqueous phase.
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Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 Downstream MEL 375
3 Results
3.1 Bioreactor cultivation
A representative example of bioreactor cultivation for the
production of MEL using Pseudozyma aphidis
DSM 14930 is shown in Fig. 2. After about 1 d, nitrate
and yeast extract as nitrogen sources were consumed,
and the course of cell protein indicated the approach to
the stationary phase. However, bio dry mass continued
to increase due to the accumulation of storage material
inside the cells. After growth had ceased, the formation
of foam cumulatively increased; therefore, impeller
speed and aeration rate had to be reduced gradually
from 300 to 250 rpm and from 720 to 100 L h 21 , respectively.
In spite of these modifications, the pO 2 was not
influenced essentially and remained at about 60%. The
reduction of impeller speed and aeration rate was not
sufficient to prevent overfoaming. For this reason, soy-
bean oil was additionally fed in various rates, both as
carbon source and as anti-foam agent. Depending on
the addition and consumption rate, different amounts of
soybean oil and fatty acids, released from soybean oil by
the lipolytic activity of P. aphidis, were detected. However,
repeated cultivations showed that after soybean oil
addition was stopped, a prolongation of the cultivation
time by 1 d was sufficient for the total assimilation of
residual substrates.
After 3 d, the first green to yellow MEL beads separated at
the bottom of the sampling bottle. The number and width
(2–10 mm) of these beads increased with time. Previous
investigations [18] showed that the MEL beads were
formed at a concentration greater than 40 g MEL L 21 and
contained high quantities of MEL .60% (wt-%) and relatively
small amounts of soybean oil ,20% (wt-%) as well
as fatty acids ,10% (wt-%). After 8 d of cultivation, 90 g
MEL L 21 was yielded.
Fig. 2. Cultivation of P. aphidis in a 30-L
bioreactor. After 1 d, the impeller speed and
aeration rate were gradually decreased
from 300 to 250 rpm and from 720 to
100 L h 21 , respectively, depending on the
foam formation. Between days 2.5 and 8,
soybean oil was fed at various rates (0.2–
0.67 g L 21 h 21 ). The first MEL beads were
observed after 3 d.
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376 U. Rau et al. Eur. J. Lipid Sci. Technol. 107 (2005) 373–380
3.2 Isolation and purification of MEL
The MEL beads could be taken as indicators for
enhanced product formation. Their consistency was
similar to highly viscous oil drops, and they could not
simply be separated by, e.g., filtration. Therefore, stepwise
conventional extraction techniques starting with the
total culture suspension and using different solvents was
first applied for isolation and purification of MEL (Fig. 3).
The extraction step with MTBE yielded on average 75%
(wt-%) MEL, 15% (wt-%) soybean oil and 10% (wt-%)
fatty acids after drying. The threefold repetition of this
extraction step was necessary for exhaustive transfer of
MEL into the organic phase. The further enrichment to
91% (wt-%) MEL and decrease to 5% (wt-%) soybean oil
and 4% (wt-%) fatty acids was achieved by subsequent
also threefold extraction, but using cyclohexane. The
resulting purified MEL fraction was a transparent, browncoloured,
highly viscous fluid at ambient temperature.
During this procedure, about 20% (wt-%) MEL was lost
compared to the mass contained in the primary culture
suspension. The quantitative analysis of the compounds
was carried out by HPLC. A complete separation of residual
soybean oil and fatty acids was achieved by using
n-hexane, methanol and water as solvent mixture with
subsequent threefold extraction by n-hexane. The purification
of MEL was documented by TLC (Fig. 3). However,
the advantage to yield pure MEL was combined with an
essential loss of recovery down to 8% (wt-%).
Different polymeric resins (Amberlite XAD-4, XAD-16,
XAD-7HP) were tested for the specific adsorption of
either MEL or fatty acids and soybean oil, in order to
Fig. 3. Scheme of the stepwise
extraction procedure by using different
solvents for isolation and
purification of MEL. The TLC
represents the three extraction
steps by n-hexane. Lanes 1–6,
organic phase; lanes 7–12, aqueous
phase; lanes 1, 2 17, 8, first
extraction; lanes 3, 4 19, 10, second
extraction; lanes 5, 6 111, 12,
third extraction. MTBE, methyl tertiary
butyl ether; FA, fatty acid; SO,
soybean oil.
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Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 Downstream MEL 377
facilitate the downstream process. As a result, all polymers
were able to accumulate MEL in different amounts
that could be eluted by MTBE or methanol. However, fatty
acids and soybean oil were also adsorbed. A specific
adsorption was not possible by the use of these resins.
Only a trend to higher adsorption capacity of MEL with
simultaneous decreasing accumulation of fatty acids and
soybean oil was observed in the order: XAD-16 . XAD-7
. XAD-4.
When the MEL-containing culture suspension was transferred
into a glass bottle, the separation of aggregated
MEL beads could be observed at the bottom as highly
viscous fluid (Fig. 4, left picture). This viscous MEL phase
and the MTBE extract of the whole culture suspension
(Fig. 3) possessed a similar composition (Fig. 4). After
sterilisation of the MEL-containing culture suspension at
121 7C for 20 min, two MEL-containing phases, a solid
sticky and an aqueous one, were formed, both fatty acid
enriched as well as soybean oil depleted (Fig. 4, right
picture). A small volume of a primary soybean oil-containing
top phase was also observed. Related to the total
mass of MEL (90 g L 21 ) yielded by MTBE extraction of the
culture suspension before heating, the MEL were distributed
after heating into the solid and aqueous phases by
89% and 11% (wt/vol), respectively. This solid phase was
easy to separate by pouring off the cell debris-containing
supernatant. If necessary, dependent on the intended
application of the MEL, the cell debris could be separated
by solving the solid phase in ethanol and subsequent filtration
using a pore width of 0.2 mm. About 11% (vol/vol)
of MEL remained suspended in the aqueous cell debris
phase and could additionally be recovered by extraction
with ethanol, centrifugation, rotary evaporation of the
solvent and vacuum drying.
Variations of time (1, 5, 15, 20 min) and temperature (100,
110, 115, 121 7C) of the culture suspension treatment
resulted in a nonessential difference of MEL content between
86.2–88.3% (wt-%) in the solid phase. Short incubation
times of 5 min led to the formation of a turbid
solid phase. The longer the time of treatment at each
temperature, the higher was the fatty acid and the lower
the soybean oil content, with a minimum of 0.3% (wt-%)
soybean oil and a maximum of 13.7% (wt-%) fatty acids
at 121 7C for 20 min (Fig. 4, grouped bars of solid phase).
Corresponding to Fig. 4, Fig. 5 shows HPLC and TLC
analyses of the MEL-containing phases before and after
heat treatment, as well as the distribution of the different
MEL. The maximum of 93% (wt-%) MEL transfer into the
solid phase with an appropriate reduction to 7% (wt-%)
MEL in the resulting aqueous phase was achieved at
110 7C for 10 min and was considered as the most effective
treatment for downstreaming the MEL. This solid
phase contained 88.3% (wt-%) MEL, 6.6% (wt-%) fatty
acids as well as 5.1% (wt-%) soybean oil and reduced the
surface tension of water/air to 31 mN m 21 (critical micelle
concentration 15 mg L 21 ).
Fig. 4. Composition of different MEL phases from a
culture suspension before (left) and after (right)
treatment at 121 7C for 20 min. The analyses were
carried out by HPLC.
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378 U. Rau et al. Eur. J. Lipid Sci. Technol. 107 (2005) 373–380
4 Discussion
Different microorganisms as, e.g., Kurtzmanomyces sp.
I-11 [12], Candida antarctica [14] and Ustilago maydis [10]
were used by other authors for shake flask production of
MEL. Kitamoto’s group succeeded in the formation of
140 g MEL L 21 by additional feeding of n-octadecane
using Pseudozyma (Candida) antarctica T 34 [19]. However,
only rare information about bioreactor production of
MEL is available. Kim et al. [15] used Candida sp. SY16 in
a 5-L bioreactor for the formation of 100 g L 21 crude MEL
phase containing only 4% (wt-%) pure MEL. Hitherto, 46
and 165 g L 21 are the highest reported yields of MEL
obtained in a bioreactor by using Pseudozyma (Candida)
antarctica ATCC 20509 [16] and Pseudozyma aphidis
DSM 70725 [18], respectively.
The choice of method for the isolation and purification of a
particular biosurfactant depends on its ionic charge, its
solubility in water, and on whether the product is cell
bound or extracellular. The methods used include solvent
extraction, adsorption followed by solvent extraction,
precipitation, crystallisation, centrifugation and foam
fractionation. Desai and Desai [20] as well as Syldatk and
Wagner [21] gave an overview of these procedures. Solvent
extraction is the most commonly used technique for
the downstream processing of biosurfactants. We also
described such a multi-step extraction (Fig. 3) for MEL
that is further on absolutely necessary if, e.g., spectroscopic
investigations are performed or an application as
pharmaceuticals is intended [3, 4]. The disadvantage of
this process is the production of huge amounts of waste
solvents that have to be recycled. Beside the costs for
bioreactor production, the recycling increases the manufacturing
costs so that an acceptance of this bioprocess
for industry is additionally inhibited.
Fig. 5. HPLC and TLC of the solid MEL
phase after heat treatment at 121 7C for
20 min. The HPLC signals correspond with
the grouped bars of the solid phase of
Fig. 4. Distribution of MEL (wt-%):
A = 40.8, B = 43.4, C = 9.7, D = 6.1. The
embedded TLC corresponds with the
grouped bars of Fig. 4: Before heat treatment,
1 = viscous MEL phase; after heat
treatment, 2 = aqueous phase, 3 = solid
phase.
Unfortunately, the specific adsorption of MEL or soybean
oil and fatty acids failed by the use of different Amberlite
resins. Only a general tendency to higher accumulation of
MEL was observed in the order: XAD-16 . XAD-7HP .
XAD-4.
The MEL can also be isolated by preparative HPLC
equipped with silica gel columns [15, 17, 22]. This is a
superior method to produce a very pure MEL mixture or
even to separate the individual MEL. However, the loss of
product is substantial, and so this is not a beneficial solution
in order to decrease the costs for the downstream
process.
A very easy, solvent-free separation of a MEL-enriched
solid phase could be achieved by just heating the culture
suspension to a temperature of 100 7C. Related to the
total MEL content of the untreated culture suspension,
the heating at 110 7C for 10 min led to the maximum
transfer of 93% (wt-%) MEL into this solid phase. The
supernatant could be poured off, which left a solid, highly
viscous mass, on average composed of 87% (wt-%) MEL
(Figs. 4, 5). The remaining 13% (wt-%) consisted of soybean
oil and fatty acids in different amounts, depending
on time and temperature throughout the treatment. Independent
of the temperature, the longer the time of treatment,
the more soybean oil was transferred to a new
phase separated at the top. Furthermore, prolonged
heating favoured the release of fatty acids from the
remaining soybean oil.
A comparison of known MEL downstream procedures is
given in Tab. 1. Unfortunately, to the best knowledge of
the authors, only two references are available with a
quantitative description of the methods. The heat treatment
attained the highest yield and is the fastest method
by far, but only on average 87% (wt-%) MEL is contained
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Eur. J. Lipid Sci. Technol. 107 (2005) 373–380 Downstream MEL 379
Tab. 1. Comparison of different methods for the downstream
processing of MEL { .
Ref. Method § Yield
[wt-%]
# Purity
[wt-%]
[22] Ethyl acetate extraction 1
preparative HPLC
79 100
[15] Ethyl acetate extraction 1
preparative HPLC
4 100
Stepwise extraction with
different solvents (Fig. 3)
8 100
Heat treatment (Fig. 4) 93 87
{ Preparative HPLC was performed with silica gel columns.
§ g MEL recovered
Yield =
g MEL before downstream 100
#
Purity is related to the mass fraction of MEL.
in the precipitated fraction. However, this solid-enriched
MEL phase should be pure enough for the most industrial
applications [5–8]. For example, pure MEL A, purified
100% (wt-%) MEL and a mixture of 88.3% MEL, 6.6%
soybean oil and 5.1% (wt-%) fatty acids reduced the surface
tension of water/air to similar data of 34.7, 26.7 and
31 mN m 21 , respectively [17].
5 Conclusion
Bioreactor production of mannosylerythritol lipids was
performed by the use of Pseudozyma aphidis
DSM 14930. Up to 93% (wt-%) MEL could be transferred
into a solid, highly viscous phase by heating the culture
suspension to 110 7C for 10 min. This phase contained on
average 87% (wt-%) MEL and could be simply isolated by
pouring off the supernatant, without producing solvent
waste. Together with the high MEL yield of 165 g L 21 ,
obtained by foam-controlled addition of soybean oil [18],
this facilitated downstream process should stimulate the
industrial production of MEL.
Acknowledgments
We thank W. Grassl for technical assistance.
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[Received: December 22, 2004; accepted: April 1, 2005]
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