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Integrated sophorolipid production and gravity separation
Authors
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
*Corresponding author: [email protected], 0161 306 4891
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Abstract
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 fermentation 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 demonstrated on three sophorolipid producing
fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter
volume required whilst maintaining sophorolipid production. Sophorolipid recoveries 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.
Keywords
Integrated separation, sophorolipid, settling, Candida bombicola, biosurfactant, glycolipid
Chemical compounds
Sophorolipid (PubChem CID: 11856871)
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Introduction
Sophorolipids are microbially produced glycolipid biosurfactants, 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 only to
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 Figure 1 [2].
Figure 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.
Sophorolipids are produced industrially by a number of companies, 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, 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 concentrations of over 300 g l−1, with
productivities of over 1 g l−1 h−1 are routinely achieved [9-11]. Sophorolipid producing
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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 hydrophobic carbon source are present [12]. The sophorolipid
production 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].
The physical form of sophorolipids is dependent on the conditions 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 fermentation 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
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lengthy inoculum preparation required for large scale production [18, 19]. There are
numerous proposed partial solutions to this problem in the literature. For example, a portion
of the broth can be removed and replaced with fresh media, allowing high productivity to be
maintained for seven 80-130 hour 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. Significant
benefits of sophorolipid separation have been shown, with a doubling of the duration of
sophorolipid production and little effect on production after 15 minutes without agitation or
aeration demonstrated 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 fermentation 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.
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Methodology
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 concentration 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 conducted 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 separator. 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 hours. Single colonies from these plates were then used to inoculate 50 ml of medium in
250 ml shake flasks, which were incubated at 25°C and 200 rpm for 30 hours. This inoculum
was diluted to an optical density of 20 at 600 nm with fresh media and 100 ml used to
inoculate the fermenter.
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Fermentations were run at 25°C, and dissolved oxygen was controlled to 30% by varying the
stirrer speed, whilst maintaining a constant aeration rate of 1 l min−1. Fermenter pH was
controlled to a value of 3.5 by the addition of 3M 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 separation 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 Figure 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.
Separation
Integrated separation of the sophorolipid from the fermentation broth was carried out using
an in house built settling column, as shown in Figure 2. The integrated arrangement of the
bioreactor and settling column is shown in Figure 3 with the settling column supported 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
observation of a sample taken from the bioreactor. When a significant layer of sophorolipid
rich phase could be seen at either the top or bottom of the sample in the universal bottle
within 2 minutes separation was initiated. Prior experiments indicated that at these conditions
separation is effective.
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During separation, which was used intermittently during fermentation, 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 bottom of the fermenter, and then in 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
experiments, 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 settling 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 column. 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 settling
vessel. The separation was stopped when the separation rate dropped below 0.5 ml min -1,
until the condition for separation was again observed.
Figure 2-Diagram of custom built sophorolipid separator used for this study. Plan view, side view and end view are
shown, with all dimensions in mm.
Figure 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.
Hydrodynamics
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To determine the effect of the sophorolipid phase on the agitation 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 fermenter at
308 h, after the start of the fermentation over a period of 12 minutes. 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=N p ρ n3 D5 (1)
where P is power, Np is power number, ρ is density, n is stirrer speed and D is stirrer
diameter.
Due to identical fermenters being used and assuming the density 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 Eq. 2 and 3;
enrichment=C p
C f(2 ) recovery=
Cp ×V p
C f ×V f(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.
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 connected to the separator. The sample was
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centrifuged at 5000 rpm for 5 minutes using a Sigma 6-16S centrifuge (Sigma laboratory
centrifuges, Germany) and the glucose in the supernatant quantified using a TrueResult®
blood glucose monitor (Nipro, Japan).
A hexane extraction to extract residual rapeseed oil followed by a triple ethyl acetate
extraction to extract sophorolipid were then applied to the whole sample, with oil
concentration 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 minutes. 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 using negative ionisation
electrospray ionisation, 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 µm filter. Flow injection analysis was used, at 0.3 ml
min-1, 50 % acetonitrile, 0.1 % formic acid, 49.9 % water, with an injection volume of 2 µl.
The viscosity of the product phase was measured using an AR2000 controlled rotational
rheometer with cone geometry (TA Instruments, USA).
Results and discussionA novel gravity sophorolipid separation technique was successfully applied to three
fermentations. Results from one fermentation without separation, fermentation 1, are
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presented alongside results from three fermentations with separation, fermentations 2, 3 and
4. Fermentations 1 and 2, without and with integrated separation 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 accumulated, with the majority of the
available sophorolipid phase separated.
Fermentations
Figure 4 shows the feeding rate of substrates for the fermentations presented, which enabled
control of the sophorolipid phase to separate 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 Figure 5, and the key metrics from these
fermentations are presented in Table I.
Figure 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).
Table I- 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 fermenter working volume and the total sophorolipid produced.
Figure 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.
Figure 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
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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 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 separator at 71.5 h in fermentation 3,
when a sophorolipid phase could be observed to settle in a sample bottle within 2 minutes.
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 relatively
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 produced 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 in line with other results in the literature,
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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 produced, 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 this reduction in product yield on biomass in
fermentation 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 dictates 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 separator. This meant only
1540 ml working volume was needed for fermentation 2 compared 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 corresponding 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 demonstrated a fairly
constant production rate throughout a fermentation until the point at which the fermentation
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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 accumulation, and run for a total of 1023 h. This enabled
the production 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 fermentation.
This could lead to a dramatic improvement in overall process productivity, by reducing the
proportion of time spent in innoculum preparation, biomass production and cleaning.
Separation
The separation results achieved in fermentations 2, 3 and 4 are shown in Table II.
Table II-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.
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 separation 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 separator 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 feeding 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.
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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 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
concentration above that demonstrated in fermentation 3 using the current technique,
however, with an increased fermenter volume, the system could operate at lower initial
sophorolipid concentrations and so give an improved enrichment.
Sophorolipid recoveries would be expected to improve significantly 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 must be recycled back to the fermenter, would be identical irrespective 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.
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 separator of 30-60 l volume for continuous separation of the
sophorolipid produced. Product recovery rate is expected to scale proportionally to the
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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 fermentation, because it does not rely on separation within the bioreactor, and
therefore the first to enable an extended sophorolipid production period at scale. The reduced
bioreactor volume, and reduced start up and cleaning costs this system could facilitate by
increasing the total sophorolipid produced per batch could significantly improve the
economics of sophorolipid production. This would make bulk application, such as for
enhanced oil recovery, a more realistic proposition.
Other glycolipid biosurfactants, notably rhamnolipids and mannosylerythritol lipids (MELs),
as well as many other bioproducts including those used as biofuels, may also form a separate,
insoluble, 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].
Effect on hydrodynamics and mass transfer
Figure 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.
Figure 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 fermentation 2 and subsequently pooled, and added to the bioreactor at 308 h. The
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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
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 industrial scale, they can give significant cost
savings.
Sophorolipid structure
Figure 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.
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
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the end of fermentation 2 shown in Figure 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 technology may have potential to act as a crude separator
of different sophorolipid forms.
Recent research has revealed the enzyme responsible for lactonisation of acidic
sophorolipids, enabling the use of genetic engineering of the genome of S.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]. Complementary 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, giving potentially dramatic
reductions in production cost and making large scale application of sophorolipid more
feasible.
ConclusionsA 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 associated 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.
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We demonstrated an 11% decrease in bioreactor working volume 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 fermentation, 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
particular mannosylerythritol lipids. With correct scaling up it is anticipated the advantages
this system offers will lead to a dramatic improvement of the economics of sophorolipid
production.
Acknowledgements
Sara Bages Estopa is acknowledged for her invaluable comments on the paper, and Reynard
Spiess and Shaun Leivers are acknowledged 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 Engineering and Physical Sciences Research Council (EP/I024905) and
the EPSRC DTA fund, which enabled this work to be conducted.
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Tables
Table I- 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 fermenter working volume and the total sophorolipid produced.
Fermentation1 2 3 4
Product separation none top bottom topDuration (h) 305 305 379 1023Yield substrate consumed (g g−1) 0.43 0.53 0.42 0.53Yield substrate fed (g g−1) 0.33 0.37 0.39 0.47Productivity (g l−1 h−1) 1.07 1.07 0.71 0.61Maximum fermenter volume (l) 1720 1544 1350 1550Total sophorolipid produced (g) 325 325 270 623Total dry cell weight (g) 16.1 21 16.5 32.1Yield product on biomass (g g−1) 20.2 15.5 16.4 19.4
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Table II-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 2111 97.1 147.7 259.5 550.6 3.73 37184 99.2 118.0 165.2 461.6 3.91 60261 83.8 89.2 96.4 540.9 6.07 87
Total 280.15 86
Total oil removed by separation (g) 8 Total cells removed
by separation (g)
below detection
limitTime (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 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
limitTime (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 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
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Figures
Figure 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.
Figure 2-Diagram of custom built sophorolipid separator used for this study. Plan view, side view and end view are
shown, with all dimensions in mm.
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Figure 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.
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Sophorolipid product
Fermentation broth
Sophorolipid depleted broth return
Sophorolipid product
Fermentation broth
Sophorolipid depleted broth return(a)
(b)
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Figure 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).
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(a)
(c)
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Figure 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.
(a)
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Figure 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.
Figure 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.
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