1. Please cite this article in press as: B.M. Dolman, et al., Integrated sophorolipid production and gravity separation, Process Biochem
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Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Integrated sophorolipid production and gravity separation
Ben M. Dolman, Candice Kaisermann, Peter J. Martin, James B. Winterburn∗
School of Chemical Engineering and Analytical Science, The Mill, The University of Manchester, Manchester, M13 9PL, UK
a r t i c l e i n f o
Article history:
Received 9 November 2016
Received in revised form
13 December 2016
Accepted 21 December 2016
Available online xxx
Chemical compounds:
Sophorolipid (PubChem CID: 11856871)
Keywords:
Integrated separation
Sophorolipid
Settling
Candida bombicola
Biosurfactant
Glycolipid
a b s t r a c t
A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been
developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fer-
mentation broth, while cells and other media components are recirculated and returned to the bioreactor.
The capability of the separation system to recover an enriched sophorolipid product phase was demon-
strated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11%
reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recov-
eries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment
of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase
enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the
fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration
could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume.
These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high
volume applications such as enhanced oil recovery economically feasible.
© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Sophorolipids are microbially produced glycolipid biosurfac-
tants, which are rapidly increasing their market share of the 27
billion USD global surfactant market [1]. While several yeast strains
are able to synthesize sophorolipids, most research and industrial
use is focused on Candida bombicola ATCC 22214, the organism used
in this study [2]. Sophorolipids consist of a hydrophilic sophorose
disaccharide bound to a hydrophobic fatty acid with a typical chain
length of 16–18 carbon atoms. The fatty acid may be joined by
an ester bond to the second glucose monomer, giving a lactonic
sophorolipid, or joined to only one glucose monomer, giving an
acidic sophorolipid due to the unbound fatty acid. These and other
differences in the fatty acid chain and acetylation of the sophorose
molecules give a range of different structures and properties. Two
common structures representing lactonic and acidic sophorolipids
are shown in Fig. 1 [2].
Sophorolipids are produced industrially by a number of compa-
nies, who often utilize sophorolipids’ detergent and low foaming
properties in a variety of formulated cleaning products [3]. The
therapeutic properties of sophorolipids have allowed them to be
commercialized in anti-dermatitis soap and other body washes,
∗ Corresponding author.
E-mail address: james.winterburn@manchester.ac.uk (J.B. Winterburn).
and in a cream to reduce oily skin by MG Intobio Co and Soliance.
There is ongoing research into potential medical applications of
sophorolipid, with anti-cancer, anti-HIV, antimicrobial and anti-
biofilm activity being investigated [4–6]. Sophorolipids also have
potential for use in low cost, high volume applications such as
bioremediation and enhanced oil recovery if production costs can
be significantly reduced [7,8].
In sophorolipid producing fermentations, product concentra-
tions of over 300 g l−1, with productivities of over 1 g l−1 h−1 are
routinely achieved [9–11]. Sophorolipid producing fermentations
begin with a cell growth phase, which typically lasts until the
nitrogen in the media is depleted, at which point the sophorolipid
production rate increases significantly, if both a hydrophilic and a
hydrophobic carbon source are present [12]. The sophorolipid pro-
duction phase normally lasts for around 200 h, at which point the
dissolved oxygen level in the fermenter cannot be maintained due
to oxygen mass transfer limitation.
This dissolved oxygen reduction is caused by the high viscosity
of the sophorolipid produced, meaning the fermentation must be
stopped and the sophorolipid recovered [12,13]. It is well known
that the presence of a separate sophorolipid phase in the bioreactor
significantly reduces the oxygen mass transfer coefficient, kLa, by
both providing a resistance to mass transfer across the air/liquid
interface and increasing the viscosity of the medium, which results
in oxygen limitation, increased stirring power requirements and
non-homogeneity in the bioreactor [12–14].
http://dx.doi.org/10.1016/j.procbio.2016.12.021
1359-5113/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

2. Please cite this article in press as: B.M. Dolman, et al., Integrated sophorolipid production and gravity separation, Process Biochem
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Fig. 1. Molecular structure of common lactonic (left) and acidic (right) sophorolipids. A lactonic bond can be seen joining the fatty acid chain to the second glucose monomer
of the sophorose in the lactonic sophorolipid, where the acidic sophorolipid has a free carboxylic acid to end its fatty acid chain.
The physical form of sophorolipids is dependent on the con-
ditions under which they are produced, which directly affect
the proportions of acidic and lactonic sophorolipids produced.
Sophorolipids typically separate from the fermentation broth as
a crystalline material if the lactonic to acidic ratio is high and the
hydrophobic carbon source concentration is low. The sophorolipids
otherwise form a viscous second phase of around 50% sophorolipid
and 50% water, which may sit below residual oil at the surface of
the broth or sink to the bottom of the bioreactor when agitation is
stopped [10,15,16].
These properties are commonly exploited at the end of a fermen-
tation to give an easy, crude separation of the sophorolipid from the
fermentation broth, either by crystal decantation, crystal filtration
or decantation of the sophorolipid gel [11,16,17]. These techniques
have not previously been used effectively to recover sophorolipids
during fermentation.
Industrially, there are a number of costs associated with
repeated batch cycles for sophorolipid fermentation, in terms of
downtime between cycles, cleaning costs and the lengthy inocu-
lum preparation required for large scale production [18,19]. There
are numerous proposed partial solutions to this problem in the lit-
erature. For example, a portion of the broth can be removed and
replaced with fresh media, allowing high productivity to be main-
tained for seven 80–130 h cycles, nevertheless removing biomass
proportionally to other components [19]. Sophorolipid settling by
gravity within a fermentation vessel or shake flask has previously
been demonstrated for small scale sophorolipid production. Signif-
icant benefits of sophorolipid separation have been shown, with a
doubling of the duration of sophorolipid production and little effect
on production after 15 min without agitation or aeration demon-
strated by Guilmanov et al. [20], and a productivity increase from
1.38 to 1.89 g l−1 h−1 shown by Marchal et al. [19]. Both studies rely
on gravity settling within the fermentation vessel or shake flask,
however, making scale up impractical due to the excessive settling
distances present if this technique were applied at industrial scale.
Effective integrated separation techniques have been developed
for other biosurfactant systems, notably foam fractionation for
hydrophobin proteins, surfactin and rhamnolipids, but there have
been no successful scalable attempts at integrated separation for
sophorolipid production [21–23].
This paper details a novel technique, based on an integrated
gravity settling column, for removing the sophorolipid phase from
the fermentation broth during fermentation, reducing the fermen-
tation volume required which allows continued substrate feeding
and provides a concentrated product phase. We also demonstrate,
for the first time, the application of this technique to extend the
production phase of a fermentation beyond 1000 h, significantly
increasing batch production.
2. Methodology
2.1. Fermentation
Sophorolipid was produced by fed batch fermentation using C.
bombicola ATCC 22214 in an Electrolab Fermac 320 fermentation
system (Electrolab, UK) with a 2 l maximum working volume, H:D
of 2, two 55 mm diameter 6-bladed Rushton-type impellers and an
initial working volume of 1 l, with a stirring rate of 200–800 rpm.
This was sufficient to disperse vegetable oil within the bioreactor.
Growth medium for the fermentations, preculture and agar plates
contained 6 g l−1 yeast extract and 5 g l−1 peptone. The initial con-
centration of glucose in all fermentations preculture and agar plates
was 100 g l−1, with an initial rapeseed oil concentration of 50 g l−1
in the fermenters, 100 g l−1 in the preculture and 0 g l−1 in the agar
plates.
Four fermentations were carried out. Fermentation 1 was con-
ducted in a conventional manner without separation. Fermentation
2 was directly comparable to fermentation 1 except that the in situ
separator was used to remove product from the top of the separa-
tor. Fermentation 3 was controlled to give a product that separated
from the bottom of the separator. Fermentation 4 was carried out
for an extended period of time to demonstrate the potential to
utilise the integrated separation to extend the fermentation period
and give higher batch production, and controlled to give separation
from the surface of the broth. C. bombicola was first transferred from
cryogenic storage (−80 ◦C) onto agar plates, and incubated at 25 ◦C
for 48 h. Single colonies from these plates were then used to inocu-
late 50 ml of medium in 250 ml shake flasks, which were incubated
at 25 ◦C and 200 rpm for 30 h. This inoculum was diluted to an opti-
cal density of 20 at 600 nm with fresh media and 100 ml used to
inoculate the fermenter.
Fermentations were run at 25 ◦C, and dissolved oxygen was con-
trolled to 30% by varying the stirrer speed, whilst maintaining a

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constant aeration rate of 1 l min−1. Fermenter pH was controlled to
a value of 3.5 by the addition of 3 M sodium hydroxide.
The feeding rates of rapeseed oil and glucose were similar for
fermentation one and two, to facilitate comparison, with different
feeding rates used for fermentation three and four, to give separa-
tion of sophorolipid to the top and bottom of the fermenter. Feeding
rates of oil were modified during the experiments to maintain a
low concentration, without limiting production, according to the
oil concentration in the samples. Glucose concentration was used
to control the relative density of the sophorolipid phase and the
bulk media to enable effective separation from either the top or
bottom of the separator, as well as being an important substrate
for sophorolipid production. The feed profiles are shown in Fig. 4.
The total sophorolipid produced was calculated by adding the
mass of sophorolipid in the fermenter and the mass of sophorolipid
removed from the fermenter using the separator. Contamination
was tested for visually using microscopy and by streak plating.
2.2. Separation
Integrated separation of the sophorolipid from the fermentation
broth was carried out using an in house built settling column, as
shown in Fig. 2. The integrated arrangement of the bioreactor and
settling column is shown in Fig. 3 with the settling column sup-
ported at an angle of 30◦ from horizontal. The separator was rinsed
with 70% ethanol before attaching to the fermenter. Sophorolipid
separation was carried out intermittently, based on visual observa-
tion of a sample taken from the bioreactor. When a significant layer
of sophorolipid rich phase could be seen at either the top or bot-
tom of the sample in the universal bottle within 2 min separation
was initiated. Prior experiments indicated that at these conditions
separation is effective.
During separation, which was used intermittently during fer-
mentation, sophorolipid rich fermentation broth was continuously
circulated from the fermenter, through the settling column and
back to the fermenter. This was pumped from the fermenter
through a stainless steel tube with an inlet 20 mm from the bot-
tom of the fermenter, and then in 8 mm external diameter silicon
tubing of 1 mm wall thickness to and from the separator. The flow
rate of media into and out of the settler was controlled to 1 ml s−1
using Matson Marlow 502 S and 503 U pumps (Watson Marlow,
UK). This flowrate was based on the results of preliminary experi-
ments, giving a residence time in the settling column of 76 s, with
a total residence time in the column and tubing of 137 s. In the set-
tling column, the sophorolipid phase separates out towards either
the top or bottom of the column, depending on the relative density
of the sophorolipid and bulk media. Initially, broth is continuously
circulated and the sophorolipid product collects in the settling col-
umn. When the sophorolipid phase accumulating in the separator
reached 50% of the height of the separator, which typically occurred
after around three minutes of separator operation, the outlet pump
was started to continuously remove the sophorolipid product phase
at a rate controlled between 0.5 and 2 ml min−1, depending on the
accumulation or reduction of the sophorolipid phase in the set-
tling vessel. The separation was stopped when the separation rate
dropped below 0.5 ml min−1, until the condition for separation was
again observed.
2.3. Hydrodynamics
To determine the effect of the sophorolipid phase on the agita-
tion requirements, the sophorolipid enriched fractions, which had
been separated from the bioreactor using the separator over the
course of fermentation 2, were pooled and returned to the fer-
menter at 308 h after the start of the fermentation over a period of
12 min. The stirrer speed and dissolved oxygen percentages were
then monitored as the fermentation control returned the dissolved
oxygen percentage to the set point. The equation for power number
is shown in Eq. (1);
P = Np n3
D5
(1)
where P is power, Np is power number, is density, n is stirrer speed
and D is stirrer diameter.
Due to identical fermenters being used and assuming the den-
sity is constant (as density changes during the fermentation are
relatively small), the power input as a function of power number
can be calculated during the fermentation. The power input was
integrated over time to determine the power input for the whole
fermentation, with the power input for fermentations equated until
the time point of the first separation.
Separation performance was measured in terms of enrichment
and recovery, which are defined in Eqs. (2) and (3);
enrichment =
Cp
Cf
(2)
recovery (%) =
Cp × Vp
Cf × Vf
× 100 (3)
where Cp is the sophorolipid concentration in the product, Cf is the
product concentration in the fermenter before separation, Vp is the
volume of product phase recovered, and Vf is the initial volume of
liquid in the fermenter.
2.4. Analytical techniques
For all analysis, 5 ml of broth was removed from the bioreactor or
5 ml product was taken from the sophorolipid collection vessel con-
nected to the separator. The sample was centrifuged at 5000 rpm
for 5 min using a Sigma 6–16S centrifuge (Sigma laboratory cen-
trifuges, Germany) and the glucose in the supernatant quantified
using a TrueResult
®
blood glucose monitor (Nipro, Japan).
A hexane extraction to extract residual rapeseed oil fol-
lowed by a triple ethyl acetate extraction to extract sophorolipid
were then applied to the whole sample, with oil concentra-
tion measured gravimetrically from the hexane extraction, and
sophorolipid measured gravimetrically from the pooled ethyl
acetate extracts[24–26]. These extracts were dried to constant
weight in weighing dishes at ambient temperature for 30 h.
Cell growth was determined by both dry cell weight and optical
density measurement. After the aforementioned hexane and ethyl
acetate extractions, 8 ml distilled water was added to the remainder
of the sample in the centrifuge tubes, which were then centrifuged
at 8000 rpm for 10 min. The supernatant was discarded and the
resulting cell pellet was resuspended in 8 ml distilled water. This
cell suspension was transferred to drying trays, which were dried
to constant weight at 90 ◦C in a drying oven. Optical density was
used as a proxy for dry cell weight when diluting the inoculum, at
a wavelength of 600 nm.
The structure of the sophorolipids produced was determined
with negative electrospray ionisation mass spectrometry, using
an Agilent 6520 QTOF mass spectrometer (Agilent, United States).
Samples were prepared by redissolving the ethyl acetate extracts,
i.e. sophorolipids, in ethyl acetate, and filtering using a 0.2 ␮m filter.
Flow injection analysis was used, at 0.3 ml min−1, 50% acetonitrile,
0.1% formic acid and 49.9 % water, with an injection volume of 2 ␮l.
The viscosity of the product phase was measured using an
AR2000 controlled rotational rheometer with cone geometry (TA
Instruments, USA).

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Fig. 2. Diagram of custom built sophorolipid separator used for this study. Plan view, side view and end view are shown, all dimensions are internal dimensions shown in
mm..
3. Results and discussion
A novel gravity sophorolipid separation technique was success-
fully applied to three fermentations. Results from one fermentation
without separation, fermentation 1, are presented alongside results
from three fermentations with separation, fermentations 2, 3 and
4. Fermentations 1 and 2, without and with integrated separa-
tion respectively, are directly comparable, to allow evaluation of
the effect of separation, but due to feeding rate control to enable
sophorolipid to be recovered from the bottom of the separator
fermentation 3 is not directly comparable. Fermentation 4 was
intended to demonstrate an extended fermentation, and so is also
not directly comparable. The separation was run periodically in
all fermentations, when sufficient sophorolipid phase had accu-
mulated, with the majority of the available sophorolipid phase
separated.
3.1. Fermentations
Fig. 4 shows the feeding rate of substrates for the fermentations
presented, which enabled control of the sophorolipid phase to sep-
arate from the surface or bottom of the separator, whilst also being
an important parameter for sophorolipid production. The progress
of the fermentations over time is presented in Fig. 5, and the key
metrics from these fermentations are presented in Table 1.
Fig. 5 shows the progress of fermentation 1, without separation,
and fermentations 2, 3 and 4, during which sophorolipid product
was separated from the fermentation broth. In fermentation 2 and
4, sophorolipid was recovered at the top of the integrated gravity
separator, and in fermentation 3 the sophorolipid was collected
from the bottom of the separator. In fermentation 2, separation
was carried out at 111, 184 and 261 h, in fermentation 3 at 72, 281,
355 and 376 h and in fermentation 4 at 86, 111, 160, 186, 232 and
540 h. No separation of the sophorolipid phase was carried out in
fermentation 1.
In fermentation 2, the glucose concentration initially rose, and
remained above 50 g l−1 for the majority of the fermentation, which
led to the sophorolipid rising to the surface of the fermentation
broth without agitation. A high glucose concentration throughout
Table 1
Key metrics for all sophorolipid producing fermentations. Fermentation 2, 3 and
4 used sophorolipid separation, with fermentation 1 included for comparison. All
metrics are the result of unique fermentations. Due to volume changes caused by
substrate addition and product removal, productivity is based on the initial fer-
menter working volume and the total sophorolipid produced.
Fermentation
1 2 3 4
Product separation none top bottom top
Duration (h) 305 305 379 1023
Yield substrate consumed (g g−1
) 0.43 0.53 0.42 0.53
Yield substrate fed (g g−1
) 0.33 0.37 0.39 0.47
Productivity (g l−1
h−1
) 1.07 1.07 0.71 0.61
Maximum fermenter volume (l) 1720 1544 1350 1550
Total sophorolipid produced (g) 325 325 270 623
Total dry cell weight (g) 16.1 21.0 16.5 32.1
Yield product on biomass (g g−1
) 20.2 15.5 16.4 19.4
fermentation 4 meant the sophorolipid was also separated from the
top of the separator during this experiment.
Sophorolipid was first separated from the bottom of the sepa-
rator at 71.5 h in fermentation 3, when a sophorolipid phase could
be observed to settle in a sample bottle within 2 min. Settling was
not possible after this until 283 h due to the high residual glucose
concentrations caused by pulse glucose feeding, which was used to
ensure good sophorolipid production. Whilst the relative density of
the sophorolipid phase and the broth also depend on other factors,
a glucose concentration of 50 g l−1 tends to represent a threshold
of sophorolipid phase separation to the surface or the bottom of
the separator. This is because higher glucose concentrations lead
to higher media densities, meaning the sophorolipid phase is rela-
tively less dense the higher the glucose concentration. After 283 h
the glucose concentration had dropped sufficiently for settling to
be used again. Lower glucose feeding could enable sophorolipid
settling throughout the fermentation, though this might impact
sophorolipid production.
Fermentations 1 and 2, which were identical apart from the
application of integrated sophorolipid separation in fermentation
2, each produced 325 g sophorolipid, with 270 g sophorolipid pro-
duced during fermentation 3. The dry cell weight production, of
16–21 g, and the yields of product on substrate, of 0.33-0.39 are

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Fig. 3. Integrated fermentation system. Bioreactor is shown on the left, with the separator in the center, and the product collection vessel on the right. Broth is pumped
from the bioreactor into the separator, and recirculated back to the bioreactor. Product is pumped from the separator into the collection vessel. This can be used for; (a) −
sophorolipid phase density higher than fermentation broth. (b)− sophorolipid phase density lower than fermentation broth.
in line with other results in the literature, as are the values for
total sophorolipid produced [9,25]. The use of the separator appears
to have little effect on the total production of sophorolipids over
the same time period, with identical values recorded for the two
comparable fermentations.
In fermentation 2 with separation, 21 g of biomass were pro-
duced, more than the 16 g of cell biomass produced in fermentation
1, with product yields on biomass of 15.5 g g−1 and 20.2 g g−1
respectively. The reason for the reduction in product yield on
biomass in fermentations 2 and 3 is not known, but with further
optimisation fermentation 4, with separation, reached a product
yield on biomass of 19.4 g g−1.
The highest working volume reached during a fermentation dic-
tates the overall fermenter volume required, and the high feeding
rates used during sophorolipid producing fermentations lead to a
large increase in the working volume required over time, much of
which is only required in the later stages of the fermentation.
The use of integrated sophorolipid separation in fermentation
2 decreased the fermenter working volume required by removing
523 ml broth from the fermenter using the separator. This meant
only 1540 ml working volume was needed for fermentation 2 com-
pared to 1720 ml in fermentation 1 without separation. This 11%
decrease in volume requirement could reduce bioreactor capital
costs. The corresponding maximum volume for fermentation 3 was
1350 ml, but was not directly comparable due to differences in
feeding rates between fermentations 2 and 3.
The overall productivity of fermentations 1 and 2 were 1.07 g
l−1 h−1 calculated at the starting volume, with a correspond-
ing productivity of 0.77 g l−1 h−1 for fermentation 3. The rate of
sophorolipid production slowed dramatically when the oil was
depleted after around 80 h in fermentations 1–3, reducing from 2 g
l−1 h−1 to 0.6 g l−1 h−1 and increasing the oil feed rate did not return
the productivity to the previous level. Many studies have demon-
strated a fairly constant production rate throughout a fermentation
until the point at which the fermentation had to be stopped due to
dissolved oxygen limitation, and with improved feeding control in
fermentation 4, a productivity of 2.0 g l−1 h−1 was maintained until
158 h [17] [12].
In fermentation 4, fermentation was continued past the point
at which fermentations usually have to be stopped due to product

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Fig. 4. Feeding profiles of glucose and rapeseed oil for all fermentations in this study. Glucose (blue), rapeseed oil (green) and total (red) shown for fermentation 1 (solid, a)
fermentation 2 (dotted, a), fermentation 3 (b) and fermentation 4 (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
accumulation, and run for a total of 1023 h. This enabled the produc-
tion of 623 g sophorolipid from a 1 l initial broth, which compares
favourably to the highest previous reported titers of around 400 g
l−1, and clearly demonstrates the capacity of integrated separation
to extend the time period of sophorolipid production in fermenta-
tion. This could lead to a dramatic improvement in overall process
productivity, by reducing the proportion of time spent in inoculum
preparation, biomass production and cleaning.
3.2. Separation
The separation results achieved in fermentations 2, 3 and 4 are
shown in Table 2.
During fermentations 2, 3 and 4 with integrated separation, the
majority of the sophorolipid was removed from the fermentation
broth, with 86% of the total sophorolipids produced separated in
fermentation 2, 74% separated in fermentation 3 and 65% separated
in fermentation 4.
Almost no cells and only 8 g of oil were removed by the separa-
tion over the course of fermentation 3, determined by gravimetric
analysis as for fermentation samples. This is because the rate of
settling of the cells was much slower than the settling of the
sophorolipid product, and oil rose to the surface of the separa-
tor rather than sinking to the bottom of the separator with the
sophorolipid. Cell removal was also negligible in fermentation 2,
though 68 g of oil was removed, which was reduced by better feed-
ing rate control in fermentation 4, where only 20 g oil was removed,
while again separating from the surface of the separator. 2.6 g cells
were removed during fermentation 4, which represents a small
proportion of the total biomass, 32.1 g.
The enrichment varied significantly between separations at
different time points, from 2.5 to 9. This is largely due to the
sophorolipid concentration present in the fermenter before the
separation, as there was little variation of the concentration in the
sophorolipid enriched product fraction, of approximately 550 g l−1.
In fermentation 2, when the sophorolipid phase was separated from
the top of the settler, a total of 68 g oil was recovered along with
the sophorolipid during the fermentation, which was the primary
reason for the variations in the concentration of the sophorolipid
phase. There is little scope to improve the product phase concentra-
tion above that demonstrated in fermentation 3 using the current
technique, however, with an increased fermenter volume, the sys-
tem could operate at lower initial sophorolipid concentrations and
so give an improved enrichment.
Sophorolipid recoveries would be expected to improve sig-
nificantly as fermentation scale is increased; in laboratory scale
experiments the separation had to be stopped as the layer of
sophorolipids at the bottom/top of the settling column became too
low, to prevent the media and cell phase being entrained in the
product stream. This minimum sophorolipid phase depth, which

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Fig. 5. Time course of fermentations using sophorolipid separation. Results for fermentation 1 (a), fermentation 2 (b), fermentation 3 (c) and fermentation 4 (d) are presented.
Dry cell (black squares) glucose (blue triangles) rapeseed oil (green circles) and sophorolipid (filled red circles) concentrations are shown. Arrows show sophorolipid separation,
total sophorolipid produced shown by open red circles. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this
article.)
Table 2
Separation results for fermentation 2, fermentation 3 and fermentation 4. All metrics are the result of unique fermentations, with the application of the novel integrated
gravity separation developed in this study.
Time (h) Sophorolipid
recovered
(g)
Sophorolipid
concentration
(g l−1
)
Total sophorolipid
present
(g)
Sophorolipid product
concentration
(g l−1
)
Enrichment Recovery at time
point
(%)
Fermentation 2
111 97.1 147.7 259.5 550.6 3.73 37
184 99.2 118.0 165.2 461.6 3.91 60
261 83.8 89.2 96.4 540.9 6.07 87
Total 280.1 86
Total oil removed by separation (g) 8 Total cells removed by separation (g) below detection limit
Fermentation 3
71.5 16.8 103.7 128.4 582.9 5.62 13
281 79.5 168.3 238.2 654.1 3.89 33
355 59.2 109.2 175.3 616.9 5.66 34
376 45.5 106.3 148.9 638.7 6.01 31
Total 201.0 74
Total oil removed by separation (g) 68 Total cells removed by separation (g) below detection limit
Fermentation 4
85.6 93.1 152.7 229.1 443.5 2.90 40
111.3 53.5 97.1 127.8 504.8 5.20 42
159.7 61.9 129.6 186.8 538.1 4.15 33
185.8 57.9 72.5 105.3 567.8 7.83 55
231.5 48.9 51.7 68.3 465.7 9.01 72
540.3 89.0 124.7 191.3 312.2 2.5 47
total 404.3 65
Total oil removed by separation (g) 20 Total cells removed by separation (g) 2.6
must be recycled back to the fermenter, would be identical irre-
spective of fermenter volume while maintaining a given size of
separator, hence a larger total fermenter volume would lead to a
large reduction in residual sophorolipid concentration.

8. Please cite this article in press as: B.M. Dolman, et al., Integrated sophorolipid production and gravity separation, Process Biochem
(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021
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Fig. 6. The effect of sophorolipid separation on agitation requirements. Dissolved oxygen and stirrer speed profiles showing the increase in stirrer speed required to maintain
the dissolved oxygen concentration after sophorolipid rich fractions separated during fermentation 2 returned to fermenter in fermentation 2 at 308 h.
Whilst the separator was designed for continuous operation
hydrodynamic considerations, in particular the turbulence caused
by inlet and outlet disturbances which become more significant
with decreasing scale, mean it could not be scaled down further to
match the sophorolipid production rates achieved in the 1 l initial
working volume fermenter. At the scale presented in this paper,
the separation occurred at a rate of around 2 ml min−1, or around
1 g min−1 sophorolipid, 30–150 times greater than the production
rate. This separation rate makes it suitable for use with a separa-
tor of 30–60 l volume for continuous separation of the sophorolipid
produced. Product recovery rate is expected to scale proportionally
to the volume of the separating column, so for a new settler design
volume can be increased while maintaining or reducing the ratio of
inertial to viscous forces, i.e. the Reynolds number. Residence time
should be increased proportionally to diameter increase, to allow
the same quantity of sophorolipid to settle or float to the surface at
the same settling/rising rate. The system could easily be connected
for steam in place sterilization at industrial scale.
This is the first study to present the design and demonstrate
the feasibility of a separation system for sophorolipid production
that could be continuously applied, having been shown in this
manuscript to separate sophorolipid whilst production continues
in the bioreactor for periods of more than one hour. It is also the
first integrated separation system applicable to large scale fermen-
tation, because it does not rely on separation within the bioreactor,
and therefore the first to enable an extended sophorolipid produc-
tion period at scale. The reduced bioreactor volume, and reduced
start up and cleaning costs of this system could significantly
improve the economics of sophorolipid production by increasing
the total sophorolipid produced per batch. This would make bulk
application, such as for enhanced oil recovery, a more realistic
proposition.
Other glycolipid biosurfactants, notably rhamnolipids and man-
nosylerythritol lipids (MELs), as well as many other bioproducts
including those used as biofuels, may also form a separate, insol-
uble, phase in a fermentation broth, and so the gravity separation
technique presented in this paper could likely also be applied to
these systems [27,28].
3.3. Effect on hydrodynamics and mass transfer
Fig. 6 shows the dissolved oxygen level and stirrer speed at the
end of fermentation 2, capturing the addition of the sophorolipid
rich fractions which were removed by separation during fermen-
tation 2 and subsequently pooled, and added to the bioreactor at
308 h. The presence of this sophorolipid phase effectively reduced
the volumetric oxygen transfer coefficient, kLa, in the fermenter,
due to its high viscosity of around 0.5 Pa.s, resulting in an increase
in stirrer speed to maintain the dissolved oxygen at the set point.
A stirring rate increase of around 75 rpm, from 500 rpm to 575 rpm
was required to maintain the desired dissolved oxygen level when
the sophorolipid product separated over the whole fermentation
Fig. 7. Mass spectrum of sophorolipid from the end of fermentation 2. Key peaks of C18:1 diacylated acidic sophorolipid at m/z 705 and C18:1 lactonic sophorolipid at m/z
687.

9. Please cite this article in press as: B.M. Dolman, et al., Integrated sophorolipid production and gravity separation, Process Biochem
(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021
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PRBI-10893; No.of Pages10
B.M. Dolman et al. / Process Biochemistry xxx (2016) xxx–xxx 9
was added. Given the high viscosity of the sophorolipid phase, it
is possible that turbulence was not attained even at the higher
agitation rate; in this instance a still higher agitation rate would
be required for proper dissolved oxygen control throughout the
bioreactor, leading to larger savings than calculated.
The mixing Power number in the turbulent regime is typically
constant, so changes in impeller speed have a cubic impact on the
mixing power. Removing the sophorolipid phase resulted in a 13%
decrease in impeller speed and a 34% decrease in mixing power
requirement by the end of the fermentation.
The relative power requirements over fermentation 1 and 2
were also compared, with an 18% reduction in power input from the
time point of the first separation observed, and a 9% improvement
when the whole fermentation is taken into account.
There would be some increased power consumption from the
pumping of the fermentation broth between the separator and the
fermenter, but it is expected this would be significantly smaller
than the agitation power at scale, as relatively low pumping flow
rates are used. If these power reductions can be achieved at indus-
trial scale, they can give significant cost savings.
3.4. Sophorolipid structure
Mass spectra of sophorolipid samples were taken to determine
the sophorolipid structures produced during the fermentation,
with the mass spectrum of the sophorolipids taken from the end
of fermentation 2 shown in Fig. 7. The main peak at m/z=705
represents a diacylated acidic C18:1 sophorolipid, with the peak
at 687 representing a diacylated C18:1 lactonic sophorolipid [29].
There were some differences in the ratios of peak heights between
sophorolipid taken from the settler and sophorolipid taken from
the fermentation broth immediately before, suggesting this tech-
nology may have potential to act as a crude separator of different
sophorolipid forms.
Recent research has revealed the enzyme responsible for lac-
tonisation of acidic sophorolipids, enabling the use of genetic
engineering of the genome of Starmerella bombicola, and robust
production and purification techniques to yield a 98% pure lactonic
or acidic sophorolipid, making the application of sophorolipid in
medicinal or personal care products much more likely [3]. Comple-
mentary to the advances in genetic engineering and purification,
this work makes significant progress in solving some of the complex
engineering challenges involved with sophorolipid production, giv-
ing potentially dramatic reductions in production cost and making
large scale application of sophorolipid more feasible.
4. Conclusions
A novel method for the integrated separation of sophorolipid
from a fermentation process has been developed. The design of the
system overcomes the production and processing difficulties asso-
ciated with in situ (i.e. in the fermenter vessel) gravity separation
of sophorolipids for scale up, and with a separator residence time
of less than two minutes the process seemed to have no impact on
further sophorolipid production by the cells. A sophorolipid phase
can be removed from the fermentation broth if the product phase
had higher or lower density than the media, with enrichments of
up to 9, an overall recovery of 86%, and up to 404 g of sophorolipid
recovered from the fermentation broth.
We demonstrated an 11% decrease in bioreactor working vol-
ume requirement when using the separator, due to the removal of
the sophorolipid product phase. Optimised feeding rates and settler
usage could further reduce the volume requirement. By using the
separator, the fermentation could be run for 1023 h, and produce
623 g sophorolipid from a 1 l initial batch, reducing the number of
fermentations required for a given product mass.
An 18% average reduction in stirrer power was demonstrated
over the course of a fermentation once sophorolipid separation was
initiated, which translates to around 9% over the entire fermenta-
tion, with a 34% decrease in power input shown by the end of the
fermentation.
The integrated separation system presented in this paper has
been developed for sophorolipid separation, but could equally be
applied to the production of other insoluble bioproducts, in par-
ticular mannosylerythritol lipids. With correct scaling up, it is
anticipated that the advantages this system offers will lead to a
dramatic improvement of the economics of sophorolipid produc-
tion.
Acknowledgements
Sara Bages Estopa is acknowledged for her invaluable comments
on the paper, and Reynard Spiess and Shaun Leivers are acknowl-
edged for their help with mass spectrometry.
The technology described in this manuscript has been filed for a
patent entitled ‘Improvements in and related to lipid production’.
The authors are grateful for financial support from the UK Engi-
neering and Physical Sciences Research Council (EP/I024905) and
the EPSRC DTA fund, which enabled this work to be conducted.
References
[1] R. Geys, W. Soetaert, I. Van Bogaert, Biotechnological opportunities in
biosurfactant production, Curr. Opin. Biotechnol. 30 (2014) 66–72.
[2] I. Van Bogaert, K. Saerens, C. De Muynck, D. Develter, W. Soetaert, E.J.
Vandamme, Microbial production and application of sophorolipids, Appl.
Microbiol. Biotechnol. 76 (2007) 23–34.
[3] S.L.K.W. Roelants, K. Ciesielska, S.L. De Maeseneire, H. Moens, B. Everaert, S.
Verweire, Q. Denon, B. Vanlerberghe, I. Van Bogaert, P. Van der Meeren, B.
Devreese, W. Soetaert, Towards the industrialization of new biosurfactants:
biotechnological opportunities for the lactone esterase gene from Starmerella
bombicola, Biotechnol. Bioeng. 113 (2016) 550–559.
[4] M.A. Díaz De Rienzo, I.M. Banat, B. Dolman, J. Winterburn, P.J. Martin,
Sophorolipid biosurfactants: possible uses as antibacterial and antibiofilm
agent, N. Biotechnol. 32 (2015) 720–726.
[5] V. Shah, G.F. Doncel, T. Seyoum, K.M. Eaton, I. Zalenskaya, R. Hagver, A. Azim,
R. Gross, Sophorolipids, microbial glycolipids with anti-human
immunodeficiency virus and sperm-immobilizing activities, Antimicrob.
Agents Chemother. 49 (2005) 4093–4100.
[6] L. Shao, X. Song, X. Ma, H. Li, Y. Qu, Bioactivities of sophorolipid with different
structures against human esophageal cancer cells, J. Surg. Res. 173 (2012)
286–291.
[7] A.E. Elshafie, S.J. Joshi, Y.M. Al-Wahaibi, A.S. Al-Bemani, S.N. Al-Bahry, D.
Al-Maqbali, I.M. Banat, Sophorolipids production by Candida bombicola ATCC
22214 and its potential application in microbial enhanced oil recovery, Front.
Microbiol. 6 (2015).
[8] L. Ławniczak, R. Marecik, L. Chrzanowski, Contributions of biosurfactants to
natural or induced bioremediation, Appl. Microbiol. Biotechnol. 97 (2013)
2327–2339.
[9] A.M. Davila, R. Marchal, J.P. Vandecasteele, Sophorose lipid fermentation with
differentiated substrate supply for growth and production phases, Appl.
Microbiol. Biotechnol. 47 (1997) 496–501.
[10] Institut Francais Du Petrole. Method of production of sophorosides by
fermentation with fed batch supply of fatty acid esters or oils. US. Patent. US
5616479A.
[11] U. Rau, S. Hammen, R. Heckmann, V. Wray, S. Lang, Sophorolipids A source for
novel compounds, Ind. Crops Prod. 13 (2001) 85–92.
[12] A.M. Davila, R. Marchal, J.P. Vandecasteele, Kinetics and balance of a
fermentation free from product inhibition: sophorose lipid production by
Candida bombicola, Appl. Microbiol. Biotechnol. 38 (1992) 6–11.
[13] N. Kosaric, F.V. Sukan, Biosurfactants:Production and Utilization-Processes,
Technologies and Economics, CRC Press, Boka Raton, 2014.
[14] U. Rau, C. Manzke, F. Wagner, Influence of substrate supply on the production
of sophorose lipids by Candida bombicola ATCC 22214, Biotechnol. Lett. 18
(1996) 149–154.
[15] H.J. Daniel, R.T. Otto, M. Reuss, C. Syldatk, Sophorolipid production with high
yields on whey concentrate and rapeseed oil without consumption of lactose,
Biotechnol. Lett. 20 (1998) 805–807.
[16] O. Stüwer, R. Hommel, D. Haferburg, H.P. Kleber, Production of crystalline
surface-active glycolipids by a strain of torulopsis apicola, J. Biotechnol. 6
(1987) 259–269.

10. Please cite this article in press as: B.M. Dolman, et al., Integrated sophorolipid production and gravity separation, Process Biochem
(2016), http://dx.doi.org/10.1016/j.procbio.2016.12.021
ARTICLE IN PRESSG Model
PRBI-10893; No.of Pages10
10 B.M. Dolman et al. / Process Biochemistry xxx (2016) xxx–xxx
[17] H.J. Daniel, M. Reuss, C. Syldatk, 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, Biotechnol. Lett. 20 (1998) 1153–1156.
[18] G.L. Kleman, W.R. Strohl, High cell density and high-productivity microbial
fermentation, Curr. Opin. Biotechnol. 3 (1992) 93–98.
[19] Institut Francals du Petrole. Process for the production of sophorolipids by
cyclic fermentation with feed of fatty acid esters or oils. US. Patent. US
5879913A.
[20] V. Guilmanov, A. Ballistreri, G. Impallomeni, R.A. Gross, Oxygen transfer rate
and sophorose lipid production by Candida bombicola, Biotechnol. Bioeng. 77
(2002) 489–494.
[21] S. Alonso, P.J. Martin, Impact of foaming on surfactin production by Bacillus
subtilis: implications on the development of integrated in situ foam
fractionation removal systems, Biochem. Eng. J. 110 (2016) 125–133.
[22] J. Beuker, A. Steier, A. Wittgens, F. Rosenau, M. Henkel, R. Hausmann,
Integrated foam fractionation for heterologous rhamnolipid production with
recombinant Pseudomonas putida in a bioreactor, AMB Express 6 (2016) 11.
[23] J.B. Winterburn, A.B. Russell, P.J. Martin, Integrated recirculating foam
fractionation for the continuous recovery of biosurfactant from fermenters,
Biochem. Eng. J. 54 (2011) 132–139.
[24] A. Daverey, K. Pakshirajan, Production, characterization, and properties of
sophorolipids from the yeast Candida bombicola using a low-cost
fermentative medium, Appl. Biochem. Biotechnol. 158 (2009) 663–674.
[25] R. Gao, M. Falkeborg, X. Xu, Z. Guo, Production of sophorolipids with
enhanced volumetric productivity by means of high cell density fermentation,
Appl. Microbiol. Biotechnol. 97 (2013) 1103–1111.
[26] Y.B. Kim, H.S. Yun, E.K. Kim, Enhanced sophorolipid production by
feeding-rate-controlled fed-batch culture, Bioresour. Technol. 100 (2009)
6028–6032.
[27] U. Rau, L.A. Nguyen, H. Roeper, H. Koch, S. Lang, Downstream processing of
mannosylerythritol lipids produced by Pseudozyma aphidis, Eur. J. Lipid. Sci.
Technol. 107 (2005) 373–380.
[28] M.C. Cuellar, L.A.M. van der Wielen, Recent advances in the microbial
production and recovery of apolar molecules, Curr. Opin. Biotechnol. 33
(2015) 39–45.
[29] K. Joshi-Navare, P. Khanvilkar, A. Prabhune, Jatropha oil derived
sophorolipids: production and characterization as laundry detergent additive,
Biochem. Res. Int. 2013 (2013) 1–11.